ISOLATION OF PROTECTIVE CULTURES (PC)

LAB with potential PC characteristics can be obtained by first classical step, i.e., pour plating the sample on selective media most popularly known as MRS-deMan, Rogosa, and Sharpe. Other media Ml7, Lactobacillus selective broth, litmus milk, tryptic soy broth (TSB) are preferred for the production of bacteriocin. After morphological examinations, typical colonies are isolated, and the isolates are identified by dedicated methods. PCR amplification and partial or complete sequencing of 16S ribosomal RNA is performed. The isolates are screened for antagonism based on inhibitory activity against food pathogen and spoilage microorganisms (Figure 14.2). Some of the sensitive test organisms that have been used by different microbiologists are:

  • Bacillus sp.;
  • Clostridium sp.;
  • E. coli;
  • Enterococcus faecalis MB 1;
  • L. casei;
  • L. plantarum;
  • Leuconostocmes enteroides;
  • Listeria innocua;
  • Listeria monocytogenes;
  • Micrococcus;
  • Salmonella typhimurium;
  • Staphylococcus aureus;
  • • Streptococcus.

Several researchers have found differences between observation of in vitro (laboratory medium) and actual results in situ (food matrix) and sometimes adversely affect the product sensory parameters. The difference is associated with the food to be protected, target microorganisms and storage conditions, etc. [18, 28, 51]. Hence, it is necessaiy that the effectiveness of isolates selected based on in-vitro screening are appraised in situ in real foods for bioprotective ability through challenging studies [25].

POTENTIAL USE OF BACTERIOCIN PRODUCING CULTURE IN FOODS

The application of bacteriocin producing LAB has been demonstrated in different foods (Table 14.2). The unique advantages of protective include natural as they are food grade bacteria, bacteriocins produced when abuse in storage temperature arises. The continuous releases of bacteriocins by live culture compensate the decomposition of bacteriocins and binding to food constituents when added in purified form.

Strategy of selection of protective cultures. (Source

FIGURE 14.2 Strategy of selection of protective cultures. (Source: Modified significantly from Leroi et al. [32].)

TABLE 14.2 Research Studies on the Effectiveness of Protective Culture LAB in Foods

Food

Cultures

Tested Against

References

Bread

Lb. pi ant arum UIG 121

Aspergillus sp. Penicillium sp.

[43]

Cereal products

Lb. plantation

Penicillium sp., Aspergillus sp.

Aflatoxin

[41]

Cheese

Lc. lactis INIA415

Acceleration of ripening

[6]

Cheese

Lc. lactis

L. monocytogenes

[28]

Cheese

Lb. gasseh K7

Cl. tyrobutyricum

[10]

Cooked bacon

Lb. sakei and Lc. lactis

Leu. mesenteroides

[12]

Cottage cheese

Lb. amylovorus DSM 19280

Penicillium expansum

[35]

TABLE 14.2 (Continued)

Food

Cultures

Tested Against

References

Fermented pork meat

Lb. plantarum PCS20

Clostridium spp.

[П]

Fermented soy milk

Lb. helviticusYMLO 14

Penicillium sp.

[9]

Fish sauce

Stop, carnosus FS19

Biogenic amines

[51]

Letuce leaf

Leuconostoc spp.

L. monocytogenes

[47]

Refrigerated pea soup

Lb. plantarum ATCC 8014

Cl. botulinum

[45]

Salmon

Lb. plantarum and C. piscicola

H,S producer and yeast and mold

[31]

Salmon

Lb. sakei CTS494

L. monocytogenes

[7]

Sausage

Lb. sakei

L. monocytogenes, Salmonella spp. and amines

[18]

Tomato puree

Lb. fennentum YML014

Yeast and mold

[1]

Yogurt

Lb. rhamnosus, Lb. zeae, Lb. harbinensis

Yeast and mold

[16]

Yogurt

Lb. casei AST 18

Penicillium sp.

[34]

Several PC of LAB cannot tolerate heat treatment; hence the addition of LAB as PC to foods should be after heat treatment. In the production of fermented products, PC are inoculated into the food as the main starter or as adjunct to starter culture meant for acidification. In this case, the acidifying bacteria should not be affected significantly by bacteriocin producing one. In postharvest and bakeiy foods, the PC or their products are sprayed on the surface. The effectiveness of PC often relies on inoculum level, concertation of metabolites, which can adversely affect the sensory acceptability of foods. Therefore, once the isolation of LAB and characterization of antimicrobials has been conducted, the results of in vitro studies should be confirmed in actual foods.

14.7.1 APPLICATIONS OF LAB IN DAIRY PRODUCTS

LAB is the most frequently used bacteria as a starter culture for acidification and flavor development in milk fermentation. Hence, scientists have studied using live bacteriocinogenic LAB for in situ production of bacteriocins during the production and storage of dairy products. Several research reports have mentioned the use of LAB to control “late blowing” caused by Clostridium spp.

in cheese. For example, the K7 bacteriocin producing Lactobacillus gasseri K7 [10], lacticin 3147 Lactococcus lactis IFPL 3593 [11], reutenn producing Lactobacillus reuteri INIAP572 [23] have shown to inhibit the spore germination and prevent “late blowing” and been proposed as an alternative approach to use of potassium nitrate. Likewise, in situ production of nisin-Z by L. diacetylactis UL 719 in a mixed culture with L. cremoris and L. lactis in Cheddar cheese was effective to inhibit the growth of Listeria itmocua throughout the ripening period (6 months). Nisin concentration of 300 IU/ml was observed [8].

In Camembert cheese, although a 31og-reduction in L. monocytogenes was observed until second week, yet a regrowth was observed for 6 weeks ripening period [36]. Numerous investigations have also studied the development of bacteriocin producing cultures to improve the maturation and quality of cheese. Bacteriocin-producing LAB may also serve as agents to induce lysis of cell-wall of starter and/or non-starter culture to cause initiation of proteolysis and the release of amino acids can further contribute to synthesis of flavor compounds. The lacticin 3147 producing L. lactis was used to hasten the ripening process of cheese and to maintain the significant lower levels of nonstarter LAB for 6 months of ripening [37].

14.7.2 APPLICATIONS OF LAB IN MARINE FOODS

Many psychro-trophic spoilage and pathogenic bacteria can survive in fresh fish. Cold smoked salmon undergoes spoilage in 3-4 weeks mainly due to microbial growth, which limits its commercial popularity. Leroi et al. [32] demonstrated a pool of LAB strains for the prevention of off-odor and acidification of cold-smoked salmon. L. monocytogenes is a major concern as it is a frequent contaminant and it can tolerate cold smoke (<30°C), salting, and remain active in the product at chilled temperatures. In this regard, the bacteriocin-producing LAB proved promising as PC isolated from seafood products. The co-culture inoculation of sakacin producing Lb. sakei with L. monocytogenes was able to demonstrate a 4-log reduction of Listeria innocua in cold-smoked salmon after 14 days of storage at 4°C [27]. Interestingly, the antilisterial activity of Lb. sakei CTS494 was not affected by the type of salmon having different physicochemical characteristics [7].

14.7.3 APPLICATIONS OF LAB IN MEAT PRODUCTS

The bacteriocin-producing LAB has shown to control foodborne pathogens in sausages. Recently, the addition of Lb. plantarum PSC 20 as protective culture was demonstrated as a feasible method to lower the L. monocytogenes counts in fermented meat [38]. Therefore, bacteriocin-producing LAB is gaining importance in the manufacture of fermented meat products to control spoilage microorganisms. The Clostridium sp. in fermented meat is controlled by the addition of nitrite [15]. Hence, antilisterial bacteriocin by PC in situ has been under focus as an alternative to control spoilage and stabilize the color of meat products. Moreover, the selection of bacteriocin-producing LAB as a potential protective culture within the autochthonous microbiota of fermented meat should be recommended, since autochthonous cultures may have better adaptability, faster growth and ensure prolonged shelf-life [15].

14.7.4 APPLICATIONS OF LABS IN FRUITS AND VEGETABLES

The bacteriocinogenic LAB are explored for biopreservation of minimally processed foods of plant origin (such as salads, sprouts, fruit juices, and sauerkr aut). Especially salads offer the advantage of convenience and freshness, which is also favorable for survival and growth of L. monocytogenes [47]. The contamination occurs due to poor handling practices during the production of these foods, and microorganisms can grow under abnormal storage temperatures. In this context, bacteriocinogenic LAB as PC can serve as a hurdle to the growth of these pathogens. In fresh-cut ready-to-eat salads, nisin-Z producing Lc. lactis and bacteriocin producing E. faecium could inhibit L. monocotogenes. Similarly, Lb. casei was able to eliminate the post-processing contaminants colifonns and enterococci in ready-to-eat vegetables after three days of storage under refrigeration [49].

The use of bacteriocinogenic LAB in the fennentation of sauerkraut and other products is beneficial in enhancing the shelf-life of the product. Incorporation of bacteriocin-producing lactococcal strains with Lew. mesenteroides or natural microflora proved valuable for sauerkraut fennentation for controlling the spoilage caused by homo-fennentative Lb. plantarum [22, 47].

 
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