Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Antibacterial Substance Produced by Streptococcus faecium under Anaerobic Culture

A facultative anaerobe isolated from Korean domestic soil produced an antibacterial substance under strict anaerobic conditions. Based on the morphological and biochemical tests, and cellular fatty acid profiles, the anaerobe was identified as Streptococcus faecium. An antimicrobial compound produced from the S. faecium was identified as 3,7,12-trihydroxy-24-cholanic acid methylester on the basis of its physico-chemical analysis. This substance had potent antibacterial activities against a test organism harboring multiple antibiotic resistance markers, and a variety of pathogenic bacteria. The isolated S. faecium produced lactic acid as well as the antibiotic compound under the anaerobic conditions.     

Comparative investigation of different methods of storage of lactic acid bacteria

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the generaLactobacillus andLactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacteriumLactococcus lactis subsp.lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Antibiotic resistance of potential probiotic bacteria of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.     

Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens

Aims: To investigate the effect of seven wine phenolic compounds and six oenological phenolic extracts on the growth of pathogenic bacteria associated with respiratory diseases (Pseudomonas aeruginosa, Staphylococcus aureus, Moraxella catarrhalis, Enterococcus faecalis, Streptococcus sp Group F, Streptococcus agalactiae and Streptococcus pneumoniae). Methods and Results: Antimicrobial activity was determined using a microdilution method and quantified as IC₅₀. Mor. catarrhalis was the most susceptible specie to phenolic compounds and extracts. Gallic acid and ethyl gallate were the compounds that showed the greatest antimicrobial activity. Regarding phenolic extracts, GSE (grape seed extract) and GSE‐O (oligomeric‐rich fraction from GSE) were the ones that displayed the strongest antimicrobial effects. Conclusions: Results highlight the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. The antimicrobial activity of wine phenolic compounds was influenced by the type of phenolic compounds. Gram‐negative bacteria were more susceptible than Gram‐positive bacteria to the action of phenolic compounds and extracts; however, the effect was species‐dependent. Significance and Impact of Study: The ability to inhibit the growth of respiratory pathogenic bacteria as shown by several wine phenolic compounds and oenological extracts warrants further investigations to explore the use of grape and wine preparations in oral hygiene.     

The antimicrobial activity of lactic acid bacteria from fermented maize (kenkey) and their interactions during fermentation

A total of 241 lactic acid bacteria belonging to Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus fermentum/reuteri and Lactobacillus brevis from various processing stages of maize dough fermentation were investigated. Results indicated that each processing stage has its own microenvironment with strong antimicrobial activity. About half of the Lact. plantarum and practically all of the Lact. fermentum/reuteri investigated were shown to inhibit other Gram-positive and Gram-negative bacteria, explaining the elimination of these organisms during the initial processing stages. Further, widespread microbial interactions amounting to 85% to 18% of all combinations tested were demonstrated amongst lactic acid bacteria within the various processing stages, i.e. raw material, steeping, 0 h and 48 h of fermentation, explaining the microbial succession taking place amongst lactic acid bacteria during fermentation. The antimicrobial effect was explained by the combined effect of acids, compounds sensitive to proteolytic enzymes and other compounds with antimicrobial activity with the acid production being the most important factor.
The pattern of antimicrobial factors was not species-specific and the safety and storage stability of fermented maize seem to depend on a mixed population of lactic acid bacteria with different types of antimicrobial characteristics. This means that introduction of pure cultures as starters may impose a risk to the product.     

Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria

Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l⁻¹ h⁻¹ of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure l-lactic acid can be obtained.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

Biological ensiling of sugarcane tops

The following lactic acid bacteria were isolated from sugarcane tops silage:Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus delbrueckii, Pediococcus cerevisiae, Leuconostoc mesenteroides andStreptococcus lactis. The isolates were grown in a synthetic medium and the final pH, sugar uptake and the effect of adding CaCO₃ and L1, L2 and L3 factors was observed. The importance of the buffering capacity of the ensiled material for the silage process with a view to the occurrence of lactic acid bacteria is discussed.     

Detection of the pediocin gene <Emphasis Type="Italic">pedA</Emphasis> in strains from human faeces by real-time PCR and characterization of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis>UVA1

Background  

Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.     

Isolation and characterization of two bacteriocins of Lactobacillus acidophilus LF221

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile. Received: 2 October 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Identification and Antimicrobial Activity of Phenylacetic Acid Produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Isolated from Fermented Soybean,Chungkook-Jang

A bacterial strain, B65-1, which showed strong antimicrobial activity, was isolated from Chungkook-Jang, a traditional Korean fermented-soybean food with antimicrobial properties. Based on carbon utilization pattern and partial 16S rRNA sequence analysis, the B65-1 strain was identified as Bacillus licheniformis. An antibiotic compound, active against bacteria and yeast such as Staphylococcus aureus, Escherichia coli, and Candida albicans, was isolated by various chromatographic procedures from culture filtrates of B. licheniformis B65-1. The purified antibiotic was identified to be phenylacetic acid, with the molecular formula C₈H₈O₂ by analyses of EI-MS and NMR. The phenylacetic acid was detected in fermented soybean made with the strain B65-1 as a starter, but was not present in extracts of nonfermented soybean. Our results indicated that the phenylacetic acid produced by B. licheniformis during fermentation of soybean is one of the main compounds of antimicrobial activity of Chungkook-Jang.     

Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20

Aims: The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro‐organisms, including Gram‐positive and Gram‐negative bacteria, yeasts and filamentous fungi. Methods and Results: Antimicrobial and antiadhesive activities were determined using the microdilution method in 96‐well culture plates. The biosurfactant showed antimicrobial activity against all the micro‐organisms assayed, and for twelve of the eighteen micro‐organisms (including the pathogenic Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were achieved for biosurfactant concentrations between 25 and 50 mg ml^?1. Furthermore, the biosurfactant showed antiadhesive activity against most of the micro‐organisms evaluated. Conclusions: As far as we know, this is the first compilation of data on antimicrobial and antiadhesive activities of biosurfactants obtained from lactobacilli against such a broad group of micro‐organisms. Although the antiadhesive activity of biosurfactants isolated from lactic acid bacteria has been widely reported, their antimicrobial activity is quite unusual and has been described only in a few strains. Significance and Impact of the Study: The results obtained in this study regarding the antimicrobial and antiadhesive properties of this biosurfactant opens future prospects for its use against micro‐organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making it a suitable alternative to conventional antibiotics.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.     

Selection of Escherichia coli-inhibiting strains of Lactobacillus paracasei subsp. paracasei

The aim of this study was to select Escherichia coli-inhibiting strains among lactic acid bacteria. On the basis of phenotypical and technological characteristics, 20 strains of lactic acid bacteria were screened from a total of 225 isolates, obtained from nine samples of artisanal Caprino d'Aspromonte cheese, made from raw goats' milk. The antagonistic activity of these 20 strains was detected in plates against three different strains of E. coli. Two strains of Lactobacillus paracasei subsp. paracasei showed a marked anti-E. coli activity against all three strains tested; the other lactic acid bacteria did not exhibit inhibiting activity. The E. coli inhibition can be ascribed to production of bacteriocin-like compounds. The use of L. paracasei subsp. paracasei strains to increase the safety of the cheeses made from raw milk is recommended because these cultures strongly inhibit E. coli, without foreseeable adverse sensory changes. Received 27 December 2001/ Accepted in revised form 28 June 2002     

Susceptibility of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> to organic acids and monoacylglycerols

Organic acids can be used as feed supplements or for treatment of poultry carcasses in processing plants. The antimicrobial activity of nineteen organic acids and two monoacylglycerols in cultures of Campylobacter jejuni CCM 6214^T (ATCC 33560) was determined using a SYBR Green-based real-time PCR assay. The IC₅₀ was a concentration at which only 50 % of a bacteria specific DNA sequence was amplified. Caprylic, capric and lauric acids were the most efficient antimicrobials among the compounds tested (IC₅₀ ≤ 0.1 mg/mL). In a weakly acidic environment (pH 5.5), the antimicrobial activity was more pronounced than at pH 6.5. At pH 5.5, oleic and fumaric acid also had clear antimicrobial activity, as did monocaprylin. The antimicrobial activity of acetic, butyric, stearic and succinic acid was low. In cells treated with fumaric acid, the potential of potassium and tetraphenylphosphonium ion-selective electrodes changed, indicating an increase in cytoplasmic and outer membrane permeability, respectively. No changes in membrane permeability were observed in cells treated with capric acid or monocaprin. Transmission electron microscopy revealed separation of the inner and outer membrane in cells treated with capric and fumaric acid, as well as cytoplasmic disorganization in cells exposed to capric acid.     

Enterococcus faecalis inhibits Klebsiella pneumoniae growth in polymicrobial biofilms in a glucose-enriched medium

Abstract

Catheter-related urinary tract infections are one of the most common biofilm-associated diseases. Within biofilms, bacteria cooperate, compete, or have neutral interactions. This study aimed to investigate the interactions in polymicrobial biofilms of Klebsiella pneumoniae and Enterococcus faecalis, two of the most common uropathogens. Although K. pneumoniae was the most adherent strain, it could not maintain dominance in the polymicrobial biofilm due to the lactic acid produced by E. faecalis in a glucose-enriched medium. This result was supported by the use of E. faecalis V583 ldh-1/ldh-2 double mutant (non-producer of lactic acid), which did not inhibit the growth of K. pneumoniae. Lyophilized cell-free supernatants obtained from E. faecalis biofilms also showed antimicrobial/anti-biofilm activity against K. pneumoniae. Conversely, there were no significant differences in planktonic polymicrobial cultures. In summary, E. faecalis modifies the pH by lactic acid production in polymicrobial biofilms, which impairs the growth of K. pneumoniae.     

Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.     

Identification of interacting mixed cultures of lactic acid bacteria by their exclusion from a model predicting the acidifying activity of non-interacting mixed cultures

A model predicting the acidifying activity of mixed cultures of lactic acid bacteria and based on the lack of interaction between the strains has been investigated to identify interacting cultures. Three mixed cultures with Streptococcus thermophilus TH3 and ST7 and Lactobacillus delbrueckii ssp. bulgaricus LB10 were grown on milk. The acidifying activities of the two mixed cultures TH3/LB10 and TH3/ST7 were predicted accurately by the model, with mean prediction errors of 7.7% and 14.1%, respectively. However, the model underestimated the acidifying activity of the mixed culture ST7/LB10, with a mean prediction error of 43.5%, which provides evidence of positive interaction between the strains ST7 and LB10 during acidification. Received: 26 April 1999 / Received revision: 16 June 2000 / Accepted: 18 June 2000