BACTERIOCIN AND ANTIBIOTICS

Misuse of antibiotics has led to drug resistance and multidrug resistance. This is seen more in clinical applications and less so in food industries. A feasible alternative is the use of antibiotics and bacteriocins together for a synergistic effect. Even though antibiotics and bacteriocins serve a similar purpose of species survival by killing competitor strains, they have quite a lot of differences. Antibiotics are secondary metabolites produced during idiophase and bacteriocins are produced during the primary growth phase. They differ in a spectrum of inhibitory activities, antibiotics have varied spectrum like narrow-spectrum or broad-spectrum while bacteriocins only inhibit closely related strains. Antibiotics have mainly clinical applications and very limited use in the food industry while bacteriocins are increasingly used in food preservation presently. They differ in biochemical characteristics, size, and molecular weight (Deraz et al., 2005; Parada et al., 2007).

Antibiotics inhibit the synthesis of nucleic acid, cell wall, and proteins of target bacteria. Bacteriocins target cell membrane and cause pore formation which leads to killing of cells. It is a receptor-mediated mechanism. A side effect of antibiotic use is drug resistance while there has been no known side effect of bacteriocin use till now. The antimicrobial activity of bacteriocins is 1000 fold more potent than eukaiyotic antimicrobial peptides because the activity is more receptor-mediated. A specific protein on the membrane of targeted cells is recognized as receptor molecules for bacteriocins to act upon. Pediocin with unr elated lactococcins A and В are specific to the receptor permease mannose- phosphotransferase (PTS). Integral membrane protein-like undecaprenyl pyrophosphate phosphatase (UppP) also serves as receptors for bacteriocins (Diep et al., 2007; Steinstraesser et al., 2011; Gabrielsen et al., 2014).

Bacteriocins are considered as “designer drugs” owing to their narrow spectrum and specificity of action against target bacterial pathogens. The first reported bacteriocin to be regarded as safe as a food preservative and which was used commercially throughout the world was Nisin. It was first approved as a bio-preservative to be used in foods like cheese spreads, pasteurized egg products against Clostridium botulinum (Riley et al., 2002). Pisciocin is another bacteriocin which has been patented to be used as a preservative in meat products and green salads against Listeria monocytogenes (Raloff, 1998; Ennahar et al., 2000). Microcin and colicins produced by E. coli H22 inhibit pathogenic Enterobacteria Klebsiella pneumoniae and Salmonella spp. (Cursino, 2006). Mersacidin from Bacillus HIL Y85 eliminated Staphylococcus aureus (MRSA) which were resistant to methicillin from the nasal epithelium of rhinitic mice (Kruszewska, 2004). Streptococcus mutans produces mutacin B-Ny266 which is found to be active against Staphylococcus aureus. It was examined by subjecting intraperitoneally to mice. Lactococcus lactis DPC3147 produces lacticin 3147 and Bacillus thuringiensis DPC6431 produces thuricin CD which inhibits the growth of Clostridium difficile. Thuricin CD has been proved to be more effective than metronidazole, vancomycin, and other antibiotics (Rea, 2010, 2011).

Bacteriocins, produced by gram-positive Firmicutes, have a circular structure containing peptides in which amino and carboxyl groups are terminally linked. They are thermostable and pH stable, resistant to many proteolytic enzymes and therefore show promise for industrial applications. They exhibit inhibition against other LAB, bacilli, Clostridia, Listeria, enterococci, staphylococci, and E. coli. They act through disruption of cell membrane integrity (Van Belkum et al., 2011). At high concentrations, circular bacteriocins like enterocin AS-48 is reported to inhibit E. coli. Some bacteriocins like carnocyclin A and Nisin are found active when gram-negative cells are treated with EDTA (ethylenediaminetetraacetic acid). EDTA alters the cell outer membrane, thereby facilitating the activity of bacteriocin against gramnegative bacteria (Stevens et al., 1991; Martin-Visscher et al., 2011).

CULTIVATION OF LACTIC ACID BACTERIA (LAB) AND BACTERIOCIN PRODUCTION

Lactobacilli are fastidious bacteria which have complex nutritional requirements. Their growth is not supported by general-purpose culture media like nutrient agar. Earlier workers had employed tomato juice for the preparation of media for culturing Lactobacilli. It was, however, discontinued because of variation in composition. De Man, Rogosa, and Shaipe (MRS) designed lactobacilli MRS agar (De Man Rogosa Sharpe agar) for culturing Lactobacilli (DeMan et al., 1960). It is an enriched and selective medium which inhibits the growth of gram-negative bacteria.

Another group of nutritionally fastidious LAB, the lactic streptococci require at least six amino acids and three vitamins in a growth medium. As these strains are homofermentative and acid-producing in nature, they also require well-buffered media for growth. A culture medium designated as Ml6 was described which supported the growth of such Streptococci. It contained phytone, a plant protein extract, peptone, and acetate, which provided buffering capacity (Lowrie et al., 1971). Buffering capacity was further improved in a medium called Ml7 wherein phosphate was replaced by glycerophosphate (Terzaghi et al., 1975). LAPTg media for isolating Lactobacilli is composed of peptone, tiyptone, glucose, yeast extract, Tween 80 and pH adjusted to 6.5 to 6.6 (Raibaud et al., 1973).

Several differential media are available for detecting and counting of Lactobacillus species from sources like yogurt, fermented milk products, probiotic products, cheese, etc. They include acidified MRS, acidified skim milk agar, Lactobacillus selection agar, Lactobacillus casei agar (LC), homofermentative heterofermentative differential medium (HHD), Lactobacillus acidophilus agar, bifidus-blood agar, reinforced clostridial agar (RCA), trehalose MRS (T-MRS), X-Glu agar, Lactobacillus selection media

(LBS), LBS medium-plus cycloheximide, facultative heterofermentative laktobazillen agar (FH agar), facultative Heterofermentative laktobazillen agar with nalidixic acid, MRS with cycloheximide, facultative heterofermentative laktobazillen agar plus vancomycin, obligate heterofermentative (OH) lactobacilli medium (OH medium) (Coeuret et al., 2003).

Minimal media or a chemically defined minimal media (CDM) is employed as a growth medium of lactobacilli while investigating the biochemical, physiological, and genetic characteristics of the microorganism. MRS is a complex medium with undefined complex organic compounds. It cannot be used for studies involving the assessment of nutritional requirements (Kim et al., 2012). Growth on minimal media helps while studying the novel or alternative metabolic pathways (Wegkamp et al., 2010). Such types of media are developed by using a single omission technique. These media are also crucial in the study of bacteriocin production and purification (Pingitore et al., 2007).

Isolation and study of Lactobacilli and Lactococci from food and dairy products, brewery, and feces is done primarily using MRS and Ml7 media. However, these media are unsuitable for large scale production as they contain beef extract, yeast extract, and peptone which are expensive components (Hayek et al., 2013). Therefore, for the purpose of production of biomass or bacteriocin, different types of media have been described in which cheap and abundantly available food byproducts, agriculture byproducts, and agro-industrial wastes are used as alternative carbon and nitrogen sources. Carbon source options which are used in the production of bacteriocin are whey, know peel, potato starch liquor, deoiled rice bran, molasses, and nitrogen sources such as com steep liquor, soya okara and vegetable waste such as pea pod (Bali et al., 2016). Agroindustry and food industry by-products utilized for bacteriocin production include whey permeate, mussel processing waste, cheese whey, octopus peptone, cull potatoes, waste potato tubers, visceral, and fish muscle residues, condensed distillers soluble, skimmed milk, soybean meal, fermented barley extract, sweet whey, etc. (Hayek et al., 2013; Bali et al., 2016).

Bacteriocin production requires the selection of suitable carbon sources, nitrogen sources, optimization of media components, metal ion requirements, surfactant concentrations, optimization of process parameters like pH, temperature, inoculum concentration, incubation period and agitation (shaking and stationary condition). Media for LAB should contain carbohydrates, nitrogen sources, fatty acids, minerals, vitamins, surfactants or emulsifiers, and buffering agents.

Besides the above requirements, induction of bacteriocin production can be done by glycerol, amino acids, a-ketoglutaric acid, pyruvic acid, and others. At times, bacteriocin also acts as a self inducer (Yi et al., 2013). The production of bacteriocin in LAB is in situ, i.e., in the medium in which they are present such as a food matrix. However, much higher amounts can be produced during in vitro optimized fermentation conditions. Such in vitro production method helps to overcome the difficulty of extraction and quantifying bacteriocin from the food matrix (Beshkova et al., 2012). These strains also aid in the improvement of texture, so used as co-cultures or starters in food, due to their biochemical activities, especially in cheese production and ripening. Starters used in cheese production belong to genera Lactococcus lactis, Lactobacillus (Lb. delbrueckii subsp. bulgaricus and L. helveticus) and Streptococcus thermopliiles (Powell et al., 2002).

In vitro bacteriocin production is usually done using brain heart infusion (BHI), De MRS, trypticase soy broth (TSB), trypticase soy broth yeast extract (TSBYE), and tryptone glucose extract (TGE). Bacteriocin production is maximum when cells are subjected to lower pH and temperature values than the growth requirements (Guerra et al., 2001; Yang et al., 2018). Bacteriocin production by Lactococcus lactis and Pediococcus acidilacticis- trains from whey resulted in less production as compared to MRS. However, enhanced bacteriocin production was obtained when the mussel-processing waste medium was supplemented with a nitrogen (N,) source (Guerra et al., 2001, 2002). This implies that bacteriocin production is largely influenced by growth conditions and media composition. Culture conditions and media compositions for bacteriocin production by different workers are given in Table 8.1.

A resting cell system which consists of non-proliferating cells is also used for the study of bacteriocin production and factors affecting production. It is easy to screen exogenous factors influencing production and study their regulation and optimization. It is an easy method of screening bacteriocin Lac-B23. Using resting cell technology, it was seen that, bacteriocin Lac-B23 yield can be increased by adding pyruvic acid, cysteine, glycine, glycerol, and Lac-B23 itself (Yi et al., 2013).

 
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