Pramod K. Gopal

Selection and Characterisation of Lactobacillus and Bifidobacterium Strains for Use as Probiotics

Abstract A large culture collection of lactic acid bacteria held at NZDRI (of over 2000 strains) was screened to select strains with functional characteristics typical of probiotic bacteria. Selection criteria employed included the ability of strains to withstand environmental conditions similar to the digestive tract as well as specific biological activites. Following an initial screening of over 200 strains selected from the collection, four strains were identified as putative probiotic strains. Three of the selected strains were of dairy origin and one was of human origin. These strains were able to survive at low pH and relatively high bile concentrations and compared favourably in these respects to two commercial probiotic strains namely Lactobacillus rhamnosus GG and Lactobacillus acidophilus LA-1. Of the 200 strains studied, a higher proportion of strains of human origin were found to be resistant to both low pH and high concentrations of bile as compared to strains of dairy origin. The four putative probiotic strains were characterised by classical microbiological techniques and molecular methodologies including DNA–DNA homology, SDS–PAGE analysis of whole cell proteins, PFGE, species-specific probes and RAPD. The strains were identified as Lb. rhamnosus HN001 (also known as DR20), Lb. acidophilus HN017, Lb. rhamnosus HN067 and Bifidobacterium. lactis HN019 (also known as DR10).

The role of autolysis of lactic acid bacteria in the ripening of cheese

Abstract The importance of autolysis of lactic acid bacteria in cheese ripening is evident from the literature. However, the mechanisms and the consequences still require investigation. The consequences of autolysis of mesophilic starters in Cheddar cheese are discussed and highlights from current physiological and genetic studies on starter autolysis are presented. The relative merits of measuring starter autolysis in cheese by viable starter cell densities, electron microscopic observations and assay of cell-free cytoplasmic enzymes are discussed for cheese studies using different starter strains and added phage to achieve different levels of autolysis. The balance of both the intact and autolysed starter cells in young curd appear to be important in cheese ripening. The intact cells are necessary for physiological reactions such as lactose fermentation and oxygen removal and possibly for a number of flavour reactions. In contrast, the main consequence of autolysed cells in cheese is to accelerate the peptidolytic reactions. The possible influences of autolysis of adventitious lactic acid bacteria during cheese ripening are discussed.

Heat and Osmotic Stress Responses of Probiotic Lactobacillus rhamnosus HN001 (DR20) in Relation to Viability after Drying

ABSTRACT The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries. In this study we observed that when prestressed with either heat (50°C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30°C in the dried form. To investigate the mechanisms underlying this stress-related viability improvement in L. rhamnosus HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing. Several proteins were up- or down-regulated after either heat or osmotic shock treatments. Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway. The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used. The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments. Carbohydrate analysis of cytoplasmic contents showed higher levels (20 ± 3 μg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 ± 3 μg/mg of protein). Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles. Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol.

Utilisation of galacto-oligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20

Two probiotic strains of bacteria Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20 were tested for their ability to utilise and grow on galacto-oligosaccharides present in a commercial hydrolysed lactose milk powder. The results clearly demonstrated that B. lactis DR10 preferentially utilises tri- and tetra-saccharides whereas Lb. rhamnosus DR20 prefers sugars with a lower degree of polymerisation, i.e., disaccharides and monosaccharides. Since galacto-oligosaccharides are non-digestible oligosaccharides, this in vitro data suggest that galacto-oligosaccharides present in milk powders are likely to promote growth of DR10 and DR20 in vivo if these strains are consumed in combination with the milk powder. Fifty four strains of lactic acid bacteria, including members from the genera Bifidobacterium and Lactobacillus, were studied for their ability to utilise lactose derived oligosaccharides. A perfect correlation was observed between the ability of a strain to utilise oligosaccharide and the presence of the lactose hydrolysing enzyme β-galactosidase. Based on these observations, a mechanism for the utilisation of galacto-oligosaccharides in genus bifidobacteria is proposed that may help to explain the ability of these organisms to out-compete other bacteria in the ecosystem of the human gastro-intestinal tract.

Potential probiotic lactic acid bacteria Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019) do not degrade gastric mucin in vitro

The mucus layer (mucin) coating the surface of the gastrointestinal tract (GIT) plays an important role in the mucosal barrier system. Any damage or disturbance of this mucin layer will compromise the hosts mucosal defence function. In the present study, the ability of three potential probiotic lactic acid bacteria (LAB) strains (Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, Bifidobacterium lactis HN019) to degrade mucin in vitro was evaluated, in order to assess their potential pathogenicity and local toxicity. The LAB strains were incubated in medium containing hog gastric mucin (HGM, 0.3%) at 37 degrees C for 48 h, following which any decrease in carbohydrate and protein concentration in the ethanol-precipitated portion of the culture medium was determined, using phenol-sulphuric acid and bicinchonic acid (BCA) protein assays, respectively. The change in molecular weight of mucin glycoproteins, following incubation with the test strains, was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In order to expose any ability of the test strains to degrade mucin visually and more directly, the test strains were also cultured on agarose containing 0.3% HGM and incubated anaerobically for 72 h at 37 degrees C. No significant change in the carbohydrate or protein concentration in mucin substrates was found following incubation with the test strains. No mucin fragments were derived from the mucin suspension incubated with test strains, and no mucinolysis zone was identified on agarose. These results demonstrate that the potential probiotic LAB strains tested here were unable to degrade gastrointestinal mucin in vitro, which suggests that these novel probiotic candidates are likely to be non-invasive and non-toxic at the mucosal interface.

Dietary Bifidobacterium lactis (HN019) enhances resistance to oral Salmonella typhimurium infection in mice.

The ability of a newly identified probiotic lactic acid bacterial strain, Bifidobacterium lactis (HN019), to confer protection against Salmonella typhimurium was investigated in BALB/c mice. Feeding mice with B. lactis conferred a significant degree of protection against single or multiple oral challenge with virulent S. typhimurium, in comparison to control mice that did not receive B. lactis. Protection included a ten‐fold increase in survival rate, significantly higher post‐challenge food intake and weight gain, and reduced pathogen translocation to visceral tissues (spleen and liver). Furthermore, the degree of pathogen translocation showed a significant inverse correlation with splenic lymphocyte proliferative responses to mitogens, blood and peritoneal cell phagocytic activity and intestinal mucosal anti‐S. typhimurium antibody titers in infected mice; all of these immune parameters were enhanced in mice fed B. lactis. Together, these results suggest that dietary B. lactis can provide a significant degree of protection against Salmonella infection by enhancing various parameters of immune function that are relevant to the immunological control of salmonellosis. Thus dietary supplementation with B. lactis provides a unique opportunity for developing immune‐enhancing probiotic dairy food products with proven health benefits.

Dose-response effect of Bifidobacterium lactis HN019 on whole gut transit time and functional gastrointestinal symptoms in adults

Abstract Objective. To assess the impact of Bifidobacterium lactis HN019 supplementation on whole gut transit time (WGTT) and frequency of functional gastrointestinal (GI) symptoms in adults. Material and methods. We randomized 100 subjects (mean age: 44 years; 64% female) with functional GI symptoms to consume a proprietary probiotic strain, B. lactis HN019 (Fonterra Research Centre, Palmerston North, New Zealand), at daily doses of 17.2 billion colony forming units (CFU) (high dose; n = 33), 1.8 billion CFU (low dose; n = 33), or placebo (n = 34) for 14 days. The primary endpoint of WGTT was assessed by X-ray on days 0 and 14 and was preceded by consumption of radiopaque markers once a day for 6 days. The secondary endpoint of functional GI symptom frequency was recorded with a subject-reported numeric (1–100) scale before and after supplementation. Results. Decreases in mean WGTT over the 14-day study period were statistically significant in the high dose group (49 ± 30 to 21 ± 32 h, p < 0.001) and the low dose group (60 ± 33 to 41 ± 39 h, p = 0.01), but not in the placebo group (43 ± 31 to 44 ± 33 h). Time to excretion of all ingested markers was significantly shorter in the treatment groups versus placebo. Of the nine functional GI symptoms investigated, eight significantly decreased in frequency in the high dose group and seven decreased with low dose, while two decreased in the placebo group. No adverse events were reported in any group. Conclusions. Daily B. lactis HN019 supplementation is well tolerated, decreases WGTT in a dose-dependent manner, and reduces the frequency of functional GI symptoms in adults.

Effects of the consumption of Bifidobacterium lactis HN019 (DR10TM) and galacto-oligosaccharides on the microflora of the gastrointestinal tract in human subjects

Abstract The effects of dietary consumption of milk derived oligosaccharides and a probiotic bacterium Bifidobacterium lactis HN019 (also known as DR10 TM ) on the microbial composition of the gastrointestinal tract of human subjects was examined using a randomized, double blind, placebo-controlled study design. Thirty subjects (age range 20–60 years) were divided randomly into three groups. After two weeks of a pre-intervention period, volunteers in group one consumed a reconstituted milk containing 2.4 g of galacto-oligosaccharides per day, volunteers in group two consumed reconstituted milk containing 3 × 10 10 CFU of B. lactis HN019 per day, and group three volunteers consumed reconstituted milk without galacto-oligosaccharides or probiotic bacteria. The feeding period continued for four weeks followed by a wash out period of two weeks. Faecal samples were collected at weekly intervals and analysed for eight major groups of microbes associated with human gastrointestinal tract. Subjects receiving reconstituted milk containing either galacto-oligosaccharides or probiotic bacteria ( B. lactis HN019) exhibited a significant increase in the faecal counts of both lactobacilli ( p p B. lactis HN019 (placebo control group). Furthermore, B. lactis HN019 survived the digestion through human gastrointestinal tract and colonised transiently. It is believed that desirable bacteria including members of the genera bifidobacterium and lactobacillus, have a positive impact on human health. The results presented in this study demonstrate that dietary supplementation with B. lactis HN019 or galacto-oligosaccharides increases the proportion of bifidobacterium and lactobacillus in the human gastrointestinal tract and hence may improve human health.

Cell surface differences of lactococcal strains

Abstract A number of cell surface properties were compared in 15 pairs of lactococcal strains in order to gain an understanding of cell surface diversity and the relationship between the acquisition of the phage-resistance phenotype and alteration of cell surface properties. Each pair comprised a parent strain and a derivative resistant to a phage (O R ) or a number of phages. Three cell surface hydrophobicity patterns were found: (1) three parent strains were more hydrophobic than their O R derivatives; (2) five O R derivatives were more hydrophobic than their parent strains; (3) there were no differences for seven strain pairs. Loosely associated cell surface material was removed without cell lysis, and concentration differences between 28 strains of 40-, 23- and 11-fold were found for the extracted protein, hexose and rhamnose, respectively. These three surface components were extracted in higher concentrations from the O R derivative for seven strain pairs and from the parent strain for three strain pairs, and no differences were observed for four strain pairs. Intracellular and extracellular lipoteichoic acid concentrations varied in four of six strain pairs studied. The extracted protein profiles determined on polyacrylamide gels and by Superose 12 chromatography and the compositions of the extracted polysaccharide were different between most of the strain pairs. In addition, the surface properties, particularly cell hydrophobicity, varied according to growth conditions for some strains. The cell-surface components showed considerable diversity within the 30 lactococcal strains studied, with multiple differences between many of the strain pairs. For example, differences in hydrophobicity, the extracellular lipoteichoic acid concentration, molecular weight profile of proteins and the amount of protein, hexose and rhamnose extracted as loosely associated cell surface material were observed between the strains of pair E8/398. No unifying theme was evident to describe the basis of changes to the cell surface in the phage-resistant derivative strains.

Autolysis of Lactococcus lactis

Abstract During cheese making, autolysis of Lactococcus lactis starter bacteria affects cheese flavour development through release of intracellular enzymes. The gene for the major autolysin in L. lactis, N-acetyl muramidase (AcmA), has been cloned and sequenced. The activity of AcmA alone, however, does not explain the huge variation in the extent of autolysis found in commercial L. lactis starter strains. Many such strains have multiple cell wall hydrolases that can be seen as different sized clearance bands in zymograms. In addition, the recently completed L. lactis subsp. lactis IL1403 genome sequence shows the presence of several open reading frames that putatively encode cell wall hydrolases having up to 42% predicted amino acid identity to AcmA. These enzymes could have roles in the autolysis of L. lactis. In this paper, we review the literature on autolysis of L. lactis and provide experimental evidence, based on Western blot and zymogram analysis, that commercial L. lactis starter strains express varying levels of AcmA and contain other cell wall hydrolases.