New Lactococcus strain with immunomodulatory activity: enhancement of Th1-type immune response

Few studies exist dealing with the probiotic activity of lactococci, which are commonly used as starter bacteria in the manufacture of many kinds of fermented dairy products. Fifteen strains of the genus Lactococcus were examined for their probiotic activities, such as immunomodulatory effects. Six strains induced the production of cytokines (IL-12, IL-6, and TNF-alpha) in macrophage-like cell line J774.1, and the highest induction was observed with Lactococcus lactis subsp. lactis G50. The cytokine induction in the J774.1 cell line was almost entirely sustained after heat-killing of the strain. Spleen cells from BALB/c mice fed G50 culture produced more IL-12 and IFN-gamma and slightly less IL-4 and IL-6 than the control (i.e., without strain G50), indicating that strain G50 can enhance Th1-type immune response in vivo. The effect of the oral administration of strain G50 on antibody response in mice was also investigated. Mice were immunized with ovomucoid (OVM), a potent egg allergen, and the antibody level in the serum was then determined. The total IgE antibody level in the group treated with strain G50 was significantly lower than that of the control. The response of OVM-specific IgG1 and IgE antibodies tended to be low in the group that was administered strain G50, compared with the response of the control group. These results suggest that strain G50 has an ability to suppress the Th2 response. Thus, Lactococcus lactis subsp. lactis G50 is a potential probiotic strain for the suppression of hypersensitive reactions caused by the Th2 response.     

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish

Aims:  We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture.
Methods and Results:  In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l -arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate.
Conclusions:  Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described.
Significance and Impact of the Study:  The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.     

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

Aims:  Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions.
Methods and Results:  A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis .
Conclusions:  The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions.
Significance and impact of the study:  This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.     

Probiotic characteristics of lactic acid bacteria isolated from kimchi

Aims: The present work was aimed at identifying strains of lactic acid bacteria (LAB) from kimchi, with properties suitable for use as starter cultures in yogurt fermentation. Methods and Results: A total of 2344 LAB strains were obtained from two different sources, one group consisted of commercial LAB strains from kimchi, and the second group consisted of those strains isolated from various types of kimchi. The LAB strains from both groups were screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that KFRI342 of the four selected strains displayed the greatest ability to reduce the growth of the cancer cells, SNU‐C4. The in vivo efficacy of strains in quinone reductase induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Strain KFRI342 was identified as Lactobacillus acidophilus by 16S rRNA sequence analysis, showed protection against tumour initiation and imparted immunostimulation as well as protection against DNA damage. Conclusions: Strain KFRI342, which showed probiotic characteristics reducing cancer cell growth, could be a suitable starter culture for yogurt fermentation because of its strong acid production and high acid tolerance. Significance and Impact of the Study: This is the first report to describe a bacterium, isolated from kimchi, Lact. acidophilus KFRI342 which has the probiotic characteristics and the acid tolerance needed for its use as a starter culture in yogurt fermentation.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc‐solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram‐positive and Gram‐negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.     

Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation

AIMS: The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. METHODS AND RESULTS: A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37 degrees C, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. CONCLUSIONS: Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.     

Phenotypic Assessment of Probiotic and Bacteriocinogenic Efficacy of Indigenous LAB Strains from Human Breast Milk

Breast milk is the combination of bioactive compounds and microflora that promote newborn’s proper growth, gut flora, and immunity. Thus, it is always considered the perfect food for newborns. Amongst their bioactives, probiotic communities—especially lactic acid bacteria (LAB)—are characterized from breast milk over the first month of parturition. In this study, seven LAB were characterized phenotypically and genotypically as Levilactobacillus brevis BDUMBT08 ({"type":"entrez-nucleotide","attrs":{"text":"MT673657","term_id":"1863149999"}}MT673657), L. gastricus BDUMBT09 ({"type":"entrez-nucleotide","attrs":{"text":"MT774596","term_id":"1873222494"}}MT774596), L. paracasei BDUMBT10 ({"type":"entrez-nucleotide","attrs":{"text":"MT775430","term_id":"1874262304"}}MT775430), L. brevis BDUMBT11 ({"type":"entrez-nucleotide","attrs":{"text":"MW785062","term_id":"2009762994"}}MW785062), L. casei BDUMBT12 ({"type":"entrez-nucleotide","attrs":{"text":"MW785063","term_id":"2009820773"}}MW785063), L. casei BDUMBT13 ({"type":"entrez-nucleotide","attrs":{"text":"MW785178","term_id":"2010296340"}}MW785178), and Brevibacillus brevis M2403 ({"type":"entrez-nucleotide","attrs":{"text":"MK371781","term_id":"1548854744"}}MK371781) from human breast milk. Their tolerance to lysozyme, acid, bile, gastric juice, pancreatic juice, and NaCl and potential for mucoadhesion, auto-aggregation, and co-aggregation with pathogens are of great prominence in forecasting their gut colonizing ability. They proved their safety aspects as they were negative for virulence determinants such as hemolysis and biofilm production. Antibiogram of LAB showed their sensitivity to more than 90% of the antibiotics tested. Amongst seven LAB, three isolates (L. brevis BDUMBT08 and BDUMBT11, and L. gatricus BDUMBT09) proved their bacteriocin producing propensity. Although the seven LAB isolates differed in their behavior, their substantial probiotic properties with safety could be taken as promising probiotics for further studies to prove their in vivo effects, such as health benefits, in humans.     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Lactococcus lactis subsp. cremoris of Plant Origin Produces Antifungal Cyclo-(Leu-Pro) and Tetradecanoic Acid

The antifungal cyclo-depeptide and the fatty acid were isolated and purified from an indigenous strain of Lactococcus lactis subsp. cremoris. Maximal activity was observed at pH 5.5 and 6.5, and at 30 °C under stationary conditions, which was detected in the culture supernatant 8 h post-inoculation in MRS broth until 22 h. The activity of antifungal compounds in the culture supernatant was sensitive to pH and temperature; and was protease-resistant. The antifungal compounds were concentrated by freeze-drying and ultrafiltration with activity retained in 1 kDa filtrates indicating low molecular weight metabolites. The compounds were further extracted by using different solvents amongst which, ethyl acetate provided the highest recovery. Antifungal compounds were separated on a silica gel column into two active fractions that were revealed to be tetradecanoic acid and cyclo-(Leu-Pro), a cyclic dipeptide, by GC–MS. Herein, we describe and attribute the biocontrol potential of L. lactis subsp. cremoris to the low molecular weight antifungal compounds isolated, which is the first report of their isolation from this strain. The broad antifungal spectrum of this candidate advocates further exploration of its biocontrol potential in managing fungal infections in different food and feed systems.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-020-00917-z.     

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.
Methods and Results:  A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes . Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively.
Conclusions:  These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells.
Significance and Impact of the Study:  This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum .     

Lactobacillus pentosus CECT 4023 T co-utilizes glucose and xylose to produce lactic acid from wheat straw hydrolysate: Anaerobiosis as a key factor

Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analyzed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate. The highest lactic acid yields based on present sugar, around 0.80 g g⁻¹, were obtained from glucose in presence of 25%, 50%, and 75% v v⁻¹ of prehydrolysate in strict anaerobiosis. Lactic fermentation of wheat straw hydrolysate obtained after enzymatic hydrolysis of the prehydrolysate yielded 0.39 g of lactic acid per gram of released sugars, which demonstrated the high potential of L. pentosus to produce lactic acid from hemicellulosic hydrolysates. Results presented herein not only corroborated the ability of L. pentosus to grow using mixtures of sugars, but also demonstrated the suitability of this strain to be applied as an efficient lactic acid producer in a lignocellulosic biorefinery approach. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2739, 2019