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4, 419432

419

Dora Beshkova Ginka Frengova

Laboratory of Applied Biotechnologies, Department of Applied Microbiology, Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Plovdiv, Bulgaria

Review

Bacteriocins from lactic acid bacteria: Microorganisms of potential biotechnological importance for the dairy industry
Bacteriocins are a heterogeneous group of ribosomally synthesized, extracellularly released, bioactive peptides or proteins displaying antimicrobial activity against other bacteria. Over the last two decades, there has been an explosion of basic and applied research on lactic acid bacteria (LAB) bacteriocins, primarily due to their potential application as biopreservatives in food and food products to inhibit the growth of food-borne bacterial pathogens. Although bacteriocins can be produced in the food matrix during food fermentation (in situ), bacteriocins by LAB can be produced in much higher amounts during in vitro fermentations under optimal physical and chemical conditions. Because of the complexity of the food matrix and the difculty of quantifying bacteriocin activities in foods, in vitro studies can be performed to simulate and study the in situ functionality of bacteriocinogenic starters. In situ bacteriocin production is most promising for a fast, widespread, and legal use of bacteriocins to achieve the desirable fermentation and a safe nal product. The bacteriocin production may be of utmost importance when bacteriocin-producing LAB are added to foods as starters or protective cultures (adjunct culture). In the current review, our interest is mainly focused on the research of in situ bacteriocin production through nding the potential of the bacteriocinogenic cultures, which have biotechnological importance for the dairy industry.
Keywords: Bacteriocins / Dairy industry / Fermented food / In situ production / Lactic acid bacteria Received: December 12, 2011; revised: February 16, 2012; accepted: April 5, 2012 DOI: 10.1002/elsc.201100127

Introduction

At present, scientic literature and the research community have generally adopted Klaenhammers [1] denition of bacteriocins, i.e. bacteriocins are a heterogeneous group of ribosomally synthesized, extracellularly released bioactive peptides or proteins displaying antimicrobial activity against other bacteria. Historically, in 1976, Tagg et al. [2] dened bacteriocins as proteinaceous compounds, synthesized by both Gram (+) and Gram () bacteria, and exhibiting inhibitory activity against species closely related to the bacteriocin producer. Information on bacteriocins was rst published in 1925, when researchers found out that a biologically active substance produced by strain Escherichia coli V appeared to have antagonistic activity against another strain of the same species (E. coli F) [3]. Later, similar antimicrobial substances produced by E. coli were found and called colicins [4].
Correspondence: Dr. Dora Beshkova (beshkova@yahoo.com), Institute of Microbiology, Laboratory of Applied Biotechnologies, 139 Ruski Blvd, 4000 Plovdiv, Bulgaria
Abbreviations: CFU, colony forming units; LAB, lactic acid bacteria
C

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 aeruginosa Pa, respectively [5]; bacteriocins, aureocin A 53 and aureocin A 70by strains of Staphylococcus aureus [6, 7]; ruminal bacteriocinsby ruminal bacterium Streptococcus 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 rst reported in 1928 [18]. This biologically active substance was later chemically dened 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] LAB are widely used in the manufacturing of fermented food and are among the best studied microorganisms. Detailed knowledge of a number of physiological traits has opened new potential applications for these organisms in the food industry, while other traits might be benecial for human health [22]. Owing to their typical association with food fermentation and also their long tradition as food-grade
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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 nonbacteriocin antibacterial compounds [24]. The persistent interest of researchers in LAB bacteriocins is prompted by their potential application as food biopreservatives, i.e. they offer a successful prospective alternative strategy for inhibiting the growth of foodborne bacterial pathogens. This opens perspectives for the use of bacteriocin-producing LAB (starters or protective cultures) in fermented foods [2547], or of (puried) bacteriocin preparations 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 biotechnological applications. This review focuses on the researchs of the production and application potential of LAB bacteriocins for the development in the biotechnology of LAB.

Classication and properties of LAB bacteriocins

The most prominent Class I bacteriocins is nisin. It is so far the most studied bacteriocin and has been permitted in several countries as food additive/biopreservative in certain processed dairy products and canned foods. Most of the bacteriocins produced by LAB are included in class II (pediocins, enterocins, lactococcins, plantaricins, acidocins). Despite the structural and physicochemical differences between bacteriocins belonging to the four separate classes, it can be assumed that the formation of pores and ion channels in the cytoplasmic membrane of the microbial cell attacked by bacteriocin is the typical mode of action of LAB bacteriocins [1,69,70]. Some of the class II bacteriocins (lactococcin A, lactococcin G, and lactacin F) require a specic receptor molecule for adsorption [7173], while nisin (class I bacteriocins) acts on liposomes and exhibits a receptor-independent action [74]. The intensity of bacteriocinogenic activity probably correlates with a specic receptor of the bacteriocin host. It is still unclear which part of bacteriocin performs the specic binding to the lipid, protein, or reactive groups of the target cell [14]. This section will not deal with the mode of action in details, but will make a brief characterization of the properties (sensitivity to enzymes, pH, and thermostability and the inhibitory spectrum) of bacteriocins produced by LAB isolated from dairy products and raw materials, as well as potential representatives to participate in starters for their in situ production [21, 26, 53, 7592]. The summarized information is presented in Table 1 .

The bacteriocin family includes a wide variety of peptides and proteins in terms of their size, microbial targets, and mechanisms of action and immunity. Several attempts have been made to classify LAB bacteriocins. Based on structural, physicochemical, and molecular properties, bacteriocins from LAB can be subdivided into three major classes [1, 59]. Nonetheless, this classication is continuously being reviewed and it is evolving with the accumulation of knowledge and the appearance of new bacteriocins [10,13,6068]. The currently accepted classication by the research community is as follows: Class I: Lantibiotics ([small], cationic, hydrophobic peptides [<5 kDa] containing unusual and posttranslationally modied amino acids [lanthionine, methyllanthionine, dehydrobutyrine, and dehydroalanine]); Type A (elongated and positively charged molecules) Subtype A1 (leader peptides are cleaved by a dedicated serin proteinase) Subtype A2 (leader peptides are cleaved by a dedicated ABC ATP-binding cassette [ATP] transporter) Type B (globular and noncharged molecules) Class II: Nonmodied, heat-stable, small, cationic, hydrophobic peptides (<10 kDa); Subclass II a (pediocin-like bacteriocinsListeria-active peptides) Subclass II b (two-peptide bacteriocins) Subclass II c (sec-dependent bacteriocins) Subclass II d (other bacteriocinsleaderless bacteriocins; antimicrobial peptides derived from larger proteins) Class III: Large, hydrophilic, heat-labile proteins (>30 kDa); Class IV: Cyclic, cationic, hydrophobic macromolecules.

In situ bacteriocin production

Althogh bacteriocins can be produced in the food matrix during food fermentation (in situ), bacteriocins by LAB can be produced in much higher amounts during in vitro fermentations under optimal physical and chemical conditions. Because of the complexity of the food matrix and the difculty of quantifying bacteriocin activities in foods, in vitro studies can be performed to simulate and study the in situ functionality of bacteriocinogenic starters. In the current review, our interest is mainly focused on the research of in situ bacteriocin production through nding the potential of the bacteriocinogenic cultures, which have biotechnological importance for the dairy industry. A characteristic of the in situ production is that the synthesis of those biologically active cultures is done in milk medium under very strict conditions (pH, temperature, time), depending on the technological parameters for obtaining milk products. In general, the bacteriocin production displayed primary metabolite kineticswith the maximum activity determined in the mid-exponential or at the end of the growth phase [25, 26, 28, 31, 37, 40, 49, 53]. In situ bacteriocin production is most promising for a fast, widespread, and legal use of bacteriocins to achieve the desirable fermentation and a safe nal product. The bacteriocin production may be of utmost importance when bacteriocin-producing LAB are added to foods as starters or protective cultures (adjunct culture). The bacteriocins may offer a competitive advantage to the producing strain and help survive this strain in a competing microbial environment, such as raw milkmaterial for obtaining various types of cheese. Cheeses made using a starter containing one or more dened Lactococcus strains usually have a uniform texture due to the known biochemical activities of their microora dur-

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Table 1. Specic properties of LAB bacteriocins.

Sensitivity to enzymes 3 Broad inhibitory spectrum to Gram (+) bacteria and some species of Gram () bacteria such as E. coli, S. typhimurium Inhibition spectrum 4 pH and thermostability 5 Active at low pH 2.0; stable to boiling for at least 5 min at pH 2.0 Reference 6 [75]

Bacteriocin-producing strain 1

Bacteriocin 2

Lc. lactis IO-1, isolated from milk product

Nisin Z

Lc. lactis NCDO 497, isolated from milk product

Nisin A

Stable at pH range (2.07.0); heat stable at acidic pH

[21, 75]

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Resistant to pepsin and sensitive to trypsin, papain, -chymotrypsin, cin, pancreatin; inactivated by proteinase K and actinase E Sensitive to proteolytic enzymes except pepsin; stable to proteinase K and actinase E Not investigated by authors Broad spectrum of activity against a wide range of Gram (+) bacteriaBacillus, Clostridium, Micrococcus, and Staphylococcus species Broad spectrum of activity toward Micrococcus luteus, Staphylococcus aureus, Bacillus cereus [76] Not investigated by authors Sensitive to proteinase K Highly stable at pH range of 6.07.0; lower stability at acidic and alkaline pH; heat stability at 40 C90 o C Heat stable at a low pH Heat stable over a pH range of 5.09.0 [77, 78] [79] Broad inhibitory spectrum to a wide range of Gram (+) bacteria Medium spectrum of inhibition against lactococci, some lactobacilli; slightly sensitive to Listeria, Bacillus, and Enterococcus species Broad spectrum of activity toward Listeria species and Gram (+) bacteria, but not against Gram () bacteria Broad spectrum of activity toward Gram (+) bacteria, some Gram () bacteria Active over a wide pH range (4.08.0) [80] Completely inactivated by all proteolytic enzymes; inactivation by catalase, -amylase, lipase not observed Not investigated by authors [81] Not investigated by authors [82] [26] Inhibition of closely related LAB, some Listeria and Staphylococcus species Narrow spectrum of activity against Listeria and Enterococcus species Antimicrobial activity against a wide spectrum of Gram (+) bacteria, but not active against lactococci; sensitive to many Gram () bacteria Active over a wide pH range (against Gram (+) bacteria), slightly more active under acidic pH; increased susceptibility at alkaline pH (against Gram () bacteria) Stable at pH range from 2.0 to 10.0, with no loss of activity at 100 C for 15 min Stable at heating up to 90 C for 30 min; complete inactivation at 121 C for 15 min Highest stability at pH range 4.06.0; unaffected by heating at 80 C for 60 min and at 100 C for 10 min Sensitive to proteolytic enzymes, except pepsin; Nonproteolytic enzymes not caused inactivation Sensitive to all proteolytic enzymes, but unaffected by treatment with catalase, phospholipase C, lysozime, Dnase, Rnase, lipase [83]

Lc. lactis Fc2, isolated from farm cheese

Nisin

Lacticin 3147

Lc. lactis DPC 3147, isolated from ker grain Lc. lactis DPC 5552, isolated from raw milk

Bacteriocin

Ent. faecium WHE 81, isolated from Munster cheese

Enterocin 81

Ent. faecalis A-4832

Enterocin AS-48

Ent. faecium 130, isolated from Mozzarella cheese Ent. faecalis 226, isolated from natural whey cultures

Bacteriocin

Enterocin 226NWC

Bacteriocins from lactic acid bacteria

Lb. plantarum WHE 92

Pediocin PA-1

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Table 1. Continued
Sensitivity to enzymes 3 Heat stable after 30 min at 121 C Inhibition spectrum 4 pH and thermostability 5 Reference 6 [84]

Bacteriocin-producing strain 1

Bacteriocin 2

D. Beshkova and G. Frengova

Lb. plantarum, isolated from homemade yogurt

Plantaricin AA135

Lb. plantarum TF711, isolated from raw goats cheese Sensitive to proteolytic and glycosidic enzymes; stable to catalase. Complete inactivation by proteolytic enzymes, -amylase; insensitive to lipase and surfactants Not investigated by authors

Plantaricin TF711

Stable to catalase treatment; completely destroyed by pepsin, trypsin, and papain Complete inactivation by all proteolytic enzymes

[85]

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Strongly inhibited Gram (+) bacteria, whereas weakly inhibited L. monocytogenes and B. subtilis; slightly sensitive to Gram () bacteria Broad inhibitory spectrum against Gram (+) bacteria, but no inhibition against Enterococcus or Listeria strains Wide spectrum of inhibition against Gram (+) bacteria Stable at pH range 1.09.0; thermostable and stable during storage at 20 C and 4 C (70% residual activity at 100 C for 30 min Relatively heat stable, retaining 25% of activity at 121 C for 30 min; stable at pH range 3.09.0 Heat stable even at 100 C and 121 C for 15 min; pH stability over a wide pH range of 3.010.0 [86] [87] Antimicrobial activity against most strains of nearly all Lactobacillus species; wide inhibitory spectrum toward several food-borne pathogens. Broad inhibitory spectrum against pathogens Heat stability from 60 C to 90 C for 10 min; stable in acidic to neutral range of pH, inactive in alkaline range at pH 9.0 [88] Sensitive to proteolytic enzymes; insensitive to -amylase and lipase Broad inhibitory spectrum against pathogens Stable in pH range 2.010.0; thermostability at 115 C for 15 min, except for BB13. [89] Broad inhibitory spectrum toward Clostridium. Listeria, Bacillus subtilis, and LAB (lactococci, lactobacilli, pediococci, enterococci) Wide antimicrobial spectrum including LAB (resistant to lactobacilli, sensitive to lactococci) and a range of various Gram (+) and Gram () bacteria Stability to heat, complete inactivation at 121 C for 30 min; pH stability at 4.06.0 [90] Sensitive to certain proteases, -chymotrypsin, protease K, pancreatin; resistant to trypsin and pepsin Complete inactivation by pancreatic proteolytic enzyme pronase and trypsin; not affected by -amylase and lipase Stable at around pH 6.0; completely inactivated at pH values between 1.03.0 and 10.012.0; heat labile. [53]

Acidocin CH5

Lb. acidophilus CH5, isolated from commercial dairy starter Lb. bulgaricus 1043, isolated from yellow cheese

Bacteriocin

Bacteriocins

Bacteriocins

Lb. plantarum (B14, C1 C4, C7, G7, G8) and Lb. rhamnosus (B13, C5, G4, G10, G18), isolated from cow, goat, buffalo milk Lb. bulgaricus (BB18, BB13), Lb. casei BCZ2, Lc. lactis BCM5, isolated from Bulgarian dairy products Lb. curvatus IFPL105, isolated from raw goats milk and artisanal cheese

Bacteriocin

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S. thermophilus ACA-DC-0001, isolated from sheeps yogurt

Thermophilin ST-1

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ing cheese manufacture and ripening. The organisms in mixedstrain starters used in the manufacture of the cheeses belong mainly to the genera Lactococcus (Lc. lactis subsp. lactis [Lc. lactis], Lc. lactis subsp. cremoris [Lc. cremoris])mesophilic, the best known or Lactobacillus (Lb. helveticus, Lb. delbrueckii subsp. bulgaricus), and Streptococcus thermophilusthermophilic, the best known, depending on the specic application [93]. The lactic fermentation of milk is required for cheese production. While some cheeses are still made from nonpasteurised milk and may even depend on the adventitious natural lactic ora (nonstarter 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 dening the quality of cheese is unclear. Their presence can have both a positive and, more frequently, negative effect, manifested in concomitant defectscalcium lactate crystal formation, off-avor 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 a signicant 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 adjunct cultures to obtain various types of cheese, can be expressed by dening the role of these cultures in the following two areas: bacteriocin-producing LAB to control adventitious microbial 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 avordiscussed in separate sections of this article.

Bacteriocin-producing LAB to control adventitious microbial populations and as protective cultures to inhibit the growth of pathogenic microorganisms in cheese

The lacticin 481-producing and lacticin 3147-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. lactis CNRZ 481 used as an adjunct to the lactococcal starter culture Lc. lactis HP, compared with the same number of bacteria in the control cheese (obtained with the standard starter culture only). At the end of the ripening period (after 6 months), the recorded decrease in the number of NSLAB was 2 log units. Other authors [28] did not nd 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. lactis DPC 3204, Lc. lactis DPC 3256), isolated from natural yogurt and producing bacteriocin, lacticin 3147. In comparison, in control, 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 detected in cheese made with bacteriocin-producing starter was

approximately 1280 AU mL1 , which remained stable over the entire ripening period. In situ production of nisin Z by Lc. diacetylactis UL 719, cocultivated with Lc. cremoris KB 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 obtained. In addition to the nisin-producing activity of the mixed starter, researchers evaluated the antagonistic effect of bacteriocin 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 reduction of 1.5 log units in experimental cheese obtained with a nisinogenic starter. At the end of ripening process in cheese produced with a nisin preparation, about 10 CFU g1 of L. innocua and 90% of the initial activity of nisin was established, compared to 104 CFU g1 and 12% for cheese produced with a nisinogenic starter. The use of Lc. diacetylactis UL 719 in a mixed starter culture 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 postcontaminant organisms, as they are usually present in low numbers. The data obtained by the authors on nisin activity in experimental Cheddar cheese produced with a nisinogenic starter (Lc. diacetylactis UL 719) are similar to those reported previously for Gouda cheese [35]. Bauksaim et al. [35] reported that Gouda cheese of good quality and safe to eat can also be obtained by the aforementioned nisin-producing culture Lc. diacetylactis UL 719, when it is added to commercial Flora Danica starter in a ratio of 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. lactis CNRZ 150 to inhibit Listeria monocytogenes during Camembert cheese manufacture and ripening. Maximum nisin concentration of ca 700 IU g1 was obtained in curd at 9.0 h (during the exponential phase of growth of bacteriocin-producing culture), then nisin concentrations decreased slowly during 924 h, and dramatically to ca 200 IU g 1 during ripening. In the presence of nisin, the numbers of L. monocytogenes decreased rapidly from 6 to 24 h and a difference of 2.4 log CFU g1 between numbers of Listeria in cheeses made with Nis+ (Lc. lactis CNRZ 150) and Nis (Lc. lactis CNRZ 1076) starter cultures was maintained throughout ripening (6 weeks). Another nisin Z-producing strain Lc. lactis IPLA 729 has been successfully applied on the inhibition of the spoilage strain of Clostridium tyrobutiricum CECT 4011, a late blowing agent, in semi-hard Vidiago cheese making [44]. The authors have obtained and studied three cheeses: experimental cheesenisinogenic culture was co-cultivated with a mesophilic starter IPLA-001 composed of Lc. diacetylactis IPLA 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; commercial cheesethe nisin producer was replaced by KNO3 as a gas-blowing preventing agent. A maximum concentration of 1600 IU nisin mL1 was measured in the experimental cheese between day 1 and day 15 of the ripening process, while at the end of the process (30 days), the activity decreased. During ripening of the three types of cheese, the following trends in the growth of

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clostridia were 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 number 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 culture that will be used in the making of Vidiago cheese, thereby improving the ability to suppress spoilage microorganisms and contributing to better organoleptic properties in comparison with commercial starters. One possibility for the improvement of the metabolic productivity of an microorganisms is genetic modication. This strategy was successfully used by Ryan et al. [28] as follows: a bacteriocinproducing transconjugant Lc. lactis DPC 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. cremoris DPC 4268; the obtained transconjugant was dened as Bac+ and Imm+ , i.e. possessing properties of lacticin 3147 production and immunity to lacticin 3147. Furthermore, on the one hand, the transconjugant had retained the lacticin 3147-producing activity of the parent strain Lc. lactis DPC 3147, while, on the other hand, it maintained the original characteristics of the commercial starter strain, i.e. the produced lactic acid levels were similar. Throughout ripening (6 months), the concentration of bacteriocin, lacticin 3147, determined in Cheddar cheese (made with a transconjugant strain as a single-strain starter) did not change and this correlated with a signicant decrease in the number of NSLAB. Later, the lacticin 3147-producing transconjugant Lc. lactis DPC 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 ripening temperatures (7 C and 12 C), the NSLAB populations in the corresponding cheeses made with the lacticin 3147-producing culture, Lc. lactis DPC 4275, increased much more slowly during ripening and reached markedly lower numbers (ca 103 CFU g1 ) at the end (240 days) of the ripening [34]). In agreement with the observations of Folkertsma et al. [95] for full fat Cheddar cheese, elevation of ripening temperature from 7 C to 12 C resulted in a more rapid developmed of NSLAB (to ca 107 CFU g1 ) in cheeses made with the nonbacteriocinogenic mixed starter ([Lc. lactis DPC 4268 + Lc. cremoris DPC 4269] or Lc. lactis DPC 4268 alone). The authors concluded that the use of the bacteriocinogenic Lc. lactis DPC 4275 can reduce the risk of avor defects in reduced fat cheese, especially when ripening is at high temperature, and should enable the production of cheeses with more predictable and consistent avor. The effectiveness of lacticin 3147 as a natural preservative was determined in Cottage cheese that was subsequently inoculated with approximately 104 L. monocytogenes Scott A g1 [33]. During the rst week of storage (at 4 C), in the experimental cheese (made with the bacteriocinproducing transconjugant Lc. lactis DPC 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. lactis DPC 4268), the initial number of inoculated pathogen cells remained essentially unchanged. Another genetically modied

starter culture Lc. lactis MM 217, capable of producing pediocin in situ, was used as a single starter culture to improve the microbiological safety of Cheddar cheese precontaminated with L. monocytogenes [32]. The nonbacteriocinogenic starter culture Lc. lactis MM 210 was used as an recipient for plasmid (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 characteristics and acid-producing activity of the parent strain. At the end of the ripening process of cheeses (6 months at 8 C), the number of live L. monocytogenes cells decreased from 3.5 log CFU g1 (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. monocytogenes, S. aureus, and E. coli O157:H7 during cheese ripening. The wild strains Lc. lactis ESI 153 and Lc. lactis ESI 515(Nis+ ) were selected by their technological and/or antimicrobial properties, used as starter cultures in cheese manufacture and were transformed to produce pediocin PA-1, as reported earlier [96]. Pediococcus acidilactici 347 (Ped+ ), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis+ ), and their respective pediocin-producing transformants Lc. lactis CL 1 (Ped+ ) and Lc. lactis CL 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. lactis CL 1 (Ped+ ) or Lc. lactis CL 2 (Nis+ , Ped+ ), there was a certain decrease in the L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157: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. lactis CL 2 (Nis+ , Ped+ ) in the respective 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 approximate concentration of 6 log CFU mL1 , which was higher than the possible contamination of natural milk. Similarly Zottola et al. [27] suggested that the use of bacteriocinogenic culture as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods. A nisinproducing transconjugant Lc. cremoris JS 102 was added as an adjunct to lactoccocal starter Lc. lactis NCDO 1404 in Cheddar cheesemaking, which contained between 400 and 1200 IU of nisin g1 cheese. The shelf life (at 22 C) of the nisin-containing cheese was signicantly greater than that of the control cheese (the cheeses were inoculated with 2000 spores of Cl. sporogenes PA 3679 during manufacture). Signicant 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 (22 C and 37 C).

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Figure 1. Overview of potential application of LAB-producing bacteriocins.

Besides lactococci as adjunct cultures during in situ bacteriocin 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 ora using combinations of lacticin 3147-producing and -resistant cultures. A stable, more resistant Lb. paracasei DPC 5337 (which was 32 times less sensitive to bacteriocin, lacticin 3147 than parental strain Lb. paracasei DPC 5336) was used as adjunct cultures in two separate trials using either Lc. lactis DPC 3147 (a natural producer) or Lc. lactis DPC 4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious NSLAB in cheddar cheese [28]. Lacticin-3147 activity was assayed during manufacture and reached approximately 12802560 AU g1 of cheese. This level of activity was maintained throughout ripening 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 avor being almost 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 a bacteriocinogenic culture (Lb. gasseri K 7) as an adjunct to the commercial starter S. thermophilus TH4DVS. In the experimental cheese, the bacteriocin-producing culture did not inhibit the thermophilic starter, but reduced the number of the nonstarter mesophilic lactobacilli to about 100 CFU g1 throughout the entire ripening (8 weeks). This inhibitory effect was observed despite the fact that no bacteriocins were found in the experimental cheese. In addition, the pH values and concentrations of organic 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 suggest the following possible explanations of this phenomenon: nonuniform bacteriocin distribution in cheese, adsorption to the caseins in the curd, or bacteriocin degradation by intracellular proteases, as reported earlier by other authors [97]. Villani et al. [26] used Enterococcus faecalis 226 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 reconstituted 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 comparison, the number of live cells of the pathogen was 9 108 in the development of L. monocytogenes as 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 monocytogenes Ohio 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). Another strain, L. monocytogenes Scott A, was not inhibited in the presence of a bacteriocin-producing culture, used either alone or as an adjunct to a commercial starter during cheese manufacture, but decreased by 1 log unit and 2 log units, respectively, after 7 and 60 days of ripening. The values of enterocin activities, determined 8 h after making the experimental cheeses (with a mixed starterbacteriocinogenic + commercial strains, and with a single starterbacteriocinogenic culture), were 2000 3000 AU g1 and 40006000 AU g1 , respectively. These results are in agreement with bacteriocin production by Ent. faecium 7C5 under Taleggio cheesemaking conditions, which still occurred at 25 h and pH 4.9 [98]. Sarantinopoulos et 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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used mixed starter (S. thermophilus ACA-DC 7, Lc. lactis ACADC 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.

Bacteriocin-producing LAB as cell lysis-inducing agents to improve cheese quality and avor

Another important aspect of using bacteriocinogenic cultures is the possibility to induce controlled lysis of a LAB starter culture 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 improved 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. cremoris HP during ripening of Cheddar cheese by adding the bacteriocinogenic culture Lc. lactis DPC 3286 (encodes the synthesis of lactococcins A, B, M) to the standard lactococcal starter. In a laboratory-scale system, it was found that the LHD (lactate dehydrogenase) activity, determined on day 180 (end of the ripening process) in the experimental 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 dehydrogenase and postproline dipeptidyl aminopeptidase in the experimental cheese, resulting in higher concentrations of free amino acids by 47% than those in the control cheese. With the successful application of the mixed culture (Lc. cremoris HP + bacteriocinogenic adjunct strain Lc. lactis DPC 3286), the authors obtained a dairy product of improved quality, reduced bitterness, 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. lactis DPC 3286), possessing the ability to lyse the second culture (Lc. cremoris HPsensitive to lactococcin A, B, and M activity), and a third culture (S. thermophilus DPC 1842acid producing and resistant to lactococcin A, B, and M activity) for pilot-scale Cheddar cheesemaking. In the experimental cheese (made with a bacteriocinogenic adjunct culture), higher (approximately two times) concentrations of free amino acids were recorded, higher release rates of intracellular LDH (by 265%), and a decrease in bitterness compared to the control cheese (made with Lc. cremoris HP alone or with a mixed starterLc. cremoris HP + S. thermophilus DPC 1842). Another bacteriocinogenic strain, Lc. lactis CNRZ 481, producing lacticin 481, was also used as an adjunct to the commercial starter culture Lc. lactis HP for pilot-scale Cheddar cheesemaking, but without impeding the production of the amount of acid required 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. lactis INIA 415 (strain containing the structural gene encoding 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 thermophilus TA 052 counts were lower (about 1 log unit) in experimental 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 experimental 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] described another alternative for Hispanico cheesemaking using a lactococcal mixed starter (lacticin 481-producing Lc. lactis INIA 639 + lacticin 481-nonproducing Lc. lactis INIA 437) to which a lactobacillus strain (Lb. helveticus LH 92) was added, which is sensitive to lacticin 481 and possesses high aminopeptidase activity. In the control cheese, about a twofold lower rate of proteolysis 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 experimental cheese, the concentrations of total free amino acids were determined to be about 2.3 times higher than those in the control cheese. As a result of using the lacticin 481-producing culture, the cheese obtain by the authors had improved quality and reduced bitterness. Martinez-Cuesta et al. [37] studied the potential of lacticin 3147-producing transconjugant Lc. lactis IFPL 3593 (used as a starter) to induce lysis of the two cultures (Lc. lactis T1 and Lb. casei IFPL 731both showed high aminopeptidase activity) added to the starter, thus achieving accelerated processes of proteolysis and ripening in semi-hard cheese, accompanied by a parallel signicant increase in its sensory characteristics intensity of aroma and cheese taste. The bacteriocin-producing transconjugant Lc. lactis IFPL 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. lactis IFPL 359. The transconjugant was dened 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 signicantly 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 adjuncts (Lc. lactis T1 and Lb. casei IFPL 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. lactis IFPL 359 [commercial starter] and adjuncts cultures [Lc. lactis T 1 + Lb. casei IFPL 731). Enterococcus species also were added as a bacteriocinproducing 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 1 Lc. lactis DPC 3147 Lc. lactis DPC 3204 Lc. lactis DPC 3256 Transconjugant Lc. lactis DPC 4275 Transconjugant Lc. lactis DPC 4275 Bacteriocin 2 Lacticin 3147 Directions for application 3 As mixed starter culture in Cheddar cheesemaking As single-strain starter culture in Cheddar cheesemaking As single-strain in reduce fat Cheddar cheesemaking Observed effects 4 No NSLAB detected in experimental cheese at the end ripening 6 months A signicant reduction in the levels of NSLAB A reduction in the number of NSLAB to 103 CFU g1 (at both ripening temperature 7 C and 12 C at the end ripening (240 days) in experimental cheese A 99.9% reduction in the counts of L. monocytogenes within 5 days at 4 C in experimental cheese A manipulation of cheese ora Reference 5 [28]

Lacticin 3147 Lacticin 3147

[28] [34]

Transconjugant Lc. lactis DPC 4275

Lacticin 3147

As single-strain starter in Cottage cheese

[33]

Lc. lactis DPC 3147 (natural producer) Lc. lactis DPC (transconjugant) Transconjugant Lc. lactis 3593

Lc. lactis CNRZ 481

Lc. lactis INIA 639

Lc. lactis INIA 415 containing the gene encoding lacticin 481 and nisin production

Lc. lactis DPC 3286

Lc. lactis DPC 3286

Lc. diacetylactis UL 719

Lc. lactis IPLA 729

Lc. lactis CNRZ 150

Transconjugant Lc. cremoris JS 102

As starter culture added individually to Lb. paracasei DPC 5337, resistant to lacticin 3147 Lacticin 3147 As starter culture to starter adjuncts An increase in values of amino peptidase activity (about two (Lc. lactis T1 and Lb. casei IFPL 731 times) and amine nitrogen (16% in semi-hard cheesemaking higher) at the end ripening (42 days) A 2 log units reduction n the counts Lacticin 481 As adjunct to the lactococcal starter of NSLAB in experimental cheese Lc. lactis HP in Cheddar at the end of ripening (6 months); cheesemaking an increase in LDH levels (vefold higher); improve the quality of cheese An increase in proteolysis (to twofold Lacticin 481 As starter along with Lc. lactis INIA higher); an increase in values of 437 and Lb. helveticus LH 92 in amino peptidase activity (to Hispanico cheesemaking 1.8-fold higher) and total amino acids (2.3-fold higher) after 25 days; a reduction in bitterness. Lacticin 481 + nisin Z As adjunct to the commercial culture An increase in secondary proteolysis and levels of total free amino acid S. thermophilus TA 052 and Lc. (1.49 and 2.34-fold higher, lactis INIA 415-2 (a respectively) on day 75. nonbacteriocinogenic mutant) in Hispanico cheesemaking An increase in proteolysis; An Lactococcin A, B, M As adjunct to the lactococcal starter increase in concentrations of total Lc. cremoris HP in Cheddar free amino acids; A reduction in cheesemaking bitterness and a cheese with improved avor and quality. Lactococcin A, B, M As adjunct to the starter Lc. cremoris An increase in LDH levels of 265%; An increase in level of total free HP + S. thermophilus DPC 1842 in amino acids (about two times); a Cheddar cheesemaking reduction in bitterness. Nisin Z As starter coculture in Cheddar A reduction in the counts of L. cheesemaking innocua to 104 CFU g1 in experimental cheese at the end of ripening (6 months) Nizin Z AS adjunct to the mesophilic starter A successfully control of the growth of Cl. tyrobutiricum in IPLA 501 (Lc. diacetylactis + experimental cheese; A Leuconostoc citreum IPLA 616) in gas-blowing preventing agent semi-hard Vidiago cheesemaking Nizin As starter culture together with Lc. A 2.4 log CFU g1 reduction in the lactis CNRZ 1076 in Camembert numbers of L. monocytogenes cheesemaking (throughout ripening6 weeks) A signicant reduction in the counts Nisin As adjunct to the lactococcal starter of L. monocytogenes and S. aureus Lc. lactis NCDO 1404 in Cheddar during storage at 23 C and 37 C. cheesemaking

Lacticin 3147

[36]

[37]

[43]

[99]

[45]

[30]

[41]

[39]

[44]

[25]

[27]

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Table 2. Continued
Bacteriocin-producing culture 1 Electrotransformant Lc. lactis MM217 Transformants Lc. lactis CL1 (Ped+ ) and Lc. lactis CL2 (Nis+ , Ped+ ) Bacteriocin 2 Pediocin PA-1 Directions for application 3 As single-strain starter in Cheddar cheesemaking As adjunct (added individually) to the commercial starter MA016 in cheesemaking Observed effects 4 A reduction in the number of L. monocytogenes to 1.0 log g1 at the end of ripening (6 months) at 8 C. A reduction in the counts of pathogens 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). Inhibition of Cl. tyrobutyricum; A reduction in the counts of NSLAB to < 100 CFU g1 throughout the entire ripening (8 weeks) Inhibition of Listeria Reference 5 [32]

Pediocin

[46]

Lb. gasseri K7

Bacteriocin

As adjunct to the commercial starter culture S. thermophilus TH4DVC in semi-hard cheesemaking

[47]

Ent. faecuim FAIR-E 198

Ent. faecalis 226 Ent. faecalis INIA 4

Ent. faecalis INIA 4

Ent. faecalis INIA 4

As adjunct to the coculture starter (S. thermophilus ACA-DC7 + Lc. lactis ACADC 52 + Lb. bulgaricus ACA-DC 8) in Greek Feta cheesemaking Enterocin 226 NWC As starter culture in Mozzarela cheesemaking Enterocin 4 As adjunct to the commercial starter (Lc. cremoris + Lc. lactis) in Manchego cheesemaking 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. Enterocin 4 As adjunct to the commercial mesophilic LD-type starter (Lc. cremoris + Lc. diacetyactis + Leuconostoc cremoris) in semi-hard Hispanico cheesemaking

Enterocin

[40]

Inhibition of L. monocytogenes

[26]

Inhibition of L. monocytogenes Ohio [29] but not of L. monocytogenes Scott A An increase in proteolysis; An increase in levels of aminopeptidase activity (about eightfold higher) on day 15. [31]

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, and a more rapid development of the characteristic cheese avor has been reported [31]. These positive effects were achieved by the authors by using the bacteriocin-producing strain Ent. faecalis INIA 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, consisting 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 LeupNA 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. faecalis INIA 4 kg1 was followed by a higher production of free amino acids and some volatile compounds (diacetil, 3-methyl-1-butanal)that are important for the organoleptic characteristics of cheese. The commercial starter (LD-type mesophilic starter culture CH-N01 consisting of Lc. lactis bio-

var. diacetylactis, Lc. cremoris, L. mesenteroides subsp. 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 period (45 days), the degree of proteolysis and concentrations of total amino acids in experimental cheese was 1.80- and 2.17fold higher than the respective values in control cheese (absence of the bacteriocinogenic strain). The aminopeptidase activity increased signicantly (twice) as a result of adding bacteriocinogenic 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.

Future prospects

The analysis of data from research work on in situ bacteriocin 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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The authors have declared no conict of interest.

Practical application
The information summarized here 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 alternative along the microbe food chain. The knowledge about in situ bacteriocin production by lactic acid bacteria (LAB) can be used both to improve the long-known applications of these microorganisms 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.

References

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 specic data reported on this. The summarized information in Table 2 can lead to the conclusion that there is a great variety of costeffective ways that can be implemented in using bacteriocinproducing 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 alternative along the microbe food chain. Bacteriocin-synthesizing LAB are preferred in their role of natural biopreservatives in food. Biological preservation implies a novel scientically based approach to improve the microbiological safety of foods and is todays response to the evergrowing consumer interest in natural 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 technological 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 specic bacteriocin production from nonstarter LAB strains to industrial strains of starter cultures, while maintaining their original 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 microorganisms 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.

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[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

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