Kay J. Rutherfurd

Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019)

Consumption of lactic acid bacteria (LAB) has been suggested to confer a range of health benefits including stimulation of the immune system and increased resistance to malignancy and infectious illness. In the present study, the effects of feeding Lactobacillus rhamnosus (HN001, DR20), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019, DR10) on in vivo and in vitro indices of natural and acquired immunity in healthy mice were examined. Mice were fed daily with L. rhamnosus, L. acidophilus or B. lactis (10(9) colony forming units) and their immune function was assessed on day 10 or day 28. Supplementation with L. rhamnosus, L. acidophilus or B. lactis resulted in a significant increase in the phagocytic activity of peripheral blood leucocytes and peritoneal macrophages compared with the control mice. The proliferative responses of spleen cells to concanavalin A (a T-cell mitogen) and lipopolysaccharide (a B-cell mitogen) were also significantly enhanced in mice given different LAB. Spleen cells from mice given L. rhamnosus, L. acidophilus or B. lactis also produced significantly higher amounts of interferon-gamma in response to stimulation with concanavalin A than cells from the control mice. LAB feeding had no significant effect on interleukin-4 production by spleen cells or on the percentages of CD4+, CD8+ and CD40+ cells in the blood. The serum antibody responses to orally and systemically administered antigens were also significantly enhanced by supplementation with L. rhamnosus, L. acidophilus or B. lactis. Together, these results suggest that supplementation of the diet with L. rhamnosus (HN001), L. acidophilus (HN017) or B. lactis (HN019) is able to enhance several indices of natural and acquired immunity in healthy mice.

Dietary Probiotic Supplementation Enhances Natural Killer Cell Activity in the Elderly: An Investigation of Age-Related Immunological Changes

Many elderly subjects are at increased risk of infectious and noninfectious diseases due to an age-related decline in lymphoid cell activity (immunosenescence). Noninvasive means of enhancing cellular immunity are therefore desirable in the elderly. Previous reports have suggested that dietary supplementation could represent an effective means of enhancing the activity of circulating natural killer (NK) cells in the elderly. In the present study, we have conducted a pre–post intervention trial to determine the impact of dietary supplementation with probiotic lactic acid bacteria (LAB) on peripheral blood NK cell activity in healthy elderly subjects. Twenty-seven volunteers consumed low-fat/low-lactose milk supplemented with known immunostimulatory LAB strains (Lactobacillus rhamnosus HN001 or Bifidobacterium lactis HN019) for a period of 3 weeks. A dietary run-in of milk alone was shown to have no significant effect on NK cells. In contrast, the proportion of CD56-positive lymphocytes in peripheral circulation was higher following consumption of either LAB strain, and ex vivo PBMC tumoricidal activity against K562 cells was also increased. Supplementation with HN001 or HN019 increased tumoricidal activity by an average of 101 and 62%, respectively; these increases were significantly correlated with age, with subjects older than 70 years experiencing significantly greater improvements than those under 70 years. These results demonstrate that dietary consumption of probiotic LAB in a milk-based diet may offer benefit to elderly consumers to combat some of the deleterious effects of immunosenescence on cellular immunity.

Immunoregulatory peptides in bovine milk.

Bovine milk is known to contain a number of peptide fractions that can affect immune function. The vast majority of immunoregulatory peptides that have been characterised are hydrolysate derivatives of major milk proteins. Recent research has also indicated that the metabolic activity of probiotic lactic acid bacteria can generate de novo immunoregulatory peptides from milk, via enzymatic degradation of parent milk protein molecules. In contrast, relatively little is known of endogenous, preformed immunoregulatory peptides in milk that may be relevant to modulating human health. The natural in vivo role of preformed and enzymatically derived peptides is likely to be one of regulation of the neonatal (bovine) gastrointestinal tract immune system, in order to modulate immune function with respect to the development of immunocompetence and avoidance of undesirable immunological responses (e.g. tolerance, and hypersensitivity to nutrients). There is scope for the further characterisation of both the origin and function of milk-derived immunoregulatory peptides, so that their potential to influence human health can be fully appraised. This review highlights our current knowledge of milk-derived immunoregulatory peptides, and outlines areas that are of relevance for further research.

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.

Acute oral toxicity and bacterial translocation studies on potentially probiotic strains of lactic acid bacteria

Three potentially probiotic lactic acid bacteria (LAB) strains, Lactobacillus rhamnosus HN001 (DR20(TM)), Lb. acidophilus HN017 and Bifidobacterium lactis HN019 (DR10()), have recently been identified and characterized. The present study was designed to evaluate the acute oral toxicity of these strains to mice, and also to investigate bacterial translocation and gut mucosal pathology in BALB/c mice fed HN019, HN001 or HN017 for 8 consecutive days at a high dose of 10(11)cfu/mouse/day. Results showed that these probiotic strains had no adverse effect on general health status, feed intake, body weight gain and intestinal mucosal morphology (villus height, crypt depth, epithelial cell height and mucosal thickness). No viable bacteria were recovered from blood and tissue samples (mesenteric lymph nodes, liver and spleen) of mice, and no treatment-associated illness or death was observed. According to these results, the oral LD(50) of HN019, HN001 and HN017 is more than 50g/kg/day for mice, and their acceptable daily intake (ADI) value is 35g dry bacteria per day for a 70-kg person. This suggests that the probiotic strains HN019, HN001 and HN017 are non-pathogenic and likely to be safe for human consumption.

Viability and dose-response studies on the effects of the immunoenhancing lactic acid bacterium Lactobacillus rhamnosus in mice.

Previous studies have indicated that the lactic acid bacterium Lactobacillus rhamnosus HN001 can enhance immune function in mice, following oral delivery. However, the influence of bacterial cell viability on immunoenhancement, and the optimum dose of HN001 required for this effect, have not been determined. In the present study, both live and heat-killed preparations of L. rhamnosus HN001 were shown to enhance the phagocytic activity of blood and peritoneal leucocytes in mice, at a dose of 109 micro-organisms daily. In contrast, only live HN001 enhanced gut mucosal antibody responses to cholera toxin vaccine. Feeding mice with 107 viable HN001/d for 14 d was shown to enhance the phagocytic capacity of blood leucocytes, with incremental enhancement observed at 109 and 1011 daily doses. In contrast, a minimum dose of 109 viable HN001/d was required to enhance the phagocytic activity of peritoneal leucocytes, and no further increment was observed with 1011 daily. This study demonstrates that L. rhamnosus HN001 exhibits dose-dependent effects on the phagocytic defence system of mice, and suggests that while the innate cellular immune system is responsive to killed forms of food-borne bacteria, specific gut mucosal immunity may only be stimulated by live forms.

Peptides affecting coagulation

Based on amino acid sequence similarities that exist between the fibrinogen gamma-chain and kappa-casein, and also functional similarities between milk and blood coagulation, considerable effort has been made to investigate the effects of milk proteins and peptides on platelet function and thrombosis. In particular, a number of peptides derived from the glycomacropeptide segment of kappa-casein, have been shown to inhibit platelet aggregation and thrombosis. KRDS, a peptide from lactoferrin has also been shown to inhibit platelet aggregation but to a lesser extent than its fibrinogen analogue RGDS. Despite their functional and structural similarities they do not act in the same way on platelet function and are thought to affect thrombus formation differently. Further investigation is needed to determine if these milk-derived bioactive peptides are released naturally following ingestion and might therefore be useful as the basis for milk-based products with anti-thrombotic properties.

Effect of dietary whey protein concentrate on primary and secondary antibody responses in immunized BALB/c mice.

Proteins derived from the whey fraction of bovine milk are known to modulate immune responses. We have previously described a rennet whey protein concentrate (WPC) that can boost intestinal tract antibody responses to orally administered T-dependent antigens. In the present study, we investigated the effects of feeding WPC to mice on specific antibody responses to several orally or parenterally administered antigens, including influenza vaccine, diphtheria and tetanus toxoids, poliomyelitis vaccine, ovalbumin and cholera toxin sub-unit. WPC-fed mice produced elevated levels of antigen-specific intestinal tract and serum antibodies against all tested antigens, compared to mice that were fed a standard chow diet. Both primary and secondary intestinal tract antibody responses were elevated by WPC feeding, while only secondary serum responses were increased in WPC-fed mice. Significant up-regulation of intestinal tract antibody was observed within 2 weeks of primary oral immunizations. A period of pre-feeding with WPC, prior to commencement of immunization, did not alter the kinetics or magnitude of immune enhancement. These results identify bovine WPC as a potentially important dietary protein supplement, capable of enhancing humoral immune responses to a range of heterologous antigens.

Probiotic supplementation to enhance natural immunity in the elderly : effects of a newly characterized immunostimulatory strain Lactobacillus rhamnosus HN001 (DR20) on leucocyte phagocytosis

Abstract Consumption of some species and strains of lactic acid bacteria (LAB) has been shown to enhance immunity in humans. In this study, the effect of dietary supplementation with Lactobacillus rhamnosus HN001 (DR20™), a newly characterized LAB strain, on immune cell function in healthy elderly subjects was investigated. The study comprised three stages, each lasting 3 weeks. In stages 1 (run-in) and 3 (washout), subjects ( n = 13) received low fat milk (LFM, 200 mL twice daily) as a base diet. In Stage 2 (supplement intervention), subjects received LFM supplemented with L. rhamnosus HN001 (1.25 × 10 8 CFU/mL). Assessment of immune function was made at the beginning, and the end of each stage. Consumption of milk supplemented with L. rhamnosus HN001 for three weeks resulted in a significant increase in the phagocytic activity of peripheral blood polymorphonuclear cells and monocytes. These responses returned to baseline following 3 weeks washout with un-supplemented milk. This suggests that L. rhamnosus HN001 is able to enhance aspects of natural immunity in humans, and could be used as a dietary adjunct for optimizing immune responsiveness in the elderly.

Immune enhancement conferred by oral delivery of Lactobacillus rhamnosus HN001 in different milk-based substrates

The probiotic Lactobacillus rhamnosus HN001 is known to enhance immunity in animal models, following oral delivery. In this study, we investigated the immunoenhancing effects of HN001 delivered to mice in different milk-based substrates, including: whole (full-fat) milk supplemented with HN001; fermented milk supplemented with HN001; or whole milk which had been part-fermented by HN001. Direct oral feeding of mice with HN001 in whole milk was shown to enhance the phagocytic activity of blood and peritoneal cells. Similar effects on phagocytosis were observed when UN001 was offered to mice in the form of a milk- or fermented milk-based diet. The degree of immune enhancement conferred by HN001 was similar whether the probiotic was used as an additive or as a fermentative agent. These studies confirm that Lb. rhamnosus HN001, derived originally from dairy food, enhances immune function following oral delivery in different milk bases.