Comparative Evaluation of Oral Administration of Probiotic Lactobacilli-fermented Milks on Macrophage Function

Six strains of lactobacilli belonging to three species (Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus helveticus) were evaluated for probiotic attributes viz. acid tolerance, bile tolerance and cell surface hydrophobicity. All the six strains exhibited probiotic attributes with considerable degree of variation. Three Lactobacillus strains selected on the basis of probiotic attributes were used for preparing three different fermented milks. In order to evaluate the effect of feeding these probiotic fermented milks on macrophage cell function, an in-vivo trial was conducted in mice for a period of 2, 5 and 8?days. The control group of mice was fed with skim milk. The phagocytic activity of macrophages increased significantly (P?<?0.05) on feeding fermented milk prepared using L. acidophilus, L. casei and L. helveticus as compared to milk group (control) on 2nd, 5th and 8th day of feeding, respectively. Likewise, the release of ??-glucuronidase and ??-galactosidase from peritoneal macrophages increased significantly (P?<?0.05) on 2nd, 5th and 8th day of feeding as compared to their respective control group (milk). The results thus depict that feeding of probiotic fermented milk enhances phagocytic activity of the macrophages.     

Cardiovascular Effects of Fermented Milk Containing Angiotensin-Converting Enzyme Inhibitors Evaluated in Permanently Catheterized, Spontaneously Hypertensive Rats

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.     

Classification of Lactobacillus helveticus Strains by Immunological Differences in Extracellular Proteinases

Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti.     

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 μg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 μg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.     

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.     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Impact of Proteolytic <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> MTCC5463 on Production of Bioactive Peptides Derived from Honey Based Fermented Milk

In this study, Lactobacillus helveticus MTCC5463 was evaluated for its proteolytic activity and production of bioactive peptides during fermentation of honey supplemented milk under specified growth conditions. Generally, lactic acid bacteria have a strong proteolytic system. However, L. helveticus MTCC5463 showed maximum proteolytic activity at 4 % level of honey supplementation compared to 6 % and control. Similarly, water soluble extract derived from fermented honey based milks exhibited different level of bioactive peptides productions during fermentation. L. helveticus MTCC5463 showed maximum peptides production at 4 % level of honey supplementation compared to control during HPLC analysis and LC–MS analysis.     

Comparative Growth Behaviour and Biofunctionality of Lactic Acid Bacteria During Fermentation of Soy Milk and Bovine Milk

The study reports the growth, acidification and proteolysis of eight selected lactic acid bacteria in skim and soy milk. Angiotensin-converting enzyme inhibition and antimicrobial profiles of skim and soy milk fermented by the lactic acid bacteria were also determined. Among eight lactic cultures (S. thermophilus MD2, L. helveticus V3, L. rhamnosus NS6, L. rhamnosus NS4, L. bulgaricus NCDC 09, L. acidophilus NCDC 15, L. acidophilus NCDC 298 and L. helveticus NCDC 292) studied, L. bulgaricus NCDC 09 and S. thermophilus MD2 decreased the pH of skim and soy milk in greater extent. Acid production (i.e. titratable acidity) by L. bulgaricus NCDC 09 and L. helveticus V3 was higher than other strains. Higher viable counts were observed in S. thermophilus MD2 and L. helveticus V3. Higher proteolysis was exhibited by S. thermophilus MD2 and L. rhamnosus NS6 in both skim and soy milk. Milk fermented by S. thermophilus (MD2) exhibited highest angiotensin-converting enzyme inhibition. Antimicrobial activities of cell-free supernatant of milk fermented by S. thermophilus MD2 and L. helveticus V3 were higher. All the tested lactic acid bacteria performed better in skim milk as compared to soy milk.     

Diversity of lactic acid bacteria and yeasts in Airag and Tarag,traditional fermented milk products of Mongolia

We used culture- and molecular-biology-based methods to investigate microbial diversity in the traditional Mongolian fermented milks “Airag” (fermented mare’s milk) and “Tarag” (fermented milk of cows, yaks, goats, or camels). By rRNA or functional gene sequencing, we identified 367 lactic acid bacteria (LAB) strains and 152 yeast strains isolated from 22 Airag and 31 Tarag samples. The total concentration of LAB in Airag (10^{7.78 ± 0.50} c.f.u. ml^–1; mean ± SD) was significantly lower (P < 0.01) than in Tarag (10^{8.35 ± 0.62} c.f.u. ml⁻¹), whereas the total concentration of yeasts in Airag (10^{7.41 ± 0.61} c.f.u. ml^-1) was significantly higher (P < 0.01) than in Tarag (10^{5.86 ± 1.29} c.f.u. ml^-1). Lactobacillus helveticus and Lactobacillus kefiranofaciens were isolated from Airag as the predominant LAB strains at levels of about 10⁷ c.f.u. ml⁻¹, whereas Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus, and Streptococcus thermophilus were the predominant isolates from Tarag at about 10⁷ c.f.u. ml⁻¹. The lactose-fermenting Kluyveromyces marxianus was isolated predominantly from Airag as its major alcoholic fermentation component. Non-lactose-fermenting yeasts such as Saccharomyces cerevisiae, Issatchenkia orientalis, and Kazachstania unispora were the predominant isolates from Tarag, at about 10⁵ c.f.u. ml⁻¹. The apparent geographic differences in the L. kefiranofaciens and S. thermophilus contents of Tarag strongly suggested that differences among the animal species from which the milk was sourced, rather than geographic distances, were the most important factors influencing the diversity of the microbial composition of traditional fermented milks in Mongolia.     

<Emphasis Type="Italic">In Silico</Emphasis> and <Emphasis Type="Italic">In vitro</Emphasis> Analysis of Novel Angiotensin I-Converting Enzyme (ACE) inhibitory Bioactive Peptides Derived from Fermented Camel Milk (<Emphasis Type="Italic">Camelus dromedarius</Emphasis>)

In this study, Lactobacillus bulgaricus NCDC (09) and Lactobacillus fermentum TDS030603 (LBF) were evaluated for their ACE-inhibitory activity and peptides production under optimized conditions from fermented camel milk (Camelus dromedarius). Lactic cultures were evaluated for their pepX activity, proteolytic activity and ACE-inhibitory activity. 09 culture exhibited higher PepX and ACE-inhibitory activity than LBF. 2% rate of inoculation and 12 h of incubation were optimized on the basis of pepX and proteolytic activity. Purified peptides from fermented camel milk were characterized by amino acids profiling through the search in BlastP, Protein information resource (PIR) databases. ACE-inhibitory activity of different peptides from fermented camel milk were also confirmed by the database of antihypertensive peptides (AHTPDB). Fermented camel milk produced by Lactobacillus cultures could be a novel source of ACE-inhibitory peptides.     

Characterization and electrotransformation of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Lactobacillus paraplantarum</Emphasis> isolated from fermented vegetables

Aims of the study were to characterize two Lactobacillus plantarum-related strains, Lact. plantarum and Lactobacillus paraplantarum isolated from fermented vegetables and, for their potential use as starter strains, compare their growth in various food matrices. Species-level identification of the strains belonging to the Lact. plantarum group was performed by multiplex-PCR with species-specific primers and generation of distinct genotypic profiles was carried out by PFGE-based DNA-fingerprinting. Growth profiles were determined in various food and feed matrices. Compared to Lact. plantarum, Lact. paraplantarum reached higher cell densities in all plant-based matrices and MRS broth. On the contrary to the good growth in plant-based matrices and MRS, poor growth was observed in unprocessed milk. Supplemented lactose did not improve the growth of either tested strain, while predigestion of milk proteins with Lactobacillus helveticus or addition of casitone proved to be an effective means to enhance growth. To find out the applicability of molecular methods, the strains were transformed with replicative plasmids by electroporation. To our knowledge, this is a first report of the electrotransformation of Lact. paraplantarum with a recombinant plasmid.     

Functionality of the S-layer protein from the probiotic strain <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.     

Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Biotherapeutic role of fermented milk

Fermented milk was used as therapy in infantile diarrhoea due to post-gastroenteritis syndrome. This treatment eliminated the disease in 4.0 days (mean value, SD=2.8; n=13) and allowed patients to return to free feeding according to their age. The weight percentile variation during treatment with fermented milk (15 days) was higher in the patients showing 3^rd degree malnutrition than in other children. Bacteriotherapy can restore faecal flora which has been lowered by diarrhoea. Our results showed that levels higher than 10⁶ UFC lactobacilli/g of faeces correlated with a healthy status of the children. Clinical applications of fermented milk with a mixture ofLactobacillus casei andLactobacillus acidophilus in the prevention of gastrointestinal disorders are possible.     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Influence of Whey Protein Concentrate on the Production of Antibacterial Peptides Derived from Fermented Milk by Lactic Acid Bacteria

In the study, growth, proteolysis and antimicrobial activity of lactic acid bacteria were evaluated in skim milk medium supplemented with different concentration of whey protein concentrate (WPC 70). Lactobacillus helveticus (V3) showed maximum pH reduction with 1% WPC. Lactobacillus rhamnosus (NS4) also produced maximum lactic acid production and viable cells counts at 1 and 1.5% WPC, respectively. However, V3 showed maximum proteolytic activity with 1.5% WPC. Streptococcus thermophilus (MD2) was found to exhibit maximum antimicrobial activity with 1.5% WPC. Peptides formed during fermentation were purified by RP-HPLC and identified using RP-LC/MS analysis. Antimicrobial peptide was identified as lactoferrin, which was found in fermented milk supplemented with 1.5% WPC by NS4.     

Molecular Identification of Potentially Probiotic Lactobacilli

The rRNA-targeted oligonucleotide probes are useful for the identification of Lactobacillus acidophilus, L. gasseri, L. johnsonii, L. crispatus, and L. amylovorus. However, the oligonucleotide probe designed for L. helveticus hybridized with nucleic acids of type strains of L. gallinarum and L. helveticus. Hence, the similarity among the 73 strains of lactobacilli was evaluated on the basis of their randomly amplified polymorphic DNA (RAPD) profiles derived from five single-primer reactions. These strains were grouped into seven clusters at a similarity level of 30%, which corresponded to six separate species of the L. acidophilus complex (L. johnsonii, L. gallinarum, L. amylovorus, L. crispatus, L. acidophilus, and L. gasseri, respectively) and L. helveticus. For the first time, strains of L. gallinarum were characterized by RAPD and PFGE analyses. The genome length in that species was estimated to be near 1.45 Mb with the summation of ApaI fragments, and near 1.95 Mb with the summation of SmaI fragments. Received: 1 July 1999 / Accepted: 2 August 1999