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Journal of the American College of Nutrition, Vol. 20, No. 2, 149-156 (2001)
Published by the American College of Nutrition

Original Research

Systemic Immunity-Enhancing Effects in Healthy Subjects Following Dietary Consumption of the Lactic Acid Bacterium Lactobacillus rhamnosus HN001

Ying-H. Sheih, MD, Bor-L. Chiang, MD, Ling-H. Wang, MD, Chuh-K. Liao, MD and Harsharnjit S. Gill, PhD

Taipei Medical College Hospital (Y.-H.S., C.-K.L.), Taipei, Taiwan
College of Medicine, National University of Taiwan (B.-L.C., L.-H.W.), Taipei, Taiwan
Milk & Health Research Centre, Massey University (H.S.G.), Palmerston North, New Zealand

Address reprint requests to: Professor Harsharnjit S. Gill, Milk and Health Research Centre, Massey University, Palmerston North, New Zealand. E-mail: H.S.Gill{at}massey.ac.nz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Objective: To determine the effects of the probiotic lactic acid bacterium, Lactobacillus rhamnosus HN001, on natural cellular immunity when delivered orally in normal low-fat milk (LFM) or lactose-hydrolyzed low-fat milk (LFM-LH).

Design: A three stage, pre-post intervention trial, spanning nine weeks.

Setting: Taipei Medical College Hospital, Taipei, Taiwan.

Subjects: Fifty-two healthy middle-aged and elderly volunteers (17 males, 35 females; median age 63.5, range 44–80).

Interventions: Stage 1 (run-in diet): 25g/200 mL reconstituted LFM powder, twice daily for 3 weeks. Stage 2 (probiotic intervention): LFM or LFM-LH, supplemented with 109 CFUs/g L. rhamnosus HN001 in each case, for 3 weeks. Stage 3 (wash-out): LFM for 3 weeks.

Measures of Outcome: In vitro phagocytic capacity of peripheral blood polymorphonuclear (PMN) leukocytes; in vitro tumoricidal activity of natural killer (NK) leukocytes.

Results: Immunological responses were unaffected by the run-in diet of LFM alone. In contrast, the relative proportion of PMN cells showing phagocytic activity increased by 19% and 15%, respectively, following consumption of HN001 in either LFM or LFM-LH; the relative level of NK cell tumor killing activity increased by 71% and 147%. In most cases these levels declined following cessation, but remained above baseline.

Conclusions: Dietary consumption of L. rhamnosus HN001, in a base of low-fat milk or lactose-hydrolyzed low-fat milk, appears to enhance systemic cellular immune responses and may be useful as a dietary supplement to boost natural immunity.

Key words: Lactobacillus rhamnosus, immune enhancement, phagocytosis, NK cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Dietary intervention is an attractive, non-invasive means of enhancing and optimizing important physiological functions, including the functioning of the immune system [1]. The ability of dietary supplementation to optimize immune function is seen as particularly important among those groups of individuals who may have an underdeveloped or poorly functioning immune system, such as infants, immunocompromised subjects and the elderly [2,3]. A suboptimally functioning immune system is thought to impact adversely on health parameters, such as the ability to combat secondary microbial infections and the ability to produce protective responses to novel foreign material (e.g., in tumor control) [4–7].

Among those dietary interventions which have been demonstrated to have an impact on the immune system, there is increasing evidence that dietary consumption of fermented foods can enhance certain key immune responses that are important in the maintenance of health [8,9]. In particular, there are a few well-defined strains of lactic acid bacteria (LAB) which have been shown to enhance immunity following dietary consumption, by acting as probiotics [10–13]. Furthermore, certain strains of probiotic bifidobacteria and lactobacilli have been shown to provide protection to infants against rotavirus diarrhea [14] and to adults against traveller’s diarrhea [15], thus suggesting that probiotics may be an effective means of disease prevention. However, not all probiotic LAB are effective in stimulating the human immune system [16], and thus the demonstration of an immunomodulatory effect is the first necessary step to identifying strains with potential benefits to human health; this is particularly true with elderly subjects, about whom there are few reported studies on the immunity-enhancing effects of probiotic LAB.

Recent studies by our group have isolated a new strain of Lactobacillus rhamnosus (L. rhamnosus HN001) from cheese [17]. L. rhamnosus HN001 has potent immune-enhancing properties following dietary delivery to laboratory animals [18,19]. Furthermore, this enhancement of immune function has been shown to correlate with enhanced resistance of mice against oral challenge with the pathogen Salmonella typhimurium [18]. However, the ability of this LAB strain to enhance important cellular immune responses in humans remains unproven.

In the present study, we investigated the effects of dietary consumption of L. rhamnosus HN001 on two important aspects of natural cellular immune responsiveness, namely non-specific polymorphonuclear (PMN) cell phagocytosis and natural killer (NK) cell tumor killing activity. Our study was conducted as a three-stage, pre/post trial using healthy middle-aged and elderly volunteers, and its main objective was to determine whether dietary consumption of L. rhamnosus HN001 could enhance natural cellular immunity. We utilized the delivery of HN001 in low-fat milk as a biologically-relevant delivery vehicle. The milk was either unaltered or lactose-hydrolyzed; the latter represents an appropriate delivery vehicle for consumers who may develop lactose intolerance. Moreover, lactose-hydrolyzed milk is rich in galacto-oligosaccharides, and thus may offer a beneficial prebiotic substrate to optimize any potential immune-enhancing effects of a milk-borne probiotic. Accordingly, the sub-objective of the present study was to determine if there was a difference in the immune status of subjects if they consumed HN001 in normal low-fat milk or lactose-hydrolyzed low-fat milk.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Preparation of Diets
Low-fat milk (LFM), containing 12% w/w total fat, was obtained in powder form as a commercial product from the New Zealand Dairy Board. Lactose-hydrolyzed LFM (LFM-LH) was obtained via standard enzymatic treatment using ß-galactosidase from K. lactis (Novo). The resultant LFM powder contained 8% (w/w) galacto-oligosaccharides, comprising predominantly tri- and tetra-saccharides (Table 1). Lactobacillus rhamnosus HN001 was obtained from the New Zealand Dairy Research Institute Culture Collection (NZDRI, Palmerston North, New Zealand). For experimental purposes, lyophilized bacteria were incorporated into LFM or LFM-LH at 109 CFUs/g powder. L. rhamnosus HN001 has been shown to exhibit no significant loss in viability due to storage (for a period of up to two years) or due to reconstitution [20].


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  		Table 1. Composition of Lactose-Hydrolyzed Low-Fat Milk Powder (per 100g Powder)

 
Blinding Method and Allocation
Milk powders or L. rhamnosus-supplemented milk powders were supplied to registered health care providers in sealed sachets. The sachets were number-coded respective to each individual trial participant; however, the treatments were known to neither the providers nor the participants. Each participant was given a supply of sachets sufficient to last each stage of the trial; replenishments were given for the next stage at regular three weekly visits to their health care provider. Each sachet contained 25g powder, with or without probiotic supplementation. Sachets were reconstituted to 200 mL immediately prior to consumption by the subjects themselves, following instructions; there was no perceptible difference in taste between reconstituted plain or probiotic-supplemented milk. Participants were made aware that the sachets might contain health-benefiting organisms, but were unaware of the trial design.

Subjects and Trial Criteria
Fifty-four healthy volunteers within the age range 44 to 80 years (median 63.5 years) were selected for this study. Prior to commencement of the trial, the selection criteria were generated from the records of participating health care providers. Inclusion criteria were general good health and mobility and agreement to conform with the trial guidelines or provide notification of non-compliance. Exclusion criteria were any recent history of acute or chronic debilitating illness, any record of milk-product intolerance and nonagreement to avoid potentially conflicting nutritional or vitamin supplements during the nine-week duration of the trial. Compliance with the dietary regimes was confirmed by the subjects, by direct report to the health care provider and return of empty sachets.

Protocol
The trial protocol was approved by the Taipei Medical College Hospital Ethics Committee. During Stage 1 of the trial (week 1 to week 3) all subjects consumed 25g/200 mL reconstituted LFM powder twice daily as a base diet run-in treatment. Subjects were randomly assigned to two groups for Stage 2 of the trial; A (n=27; 9 M, 18 F) and B (n=27; 9 M, 18 F). In Stage 2 (week 4 to week 6) subjects consumed L. rhamnosus-supplemented milk:

In Stage 3 (week 7 to week 9) all subjects returned to non-supplemented reconstituted LFM as a three week wash-out period (Fig. 1).



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  		Fig. 1. Flow-chart outlining the trial design and timing of immune measurements.

 
Blood Collection and Isolation of Leukocytes
For blood collection, subjects fasted overnight and samples were collected at 8 am the following morning (local time). Peripheral blood samples were withdrawn from subjects by venipuncture at four time points throughout the trial (Fig. 1):
  1. Week 0 (immediately prior to commencement of the trial, to provide baseline immune measurements);
  2. At the end of Week 3 (after all subjects had consumed LFM for three weeks)
  3. At the end of Week 6 (after subjects had consumed HN001-supplemented milks for three weeks);
  4. At the end of week 9 (after all subjects had ceased consumption of HN001-supplemented milks, and had returned to LFM for the final three weeks of the trial).

Bloods were routinely processed within two hours of collection. Mononuclear cells and polymorphonuclear cells (PMNs) were separated from heparinised whole blood via centrifugation over isotonic Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) or 4.5% dextran followed by Ficoll-Hypaque, respectively. Cell types were prepared separately and washed via centrifugation in PBS. Prior to in vitro experimentation, cells were counted (trypan blue dye exclusion) and resuspended to the desired concentration in RPMI 1640 medium (Sigma, St. Louis) supplemented with 10% fetal calf serum.

Immunological Measurements
Measurements were undertaken by experienced laboratory personnel in a clinical immunology laboratory, using equipment and standardized procedures as outlined previously [21]. In the PMN cell fraction, phagocytic activity was assessed via membrane-bound NADPH-oxidase activity, as measured by the reduction of nitroblue tetrazolium (NBT; Sigma) [22]. Briefly, PMN cells in suspension were stimulated with 100 ng/mL phorbol-myristate acetate (PMA; Sigma) in the presence of 0.6 mg/mL NBT for 30 minutes at 37°C. After incubation, cells were centrifuged onto glass slides using a cytospin; the percentage of cells showing NBT reduction activity was estimated for each sample using low-power microscopy.

In the mononuclear cell fraction, the activity of natural killer (NK) cells was assessed by specific target lysis against chromium-labeled K562 tumor cells. Briefly, 104 target 51Cr-labelled K562 cells were mixed with effector PBMCs in 96 well round-bottomed tissue culture plates at an effector:target ratio of 100:1 (a ratio which had been determined previously as optimal for differentiating positive results from background). Chromium release was assessed after four hours of incubation at 37°C by measuring gamma-emission, and specific lysis was determined as:

where E is the amount of chromium released in the presence of effector cells, S is the spontaneous release in medium alone, and T is the total amount of chromium released by adding HC1 to lyse 100% of target cells.

Statistical Analysis
Repeat measures analysis of variance (ANOVA) was used to analyze changes in immune measurements due to treatment over time. Significant differences between paired sets of time-point data were identified by Dunnett’s post hoc test for each group. A probability value of <0.05 was considered sufficient to reject the null hypothesis of no treatment effect. To compare the magnitude of any HN001-specific changes between the Stage 2 test diets, post-HN001 data were re-expressed as the relative (percentage) change in response between time points 2 and 3 of the trial for each individual; Mann-Whitney U tests were then used to compare the magnitude of relative changes in response to HN001 consumption for Group A versus Group B.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Clinical Observations
Of the fifty-four subjects initially enrolled in the trial, two withdrew from Group A during Stage 2 of the trial for personal reasons (work commitments and an unforseen requirement for invasive surgery, respectively). Fifty-two subjects completed the trial, 25 from Group A (8 males, 17 female; median age 67, range 45–74); and 27 from Group B (9 male, 18 female; median age 60, range 44–80) (Fig. 1). Throughout the duration of the study, there were no reports of adverse effects on health and no general health problems were recorded among the subjects.

Immune Measurements
Significant changes in immune responses over time were detected among both groups of subjects. There were no significant changes between immune measurement 1 (baseline week 0) and measurement 2 (after three weeks’ run-in consumption of LFM). Significant increases in PMN cell phagocytic activity and NK cell killing activity were observed at immune measurements 3 and 4 (i.e., following three weeks’ consumption of L. rhamnosus HN001 and after three weeks’ return to LFM, respectively). In absolute terms, the mean percentage of PMN cells displaying phagocytic activity increased from 75 to 89 following consumption of HN001 in LFM and from 73 to 84 following consumption of HN001 in LFM-LH. The mean percentage of specific target cell lysis by NK cells increased from 12 to 21 following consumption of HN001 in LFM and from 10 to 25 following consumption of HN001 in LFM-LH.

PMN Cell Activity
The proportion of PMN cells displaying phagocytic activity increased significantly following consumption of HN001 in either LFM or LFM-LH (Fig. 2). Responses were significantly higher at both week 6 and week 9 in comparison to either the respective baseline values (week 0) or the values at week 3 for each group (p < 0.01 in each case). In both groups, the overall responses were highest at week 6. There was no significant difference in the relative increase in PMN cell activity due to consumption of HN001 (i.e., the degree of increase between week 3 and week 6) between Group A and Group B subjects (mean relative increases were 18.7% and 15.1%, respectively).



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  		Fig. 2. Effect of dietary consumption of milk or L. rhamnosus HN001-supplemented milk on PMN cell phagocytic activity. Group A: L. rhamnosus HN001 in LFM between weeks 3 and 6. Group B: L. rhamnosus HN001 in LFM-LH between weeks 3 and 6. Healthy adult and elderly subjects consumed low-fat milk for 3 weeks, followed by milk supplemented with L. rhamnosus HN001 for 3 weeks, before returning to non-supplemented low-fat milk for the final 3 weeks of the trial. Immune measurements were taken at the start of the trial (week 0), after 3 weeks’ consumption of LFM (week 3), after 3 weeks’ consumption of L. rhamnosus HN001 in LFM (upper graph) or L. rhamnosus HN001 in lactose-hydrolyzed LFM (lower graph) (week 6) and after 3 weeks’ return to non-supplemented LFM (week 9). Data refer to mean (+ standard deviation) percentages of PMN cells displaying NBT reduction activity at each time point.*=significantly higher levels of phagocytic activity in comparison to respective values at week 0 or week 3 (p < 0.01).

 
NK Cell Killing
The degree of specific target cell lysis by NK cells increased significantly following consumption of HN001 in either LFM or LFM-LH (Fig. 3). Responses were significantly higher at both week 6 and week 9 in comparison to respective week 0 or week 3 values for each group (p < 0.01 in each case). Overall responses were highest at week 9 in Group A and at week 6 in Group B. The relative increase in NK cell activity due to consumption of HN001 was higher among the Group B subjects (mean 147% increase) than among Group A subjects (mean 71.3% increase); however, this difference was not statistically significant (p=0.176).



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  		Fig. 3. Effect of dietary consumption of milk or L. rhamnosus HN001-supplemented milk on NK cell tumor killing activity. Group A: L. rhamnosus HN001 in LFM between weeks 3 and 6. Group B: L. rhamnosus HN001 in LFM-LH between weeks 3 and 6. Healthy adult and elderly subjects consumed low-fat milk for 3 weeks, followed by milk supplemented with L. rhamnosus HN001 for 3 weeks, before returning to non-supplemented low-fat milk for the final 3 weeks of the trial. Immune measurements were taken at the start of the trial (week 0), after 3 weeks’ consumption of LFM (week 3), after 3 weeks’ consumption of L. rhamnosus HN001 in LFM (upper graph) or L. rhamnosus HN001 in lactose-hydrolyzed LFM (lower graph) (week 6) and after 3 weeks’ return to non-supplemented LFM (week 9). Data refer to mean (+ standard deviation) percentages of specific target cell lysis at each time point.*=significantly higher levels of killing activity in comparison to respective values at week 0 or week 3 (p < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Dietary intervention, such as supplementation with micronutrients or total protein supplementation, has been demonstrated previously as an effective strategy to enhance cell-mediated immune responses in healthy elderly subjects [2,7,23]. There is also evidence that dietary consumption of defined strains of probiotic LAB can enhance cellular immune responses in adult subjects. Lactobacillus bulgaricus (strain 100158), Streptococcus thermophilus (strain 001158 [S85]) or L. brevis subsp. coagulans have each been shown to enhance cytokine production [10,12], while L. acidophilus (strain La1) and Bifidobacterium bifidum (strain Bb12) have been shown to enhance PMN and mononuclear leukocyte phagocytosis [13]. Our study has suggested that L. rhamnosus HN001, a strain demonstrated previously to modulate immune responses in animals models, can also enhance PMN cell phagocytosis and NK cell killing activity, following dietary consumption by healthy middle-aged and elderly subjects. This result is important, since cellular immune responses are known to decline with increasing age, and therefore dietary interventions that can enhance cellular immunity might be of significant value in combating the deleterious effects of aging on the immune system. Lesourd [24] has identified diminished immune function among malnourished elderly individuals as a significant risk factor contributing to hyporesponsiveness to vaccination. Furthermore, Chandra [2] demonstrated that immune enhancement following micronutrient supplementation promoted enhanced immunoresponsiveness to influenza vaccination in healthy elderly individuals and a concomitant decrease in the mean duration of infection-related morbidity was recorded.

The demonstration here that L. rhamnosus HN001 may be effective in stimulating circulating leukocytes implies that dietary consumption of this strain could play an important role in enhancing systemic immune responses in adults, with the possibility of enhanced immune effector responses at extra-intestinal tissue sites. Of the immune responses that were enhanced by dietary consumption of HN001, phagocytic activity by PMN cells constitutes an important anti-microbial defense mechanism by these cells, while NK cell killing activity is thought to be important in the control of viral-infected cells [25]. It is therefore possible that enhanced performance by these cells, following consumption of HN001, could lead to an increased ability of consumers to fight infectious diseases, although this remains to be determined. We have previously reported enhanced resistance of mice to Salmonella typhimurium following consumption of HN001 [18], while other immune-enhancing LAB strains have been shown to confer protection against infection with gut pathogens including Salmonella, Escherichia coli or Shigella sonnei in animal models [26,27]. In human studies, B. bifidum and Streptococcus thermophilus (undefined strains) and L. rhamnosus (strain GG) have been shown to confer protection against diarrheal infection [14,15].

In most cases in this study, immune responses declined following cessation of HN001 consumption, yet remained significantly elevated above baseline (pre-trial) levels at the final immune measurement (week 9). This carry-over effect has been recorded previously for other immunity-enhancing strains of LAB, including L. acidophilus Lal and B. bifidum Bb12 [13]. Previous research by our own group has identified significant carry-over effects following cessation of consumption of the immunity-enhancing strain B. lactis HN019 by healthy elderly subjects [28], and this was attributed to the ability of B. lactis HN019 to colonize the human intestine and provide sustained immunity-enhancement. Both B. lactis HN019 and L. rhamnosus HN001 have been demonstrated to possess biological properties suitable for survival in the human gut [17], and both strains have been proven to act in a probiotic capacity via gut colonization and immunity-enhancing effects [20,28; P.K. Gopal and H.S. Gill, unpublished results 1999].

In our study, two types of milk were utilised as a delivery vehicle for L. rhamnosus HN001, namely low fat milk and lactose-hydrolyzed LFM. The rationale for the use of lactose-hydrolyzed milk was two-fold: first, to determine whether immunity-enhancement by HN001 was efficacious when delivered in a milk formulation specifically modified to be favorable toward lactose-intolerant consumers; and secondly, to determine whether a medium rich in oligosaccharides could promote the immunity-enhancing effects of HN001. Natural oligosaccharides have been suggested previously to selectively promote the growth of potentially probiotic LAB [29], and previous studies by our group have indicated that delivery of B. lactis HN019 in oligosaccharide-rich milk promotes strong immunity-enhancing effects [28]. Results from the present study have confirmed the first point, i.e., that HN001 delivered in lactose-hydrolyzed milk retains its immunity-enhancing benefits in healthy adult and elderly subjects. However, while immunoresponses tended to be higher among Group B subjects who consumed HN001 in lactose-hydrolyzed milk, differences in the overall magnitude of immunity-enhancing effects between the probiotic delivered in LFM or in LFM-LH were not significant at the 5% level. This contrasts with results obtained from a recent trial utilising B. lactis HN019, where the immunity-enhancing benefits of this strain were found to be significantly (p < 0.05) augmented following consumption of the LAB in lactose-hydrolyzed milk, using similar group sizes to this study [30]. These results suggest that immunity-enhancing effects by different strains of health-promoting dietary LAB are likely to be operating in response to different conditions and that in some cases the delivery medium can be used to optimize the health benefits of the probiotic. However, further trials would be necessary to confirm this—sample size estimations, based on results of the present study, have indicated that future trials would require in excess of 250 subjects in order to achieve >90% power for detecting a significant difference in response between subjects who consume HN001 in normal low fat milk or lactose-hydrolyzed low-fat milk.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Results of the present study have suggested that oral delivery of L. rhamnosus HN001 in a milk base can enhance indices of natural immunity in middle-aged and elderly consumers, but that delivery of the probiotic in an oligosaccharide-enriched milk base does not appear to strongly potentiate the immunomodulatory effects. While probiotic-immunomodulation may increase resistance to disease, further clinical studies employing randomized, placebo-controlled designs will be necessary to confirm both immune-enhancement and disease protection.


   ACKNOWLEDGMENTS
 
Financial support for this research was provided by the New Zealand Dairy Board. NZDB holds intellectual property rights for L. rhamnosus HN001; none of the authors hold share or stock investments in NZDB.

Received March 10, 2000. Accepted January 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Gill HS, Darragh AJ, Cross ML: Optimizing immunity and gut function in the elderly. J Nutr Health Aging, in press, 2001.
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  11. Kaila M, Isolauri E., Soppi E: Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr Res 32: 141–144, 1992.[Medline]
  12. Kishi A, Uno K, Matsubara Y: Effect of the oral administration of Lactobacillus brevis subsp. coagulans on interferon-{alpha} producing capacity in humans. J Am Coll Nutr 15: 408–412, 1996.[Abstract]
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