afab volume 4 issue 2

Volume 4, Issue 22014

ISSN: 2159-8967www.AFABjournal.com

72 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 2 - 2014 73

Sooyoun Ahn University of Florida, USA

Walid Q. Alali University of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Debabrata Biswas University of Maryland, USA

Claudia S. Dunkley University of Georgia, USA

Michael Flythe USDA, Agricultural Research Service

Lawrence Goodridge McGill University, Canada

Leluo Guan University of Alberta, Canada

Joshua Gurtler ERRC, USDA-ARS, USA

Yong D. Hang Cornell University, USA

Armitra Jackson-Davis Alabama A&M University, USA

Divya Jaroni Oklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael Johnson University of Arkansas, USA

Timothy Kelly East Carolina University, USA

William R. Kenealy Mascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

Woo-Kyun Kim University of Georgia, USA

M.B. Kirkham Kansas State University, USA

Todd Kostman University of Wisconsin, Oshkosh, USA

Y. M. Kwon University of Arkansas, USA

Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain

Melanie R. Mormile Missouri University of Science and Tech., USA

Rama Nannapaneni Mississippi State University, USA

Jack A. Neal, Jr. University of Houston, USA

Benedict Okeke Auburn University at Montgomery, USA

John Patterson Purdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos Rostagno LBRU, USDA-ARS, USA

Roni Shapira Hebrew University of Jerusalem, Israel

Kalidas Shetty North Dakota State University, USA

EDITORIAL BOARD


EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA

EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA

Philip G. CrandallUniversity of Arkansas, USA

Janet Donaldson Mississippi State University, USA

Ok-Kyung KooKorea Food Research Institute, South Korea

MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium

TECHNICAL EDITORJessica C. ShabaturaFayetteville, USA

ONLINE EDITION EDITORC.S. ShabaturaFayetteville, USA

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EDITORIAL STAFF


Antibiotic Use in Livestock ProductionBroadway, P. R., J. A. Carroll, and T. R. Callaway

76

Effects of Co-nutrients in Foods and Bioremediation in the Environment on Methylmercury

P. G. Crandall, C. A. O’Bryan

86

Alternative antimicrobial supplements that positively impact animal health and food safety Broadway, P. R., J. A. Carroll, and T. R. Callaway

109

Human Health Benefits of Isoflavones from Soybeansk. Kushwaha, C. A. O’Bryan, D. Babu, P. G. Crandall, P. Chen, and S.-O. Lee

122

REVIEW

Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure CompostingM.C. Erickson, J. Liao, X. Jiang, and M.P. Doyle

96

ARTICLES

Instructions for Authors147

Introduction to Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

TABLE OF CONTENTS


www.afabjournal.comCopyright © 2014

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Antibiotic usage is a useful and commonly implemented practice in livestock and production ag-

riculture that has progressively gained attention in recent years from consumers of animal products due

to concerns about human and environmental health. Sub-therapeutic usage of antibiotics has led to a

concern that prophylactic supplementation leads to antimicrobial resistance, and this particular practice

has come under public scrutiny. The consumer and media misconceptions about antibiotic usage and pro-

duction strategies utilized in livestock production have caused a shift in consumer demands. Antibiotics

directly and indirectly affect the livestock industry by treating illness and promoting the overall health of

the animal, which may enhance production parameters such as growth and profitability. However, pending

legislation threatens to eliminate the current antibiotic usage strategies implemented by producers. This

review will address the historical and current use of antibiotics as it pertains to production animal agricul-

ture to summarize how antibiotics promote animal health and growth performance.

Keywords: Antibiotic, livestock, animal health, review

INTRODUCTION

Antibiotic usage in meat animal production is

a hotly debated issue in the livestock industry that

has acquired more attention as consumers seek to

place more “natural” and “safer” products on their

Correspondence: Todd Callaway, [email protected]: +1-979-260-9374 Fax: +1-979-260-9332.

table (Gilbert and McBain, 2003). Consumer percep-

tion can greatly influence food animal production as

has been recently observed for some common food

production practices; such as lean finely textured

beef (“pink slime”) which was removed from meat

formulations of producers due to negative media at-

tention and consumer perception (Flock, 2012). The

use of gestation crates in swine production has also

drawn increasing attention, leading to the refusal of

REVIEWAntibiotic Use in Livestock Production

P. R. Broadway1, J. A. Carroll2, and T. R. Callaway3

1Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX2Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX

3Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, USDA, College Station, TX

“Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or exclusion of others that may

be suitable.” USDA is an equal opportunity provider and employer

Agric. Food Anal. Bacteriol. 4: 76-85, 2014


some retailers and restaurants to purchase pork from

producers that utilize gestation crates (Food Safety

News, 2013). Furthermore, antibiotic usage in ani-

mals for health benefits and growth promotion has

continued to be a concern of the American consum-

er in recent years. In response to similar concerns,

the European Union (EU) banned sub-therapeutic

supplementation of animal feeds with antibiotics

(Pradella, 2006). Recently, the U.S. Food and Drug

Administration issued a guidance directive on the

judicious use of antibiotics in food animals, and this

measure has led some to believe that this is phase

one of an agenda to remove sub-therapeutic antibi-

otic use from livestock production.

The gastrointestinal tract of animals is populated

with a complex microbial ecosystem that is essen-

tial for the function, growth, and overall health of

the animal (Chaucheyras-Durand and Durand, 2010).

Many livestock producers currently utilize feeding

and production strategies, including the use of an-

tibiotics, that alter the microbial ecology of the gas-

trointestinal tract of the animal to benefit the overall

health and production efficiency of their animals. As

a bonus to the consumer, some of these strategies

may also help eliminate or reduce foodborne patho-

gens that may contaminate the food supply (Perl-

man, 1973). If and when sub-therapeutic antibiotic

use in food animals is banned in the U.S., alternative

strategies must be implemented to replicate these

positive effects in order for the livestock industry to

remain viable.

CURRENT USE OF ANTIBIOTICS IN LIVE-STOCK

Antibiotics are used in the livestock industry for

a variety of reasons including treatment of disease,

prophylaxis, as well as improving feed efficiency

and overall growth performance (Berge et al., 2005;

Brown et al., 1975). While antibiotics do not make

label claims that suggest alteration of growth param-

eters in livestock, the association between their use

and growth promotion has been reported in many

species such as cattle, swine, and poultry for over 50

years (Moore et al., 1946; Jukes et al., 1950; Rogers et

al., 1995; Salinas-Chavira et al., 2009). Performance

parameters can be quantitatively measured in a va-

riety of ways including, but not limited to: mortality,

weight gain, meat/milk quality, and feed efficiency.

While the mode of action by which antibiotics im-

prove feed efficiency has not been fully elucidated,

growth performance may be enhanced due to de-

creased inflammation in the small intestine (Feighner

and Dashkevicz, 1987; Eyssen and DeSomer, 1963).

To further explain how antibiotics may work in con-

junction to promote animal health and food safety,

McCracken and Gaskins (1999) indicated that the de-

velopment of the intestinal immune system occurs

in conjunction with the development of the normal

microflora of the animal; however chronic stimula-

tion of the immune system may decrease the amount

of protein available for growth (Gordon et al., 1963).

Studies comparing germ-free and conventionally

raised animals have demonstrated this phenomenon

and have reported alterations in immune function of

these animals in conjunction with the development

of the intestinal microflora (McCraken and Lorenz,

2001). Thinning of the intestinal epithelium in con-

junction with the use of antibiotics may be the result

of decreased microbial production of polyamines and

volatile fatty acids (VFAs) that enhance intestinal cell

growth and activity (Ferket et al., 2002). Ferket et al.

(2002) states that intestinal mucosal thinning that may

occur with the use of antibiotics may increase energy

availability for growth because the animal does not

have to maintain a larger intestinal mucosal layer.

Cattle

Antibiotics have been used for decades in cattle,

and some of the most commonly used antibiotics in

the feedlot setting are a class of compounds known

as ionophores (Russell and Strobel, 1989). Iono-

phores were approved for use in ruminants in the

1970s (Russell and Strobel, 1989). The ionophore

monensin was fed to chickens as a coccidiostat, and

the manure from these poultry houses was spread

on cattle pastures as a fertilizer. Cattle grazing these

pastures grew more rapidly than cattle grazing pas-


tures fertilized with manure from poultry houses

where the chickens were not fed monensin (Callaway,

2013). As a result, the ionophore monensin was di-

rectly incorporated into cattle rations beginning in

the 1970’s, and this compound has been reported

to enhance growth performance through a variety

of modifications of the ruminal microbiome (Raun et

al., 1976; Callaway et al., 2003). Ionophores primar-

ily inhibit bacteria with Gram positive physiology, in-

cluding lactic acid bacteria, and this improves growth

efficiency, average daily gain (ADG), reduces waste-

ful protein degradation (by hyperammonia produc-

ing bacteria), reduces methanogenesis, and reduces

ruminal acidosis via lower lactate production (Russell

and Strobel, 1988). Ionophores have been reported

to reduce liver abscesses by inhibiting epithelial ke-

ratinization caused by lactic acidosis and subsequent

Fusobacterium necrophorum infections (Nagaraja

and Chengappa, 1998; Lechtenberg et al., 1998).

While compounds such as ionophores alter the

microbial ecology of the gastrointestinal tract to pro-

mote overall health and performance, other antibi-

otics are used to treat specific bacterial disease and

illness. Some of these antibiotics may also elicit a

dual effect, promoting both health and performance

in the animals. Bovine respiratory disease (BRD) is

the most common and expensive disease present in

American cattle, and the use of antibiotics to treat/

prevent this disease is a great example of this dual

effect of antibiotics (Smith, 1998; Snowder et al.,

2006). Bovine respiratory disease is a complex dis-

ease caused by exposure to various viral (e.g., Infec-

tious Bovine Rhino-tracheitis, Bovine Viral Diarrhea,

Bovine Respiratory Syncytial Virus, and Parainfluenza

Virus) and/or bacterial (e.g.., Pasteurella hemolytica,

Pasteurella multocida, Haemophilussomnus, Myco-

plasmasp. and Actinomycespyogenes) pathogens.

Bovine respiratory disease may be mitigated in a

number of ways including vaccination, management

practices, and antibiotic treatments to prevent and/

or treat the disease. Addition of chloratetracycline

and sulfamethazine to treat enteritis, coccidiosis, and

bovine respiratory disease (BRD) in the ration of cattle

arriving at the feed lot was also reported to increase

ADG while decreasing the risk of bovine respiratory

disease for the first 28 days at the feedlot (Guillermo

and Berg, 1995; Smith et al., 1993). Another com-

monly used antibiotic in beef production is Tilmico-

sin which is a broad spectrum antibiotic used to treat

and prevent BRD. Tilmicosin works to inhibit protein

synthesis of bacteria such as Pasteurella hemolytica

that may lead to the onset of BRD. Treatment of cat-

tle upon arrival into feedlots with Micotil®, a solution

of Tilmicosin, was shown to decrease BRD symptoms

and increase dry matter intake (Galyean et al., 1995).

Antibiotics are also used in livestock to prevent

specific physiologic disorders such as ruminal lactic

acidosis, a common problem in grain fed cattle that

can be chronic or acute and range from moderate to

severe (Nagaraja and Titgemeyer, 2007; Slyter, 1976;

Muir et al., 1981; Nagaraja et al., 1982). Ruminal aci-

dosis is the accumulation of lactate in the rumen re-

sulting in a lowered pH that decreases animal growth

performance parameters, and leads to the devel-

opment of other health problems such as laminitis,

bloat, and liver abscesses (Nagaraja and Chengappa,

1998; Nocek, 1997; Enemark, 2008). In acute clinical

lactic acidosis, D-lactate is the acid primarily respon-

sible for this condition (Dunlop, 1965); however, the

role of lactate in sub-acute acidosis is not fully under-

stood (Enemark, 2009). The onset of acidosis is linked

with feeding readily fermentable carbohydrates that

are commonly associated with a high concentrate ra-

tion as would normally be fed in the cattle feedlot

or swine finishing production systems (Owens et al.,

1998; Russell and Hino, 1985).

Antibiotics/antimicrobials and other feedstuffs

have been reported to be effective strategies to pre-

vent the onset of ruminal acidosis (Owens et al.,1998;

Callaway et al., 2003). Antibiotics may decrease the

incidence of liver abscesses in cattle which may be

the result of ruminal acidosis and may predict carcass

performance (Rogers et al., 1995; Brown and Law-

rence, 2010). Virginamycin is an antibiotic used to

prevent necrotic enteritis in cattle and has also been

reported to increase the gain to feed ratio in cattle

(Salinas-Chavira et al., 2009). Rogers et al. (1995) re-

ported an increase in ADG and feed conversion, and

a decrease in liver abscesses in cattle fed virginamy-

cin.


Swine

As in ruminants, such as cattle, antibiotics are used

in swine production for many of the same reasons.

These pharmaceuticals are used in swine for both pro-

phylactic and treatment therapies, and in some cases,

these antibiotics can also effect performance parame-

ters. Jensen et al. (1955) reported increased gains and

feed conversion in swine fed the antibiotic aureomy-

cin. While aeuromycin was also initially reported to en-

hance reproductive performance in swine (Yestal et al.,

1952), subsequent work by Davey et al. (1955) reported

no difference in reproductive performance when swine

were fed various concentrations of the antibiotic. Via-

bility and performance of newborn and suckling piglets

was also unaffected when swine were supplemented

with aureomycin (Davey et al., 1955). Aureomycin was

further reported to increase profitability by increasing

belly weight and decreasing backfat thickness (Perry et

al., 1953). Zimmerman (1986) reported that antibiot-

ics such as chloratetracyline, furazolidone, lincomycin,

salinomycin, tylosin, and virginamycin may improve

average weight gain by approximately 15%. Addition-

ally, Zimmerman (1986) reported that combined use

of chloratetracycline, penicillin, and sulfamethazine

(2:1:2) increased ADG in starter pigs by 25%. Multiple

studies in swine also indicate that treatment by any of

the aforementioned antibiotics can increase farrowing

rate (Zimmerman, 1986; Ruiz et al., 1968; Anderson,

1969; Hays 1978). Litter size may also be increased with

the addition of a combination of antibiotics (Zimmer-

man, 1986; Ruiz et al., 1968; Hays 1978). The antibiotics

penicillin and streptomycin increased the growth rate

of swine fed to market weight (Bridges et al., 1952).

Penicillin and streptomycin used in conjunction are still

approved for use in the swine industry, as well as bo-

vine, equine, and ovine species, to treat bacteria such

as Arcanobacterium, Klebsiella pneumonia, Listeria

spp., Mannheimia haemolytica, Pasteurella, Staphy-

lococcus, and Salmonella (Norbrook Laboratories,

2013). Tylosin is another antibiotic approved for use in

swine that can be provided via intramuscular injection,

feed, or water, and is effective in preventing and con-

trolling porcine proliferative enteropathy (ileitis; Para-

dis, 2004; Marseller et al., 2001; McOrist et al., 1997).

Tylosin supplemented in the drinking water of swine

for 17 days decreased clinical signs of gastrointestinal

infection and promoted growth performance (Paradis

et al., 2004). Tylosin-supplemented swine showed no

clinical or pathological signs of proliferative enteropa-

thy (ileitis) after experimental infection with Lawsonia

intracellularis (McOrist et al., 1997). The mitigation of

disease in concert with enhanced growth and repro-

ductive performance as a result of antibiotic usage in

swine help make the use of antibiotics a profitable pro-

duction strategy (Zimmerman, 1986).

Poultry

Antibiotic usage is an extremely important as-

pect of poultry production and has been used in

production and researched extensively since the

1950s (Feighner and Dashkevicz, 1987). Antibiotics

used in poultry production are believed to be effec-

tive growth promotants due to the alterations they

induce in the microflora of the gastrointestinal tract

(Feighner and Dashkevicz, 1987). This theory is sup-

ported by experiments that report germ-free chick-

ens grow more efficiently than commercially raised

poultry, and germ-free animals do not grow faster

when given antibiotics with growth promoting capa-

bilities (Coates et al., 1963; Forbes and Pank, 1959).

In poultry, antibiotic feeding has been reported to

increase weight gain and feed conversion efficiency

(feed/gain; Feighner and Dashkevicz, 1987; Bunyan

et al., 1977). Feed efficiency has been reported to

be improved in poultry supplemented with antibiot-

ics by reducing microbial populations in competition

for nutrients and reduction of pathogenic bacteria

(Feighner and Dashkevicz, 1987; Eyssen and de-

Somer, 1963; Barnes et al., 1978). Studies have re-

ported that ammonia production by bacteria in the

GI tract of monogastrics may suppress growth (Dang

and Visek, 1960; Harbers et al., 1963; Visek, 1978).

Deconjugation of bile salts may also play a role in

growth suppression due to Streptococcus faecium

in the small intestine; however, the use of antibiotics

has been reported to reduce attachment of this bac-

terium to intestinal epithelia (Cole and Fuller, 1984;

Fuller et al., 1984; Fuller et al., 1983).


Table 1. Some antibiotics used in animal agriculture that may be used to promote overall animal health and impact pathogen colonization and shedding

Some Antibiotics used in Animal Agriculture By Species

Cattlea Swineb Poultryc Aquacultured

Amoxicillin Apramycin Ardacin Amoxicillin

Ampicillin Bacitracin Avilamycin Ampicillin

Enroflaxin Bambermycin Avoparcin Chloramphenicol

Erythromycin Carbadox Bacitracin manganese Cortimoxazole

Florfenicol Chloratetracycline Erythromycin Enroflaxin

Oxytetracycline Furazolidone Lincomycine Erythromycin

Penicillin Lincomycin Mocimycin Florfenicol

Sulfadimethoxine Nosiheptide Neomycin Furazolidine

Tilmicosin Salinomycin Nosiheptide Nitrofurans

Tylosin Tiamulin Penicillin Oxolinic acid

Tylosin Soframycin Oxytetracycline

Virginamycin Tetracycline Sarafloxacin

Tylosin Streptomycin

Virginamycin Sulphadizine

Trimethoprim-

Sulfamethoxazole

aCurrin and Whittier, 2009b Zimmerman, 1986cCastanon, 2007dDefoirdt et al., 2011


The intestinal epithelia in poultry and other spe-

cies play a large role in the growth capabilities of

animals, and antibiotics can alter the intestinal micro-

flora as well as the intestinal epithelia of animals to

promote growth. As mentioned previously, thinner

intestinal epithelia may result in more efficient nutri-

ent uptake and absorption (Eyessen and deSomer,

1963; Ford and Coates, 1971; Siddons and Coates,

1972; Sieburth et al., 1951). Also, antibiotics reduce

populations of bacteria in the intestines, thereby

making more nutrients available for animal growth

(Eyssen, 1962; Monson et al., 1954). When antibiotics

reduce the microbial population in the GI tract, they

may inherently reduce pathogens responsible for dis-

ease or subclinical infections (Eyssen and deSomer,

1963a; Eyssen and deSomer, 1963b; Eyssen and de-

Somer, 1967; Sieburth et al., 1951). The combination

of all these effects elicited by antibiotics provides a

possible explanation as to why antibiotics enhance

growth performance and feed efficiency.

Aquaculture

As in mammalian production, antibiotics also play

a critical role in the aquaculture industry. Diseases in

production aquaculture are estimated to cause losses

of approximately 3 billion dollars per year globally

(Subasinghe, et al., 2001). There are more than 100

known pathogens to fish; however, some of these are

opportunistic pathogens (Alderman and Hastings,

1998). One of the main bacterial culprits are Vibrio

bacteria (harveyi, cambellii, and parahaemolyticus;

Defoirdt et al., 2007). While these pathogens are

detrimental to the health of the aquaculture, some

bacteria such as Vibrio cholera and vulnificus, may

cause human disease as well (Thompson et al., 2004).

Some of the antibiotics used in aquaculture are chlor-

amphenicol, gentamycin, trimethorprim, tiamulin,

tetracyclines, quinolones, and sulfonamides (Table 1;

Defoirdt et al., 2007). Most of these antibiotics are in-

corporated into the feed of the aquaculture at speci-

fied dosages with required withdrawal times (Alder-

man and Hastings, 1998). However, countries around

the world have vastly different regulations regarding

the administration, dosage, withdrawal, and control

of antibiotics in aquaculture (Alderman and Hastings,

1998). As in many food-producing species, antibiotic-

resistant bacteria such as Aeromonas salmonicida, A.

hydrophila, Vibrio anguillarum, Pseudomonas fluores-

cens, Pasteurella piscida, and Edwardsiella tarda have

been documented in aquaculture species (Aoki, 1988).

SUMMARY

Antibiotics are an important part of agriculture

and food production originating from the cattle,

swine, poultry, and aquaculture industries, and much

research has been conducted to determine the ef-

ficacy and safety of these pharmaceuticals. These

compounds are used not only to treat disease, but

can also be used effectively as a prophylactic treat-

ment. Such strategies to control pathogens in food-

producing animals may, in some cases, improve

growth performance parameters while simultaneous-

ly promoting the overall health of the animal. Thus,

antibiotics are a critical player in the profitability of

agriculture in the U.S. and throughout the world and

play a vital role in feeding the ever growing world

population. However, an ever changing population

and shifts in consumer demand have placed pres-

sure on the agricultural industry and governments

to reduce and/or eliminate the use of antibiotics in

food production. While this potential change could

possibly be detrimental to current management

strategies, there are potential alternatives to antibi-

otics that have been extensively researched in live-

stock to promote health, performance, profitability,

and food safety.

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ABSTRACT

Mercury, a potentially toxic metal, is present in the environment as a result of both natural processes and

from man-made sources. The amount of mercury mobilized and released into the biosphere has increased

significantly since the beginning of the industrial age. Inorganic mercury deposits in water and bottom sed-

iments where it is subject to bacterial conversion to methylmercury, which bioaccumulates in the aquatic

food chain with sometimes tragic consequences. This review discusses the production of methylmercury

in the environment and exposure to and health effects for humans. We also discuss current knowledge of

other nutrient interactions with methylmercury in the diet as well as possible methods for bioremediation

of methylmercury in the environment.

Keywords: Methylmercury, Minamata disease, mercury poisoning, biomagnification, bioaccumula-

tion, bioavailability, bioremediation

INTRODUCTION

The element mercury is a non-essential trace el-

ement that is toxic to humans and animals. At the

fifty-third meeting of the Joint FAO/WHO Expert

Committee on Food Additives (JECFA, 2000) an up-

date on the toxicity risks from methylmercury was

summarized and a provisional tolerable weekly in-

take of methylmercury for the general population

Correspondence: Philip G. Crandall, [email protected]: +1 -479-575-7686 Fax: +1-479-575-6936

(3.3 µg/kg body weight) was reaffirmed with the ad-

monition that pregnant women and nursing mothers

may be in a higher risk category. The US Environ-

mental Protection Agency has also calculated a ref-

erence dose (RfD) level for methylmercury, which is

EPA’s estimate of the maximum acceptable daily ex-

posure to humans that is not likely to cause harmful

effects during a lifetime. The RfD for methylmercury

was last revised by EPA 2001 and is currently 0.1 µg/

kg of body weight per day (Environmental Protec-

tion Agency, 2014), which is appreciably higher than

the JECFA recommendations. In the environment,

REVIEWEffects of Co-nutrients in Foods and Bioremediation

in the Environment on Methylmercury

P. G. Crandall, and C. A. O’Bryan

1 Department of Food Science, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704



particularly lakes, waterways and wetlands, mercury

can be converted from its elemental state to a high-

ly toxic, organic compound called methylmercury

through biogeochemical interactions. Once ingest-

ed methylmercury can easily cross the blood-brain

and placental barriers and high levels of exposure

may cause severe and irreversible damage, partic-

ularly to the fetal central nervous system (Clarkson

and Magos, 2006). Methylmercury concentrations

in water, soil, and sediments are usually very low es-

pecially when compared to the less toxic inorganic

form (Zhang et al., 2010a; 2010b). However, methyl-

mercury can accumulate (bioaccumulation) and be

magnified (biomagnification) in aquatic food webs

and even some terrestrial plants, for instance rice

(Zhang et. al., 2010b), eventually posing a serious

threat to humans through the consumption of fish

and/or rice (Zhang et al., 2010a). See Figures 1 and 2

for additional information.

EXPOSURE TO METHYLMERCURY

The main source of methylmercury contamina-

tion to humans is fish, a highly nutritious food with

known benefits for human health. The Food and

Drug Administration just completed and published

a 10 year study on the levels of methylmercury con-

tamination in the domestic fish supply (Tables 1 and

2; FDA, 2013). Fish are also a vital cultural and eco-

nomic commodity for many communities around the

world. All fish, however, do not have similar amounts

of mercury because of bioaccumulation of methyl-

mercury through the many levels of the aquatic food

chain. Concentrations of total mercury vary widely

across fish and shellfish species, with the mean val-

ues differing by as much as 100-fold (Keating et al.,

1997). Methylmercury is bound to proteins, as well

as to free amino acids, that are components of mus-

cle tissues, and is not removed by any cooking or

Figure 1. Mercury enters the food chain via manmade and natural emissions and is transformed into methylmercury in the lakes and oceans where it accumulates in fish (Environmental Protec-tion Agency, 2014).


Figure 2. Mercury bioaccumulation. Methylmercury enters the base of the food web and is bio-magnified at each successive level of the food chain. Highest levels are found in predators at the top of the aquatic food web (USGS, 2013).

Table 1. Fish and shellfish with the highest levels of mercury (FDA, 2013)

SPECIESMERCURY CONCENTRATION (PPM) NO. OF

SAMPLESMEAN MEDIAN STDEV MIN MAX

MACKEREL KING 0.730 N/A N/A 0.230 1.670 213

SHARK 0.979 0.811 0.626 ND 4.540 356

SWORDFISH 0.995 0.870 0.539 ND 3.220 636

TILEFISH (Gulf of Mexico)

1.450 N/A N/A 0.650 3.730 60

Mercury was measured as Total Mercury

N/A-data not available


Table 2. Fish and shellfish with lower levels of mercury (FDA, 2013)

SPECIESMERCURY CONCENTRATION (PPM) NO. OF

SAMPLESMEAN MEDIAN STDEV MIN MAX

ANCHOVIES 0.017 0.014 0.015 ND 0.049 14

BUTTERFISH 0.058 N/A N/A ND 0.36 89

CATFISH 0.025 0.005 0.057 ND 0.314 57

CLAM * 0.009 0.002 0.011 ND 0.028 15

COD 0.111 0.066 0.152 ND 0.989 115

CRAB 1 0.065 0.050 0.096 ND 0.610 93

CRAWFISH 0.033 0.035 0.012 ND 0.051 46

CROAKER ATLANTIC (Atlantic) 0.065 0.061 0.050 ND 0.193 57

FLATFISH 2* 0.056 0.050 0.045 ND 0.218 71

HADDOCK (Atlantic) 0.055 0.049 0.033 ND 0.197 50

HAKE 0.079 0.067 0.064 ND 0.378 49

HERRING 0.084 0.048 0.128 ND 0.560 26

JACKSMELT 0.081 0.050 0.103 0.011 0.500 23

LOBSTER (Spiny) 0.093 0.062 0.097 ND 0.270 13

MACKEREL ATLANTIC (N.Atlantic) 0.050 N/A N/A 0.020 0.160 80

MACKEREL CHUB (Pacific) 0.088 N/A N/A 0.030 0.190 30

MULLET 0.050 0.014 0.078 ND 0.270 20

OYSTER 0.012 ND 0.035 ND 0.250 61

PERCH OCEAN * 0.121 0.102 0.125 ND 0.578 31

POLLOCK 0.031 0.003 0.089 ND 0.780 95

SALMON (CANNED) * 0.008 ND 0.017 ND 0.086 34

SALMON (FRESH/FROZEN) * 0.022 0.015 0.034 ND 0.190 94

SARDINE 0.013 0.010 0.015 ND 0.083 90

SCALLOP 0.003 ND 0.007 ND 0.033 39

SHAD AMERICAN 0.045 0.039 0.045 0.013 0.186 13

SHRIMP * 0.009 0.001 0.013 ND 0.050 40

SQUID 0.023 0.016 0.022 ND 0.070 42

TILAPIA * 0.013 0.004 0.023 ND 0.084 32

TROUT (FRESHWATER) 0.071 0.025 0.141 ND 0.678 35

TUNA (CANNED, LIGHT) 0.128 0.078 0.135 ND 0.889 551

WHITEFISH 0.089 0.067 0.084 ND 0.317 37

WHITING 0.051 0.052 0.030 ND 0.096 13

Mercury was measured as Total Mercury except for species (*) when only methylmercury was analyzed. ND-mercury concentration below detection level (Level of detection = 0.01 ppm)1Includes: Blue, King, Snow2Includes: Flounder, Plaice, Sole


cleaning processes that do not destroy muscle tis-

sues. In addition to fish, rice cultivated in areas con-

taminated with mercury can contain relatively high

levels of methylmercury (Horvat et al., 2003; Zhang

et al., 2010b). Other food sources of methylmercury

have been reported including organ meats of ter-

restrial animals (Ysart et al., 2000), and chicken and

pork (Lindberg et al., 2004), probably as a result of

the use of fish meal as livestock feed. Persons living

in certain communities also have higher methylmer-

cury exposure because they consume the flesh of

fish-eating marine mammals (Grandjean et al., 1995;

Van Oostdam et al., 2005).

MERCURY IN THE ENVIRONMENT

Mercury is found in the environment in three forms,

elemental mercury, inorganic compounds and or-

ganic compounds; each form has specific solubility,

chemical reaction, and toxicity characteristics (Clark-

son, 2002; Goldman and Shannon, 2001). Elemental

mercury is released via degassing from the crust and

oceans of the earth, and the combustion of fossil fuels

releases elemental mercury to the environment (ATS-

DR, 1999). Additional mercury is released from indus-

trial waste; the total amount of mercury released each

year from all sources may add up to as much as 9000

tons each year (ATSDR, 1999; Trasande et al., 2005).

Mercury is deposited in surface waters from both

industrial and naturally-occurring atmospheric sourc-

es where it can attach to particles suspended in the

water. These particles eventually settle into the sedi-

ment where the mercury can be “methylated” dur-

ing a complex chemical process facilitated by anaer-

obic organisms, thus forming methylmercury. Many

factors dictate the occurrence rate of the methyla-

tion process. For example, studies have shown that

water with a lower pH and higher dissolved organic

carbon content generally results in higher levels of

methylation (United States Geological Survey, 2009).

Methylmercury is biomagnified in the aquatic food

chain from bacteria, to plankton, through macro-

invertebrates, to herbivorous fish, to fish-eating fish

(Wiener et al., 2003). Humans and other fish eating

mammals, such as otters and whales, which consume

fish from the top of the aquatic food chain receive

the methylmercury that has bioaccumulated through

this process (Mergler et al., 2007).

HEALTH EFFECTS OF METHYLMERCURY IN HUMANS

When methylmercury is ingested it is readily and

completely absorbed by the gastrointestinal tract.

Methylmercury is complexed with the amino acid

cysteine and with proteins and peptides contain-

ing cysteine; this complex is then recognized by

the amino acid transporting system of the body as

methionine, another essential amino acid (Kerper

et al., 1992). Because this complex is recognized by

the body as an essential amino acid, it is transport-

ed freely throughout the body including across the

blood–brain barrier and across the placenta, where

it is absorbed by the developing fetus. Since the

methylmercury is so strongly bound to proteins and

because the complex is recognized as an amino acid

it is not readily removed from food or from the body

(Carrier et al., 2001).

There are several studies that suggest that meth-

ylmercury causes developmental delays in children

exposed before birth, including attention defi-

cits, loss of IQ points and decreased performance

in tests of language skills and memory (Rice et al.,

2003). There is insufficient data to make a causal

link between pre-natal exposure to methylmercury

from the mother’s diet and autism in spite of the

expressed concerns of the public (van Wijngaarden

et al., 2013). In adults, ingestion of methylmercury

has been linked to increased risk of cardiovascular

disease including heart attack (Salonen et al., 1995;

Guallar et al., 2002; Choi et al., 2009), and there is

some evidence that methylmercury can cause auto-

immune diseases in sensitive individuals (Hultman

and Hansson-Georgiadis, 1999).

In addition to chronic exposure to methylmercury

there have been several episodes of acute expo-

sure in which large numbers of people were severely

poisoned by food contaminated with high levels of


methylmercury. The most widely known incident is

probably the dumping of industrial waste that re-

sulted in the pollution of water and fish and subse-

quent mass poisonings in Minamata and Niigata,

Japan (Harada, 1995). Another such episode took

place in Iraq in 1971; wheat treated with methylmer-

cury was shipped to Iraq as seed grain not intended

for human consumption. Due to a number of factors,

including foreign-language labeling and late distri-

bution within the growing cycle, this toxic grain was

consumed as food by Iraqi residents in rural areas.

The recorded death toll was 650 people, but figures

at least ten times greater have been suggested,

making this the largest mercury poisoning disaster

(Bakir et al., 1973). These episodes resulted in neu-

rological symptoms including loss of feeling, loss of

physical coordination, difficulty in speech, narrowing

of the visual field, hearing impairment, blindness,

and death. Children who had been exposed in-utero

through their mothers’ ingestion were also affected

with a range of symptoms including motor difficul-

ties, sensory problems and mental retardation.

REDUCING BIOACESSABILITY

The protective effect of selenium against methyl-

mercury toxicity has been hypothesized for a num-

ber of years (Pařízek and Oštádalová, 1967; Skerfv-

ing, 1978; Cuvinaralar and Furness, 1991; Raymond

and Ralston, 2004; Falnoga and Tusek-Znidaric, 2007;

Yang et al., 2008; Khan and Wang, 2009). The protec-

tive effects of selenium against methylmercury toxic-

ity in fetal brain development have now been con-

firmed but only in animal studies (Beyrouty and Chan,

2006; Sakamoto et al., 2013). Yang, et al. (2008) and

Khan and Wang (2009) have summarized the several

physiologic/biochemical mechanisms proposed to

explain the antagonism between methylmercury and

selenium. It seems likely that the molecular mecha-

nism involves the formation of insoluble, equimolar,

and biologically unavailable mercury selenide pre-

cipitates, since approximately 1:1 molar ratios of se-

lenium and mercury have been observed in marine

mammals, sea birds and humans (Chen et al., 2006;

Khan and Wang, 2009; Li et al., 2012) . Shim et al.

(2009) found that phytochemical rich foods, spe-

cifically green tea extract, black tea extract, and soy

protein significantly reduced mercury bioaccessibil-

ity by 82 to 92%, 88 to 91%, and 44 to 87%, respec-

tively. Wheat bran decreased bioaccessibility by 84%,

oat bran by 59 to 75% and psyllium by 15 to 31% at

amounts greater than 500 mg (Shim et al., 2009). Evi-

dence also exists that suggests the developmental

and cardiovascular toxicity of methylmercury may be

mediated by co-exposures to omega-3 fatty acids,

in particular docosahexaenoic acid (DHA) (Jin et al.,

2007). Nøstbakken et al. (2012) found that omega-3

lessened methylmercury toxicity, either by decreas-

ing programmed cell death (eicosapentaenoic acid)

or by reducing methylmercury uptake (DHA).

BIOREMEDIATION

Bioremediation is a waste management tech-

nique that involves the use of organisms to remove

or neutralize pollutants from a contaminated site.

The use of microbial biomass for bioremediation

of toxic metals has been pursued for a number of

years (Akthar et al., 1995, 1996; Akthar and Mohan,

1995; Gupta et al., 2000; Karna et al., 1999; Pethkar et

al., 2001; Puranik and Paknikar, 1997; Volesky, 1987).

Both live and non-living microbial biomass has been

studied for removal of toxic metal ions but many re-

searchers believe that non-living or processed bio-

mass is a better choice. Non-living biomass does not

have toxicity limitations as would living cells, nutri-

ents are not needed for growth of biomass and since

non-living biomass acts as an ion exchanger the pro-

cess is rapid (Paknikar et al., 2003).

The cell wall polymers of fungi are known to have

functional groups such as amino, amide, hydroxyl,

carboxyl, sulfhydryl and phosphate which have been

implicated in metal binding (Akhtar et al., 1995; Gup-

ta et al., 2000). Karunasagar et al. (2003) studied the

effectiveness of a biosorbent prepared from biomass

of Aspergillus niger for removal of methylmercury

from dilute solutions. They determined that removal

of methylmercury from spiked ground water samples


was efficient and not influenced by other ions, and

that the biosorbent was reusable for up to six cycles

without appreciable loss of binding capacity.

Live bacteria provide another means of methylmer-

cury bioremediation (Barkay, et al., 2003; Barkay and

Wagner-Dobler, 2005; Chen and Wilson, 1997; Miller,

1999; Nascimento and Chartone-Souza, 2003). Bac-

teria can break down mercury compounds through

the acquisition of a transferable genetic element

known as the mer operon (Omichinski2007). The

mer operon is a dedicated set of mercury-resistant

genes that are self-regulated by the DNA-binding

protein MerR; bacteria resistant to methylmercury

code for proteins that regulate mercury transport

(MerA, MerP, MerT) and mercury degradation (MerA

and MerB) (Osborn et al., 1996; Sahlman et al., 1997;

Silver and Phung, 1996; Wilson et al., 2000). Chien

et al. (2010) made the point that there are substrate

specificities among the MerB enzymes, elucidating

the necessity for selecting the appropriate bacterial

strains or MerB enzymes to apply them in bioreme-

diation engineering for cleaning up specific mercury

contaminants.

Meagher (2000) engineered MerA and MerB into

plants to remediate methylmercury contamination.

Their theory was that remediation using plants is

potentially more robust than bacterial remediation,

because plants use solar energy, have roots that

penetrate contaminated sediments, and accumu-

late a large aboveground biomass. There are actu-

ally a few well-characterized plant species used to

clean up contaminated wetland ecosystems (Mea-

gher, 2000). Plants such as cottonwood trees (Lyyra

et al., 2007) and tobacco (Heaton et al., 2005) have

been modified to express either MerB or both MerB

and MerA; the plants converted the methylmercury

to ionic mercury or elemental mercury, respectively;

however, the elemental mercury was released into

the atmosphere, where it may still pose a risk.

CONCLUSIONS

Bioremediation is considered to have advantages

over conventional techniques such as chemical pre-

cipitation, conventional coagulation, adsorption by

activated carbons, adsorption by natural materials,

ion exchange, or reverse osmosis. Mercury-resistant

bacteria possess the mer operon enabling them to

convert the toxic forms of mercury to nontoxic forms.

Those possessing the merB gene are more valuable

as they can detoxify methylmercury along with other

organic mercurial compounds and inorganic mercury

to nontoxic, volatile mercury. Bacteria harboring the

merB gene and genetically modified organisms pos-

sessing the mer operon including merB are promis-

ing tools for use in bioremediation of methlymercury.

However, the cons for the bacterial-based or plant-

based processes may include production of large

volumes of mercury-loaded biomass, the disposal of

which is problematic.

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ABSTRACT

Land application is a common method for disposal of manure and litter that accumulate during poul-

try production; however, zoonotic pathogens residing in the manure may contaminate either directly or

indirectly ready-to-eat produce crops. Aerobic composting of animal manure is a beneficial process treat-

ment that inactivates these pathogens. Although heat is considered to be the primary contributing factor

to inactivation, ammonia and volatile acids may also serve antimicrobial roles during composting. This

study was designed to determine the relative contributions of chemicals and heat to the inactivation of

Salmonella and Listeria monocytogenes in chicken manure-based compost mixtures formulated to give

initial carbon:nitrogen (C:N) ratios of 20:1, 30:1 and 40:1. The different initial C:N ratio formulations of the

compost mixtures had no effect on pH or the cumulative heat generated. In general, there was within all

compost mixtures an initial decline in pH followed by an increase in pH that coincided with an increase

in temperature. Levels of ammonia and volatile acids were higher in compost mixtures formulated to an

initial C:N ratio of 20:1 than in other C:N formulations. The inactivation rates of Salmonella and L. monocy-

togenes within 20:1 C:N formulations were higher than in other formulations. Regression models derived

from the data revealed that volatile acid levels, in addition to heat, played a major role in pathogen inac-

tivation. Therefore, it may be advantageous to formulate compost mixtures containing chicken litter to an

initial C:N of 20:1 to take advantage of the antimicrobial activity of volatile acids generated when sub-lethal

temperatures occur.

Keywords: manure, litter, composting, chicken, heat, ammonia, volatile acids, pH, Listeria monocy-togenes, Salmonella

Correspondence: M.C. Erickson, [email protected]: +1 -770-412-4742 Fax: +1-770-229-3216

Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure Composting

M.C. Erickson1*, J. Liao1, X. Jiang2, and M.P. Doyle1

1Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin, GA

2Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC



INTRODUCTION

Poultry production in the United States is a

major enterprise having had a combined value

in broiler, egg, and turkey production of $38 bil-

lion in 2012 (USDA, 2012). Although meat and

eggs are the major outputs from this enterprise,

a substantial amount of manure (generated from

layer and turkey operations) and litter (mixture of

manure, bedding material, wasted feed, feathers,

and soil generated from broiler operations) is also

produced. For example, the estimated tons of

manure produced from poultry operations in the

U.S. in 2007 was 81 million tons (US EPA, 2013).

To dispose of this waste, land application has of-

fered the best solution (Moore et al., 1998; Ritz and

Merka, 2013). Poultry manure can harbor zoonotic

pathogens such as Salmonella, Listeria monocy-

togenes, and Campylobacter (Chinivasagam et

al., 2010; Hutchison et al., 2004, 2005), and if ap-

plied to fields growing ready-to-eat produce, these

pathogens may contaminate those crops. Once

excreted from the animal, pathogen survival is de-

pendent on storage conditions (Goss et al., 2013;

Leifert et al., 2008; Ziemer et al., 2010). If left un-

disturbed, Williams and Benson (1978) determined

that Salmonella Typhimurium survived for at least

18 months in chicken litter at 11 or 25°C, and 13

days at 38°C. Decimal reduction times for S. Ty-

phimurium in poultry manure are not only affected

by storage temperature, but also by the type of

matrix, being greater in manure slurries compared

to manure piles (Himathongkham et al., 2000). Ac-

cording to USDA, only 5% of all U.S. cropland in

2006 was fertilized with manure, with most chicken

manure being applied to peanut and cotton fields

(MacDonald et al., 2009). Although this mode of

disposal would appear to have a minimal food

safety risk, natural waterways and irrigation ponds

in the Southeastern U.S. have been found contami-

nated with Salmonella and Campylobacter, espe-

cially after precipitation events (Gu et al., 2013b;

Haley et al., 2009; Luo et al., 2013) and likely oc-

curred from pathogen runoff of peanut and cotton

fields amended with poultry manure. These water

sources are frequently used to irrigate fields grow-

ing ready-to-eat produce (Gu et al., 2013a), hence

animal manure should be treated to inactivate

pathogens prior to land application.

A treatment that is often recommended to inac-

tivate vegetative bacterial pathogens in manures is

thermophilic aerobic composting. In this process,

manure is mixed with one or more carbon amend-

ments to produce a nutrient-rich environment

favorable for the metabolism of thermophilic mi-

croorganisms. The major factor responsible for in-

activating pathogens in such systems is heat gener-

ated by the metabolic activity of these thermophilic

microorganisms (Erickson et al., 2010; Wichuk and

McCartney, 2007)). As a result, process conditions

that are based on time and temperature have been

promulgated in regulations or guidelines world-

wide (Hogg et al., 2002). For example, guidelines

within the U.S. include either a minimum temper-

ature of 55°C for 3 days in aerated static piles or

in-vessel systems or 55°C for 15 days in windrow

systems (narrow trapezoidal elongated rows) dur-

ing which time the piles must be turned a minimum

of 5 times to ensure that all material is subjected to

the necessary thermal conditions (US EPA, 1999).

Although heat is the primary mechanism for in-

activating pathogens during aerobic composting,

temperature stratification within static piles can re-

sult in extended survival of pathogens at the sur-

face as well as extended survival of pathogens at

interior sites of piles composted during the winter

(Berry et al., 2013; Erickson et al., 2010; Shepherd

et al., 2007). In addition, exposure of the pathogen

to nonlethal heat or selected moisture conditions

could lead to metabolic alterations in the patho-

gen that makes them more resistant to the thermal

conditions encountered during the thermophilic

phase of composting (Chen et al., 2013; Shepherd

et al., 2010; Singh et al., 2011, 2012). As evidence of

this potential activity, Salmonella, Escherichia coli

O157:H7, and Listeria survived in poultry manure-

based compost piles when exposed to tempera-

tures above 55°C for more than 8 days (Hutchison

et al., 2005). Hence, other factors, either chemical

or biological, may provide a greater contribution


to pathogen inactivation under those conditions.

For example, accumulation of free ammonia in

poultry manure has been reported to contribute to

inactivation of S. Typhimurium and E. coli O157:H7

in poultry manure (Himathongkham et al., 2000).

Alternatively, volatile acids generated during the

early phase of composting in cow manure systems

formulated to have an initial carbon:nitrogen (C:N)

ratio of 20:1 were suggested to be bactericidal

agents effective against Salmonella but not Liste-

ria (Erickson et al., 2009a,b). Given that there are

differences in microbial and raw material composi-

tion between cow and chicken manure (De Bertoldi

et al., 1987; Lynch, 1987; Wang et al., 2007), it was

the objective of this study to determine the relative

contributions of heat, volatile acids, and ammonia

to the inactivation of Salmonella and L. monocyto-

genes in chicken manure-based compost mixtures

formulated to C:N ratios ranging from 20:1 to 40:1.

MATERIALS AND METHODS

Pathogen Strains and Their Preparation for Experimental Trials

Five strains of Listeria monocytogenes (101M,

12443, F6854, G3982, and H7550) from the culture

collection housed at the Center for Food Safety, Uni-

versity of Georgia were used for these studies. In

addition, three strains of Salmonella enterica serovar

Enteritidis (ME-18, H4639, and H3353) and one strain

of S. enterica serovar Newport (11590) were also

used from the culture collection. All strains had been

labeled with the green-fluorescent plasmid (GFP),

but for L. monocytogenes strains, the plasmid also

contained an erythromycin-resistant marker, whereas

Salmonella strains contained an ampicillin-resistant

marker. Previously, plasmid stability of these GFP-

labeled strains was reported to range from 8 to 52%

and 15 to 77% plasmid loss after 20 generations for

the L. monocytogenes and Salmonella strains, re-

spectively (Ma et al., 2011).

To prepare the pathogen strains for challenge

studies, frozen cultures of L. monocytogenes and Sal-

monella were thawed and individually streaked onto

plates containing brain heart infusion agar (Becton

Dickinson, Sparks, MD) with 8 µg/mL of erythromy-

cin (BHIA-E) and tryptic soy agar (Difco Laboratories,

Detroit, MI) with 100 µg/mL ampicillin (TSA-A), re-

spectively. Following incubation of plates at 37°C for

ca. 24 h, individual colonies were removed and sub-

sequently streaked onto a second plate and held at

37°C for an additional 24 h. From this second set of

plates, individual colonies of L. monocytogenes and

Salmonella were removed and inoculated into 10

ml of brain heart infusion broth (Becton Dickinson)

containing 8 µg/mL erythromycin (BHIB-E) and tryp-

tic soy broth containing 100 µg/mL ampicillin (TSB-

A), respectively. These suspensions were incubated

for ca. 24 h at 37°C with agitation (150 rpm) before

harvesting the bacteria by centrifugation (4,050 x g,

15 min, 4°C). The pelleted cells were washed three

times in 0.1% peptone water (Difco) and resediment-

ed by centrifugation before reconstituting in 0.1%

peptone water to an optical density at 630 nm of

ca. 0.5 that corresponded to a concentration of ca.

109 CFU/mL. The five strains of L. monocytogenes

were then combined in equal proportions to make

one 5-strain stock culture mixture, whereas the four

strains of Salmonella were combined for one 4-strain

stock culture mixture. Each of these stock culture

mixtures was then diluted 10-fold with deionized wa-

ter for mixtures of 108 CFU/mL that were used to spray

chicken litter. Immediately after preparation of the

spray mixtures, L. monocytogenes and Salmonella

was enumerated by plating serial dilutions (1:10) on

modified oxford medium (Acumedia Manufacturers,

Lansing, MI) containing 10 mg/mL buffered colistin

methanesulfonate, 20 mg/mL buffered moxalactam

solution, and 8 µg/mL erythromycin (MOX-E) and

TSA-A, respectively. Salmonella colonies emitted a

bright green fluorescence when plates were held un-

der a handheld UV light (365 nm) and the fluorescent

colonies were counted as Salmonella. Fluorescent L.

monocytogenes colonies were smaller than Salmo-

nella colonies and required a Leica MZ16 FA stereo

fluorescence microscope (Bannockburn, IL) for visu-

alization and counting.


Compost Ingredients, Preparation, and Experimental Design

Fresh chicken litter was collected from a broiler

production facility located in Orchard Hill, GA.

Batches were collected at different times for each

independent replicate trial. Following transport, the

litter was mixed thoroughly and a portion of the lit-

ter was removed for compositional analysis. The re-

mainder of the litter was frozen to kill insect eggs and

then held at -20°C until which time it was ready to be

composted. Wheat straw and cottonseed meal were

purchased from a local feed store and served as the

major carbon sources for the compost mixtures.

Chicken litter was added to a 28-L sanitized bowl

and sprayed manually using a spray bottle with both

the Salmonella and L. monocytogenes inocula for

populations approximating 107 CFU/g. This inocu-

lated mixture was then mixed with a Hobart mixer

(model D320:0.75 h.p.). Wheat straw, cottonseed

meal, and water were then added in such quantities

that compost mixtures had an initial moisture con-

tent of 60% and a C:N ratio of either 20:1, 30:1, or

40:1. Immediately after mixing, the compost mix-

tures were sampled for chemical and microbiologi-

cal analysis. The remainder of the compost mixture

was then placed in one of three bioreactors.

In this experimental study, three independent tri-

als were conducted wherein each trial consisted of

three bioreactor systems containing one compost

mixture each of the 20:1, 30:1 and 40:1 C:N ratio mix-

tures. The compost mixtures were composted for up

to 6 days and were sampled on days 1, 2, 3, and 6 to

measure microbiological and chemical parameters.

Composting Apparatus and Sampling

Bioreactors (46 cm high x 32 cm diameter) were

constructed from PVC plastic pipe. Tightly fitting

PVC covers had holes drilled into their center such

that the bottom cover hole allowed condensate to

drip into an attached bottle and the top cover hole

allowed compressed air (155 ml/min) to be delivered

to the system. Within the biochamber, a perforated

shelf was supported 5 cm above the bottom. Two

sampling ports (3 cm diameter) at heights of 6 to

9 cm and 10 to 13 cm above the PVC shelf and a

hole (0.5 cm diameter) at a height of 6.5 cm above

the shelf for insertion of a thermocouple wire were

drilled into the sides of the bioreactors. Bioreac-

tors were housed within a Precision 30 Mechani-

cal Convection incubator (Thermo Fisher Scientific,

Waltham, MA) that was maintained at a temperature

of 40°C. Trapped air in the incubator was vented to

a filtered exhaust system.

Compost material (ca. 5 kg) was placed into each

bioreactor after which a type T thermocouple wire

was inserted through the small hole to a site desig-

nated as the bottom center (16 cm from bioreactor

wall). An additional thermocouple was inserted to

a depth of 10 cm into the top center of the com-

post mixture. All thermocouples (two per bioreac-

tor) were connected to a multi-channel HotMux

data logger (DCC Corp., Pennsauken, NJ) that was

programmed to record temperatures at the 6 loca-

tions at 30-min intervals. Cumulative heat > 40°C

(degree-days) was calculated as the product of time

(days) and temperature (°C above the ambient in-

cubator temperature of 40°C). Oxygen levels in the

bioreactor system were measured on all sampling

days using a Demista OT-21 oxygen probe (Arling-

ton Heights, IL) prior to removing duplicate samples

(25 g) with a sanitized grabbing tool at both the bot-

tom center and top center locations.

Chemical and Microbiological Analyses

All compost ingredients (chicken litter, wheat

straw, and cottonseed meal) as well as the initial

compost mixtures were analyzed for carbon, nitro-

gen, and moisture contents. Carbon content was

determined on the basis of ash content obtained af-

ter combustion of samples at 550°C. The University

of Georgia’s Soil Testing Laboratory (Athens, Geor-

gia) was used for analysis of nitrogen content via a

macro-Kjeldahl method. Moisture levels were based

on residual weights of vacuum dried samples.

Ammonia concentrations in compost samples


(5 g) was determined with a phenol-hypochlorite

spectrophotometric procedure (Weatherburn, 1967),

whereas the Hach spectrophotometric Method 8196

test kit (Loveland, CO) as adapted by Montgomery

et al. (1962) was used to measure volatile acid con-

centrations in compost samples. Measurement of

pH was made with an Acumet Basic pH meter (Fisher

Scientific, Pittsburgh, PA) on compost samples (5 g)

dispersed in 250 ml of deionized water.

Salmonella and L. monocytogenes were enumer-

ated by direct plate counts (limit of detection was 2

log CFU/g) or detected by selective enrichment cul-

ture (limit of detection was 1 log CFU/g). In either

case, compost samples (5 g) placed in a Whirl-Pak

bag were first pummeled in a Stomacher 400 Circu-

lator (Seward Ltd., West Sussex, UK) for 1 min after

adding 45 mL of 0.1% peptone water. Diluted (1:10)

aliquots of this homogenate were applied to either

TSA-A plates to enumerate Salmonella or MOX-E

plates to enumerate L. monocytogenes. Enrichment

cultures of Salmonella and L. monocytogenes con-

sisted of adding 1 mL of the homogenate to 9 ml

of selective enrichment medium (TSB-A or BHIB-E,

respectively) and incubating this mixture for 24 h at

37°C. Aliquots of these enriched samples were then

streaked onto TSA-A or MOX-E plates to determine

the presence or absence of fluorescent Salmonella

or L. monocytogenes colonies, respectively.

Statistical Analyses

The StatGraphics Centurion XVI software, version

16.1.03 (StatPoint Technologies, Inc., Herndon, VA)

was used for statistical analysis of the collected data;

however, pathogen populations were first converted

to logarithmic values prior to conducting these op-

erations. When samples did not yield any colonies

during plate count enumeration but did have fluo-

rescent colonies on plates streaked from enrichment

cultures, a value of 1.0 log CFU/g, corresponding

to the limit of detection by enrichment culture, was

assigned to that sample. Otherwise, samples yield-

ing negative results for both plate counts and en-

richment cultures were assigned a value of 0.0 log

CFU/g. After conversion of enrichment culture data,

all data were subjected to general linear models

analysis of variance (GLM ANOVA) to determine the

significance of experimental variables over all sam-

pling times examined in the study. To differentiate

treatments at individual sampling times, the data

were subjected to one-way ANOVA and when sta-

tistical differences were observed (P < 0.05), sample

means were differentiated using the least significant

difference test. Multiple linear regression analysis

was also conducted on data from each sampling

day and treatment in an attempt to relate the total

pathogen loss in the mixtures to the independent

variables of pH, cumulative heat, and concentrations

of volatile acids and ammonia.

RESULTS AND DISCUSSION

Chicken litter, collected from broiler houses, was

mixed with wheat straw, cottonseed meal, and water

in combinations to give mixture treatments varying in

their initial C:N ratio. Following analysis of these ini-

tial compost mixtures, the C:N ratios that were mea-

sured for the 3 independent replicate trials averaged

20.6 ± 1.7, 32.4 ± 2.4, and 43.6 ±1.7, respectively.

Initial moisture contents in the 20:1, 30:1, and 40:1

C:N ratio formulations were 62.7 ± 1.9, 60.8 ± 1.7,

and 60.5 ± 2.8%, respectively. Continued monitor-

ing of moisture contents on days 2 and 6 revealed

that compost mixtures were generally above 40%

moisture during this time and thus aerobic microbial

activity would not have been inhibited (Rynk, 1992).

Oyxgen concentrations during composting were also

well above the 5% level that is considered to limit

aerobic microbial activity (Rynk, 1992).

All compost mixtures were initially characterized as

slightly alkaline (Table 1). After one day of compost-

ing, the pH of all mixtures had decreased from 1.5 to

2.2 units and declines were greater as the C:N ratio of

the compost formulation decreased. After this point in

time, the pH of all mixtures increased. Overall, there

were no significant differences in pH with the different

C:N ratio treatments throughout the composting pe-

riod (P < 0.05).


Table 1. pH (mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Table 2. Volatile acid concentrations (mg/g, mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

1 Levels followed by a different letter are significantly different (P < 0.05)

Initial C:N ratio

Days 20:1 30:1 40:1

0 7.69 ± 0.48 c-e1 7.89 ± 0.19 d-f 7.79 ± 0.22 d-f

1 5.50 ± 0.39 a 5.96 ± 1.36 a 6.30 ± 1.37 ab

2 6.47 ± 1.68 a-c 7.52 ± 2.00 d 7.40 ± 1.80 cd

3 8.09 ± 0.95 d-f 7.37 ± 1.48 cd 7.26 ± 1.58 b-d

6 9.00 ± 0.22 f 8.55 ± 0.78 ef 8.12 ± 1.05 d-f

Initial C:N ratio

Days 20:1 30:1 40:1

0 5.49 ± 2.37 a-c1 4.27 ± 1.32 a 3.68 ± 1.32 a

1 11.68 ± 3.52 d-g 12.42 ± 3.26 fg 9.08 ± 4.04 c-e

2 12.08 ± 3.59 e-g 11.34 ± 4.91 d-g 8.79 ± 3.88 b-d

3 13.26 ± 6.88 g 9.76 ± 4.49 d-f 5.79 ± 4.28 ab

6 8.80 ± 4.20 b-d 4.79 ± 2.67 a 4.85 ± 2.99 a



These results were in contrast to those that were

observed when compost mixtures were formulated

to different C:N ratios with dairy manure as the ni-

trogen source (Erickson et al., 2009a, b). In those

studies, compost mixtures formulated to a C:N ratio

of 40:1 did not decline in pH during the first day of

composting.

Volatile acids, including acetate, butyrate, and

propionate, are produced during the early phases of

aerobic composting and digestion (Beck-Friis et al.,

2003; Ugwuanyi et al., 2005a,b) and may be poten-

tial contributors to the pH declines observed in this

study during the first day of composting of chicken

litter. This suggestion was corroborated by the ob-

served increase in volatile acid levels that occurred

in the chicken litter compost mixtures during the first

day (Table 2). The greatest increase in volatile acid

concentrations was observed in the 20:1 C:N com-

post mixtures, whereas the least increase occurred

in the 40:1 C:N compost mixture. Furthermore, as

composting continued, volatile acid levels declined

in all compost mixtures, but the decrease was slower

in 20:1 C:N compost mixtures than in the 40:1 C:N

compost mixtures. Generally, facultative anaerobic

microorganisms produce volatile acids in response

to low oxygen concentrations (Brinton, 1998); how-

ever, it would appear that the nutrient conditions

provided in the 20:1 C:N compost mixtures were

more conducive than the other compost mixture for-

mulations for generating such compounds.

Ammonia is another common byproduct pro-

duced during the degradation of chicken manure or

chicken litter (Bush et al., 2007; Himathongkham et

al., 2000). There were significant differences in the

ammonia concentrations of the different formula-

tions of the chicken compost mixtures (Table 3).

Specifically, when all sampling days were taken into

account, the 20:1 C:N ratio compost mixture had the

highest ammonia concentrations, whereas the low-

est levels were in the 40:1 C:N ratio compost mix-

tures (P < 0.05). In addition, during the composting

process, the ammonia concentrations were continu-

ously shifting, with maximal levels found in the 20:1

C:N mixtures on day 3, whereas maximal levels in

Table 3. Ammonia concentrations (µg/g, mean ± S.D.) in compost mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio

Days 20:1 30:1 40:1

0 131.6 ± 24.7 a-c1 114.2 ± 23.8 ab 99.7 ± 30.2 ab

1 218.6 ± 94.3 c-e 215.9 ± 74.2 c-e 132.5 ± 23.4 b

2 271.0 ± 98.9 e 217.0 ± 56.4 c-e 145.3 ± 19.9 bc

3 379.4 ± 158.6 f 185.8 ± 47.1 b-d 123.9 ± 73.8 ab

6 246.6 ± 37.9 de 168.8 ± 216.7 bc 54.3 ± 57.3 a



Table 4. Cumulative metabolic heat > 40°C (degree-days1, mean ± S.D. )during composting of mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

Initial C:N ratio

Days 20:1 30:1 40:1

1 2.90 ± 2.00 a2 2.84 ± 2.36 a 3.48 ± 3.17 a

2 6.42 ± 4.53 ab 9.14 ± 10.67 a-c 9.16 ± 7.79 a-c

3 15.96 ± 10.02 cd 16.72 ± 16.09 b-d 19.05 ± 11.17 d

1 Accumulated product of temperature (°C above the ambient incubator temperature of 40°C) and

composting time (days) 2 Levels followed by a different letter are significantly different (P < 0.05)

Table 5. Fate of Salmonella and L. monocytogenes populations (log CFU/g, mean ± S.D.) in com-post mixtures formulated with chicken litter, wheat straw, and cottonseed meal to different initial C:N ratios

1 Populations for each pathogen followed by a different letter are significantly different (P < 0.05).

Salmonella L. monocytogenes

Day 20:1 C:N 30:1 C:N 40:1 C:N 20:1 C:N 30:1 C:N 40:1 C:N

0 7.43 ± 0.25 f 7.22 ± 0.26 f 7.38 ± 0.44 f 7.53 ± 0.17 g 7.21 ± 0.12 g 7.14 ± 0.13 g

1 3.64 ± 0.83 d 3.73 ± 1.96 d 5.31 ± 1.16 e 3.49 ± 1.22 e 3.79 ± 1.09 e 4.90 ± 0.97 f

2 1.58 ± 1.82 bc 0.48 ± 0.89 a 2.57 ± 2.33 c 1.21 ± 1.48 c 0.99 ± 0.88 bc 2.40 ± 1.83 d

3 0.28 ± 0.66 a 0.28 ± 0.66 a 1.82 ± 2.42 c 0.00 ± 0.00 a 0.28 ± 0.66 ab 1.12 ± 1.05 c

6 0.00 ± 0.00 a 0.00 ± 0.00 a 0.57 ± 0.84 ab 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a


both the 30:1 and 40:1 C:N mixtures were detected

on day 2. The increased generation of both ammo-

nia and volatile acids in compost mixtures initially

formulated to low C:N ratios agrees with the models

presented by Delgado-Rodríguez et al. (2010) that

demonstrated a higher level of volatile compounds

present during municipal solid waste composting at

low C:N ratios.

Temperatures during composting of the mixtures

were monitored throughout the period when sam-

ples were collected from the bioreactors. To assess

the cumulative heat exposure above the ambient

incubator temperature of 40°C, time-temperature

curves were integrated using 40°C as the baseline.

Results for the first 3 days of composting, expressed

as cumulative heat > 40°C (degree days), are pre-

sented in Table 4 and, although heat generation in

compost mixtures was slightly greater as the C:N ra-

tio increased, it was not significantly different (P >

0.05). Heat generation within each bioreactor was

fairly homogeneous, as location was not a significant

factor affecting the cumulative levels (P > 0.05). In

contrast, over the three independent replicate trials,

the level of heat accumulated in the compost mix-

tures was significantly different from each other (P <

0.05). As the manure source for each of these inde-

pendent trials was collected at separate times from

the broiler houses, a plausible explanation is that

the chicken litter had been collected in the houses

at different periods of time before being removed

for composting. Aged manure used in composting

mixtures produces less heat than fresh manure (Berry

et al., 2013; Li et al., 2008). Such variability in manure

age and subsequent variability in heat generation

in this study would likely have contributed to an in-

ability to detect a significant effect of C:N ratio on

heat generation. A similar situation was also likely

responsible for the inconsistent response of heat

generation in compost mixtures formulated to dif-

ferent C:N ratios when using dairy manure (Erickson

et al., 2009b). In that study, no statistical differences

occurred in the heat generated for the different C:N

formulations in the bioreactor trials inoculated with

E. coli O157:H7, whereas in bioreactor trials inocu-

lated with L. monocytogenes, 20:1 formulations were

statistically different for the 30:1 C:N formulations.

During composting of chicken litter with different

C:N ratio mixtures, pathogen levels were monitored

(Table 5). Using ANOVA on data collected shortly

after composting was initiated (days 1 and 2 only),

it was revealed that the C:N ratio had a significant

effect on inactivation of L. monocytogenes and Sal-

monella (P < 0.05). For both pathogens, the levels

of the pathogen were higher in mixtures formulated

to a C:N ratio of 40:1 than in those mixtures formu-

lated to either 20:1 or 30:1. Slower inactivation had

been observed previously for Salmonella in dairy

manure compost mixtures of formulations having a

C:N ratio of 40:1 compared to ratios of 20:1 and 30:1

(Erickson, 2009a), whereas the C:N ratio in dairy ma-

nure formulations did not affect the inactivation of

L. monocytogenes (Erickson et al., 2009b). Pathogen

inactivation, however, was not log-linear, but was

characterized as biphasic. Hence, to determine if

the C:N treatment affected inactivation during tail-

ing, the number of days to complete inactivation of

the pathogen was recorded for each replicate trial.

For Salmonella, the days to complete inactivation

ranged from 2 to 4, 2 to 5, and 3 to 8 for 20:1, 30:1,

and 40:1 C:N formulations, respectively, whereas the

days to complete inactivation of L. monocytogenes

ranged from 2 to 3, 2 to 4, and 4 to 5, respectively.

Given that only three values for each treatment were

available, ANOVA applied to the days to inactivation

data failed to reveal any significant effect by the C:N

ratio of the mixture for either pathogen (P > 0.05).

Despite this negative response, there is a trend of

increasing days to inactivation with an increasing ini-

tial C:N ratio of the compost mixture and if explored

in the future with a larger number of trial replicates,

could prove to be significant.

Comparison of Salmonella and L. monocytogenes

responses in the composting mixtures revealed no

significant differences in the rate of inactivation or

days to inactivation (Table 5, P > 0.05). The similar-

ity in responses contrast to those reported for com-

posting of rural sewage sludge with straw (Pourcher

et al., 2005) and composting of swine manure (Gre-

wal et al., 2007), in which L. monocytogenes persist-

ed for longer periods of time than Salmonella. Dif-


ferences in comparative pathogen response in this

study and others may have arisen due the different

isolates used or to the different formulations used

for composting.

To understand the contribution of potential chem-

ical and physical factors to pathogen losses during

composting on any one sampling day, models were

derived using backward stepwise regression. Both

cumulative heat and levels of volatile acids were fac-

tors included in those models, described below, and

explained 19.7% and 28.9 % of the variability in the

data for Salmonella and L. monocytogenes, respec-

tively.

Salmonella losses = 1.455 + (0.121*cumulative

heat>40°C) + (0.221* volatile acid concentration)

(P = 0.0123)

L. monocytogenes losses = 0.076 + (0.215*cumu-

lative heat>40°C) + (0.317* volatile acid concentra-

tion)

(P = 0.0018)

These models reveal that volatile acids, in addi-

tion to heat, have a bactericidal role in chicken litter

compost mixtures, particularly in those formulations

(i.e. 20:1 C:N compost mixtures) in which high con-

centrations of volatile acids are produced.

CONCLUSIONS

In summary, chicken litter was mixed with wheat

straw and cottonseed meal to give formulations hav-

ing initial C:N ratios of 20:1, 30:1 or 40:1. Although

the pH decreased in all formulations during the first

day of composting, the C:N ratio of the formulation

did not have a significant effect on pH (P > 0.05) nor

did it have a significant effect on the cumulated heat

generated in the mixtures during composting (P >

0.05). In contrast, the different C:N formulations did

have a significant effect on ammonia concentrations

and volatile acids produced during composting, with

the greatest amounts of these antimicrobials being

generated in the 20:1 C:N compost mixtures and the

least in the 40:1 C:N compost mixtures (P < 0.05).

Moreover, in the 20:1 C:N compost mixtures, the

inactivation rates of both Salmonella and L. mono-

cytogenes were higher as well as the days required

to achieve complete inactivation were in general

sooner, than in compost mixtures formulated to ei-

ther 30:1 or 40:1. Multiple linear regression models

that were derived from fitting pathogen losses to cu-

mulative heat and volatile acid levels were significant

and explained 20 to 29% of the variability in the data.

Hence, in conditions where heat may be insufficient

to inactivate pathogens (winter composting or at the

surface of unturned static compost piles), it may be

advantageous to formulate the initial C:N ratio of

chicken litter compost mixtures to values approach-

ing 20:1, as higher volatile acid concentrations in

these mixtures provide additional antimicrobial ac-

tivity.

ACKNOWLEDGEMENTS

The project was supported by the National Re-

search Initiative of the USDA Cooperative State Re-

search, Education, and Extension Service, grant #

2008-35201-18658.

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ABSTRACT

Recently, a vast array of potential antibiotic alternatives have been introduced and researched in the

livestock industry as a means to provide livestock producers with products that will positively impact animal

health and performance. Some of these products may be used in conjunction with current antibiotic usage

strategies, and some of these products may be used to completely replace some antibiotics in livestock

production. These innovative antibiotic alternatives include direct fed microbials (DFM), yeast extracts,

bacteriocins, bacteriophages, phytochemicals, and various acids. Many of these products have the ability

to promote animal health and improve growth performance simultaneously, and some of these compounds

may additionally enhance food safety through pre-harvest pathogen reduction. Antibiotic alternatives may

be essential tools for livestock production in the future should legislation arise that inhibits prophylactic

usage of conventional antibiotics and as a means to appeal to shifting consumer demands. Furthermore, it

is also possible that these alternatives can be used as an additional supplement to incorporate into current

practices and strategies in livestock production to maximize the potential to enhance both animal health

and growth performance. This review will discuss potential alternative antimicrobial supplements in animal

agriculture and their impact on animal health, performance and pathogen reduction.

Keywords: Antibiotic, livestock, animal health, review

Correspondence: Todd Callaway, [email protected]: +1-979-260-9374 Fax: +1-979-260-9332.

REVIEWAlternative Antimicrobial Supplements

That Positively Impact Animal Health and Food Safety

P. R. Broadway1, J. A. Carroll2, and T. R. Callaway3

1Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX2Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX

3Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, USDA, College Station, TX

“Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or exclusion of others that may

be suitable.” USDA is an equal opportunity provider and employer



INTRODUCTION

Internet news sources and social media play in-

creasing roles in publicizing livestock production

practices and influence public opinion and percep-

tion, both fairly and unfairly. Recently, internet me-

dia sources have published propaganda to condemn

the use of antibiotics in meat and poultry (Consumer

Reports, 2012, 2013). Consumer-driven changes to

the market due to shifting consumer demands mean

that the beef, pork and poultry industries are faced

with increasing challenges to profitability and must

search for alternatives to remain profitable while

still remaining environmentally friendly. Thus, the

animal production industry needs potential alter-

native strategies to supplement or replace current

antibiotic implementation practices. Furthermore,

as consumer purchasing trends continue to gravi-

tate towards “natural” and “organic” products, the

livestock industry needs to be equipped to survive

in an environment where sub-therapeutic concentra-

tions of antibiotics supplemented as feed additives

are absent. Consumer demand and public concern

have previously been shown to influence antibiotic

legislation. For example, the European Union (EU)

banned sub-therapeutic supplementation of animal

feeds with antibiotics (Pradella, 2006). Recently, the

U.S. Food and Drug Administration (FDA) issued a

guidance to further regulate antibiotic usage in food

animals. While this directive does not eliminate the

use of antibiotics, some believe this is the beginning

of the end for antibiotic usage in food animals. For-

tunately, substantial research has been conducted

regarding the use of antibiotics in food animals, and

many alternatives have been proposed and evalu-

ated in regard to meeting the shifting consumer

demands without impinging on the health, welfare,

profitability, or wholesomeness of the food supply.

The gastrointestinal (GI) tract of animals is popu-

lated with a complex microbial ecosystem that is es-

sential for the function, growth, and overall health of

the animal (Chaucheyras-Durand and Durand, 2010);

therefore, any potential alternatives to antibiotics

must support this symbiotic relationship. Evidence

across multiple species generally supports the posi-

tive effects antibiotics exert on growth promotion,

growth rates, and overall animal health, thus produc-

ers have adopted the use of these pharmaceuticals

over the years as a strategy to increase profitabil-

ity, performance and animal health. Antibiotics are

typically defined as compounds that inhibit bacteria

(antibacterials), while antimicrobials are compounds

that inhibit microorganisms. Many livestock produc-

ers currently utilize antibiotic/antimicrobial feeding

and production strategies that alter the microbial

ecology of the GI tract of the animal to benefit the

overall production efficiency of their animals, as well

as strategies that can eliminate or reduce foodborne

pathogens that may contaminate the food supply

(Perlman 1973). However, these positive effects must

be replicated by strategies that fill in the gap left if

and when antibiotic use in food animals in banned in

the U.S. While strategies such as genetic selection,

selective breeding, and other management prac-

tices are utilized to promote health and profitability

along with antibiotics, this review will focus primarily

on feed additives.

ALTERNATIVES TO ANTIBIOTICS IN LIVE-STOCK PRODUCTION

Due to recent developments in feed additives,

direct fed microbials (DFM), and pharmaceuticals,

producers now have multiple options available to

enhance the natural microbial ecology of the animal,

to prevent illness and improve production efficiency.

While numerous alternatives to antibiotic use have

been researched in animal production systems (Table

1), to date, there has been no “silver bullet” identi-

fied, nor is one likely to be found. While it has been

demonstrated that many of the currently used antibi-

otics work across species, the results associated with

the use of antibiotic alternatives have been inconsis-

tent at times with varied results generated from spe-

cies to species. Additionally, most alternatives have

been evaluated as potential prophylactic treatments,

and they may not be effective in treating actual illness

or disease. Often times, the cost of antibiotic alter-

natives are offset by increases in performance; how-


ever, depending on different management and pro-

duction strategies, these alternatives have not been

demonstrated to be as effective as antibiotics. Each

strategy has specific advantages and limitations in-

cluding: effectiveness, production stage/system, ani-

mal age, animal type, changes in performance vari-

ables, cost, and labor. All of these variables must be

explored before implementing antibiotic alterative(s)

in a livestock production setting.

Direct-Fed Microbials (DFM)

One such strategy that may be a potential alterna-

tive to antibiotics is known as competitive enhance-

ment. Callaway et al. (2008) defines competitive

enhancement strategies as introducing live cultures

of bacteria or fungi into the GI tract that provide a

competitive advantage to commensal organisms

that can, in turn, exclude pathogenic bacteria (e.g.,

supplementing with a probiotic, or addition of a pre-

biotic). Probiotic supplements fed to livestock are

defined by Chaucheyras-Durand and Durand (2010)

as “live microorganisms that possess the ability to

evoke positive health benefits (at appropriate dos-

age concentrations) in the animal to which the micro-

organism was administered”; however, when used to

treat or prevent disease, this definition is not identi-

cal to the definition provided by the Food and Drug

Administration (FDA). Prebiotics are defined as feed

ingredients that benefit the host by selectively stim-

ulating the growth or activity of bacteria (Gibson and

Table 1. Alternatives to antibiotics that may be used in food animals that may be used to pro-mote overall animal health and/or impact pathogen colonization and shedding

DFM1 Bacteriophages Phytochemicals Acids

Bacillus Finylase Citrullene Acetic acid

Lactobacillus Citrus pulp Caproic acid

Lactococcus Curcumin Formic acid

Streptococcus Eugenol Fumarate

Yeast Flavonoids Malate

Yeast cell wall Limonene Propionic acid

Linalool Sodium Chlorate

Piperin

Thymol

1Direct-Fed Microbials


Roberfroid, 1995). A combination of prebiotic ingre-

dients is referred to as synbiotics (Patterson and Bur-

kholder, 2003). Fuller et al. (1989) explained that the

fully developed GI tracts use its microbial ecosystem

symbiotically to fill voids in microbial populations

and thereby inhibit the colonization of pathogens.

However, even when the GI tract is fully developed

and contains a healthy microflora, some pathogens

may be able to attach and cause illness. When a GI

flora ecosystem is fully developed and the system is

in a well-balanced state, the bacteria attach to the

intestinal epithelium and can possibly physically in-

hibit the binding and colonization of pathogens that

may be introduced into the gut (Collins and Gibson,

1999; Lloyd et al., 1977). Numerous compounds are

produced by bacteria in the gut such as volatile fatty

acids (VFA) that may inhibit the growth of and/or kill

pathogens introduced to the animal (Wolin, 1969;

Walsh et al., 2008).

Direct-fed microbials have recently gained inter-

est from the livestock feed industry as a tool to in-

crease performance while simultaneously serving as

a replacement for antibiotics (Ghorbani et al., 2002).

These DFM products have been shown to enhance

the formation of a healthy microbial community

within the GI tract of the animal (Fuller, 1999). As

with antibiotics, addition of DFM in the diet of dairy

cows has been shown to reduce the risk of ruminal

acidosis (Nocek et al., 2000). While the mode of ac-

tion that reduces acidosis with the inclusion of lac-

tate producing bacteria is not fully understood, the

phenomena may be the result of changes in fermen-

tation and microbial populations (Ghorbani et al.,

2002). Enhanced performance parameters such as

feed efficiency and ADG have also been reported

in feedlot cattle supplemented with DFM (Swinney-

Floyd et al., 1999; Rust et al., 2000).

Some bacteria and DFM feed additives produce

compounds called bacteriocins which are proteins

synthesized by bacteria that inhibit the growth of

other bacteria occupying the same environmental

niche (Jack et al., 1995). Such bacteriocins have been

isolated from the rumen of cattle (Wells et al., 1997;

Russell and Mantovani, 2002). Bacteriocins, such as

those commonly produced by Lactococcus bacte-

ria have the ability to inhibit pathogens such as E.

coli (Russell and Mantovani, 2002) that pose a risk

to human health. Brashears et al. (2003) reported

that supplementing feedlot cattle with a Lactobacil-

lus DFM was effective in reducing E. coli O157:H7

in fecal samples as well as reducing prevalence at

slaughter.

As in cattle, Lactobacillis has been shown to in-

hibit multiple strains of Salmonella in poultry (Jin et

al., 1996). A combination of L. acidophilus and Strep-

tococcus faecium cultures were shown to reduce col-

onization of Campylobacter jejuni (Morishita et al.,

1997). Reductions in E. coli and Salmonella strains

have also been observed when poultry were supple-

mented with Bacillus subtilis spores (LaRagione et

al., 2001; Laragione and Woodward, 2003). While

bacteriocins may be beneficial in most cases, some

bacteria may exhibit resistance to specific bacterio-

cins (Russell and Mantovani, 2002). Colicin E1, a bac-

teriocin produced by E. coli, has been shown to be

effective against pathogens that may be present in

young swine that contribute to diarrheal symptoms

(Cutler et al., 2007). Reduction/elimination of these

pathogens through the use of Colicin E1 may be an

effective tool to improve health and performance

(Cutler et al., 2007). In addition to pre-harvest patho-

gen reduction applications, Colicin E1 has also been

used successfully when applied directly to beef car-

casses to inhibit E. coli O157:H7 (Patton et al., 2008).

Researching probiotic supplementation in swine

has yielded inconsistent results (Turner et al., 2001).

Some studies have shown no differences in the

growth and performance of swine fed Lactobacilli

(Harper et al., 1983), while other studies reported en-

hanced growth and profitiability (Jasek et al., 1992;

Gombo et al., 1995). Bacillus spp. feed supplemen-

tation has also been shown to decrease incidence

of disease, reduce E. coli shedding and improve

feed efficiency in swine (Bonomi, 1992; Kyriakis et

al., 1999; Succi et al., 1995). Other probiotics that

have exhibited positive effects in growing and finish-

ing swine are Streptococcus spp. (Turner et al., 2001).

Multiple studies with Streptococcus cultures have

suggested that supplementation with these probi-

otics enhance growth and feed conversion in swine


(Kumpercht and Zobac, 1998; Roth and Kirchgessner,

1986; Underahl, 1983).

Another probiotic that may be utilized in swine is

a yeast culture. Yeast cultures fed to swine have been

reported to increase growth performance (Bertin et

al., 1997; Maloney et al. 1998; Matthew et al., 1998).

Yeast DFM may enhance digestion and maintain the

microbial ecosystem of the GI tract in swine (Van

Heugten et al., 2003), thereby making yeast DFM a

possible antibiotic alternative during the weaning

phase of young swine by inhibiting colonization of

pathogens and improving performance (Anderson

et al., 1999). Yeasts have also been used in cattle

as antibiotic alternatives (Jouany and Margavi, 2007).

For example, supplementation of live yeast probi-

otics in dairy cattle has been reported to increase

milk production and dry matter intake (Jouany, 2006;

Sniffen et al., 2004; Stella et al., 2007).

Yeast cell wall products are another type of feed

supplement that have been fed to livestock as a

means to improve animal performance, and to elimi-

nate the pathogenicity of certain bacteria. Yeast cell

wall components have been reported to function as

immunomodulators that activate immune compo-

nents such as macrophages, neutrophils, and other

immunocompetent cells (Eicher et al., 2006; Onder-

donk et al., 1992; Seljelid et al., 1987). Approximate-

ly half of the yeast cell wall is composed of biological

response modifiers (Bohn and BeMiller, 1995), and

these components have antibacterial (Kogan et al.,

1989), antimutagenic (Kogan et al., 2005), antioxi-

dant, and antitumor (Khalikova et al., 2005) activities

that may promote animal health. Kogan and Kocher

(2007) suggested that yeast cell wall polysaccharides

may prevent bacterial attachment of pathogens to

the mucosal epithelium in swine. Multiple studies

conclude that yeast products may protect swine from

bacterial infections while improving performance

parameters such as weight gain (Lemieux et al.,

2003; Rozeboom et al., 2005). Yeast cell wall prod-

ucts have also been shown to improve metabolism

in heifers during an endotoxin immune challenge

without degradation of carcass tissues (Burdick San-

chez et al., 2013). With respect to animal health and

food safety, yeast products have been reported to

help alleviate infections caused by E. coli (Buts et

al., 2006), Salmonella (Mahzounieh et al., 2006), and

Clostridium (Katz, 2006) in lab animals. In poultry,

supplementation with yeast cell wall products has

yielded inconsistent results with respect to growth

performance and pathogen reduction (Griggs and

Jacob, 2005).

Bacteriophages

Bacteriophages are viruses found commonly in

the GI tract and environment that prey specifically on

bacteria, including pathogenic bacteria. Bacterio-

phages can bind to specific bacterial receptors, in-

ject phage DNA, take control of that cell, reproduce,

and release new phages that lyse (or rupture) the host

bacterial cell, thus resulting in bacterial cell death

(Guttman et al., 2004 Kutter and Sulakvelidze, 2005).

However, there are limitations to the use of bacte-

riophages. Just as bacteria can become resistant to

antibiotics, bacteria may also become resistant to

bacteriophages (Sklar and Joerger, 2000; Smith and

Huggins, 1982; Smith et al., 1987). Another concern

with the use of a bacteriophage is the passage of the

supplement through the GI tract. Factors such as pH,

viscosity, and microbial populations may influence

the survivability and effectiveness of bacteriophage

therapies (Hurley et al., 2008). Hurley et al. (2008)

reported no reduction in fecal Salmonella shedding

when fed to 28-day old chickens. Sklar and Joerger,

(2000) reported minimal reductions in Salmonella

populations of chickens when fed bacteriophages,

and these researchers hypothesized that the intra-

cellular nature of Salmonella may prevent phage at-

tachment. When isolated and fed to feedlot cattle,

researchers concluded that bacteriophages could

be beneficial in a pre-harvest pathogen reduction in-

tervention strategy to combat food pathogens such

as E. coli (Callaway et al., 2008; Johnson et al., 2008).

Additionally, phage therapy has also been reported

successful in pathogen reduction in swine (Wall et

al., 2010) and sheep (Bach et al., 2003). In fact, Roz-

ema et al. (2009) reported that supplementation of

feedlot cattle with bacteriophages for the control

of E. coli O157:H7 shedding may be an alternative


pre-harvest intervention that could be utilized to

promote food safety in finishing cattle (Dini and De

Urraza 2010). Also a product called Finalyse® has

been developed for use as a hide spray to reduce E.

coli O157:H7 on hides of cattle before they enter the

slaughter plant, and FDA has issued no objection or-

der to the use of phage (Coffey et al., 2011).

Phytochemicals

Phytochemicals, such as citrus pulp, are being ex-

plored as replacements for sub-therapeutic antibiot-

ics supplementation in animal feeds. Citrus pulp and

peel are low cost byproduct feedstuffs with good

nutritive values (high TDN) that have been used in

beef and dairy production for many years (Arthing-

ton et al., 2002). Citrus pulp and peel contain es-

sentials oils, including but not limited to citrullene,

linalool, and limonene, that are bactericidal and can

alter the microbial ecology of the GI tract (Lota et

al., 2002; Fisher and Phillips, 2006; Viuda-Martos et

al., 2008). Recent studies have demonstrated that

feeding citrus pulp and peel to cattle, sheep, and

swine can reduce populations of E. coli O157:H7 and

Salmonella Typhimurium (Nannapaneni et al., 2008;

Callaway et al., 2008; Callaway et al., 2011). While

citrus pulp may have antipathogenic effects in cattle,

Broadway et al. (2013) reported that there were only

minimal changes to the bacterial ecology in the ru-

men of cattle supplemented with dried citrus pulp,

thereby concluding that citrus pulp may be used an

alternative agent to prevent colonization and shed-

ding of foodborne pathogens without significantly

altering ruminal microbial ecology and digestibility.

Essential oils have also been researched in poultry

to control pathogens (Griggs and Jacob, 2005). Thy-

mol, eugenol, curcumin, and piperin are some of the

essential oils found in thyme, clove, turmeric, and

black pepper, and these products have been shown

to inhibit enteridis causing Clostridium perfringens

in poultry (Mitsch, et al., 2004). Other pathogens

such as Salmonella and E. coli have been reduced,

in vitro, when cultured with thymol (Marino et al.,

1999; Karapinar and Aktug, 1987; Helander et al.,

1998) and thyme (Aktug and Karapinar, 1986). Other

phytochemicals including garlic (Singh and Shukla,

1984), cinnamon (Hernandez et al., 2004) and black

pepper (Dorman et al., 2000) have been reported to

reduce pathogen populations and enhance metabo-

lism in some cases.

Phytochemicals found in the seaweed such as

A. nodosum have been reported to reduce E. coli

prevalence of feces and on the hides of cattle at har-

vest (Behrends et al., 2000). Feeding this same com-

pound to swine was reported to enhance growth pa-

rameters but was unsuccessful in treating Salmonella

infections (Turner, 2001). Other plant extracts called

Saponins have been reported to alter ruminal micro-

flora (Killeen et al., 1998), and have been reported to

prevent the growth of E. coli (Sen et al., 1998).

Other naturally occurring phytochemicals, such as

flavonoids, may be included in a production strat-

egy to promote overall animal health and decrease

pathogen shedding into the food supply (Holiman

et al., 1996; Mandalari et al., 2007). Flavonoids are

found in plant tissues and bark, and display some an-

tioxidant capability (Pietta, 2000). Flavonoids have

also been reported to decrease the viability of patho-

genic bacteria such as E. coli and Salmonella, as

well as Candida albicans and Sacchromyces species

(Mandalari et al., 2007; Sohn et al., 2004; Friedman,

2007). Therefore, flavonoids may be an alternative to

antibiotics that could positively impact the health of

the animal while promoting food safety through the

reduction of pathogenic microorganisms.

Acids

Inorganic and organic acids, as well as inorganic

compounds, are another potential natural alterna-

tive that could be incorporated into livestock pro-

duction. Acids are used to eliminate foodborne

pathogens in food production, and they also may be

beneficial in live animals to decrease or eliminate the

presence of pathogens and improve digestion. Or-

ganic acids may target the cell wall and membrance

and interfere with bacterial metabolism, and inter-

nalization of dissociated acid components into the

cytoplasm also alters pH and interferes with cellular

metabolism (Ricke, 2003). However, the mechanisms


by which acids are bacteriocidal have not been ful-

ly elucidated and may target a variety of bacterial

components and metabolic processes (Ricke, 2003).

Organic acids such as lactic acid in diets have been

reported to decrease the incidence of pathogens

in livestock (Byrd et al. 2001). Other acids such as

propionate have been used to improve the ruminal

fermentation, and research pertaining to malate/fu-

marate reported increased lactic acid utilization by

Megasphaera and Selenomonas ruminantium which

resulted in similar impacts as those seen when utiliz-

ing ionophores (Martin and Nisbet, 1990; Nisbet and

Martin, 1990; Nisbet and Martin, 1993; Waldrip and

Martin, 1993). Propionic acid and formic acid fed

in combination has also been researched as a feed

supplement in broilers, and the acid was reported to

decrease populations of Salmonella in experimental-

ly-infected birds (Hinton and Linton, 1988). Similarly,

caproic acid was also shown to decrease Salmonella

Enteridis in chickens (Van Immerseel et al. 2004). In

addition to Salmonella inhibition, supplementing a

combination of organic acids such as formic, acetic,

and propionic acids were reported to reduce the

growth of Campylobacter (Chaveerach et al., 2004).

Another antibiotic alternative that may reduce

the prevalence of foodborne pathogens is sodium

chlorate, an organic product shown to inhibit nitrate

reductase positive bacteria. Callaway et al. (2002),

and Anderson et al. (2000, 2002) reported that E. coli

populations could be reduced without significant

changes in the microbial ecology of the rumen of

cattle. Byrd et al. (2003) reported that water treat-

ment with sodium chlorate was able to decrease the

prevalence of S. Typhimurium in broilers. Addition-

ally, sodium chlorate has been reported to decrease

E. coli O157:H7 populations in the GI tract of inocu-

lated swine (Anderson et al., 2001a), Sodium chlo-

rate has also been reported to decrease Salmonella

in swine prior to harvest (Anderson et al., 2001b).

Other studies have reported chlorate to inhibit the

survival of Salmonella while not interfering with po-

tentially beneficial species in the GI tract (Anderson

et al., 2001a,b; Byrd et al., 2003; Jung et al., 2003);

however, chlorate is still awaiting FDA approval for

use in food animals.

CONCLUSION

As more increasingly negative attention surrounds

the use of antibiotics in livestock production, and as

consumers increase the pressure for future legisla-

tion regarding livestock production practices, more

research and product development is needed to find

suitable alternatives to the use of antibiotic supple-

mented feed for growth promotion and health ben-

efits to meet the demand of an ever growing and

evolving consumer population. There are many

currently available or near-market ready products

that can assist livestock producers in the control of

bacterial populations within their animals to assist in

the maintenance of growth and production perfor-

mance parameters while simultaneously improving

the safety of food products for the consumer. How-

ever, many factors must be taken into consideration

when selecting a particular product as an alternative

to antibiotics, and there are advantages and limita-

tions to each product. Therefore, producers must

select product(s) that best suit their specific opera-

tional needs and are economically feasible.

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ABSTRACT

Isoflavones are a group of chemicals that are found in legumes, predominantly in soybean and soy prod-

ucts. Soy isoflavones have been a component of the diet of certain populations for centuries. Many health

claims have been made for isoflavones including: cancer prevention, alleviation of menopausal symptoms,

positive effects on bone health and a lowering of blood lipids leading to lowered susceptibility to cardio-

vascular disease. However, because of their estrogenic activity some negative effects of isoflavones have

been postulated. This review examines the literature associated with benefits as well as the negative effects

of consumption of soy isoflavones. Results in some studies are limited or conflicting, but when viewed in

its entirety, the current literature supports the safety of isoflavones as typically consumed in diets based on

soy containing products.

Keywords: Isoflavones, soybeans, soy products, health benefits, cancer prevention, bone metabo-

lism, blood lipids

INTRODUCTION

Soybeans are legumes, plants that form root nod-

ules containing nitrogen-fixing soil bacteria (Rhizo-

bia) in a symbiotic relationship. The soybean plant

releases chemical signals, called isoflavonoids, to

attract the nitrogen fixing bacteria (Rolfe, 1988). Iso-

flavonoids, also known as isoflavones, are produced

by the same pathway that produces flavonoids, the

Correspondence: Philip G. Crandall, [email protected]: +1 -479-575-7686 Fax: +1-479-575-6936

phenylpropanoid pathway. The phenylpropanoid

pathway begins with phenylalanine and naringenin

being converted into the isoflavone genistein by two

enzymes, isoflavone synthase and a dehydratase,

that are found only in legumes (Deavours and Dixon,

2005). Naringenin chalcone, another intermediate

is converted to daidzein by the sequential action of

two other legume-specific enzymes, chalcone reduc-

tase and type II chalcone isomerase as well as iso-

flavone synthase (Deavours and Dixon, 2005). Within

the soybean, isoflavones are bound to a sugar mol-

ecule (glycosidic form) but fermentation or digestion

REVIEWHuman Health Benefits of Isoflavones From Soybeans

K. Kushwaha1, C. A. O’Bryan1,3, Dinesh Babu1§, P. G. Crandall1,3, P. Chen2, and S.-O. Lee1

1 Department of Food Science, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704

2Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR 727013Center for Food Safety, University of Arkansas, Fayetteville, AR 72704

§Present address: Food Safety Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209



Table 1. Total isoflavones. daidzein and genistein of selected soyfoods. expressed in mg/100g. Values have been taken from USDA database (USDA. 2008).

Food product Total isoflavones Daidzein Genistein

Soy flour, full-fat 177.89 71.19 96.83

Soy flour, textured 148.61 59.62 78.90

Soy flour, defatted 131.19 57.47 71.21

Soybeans 128.34 46.46 73.76

Soy protein concentrate, aqueous washed 102.07 43.04 55.59

Soy protein isolate 97.43 33.59 59.62

Natto 58.93 21.85 29.04

Soybean chips 54.16 26.71 27.45

Tofu, fried 48.35 17.83 28.00

Tempeh 43.52 17.59 24.85

Miso 42.55 16.13 24.56

Soybean sprouts 40.71 19.12 21.60

Tofu, soft 29.24 8.59 20.65

Tofu, silken 27.91 11.13 15.58

Soy infant formula. powder 25.00 7.23 14.75

Tofu, firm 22.70 8.00 12.75

Soy hot dog 15.00 3.40 8.20

Okara 13.51 5.39 6.48

Soy protein concentrate, alcohol extracted 12.47 6.83 5.33

Bacon, meatless 12.10 2.80 6.90

Soy milk 9.65 4.45 6.06

Vegetarian burger 9.30 2.95 5.28

Soy cheese, Mozzarella 7.70 1.10 3.60

Soy cheese, Cheddar 7.15 1.80 2.25

Soy drink 7.01 2.41 4.60


results in the release of the sugar molecule from the

isoflavone, leaving the isoflavone as an aglycone

(Zubik and Meydani, 2003). Soy isoflavone glyco-

sides include genistin, daidzin, and glycitin, while the

aglycones include genistein, daidzein, and glycitein

(Figure 1). The typical composition for soybeans is

40% diadzin/diadzein, 50% genistin/genestein and

10% glycetin/glycitein (Murphy et al., 1999).

People from countries that consume large

amounts of soy foods are reported to have an im-

proved chronic disease burden compared with coun-

tries consuming very little soy (Boring et al., 1994;

Thom et al., 1992). The average isoflavone intake

from soy food by Asian women ranges from 25 to

50 mg/day (Messina et al., 2006) compared to non-

Asian women who take in less than 2 mg/day (Van

Erp-Baart et al., 2003; de Kleijn et al., 2001). Health

benefits attributed to soy isoflavones include low-

ering blood pressure ( Hooper et al., 2008), preven-

tion of coronary heart disease (Nagata et al., 1998;

Smit et al., 1999), better bone health ( Ho et al., 2001;

Mei et al., 2001; Horiuchi et al., 2000; Tsuchida et al.,

1999) relief of menopausal symptoms (Messina,1999)

and decreased risk of certain types of cancer such as

breast and prostate (Severson et al., 1989; Jacobsen

et al., 1998; Lee et al., 1991; Wakai et al., 1999). Since

isoflavones are capable of exerting estrogen-like ef-

fects they are often referred to as phytoestrogens

(Lampe, 2003). Since isoflavones structurally closely

resemble esterogenic steroids of animals they are

able to bind to both estrogen receptors alpha (ERα)

and beta (ERβ) (Figure 2) (Kuiper et al., 1997; Kuiper

et al., 1998). This review will look at the evidence, pro

and con, regarding the health benefits of isoflavones

derived from soy.

CONSUMPTION OF SOYFOODS

Soybeans and soy products represent one of the

richest and cheapest sources of protein (Codina et

al., 2003). Soybeans have been used as a major part

of the diet in Asian countries and some other parts

of the world for more than 5,000 years. Soy foods

Figure 1. Chemical structures of soybean isoflavones (Naya and Imai, 2013).

Reproduced with permission of Nayga and Imai (2013), available from http://www.intechopen.com/books/

soybean-bio-active-compounds/recent-advances-on-soybean-isoflavone-extraction-and-enzymatic-modifi-

cation-of-soybean-oil


eaten in the Asian countries are often fermented by

microorganisms; for example miso which is added to

soups and stews in Japan, soy paste used in Korea

and tempeh, with a meat-like texture in Indonesia

(Synder and Kwon, 1987a). Soy sauce is perhaps the

most familiar soy product which is made either by

a long fermentation process or by acid hydrolysis;

this acid hydrolyzed product contains no isoflavones

(Luh, 1995). Soy milk is made by extracting the pro-

teins and lipids in soybeans with boiling water; soy-

milk may then be curdled to prepare tofu, which is

pressed to remove water and can be fried or added

to numerous other dishes (Snyder and Kwon, 1987a).

Soybean-containing foods have become more

popular in the United States, especially after October

1999 when the Food and Drug Administration (FDA)

approved a health claim for soy proteins for reduc-

ing heart disease (FDA, 1999). However, soy foods in

the United States are generally quite different from

the forms of soy consumed in Asia. Soybeans grown

in the United States are utilized mostly as a source

of edible oil. After extraction the defatted soy flour,

which is high in protein, is used in many bread and

cake products, particularly in doughnuts (Snyder and

Kwon, 1987b). Alternatively the soy flour is washed

with water to remove soluble carbohydrates creating

soy protein concentrate, which is even higher in pro-

tein. When the soy flour is extracted with hot, aque-

ous 65% alcohol it forms a different type of soy pro-

tein concentrate which contains no isoflavones. Both

of these soy protein concentrates can be extruded

to form textured soy protein, a meat-like product.

The proteins in soy flour may be solubilized with a

mild alkaline extraction followed by a precipitation

at a low pH to produce soy protein isolate (SPI). This

SPI is widely found in canned foods or is used by

athletes as a source of protein. There are also new

“soy” foods such as soy cheese, soy ice cream and

soy yogurt.

EFFECT OF PROCESSING ON ISOFLA-VONE CONTENT OF FOODS

Processing has a substantial influence on the

amount and form of isoflavones in soyfood products.

During the course of processing, some isoflavones

may be lost and the chemical composition may also

change. Soybeans for oil production have the hulls

removed and the remainder of the beans after the

fat is removed is pressed into flakes and then ground

into soy flour. The isoflavone content of the soy flour

Figure 2. Comparison between isoflavone and estrogen molecule showing similarity in conforma-tion (Setchell and Cassidy, 1999).

Reproduced with permission of American Society for Nutrition


is about the same as whole soybeans, indicating that

defatting and milling causes little loss or transforma-

tion of isoflavones; soy oils contain only traces of iso-

flavones because of the highly polar nature of the

isoflavones leading to an inability to partition into

the lipophilic oil during soy oil extraction (Coward

et al., 1998; Setchell, 1998). Production of soy pro-

tein concentrate using alcohol washing causes the

loss of most of the isoflavones, whereas a substantial

amount of isoflavones are retained after water wash-

ing (Wang and Murphy, 1996). More isoflavones are

retained in SPI than in soy protein concentrate (Wang

and Murphy, 1996). Fermented soybean products

such as miso and natto are reported to have higher

isoflavone content than in products such as soymilk

and tofu, which is thought to be due to the action of

bacteria during fermentation (Fukutake et al., 1996).

Extruded soy products such as cereals have a lower

amount of isoflavones due primarily to the heat and

loss of moisture during the extrusion (Mahungu et

al., 1999).

ISOFLAVONE METABOLISM

After the soybean is eaten, the glycosidic forms of

the isoflavones undergo hydrolysis due to the action

of the brush border and bacterial β-glucosidases to

remove the sugar moiety; the aglycone form is then

either absorbed or undergoes further metabolism

by intestinal bacteria in the large bowel (Chen et

al., 2003; Setchell et al., 2003). The isoflavone daid-

zein is usually metabolized to dihydrodaidzein or O-

desmethylangolensin (Bowey et al., 2003; Setchell,

1998; Yuan et al., 1995; Zubik and Meydani, 2003). In

a small number of persons daidzein may also be me-

tabolized in the intestine to equol, a metabolite that

has greater estrogenic activity than daidzein (Muth-

yala et al., 2004). Equol exists in 2 stereoisomers,

R or S, which differ significantly from each other in

terms of their binding affinities with estrogen recep-

tor (ER) (Muthyala et al., 2004). The S isomer has a

high binding affinity for both receptors but prefers

ERβ, whereas the R isomer binds weakly and prefers

ERα; however, both isomers have a higher affinity for

both ERs than does the precursor daidzein (Muth-

yala et al., 2004). Human gut microflora metabolize

daidzein to produce only the S isomer (Setchell et

al., 2005). Studies that measured urinary equol ex-

cretion after soy consumption indicated that only

about 33% of individuals from Western populations

metabolize daidzein to equol (Setchell et al., 2002).

The prevalence of equol producers appears to be

higher in Asian populations than in non-Asian (Arai

et al., 2000; Wu et al., 2006; Cassidy et al., 2006) and

appears to be linked to the gastrointestinal normal

flora in these individuals (Setchell et al., 2002).

The role of gut microflora in the production of

equol was elucidated in experiments with germ-

free rats which, when fed daidzein, did not produce

equol; when the rats were inoculated with fecal flora

from equol producers they were able to produce

equol from daidzein (Bowey et al., 2003; Axelson

and Setchell, 1981). A number of bacterial species

capable of converting daidzein to the S isomer of

equol in vitro have been isolated from both food

and human gut flora including, Lactococcus garviae

from Italian cheese (Fortina et al., 2007), 6 strains of

bacteria belonging to the Coriobacteriaceae fam-

ily from tofu brine (Abiru et al., 2013), Eggerthella

spp strain YY791 (Yokoyama and Suzuki, 2008) and

YY7918 (Yokoyama et al., 2011), and Slackia isoflavo-

niconvertens (Schroder et al., 2013) from human gut

flora. Many researchers have proposed that equol

producers may have improved disease risk patterns

as compared with non-producers (Fujioka et al.,

2004; Kurahashi et al., 2008; Lampe, 2009; Magee,

2011; Setchell et al., 2002; Wu et al., 2007). There

is much evidence that suggests that equol produc-

ers have a lower breast cancer risk as compared with

non-producers (Atkinson et al., 2003; Duncan et al.,

2000; Falk et al., 2005; Ursin et al., 1999).

DISEASE PREVENTION ACTIVITIES

Isoflavones and Bone Health

Loss of bone mass, known as osteoporosis, poses

a major human health threat by contributing to bone


fractures in those afflicted. Worldwide, osteoporosis

causes more than 8.9 million fractures annually, re-

sulting in an osteoporotic fracture every 3 seconds

(Johnell and Kanis, 2006). Osteoporosis is estimated

to affect 200 million women worldwide; approxi-

mately 10% of women aged 60, 20% of women aged

70, 40% of women aged 80 and 67% of women aged

90 are afflicted (Kanis, 2007). One of the means by

which isoflavones promote bone health is hypoth-

esized to be because of their affinity for ERβ since

bone tissue contains large amounts of ERβ (Mes-

sina, 1999). Arjmandi et al. (1998) found that there

was a significant increase in insulin-like growth fac-

tor 1 (IGF-1) mRNA in isoflavone treated groups

compared to control groups; since IGF-1 mRNA

stimulates bone formation, this is another possible

mechanism for the positive role of isoflavone in bone

health. Isoflavones have also been postulated to

prevent osteoporosis because of their similar struc-

ture to ipriflavones, which inhibit bone resorption in

humans (Tsuda et al., 1986; Brandi, 1997).

There is inconsistent data available on the ben-

eficial effect of soy isoflavones on bone density in

human studies due to the small subject number and

short duration of the soy consumption, a larger ef-

fect on bone density was observed in animal models

using higher doses (Arjmandi et al. 1998). From epi-

demiological studies as well as clinical trials, Messina

et al., (2004) showed that Asian women who take in

more soy isoflavones have higher bone mineral den-

sity and have a low rate of hip fracture compared to

non-Asians, and concluded that isoflavones reduce

bone loss in postmenopausal women.

Isoflavones and Cancer Prevention

Cancer affects persons of every socioeconomic

level and every area of the world; cancer accounts for

one in every eight deaths worldwide – more than HIV/

AIDS, tuberculosis, and malaria combined (American

Cancer Society, 2013). An estimated 14.1 million new

cancer cases and 8.2 million cancer-related deaths

occurred in 2012 (International Agency for Research

on Cancer, 2013). The most common causes of can-

cer death were cancers of the lung, liver, and stom-

ach (International Agency for Research on Cancer,

2013). It is estimated that more than two-thirds of hu-

man cancers could be prevented by modification of

lifestyle including dietary modification (Haque et al.,

2010). The U.S. National Cancer Institute has been

actively investigating the anticancer effects of soy-

beans since 1991 (Messina and Barnes, 1991).

Akiyama et al., (1987) demonstrated that genistein

was a specific inhibitor of a tyrosine-specific protein

kinase, an enzyme that is often overexpressed in

cancer cells. Constantinou et al. (1990) found that

genistein suppressed growth and induced differen-

tiation in leukemia cells. Later, genistein was found

to inhibit multiple protein tyrosine kinases relevant

to cancer cell proliferation (Bektic et al., 2005). Ra-

biau et al. (2010) treated human prostate cancer cells

with genistein or daidzein and found that they down

regulated growth factors involved in proliferation of

new blood vessels in tumors. In a study reported in

2011, prostate cancer patients scheduled for radical

prostatectomy were randomly assigned to receive a

placebo or 30 mg genistein daily for 3 to 6 weeks

before surgery. Among the patients who received

genistein, serum prostate specific antigen (PSA) lev-

els decreased by 7.8%, whereas serum PSA levels

increased by 4.4% in patients who received the pla-

cebo (Lazarevic et al., 2011).

Consumption of soy isoflavones is higher in Asian

diets as compared to Western; daily consumption of

soy isoflavones in Japan ranges from 26 to 54 mg,

compared to 0.5 to 3 mg in the United States (Naga-

ta, 2010). Breast cancer incidence increased by more

than 20% between 2008 and 2012 and mortality from

breast cancer increased by 14%; it is the most com-

mon cause of cancer deaths among women and the

most frequently diagnosed cancer in women in 140

of 184 countries (International Agency for Research

on Cancer, 2013). Some epidemiologic evidence

suggests that soy consumption early in life and

through puberty reduces breast cancer risk (Mes-

sina and Hilakivi, 2009) and this has been supported

by animal studies which suggest that soy intake is

protective at specific stages of development but not

at other points (Warri et al., 2008). Lamartiniere et

al., (2000) briefly exposed young rodents to dietary


supplements of genistein and found that it reduced

mammary cancers indicating early intake of soy iso-

flavones protects against breast cancer. Animal stud-

ies demonstrated that when carcinogenic rodents

were fed with isoflavone-rich soy protein or isolated

isoflavones, mammary carcinogenesis was inhibited

by 25 to 50% (Magee and Rowland, 2004; Messina

and Loprinzi, 2001) indicating that isoflavones do

have an antiesterogenic effect. Pisani et al., (1999)

and Kitamura et al. (2002) reported low risk of breast

cancer in the Asian countries where soy is commonly

consumed as isoflavone might exert an antiestero-

genic effect on breast tissue.

Adults who consumed large amounts of soy as ad-

olescents have been determined to have a lower risk

for breast cancer as compared to adults who did not

consume large amounts of soy in adolescence (Shu

et al., 2001). Wu et al. (1996) found that among Asian

Americans, tofu consumption protected against both

pre- and postmenopausal breast cancer. Another

study reported by Chinese epidemiologists wherein

adolescents had high soy consumption which re-

sulted in a 50% reduction in adult breast cancer risk

whereas adult intake did not impact these findings

(Shu et al., 2001). Similarly, a U.S. case-control study

involving Asian Americans reported that high soy

consumption during both adolescence and adult-

hood was associated with a one-third reduction in

risk whereas high adult intake alone was not protec-

tive (Wu et al., 2002). Both dosage and timing of

exposure to soy isoflavones appear relevant to their

potential chemopreventive effect. Two meta-analy-

ses (Dong and Qin, 2011; Wu et al., 2008) found soy

intake to be significantly associated with reduced

risk of breast cancer in Asian but not Western human

populations, which may be explained by both higher

soy intake among Asians and their tendency to con-

sume soy from an early age.

Several studies have shown mixed results regard-

ing the effect of isoflavones supplements on the

proliferation of breast cells in breast cancer patients.

In 2007, a Japanese collaborative cohort study sug-

gested that consumption of soy foods such as tofu,

boiled beans, and miso soup has no protective ef-

fects against breast cancer (Nishio et al., 2007). In

multiple trials no effects on breast proliferation or

mammographic density were observed for isofla-

vones and considerable epidemiologic data shows

either no effect or only a modest protective role of

soy/isoflavone intake on breast cancer risk (Messina

and Wood, 2008). Studies conducted by Caan et al.,

(2011) and Guha et al., (2009) reported that soy con-

sumption had no adverse effects on breast cancer

survivors. Furthermore, they suggest that soy con-

sumption at levels comparable to those among Asian

populations does not detract from the benefits of

tamoxifen therapy, and may even offer some protec-

tion against recurrence and cancer-related death.

The American Cancer Society stated that con-

sumption of soy foods would not decrease survival

nor increase recurrence of cancers, but there was

not enough evidence to make a statement about

isoflavone supplements (Rock et al., 2012). However,

in another study conducted by a U.S.- Chinese re-

search team, researchers monitored and measured

intake of soy isoflavones over the course of seven

years; they determined that soy isoflavones signifi-

cantly reduced risk of cancer recurrence in patients

who consumed at least 10 mg of isoflavones (ap-

proximately 3 g of soy protein) per day (Nechuta et

al., 2012). Bloedon et al. (2002) and Allred et al. (2001)

found that soy protein and isoflavones stimulated

the growth of mammary tumors in ovariectomized

mice implanted with estrogen-sensitive breast can-

cer cells. In contrast Zhou et al., (2004) demonstrated

that isoflavones could inhibit the growth of tumors in

mice when intact ovaries were implanted with these

same types of cells.

Messina et al., (2006) suggested that a daily dose

of 120 mg isoflavones may be useful in prostate can-

cer prevention, but recommended consumption of

soy foods rather than isolated isoflavones supple-

ments, because other soy components such as soy

protein, fiber and saponins may offer additional

health benefits. Nagata et al., (2007) studied the ef-

fect of dietary isoflavone against prostate cancer in

Japanese males and found that inclusion of dietary

isoflavone might be an effective dietary protective

factor against prostate cancer in Japanese men. In

this study male subjects in the highest category of


isoflavones intake (greater than 90 mg/day) exhib-

ited a 58% lower prostate cancer risk than male sub-

jects in the lowest category (less than 30 mg/day). In

one study, early-stage prostate cancer patients were

randomly assigned to receive a soy protein supple-

ment (60 mg/day isoflavones) or a placebo daily for

12 weeks. Patients who received the soy protein sup-

plement exhibited greater decreases in total serum

PSA and free testosterone than did patients who re-

ceived the placebo, but these differences were not

statistically significant (Kumar et al., 2004). In two

small studies prostate cancer patients were fed soy

isoflavone and in these studies there appeared to be

a decrease in the rate of the rising serum PSA con-

centration associated with prostate tumor growth

(Fischer et al., 2004; Hussain et al., 2003). A trial of

soy milk supplementation (141 mg/day isoflavones)

in men with PSA recurrent prostate cancer found

that PSA levels increased by an average of 20%

over a 12-month period compared to a 56% yearly

increase prior to the study (Pendleton et al., 2008).

Hamilton-Reeves and coworkers at the University of

Minnesota, (2007) examined the potential protective

effect of soy protein isolate, with low and high lev-

els of isoflavones, on prostate cancer risk in men at

high risk for developing the advanced form of pros-

tate cancer. They found that soy protein isolate con-

sumption suppressed androgen receptor expression

in the prostate and could be beneficial in prevent-

ing prostate cancer. Kumar et al., (2007) treated 53

prostate cancer patients with 80 mg purified isofla-

vones or a placebo for 12 weeks. Although plasma

isoflavones increased with no observed clinical tox-

icity, there was no modulation of serum sex hormone

binding globulin, total estradiol, or testosterone in

the isoflavone-treated group compared to placebo.

The study establishes the need to explore other po-

tential mechanisms by which prolonged and consis-

tent purified isoflavone consumption may modulate

prostate cancer risk.

A meta-analysis of eight studies performed by

Yan and Spitznagel (2009) found that isoflavone con-

sumption was associated with a reduction in risk of

prostate cancer, but the association was not statisti-

cally significant. Similar results were reported by Mi-

yanaga et al., (2012) wherein isoflavones had no influ-

ence on the level of PSA, but biopsies showed that

isoflavone intake reduced the incidence of prostate

cancer, but that this difference was not statistically

significant. Several epidemiologic studies have also

shown no association between high consumption of

fermented soy foods and prostate cancer (Hwang et

al., 2009). Isoflavones are purported to slow prostate

cancer growth and cause cancer cells to die (Fot-

sis et al., 1993). Supplementation with soy protein

or soy isoflavone decreased the markers of cancer

development and progression in prostate cells in-

cluding PSA, testosterone, and androgen receptor

in patients with prostate cancer (Kumar et al., 2004;

Dalais et al., 2004) or in men at high risk for develop-

ing advanced prostate cancer (Hamilton-Reeves et

al., 2007).

Isoflavones and Cardiovascular Disease

In 2010, cardiovascular disease (CVD) was the lead-

ing cause of death responsible for 597,689 deaths in

the U.S. (CDC, 2013). An estimated 30% of all global

deaths in 2008 were from CVD (WHO, 2011a); of these

deaths, an estimated 7.3 million were due to coro-

nary heart disease and 6.2 million were due to stroke

(WHO, 2011b). In order to reduce coronary heart dis-

ease it is recommended that saturated fat should be

replaced with polyunsaturated fatty acids. Soy foods

are ideal for this replacement since they contain the

omega-6 polyunsaturated fatty acid (PUFA) linoleic

acid, which comprises about 55 percent of the total

fat in soybeans and which reduces blood cholesterol

levels (Slavin et al., 2009; Jenkins et al., 2002). Jen-

kins et al. (2011) recommended a diet supplemented

with cholesterol-lowering foods including soyfoods

like soymilk and soy meat alternatives, oats, nuts and

plant sterols for adults with high cholesterol. This diet

lowered low density lipoprotein (LDL) cholesterol by

13.8% compared with a decrease of only 3% in those

that followed a standard low saturated fat diet. Other

epidemiological studies have also suggested that

Asian populations consuming large amounts of soy

have lower rates of cardiovascular disease than West-

ern populations (Zhang et al. 2003).


Research suggests that soy protects against ath-

erosclerosis by lowering cholesterol, or by increas-

ing blood levels of nitric oxide which helps in blood

vessel dilation, inhibits oxidative damage caused by

cholesterol and prevents the adhesion of white cells

to the vascular wall. (Cena and Steinberg, 2011). A

combination of soy protein and isoflavones appear

to exhibit the strongest hypocholesterolemic effects

compared to isolated soy protein or soy isoflavones

alone (Mortensen et al., 2009). Research studies

suggest that soy protein decreases postprandial

triglyceride levels, which is increasingly viewed as

important for reducing coronary heart disease risk

(Zhan and Ho, 2005). Whole soy can promote a 3%

to 5% reduction in blood cholesterol (Lichtenstein et

al. 2006; Zhan and Ho 2005). Henrotin et al., (2003)

concluded that soy protein led to increased blood

levels of L-arginine (the amino acid that the human

body uses to produce nitric oxide) and nitric oxide

metabolites. Isoflavones increase endothelial nitric

oxide production, enhancing vasodilation and im-

proving blood flow (Taku et al., 2007; Zhan and Ho,

2005). Specific findings include beneficial effects on

lipids and lipoproteins, with a decline in total cho-

lesterol (9%), LDL cholesterol (13%), and triglycer-

ides (11%) and an increase in high density lipopro-

tein (HDL) cholesterol (2.4%) (Anderson et al., 1995).

Isoflavones found in soybean and soy foods provide

cardiovascular health benefits by neutralizing free

radicals that cause oxidative damage to cells thus

improving arterial elasticity, a vascular function that

normally decreases with age, helping to reduce LDL

cholesterol levels (Steinberg et al. 2003). Otherwise,

these free radicals within blood vessels can oxidize

circulating LDL cholesterol, starting a cascade of in-

flammatory events that ultimately increases the risk

of developing heart disease. In a study conducted

by Candy (1996) 61 middle-aged men that had been

diagnosed as having a high risk of developing cor-

onary disease were asked to consume soy protein

(20 g) and soy isoflavone (80 mg) for five weeks;

those consuming soy showed significant reductions

in both diastolic and systolic blood pressure com-

pared to those who were given a placebo diet con-

taining olive oil. In the mid-1990s, a meta-analysis of

29 clinical trials found that compared to animal pro-

tein, soy protein significantly reduced blood levels

of several lipids (total cholesterol, LDL cholesterol

and triglycerides) (Anderson et al. 1995). There is ad-

ditional data suggesting isoflavones have indepen-

dent coronary benefits. In several studies isoflavones

have been shown to enhance endothelial function

(Walker et al., 2001; Squadrito et al., 2002, 2003) and

systemic arterial elasticity (Nestel et al., 1997, 1999);

both of these measures are considered to be indi-

cators of coronary health (Bonetti et. al., 2003; Her-

rington et al., 2004).

In several research studies it has been shown that

on average, soy protein lowers LDL cholesterol ap-

proximately 4% (Sacks et al., 2006; Zhan and Ho,

2005). Each one percent reduction in cholesterol low-

ers coronary heart disease risk at least 2% (Law et al.,

1994). A meta-analysis conducted by Weggemans

and Trautwein (2003) found that soy protein slightly

raised HDL cholesterol levels leading tham to con-

clude that as a result of the changes in lipid levels,

soy could reduce heart disease risk by as much as 20

percent. Furthermore, there is evidence to suggest

that soyfoods may decrease blood pressure (Rivas et

al., 2000) and increase LDL cholesterol particle size

(Desroches et al., 2004). Dong et al., (2011) analyzed

27 clinical (human intervention) studies and found

that on average, soy lowered blood pressure about

2 ½ points. Li et al. (2010) studied the effect of oral

isoflavone supplementation on vascular endothelial

function in postmenopausal women and concluded

that isoflavone helps to improve endothelial func-

tion.

Zhan and Ho (2005) reported that the inclusion of

soy in the diet can decrease blood levels of LDL cho-

lesterol. A meta-analysis of 7 studies found that soy

protein that contained enriched isoflavones, and in

comparison with animal protein without isoflavones,

were associated with a significant decrease in serum

total cholesterol (0.32 mmol/L or 5.69%) in the hyper-

cholesterolemic subcategory and LDL cholesterol

(0.18 mmol/L or 4.98%) in the total human popula-

tion. It was also reported in this study that a signifi-

cant increase in serum HDL cholesterol (0.04 mmol/L

or 3.00%) occurred in the total population. Similarly


a study by Steinberg et al., (2003) demonstrated that

consuming intact soy protein and isoflavone may im-

prove vascular function better than the consumption

of either component alone. Gardner et al., (2007)

compared cholesterol levels between those who

drank soymilk and those who drank 1% dairy milk as

part of an overall diet containing moderate fat (35%

of calories). In only four weeks, the soymilk group

exhibited a 5% reduction in LDL cholesterol, a sta-

tistically significant advantage over those who con-

sumed dairy milk.

ROLE OF ISOFLAVONES IN REDUCING MENOPAUSAL SYMPTOMS AND HOT FLASHES

Hot flashes are the primary reason that women

seek medical attention for menopausal symptoms

(Tice et al., 2003). A low incidence of hot flashes and

other menopausal symptoms in Japanese women

is believed to be due to the estrogen-like effects of

soy isoflavones (Kuiper et al., 1998; Lock, 1992; 1994).

Women fed with soy flour (45 g) daily demonstrated

a reduction (40%) in menopause symptoms (Allaoua

et al., 2005). Conversely, other studies have reported

no beneficial effect of soy intake on menopausal

symptoms. A meta-analysis of 25 trials involving

2,348 participants published between 1966 and 2004

concluded that soy phytoestrogens did not improve

hot flashes or other menopausal symptoms (Krebs

et al., 2004). The isoflavone daidzein has been re-

ported to reduce hot flashes in menopausal women.

The chemical structure of daidzein is very similar to

the human body’s own estrogen. A study was con-

ducted by Khaodhiar et al., (2008) on 190 women

ranging in age from 38 to 60 years in various stages

of menopause, who had 4 to 14 hot flashes daily. The

women were given either one or two concentrations

of diadzein rich isoflavone-aglycone. The number of

hot flashes in the diadzein rich isoflavone groups was

reduced by 52% and 51% at the end of 12 weeks,

while the placebo group experienced a 39% reduc-

tion. A few studies using higher doses of isoflavone

(50 to 80 mg/day), enrolling women with more vaso-

motor symptoms at baseline (4 to 7 symptoms/day)

and with larger sample sizes, have exhibited mildly

beneficial effects on self-reported frequency and se-

verity of vasomotor symptoms (Albertazzi et al. 1998,

Washburn et al. 1999). Han et al., (2002) reported a

26% decrease in hot flash frequency in a group who

consumed 100 mg/day of isoflavone as compared to

a group who receive a placebo. Isoflavones from soy

have also received attention as a possible alterna-

tive to conventional hormone replacement therapy

(HRT) (Brandi, 1999; Eden, 2001; Elkind-Hirsch, 2001;

Glazier and Bowman, 2001; Vincent and Fitzpatrick,

2000). Since there is a chemical similarity to the fe-

male sex hormone estrogen, isoflavones have been

used in studies for relief of menopausal symptoms

(Adlercreutz et al., 1992; North and Sharples, 2001).

A ROLE FOR ISOFLAVONES IN OBESITY

Obesity is a state of excessive fat accumulation

in the body, especially in abdominal adipose tissue,

and is closely linked to metabolic disorders, which

include diabetes, cardiovascular disease, nonal-

coholic fatty liver disease, dyslipidemia, and other

health problems (Després et al., 2008; Lois et al.,

2008;). Nearly 20% of women aged 55 to 65 years

suffer an increase in glucose tolerance and insulin

resistance, thought to be due to estrogen defciency

(Gaspard et al., 1995; Tufano et al., 2004). The effect

of soy isoflavone supplementation on postmeno-

pausal women has yielded mixed responses in sev-

eral clinical trials due to differences in dose, duration

of isoflavone supplementation, body weight, physi-

cal status of individual, and variability of experimen-

tal designs (Zhang et al., 2013). Some researchers

have suggested a reduction in body weight (Sites et

al., 2007; Gardner et al., 2001), fasting blood glucose

(Villa et al., 2009; Crisafulli et al., 2005), blood fat lev-

els (Reynolds et al, 2006) and insulin level (Villa et al.,

2009; Crisafulli et al., 2005) as possible causes. How-

ever, other researchers did not reach this same con-

clusion (Charles et al., 2009; Khaodhiar et al., 2008).


EFFECT OF ISOFLAVONES ON THYROID FUNCTION

Concerns have been expressed that soy iso-

flavone intake adversely affects thyroid function

(Messina and Redmond, 2006). The thyroid gland

releases two primary hormones T4 and T3 (thyrox-

ine and trio-iodothyronine, respectively) in a ratio

of roughly 80:20 in response to the signal sent by

thyroid stimulating hormone (TSH). Several studies

have reported the interaction of soy isoflavones with

thyroid function. Isoflavones lead to immune dys-

function by causing potent stimulation of T cell and

B cell mediated immunity due to induced structural

changes in thyroid peroxidase (Chen and Rogan,

2004). A study in premenopausal women (Duncan et

al., 1999) demonstrated a decline in free T3 levels

with a high isoflavone diet. Another study conduct-

ed with post-menopausal women demonstrated a

rise in T4 with 56 mg isoflavones/day and a rise in

T3 and TSH with 90 mg isoflavones/day at 6 months

compared with controls, but these alterations were

considered clinically non-significant (Persky et al.,

2002). Several other studies revealed non-significant

changes in thyroid profile with isoflavones in meno-

pausal women (Borchers et al., 2008; Duncan et al.,

1999; Teas et al., 2007). The precise reason for such

reported variations is unclear although differences

in doses, dosage forms, isoflavone composition or

the duration of treatment may all be considered im-

portant factors. Milerová et. al., (2006) reported on a

study that looked at thyroid hormones and thyroid

autoantibodies, along with blood levels of daidzein

and genistein. The study focused on children with-

out overt thyroid disease, who were not iodine de-

ficient. They found a significant positive association

of genistein with thyroglobulin autoantibodies and a

negative correlation with thyroid volume. They con-

cluded that even small differences in soy phytoes-

trogen intake may influence thyroid function, which

could be important when iodine intake is insufficient.

Studies by Huang et al., (2005) and Xiao et al.,

(2004) have shown that isoflavones suppress the

binding ability of hepatic thyroid hormone recep-

tor to the thyroid hormone response element of the

target genes. Similarly, in vitro studies by Divi et al.,

(1996; 1997) have shown that isoflavones inhibit thy-

roid peroxidase. Genistein and daidzein block thy-

roid peroxidase-catalyzed tyrosine iodination by act-

ing as alternate substrates (Divi et al., 1997). Chang

and Doerge (2000) reported that consumption of soy

could cause goiter only in animals or humans con-

suming diets marginally adequate in iodine or who

were predisposed to develop goiter and in most

cases dietary supplementation with adequate iodine

can reverse the disorders (Schone et al., 1990). How-

ever, study by Poirier et al., (1999) reported that even

the feeding of genistein to rats, which as an animal

species are very sensitive to goitrogenic agents,

does not disrupt normal thyroid functioning.

CONCLUSIONS

Consumers in the U. S. have become aware of

the potential health benefits of soy, and as a result

consumption in the U. S. has increased although it

still remains far below that in Asia. There is a great

deal of evidence to support the beneficial effects

of soy isoflavones in the prevention of bone loss in

postmenopausal women. Although isoflavones have

been demonstrated to positively impact the bio-

markers of prostate cancer, their potential benefits

have not been substantiated in clinical trials. Benefi-

cial effects of isoflavones for relieving menopause

symptoms and prevention of breast cancer have not

been proven and the antithyroid actions of soyfoods

appear to be consistent in both animals and hu-

mans. The inconsistency of results from animal and

human studies may be partially due to variation in

the bioavailability of the isoflavones. Varying levels

and duration of isoflavone consumption have also

been shown to be of importance in whether soy-

foods have a beneficial role. In addition, the role of

the gut microflora may be particularly important in

the production of equol from daidzein, since equol

may actually confer more health benefits than daid-

zein. Much more research needs to be carried out in

this area, in order to understand how soy can have

health benefits in the broader population.


ACKNOWLEDGEMENTS

The writing of this review was supported in part by

a grant from the Arkansas Soybean Board.

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docrinol. Invest. 27:648–653.

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77:1459– 1465.


The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

VOLUME 4 ISSUE 1

Preventing Post-Processing Contamination in a Food Nugget Processing Line When Lan-guage Barriers ExistJ. A. Neal, C. A. O’Bryan and P. G. Crandall

20

A Personal Hygiene Behavioral Change Study at a Midwestern Cheese Production PlantJ. A. Neal, C. A. O’Bryan and P. G. Crandall

13

Behavioral Change Study at a Western Soup Production Plant

C. A. O’Bryan, J. A. Neal, and P. G. Crandall

27

Salmonella in Cantaloupes: You Make Me Sick!B. A. Almanza

35

The Hurricane Sandy DilemmaB. A. Almanza

43

Introduction Special IssueP. G. Crandall

8

Case Studies

Instructions for Authors61

Introduction to Authors

Intellect-u-ale: A Smart Approach to Quality Assurance in a Micro-BreweryA. J. Corsi, M. Goodman, and J. A. Neal

50


MANUSCRIPT SUBMISSION

Authors must submit their papers electronically

([email protected]). According to instruc-

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com. Authors who are unable to submit electroni-

cally should contact the editorial office for assistance

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INSTRUCTIONS TO AUTHORS

• Aerobic microbiology

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• Antibiotics

• Antimicrobials

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• Food control

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• Food Safety

• Foodborne pathogens

• Gastrointestinal microbiology

• Microbial education

• Microbial genetics

• Microbial physiology

• Modeling and microbial kinetics

• Natural products

• Phytoceuticals

• Quantitative microbiology

• Plant microbiology

• Plant pathogens

• Prebiotics

• Probiotics

• Rumen microbiology

• Rapid methods

• Toxins

• Veterinary microbiology

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• Water microbiology

CONTENT OF MANUSCRIPT

We invite you to consider submitting your re-

search and review manuscripts to AFAB. The jour-

nal serves as a peer reviewed scientific forum for to

the latest advancements in bacteriology research

on Agricultural and Food Systems which includes

the following fields:


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expressed in this viewpoint is the authors alone and

does not necessarily represent the opinion of AFAB

or the editorial board.

COPYRIGHT AGREEMENT

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permits and is available online (www.afabjournal.com).


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PEER REVIEW PROCESS

Authors will be requested to provide the names

and complete addresses including emails of five (5) potential reviewers who have expertise in the research

area and no conflict of interest with any of the authors.

Except for manuscripts designated as Rapid Commu-

nication each reviewer has two (2) weeks to review

the manuscript, and submit comments electronically

to the editorial office. Authors have three (3) weeks

to complete the revision, which shall be returned to

the editorial office within six (6) weeks after which the

authors risk having their manuscript removed from

AFAB files if they fail to ask the editorial office for

an extension by email. Deleted manuscripts will be

reconsidered, but they will have to be processed as

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sessed upon submission. Once reviewed, the author

will be notified of the outcome and advised accord-

ingly. Editors handle all initial correspondence with

authors during the review process. The editor-in chief

will notify the author of the final decision to accept or

reject. Rejected manuscripts can be resubmitted only

with an invitation from the editor or editor-in chief. Re-

vised versions of previously rejected manuscripts are

treated as new submissions.

PRODUCTION OF PROOFS

Accepted manuscripts are forwarded to the edito-

rial office for technical editing and layout. The manu-

script is then formatted, figures are reproduced, and

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Corrections must be returned by e-mail. Changes

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es. Author alterations to proofs exceeding 5% of the

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correspondence of proofs must be agreed to by the

editorial office and the author within 48 hours or proof

will be published as is and AFAB will assume no re-

sponsibility for errors that result in the final publication.

PUBLICATION CHARGES

AFAB has two publication charge options: conven-

tional page charges and rapid communication. The

current charge for conventional publication is $25 per printed page in the journal. There is no additional

charge for the publication of pages containing color

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munication, authors will pay the rapid communication

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in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one

week and $100 per journal page.

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If you are wishing to obtain a physical hard copy of

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and $150/page for color prints. You may order your

offprints at any time after publication on our website.

Scientific conference organizers may be expected to

agree to a set number of offprints as a part of their

agreement with AFAB.


MANUSCRIPT CONTENT REQUIREMENTS

Preparing the Manuscript File

Manuscripts must be written in grammatically

correct English. AFAB offers a fee based language

service upon request ([email protected]).

Manuscripts should be typed double-spaced, with

lines and pages numbered consecutively. All docu-

ments must be submitted in Microsoft Word (.doc or

.docx, PC or Mac). All special characters (e.g., Greek,

math, symbols) should be inserted using the sym-

bols palette available in this font. Tables and figures

should be placed in separate sections at the end of

the manuscript (not placed in the text). Failure to fol-

low these instructions will cause delays of the pro-

cessing and review of the manuscript.

Title Page

At the very top of the title page, include a title of

not more than 100 characters. Format the title with

the first letter of each word capitalized. No abbre-

viations should be used. Under the title, the authors

names are listed. Use the author’s initials for both first

and middle names with a period (full-stop) between

initials (e.g., W. A. Afab). Underneath the authors, a

list affiliations must be listed. Please use numerical

superscripts after the author’s names to designate

affiliation. If an authors address has changed since

the research was completed, this new information

must be designated as “Current address:”. The cor-

responding author should be indicated with an aster-

isk e.g., * Corresponding author. The title page shall

include the name and full address of the correspond-

ing author. Telephone and e-mail address must also

be provided for the corresponding author, and email-addresses must be provided for all authors.

Editing

Author-derived abbreviations should be defined

at first use in the abstract and again in the body of

the manuscript. If abbreviations are extensive au-

thors may need to provide a list of abbreviations

at the beginning of the manuscript. In vivo, in vitro

and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and

merge such paragraphs appropriately. Authors must

not begin sentences with “Figure or Table shows…”

as these are inanimate objects and cannot “show”

anything. When number are reported in text or in ta-

bles, always put a zero in front of decimal numbers:

“0.10” instead of “.10”.

MANUSCRIPT SECTIONS

Abstract

The abstract provides an abridged version of the

manuscript. Please submit your abstract on a sepa-

rate page after the title page. The abstract should

provide a justification of your work, objectives, meth-

ods, results, discussion and implications of study or

review findings . Your abstract must consist of com-

plete sentences without references to other work or

footnotes and must not exceed 250 words. On the

same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-

ing. Ideally, these keywords should include signifi-

cant words from the title.

Introduction

The introduction should clearly present the foun-

dation of the manuscript topic and what makes the

research or the review unique. The introduction

should validate why this topic is important based on

previously published literature, and the relevance of

the current research. Overall goals and project ob-

jectives must be clearly stated in the final sentence

of the last paragraphs of the introduction.

Materials and Methods

Information on equipment and chemicals used

must include the full company name, city, and state

(country if outside the United States or Province if

in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].


Variability, Replication, and Statistical Analysis

To properly assess biological systems indepen-

dent replication of experiments and quantification

of variation among replicates is required by AFAB.

Reviewers and/or editors may request additional

statistical analysis depending on the nature of the

data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

Discussion

The discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

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manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

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need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

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tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

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