Patricia L. Conway

Evaluation of microencapsulation of a Bifidobacterium strain with starch as an approach to prolonging viability during storage

Aims: To optimize a spray coating process for the production of encapsulated microspheres containing viable Bifidobacterium cells and to determine whether the readily gelatinized modified starch coating used in this study improved bacterial survival in foods or under acid conditions.

The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract

The possibility of using high amylose maize starch granules as a delivery system for probiotic bacteria has been investigated using Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B which were isolated from a healthy human. The Bifidobacterium cells were able to adhere to the amylomaize starch granules and were also able to hydrolyse the starch during growth. Initially, in vitro studies were carried out by studying the survival of strains Bifidobacterium LaftiTM 8B and LaftiTM 13B when exposed to pH 2·3, 3·5 and 6·5 as well as 0·03 and 0·05% w/v bile acids. Both strains were grown either in the absence or presence of high amylose maize starch granules, then mixed with the high amylose maize starch granules and exposed to acidic buffers or bile acid solutions. It was shown that growth in and the presence of high amylose maize starch granules led to enhanced survival of strains LaftiTM 8B and LaftiTM 13B. Subsequently, survival in vivo was monitored by measuring the faecal level of Bifidobacterium LaftiTM 8B after oral administration of the strain to mice. A sixfold better recovery of strain LaftiTM 8B from mice faeces after oral dosage was noted for cells grown in amylose‐containing medium compared with controls. It was concluded that high amylose maize starch granules contributed to enhanced survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B.

Manipulation of colonic bacteria and volatile fatty acid production by dietary high amylose maize (amylomaize) starch granules

Aims: To study the effects of amylomaize starch and modified (carboxymethylated and acetylated) amylomaize starches on the composition of colonic bacteria and the production of volatile fatty acids, in mice. 
Methods and Results: Balb/c mice were fed with experimental diets containing various amount of amylomaize and modified amylomaize starches. Colonic bacterial populations and short‐chain fatty acids were monitored. Results showed that the increases in indigenous bifidobacteria were detected in mice fed all starches tested; however, the highest numbers were observed in the group fed with 40% unmodified amylomaize starch. The starch type influenced the populations of indigenous Lactobacillus, Bacteroides and coliforms. High Lactobacillus numbers were achieved in the colon of mice fed with high concentration of amylomaize starch. Acetylated amylomaize starch significantly reduced the population of coliforms. In addition, orally dosed amylomaize utilizing bifidobacteria reached their highest levels when fed together with amylomaize or carboxymethylated amylomaize starch and in both cases butyrate levels were markedly increased. 
Conclusions: These results indicate that different amylomaize starches could generate desirable variation in gut microflora and that particular starches may be used to selectively modify gut function. 
Significance and Impact of Study: Amylomaize starch appeared to enhance the desirable composition of colonic bacteria in mice, and suggested it possessed the potential prebiotic properties. Therefore, resistant starch and its chemical derivatives may exert beneficial impacts to the human colon.

Effect of Bifidobacterium breve M-16V supplementation on fecal bifidobacteria in preterm neonates - A randomised double blind placebo controlled trial

Background Probiotic supplementation significantly reduces the risk of necrotising enterocolitis (NEC) and all cause mortality in preterm neonates. Independent quality assessment is important before introducing routine probiotic supplementation in this cohort. Aim To assess product quality, and confirm that Bifidobacterium breve (B. breve) M-16V supplementation will increase fecal B. breve counts without adverse effects. Methods and Participants Strain identity (16S rRNA gene sequencing), viability over 2 year shelf-life were confirmed, and microbial contamination of the product was ruled out. In a controlled trial preterm neonates (Gestation <33 weeks) ready to commence or on feeds for <12 hours were randomly allocated to either B. breve M-16V (3×109 cfu/day) or placebo (dextrin) supplementation until the corrected age 37 weeks. Stool samples were collected before (S1) and after 3 weeks of supplementation (S2) for studying fecal B. breve levels using quantitative PCR (Primary outcome). Secondary outcomes included total fecal bifidobacteria and NEC≥Stage II. Categorical and continuous outcomes were analysed using Chi-square and Mann-Whitney tests, and McNemar and Wilcoxon signed-rank tests for paired comparisons. Results A total of 159 neonates (Probiotic: 79, Placebo: 80) were enrolled. Maternal and neonatal demographic characteristics were comparable between the groups. The proportion of neonates with detectable B. breve increased significantly post intervention: Placebo: [S1:2/66 (3%), S2: 25/66 (38%), p<0.001] Probiotic: [S1: 29/74 (40%), S2: 67/74 (91%), p<0.001]. Median S1 B. breve counts in both groups were below detection (<4.7 log cells.g−1), increasing significantly in S2 for the probiotic group (log 8.6) while remaining <4.7 log in the control group (p<0.001). There were no adverse effects including probiotic sepsis and no deaths. NEC≥Stage II occurred in only 1 neonate (placebo group). Conclusion B. breve M-16V is a suitable probiotic strain for routine use in preterm neonates. Trial Registration Australia New Zealand Clinical Trial Registry ACTRN 12609000374268

Association of Lactobacillus spp. with Peyer's Patches in Mice

ABSTRACT Sixteen strains of Lactobacillus isolated from humans, mice, and food products were screened for their capacity to associate with Peyers patches in mice. In preliminary experiments, in vitro binding to tissue pieces was assessed by scanning electron microscopy, and it was demonstrated qualitatively that 5 of the 16 strains showed some affinity for the Peyers patches, irrespective of their association with the nonlymphoid intestinal tissue. Lactobacillus fermentum KLD was selected for further study, since, in addition to its intrinsically high adhesion rate, this organism was found to exhibit a preferential binding to the follicle-associated epithelium of the Peyers patches compared with its level of binding to the mucus-secreting regions of the small intestine. Quantitative assessment of scanning electron micrographs of tissue sections which had been incubated with L. fermentum KLD or a nonbinding control strain, Lactobacillus delbruckii subsp.bulgaricus, supported these observations, since a marked difference in adhesion was noted (P < 0.05). This preferential association of strain KLD with the Peyers patches was also confirmed with radiolabeled lactobacilli incubated with intestinal tissue in the in vitro adhesion assay. Direct recovery of L. fermentum KLD from washed tissue following oral dosing of mice revealed a distinct association (P < 0.05) between this organism and the Peyers patch tissue. In contrast, L. delbruckii subsp. bulgaricus showed negligible binding to both tissue types in both in vitro and in vivo adhesion assays. It was concluded that L. fermentum KLD bound preferentially to Peyers patches of BALB/c mice.

Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to mice.

The indigenous gastrointestinal microbiota acts as an integral defense against the colonisation of orally introduced microbes. Whilst this can be important in host protection, some introduced species, including lactobacilli, can have a positive impact on existing microbial communities. The interaction of a candidate probiotic strain of Lactobacillus fermentum within the gastrointestinal tract was monitored in a mouse model and its effect on the indigenous microbiota observed. L. fermentum KLD was administered via oro-gastric doses to mice with both a specific pathogen-free (SPF) and an ampicillin-depleted gut microbiota, containing no detectable lactobacilli. Its persistence was monitored by detection in faecal homogenates using culturing methods and polymerase chain reaction with L. fermentum specific primers. Microbial population shifts were observed using denaturing gradient gel electrophoresis (DGGE). L. fermentum KLD was detected within the gastrointestinal tract of SPF mice for up to 36 h, and for greater than 11 days in the ampicillin-treated mice. The administration resulted in substantial changes within the host Lactobacillus levels, determined by DGGE of 16S rDNA from faecal samples. Denaturing gradient profiles, from faecal samples collected at a range of pre- and post-dose intervals of groups of 10 SPF mice, indicated that several other constituents of the gastrointestinal community also fluctuated following dosing. These included Bifidobacterium and Eubacterium, which increased following KLD administration. The indigenous microbiota affected the persistence of L. fermentum KLD and in SPF mice the administration of this strain induced significant shifts in the indigenous microbial community.

Isolation of human faecal bifidobacteria which reduce signs of Salmonella infection when orogastrically dosed to mice

A. HENRIKSSON AND P.L. CONWAY. 2001.

Presence of F107, 2134P and Av24 fimbriae on strains of Escherichia coli isolated from Swedish piglets with diarrhoea

A total of 109 Escherichia coli isolates from piglets with diarrhoea, that had previously been shown to be enterotoxin producers, but negative for the adhesive fimbriae K88, K99, 987P and F41 were tested for the presence of more recently characterised fimbriae. Testing was done by immunodot assay with absorbed polyclonal antisera against Av24 and F107 fimbriae, and unabsorbed polyclonal antiserum and monoclonal antiserum against 2134P fimbriae. Strains were also tested by polymerase chain reaction for the presence of genes encoding the major subunit of F107 fimbriae. After elimination of possible non-specific reactions, antisera testing produced 10 strains positive with all 4 antisera, 1 strain that reacted with all antisera except F107, 2 strains that reacted with all antisera except the 2134P monoclonal, 3 strains that reacted with 2134P polyclonal and F107 and 2 that reacted with F107 only. The PCR testing confirmed the results of the antisera, but also produced an additional 14 positive strains, giving a total of 30% of the strains tested reacting positively by PCR. Furthermore, all 33 isolates positive by PCR came from pigs that were older than 1 week, which is 45% of the 72 isolates tested which came from older pigs.

Probiotics in very preterm infants: the PiPS trial

We respectfully disagree with the conclusions made by Kate Costeloe and colleagues (Feb 13, p 649). There is a signifi cant discrepancy regarding the dose of Bifidobacterium breve BBG-001 (appendix). The authors claim that despite the decay during the trial, the administered dose remained above the recommended threshold (108–109 colony-forming units [CFU], ie, 100 million–1 billion CFU). However, the dose of 100 million CFU is far below the dose administered in their pilot study (1 billion CFU) and the dose used by Hiroyuki Kitajima and colleagues (0·5 billion CFU) that had suggested benefits. Despite this low dose, infants colonised with B breve had clinically significant benefits for all outcomes. Our unadjusted analysis based on their raw data suggests statistically signifi cant benefi ts (table). Hence, their conclusion against BBG-001 is not justifi ed, as signifi cant benefi ts of this strain have not been ruled out. If a minimum guaranteed dose at or above that used in the pilot study was assured throughout the trial, signifi cant differences might have occurred between the two groups despite cross contamination. The 95% CI for the primary analysis supports this possibility. A truly large trial detecting a clinically signifi cant minimum eff ect size (eg, 20%) would need several thousand participants at least. Costeloe and colleagues question the validity of meta-analyses, which is inappropriate considering the limitations of PiPS trial and the increasing agreement about protection by probiotics, via shared beneficial pathways. Given the significant cross contamination (49%), analysis of the intention-totreat population was a futile exercise. Satsuki Totsu reported benefits of Bifidobacterium bifidum in preterm infants in a cluster randomised trial. In cluster randomised controlled rials, patients allocated to the supplement do not have the issue of cross contamination.

Strain dependent protection conferred by Lactobacillus spp. administered orally with a Salmonella Typhimurium vaccine in a murine challenge model

Consumption of Lactobacillus spp. has been shown to enhance immune responses in mice. This study examined the immuno-adjuvant capacity of two strains: Lactobacillus acidophilus L10 and Lactobacillus fermentum PC2, in the induction of protective humoral immunity in a Salmonella Typhimurium vaccine challenge model. Briefly, BALB/c mice were divided into four groups. Three groups of mice received S. Typhimurium vaccine (10(8) colony forming units (CFU) per dose) on days 0 and 14. In addition to the vaccine, five doses (10(8) CFU per dose) of either L. acidophilus L10 or L. fermentum PC2 were also administered to a group. All mice were challenged with viable S. Typhimurium on day 28. On day 10 post challenge, the study was terminated and microbial and immunological parameters were assessed. Mice dosed with L. fermentum PC2 in addition to the vaccine had a significantly enhanced S. Typhimurium humoral response. The mice in this group had high levels of lactobacilli in the feces and in association with the Peyers patches, no detectable levels of either lactobacilli or S. Typhimurium in the spleen, and no detectable weight loss. Mice given L. acidophilus L10 with the vaccine were unable to exhibit elevated S. Typhimurium specific humoral responses. However, there was no detectable S. Typhimurium in the spleens of this group. Interestingly, translocation of lactobacilli into the spleen was observed as well as a slight weight loss was noted in mice that received the L. acidophilus L10 with the vaccine. This study shows that, the L. fermentum PC2 had a greater capacity than the L. acidophilus L10 to act as an oral adjuvant in a S. Typhimurium oral vaccine program and afforded greater protection against a live S. Typhimurium challenge.