Obtaining Bacteriocins by Chromatographic Methods

Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr+ or Gr- bacteria [1]. Their low toxicity as well as the absence of allergenicity and reactogenicity is confirmed by testing selected bacteriocins [2] [3]. Bacteriocins can be widely used as preservatives and antibiotic alternatives in medicine. Nisin, a Streptococcus lactis-derived bacteriocin, has been in practice in food industry for a long time. A relevant product contains about 2.5% of nisin. For medical use (e.g., when injected into the blood stream), highly purified drugs are required. However, the yield of bacteriocins accounts for no more than a few percents from the total activity in the culture liquid. In this paper, we propose methods (by example of two B. subtilis strains), allowing to increase the yield up to ~80%. It is believed that other bacteriocins may be purified by these methods and with the same yield.

Potential applications of lactic acid bacteria and bacteriocins in antimycobacterial therapy

Tuberculosis(TB) is a communicable disease caused by Mycobacterium tuberculosis(M. tuberculosis). WHO estimated that 10.4 million new(incident) TB cases worldwide in year 2016. The increased prevalence of drug resistant strains and side effects associated with the current anti-tubercular drugs make the treatment options more complicated. Hence, there are necessities to identify new drug candidates to fight against various sub-populations of M. tuberculosis with less or no toxicity/side effects and shorter treatment duration. Bacteriocins produced by lactic acid bacteria(LAB) attract attention of researchers because of its "Generally recognized as safe" status. LAB and its bacteriocins possess an effective antimicrobial activity against various bacteria and fungi. Interestingly bacteriocins such as nisin and lacticin 3147 have shown antimycobacterial activity in vitro. As probiotics, LAB plays a vital role in promoting various health benefits including ability to modulate immune response against various infectious diseases. LAB and its metabolic products activate immune system and thereby limiting the M. tuberculosis pathogenesis. The protein and peptide engineering techniques paved the ways to obtain hybrid bacteriocin derivatives from the known peptide sequence of existing bacteriocin. In this review, we focus on the antimycobacterial property and immunomodulatory role of LAB and its metabolic products. Techniques for large scale synthesis of potential bacteriocin with multifunctional activity and enhanced stability are also discussed.

Partial Characterization of Bacteriocins from Two <i>Pediococcus acidilactici</i>Strains Isolated during Traditional Sorghum Beer Processing in Côte d’Ivoire

Two lactic acid bacteria strains (At1BEAE22 and At344E21) isolated during tchapalo production were identified on the basis of phenotypic analyses. Bacteriocins produced by these strains were tested for their antimicrobial activities using well diffusion agar method. Heat resistance, pH sensitivity and enzyme treatments were also analyzed. Results showed that both lactic acid bacteria strains were identified as Pediococcus acidilactici. Their bacteriocins inhibited growth of Lactobacillus delbrueckii F/31, Listeria innocua ATCC 33090, Enterococcus faecalis, Enterococcus faecalis ATCC 29212, Streptococcus sp, Enterococcus faecalis CIP 105042 and Enterococcus faecium ATCC 51558. These bacteriocins were heat stable at 60&degC for 30 min for all indicator bacteria. However, they remained active only against Lactobacillus delbrueckii and Listeria innocua at 121&degC for 60 min. Moreover, they were active in a wide range of pH (3 to 9) with a maximum activity observed at pH 5 and 6 on all indicator bacteria. But, bacteriocin from Pediococcus acidilactici At34E21 was more stable at acidic pH than basic one. The fact that the bacteriocin was inactivated by proteinase K and α-chymotrypsin indicated its proteinaceous nature, a general characteristics of bacteriocins.

Bacteriocins as antimicrobial and preservative agents in food:Biosynthesis,separation and application

Emerging evidence shows the trend of using safe and natural preservatives like bacteriocins in food processing.The increasing demand by the food industry to extend keeping quality and prevent spoilage of various food products has appealed for new preservatives and new methods of conservation.Further,to discover the novel spectrum of antimicrobial compounds that could effectively battle food-borne pathogens,bacteriocins have been intensively studied in the last few years.Recently,great attention has been paid to the application of lactic acid-bacterial bacteriocins targeting food spoilage-causing or pathogenic microorganisms with no significant side effects.Different mechanisms of action like pore-formation,retarding cell-wall/nucleic acid/protein synthesis have been proposed and described for diverse bacteriocins.Therefore,the syntheses,purification of bacteriocins in addition to their applications as antimicrobial and preservative agents in food processing are reviewed in detail.The review will also discuss the preservative effect of bacteriocins and their combinations having multiple modes of action(hurdle approach)to reduce the microbial load as well as antimicrobial resistance.

Retrospecting the concept and industrial significance of LAB bacteriocins

Ever increasing demand for natural products with minimum chemical additives has necessitated the search for innovative green,potent,environment friendly,natural substitutes for prolonging the shelf life of food products.The lactic acid bacteria(LAB)are a group of Gram-positive microorganisms with great biotechnological potential in the food industry.To reduce pathogen invasion,they can produce inhibitory compounds(bacteriocins,organic acids,hydrogen peroxide,diacetyl,and carbon dioxide).LAB-bacteriocin are proteinaceous antimicrobial molecules with a diverse genetic origin,post-translationally modified or not,that can help the producer organism to outcompete other bacterial species.In addition,they are emerging as a novel wave of potential antibiotics with potent in vitro and in vivo activities.Moreover,LAB-bacteriocin serves as an effective method of natural bio preservation in the food industry and enables the reduction of other costly or user-unfriendly treatments while increasing the product self-life.Therefore,this article provides a recapitulation of classification of bacteriocins,potential of LAB to produce different bacteriocins,their biology,and purification methods.Additionally,it will give a critical snapshot of industrial applications of LAB-bacteriocin as a starter culture,preservative,antibiotic,and probiotics,etc.

细菌素Bacteriocin E50-52(H)基因设计及其毕赤酵母表达载体构建

设计及合成细菌素Bacteriocin E50-52(H)基因,克隆到组成型分泌表达质粒pGAPZαA中构建重组质粒pGAPZα-Bacteriocin E(H),经PCR、测序验证正确后,电转化整合到毕赤酵母基因组,基因组PCR、测序验证结果表明成功构建了细菌素组成型重组表达载体,为在毕赤酵母中表达奠定了基础。

Role of microbial dysbiosis in the pathogenesis of esophageal mucosal disease:A paradigm shift from acid to bacteria?

Genomic sequencing,bioinformatics,and initial speciation(e.g.,relative abundance)of the commensal microbiome have revolutionized the way we think about the“human”body in health and disease.The interactions between the gut bacteria and the immune system of the host play a key role in the pathogenesis of gastrointestinal diseases,including those impacting the esophagus.Although relatively stable,there are a number of factors that may disrupt the delicate balance between the luminal esophageal microbiome(EM)and the host.These changes are thought to be a product of age,diet,antibiotic and other medication use,oral hygiene,smoking,and/or expression of antibiotic products(bacteriocins)by other flora.These effects may lead to persistent dysbiosis which in turn increases the risk of local inflammation,systemic inflammation,and ultimately disease progression.Research has suggested that the etiology of gastroesophageal reflux disease-related esophagitis includes a cytokine-mediated inflammatory component and is,therefore,not merely the result of esophageal mucosal exposure to corrosives(i.e.,acid).Emerging evidence also suggests that the EM plays a major role in the pathogenesis of disease by inciting an immunogenic response which ultimately propagates the inflammatory cascade.Here,we discuss the potential role for manipulating the EM as a therapeutic option for treating the root cause of various esophageal disease rather than just providing symptomatic relief(i.e.,acid suppression).

Study Bacteriocin Production and Optimization Using New Isolates of <i>Lactobacillus</i>spp. Isolated from Some Dairy Products under Different Culture Conditions

Lactobacilli belong to the group of lactic acid bacteria (LAB), that have several distinguished abilities such as production of lactic acid, enzymes such as β-Galactosidase and natural antimicrobial substances called bacteriocins. Bacteriocin is a biopreservative agent potential of suppressing growth of some contaminant bacteria in food industry but its commercial availability is limited and costly. The study aimed to select isolates of Lactobacillus spp. potential for producing bacteriocins to suppress the growth of Escherichia coli ATCC 25922 and Bacillus subtilis NCIB3610, and to optimize the process of bacteriocin production. Results obtained in this study showed that L. acidophilus isolate CH1 was selected as the best candidate for bacteriocin among the four isolates that tested. The largest amounts of the bacteriocins were synthesized only in MRS medium was supplemented with K2HPO4 (1.0%), Tween 80 (1%), Beef extract (1%), glucose, cyctein and peptone extract (1%). The optimization of culture conditions for bacteriocin production areas showed that corn steep liquor medium was the best medium for all isolates against Bacillus subtilis while no effect was observed on Escherichia coli ATCC 25922 except when used MRS medium. The optimum conditions for bacteriocin production were pH 6.0, temperature 34?C with 4% Phenyl acetamide showing the greatest growth inhibition areas.

Safety Properties and Probiotic Potential of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895

This study reports on the safety and putative probiotic properties of Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933. According to the bacterial reverse mutation (Ames) test, cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 were not mutagenic. The two strains co-aggregated with Escherichia coli and Pseudomonas aeruginosa, and cell-free supernatants inhibited the growth of Streptococcus intermedius and Porphyromonas gingivalis. Endospores of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 were tolerant to 0.3% (w/v) bile salts and survived incubation for 4 h in MRS broth at pH 2.0 to 3.0. The ability of the two strains to produce antimicrobial compounds potentiates their application in health care formulations, personal care products, food and animal feed.

Isolation and Characterization of Enterococci Bacteriocinic Strains from Tunisian Fish Viscera

A collection of lactic acid bacteria isolates from fish viscera was studied and investigated regarding to their functional properties and safety aspects. From these, three isolates GM1, GM2 and GM3 were identified as Enterococcus feacium species using molecular methods. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with restriction enzyme HaeIII separated these isolates into distinctive group which suggest genotypic variability within enterococci strains isolated from fish viscera. The three strains GM1, GM2 and GM3 exhibited antimicrobial activity. Indeed strains have been shown to produce bacteriocins with inhibitory effect against food spoilage bacteria and pathogenic fish including Carnobacterium maltaromaticum. The molecular mass of bacteriocin, as calculated by tricine-SDS-PAGE, was found to be 4.5 kDa. All isolates were tested positive upon PCR amplification of enterocin A structural gene. Investigations of antibiotic resistance show that the isolates were mostly sensitive to several antibiotics (ampicillin, penicillin, tetracycline, gentamycine) and resistance to rifampicin. All isolates grow in esculin azide agar as a selective medium for enumeration of probiotic enterococci. This study suggests that our strains can be employed as probiotic or to improve the safety of food products.

One-Step Procedure for Direct Purification of Pediocin-Like Bacteriocins and Cationic Antimicrobial Peptides from Complex Culture Medium on an Analytical, Semipreparative, and Preparative Scale. A New Type of Liquid Chromatography—Colloidal High-Performance Liquid Chromatography (CHPLC)

A simple time- and labor-saving one-step procedure suitable for analytical-, semipreparative-, and preparative-scale preparation of bacteriocins and other cationic antibacterial peptides (for example as lysozyme) has been developed. Suspensions of bacterial cells, other colloidal- and fast-particle materials, and dissolved anionic molecules passed through the column;however, cationic bacteriocins were separated by chromatography. The purity of the target substance at more than 97% was achieved by the judgment of spectral, chromatographic, and antimicrobial activity. A many-fold concentration increase was achieved for bacteriocins, lysozyme, and bacitracin (a commercial antibiotic) in comparison with the starting solutions (see the text below). In the experiments, homemade cation-exchange columns have been used. The columns have been prepared by the grafting of glass fibers with a diameter of 0.2 μm. In this paper, applications of a new type of liquid chromatography—colloidal high-performance liquid chromatography (CHPLC)—were demonstrated.

Physical chemical and biological characterization of a new bacteriocin produced by Bacillus cereus NS02

Objective:To screen the bacteriocinogenic isolate from buffalo milk and to characterize it on physical,chemical and biological aspects for the application in biopreservation.Methods:Bacillus cereus(B.cereus)was isolated and assessed for its baceteriocinogenic activity.Bacteriocin was produced and purified by ammonium sulphate precipitation,dialysis and gel filtration chromatography.Purified bacteriocin was used to clieck its antimicrobial activity against food borne bacteria.Effect and stability of bacteripcin was determined with the respect to temperature,pH,enzymes,organic solvents and chemicals.Bacteriocin was also subjected to SDS PAGE analysis to determine its molecular weight.In addition,functional groups exist in the bacteriocin was determined by FTIR analysis.Results:B.cereus was identified by 16S rRNA sequence analysis.Bacteriocin showed increased activity against all the bacteria used and its activity unit was found to be 51,200 AU/mL.It was stable to high temperature(100℃)and wide range of pH(3-10),sensitive to proteolytic enzymes and resistant to nonprotcolytic enzymes.It was low molecular weight(3.5-6 kDa)protein and FTIR study revealed the presence of amide group and NH stretching,Conclusions:Bacteriocin produced in this study possesses the highest antimicrobial activity against both gram positive and gram negative bacteria thereby it has immense application as biopreservative agent.FTIR proved its peptide nature.

Antibacterial Activity of Lactic Acid Bacteria and Extraction of Bacteriocin Protein

Biopreservation systems in foods are of increasing interest for industry and consumers. Bacteriocin producing Lactobacillus spp. is considered Generally Recognize as Safe (GRAS), useful to control the fast development of pathogens and spoiling microbes in food and feed. Lactobacillus spp. was isolated from traditional winter fermented vegetable cucumber & carrot by the use of selective media. Especially De Man, Rogosa and Sharpe (MRS) Agar media were used to isolate the Lactobacillus species. Morphologically identified by gram staining & colony morphology. Biochemically recognized by catalase, oxidase, MRVP & carbohydrate fermentation test. Antimicrobial activity of Lactobacillus spp. was confirmed by Well Diffusion Method. Molecular characterization of bacteriocin protein and molecular weight determined by SDS PAGE method. The isolate was found to be facultative anaerobic, Gram positive, and catalase negative. The result of antimicrobial activity measured by the Arbitrary Unit (AU/ml) of zone of the inhibition. Six isolates found from the sample but most activities exhibited isolate 4 against Bacillus megaterium (55 mm) zone of diameter. The molecular weight of the washed bacteriocin was calculated to be about 40 kDa (Isolate 1) and 15 kDa & 30 kDa (Isolate 5). Bacteriocin protein reduces chemical preservatives and uses in future as biopreservative in food industry.

Prospective use of bacteriocinogenic Pediococcus pentosaceus as direct-fed microbial having methane reducing potential

Direct-fed microbials（DFM）, generally regarded as safe status, are successfully used in improving rumen ecology, gastro-intestinal health, feed efficiency, milk production and growth rate in ruminants. On the other hand, methanogenesis in rumen, which accounts for a significant loss of ruminant energy and increased greenhouse gas in environment, is of great concern, therefore, use of DFM for improving productivity without compromising the animal health and ecological sustainability is encouraged. The present study was conducted to investigate the methane reducing potential of bacteriocinogenic strain Pediococcus pentosaceus-34. Since, the culture showed no hemolysis on blood agar and DNase activity, hence, it was considered to be avirulent in nature, a prerequisite for any DFM. The culture also showed tolerance to pH 5.0 for 24 h with 0.5% organic acid mixture, whereas when given a shock for 2 h at different p H and organic acids concentrations, it showed growth at pH 3.0 and 4.0 with 0.1 and 1.0% organic acids, respectively, as having good animal probiotics attributes. The total gas production was significantly（P〈0.05） higher in live pedicoccal culture（LPC） and dead pedicoccal culture（DPC） both with wheat straw, when compared to the control. In sugarcane bagasse, gas production was significantly lower（P〈0.05） with LPC compared to the control and DPC both. Methane was reduced by the inclusion of LPC in sugarcane bagasse（0.07 mL CH4 mg–1 dry matter digestibility） with no effect on other rumen fermentation parameters. However, with wheat straw and LPC total gas, in vitro dry matter digestibility, total volatile fatty acids increased significantly but no reduction in methane production was observed in comparison to the control. Therefore, further research is warranted in this direction, if the bacteriocinogenic strains can be used as DFM for ruminants to improve the ruminant productivity.

Effect of bacteriocin-producing Pediococcus acidilactici strains on the immune system and intestinal flora of normal mice

This study was performed to determine the effects of bacteriocin-producing and non-bacteriocin-producing Pediococcus acidilactici strains on the immune system and intestinal flora of normal mice.Two P.acidilactici strains with antibacterial activity(P.acidilactici CCFM28 and CCFM18)were obtained based on the inhibition-zone assay.The produced components were identified as bacteriocins through protease treatment,pH adjustment and hydrogen peroxide treatment.Bacteriocin-producing and non-bacteriocin-producing P.acidilactici strains(P.acidilactici CCFM28,CCFM18 and NT17-3)caused significant changes in serum immune factors and intestinal flora of normal mice.After 14 days of intervention,the relative abundance of Firmicutes was significantly decreased,but that of Proteobacteria was significantly increased at the phylum level.At the genus level,the administration of three P.acidilactici strains resulted in the downregulation of Blautia and the upregulation of Ruminococcus and Lactobacillus.Furthermore,there were also different regulations on some probiotic strains,such as Bifidobacterium,Coprococcus and Akkermansia,which were closely related to the antibacterial ability of the bacteriocin and the type of strain.The results indicated that the intervention of different P.acidilactici strains could differently change the structure of intestinal flora in normal mice,which provided theoretical guidance for the selective use of bacteriocin-producing strains for health regulation in the future.

Use of Bacteriocin Producing <i>Lactococcus lactis</i>subsp. <i>lactis</i>LABW4 to Prevent <i>Listeria monocytogenes</i>Induced Spoilage of Meat

A bacteriocin producing strain of Lactococcus lactis subsp. lactis LABW4 was isolated from naturally fermented milk product which exhibited strong antibacterial activity against Listeria monocytogenes MTCC657, a food spoilage psychrophilic organism. Both cell free and heat killed supernatants of LABW4 were effective to produce zones of inhibition against L. monocytogenes in vitro. The antibacterial metabolite(s) of LABW4 showed strong cidal effect on the growth of L. monocytogenes. Meat samples, mixed with heat killed supernatant of LABW4 when inoculated with Listeria, remain fresh up to 25 days in refrigerated condition whereas spoilage started immediately after 24 hours of inoculation for control sets. Enhancement of Lactate dehydrogenase of L. monocytogenes upon treatment with LABW4 cell free supernatant suggested its lytic mode of action. Cell lysis or degradations were also supported by scanning electron micrograph of treated cells.

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.

Antibiotic and Bacteriocin Sensitivity of <i>Listeria monocytogenes</i>Strains Isolated from Different Foods

This study aimed to determine the antibiotic and bacteriocin sensitivity of Listeria monocytogenes strains isolated from animal derived foods. With disc diffusion assay, all fourteen L. monocytogenes strains were suscepti-ble to the antibiotics, including penicillin G, vancomycin, tetracycline, chloramphenicol, rifampicin, erythromycin, gentamicin and trime- thoprim. However, the percentages of fosfomycin and streptomycin resistances were 92.9% and 7.1%, respectively. Multiple resistances were not observed among the tested strains. The results of well diffusion assays showed that all strains were inhibited by the cell-free supernatant of a bacteriocin-producing strain, Pediococcus acidilactici 13, with the inhibition zones ranging from 16.00 to 24.50 mm. These results provide useful information on antibiotic resistance of L. monocytogenes strains isolated from foods, and can potentially be used to develop bacteriocin-based interventions to guard against the hazards associated with L. monocytogenes in ready-to-eat meat and poultry products.

<i>Lactobacillus</i>sp.—A Threat to Pathogenic Microorganisms and Tumor Cells

Beneficial bacteria, often used as probiotics, play efficient role in providing protection against pathogenic microorganisms in humans. Probiotic bacteria like many Lactobacillus sp. (L. acidophilus, L. casei etc.), Bafidobacterium sp. Streptococcus thermophiles, Bacillus coagulans, etc. are beneficial and nowadays are used as supportive therapeutics. Even Lactobacillus is highlighted for showing the anticancer effects on some human cancer cells (cervical, gastric, colon, breast cancer). Some specific antibiotics like peptide antibiotic, Acidolin;some specific bacteriocins and some metabolites like H2O2, acetic acid, lactic acid produced by Lactobacillus sp. kill or inhibit other microorganisms mainly gram positive and gram negative bacteria, including both enteropathogens and spore formers. Proteins like Enterolisin A, Labyrintho peptin A2, etc. show promising anti-cancer activities. Lactobacillus has effective role in clinical and industrial fields. Clinically it is used to manufacture medicines, some hormones and industrially used for broad fermentations, etc. Studies are going on to use Lactobacillus in more broad ways, by improving strains, increasing specificity, making more effective and to find out some other characteristics to prepare them as more natural therapeutic modality.

Isolation and Characterization of Lactic Acid Bacteria Producing Bacteriocin like Inhibitory Substance (BLIS) from “Gappal”, a Dairy Product from Burkina Faso

Indigenous fermented foods are known for their nutritional and functional properties but they are often spoiled by pathogenic bacteria that can constitute a food safety problem. “Gappal” is a no-thermal treat food based on millet dough and milk and its production conditions can constitute a food safety problem. The aim of this study was to screen and identify LAB producing Bacteriocin-like inhibitory substances using a matrix similar to “Gappal”. The detection of potential BLIS was first performed using overlaid method after enrichment of samples in whey and millet dough. The isolates demonstrating inhibiting area were preselected, purified and tested for the presence of antibacterial properties using their neutralized cell-free culture supernatant and subsequently treated with catalase in combination with protease, pepsin or trypsin. The antimicrobial effect of two isolates (Gbf48 and Gbf50) after growth on MRS broth over 12 h at 30?C were active against E. faecalis ATCC 19433, M. luteus ATCC 49732, S. aureus ATCC 2523, L. monocytogenes, B. megaterium, B. sphaericus and B. cereus with an activity of 2560 AU/mL. The 16S RNA gene sequencing identification indicated that these isolates are Pediococcus acidilactici. Gbf 48 and Gbf 50 could be used to improve preservative factors for a controlled fermentation of non thermal treatment fermented food for their potential of acidification adds to BLIS production.