Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

Antagonistic Action of Lactobacilli and Bifidobacteria in Relation to Staphylococcus aureus and Their Influence on the Immune Response in Cases of Intravaginal Staphylococcosis in Mice

The antibacterial activity of Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2 strains or their various compositions in relation to Staphylococcus aureus in vitro and on models of experimental intravaginal staphylococcosis of mice was determined. It was found that under the influence of these strains and their various compositions, the in vitro growth of Staph. aureus was inhibited, and the number of colonies of Staph. aureus plated from the vagina of infected mice was significantly reduced. The antibacterial activity of these strains separately and in compositions correlated with their ability to improve the performance of the immune response. These strains were the most effective in the following compositions: Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2. Strains of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. bifidum VK2, and B. longum VK1 are prospective components of future probiotic drugs efficient in treating staphylococcosis and for immunity correction.     

Thin layer chromatographic determination of organic acids for rapid identification of bifidobacteria at genus level

This study presents a simple and fast method for the identification of bifidobacteria using a thin layer chromatographic (TLC) analysis of the short chain fatty acids in a culture broth. When the chromatogram was sprayed with the indicator solution (methyl red-bromophenol blue in 70% ethanol), lactic acid exhibited two red spots, and acetic acid, propionic acid, and butyric acid all produced blue spots. Succinic acid and citric acid produced yellow and dark yellow spot, respectively. In addition, these organic acids showed different R(f) values. The total time taken to analyze the organic acids in the 10 bacterial culture broths using the proposed method was approximately 50 min. The proposed TLC method was used to analyze the organic acids in culture broths of the following strains, five Bifidobacterium species. (Bifidobacterium longum, B. breve, B. infantis, B. bifidum, and B. adolescentis) and five other lactic acid bacteria strains (Lactobacillus casei, L. bulgaricus, L. acidophilus, Streptococcus thermophilus, and S. lactis). Both spots of lactic acid and acetic acid were detected on all the TLC plates from the five bifidobacterial culture broths. The five other lactic acid bacterial culture broths, however, only exhibited lactic acid spots. Accordingly, the proposed TLC method would appear to be a useful tool for rapid identification of Bifidobacterium spp. at the genus level.     

Selective enumeration of Lactobacillus casei in yoghurt-type fermented milks based on a 15°C incubation temperature

A procedure was developed to enumerate selectively Lactobacillus casei populations in yoghurt-type fermented milks that can also contain strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium infantis. Commercial LBS agar was acidified to pH 5.4, and the plates were incubated at 15°C for 14 days under anaerobic conditions. Acidification prevented the development of streptococci, and incubation at 15°C limited the development of the lactobacilli and the bifidobacteria. L. casei formed colonies on HHD medium which were different from those obtained with L. bulgaricus. Counts of L. casei on HHD confirmed results obtained on LBS - pH 5.4 medium and incubated at 15°C. L. casei did not form colonies on M17, nor did L. acidophilus or L. bulgaricus.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Selective enumeration of Lactobacillus casei from yogurts and fermented milk drinks

A selective medium (LC agar) was developed for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pour-plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. Growth of S. thermophilus was prevented by adjusting pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar. © Rapid Science Ltd. 1998     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

Use of hydrolysates from Atlantic cod (Gadus morhua L.) viscera as a complex nitrogen source for lactic acid bacteria

Hydrolysates of cod viscera were tested as an alternative to commonly used complex nitrogen sources (peptones and/or extracts) for the type strains of the lactic acid bacteria Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus sakei and Pediococcus pentosaceus. Comparative studies with MRS-like media containing different nitrogen sources showed that all the fish hydrolysates performed equally well or better than commercial extracts/peptones for all selected lactic acid bacteria.     

Fermentation of fructans by epiphytic lactic acid bacteria

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus . In the species Lact. paracasei subsp. paracasei , all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum ), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

Manufacturing of fermented goat milk with a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus in a controlled bioreactor

AIMS: This work was undertaken to study the feasibility and the characteristics of a fermented product made of goat milk, using a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus under controlled conditions, and to determine their survival in the fermented milk during refrigerated storage. METHODS AND RESULTS: Goat milk was inoculated with Lact. acidophilus and Bif. animalis mixed starter, fermented in a glass bioreactor with controlled temperature (37 degrees C) and anaerobiosis, and monitored for growth and acidification. The fermented milk was then stored for 10 days under refrigeration, and monitored daily for starter microflora survival and pH changes. Lact. acidophilus viable counts reached a maximum of 7.1 x 10(8) colony-forming units (CFU) ml(-1), and Bif. animalis a maximum of 6.3 x 10(7) CFU ml(-1) by 20 h of fermentation. During refrigerated storage, both strains exhibited a good survival, with viable numbers remaining essentially constant throughout the experiment, whereas the pH of the fermented milk dropped slightly. CONCLUSIONS: Mixed cultures of Bif. animalis and Lact. acidophilus may be used to produce fermented goat milk with high counts of both probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Goat milk fermented with Bif. animalis and Lact. acidophilus can be manufactured as an alternative probiotic dairy product.     

Different inhibition of Lactobacillus delbrueckii subsp. bulgaricus by D- and L-lactic acid: effects on lag phase, growth rate and cell yield

Inhibition of Lactobacillus bulgaricus by lactic acids at constant pH is governed by two factors: the total concentration of lactic acids and the D/L ratio of lactic acid stereoisomers. L-Lactic acid is more inhibitory than the D-isomer, the latter being the only isomer produced by Lact. bulgaricus. The major inhibitory effect is an extremely prolonged lag phase (2–39 h for the range 0–25 g 1^-1 added lactic acids). Added lactic acids reduce the maximum cell concentration obtained in pH controlled fermentations, but have virtually no effect on maximum specific growth rate and on the yield of biomass on produced lactic acid. The added lactic acids have no effect on the growth-related ATP consumption nor on the non-growth related ATP consumption. The culture is heterogeneous since a part of the culture is unable to grow in the presence of L-lactic acid. A different inhibitory effect of D- and L-lactic acid has implications for yoghurt manufacture and potential for improved food preservation.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.     

Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacilus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100°C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.     

Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk

Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif. adolescentis and Bif. bifidum), Lactobacillus acidophilus and a mixed culture of Lact. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria. The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another. Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact. delbrueckii subsp. bulgaricus, Strep. salivarius subsp. thermophilus and Lact. acidophilus. However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents. The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures.     

Attachment of Lactobacillus casei strain GG to human colon carcinoma cell line Caco-2: comparison with other dairy strains

The degree of adhesion of Lactobacillus casei strain GG to human Caco-2 cell line was investigated. Assessment of adhesion was compared to the adhesion of enterotoxigenic human Escherichia coli strain H 10407 and enterotoxigenic bovine E. coli strain B44 (non-adhesive). Freeze-dried Lactobacillus GG or samples from dairy products had medium to strong binding to the Caco-2 cell line. Lactobacillus acidophilus (NCFB 1748) and L. bulgaricus showed no adhesion to the cell line while four tested Bifidobacterium strains had no or very weak adhesion to the Caco-2 cell line.     

Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species

AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim. The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes. METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact. paracasei, Lact. rhamnosus and Lact. plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1. The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated. E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact. acidophilus. MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact. paracasei, Lact. rhamnosus and Lact. plantarum, respectively. With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact. acidophilus were 0.064-1 microg ml(-1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim. The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains.