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Influence of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas* Corresponding author. Violeta Pardío Sedas: Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, Avenida Miguel Ángel de Quevedo s/n esquina Yáñez, Colonia Unidad Veracruzana, Veracruz, Veracruz, México CP. 91710, Telephone: 52-229-9342075 ext. 24125, fax: ext. 24104, E-mail: [email protected], [email protected] Departamental-Institutional contact: Ms Yadira Sarmiento. Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, Avenida Miguel Ángel de Quevedo s/n esquina Yáñez, Colonia Unidad Veracruzana, Veracruz, Veracruz, México CP. 91710, Tel: 52-229-9342075 ext. 24103, fax: 24104, E-mail: [email protected] Running title: Influence of ozone on physical properties of oysters Word count: 3890 Number of tables: 3 Number of figures: 1 Number of references: 29 Words in references: 782

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Page 1: Influence of ozone depuration on the physical properties … of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas*

Influence of ozone depuration on the physical properties of fresh

American oysters (Crassostrea virginica)

Violeta Pardío-Sedas*

Corresponding author. Violeta Pardío Sedas: Facultad de Medicina Veterinaria y

Zootecnia, Universidad Veracruzana, Avenida Miguel Ángel de Quevedo s/n esquina

Yáñez, Colonia Unidad Veracruzana, Veracruz, Veracruz, México CP. 91710,

Telephone: 52-229-9342075 ext. 24125, fax: ext. 24104, E-mail: [email protected],

[email protected]

Departamental-Institutional contact: Ms Yadira Sarmiento. Facultad de Medicina

Veterinaria y Zootecnia, Universidad Veracruzana, Avenida Miguel Ángel de Quevedo s/n

esquina Yáñez, Colonia Unidad Veracruzana, Veracruz, Veracruz, México CP. 91710, Tel:

52-229-9342075 ext. 24103, fax: 24104, E-mail: [email protected]

Running title: Influence of ozone on physical properties of oysters

Word count: 3890

Number of tables: 3

Number of figures: 1

Number of references: 29

Words in references: 782

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Abstract

Ozone depuration is a nonthermal technology applied to moderated sewage-contaminated

oysters to increase the supply of safe and nutritious bivalves. Ozone reacts with proteins

causing peptide bond cleavage and a range of amino acid side chain modifications.

Collagen contributes to the texture and tenderness of oyster meat and although scientific

information is available in relation to the action of ozone on proteins, little is known about

the change in physical properties of proteins in food systems after an ozonization process

that may affect the textural properties of the food product. Ozone treatment diminished

oyster quality. Texture of oyster meat was positive correlated (r = 0.550) with proteolysis

as Free amino acid content increased and firmness of oyster meat decreased; texture and

shear force were negative correlated (r = -0.724), as shear force values decreased and

panelist detected a softer meat. Meanwhile, WB shear force was negatively correlated (r = -

0.469) to proteolysis as Free amino acid content increased and WB shear force diminished

by ozone depuration.

Kew words: American oyster, ozone depuration, proteolysis, shear force, texture attribute

List of abbreviations: FAA (Free Amino Acid)

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Introduction

Globally, seafood provides more than 3 billion people with at least 15% of their average

per-capita intake of animal protein (FAO, 2011). Nevertheless, bivalve mollusks (oysters,

clams, mussels, cockles, and scallop) are highly perishable and risky, and may cause human

illnesses because: 1) their immobility and their habitat in coastal estuaries render them

susceptible to fecal contamination and environmental pollution in the water; 2) their filter

feeding behavior leads to the accumulation of pathogens naturally present in growing areas

contaminated with polluted waters; 3) consumers often eat the shellfish whole and raw,

including the gastrointestinal tract where pathogens are concentrated. Raw fresh oysters are

sold either as whole in-shell unpackaged or shucked packaged in polyethylene pouches

along with tap water, being the shelf-life about 6 days under refrigeration.

To improve and ensure their microbiological safety raw shellfish are depurated by

removing primary human pathogens and the load of commensal bacteria that may also be

pathogenic to humans such as Vibrios, reducing the likelihood of transmitting infectious

agents to consumers, and to prevent recontamination with foodborne pathogens and

spoilage organisms. Depuration is one of the major treatment processes applied to

moderated sewage-contaminated shellfish bivalves to increase the availability and supply of

safe and nutritious oysters, mussels, and clams eaten raw. The depuration process exploits

the natural physiological functions of the gastrointestinal tract of live animals and the

success of the process is dependent on the animal wellbeing, the health status, the

physiological activity, and pumping rate and behavioral responses (Schneider et al., 2009).

Although markets have been growing for the treated seafood, a substantial number

of consumers still prefer live, whole raw mollusks or slightly cooked. This strong growing

consumer’s demand for high-quality foods minimally processed that are additive-free, with

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their natural freshness and sensorial characteristics, an extended shelf-life, and are safe

products without quality degradation has led to the development of several novel and

innovative food processing methods of microbial control known as Nonthermal

technologies introduced to replace or complement the well-established thermal processes,

such as the emerging technologies ohmic heating, ultraviolet light, and ozonization (Novak

and Yuan, 2007).

How composition is altered

The main purpose of depuration is to maintain the shelf life and to reduce or minimize de

loss of flavor and texture during storage in highly perishable fresh shellfish, as bivalve

mollusks (Cheney, 2010). To increase the efficacy of depuration it is combined with

disinfectants as ozone. Ozone (O3) is very unstable both in the gaseous phase and in

solution, decomposing into highly reactive free radicals as hydroxy (• OH), hydroperoxy (•

HO2), and superoxide (• O2). The powerful and effective antimicrobial activity comes from

the molecule’s inherent instability, attributed to the great oxidizing power of these free

radicals, killing microorganisms through oxidation of their cell membranes (Kim et al.,

1999). Considered as generally recognized as safe (GRAS) as an antimicrobial agent for

use as a disinfectant or sanitizer for foods (USDA, 1997), ozone has a high germicidal

effectiveness against a wide range of pathogenic organisms including both Gram-positive

and Gram-negative bacteria, viruses, fungi, and protozoa (Skall and Olesen, 2011).

Microbiological, biochemical, and sensory changes are associated with the

deterioration of mollusks quality that depends on many factors, such as handling and

storage, intrinsic factors of the animals (species, age and size, fat composition, feeding and

physiological status), and the qualitative and quantitative compositions of the initial

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microbial flora (Gram and Hus, 2000). Ozone depuration may promote a faster

physiological deterioration, biochemical changes and microbial degradation of oyster even

when only slight processing operations are used.

Limited data is available in the literature on the application of ozone to molluscan

shellfish preservation, and different process conditions and concentrations of ozone have

been employed to depurate several molluscan shellfish. In order to study the influence of

ozone on the physical characteristics of American oyster (Crassostrea virginica), 750

organisms (5 - 6 g, 9 cm long) collected by diving from banks of harvesting areas in a

coastal lagoon system, were immediately transported to the laboratory in coolers at 4°C

(NOM-109-SSA1-1994) (SSA, 2012). Oysters were scrubbed and rapidly washed by high-

pressure water spray to remove fouling organisms, mud and surface debris, and attached

algae adhering to the shell, damaged o gaping animals were discarded, and immediately

placed in the recirculating seawater system (9 L/min, 270/00 salinity, 90% dissolved oxygen,

25.0°C, and pH 8.0) within 2 h of harvesting and depurated with clean artificial seawater

during 2-4 hours to promote oyster activity. Afterwards, seawater was disinfected by ozone

generated by corona discharge at 0.2, 0.4, and 0.6 mg/L. Each tank was loaded with 250

oysters previously estimated and placed into plastic mesh baskets as single overlapping

layer. Duplicate samples of 30 oysters from aleatory locations in each tank were collected

at 0, 2, 4, and 6 h for microbial analyses within 2 h of collection. According to Table 1, E.

coli levels declined in the first 2 h of depuration, and after 4 h the MPN decreased 88, 50,

and 64% of its original concentration, and 88, 50 and 82% after 6 h of depuration when 0.2,

0.4, and 0.6 mgO3/L were applied, respectively.

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E. coli in oyster control samples decreased 33.3% during 4 h of depuration, and

increased to 650 MPN/100g at 6 h. Meanwhile, Vibrio cholerae non O1 was not detected

after 6 h of depuration with 0.2 mg/L and after 4 h of depuration with 0.4 and 0.6 mg/L,

reaching the legally bacteriological limit of no detection of V. cholerae non O1 in 50 g of

oyster flesh. As it can be observed, oysters released E. coli rapidly after 2 h of depuration

and reached the legally bacteriological limits of E. coli ˂ 230 MPN/100 g (NOM-242-

SSA1-2005), indicating that E. coli was more sensitive than V. cholera non O1. Schneider

et al. (1991) reported that 3 mg/liter ozonated recirculated artificial seawater applied to

Mercenaria campechiensis, reduced V. vulnificus in the shellfish meats by an average of 2

log units. E. coli in Mytilus galloprovincialis experimentally contaminated was reduced

42% of its original concentration with 50 mg O3/h after 5 h of depuration and V. cholerae

O1 was reduced by 1 log after 24 h of depuration, level that remained for 44 h (Croci et al.,

2002); according to Meloni et al. (2008) E.coli counts in Mytilus galloprovincialis

decreased after 8 h - depuration, meanwhile Vibrios declined at slower rate. Maffei et al.

(2008) working in ozone open-circuit seawater-disinfection system at the Bivalve

Depuration Center (Cattolica, Rimini, Italy) reported a mean E. coli reduction (62%) during

depuration of Chamelea gallina, however V. parahaemolyticus and V. alginolyticus were

not eliminated. Cozzi et al. (2009) depurated blue mussels (Mytilus galloprovincialis) using

a combined system of UV light (55 watt) and ozone (50 mg/h). After 24 h of treatment both

E. coli and V. parahaemolyticus concentrations decreased by approximately 2 Log while V.

vulnificus decreased slower only by 1 Log. After 72 h the depuration process reduced E.

coli and Vibrio contamination to level close or below the detection limit of the methods (10

CFU/g). When Borazjani et al. (2003) depurated whole in-shell oysters (Crassostrea

virginica) for 30 min with 2 to 4 mg O3/L, V. vulnificus counts in oyster’s meats were not

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reduced. Rong et al. (2010) treated oysters with 5 μg liter−1

ozonated water for 2 min and

resulted in a less than 1-log decrease of indigenous microbiota.

This findings confirming that E. coli is an inadequate index of microbiological

safety of molluskan shellfish not only because its presence does not correlate with the

occurring Vibrios, but also as a means of assessing the efficacy of the depuration process.

In consequence, adoptions of shorter treatment times for mollusks with high initial loads of

Vibrios could lead to an insufficient reduction to guarantee the safety of consumers with

lower than normal thresholds of infection, representing a potential health hazard.

While the destruction of bacteria by ozone has been studied extensively, relatively

little information is available on the effect of ozone on various food components. Oyster is

one of the most important and abundant harvested bivalves and highly valued worldwide

for its rich nutrition and desirable taste. The popularity of oysters in the diet continues to

increase because they constitute a healthy low-fat contribution of proteins, healthy

polyunsaturated fatty acids, and phytosterols into a balanced diet. Depending on sex,

maturity, water temperatures, food supply, stress and other environmental parameters, 100

g of raw oyster contains 77.4 – 90.2 g moisture, 5.6 – 10.9 g protein, 1.9- 4.7 g

carbohydrate, 0.7 – 2.4 g total fat, 0.7 – 2.9 g ash, 37 – 58 mg cholesterol and 75 calories. It

provides 100% of vitamin B12 and zinc and about 35% iron (Sáenz, 2007). As fresh

seafood, oysters are more perishable than other high-protein foods and have a short shelf

life. Fresh oysters are popular consumed raw as they have more delicate flavor and texture

than cooked oysters and retain more nutrients. For a consumer, ‘eating quality’ of shellfish

is associated with freshness. Fresh shellfish product indicates typical aquatic fresh odour

and flavor, brilliant appearance and colour, and texture characteristics of the species in

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good condition; thus, fresh shellfish products should be marketed as fast as possible since

their shelf life is very short (Gram and Huss, 2000).

The possible pro-oxidant effect of ozone on shellfish constituents has not been

extensively studied up to now, although previous reports have shown a potential negative

effect on phospholipids, polyunsaturated fatty acids, and membrane proteins (Cataldo,

2003). Given the high reactivity of ozone, it is difficult to predict its reaction as it may

oxidize or ionize a substrate or spontaneously decompose to oxygen and free radicals.

Ozone is not universally beneficial and in some cases may promote surface oxidation,

discoloration, or even development of undesirable odors, resulting in degradation of the

color, texture and flavor of the food (Khadre et al., 2001); moreover, ozone treatments may

negatively affect the nutritional quality of the product. Unfortunately, little is known about

the changes in physical, biochemical or sensory properties of bivalve mollusks.

Aquatic multicellular animals contain collagen in the edible tissues such as muscle,

bone and skin. A close relationship has recently been reported between texture and collagen

content for the muscles of many aquatic species, suggesting the importance of this protein

in association with physical properties of marine foods (Mizuta et al., 2005). Information of

muscle collagens of bivalve mollusks is still limited for the biochemical properties and

components. The mantle of a bivalve is a thin, sheet-like membranous organ that covers the

inside of the animal's shell and biomineralization of shell is considered as its primary

function. Collagen fibre is mainly distributed in the muscle connective tissues (epimysium,

perimysium, and endomysium) of both mantles and adductors, and in the inner connective

tissue matrix of the mantles. The collagen contents of the edible tissues varied

considerably; the mantles have much higher collagen content than the adductors (Mizuta et

al., 2004). Mizuta et al. (2005) reported that the major collagen in mantle of the oyster

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Crassostrea gigas have a heterotrimer structure (1)2, with a content of 1.1% of protein,

and the major collagen showed relatively low content of alanine and high content of

hydroxylysine.

Several studies have been carried out to investigate the basic interaction of ozone

with different proteins and the interactions of specific amino acids in proteins. Although all

amino acids are potential targets for oxidation by reactive oxygen, the major aromatic

amino acids tyrosine, tryptophan, phenylalanine, the sulphur containing amino acids

cysteine, methionine as well as the aliphatic amino acids arginine, lysine, proline and

histidine appear especially sensitive to oxidation (Cataldo, 2003). Ozone reacts with

proteins and causes the oxidation of the polypeptide backbone of the protein, peptide bond

cleavage, protein–protein cross-linking and a range of amino acid side chain modifications.

Helices, sheets, coils, and folded branches which define the secondary and tertiary structure

of proteins seem to be modified by the reaction of ozone with proteins (Hicran et al., 2012).

The sensitivity of a given kind of amino acid residue to oxidation by ozone varies from one

protein to another and this variability is due to differences in the structures of the proteins

(Sharma and Graham, 2010). The oxidation of amino acids may induce an increase local

flexibility or rigidity to the protein chain causing an alteration of its secondary and tertiary

structure. As a result of ozone attack the protein molecules undergo changes in their visual

folding and binding ability which influence their functionality (Cataldo, 2003).

Curran et al. (1984) reported that oxygen-derived free radicals from ozone can (i)

directly degrade soluble collagen and (ii) at low levels can potentiate the destructive role of

proteinases by increasing the susceptibility of collagen to degradation by proteinases. These

authors established that oxidants can directly degrade soluble collagen, and at low levels

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can modify collagen, making it susceptible to proteolytic degradation. Fujimori (1985)

showed that upon prolonged exposure even at low levels, the initial modification of the

most ozone-sensitive collagen regions may be followed by further extensive oxidation.

Other ways in which composition is altered

Although scientific information is available in relation to the action of ozone on proteins,

little is known about the change in physical properties of proteins in food systems after an

ozonization process that may affect the textural properties of the food product. To

understand the effect of ozone in oyster texture when used as an alternative processing

technology to improve the quality of fresh oysters, the effect of ozone on proteins was

detected by the proteolysis, Warner-Bratzler shear force, and texture analysis of oyster

meats (Table 2).

Ozone depuration with 0.4 mg/L concentration did not produce significant changes

(P˃0.05) in lysine (LYS) levels between control fresh oysters before (113.38 mg/g) and

after 6 h of depuration (112.48 mg/g); however LYS levels increased significatively

(P˂0.05) from 113.38 to 133.42 mg/g after 6 h of depuration, being ozonated samples

significatively (P˂0.05) higher than control samples (133.42 and 112.48 mg/g,

respectively), indicating an increase of Free Amino Acid (FAA) as ozone reacted with

proteins causing proteolysis. In the case of 0.6 mg/L depuration, LYS levels between

control oyster samples before and after 6 h depuration (119.10 and 117.86 mg/g) were not

statistically different (P˃0.05); nevertheless, LYS levels of ozonated oyster samples (96.95

mg/g) were significatively higher than fresh control oysters (119.10 mg/g) and control

oysters (117.87 mg/g) after 6 h of depuration. It can therefore be surmised that the protein

quality of oysters was affected by the ozone-depuration processes.

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The Warner-Bratzler shear force values for oyster muscle and mantle of fresh oyster

samples (0.430 kgf) were significantly higher (P˂0.05) than shear values for ozonated

samples (0.402 kgf) after 6 h of depuration with 0.4 mg/L. Similar trend was observed

when oysters were depurated with 0.6 mgO3/L, being the shear force values for fresh oyster

meat (0.460 kgf) significantly higher (P˂0.05) than ozonated oyster shear values (0.422

kgf), and both ozonated oyster meats with 0.4 and 0.6 mgO3/L were significantly lower than

control oyster meats after 6 h of depuration. Depuration with 0.4 mg/L produced a 6.5%

decrease in the shear force values and 0.6 mg/L treatment produced a decrease of 8.3%.

According to Curran et al. (1984), at low levels of ozone, collagen and elastin can be

modified, making it susceptible to proteolytic degradation, generating peptides and amino

acids. The intermolecular cross-links in collagen, the main constituent of fish and shellfish

connective tissue, are thought to be responsible for the stability, physical strength and

mechanical properties of the connective tissue and other components of the extracellular

matrix (Hultmann and Rustad, 2004). Thus, the breakdown of the connective tissue by

ozone may lead to undesirable textural changes in the oyster and the decreased shear

strength may have been due to the denaturation of the protein fraction as FAA content

increased, as proteins are broken down to peptides and FAA by ozone. Moreover, seafoods

have less extensive cross-link formation in collagenous tissue in comparison with their

mammalian counterparts. Additionally, the nutritional quality of the protein may be

changed as FAA content increased and the quantity of the essential amino acids in food is

connected to the nutritional quality of protein.

When oysters were depurated with 0.4 and 0.6 mgO3/L of ozone, the sensory scores

for texture of control and ozonated samples with 0.4 mg/L were significantly different

(P˂0.05) after 6 h of depuration. Texture scores of ozonated oysters were significantly

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higher than fresh and depurated-control oysters, indicating that panelists detected a softer or

slightly firm consistency. When oysters were depurated with with 0.6 mg/L, a significantly

higher (P˂0.05) increase in texture scores was observed. The panelists detected a softer

texture (3.45 less firm and elastic) in ozonated oysters with 0.6 mg/L than fresh, control,

and ozonated oyster samples with 0.4 mg/L (2.60), although the loosing pattern of firmness

was observed in both ozonated oyster treatments. This texture degradation phenomenon

may be due to protein denaturation as results of ozone treatment and noted by the panelists.

Although the Eastern oyster or Crassostrea virginica is one of the most

economically and ecologically important shellfish species along the eastern seaboard, very

few scientific studies have been conducted to evaluate its sensory characteristics. Besides

aroma, texture is considered to be one of the important quality attributes of seafoods which

determines consumer acceptance and, hence, the marketability of these products. Collagen,

because of its occurrence and characteristics, plays a factor in the tenderness of the oyster

meat. The content of collagen is important to consumers because when oyster has more

collagen it will have a crunchy, fleshy, chewier texture, wonderfully plump and meaty as

showed in Figure 1.

Correlations were obtained to examine possible relationships between the texture

attribute, proteolysis, and the instrumental texture measurements. As it can be observed in

Table 3, texture attribute for oyster meat (mantle and adductor muscle) shows a pattern of

positive correlation (r = 0.550) with proteolysis, indicating that panelist detected a diminish

in the chewiness of the meat oyster due to the protein denaturation as a result of ozone

treatment. Negative correlations were obtained between texture and shear force (r = -

0.724), indicating that instrumental values of shear force decreased as the panelist detected

a softer meat ever since samples were scored by panelists using a 10-cm line scale starting

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from 0 (firm) to 10 (friable) to evaluate the changes in texture. Meanwhile, WB shear force

was negatively correlated (r = -0.469) to proteolysis as FAA content increased after

ozonation treatment and WB shear force diminished.

According to these findings, the Warner-Bratzler shear force measurement may be

useful for quality control purposes for determining firmness in oyster, and FAA content

could be considered as indicator of freshness and quality of bivalve mollusks, as objective

quality indicators are useful when they can foretell the shelf life before a fresh shellfish

becomes spoiled.

Analytical Techniques

A variety of biochemical, physical, and microbiological methods have been used to assess

freshness, although the sensory evaluation is still the most satisfactory method to achieve

such a goal.

The requirement that purified shellfish must meet (MPN E. coli <230 /100g of

shellfish) and the presence of V. cholera, V. parahaemolyticus, and V. vulnificus detection

are generally performed according U.S. Food and Drug Administration methodology and

expressed as percent of isolation (FDA, 2004). Presumptive V. cholerae isolates could be

confirmed with polyvalent antisera Serobac (Difco Laboratories Ltd., Franklin Lakes, NJ,

USA) to identify the O1 serogroup, and the serotype is identified using Inaba and Ogawa

specific antisera. If there is no agglutination with the polyvalent antisera, the presence of V.

cholerae non O1 is reported.

Unlike other muscle foods, seafoods have less connective tissue content and less

extensive cross-link formation in collagenous tissue in comparison with their mammalian

counterparts. These intermolecular cross-links in collagen, the main constituent of shellfish

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connective tissue, are thought to be responsible for the stability, physical strength and

mechanical properties of the connective tissue and other components of the extracellular

matrix (Hultmann and Rustad, 2004). The effect of ozone on proteins could be carried out

by the proteolysis of oyster meats measured by using TNBS method based on the reaction

of primary amino groups with trinitro-benzene-sulfonic acid (TNBS) reagent (Adler-Nissen

1979). The absorbance is read against water at 340 nm and results are reported as mg

Lysine/g dry weight. Another physical characteristic is texture, measured by cutting

strength of the ventral part of the shucked oyster meat with a texture analyzer (TA-XT2i,

Stable Micro Systems, Goldaming, UK) equipped with a Warner Bratzler blade operating at

a speed of 4.00 mm/s, a cutting distance of 35 mm, and a force penetration of 5.0 g. Cutting

strength is taken as the mean of three measurements on each oyster per treatment and per

trial and expressed as the force (kgf) required for cutting 1 mm into oyster tissue. The effect

of ozone on texture could be determined by descriptive sensory analysis as well. Candidates

for this type of panel are selected on the basis of both availability and liking of seafood. To

assess abilities of candidates, candidates are subjected to sequential analysis (p0 = 0.30, p1 =

0.60, and < 0.05). Selected panelists are trained on randomly selected days in 30 min

training sessions. They are oriented with the fresh, ozonated, and rotten samples. During 1-

h training sessions, panelists are asked to generate a list of texture descriptors. To evaluate

the changes in texture, samples of treatments and control are compared in terms of degree

of deviation of intensity from the fresh sample attributes using a 10-cm line scale with

three-anchor points: 0 for firm (elastic and chewy), 5 for soft (mild, slightly firm), and 10

for friable (mushy, lax). The analyses are performed once a week in individual booths for

sensory panel and samples are scored under red light. The samples must be shucked in

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aseptic conditions and kept over crushed ice until served to the panelists in individually

plastic capped cups, coded with a 3-digit numbers, and presented to panelists in one row in

random order. Each judge receive three dishes in each session trial: the raw fresh sample

(reference), the depurated raw control samples, and ozonated oyster samples in order to

evaluate the degree of firmness. Distilled water and soda crackers are used as palate

cleansers and the panelists are instructed to discard the samples after every evaluation

(Pardío et al., 2011).

Summary points

- Ozonization represents an alternative technique for enhancing the value of raw

oysters. From public health perspective, oysters depurated with 0.4 and 0.6 mg O3/L

during 6h may be safe for raw oyster consumers. These findings are of importance

as V. cholerae non O1 populations were decreased to non-culturable levels.

- At 0.4 and 0.6 mg O3/L, ozone may influence the functional properties of protein as

a result of structural changes by protein hydrolysis, which depended on the amount

of ozone. This denaturation was detected by panelists as a decrease in firmness of

the meat oyster.

- Correlation results suggest that denaturation of muscle proteins during ozone-

depuration have effect on FAA, shear force and texture. Thus, ozone should be

carefully dosed to avoid undesired oxidation of proteins

- According to these findings, FAA content could be considered as indicator of

freshness and quality of bivalve mollusks.

- Further investigations are needed in order to determine the changes in structural and

functional properties of oyster proteins.

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Titles for all Figures and Tables

Table 1. Effect of ozone depuration on American oyster naturally contaminated with

Escherichia coli and Vibrio cholera.

Table 2. Effect of ozone depuration on proteolysis, WB shear force (kgf), and texture of

American oyster samples.

Table 3. Correlation matrix of the proteolysis, shear force, and texture for oyster meat

depurated with 0.4 and 0.6 mg/L of ozone.

Fig. 1 The meat of an oyster sample fresh and depurated 6 h with 0.4 and 0.6 mg/L of

ozone.

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Table 1.

E. coli

(MPN/100 g)

V. cholerae non O1/non O139

(% isolation) Ozone concentration (mg O3/L) Ozone concentration (mg O3/L)

Time

(hour) Control

0.2

0.4

0.6

Control

0.2

0.4

0.6

Fresh 600a 340

b 85c 135

c 100 100 100 100

0 600a

340b

40c

110c

100 100 100 100

2 200a

60b

20c

40c

100 100 100 100

4 200a

40b

20c

70b

100 100 0 0

6 650a

40b

20c

20c

100 0 0 0

Means with different letter are significantly different (P ˂ 0.05) among columns.

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Table 2.

Proteolysis (mg Lysine/g d.w.)

Time

(hour) Control

Ozonated

0.4 mg/L

Control

Ozonated

0.6 mg/L

fresh 113.38 ± 0.63a,y

113.38 ± 0.63a,y

119.10 ± 2.45a,y

119.10 ± 2.45a,y

6

112.48 ± 0.63a,y

133.42 ± 1.84b,z

117.86 ± 2.59a,y

127.00 ± 2.00c,z

Warner-Bratzler Shear Force (kgf)

Time

(hour) Control

Ozonated

0.4 mg/L

Control

Ozonated

0.6 mg/L

fresh 0.430 ± 0.96a,y

0.430 ± 0.96a,y

0.460 ± 0.51b,y

0.460 ± 0.51b,y

6

0.428 ± 0.11a,y

0.402 ± 0.22b,z

0.459 ± 0.35a,y

0.422 ± 0.36c,z

Texture scores

Time

(hour) Control

Ozonated

0.4 mg/L

Control

Ozonated

0.6 mg/L

fresh 1.75 ± 0.21a,y

1.75 ± 0.21a,y

1.60 ± 0.11b,y

1.60 ± 0.11b,y

6

1.60 ± 0.14a,y

2.60 ± 0.28b,z

1.59 ± 0.15a,y

3.45 ± 0.21c,z

a, b

Means with different letter are significantly different (P ˂ 0.05) among columns

x,y

Means with different letter are significantly different (P ˂ 0.05) among rows

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Table 3.

Variables Proteolysis

(mg Lysine/g d.w.)

WB Shear force

(kgf) Texture attribute

Proteolysis

(mg Lysine/g d.w.) 1.00 -0.469 0.550

WB Shear force

(kgf) -0.469 1.00 -0.724

Texture attribute 0.550 -0.724 1.00

Values in bold are different from 0 with a significance level = 0.05

Fig. 1

Fresh 0.4 mg/L 0.6 mg/L

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======================

Professor Victor R. Preedy

Dept Nutrition and Dietetics

School of Medicine

Kings College London

Email: <[email protected]>

We wish to invite you to contribute a chapter to a new book which documents how biologically

active components in foods alter at various stages of processing, production or other steps such

pre- and post harvesting, up to the point of consumption. The book Food Processing and Impact

on Active Components: A Modern Approach will be published by Academic Press, an imprint of

Elsevier, the world's leading publisher of academic books (see www.academicpress. com) and will

be edited by myself. Kings College London is one of the worlds leading universities and is

"University of The Year" (see www.kcl.ac.uk). We enclose a Preface and a List of Chapters.

It has been suggested that you contribute an article which is entitled above.

Of course, we are happy for you to suggest alternative titles or other areas associated with how

biologically active components in foods alter. Should you kindly accept our invitation to

participate, the deadline for submission is 1st December 2012. We can extend the delivery date if

this is not suitable, please let us know. The contributions are a minimum of 3500 words and a

maximum of 5200 words plus illustrations.

If you agree then you will be contributing to what is arguably the most significant academic book

on anti-oxidants in foods-in this instance beverages. The Publisher will give each senior

Contributor one (1) print copy, and each non-senior Contributor one (1) electronic copy, of the

volume of the Work in which his or her contribution appears, free of charge. In addition, all

Contributors to the Work shall receive one (1) electronic offprint of his or her contribution, free of

charge.

Can you please reply, by email only, to < [email protected]> ideally within seven

days of this invitation. Please include your postal address, fax and telephone numbers. Please

correct any inaccuracies in the address and email we have for you above.

In your email reply please feel free to suggest alternative and additional titles. Co-authors are

welcome: there is no need to ask. Upon acceptance of this invitation, we will send you a copy of

the Author Guidelines and other instructions by email so you can begin your writing.

We will send this invite with and without attachments to avoid any spam filters. Thank you very

much for your time and consideration.

Page 26: Influence of ozone depuration on the physical properties … of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas*

With best wishes and regards,

Professor Victor R. Preedy

The Editorial Office

Food Processing and Impact on Active Components: A Modern Approach

Kings College London,

< [email protected]>

======================

Professor Victor R. Preedy

Dept Nutrition and Dietetics

School of Medicine

Kings College London

Email: <[email protected]>

Dra. Violeta T. Pardío Sedas, Investigador T.C., Responsable del Laboratorio de Toxicología,

Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, Tel/Fax: 52-229-9342075

y 934-4053 ext. 24125, E-mail:[email protected],

Dear Prof Pardío

Thank you for your email and I am delighted you have decided to contribute to this potentially

prestigious publication. We will arrange to send you instructions soon by email.

Please remember that this is a book on processing rather than a book on health effects or

descriptive contents. Thus you need to describe how components alter in response to processing:

such as cultivars, heat and non-heat treatments, storage, extraction, purification, filtration,

packaging, and so on. Perhaps sensory aspects should not be in the title.

Co-authors are welcome: there is no need to ask.

If you know of any colleagues who may want to contribute a chapter to Processing and Impact on

Active Components in Food please let me know soon

With best wishes and kind regards

Victor Preedy

Processing and Impact on Active Components in Food

Dept Nutrition and Dietetics

School of Medicine

Kings College London

Email: <[email protected]>

Page 27: Influence of ozone depuration on the physical properties … of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas*

16-Nov-2012

Dr. Violeta T. Pardío Sedas Facultad de Medicina Veterinaria y Zootecnia Universidad Veracruzana Mexico Dear Dr Sedas : Thank you for agreeing to contribute to PROCESSING AND IMPACT ACTIVE COMPONENTS IN FOOD (the “Work”) edited by Victor R. Preedy (the “Editor”). This Agreement between you and Elsevier Inc. regarding your contribution titled “Physical and sensorial assessment of fresh american oysters (Crassostrea virginica) depurated with ozone” outlines our respective obligations and rights. A. You agree that your contribution will be the agreed upon length, containing the content and substance (including any illustrative materials), as instructed by the Editor; your contribution will be submitted in the form and manner and by the date agreed upon, and you will keep one complete copy of the contribution to ward against loss. If requested, you also agree to review proofs of the contribution in the designated time period, making any necessary revisions and answering queries, to help ensure the accurate publication of your contribution. You shall also deliver with the manuscript the relevant illustrations (meaning photographs, drawings, sketches, diagrams, charts, maps, tabular matter and any other accompanying material), along with captions for all such illustrations and shall obtain the rights to use such Illustrations in the contribution. B. To facilitate publication of your contribution, you assign to us the copyright and all other rights in and to the contribution. This assignment of rights means that you have given us the exclusive right to publish and reproduce the contribution, or any part of the contribution, in print and all other forms of media, in any edition, revision, or other form, in all languages, throughout the world, and the right to license others to do the same. We may list your name and affiliation for credit, promotional and advertising purposes associated with your contribution in such manner which, in our judgment, fairly reflects your contribution. We may, but we are under no obligation to request your participation in any subsequent editions of the Work, and we may use all or any or your contribution in any such revisions and/or editions or in any other manner or form without additional compensation other than expressly provided in this Agreement. C. As the author of your contribution you may publish a summary of the contribution on your personal or your institution’s website and may make copies of up to 10% of the contribution for your classroom use. You also retain the right to draw upon the information and materials contained in the contribution for your continued professional and educational use, such as for classroom and meeting lectures and in the preparation of journal articles or book chapters. We do, however, require that in all cases you appropriately cite the Work and the Publisher as the copyright holder of the contribution. D. We will provide the senior author of the contribution with one (1) free print copy of the Work, and each contributor with one (1) free electronic copy of the Work. Each contributor may purchase additional copies of the Work, and other books published by the Publisher in print form (excluding the Publisher’s Major Reference Works program) directly from us at a discount of 25%, all for personal use and not for resale.

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Page 29: Influence of ozone depuration on the physical properties … of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas*

Dear Contributor:

I am delighted to learn from Dr. Preedy that you have agreed to contribute to Processing and Impact

on Active Components in Food As the developmental editor working on this outstanding text, I will

do everything I can to make the process of submitting a new chapter as easy as possible. Please contact

me with questions or problems any time:

Carrie Bolger at [email protected]

For this edition, we will be using a website, Electronic Manuscript Submission System (EMSS), to help

you manage your chapter. If you have never logged in to the website before for another project:

The website address is: http://editorial.elsevier.com

Enter your current email address.

New Users: Your password is your last name in all lowercase letters.

(Note, if you are a MAC user, we recommend using Firefox as your web browser)

If you have been on the site before, you have already changed your password. If you cannot

remember your password and need a new one, you can request one from the login page, or contact me.

Upon receipt of this email, please log on to the site. Once you log on, you will be asked to change your

password and to enter your mailing address and affiliations before you can do anything else on the site.

Next, click on the drop down menu and select Book Title. You can access your chapter page by clicking

on your chapter title. You can access the author instructions by clicking on "author instructions" in the

grey bar across the top of the page.

Your final chapter is due within 4 weeks of receiving this letter. If you have any questions relating to

the content of your chapter, please contact the Editorial Office at

<[email protected]>. If you have any non-content related questions, or problems

with the site, please contact me. Thanks and I look forward to working with you.

I am looking forward to working with you on this edition and if at any time, you have questions or

problems with anything at all, please contact me - I am here to help!

Sincerely,

Carrie BolgerCarrie BolgerCarrie BolgerCarrie Bolger Editorial Project Manager

Life Sciences

Elsevier

525 B Street, Suite 1800

San Diego, CA 92101

(p) 619-699-6747, (f) 619-699-6715

[email protected]

Page 30: Influence of ozone depuration on the physical properties … of ozone depuration on the physical properties of fresh American oysters (Crassostrea virginica) Violeta Pardío-Sedas*

De: "Bolger, Carrie (ELS-SDG)" <[email protected]>

Para: Violeta Pardio <[email protected]>

Enviado: Miércoles, 13 de febrero, 2013 13:25:24

Asunto: RE: ISBN

Hi Violeta,

The ISBN is 978-0-12-404699-3. The book is not in print. We are still waiting for the

remaining chapters to be submitted.

Please let me know if you have any questions.

Kind regards,

Carrie

From: Violeta Pardio [mailto:[email protected]]

Sent: Wednesday, February 13, 2013 4:56 PM

To: Bolger, Carrie (ELS-SDG)

Subject: Re: ISBN

Hi Carrie,

Do you know by chance the ISBN of the book Processing ad Impact on Active Components

in Food, Preddy V. (ed.).?

Kind regards,

Violeta

Dra. Violeta T. Pardío Sedas

Investigador T.C.

Responsable del Laboratorio de Toxicología

Facultad de Medicina Veterinaria y Zootecnia

Universidad Veracruzana

Tel/Fax: 52-229-9342075 y 934-4053 ext. 24125