bacteriocin of lab importance for dairy industry
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Eng. Life Sci. 2012, 12, No. 4, 419432 419
Dora Beshkova
Ginka Frengova
Laboratory of Applied
Biotechnologies, Department of
Applied Microbiology, Bulgarian
Academy of Sciences, TheStephan Angeloff Institute of
Microbiology, Plovdiv, Bulgaria
Review
Bacteriocins from lactic acid bacteria:Microorganisms of potential biotechnological
importance for the dairy industryBacteriocins are a heterogeneous group of ribosomally synthesized, extracellularlyreleased,bioactive peptides or proteinsdisplayingantimicrobialactivity against otherbacteria. Over the last two decades, there has been an explosion of basic and appliedresearch on lactic acid bacteria (LAB) bacteriocins, primarily due to their potentialapplication as biopreservatives in food and food products to inhibit the growth offood-borne bacterial pathogens. Although bacteriocins can be produced in the foodmatrix during food fermentation (in situ), bacteriocins by LAB can be producedin much higher amounts during in vitro fermentations under optimal physical andchemical conditions. Because of the complexity of the food matrix and the difficultyof quantifying bacteriocin activities in foods, in vitro studies can be performed tosimulate and study the in situ functionality of bacteriocinogenic starters. In situbacteriocin production is most promising for a fast, widespread, and legal use ofbacteriocins to achieve the desirable fermentation and a safe final product. Thebacteriocin production may be of utmost importance when bacteriocin-producingLAB are added to foods as starters or protective cultures (adjunct culture). In thecurrent review, our interest is mainly focused on the research of in situ bacteriocinproduction through finding the potential of the bacteriocinogenic cultures, whichhave biotechnological importance for the dairy industry.
Keywords: Bacteriocins / Dairy industry / Fermented food / In situ production / Lactic acidbacteria
Received: December 12, 2011; revised: February 16, 2012; accepted: April 5, 2012DOI: 10.1002/elsc.201100127
1 Introduction
At present, scientificliterature and the researchcommunity have
generally adopted Klaenhammers [1] definition of bacteriocins,
i.e. bacteriocins are a heterogeneous group of ribosomally syn-
thesized, extracellularly released bioactive peptides or proteins
displaying antimicrobial activity against other bacteria.Histori-
cally, in 1976, Tagg et al. [2]defined bacteriocinsas proteinaceous
compounds, synthesized by both Gram (+) and Gram () bac-
teria, and exhibiting inhibitory activity against species closely
related to the bacteriocin producer. Information on bacteriocins
was first published in 1925, when researchers found out that a
biologically active substance produced by strain Escherichia coli
V appeared to have antagonisticactivity against another strain of
the same species (E. coliF) [3]. Later, similar antimicrobial sub-
stances produced byE. coliwere found and called colicins [4].
Correspondence: Dr. Dora Beshkova ([email protected]), In-
stitute of Microbiology, Laboratory of Applied Biotechnologies, 139
Ruski Blvd, 4000 Plovdiv, Bulgaria
Abbreviations: CFU, colony forming units; LAB, lactic acid bacteria
Bacteriocins can be produced by different species of Gram (+)
or Gram () bacteria: bacteriocins, bacillocin Bb, and pyocin Pa
produced by the soil-associated bacteria Bacillus brevis Bb and
Pseudomonas aeruginosaPa, respectively [5]; bacteriocins, aure-
ocin A 53 and aureocin A 70bystrains ofStaphylococcus aureus
[6, 7]; ruminal bacteriocinsby ruminal bacterium Streptococ-
cus bovis [8]; bacteriocinby purple nonsulfur phototrophic
bacteria, Rhodobacter capsulatus ATCC 17016 [9]; differently
called bacteriocinsby representatives of the lactic acid bacteria
(LAB) [1017]. Bacteriocin synthesis by LAB was first reportedin 1928 [18]. This biologically active substance was later chem-
ically defined as a polypeptide [19] and given the name nisin
[20, 21]. There is a growing interest in bacteriocins produced by
representatives of different LAB genera and new data have been
continuously reported.[1017] LABare widelyused in theman-
ufacturing of fermentedfoodand areamong thebest studied mi-
croorganisms. Detailed knowledge of a number of physiological
traits has opened new potential applications for these organ-
isms in the food industry, while other traits might be beneficial
for human health [22]. Owing to their typical association with
food fermentation and also their long tradition as food-grade
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420 D. Beshkova and G. Frengova Eng. Life Sci. 2012, 12, No. 4, 419432
bacteria, LAB are generally recognized as safe (GRAS) [23].
LAB can exert a biopreservative or inhibitory effect against
other microorganisms as a result of competition for nutrients
and/or of the production of antagonistic compounds such as
organic acids (mainly lactic acid), ethanol, aroma compounds,
fatty acids, hydrogen peroxide, bacteriocins, and nonbacteri-
ocin antibacterial compounds [24]. The persistent interest of
researchers in LAB bacteriocins is prompted by their potentialapplication as food biopreservatives, i.e. they offer a successful
prospective alternative strategy for inhibitingthe growth of food-
borne bacterial pathogens. This opens perspectives for the use
of bacteriocin-producing LAB (starters or protective cultures) in
fermented foods [2547], or of (purified) bacteriocin prepara-
tions in both fermented and nonfermented foods [27,4858] to
improve food quality, naturalness, and safety.
The fact that bacteriocins are active against numerous
foodborne and human pathogens, are produced by GRAS
microorganismsLAB, and are readily degraded by proteolytic
host systems makes them attractive candidates for biotechno-
logical applications. This review focuses on the researchs of the
production and application potential of LAB bacteriocins for thedevelopment in the biotechnology of LAB.
2 Classification and properties of LABbacteriocins
The bacteriocin family includes a wide variety of peptides and
proteins in terms of their size, microbial targets, and mecha-
nisms of action and immunity. Several attempts have been made
to classify LAB bacteriocins. Based on structural, physicochem-
ical, and molecular properties, bacteriocins from LAB can be
subdivided into three major classes [1, 59]. Nonetheless, this
classification is continuously being reviewed and it is evolvingwith the accumulation of knowledge and the appearance of new
bacteriocins [10,13,6068].The currentlyaccepted classification
by the research community is as follows:
Class I: Lantibiotics ([small], cationic, hydrophobic peptides
[
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Eng. Life Sci. 2012, 12, No. 4, 419432 Bacteriocins from lactic acid bacteria 423
ing cheese manufacture and ripening. The organisms in mixed-
strain starters used in the manufacture of the cheeses belong
mainly to the genera Lactococcus(Lc. lactissubsp. lactis[Lc. lac-
tis], Lc. lactis subsp. cremoris [Lc. cremoris])mesophilic, the
best known or Lactobacillus(Lb. helveticus, Lb. delbrueckiisubsp.
bulgaricus), and Streptococcus thermophilusthermophilic, the
best known, depending on the specific application [93]. The
lactic fermentation of milk is required for cheese production.While some cheeses arestill made from nonpasteurised milk and
may even depend on the adventitious natural lactic flora (non-
starter LABNSLAB) for the fermentation, most are produced
on a commercial scale using the appropriate starter culture. The
contribution of NSLAB to the formation of the organoleptic
characteristics defining the quality of cheese is unclear. Their
presence can have both a positive and, more frequently, negative
effect, manifested in concomitant defectscalcium lactate crys-
tal formation, off-flavor development, and slit formation. One
of the most promising strategies to control NSLAB populations
is to employ well-characterized LAB, as adjunct cultures that
suppress the emergence of wild nonstarter cultures [94]. With
regards to this, the live bacteriocin-producing cultures can be asignificant alternative for improving the food safety and quality
of dairy products. The effect of in situ bacteriocin production
achieved using bacteriocinogenic dairy cultures as starter or ad-
junct cultures to obtain various types of cheese, can be expressed
by defining the role of these cultures in the following two ar-
eas: bacteriocin-producing LAB to control adventitious micro-
bial populations and as protective cultures to inhibit the growth
of pathogenic microorganisms in cheese; bacteriocin-producing
LAB as cell lysis-inducing agents to improve cheese quality and
flavordiscussed in separate sections of this article.
4 Bacteriocin-producing LAB to controladventitious microbial populations and asprotective cultures to inhibit the growthof pathogenic microorganisms in cheese
Thelacticin481-producing andlacticin3147-producing cultures
have been used successfully to improve the quality of Cheddar
cheese through the inhibition of NSLAB [28, 43]. O `Sullivan
et al. [43] observed a reduction of 4 log units in the number
of NSLAB after 4 months of ripening in experimental Cheddar,
with lacticin 481-producing strain Lc. lactisCNRZ 481 used as
an adjunct to the lactococcal starter culture Lc. lactisHP, com-
pared with the same number of bacteria in the control cheese
(obtained with the standard starter culture only). At the endof the ripening period (after 6 months), the recorded decrease
in the number of NSLAB was 2 log units. Other authors [28]
did not find NSLAB throughout the ripening period (6 months)
in experimental Cheddar cheese prepared with a mixed starter
culture comprising three strains (Lc. lactis DPC 3147, Lc. lac-
tisDPC 3204, Lc. lactisDPC 3256), isolated from natural yogurt
and producing bacteriocin, lacticin 3147. In comparison, in con-
trol, cheese produced with the commercial cheesemaking strain
Lc. cremoris DPC 4268, the number of living NSLAB cells was
107.5 CFU g1 after 4 months. The amount of bacteriocin de-
tected in cheese made with bacteriocin-producing starter was
approximately 1280 AU mL1, which remained stable over the
entire ripening period.
Insitu production of nisinZ byLc. diacetylactisUL 719,cocul-
tivated with Lc. cremorisKB and Lc. lactis(at a ratio of 1.0:1.5:1.5
vol.) in Cheddar cheese, was determined throughout ripening
(6 months) [39]. Nisin concentration of 306 IU mL1 was ob-
tained. In addition to the nisin-producing activity of the mixed
starter, researchers evaluated the antagonistic effect of bacteri-ocin against the growth of Listeria innocua. A reduction of 3.0
log units in the level of living cells of the pathogen was found
in experimental cheese produced with encapsulated nisin, and a
reductionof 1.5log units in experimental cheese obtained with a
nisinogenic starter. At the end of ripening process in cheese pro-
duced with a nisin preparation, about 10 CFU g1 ofL. innocua
and90% of theinitial activity of nisin wasestablished, compared
to 104 CFU g1 and 12% for cheese produced with a nisinogenic
starter. The use ofLc. diacetylactisUL 719 in a mixed starter cul-
ture did not appear to be the best strategy to inhibit L. innocua
in Cheddar cheese. However, a mixed culture containing a nisin
Z-producing strain might be used for controlling postcontami-
nant organisms, as they are usually present in low numbers. Thedata obtained by the authors on nisin activity in experimental
Cheddar cheese produced witha nisinogenic starter (Lc. diacety-
lactisUL 719) aresimilar to those reported previously forGouda
cheese [35]. Bauksaim et al. [35] reported that Gouda cheese of
good qualityandsafe toeat canalsobe obtained bythe aforemen-
tioned nisin-producing culture Lc. diacetylactisUL 719, when it
is addedto commercialFloraDanicastarterin a ratioof 0.6:1.4%.
In the experimental cheese, a maximum nisin concentration of
512 IU g1 was recorded (after 6 weeks), followed by a decrease
in the activity to 128 IUg1 after 27 weeks, and up to 32 IU g1
after 45 weeks of ripening. Maisnier-Patin et al. [25] evaluated
the potential of another nizin-producing starter Lc. lactisCNRZ
150 to inhibit Listeria monocytogenesduring Camembert cheese
manufacture and ripening. Maximum nisin concentration of ca700IUg1 was obtained in curd at 9.0h (during the exponential
phase of growth of bacteriocin-producing culture), then nisin
concentrations decreased slowly during 924 h, and dramati-
cally to ca 200 IU g1 during ripening. In the presence of nisin,
the numbersofL. monocytogenesdecreased rapidlyfrom6 to24 h
and a difference of 2.4 log CFU g1 between numbers of Liste-
ria in cheeses made with Nis+ (Lc. lactisCNRZ 150) and Nis
(Lc. lactisCNRZ 1076) starter cultures was maintained through-
out ripening (6 weeks). Another nisin Z-producing strain Lc.
lactisIPLA 729 has been successfully applied on the inhibition
of the spoilage strain ofClostridium tyrobutiricum CECT 4011, a
lateblowingagent,in semi-hard Vidiagocheesemaking[44]. The
authors have obtained and studied three cheeses: experimental
cheesenisinogenic culture was co-cultivatedwith a mesophilic
starter IPLA-001 composed of Lc. diacetylactisIPLA 8381 and
Leuconostoc citreum IPLA 616; control cheeseinstead of the
nisin Z-producing culture, an acidifying nonbacteriocinogenic
strain Lc. lactis IPLA 947 was used in the mixed starter; com-
mercial cheesethe nisin producer was replaced by KNO 3 as
a gas-blowing preventing agent. A maximum concentration of
1600 IU nisin mL1 was measured in the experimental cheese
betweenday 1 andday15 of theripeningprocess, whileat theend
of the process (30 days), the activity decreased. During ripeningof thethreetypes of cheese, thefollowing trendsin thegrowth of
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424 D. Beshkova and G. Frengova Eng. Life Sci. 2012, 12, No. 4, 419432
clostridiawere observed, and the following values for the number
of live cells were determined: the number decreased from 1.2
106 to 1.3 103; the number increased to 3.5 107; the num-
ber increased to 1.99 109 for the experimental, control, and
commercial cheese, respectively. The authors concluded that the
inclusion of the nisin-producing strain IPLA 729 in the mixed
starter IPLA-001 made it possible to prepare a balanced cul-
ture that will be used in the making of Vidiago cheese, therebyimproving the ability to suppress spoilage microorganisms and
contributing to better organoleptic properties in comparison
with commercial starters.
Onepossibilityfor the improvementof themetabolic produc-
tivityof an microorganisms is genetic modification.This strategy
wassuccessfully used by Ryan et al.[28] as follows: a bacteriocin-
producing transconjugant Lc. lactisDPC 4275 was obtained by
transferring a 63-kb plasmid, pMRC01 (encodes bacteriocin,
lacticin 3147 production) from strain Lc. lactis DPC 3147 to
the commercial Cheddar cheesemaking starter Lc. cremorisDPC
4268; the obtained transconjugant was defined as Bac+ and
Imm+, i.e. possessing properties of lacticin 3147 production and
immunity to lacticin 3147. Furthermore, on the one hand, thetransconjugant had retained the lacticin 3147-producing activ-
ity of the parent strain Lc. lactisDPC 3147, while, on the other
hand, it maintained the original characteristics of the commer-
cial starter strain, i.e. theproduced lactic acid levels were similar.
Throughout ripening (6 months), the concentration of bacteri-
ocin, lacticin 3147, determined in Cheddar cheese (made with a
transconjugant strain as a single-strain starter) did not change
and this correlated with a significant decrease in the number
of NSLAB. Later, the lacticin 3147-producing transconjugant
Lc. lactisDPC 4275 was used by other authors as a component
of a starter culture not only for obtaining reduced fat cheddar
[34], but also for obtaining Cottage cheese [33]. At both ripen-
ing temperatures (7Cand12C), theNSLAB populations in the
corresponding cheeses made with the lacticin 3147-producingculture, Lc. lactisDPC 4275, increased much more slowlyduring
ripening andreachedmarkedly lower numbers (ca103 CFUg1)
at theend (240 days)of theripening[34]). Inagreement with the
observations of Folkertsma et al.[95] forfull fatCheddar cheese,
elevation of ripening temperature from 7C to 12C resulted in
a more rapid developmed of NSLAB (to ca 107 CFU g1) in
cheeses made with the nonbacteriocinogenic mixed starter ([Lc.
lactisDPC 4268+ Lc. cremorisDPC4269] or Lc. lactisDPC 4268
alone). The authors concluded that the use of the bacteriocino-
genic Lc. lactisDPC 4275 can reduce the risk of flavor defects in
reduced fat cheese, especially when ripening is at high tempera-
ture, and shouldenable theproductionof cheeses with morepre-
dictable and consistent flavor. The effectiveness of lacticin 3147
as a naturalpreservative wasdetermined in Cottage cheese that
was subsequently inoculated with approximately 104 L. mono-
cytogenesScott A g1 [33]. During the first week of storage (at
4C), in the experimental cheese (made with the bacteriocin-
producing transconjugant Lc. lactisDPC 4275 as a starter), the
concentration of lacticin 3147 was determined to be 2560 AU
mL1. Furthermore, a decrease of 99.9% was registered in the
number of L. monocytogenes in the same cheese within 5 days,
whereas in the control cheese (made with commercial starter Lc.
lactisDPC4268), theinitial numberof inoculated pathogencells
remained essentially unchanged. Another genetically modified
starter culture Lc. lactisMM 217, capable of producing pediocin
in situ, was used as a single starter culture to improve the mi-
crobiological safety of Cheddar cheese precontaminated with
L. monocytogenes[32]. The nonbacteriocinogenic starter culture
Lc. lactisMM 210was used as an recipient forplasmid (pMC117)
coding for pediocin PA-1 production. The electrotransformed
derivate of strain MM 210 containing pMC117, was named Lc.
lactis MM 217. This strain retained the growth characteristicsand acid-producing activity of the parent strain. At the end of
theripeningprocess of cheeses (6 months at 8C),the number of
live L. monocytogenescellsdecreasedfrom3.5logCFUg1 (initial
concentration) to about 1.0 log CFU g1 in experimental cheese
(made with a bacteriocin-producing starter culture), compared
to a recorded number of pathogen cells of 4.0 log CFU g1
in the control cheese (made with a nonbacteriocin-producing
starter culture). The level of pediocin activity decreased from
approximately 64,000 AU g1 after 1 day to 2000 AU g1 after 6
months. Rodriguez et al. 2005 [46] evaluated the antimicrobial
activity of bacteriocin-producing transformants (Lc. lactis CL
1 [Ped+]-pediocin-producing strain and Lc. lactis CL 2 [Nis+,
Ped
+
] nisin-and pediocin-producing strain) against L. monocy-togenes, S. aureus, and E. coliO157:H7 during cheese ripening.
The wild strains Lc. lactisESI 153 and Lc. lactisESI 515(Nis+)
were selected by their technological and/or antimicrobial prop-
erties, used as starter cultures in cheese manufacture and were
transformed to produce pediocin PA-1, as reported earlier [96].
Pediococcus acidilactici347 (Ped+), Lc. lactis ESI 153, Lc. lactis
ESI 515 (Nis+), and their respective pediocin-producing trans-
formants Lc. lactisCL 1 (Ped+) and Lc. lactisCL 2 (Nis+, Ped+)
were added individually as adjunct to the commercial starter
culture MA 016. In the experimental cheeses containing one of
the above bacteriocinogenic cultures, bacteriocin activity was
established during their ripening (30 days). After 30 days, in the
presence of the bacteriocinogenic cultures Lc. lactisCL 1 (Ped+)
or Lc. lactisCL 2 (Nis+, Ped+), there was a certain decrease inthe L. monocytogenescounts by 2.97 and 1.64 log units, S. aureus
by 0.98 and 0.40 log units, and E. coliO157:H7 by 0.84 and 1.69
log units when compared with control cheese (made without
adjunct culture). The combination of the bacteriocins nisin and
pediocin, synthesized by Lc. lactisCL 2 (Nis+, Ped+) in the re-
spective experimental cheeses, revealed a higher inhibitory effect
only against the pathogen E. coli. All cheeses investigated by the
authors were inoculated with each of the given pathogen in an
approximateconcentrationof 6 log CFU mL1,whichwashigher
than the possible contamination of natural milk. Similarly Zot-
tola et al.[27] suggestedthat theuse of bacteriocinogenic culture
as an ingredientin pasteurized process cheese or coldpack cheese
spreads couldbe an effectivemethod of controllingthe growthof
undesirable microorganisms in these processed foods. A nisin-
producing transconjugant Lc. cremoris JS 102 was added as an
adjunct to lactoccocal starter Lc. lactisNCDO 1404 in Cheddar
cheesemaking, which contained between 400 and 1200 IU of
nisin g1 cheese. The shelf life (at 22C) of the nisin-containing
cheese was significantly greater than that of the control cheese
(the cheeses were inoculated with 2000 spores of Cl. sporogenes
PA 3679 during manufacture). Significant reduction in numbers
of the nonspore-forming test microbes (L. monocytogenes V 7
and S. aureus 196 E) were observed in experimental cheeses at
both temperatures (22C and 37C).
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Eng. Life Sci. 2012, 12, No. 4, 419432 Bacteriocins from lactic acid bacteria 425
Figure 1. Overview of potential application ofLAB-producing bacteriocins.
Besides lactococci as adjunct cultures during in situ bacteri-
ocin production, the quality of the cheese may be improved by
also using lactobacilli [36,47] and enterococci [26,29,40]. Ryan
et al. [36] applied one strategy for manipulation of cheese flora
using combinations of lacticin 3147-producing and -resistant
cultures. A stable, more resistant Lb. paracaseiDPC 5337 (which
was 32 times less sensitive to bacteriocin, lacticin 3147 than
parental strain Lb. paracaseiDPC 5336) was used as adjunct cul-
tures in two separatetrialsusingeither Lc. lactisDPC3147(anat-
ural producer) or Lc. lactisDPC 4275 (a lacticin 3147-producing
transconjugant) as the starter. These lacticin 3147-producing
starters were previously shown to control adventitious NSLABin cheddar cheese [28].Lacticin-3147activity wasassayed during
manufacture and reached approximately 12802560 AU g1 of
cheese. This level of activity was maintained throughout ripen-
ing and correlated with a reduction in the growth rate of NSLAB
in the control cheeses. The authors deducted that the resistant
adjunct strain formed the dominant Lactobacillus population
(levels of 107 CFU g1, in contrast to the sensitive strains
levels 100- to 1000-fold lower) in the experimental cheeses that
were with improved quality and with the bitter flavor being al-
most undetectable. Bogovic Matijasic et al. [47] implemented
a model for inhibiting the growth of Cl. tyrobutyricum, with
the pathogen inoculated in advance (at a concentration of 2.5
103
spores mL1
) in semi-hard cheese, obtained by using abacteriocinogenic culture (Lb. gasseriK 7) as an adjunct to the
commercial starter S. thermophilusTH4DVS. In the experimen-
tal cheese, the bacteriocin-producing culture did not inhibit the
thermophilic starter, but reduced the number of the nonstarter
mesophilic lactobacilli to about 100CFU g1 throughout the en-
tire ripening (8 weeks). This inhibitory effect was observed de-
spite thefact that nobacteriocins were found in theexperimental
cheese. In addition, the pH values and concentrations of or-
ganic acids (factors contributing to the antimicrobial properties
of LAB) were similar in the cheeses produced in the presence
or absence of a bacteriocin-producing culture. The authors sug-
gest the following possible explanations of this phenomenon:
nonuniform bacteriocin distribution in cheese, adsorption to
the caseins in the curd, or bacteriocin degradation by intracel-
lular proteases, as reported earlier by other authors [97]. Villani
et al. [26] used Enterococcus faecalis226 NWC (culture produced
the bacteriocin, enterocin 226 NWC) as a starter in Mozzarella
cheesemaking from water buffalo milk. When L. monocytogenes
was cocultured with a bacteriocin-producing culture in recon-
stituted skim milk, the live cells of the pathogen decreased to a
level of 1.5 107 and were completely destroyed after 7 and 72 h
of incubation of the mixed culture, respectively. For compari-son, the number of live cells of the pathogen was 9 108 in
the development of L. monocytogenesas a monoculture. Later,
Nunez et al. [29] reported that another enterocin-producing
strain Ent. faecalis INIA 4 was successfully used for Manchego
cheesemaking from raw ewes milk. Listeria monocytogenesOhio
counts decreased by 3 log units after 8 h, and by 6 log units
after 7 days in cheese made with an enterocinogenic culture,
whereas no inhibition was recorded after 60 days in control
cheese made with commercial starter (Lc. cremoris+ Lc. lactis).
Anotherstrain, L. monocytogenesScottA, wasnotinhibited in the
presence of a bacteriocin-producing culture, used either alone
or as an adjunct to a commercial starter during cheese manu-
facture, but decreased by 1 log unit and 2 log units, respectively,after 7 and 60 days of ripening. The values of enterocin activi-
ties, determined 8 h after making theexperimental cheeses (with
a mixed starterbacteriocinogenic + commercial strains, and
with a single starterbacteriocinogenic culture), were 2000
3000 AU g1 and 40006000 AU g1, respectively. These re-
sults are in agreement with bacteriocin production by Ent. fae-
cium 7C5 under Taleggio cheesemaking conditions, which still
occurred at 25 h andpH 4.9[98]. Sarantinopouloset al.[40]have
investigated the possibility of the use of bacteriocinogenic strain
Ent. faecium FAIR-E 198 as a adjunct culture to the traditionally
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426 D. Beshkova and G. Frengova Eng. Life Sci. 2012, 12, No. 4, 419432
used mixed starter (S. thermophilusACA-DC 7, Lc. lactisACA-
DC 52, and Lb. bulgaricus ACA-DC 84) to manufacture Greek
Feta cheese. This strain was isolated from Greek Feta cheese and
demonstrated an antagonistic effect against Listeria.
5 Bacteriocin-producing LAB as celllysis-inducing agents to improve cheesequality and flavor
Another important aspect of using bacteriocinogenic cultures
is the possibility to induce controlled lysis of a LAB starter cul-
ture or NSLAB in their presence, and subsequent intracellular
release of proteinases and peptidases, resulting in rapid onset
of proteolysis, i.e. a new alternative is proposed for acceleration
of cheese ripening aimed at obtaining dairy products with im-
proved organoleptic characteristics [30,31,37,38,4143,45,99].
Morgan et al. [30] described a method for increasing the rate
of lysis of the commercial starter culture Lc. cremorisHP during
ripening of Cheddar cheese by adding the bacteriocinogenic cul-ture Lc. lactisDPC3286 (encodesthe synthesis of lactococcins A,
B, M)to thestandardlactococcal starter.In a laboratory-scale sys-
tem, it was found that the LHD (lactate dehydrogenase) activity,
determined onday180 (end ofthe ripeningprocess) inthe exper-
imental cheese (made with bacteriocinogenic adjunct culture),
was 66% higher than the activity in the control cheese (obtained
with the lactococcal starter). In addition, higher values were also
recorded for both enzymesglucose-6-phosphate dehydroge-
nase and postproline dipeptidyl aminopeptidase in the experi-
mental cheese, resulting in higher concentrations of free amino
acids by 47% than those in the control cheese. With the suc-
cessful application of the mixed culture (Lc. cremorisHP+ bac-
teriocinogenic adjunct strain Lc. lactisDPC 3286), the authors
obtained a dairy product of improved quality, reduced bitter-ness, and higher grading scores. The use of the two-component
culture on a pilot scale, however, created a problem by killing
the acid-producing strain included in the mixed starter in the
making of Cheddar cheese. Later, the same authors (41) resolved
this problem by applying a new three-component mixed culture
consisting of the following cultures: a lactococcin A, B, and M
strain producer (Lc. lactisDPC 3286), possessing the ability to
lyse thesecondculture(Lc.cremorisHPsensitiveto lactococcin
A, B, and M activity), and a third culture ( S. thermophilusDPC
1842acid producing and resistant to lactococcin A, B, and M
activity) for pilot-scale Cheddar cheesemaking. In the experi-
mental cheese (made with a bacteriocinogenic adjunct culture),
higher (approximately two times) concentrations of free aminoacids were recorded, higher release rates of intracellular LDH
(by 265%), and a decrease in bitterness compared to the con-
trol cheese (made with Lc. cremoris HP alone or with a mixed
starterLc. cremorisHP+ S. thermophilusDPC 1842). Another
bacteriocinogenic strain, Lc. lactis CNRZ 481, producing lac-
ticin 481, was also used as an adjunct to the commercial starterculture Lc. lactisHP for pilot-scale Cheddar cheesemaking, but
without impeding the production of the amount of acidrequired
for this type of cheese [43], in contrast to lactococcin A, B, and
M strain-producer Lc. lactis DPC 3286, which was reported to
have a negative effect in this context [41]. In the experimental
cheese (made with a mixed starterbacteriocinogenic culture
+ commercial starter culture), 45 times higher levels of LDH
were determined compared with those in the control cheese
(made with a commercial starter culture alone). Other authors
[45] also reported that addition of a bacteriocinogenic strain
Lc. lactisINIA 415 (strain containing the structural gene encod-
ing lacticin 481 and nisin Z production) as adjunct culture to
commercial S. thermophilus TA 052 and Lc. lactis INIA 415-2(a nonbacterioconogenic mutant) is a successful alternative for
the acceleration of proteolysis of Hispanico cheese. Streptococcus
thermophilusTA 052 counts were lower (about 1 log unit) in ex-
perimental cheese (made with bacteriocinogenic culture) on day
15 of ripening. From day 25 to day 75 (end of ripening), in the
presence of the bacteriocinogenic culture, i.e. in the experimen-
tal cheese, the total free amino acid concentrations were about
2.5 times higher than those in the control cheese (made without
a bacteriocinogenic adjunct culture). Later, Garde et al. [99] de-
scribed another alternative for Hispanico cheesemaking using a
lactococcal mixed starter (lacticin 481-producing Lc. lactisINIA
639 + lacticin 481-nonproducing Lc. lactisINIA 437) to which
a lactobacillus strain (Lb. helveticus LH 92) was added, whichis sensitive to lacticin 481 and possesses high aminopeptidase
activity. In the control cheese, about a twofold lower rate of pro-
teolysis was recorded, and about 1.8 times lower values for the
activity of cell-free aminopeptidase was determined compared
with the values obtained in the experimental cheese after 25
and 7 days of ripening, respectively. After 25 days, in the experi-
mental cheese, the concentrations of total free amino acids were
determined to be about 2.3 times higher than those in the con-
trol cheese. As a result of using the lacticin 481-producing cul-
ture, the cheese obtain by the authors had improved quality and
reduced bitterness. Martinez-Cuesta et al. [37] studied the po-
tential of lacticin 3147-producing transconjugant Lc. lactisIFPL
3593 (used as a starter) toinduce lysis of thetwo cultures (Lc. lac-
tisT1and Lb.caseiIFPL 731bothshowedhigh aminopeptidaseactivity)addedto thestarter, thusachieving accelerated processes
of proteolysis and ripening in semi-hard cheese, accompanied
by a parallel significant increase in its sensory characteristics
intensity of aroma and cheese taste. The bacteriocin-producing
transconjugant Lc. lactisIFPL 3593 was obtained by transferring
a 46-kb plasmid, pBAC 105 (encodes bacteriocin, lacticin 3147
production) from strain Lc. lactis IFPL 105 to the commercial
cheesemaking starter Lc. lactisIFPL 359. The transconjugant was
defined as Bac+ and Imm+, i.e. possessing properties of lacticin
3145 production and immunity to lacticin 3147. The values of
X-prolyl-dipeptidyl aminopeptidase activity were significantly
higher (about two times) in experimental cheese (made with the
bacteriocinogenic culture, as starter). In addition, detection of
intracellular activity and loss of cellular viability of starter ad-
juncts (Lc. lactisT1 and Lb. caseiIFPL 731) were simultaneous.
The concentration of amine nitrogen in experimental cheese on
day 45 (end of ripening) was 16% higher than in the control
cheese (made with nonbacteriocinogenic parental strain Lc. lac-
tisIFPL 359 [commercial starter] and adjuncts cultures [Lc. lactis
T 1 + Lb. caseiIFPL 731).
Enterococcus species also were added as a bacteriocin-
producing adjunct to commercial starter culture in cheese
making [31, 38]. A feasible and a cost-effective method for
increasing the rate of starter lysis during semi-hard Hispanico
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Table 2. Bacteriocin-producing LABdirection for their use in different cheesemaking.
Bacteriocin-producing culture Bacteriocin Directions for application Observed effects Reference
1 2 3 4 5
Lc. lactisDPC 3147 Lc. lactisDPC
3204 Lc. lactisDPC 3256
Lacticin 3147 As mixed starter culture in Cheddar
cheesemaking
No NSLAB detected in experimental
cheese at the end ripening 6
months
[28]
Transconjugant Lc. lactisDPC 4275 Lacticin 3147 As single-strain starter culture inCheddar cheesemaking
A significant reduction in the levelsof NSLAB
[28]
Transconjugant Lc. lactisDPC 4275 Lacticin 3147 As single-strain in reduce fat
Cheddar cheesemaking
A reduction in the number of NSLAB
to 103 CFU g1 (at both ripening
temperature 7C and 12 C at the
end ripening (240 days) in
experimental cheese
[34]
Transconjugant Lc. lactisDPC 4275 Lacticin 3147 As single-strain starter in Cottage
cheese
A 99.9% reduction in the counts of L.
monocytogeneswithin 5 days at
4C in experimental cheese
[33]
Lc. lactisDPC 3147 (natural
producer) Lc. lactisDPC
(transconjugant)
Lacticin 3147 As starter culture added individually
to Lb. paracaseiDPC 5337,
resistant to lacticin 3147
A manipulation of cheese flora [36]
Transconjugant Lc. lactis3593 Lacticin 3147 As starter culture to starter adjuncts
(Lc. lactisT1 and Lb. caseiIFPL 731
in semi-hard cheesemaking
An increase in values of amino
peptidase activity (about two
times) and amine nitrogen (16%higher) at the end ripening (42
days)
[37]
Lc. lactisCNRZ 481 Lacticin 481 As adjunct to the lactococcal starter
Lc. lactisHP in Cheddar
cheesemaking
A 2 log units reduction n the counts
of NSLAB in experimental cheese
at the end of ripening (6 months);
an increase in LDH levels (fivefold
higher); improve the quality of
cheese
[43]
Lc. lactisINIA 639 Lacticin 481 As starter along with Lc. lactisINIA
437 and Lb. helveticusLH 92 in
Hispanico cheesemaking
An increase in proteolysis (to twofold
higher); an increase in values of
amino peptidase activity (to
1.8-fold higher) and total amino
acids (2.3-fold higher) after 25
days; a reduction in bitterness.
[99]
Lc. lactisINIA 415 containing the
gene encoding lacticin 481 and
nisin production
Lacticin 481+ nisin Z As adjunct to the commercial culture
S. thermophilusTA 052 and Lc.
lactisINIA 415-2 (a
nonbacteriocinogenic mutant) in
Hispanico cheesemaking
An increase in secondary proteolysis
and levels of total free amino acid
(1.49 and 2.34-fold higher,
respectively) on day 75.
[45]
Lc. lactisDPC 3286 Lactococcin A, B, M As adjunct to the lactococcal starter
Lc. cremorisHP in Cheddar
cheesemaking
An increase in proteolysis; An
increase in concentrations of total
free amino acids; A reduction in
bitterness and a cheese with
improved flavor and quality.
[30]
Lc. lactisDPC 3286 Lactococcin A, B, M As adjunct to the starter Lc. cremoris
HP + S. thermophilusDPC 1842 in
Cheddar cheesemaking
An increase in LDH levels of 265%;
An increase in level of total free
amino acids (about two times); a
reduction in bitterness.
[41]
Lc. diacetylactisUL 719 Nisin Z As starter coculture in Cheddar
cheesemaking
A reduction in the counts ofL.
innocuato 104 CFU g1 in
experimental cheese at the end of
ripening (6 months)
[39]
Lc. lactisIPLA 729 Nizin Z AS adjunct to the mesophilic starter
IPLA 501 (Lc. diacetylactis+
Leuconostoc citreum IPLA 616) in
semi-hard Vidiago cheesemaking
A successfully control of the growth
ofCl. tyrobutiricum in
experimental cheese; A
gas-blowing preventing agent
[44]
Lc. lactisCNRZ 150 Nizin As starter culture together with Lc.
lactisCNRZ 1076 in Camembert
cheesemaking
A 2.4 log CFU g1 reduction in the
numbers ofL. monocytogenes
(throughout ripening6 weeks)
[25]
Transconjugant Lc. cremorisJS 102 Nisin As adjunct to the lactococcal starter
Lc. lactisNCDO 1404 in Cheddar
cheesemaking
A significant reduction in the counts
ofL. monocytogenesand S. aureus
during storage at 23C and 37C.
[27]
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428 D. Beshkova and G. Frengova Eng. Life Sci. 2012, 12, No. 4, 419432
Table 2. Continued
Bacteriocin-producing culture Bacteriocin Directions for application Observed effects Reference
1 2 3 4 5
Electrotransformant Lc. lactis
MM217
Pediocin PA-1 As single-strain starter in Cheddar
cheesemaking
A reduction in the number of L.
monocytogenesto 1.0 log g1 at the
end of ripening (6 months) at 8C.
[32]
Transformants Lc. lactisCL1 (Ped+)and Lc. lactisCL2 (Nis+, Ped+)
Pediocin As adjunct (added individually) tothe commercial starter MA016 in
cheesemaking
A reduction in the counts ofpathogens as follows: L.
monocytogenesto 1.64 log Units;
S. aureusto 0.40 log Units and E.
colito 0.84 log Units on the end
of ripening (30 days).
[46]
Lb. gasseriK7 Bacteriocin As adjunct to the commercial starter
culture S. thermophilusTH4DVC
in semi-hard cheesemaking
Inhibition ofCl. tyrobutyricum; A
reduction in the counts of NSLAB
to< 100 CFU g1 throughout the
entire ripening (8 weeks)
[47]
Ent. faecuim FAIR-E 198 Enterocin As adjunct to the coculture starter (S.
thermophilusACA-DC7+ Lc.
lactisACADC 52+ Lb. bulgaricus
ACA-DC 8) in Greek Feta
cheesemaking
Inhibition ofListeria [40]
Ent. faecalis226 Enterocin 226 NWC As star ter culture in Mozzarelacheesemaking
Inhibition ofL. monocytogenes [26]
Ent. faecalisINIA 4 Enterocin 4 As adjunct to the commercial starter
(Lc. cremoris+ Lc. lactis) in
Manchego cheesemaking
Inhibition ofL. monocytogenesOhio
but not ofL. monocytogenesScott A
[29]
Ent. faecalisINIA 4 Enterocin 4 As adjunct to the commercial
mesophilic CH-N01-type starter
(Lc. lactis+ Lc. diacetylactis+ Lc.
cremoris+ Leuconostoc cremoris)
in semi-hard Hispanico
cheesemaking.
An increase in proteolysis; An
increase in levels of
aminopeptidase activity (about
eightfold higher) on day 15.
[31]
Ent. faecalisINIA 4 Enterocin 4 As adjunct to the commercial
mesophilic LD-type starter (Lc.
cremoris+ Lc. diacetyactis+
Leuconostoc cremoris) in semi-hard
Hispanico cheesemaking
An increase in proteolusis (1.8-fold
higher) and levels of total free
amino acids (2.17-fold higher); A
reduction in level of hydrophobic
peptides and bitterness in
experimental cheese at the end of
ripening (45 days)
[38]
cheese ripening, anda more rapid developmentof thecharacter-
istic cheese flavor has been reported [31]. These positive effects
were achievedby the authors by usingthe bacteriocin-producing
strain Ent. faecalisINIA 4 (at a low inoculation level0.003%
v/v) to the commercial LD-type (cultures producing diacetyl
and carbon dioxide) mesophilic starter culture CH-N01, con-
sisting of Lc. lactis, Lc. lactis biovar. diacetylactis, Lc. cremoris,
Leuconostoc mesenteroides subsp. cremoris to make Hispanico
cheese. From 3 to 15 days, released aminopeptidase generally
double in experimental cheeses (with bacteriocinogenic culture,
as adjunct), reaching values for activity on Lys-pNA and Leu-
pNA up to 9.8- and 6.4-fold higher, respectively, than in control
cheese. Similarly, Oumer et al. [38] concluded that early lysis of
starter cells in Hispanico cheese made from mixture of cows`and
ewes` milks (4:1) inoculated with 1.0 g bacteriocin-producing
culture Ent. faecalisINIA4kg1 wasfollowed by a higher produc-
tion of free amino acids and some volatile compounds (diacetil,
3-methyl-1-butanal)that are important for the organolep-
tic characteristics of cheese. The commercial starter (LD-type
mesophilic starter culture CH-N01 consisting of Lc. lactisbio-
var. diacetylactis, Lc. cremoris, L. mesenteroidessubsp. cremoris)
lost viability more rapidly in experimental cheese (made with
the bacteriocinogenic strain), which reached counts of up to 6
107 CFU g1 during ripening. At the end of ripening pe-
riod (45 days), the degree of proteolysis and concentrations of
total amino acids in experimental cheese was 1.80- and 2.17-
fold higher than the respective values in control cheese (absence
of the bacteriocinogenic strain). The aminopeptidase activity
increased significantly (twice) as a result of adding bacteriocino-
genic culture to milk. Inoculating milk with Ent. faecalis INIA
4 reduced the level of hydrophobic peptides that are associated
with bitterness in the experimental cheese.
6 Future prospects
The analysis of data from research work on in situ bacteri-
ocin production could not exclude any mention of the potential
for application of bacteriocin-synthesizing lactic acid cultures
(LAB), which are of great biotechnological importance for the
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Eng. Life Sci. 2012, 12, No. 4, 419432 Bacteriocins from lactic acid bacteria 429
Practical application
The information summarized here can lead to the conclu-sion that there is a great variety of cost-effective ways thatcan be implemented in using bacteriocin-producing cul-tures as starters, as adjunct cultures for fermented foods
and as protective cultures to the surface of food products.Despite the large variety, they all possess potential prop-erties for improving food quality and safety, and are anantimicrobial alternative along the microbe food chain.
The knowledge about in situ bacteriocin production bylactic acid bacteria (LAB) can be used both to improve thelong-known applications of these microorganisms and tocreate new aspects of applications of these cultures in thefood industry. Some properties of LAB can be the key linkto the health effects in nutritious foods. For some of thephysiological properties found in LAB, it has been provedthat these organisms areexcellentfor progressive research.Moreover, LAB have been increasingly used as a model
organism for future physiological and genetic research.
dairy industry. Figure 1 schematically represents the possible
potential applications of LAB bacteriocins, and Table 2 shows
directions for using bacteriocinogenic cultures in making milk
products (different types of cheese) on the basis of the specific
data reported on this. The summarized information in Table 2
can lead to the conclusion that there is a great variety of cost-
effective ways that can be implemented in using bacteriocin-
producing cultures as starters, as adjunct cultures for fermented
foods, and as protective cultures to the surface of food products.
Despite the large variety, they all possess potential properties for
improving food quality and safety, and are an antimicrobial al-
ternative along the microbe foodchain. Bacteriocin-synthesizingLAB are preferred in their role of natural biopreservatives in
food. Biological preservation implies a novel scientifically based
approach to improve the microbiological safety of foods and is
todays response to the evergrowing consumer interest in nat-
ural foods without chemical preservatives. In conclusion, the
efforts of researchers should be directed toward selection of new
LAB strains whose features satisfy both the relevant technologi-
cal requirements for a standard starter culture in making dairy
products, as well as producing bacteriocins with antimicrobial
activity, acting as "bioconservatives," and providing quality and
safe food products. In this respect, the successful strategies will
include genetic engineering to transfer genes encoding a specific
bacteriocin production from nonstarter LAB strains to indus-trial strains of starter cultures, while maintaining their origi-
nal technological features to obtain a quality end product. The
knowledge about in situ bacteriocin production by LAB can be
used both to improve the long-known applications of these mi-
croorganisms and to create new aspects of applications of these
cultures in the food industry. Some properties of LAB can be the
key link to the health effects in nutritious foods. For some of the
physiological properties found in LAB, it has been proved that
these organisms are excellent for progressive research. Moreover,
LAB have been increasingly used as a model organism for future
physiological and genetic research.
The authors have declared no conflict of interest.
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