Research Article - Journal of Food Technology and Preservation (2021) Volume 5, Issue 3

Microbial production of bacteriocins and its applications in food preservation: A review.

Tanushree Ghosh¹, Aloke Purkait², Binoy Garai³, Argha Banerjee^3*

¹Department of Microbiology, University of Kalyani, Kalyani-741235, Nadia, West Bengal, India

²Department of Soil Science and Agricultural Chemistry, Palli Siksha Bhavana (Institute of Agriculture), Visva- Bharati, Sriniketan-731236, Birbhum, West Bengal, India

³Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India

Corresponding Author:

Argha Banerjee
Department of Plant Pathology
Bidhan Chandra Krishi Viswavidyalaya
Mohanpur-741252
Nadia
West Bengal
India
E-mail: arghyabanerjee18@gmail.com

Accepted date: 19 March, 2021

Citation: Garai B, Purkait A, Ghose T ,et al.Microbial production of bacteriocins and its applications in food preservation: A review. J Food Technol Pres 2021;5(3):5-13.

Abstract

Bacteriocins, a heterogeneous group of bioactive bacterial peptides or proteins, are ribosomally synthesized that inhibit or kill other related or unrelated microorganisms. They are industrially attractive molecules because of their great potential as natural preservatives in food, feed and cosmetic industries and other fields such as feeds, organic fertilizers, environmental protection and personal care products. For decades, synthetic chemical preservatives for the preservation of food are successful to some extent, their quality is not as satisfying as fresh food. However, heavy dependence on these drugs has led to significant drawbacks which propel the continuous development of bacteriocins as an herbal bactericide. Their narrow target range, high activity, surprising stability and low toxicity position them as viable alternatives or complements to existing chemical preservatives. Since they pose no health risk concerns, soon bacteriocins are largely dependent on their use as safe food preservatives. This article gives an overview of the classification of bacteriocins, isolation and characterization, and mode of action. Besides, this article stated the potential applications of bacteriocin in the bactericidal formulations sector. Special emphasis has been provided on explaining the beneficial aspects of NISIN.

Keywords

Bacteriocin, Nisin,Bio-preservation,Formulation,Herbal biocide.

Introduction

In the production of food, it’s crucial to take proper measures for ensuring its safety and stability during self-life. Food preservation is carried out to maintain the quality of raw material and physicochemical properties as well as the functional quality of the food commodities whilst providing safe and stable products Food deterioration by foodborne pathogens during storage is one of the major concerns in society [1]. A report by the Food and Agriculture Organization (FAO) of the United Nations has stated that one-third of the foods produced for human consumption are either spoiled or wasted [2].The use of synthetic chemical preservatives has increased over the past few decades and also considerable scientific data have emerged on the intolerance of food additives with various health issues [3]. Consumers are becoming increasingly aware of the human health risk posed by the use of synthetic chemical preservatives in foods [4]. Hyperactivity and other neurophysiological issues have been reported in children as regards/due to the consumption of chemical preservatives [5]. Some of these preservatives are carcinogenic while others are known to cause various side effects, which included breathing difficulties, obesity, cancer heart damage and other health implications [6]. The use of microorganisms and their metabolites for the preservation of foods began in prehistory. Lactic Acid Bacteria (LAB) are generally recognized as safe for this purpose [7]. Moreover, natural antimicrobials could be an effective way to prevent or minimize food spoilage and/or foodborne outbreaks as an alternative to chemical preservatives [8]. Bacteriocins are a large family of ribosomally synthesized proteinaceous toxins produced by bacteria and Archaea that have antimicrobial activity against bacteria closely related to the producer strain [9]. Exceptionally few bacteriocins along with their native antibacterial property also exhibit additional anti-viral and antifungal properties [10]. These are antimicrobial peptides, which can be considered safe since they can be easily degraded by proteolytic enzymes of the mammalian gastrointestinal tract [4]. The aim of this review is to introducing the brief history of bacteriocins and emphasizing the latest progress in research, development and applications of its in food preservation. Literature review on bacteriocins showed that a significant amount of research has been done on screening and isolation of bacteriocin producing bacteria from the genera of Lactic Acid Bacteria (LAB) probably due to their GRAS feature and immediate applications in food industries.

Source and Analysis of Bacteriocins

Bacteriocins from Gram-positive bacteria especially from Lactic Acid Bacteria (LAB) have been thoroughly investigated considering their great biosafety and broad industrial applications. In the bacterial community, many Gram-positive, negative and archaea bacteria are known to produce bacteriocins [11]. Also, most bacteriocin producers belong to Lactic Acid Bacteria (LAB), a group that occurs naturally in foods and have a long history of safe use in the dairy industry. Bacterial cells that produce bacteriocins are resistant to their antimicrobial peptides, which is mediated by the specific immunity proteins produced by the host cells. Since they pose no health risk concerns, bacteriocins, either purified or excreted by bacteriocin-producing strains, are a great alternative to the use of chemical preservatives in dairy products [4]. Bacteriocins, such as nisin is accepted to safe for use a food preservative in vegetables, dairy, meats and other food products as they inhabit the contamination of microorganism during the production process [12].If bacteriocins produced by one bacterium are inhibitory to other bacteria belonging to the same species, generally they are considered as narrow-spectrum bacteriocins [13]. On contrary, if they are inhibitory against bacteria belonging to another genus, they are considered broad-spectrum bacteriocins. The history of bacteriocins extends to the early 1920s. The first report on bacteriocin production was from E. coli in 1925 and this peptide was named colicins to reflect the microbial source. While their antimicrobial activity was first discovered in 1928, bacteriocins were not used in food products until 1951. In the 1960s, the first bacteriocin called nisin, which is produced by Lactococcus lactis subsp. lactis, was purified and recognized as a food preservative by FAO/WHO in 1969 (FAO and WHO., 2015). To obtain the active protein fractions, crude bacteriocin substance was purified by Fast Protein Liquid Chromatography (FPLC) using a strong anion exchange column [14]. Tricinesodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purified bacteriocin. The amino acid analysis was performed by High-Performance Liquid Chromatography (HPLC). Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and mass spectroscopy techniques were performed to estimate the approximate molecular mass of the compound [15].

Need for bacteriocin

Over the past few decades the usage of synthetic chemical preservatives has increased and also significant scientific data have emerged on the intolerance of food additives with various health issues [3]. Hyperactivity and other neurophysiological matters have been stated in children as regards/due to the intake of chemical preservatives [5]. Many of these preservatives are carcinogenic while others are identified to cause several side effects, which involved breathing difficulties, heart damage and other health effects (Table 1).

Table 1: Name of chemical preservatives and their side effect on human health.

The adverse effect of frequently used preservatives can be overcome by the application of bio preservatives. This approach involves the usage of natural microbiota or their antimicrobial products for extending the shelf-life and enhancing the food commodities' safety [16].The term bacteriocin was introduced to denote a toxic protein or peptide produced by any type of bacteria that is active on related bacteria but does not harm the producing cell. This expression was inspired by analogy with colicin, the first bacteriocin described that was produced by Escherichia coli [17]. Bacteriocins, produced by microbes are natural antimicrobial peptides, prevent other microbes to compete with them for nutrients.

Classification of Bacteriocins

Bacteriocins are classified into different groups (Table 2). Class I bacteriocins (lantibiotics) are small peptides that undergo extensive post-translational modification to produce the active peptide. Nisin, the most studied bacteriocin, belongs to class I bacteriocins, which are active against a broad spectrum of food spoilage and pathogenic bacteria, including Listeria monocytogenes. Class II bacteriocins are heat-stable, low molecular weight, membrane-active peptides. Members of class III are large heat-labile proteins, and a fourth class (complex bacteriocins) has also been suggested, requiring nonprotein moieties for activity [18].

Table 2: Classification of bacteriocins.

Bacteriocins are produced both by Gram-positive (lantibiotics) and gram-negative microorganisms (colicins, microcins) and few archaea. It is a group of gram-positive, non-motile, nonspore- forming have low proportions of G+C in their DNA. Lactic acid bacteria produced lactic acid in either homofermentative or hetero-fermentative pathway. Some genera that belong in this group are Lactobacillus, Lactococcus, Enterococcus, Carnobacterium, Streptococcus etc. LAB isolated from many food and animal sources showing inhibitory activity against Staphylococcus aureus and Salmonella typhimurium Based on amino acid compositions, types of post-translational modifications and sizes many classifications are available on bacteriocins. Bacteriocins can differ significantly both in size and in chemical properties, ranging from small peptides (the Gram-positive bacteriocins and microcins are peptides below 10 kDa) to large proteins (colicins are 30-80 kDa proteins). The great heterogeneity of bacteriocins and broad scope for biomedical and food applications collectively lead to the continuous evolution of new classification approaches for the Gm+ bacterial bacteriocins.

Classification of gram positive bacteriocins

Based on the biochemical and genetic characteristics, Grampositive bacteriocins were classified into 3 classes. (Grampositive bacteriocins have been classified into four groups: class I, post-translationally modified bacteriocins that contain modified amino acids, such as lanthionine, methyl lanthionine, and dehydrated amino acids, and that are collectively known as lantibiotics; class II, small (<10000) heat-stable, non-modified bacteriocins; class III, large (>10000) heat-labile bacteriocins; and class IV, complex bacteriocins, carrying lipid or carbohydrate moieties. Class I, II, and III Gram-positive bacteriocins are further subdivided according to other characteristics. The most studied are class I and class II, many of them being produced by Lactic Acid Bacteria (LAB), also known as LAB bacteriocins [17]. Classes I and II have 2 and 3 subclasses, respectively. Due to a lack of systematic organization. A modern classification based on the structural similarity, phylogenetic evolution and consensus motif sequence. Based on the modern classification, bacteriocins are classified into 12 groups. Groups 1, 3 and 4 are further subdivided into subgroups a and b.

Classification of gram- bacteriocins

Bacteriocins produced by Gram-negative bacteria are grouped into 2 categories, colicins and microcins. Genes encoding these bacteriocins can be present either on the plasmid or chromosome as a subset of three genes, bacteriocin encoding gene, immunity protein genes and lysis genes involved in the release of bacteriocins from cells. Colicins are classified into 2 classes if based on translocation and into 3 classes if based on their mode of action. Microcins are grouped into classes I and II. Class II is further subdivided into 2 subclasses IIa and IIb.

Classification of archaea bacteriocins

Many archaeal members have been reported to produce bacteriocins. To the best of our knowledge, to date two types of archaeocins, halocins and sulfolobicins, have been identified and described in the literature. Generally, halocins are produced by the members of halo bacteria further classified. Smaller microhalocins have a smaller size as low as 3.4 kDa and the larger ones have 35 kDa. They are secreted during late exponential to early stationary phase. Sulfolobicins is produced by Sulfolobus islandicus member of the crenarchaeal phylum. This bacterium grows at pH 2-4 and 65-85 °C. Sulfolobicins are narrow-spectrum bacteriocins that inhibit the growth of closely Sulfolobus and its allies. These bacteriocins are intracellular and membrane-associated.

NISIN

Nisin, the most studied bacteriocin was developed in the early 1960s and is currently use as a safe additive in certain food products for food preservation, certain bacteriocin-producing lactic acid bacteria, which are generally recognized as safe microorganisms, or their extracellular extracts are receiving increased attention as protective cultures or antimicrobial extracts in minimally processed food products. It belongs to class I bacteriocins and is composed of 34 amino acids long cyclic polypeptide chain with a molecular mass of 3500 Da. It is active against a broad spectrum of food spoilage and pathogenic bacteria, including Listeria monocytogenes. It exists in two variants, variant A contains histidine at position 27 and variant Z has asparagine[19]. Nisin got approval from the US FDA in 1988 for its application in cheese spread. It is used in >50 countries as a food preservative. The acceptance of nisin as a natural preservative in US has increased the market of this compound. EU market has a reservation on its designation as a natural preservative. Generally, industrial- scale production of nisin is conducted using L.lactis strains. Fermentation studies on the nisin production indicate that it follows primary metabolite kinetics producing the bacteriocin during the growth phase and declines completely after entering the stationary phase [20]. Till now, nisin is the only bacteriocin that has been approved for applications in the food industry.

Crystal structure of NISIN: Crystal structure of these predator proteins would provide practical evidence in understanding the mode of action of these proteins over the sensitive cells (Figure 1). Generally, Edman degradation, NMR spectroscopy and tandem MS are the tools applied to elucidate the structures. Chemical modification of modified residues makes the treated bacteriocins more user-friendly for structural elucidation. Nisin inhibits the bacterial cells by its unique capability of creating pores on the bacterial cell wall. It inhibits the cell wall synthesis by binding to the lipid II, the precursor molecule for the cell wall synthesis. Inhibition of cell wall increases the membrane permeabilization. The crystal structure of nisin-lipid II complex has been resolved using highresolution NMR spectroscopy.

Figure 1: Crystaline structure of Nisin (describe about the A and B).

The primary structure of nisin is simple, which contains five lanthionine rings A, B, C, D and E. The interface between nisin-lipid II complex has 36 intermolecular NOEs (Nuclear Overhauser Effect) between first 1-10 amino acid residues of nisin, MurNAc (N-Acetyl muramic acid) and isoprene unit of lipid II molecule. The presence of additional two more intermolecular hydrogen bonds was experimentally confirmed. The NH2 terminal part of nisin binds to the lipid II complex reducing the intrinsic flexibility with a distorted tail. The pyrophosphate moiety of lipid II molecule is coordinated by two NH2 terminal lanthionine rings A and B of nisin molecule forming a cage-like structure. This cage-like structure allows the formation of 5 intermolecular hydrogen bonds between backbone amides of nisin and the pyrophosphate group of lipid II thereby inhibiting the formation of cell wall. Additionally, the structure of bacteriocins will provide information on the post-translational modifications and their metabolic synthetic pathways.

Bacteriocins vs. Antibiotics

Bacteriocins are often confused in the literature with antibiotics. This would limit their use in food applications from a legal standpoint. In some countries, it is critical to make the distinction between bacteriocins and antibiotics.

The main differences between bacteriocins and antibiotics are summarized in Table 3. Bacteriocins, which are distinguishable from clinical antibiotics, should be safely and effectively used to control the growth of target pathogens in foods. This review will differentiate bacteriocins from antibiotics based on synthesis, mode of action, antimicrobial spectrum, and toxicity and resistance mechanisms. Recognizing that bacteriocins are different from antibiotics, Abiological food preservatives since bacteriocins, unlike antibiotics, are not used for medicinal purposes.

Table 3: Bacteriocins vs. Antibiotics.

Bacteriocins with Antiviral and Antifungal Properties

Antiviral bacteriocins

The purified protein has a molecular mass of 4 kDa. The protein showed antimicrobial activity over E. faecalis SA-74 but also an antiviral activity. The 50% of egg lethal dose (ELD50) of a new castle disease virus in the presence of staphylococcal reduced from 10-9 to 10-4. The bacteriocin did not show any antiviral activity over single-stranded RNA poliovirus E. mundtii ST4V was isolated from soya beans. The isolated strain secretes a 3950 kDa broad-spectrum bacteriocin that is active against Gm+, Gm- bacteria and virus. Bacteriocin ST4V was able to retain its antimicrobial and antiviral activity even after treatment with proteinase K, pronase, pepsin, trypsin and α-amylase. A potential explanation for the mode of action of this antiviral protein is the aggregation of viral particles in the host cells, blockage of viral receptors preventing the entry of the virus into host cells, or inhibition of key steps involved in the viral replication. E. faecium CRL35 expresses 3.5 kDa CRL35, which has been shown to have antiviral properties against thymidine kinase positive (tk+) and negative (tk-) HSV-1 and 2. Enterocin was able to inhibit the viral replication both in vero and BHK-21 cell lines at 100 μg/mL.

Antifungal bacteriocins

Burkholderia sp. T-34 ( Accession No. NITE BP - 477), Burkholderia sp. RO-1 (receipt number NITE ABP-698), B. purantari JCM5492 and B. phenazinium JCM10564 can produce antifungal bacteriocins in potato dextrose medium. Ultrafiltration of the culture supernatants showed that the molecular mass of the antifungal protein was 10 kDa. The protein shows its anti-fungal properties at pH 7 on different species of Aspergillus, Rhizopus, Mucor and Pencillium. The bacteriocin inhibits the fungi by stopping the germination of spores and elongation of hypa.

Methods for Isolating Bacteriocin Producers

Agar diffusion bioassay

Screening of bacteriocin-producing LAB from different milk products were conducted by this method. Listeria innocua (ATCC 33090) and L. sakei (ATCC 15521) were used as indicator bacteria. L. innocua was grown in Brain Heart Infusion broth and Latilactobacillus sakei has grown anaerobically MRS broth at 37 °C. One mL of indicator organism (5 × 105 CFU/mL) was mixed into MRS soft agar and allowed to solidify. After solidification of agar three wells were cut and each well was filled with 35 μL of cell-free supernatant (CFS) of LAB, CFS neutralized with NaOH and neutralized CFS treated with catalase. After 24 h of incubation, the zone of inhibition on the agar plates was measured using an electronic caliper.

Double patch plate method for confirmation of Bac+ colonies

Colonies that were preliminarily screened for bacteriocin production were confirmed for bacteriocin production using the double patch plate confirmation assay. Colonies screened for bacteriocin production were carefully collected and inoculated in two different MRS plates. After 4 h of growth one plate was overlaid with soft agar containing the indicator organism and incubated till indicator lawn grown to completion. Bacteriocin-Producing Colonies were confirmed by the formation of a “zone of inhibition” on the top softest agar layer present on the plate. The confirmed bacteriocin colonies were collected from the duplicate plate into MRS broth for growth to make a glycerol stock for preservation and future studies.

Isolation of novel bacteriocin producers

Leuconostoc gelidium stain UAL187 was isolated from the packed meat. It secretes bacteriocins leucocin A, which is active against LAB, Listeria and Enterococcus faecalis. Leucocin A coding sequence is located on the plasmid pLG7.6. Leucocin was purified from the culture supernatant with a high purity yielding 2.06 mg of protein and this bacteriocin was made up of 37 amino acids. Brochothrix campestris ATCC 43754 was isolated from soil. It secretes bacteriocin Brochocin-C. Brochocin-coding sequence was located from the chromosomal DNA. The bacteriocin was active against Clostridium botulinum and a wide variety of Gm+bacteria. Brochocin-C was stable at 121 °C for 15 min and inactivated by enzyme treatment. E. faecium 900 produces enterocin 900. The protein expression gene is located on its chromosome. Enterocin is 71 amino acids long peptide active against a wide range of Gm+ and Gm- bacteria.

Effectiveness of bacteriocins in food systems

Though results obtained from broth systems show bacteriocins inhibit target organisms, applied studies must be done to confirm their effectiveness in food. The chemical composition and the physical conditions of food can have a significant influence on the activity of the bacteriocin. Nisin, for example, is 228 times more soluble at pH 2 than at pH 8. Since lactic acid bacteria are commonly used as starter cultures in food fermentations, investigators have explored the use of bacteriocin producers as starter cultures.In some cases, natural bacteriocin producers, such as Lactobacillus plantarum, Pediococcus acidilactici and Enterococcus faecalis are and have been used in such studies. found that counts of L. monocytogenes Ohio in Manchego cheese inoculated with a bacteriocin-producing Ent. faecalis strain decreased by 6 logs in 7 days, whereas the survival of the organism in cheese made with the commercial starter culture was not affected. Similarly, the surviving number of L. monocytogenes found in a naturally contaminated salami sausage decreased when the product was inoculated with the bacteriocin producer Lactiplantibacillus plantarum MCS1.

Bacteriocins have been directly added to foods such as cheese to prevent Clostridium and Listeria. Nisin inhibits the outgrowth of C. botulinum spores in cheese spreads and it is approved as a food additive in the United States for this purpose ZUS. Food and Drug Administration 1988. Nisin has many applications in foods Tables 4 and 5 and is approved for use in various foods throughout the world.

Table 4: Application of Nisin on different food commodities.

Table 5: Bacteriocins as food preservatives: examples of suggested application.

Application of bacteriocins

The applications of antimicrobial proteins extracted from various bacteria have been studied for their biopreservation potential towards various food samples [21-23]. Though, bacteriocins have applications in many food systems, foods should not be preserved by bacteriocins alone but rather as part of a system with multiple hurdles. Since LAB are commonly found in meat, bacteriocins produced by these bacteria have been explored and isolated. Though most bacteriocins have been isolated from food-associated LAB, they are not necessarily effective in all food systems. However, several bacteriocins certainly do have potential in food applications when used under the proper conditions. One of the best studied examples is the use of nisin in meat systems. Nitrates are commonly used to prevent clostridial growth in meat; however, safety concerns regarding the presence of nitrites have prompted the food industry to look for alternative methods of preservation. Nisin or its combination with lower levels of nitrate can prevent the growth of Clostridium Leucocin A, enterocins, sakacins and the carno bactericins A and B prolong the shelf life of fresh meat. The most promising results in meats were obtained using pediocin PA-1 produced by Pediococcus acidilactici, pediocin PA-1 immediately reduces the number of target organisms. Pediocin PA-1 is active against the foodborne pathogen L.monocytogenes and Lactob curvatus, a spoilage organism. In the Lacob. curvatus study, however, pediocin PA-1 is less active than nisin in the model meat system, and neither preservative is effective when used in a commercially manufactured meat product. Pediocin AcH (PA-1). Successfully controlled the growth of L. monocytogenes in raw chicken in another study. Recently, Seo and Kang, (2020) developed bacteriocins purified from Pediococcus acid ilactici could be effective anti-biofilm agents to control Salmonella typhimurium contamination in food matrices and food processing facilities. Bacteriocin formulation produced from L.plantarum subsp plantarum [24] isolated from the bacon that can inhibit the growth of L. monocytogenes. However, a synergistic effect was observed for a composition containing both bacteriocin from Zhang-LL strain and nisin and inhibited L. monocytogenes by 1000-fold at 4°C for 13 days than the controls having individual bacteriocins. The formulation can be further assessed to preserve milks and milk products, fruits, vegetables and instant foods [24,25]. Barman et al. formulated food-grade bacteriocin from Lactococcus lactis isolated from the homemade buttermilk. The bacteriocin of the isolates had an antibacterial spectrum both against Gram-positive as well as Gram-negative pathogenic bacterial strains. The inhibition of Gram-negative bacteria by bacteriocin of LAB is an unusual phenomenon. It exhibits bacteriocidal action on Staphylococcus aureus MTCC96 and Burkholderia sp. MTCC741. Baljinder et al. reported L.fermentum HV6b MTCC produces class IIa bacteriocin peptide fermenticin HV6b. It can inhibit the growth of Bacteroides, Gardnerella vaginalis, Mabiluncus, Staphylococci and Streptococci which causes vaginal infections in humans. Fermenticin HV6b has a unique sperm immobilization and spermicidal activity. A new formulation containing L.fermentum HV6b or fermenticin HV6b alone or in combination can be used in the production of vaginal creams that can protect the human vagina from microbial infections and also work as contraception. Kaya and Simsek,produced bacteriocin that showed specific-antimicrobial activity to foodborne pathogens were isolated and the success of the cocktail of Pathogen-Specific Bacteriocins (PSBs) in milk were demonstrated.PSB producers were isolated from 250 different foods by a new approach that screening their antimicrobial activity against a mixture of five different strains of each Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus. Lactobacillus plantarum PFC339, Enterococcus faecalis PFC340, Lactobacillus delbrueckii subsp.lactis PFC341 were identified that inhibited the growth of pathogen strains respectively, but not lactic acid bacterial strains. Patented inventories about the potential applications of biodiesel in the pesticide formulations sector are listed in Table 6.