Safety evaluation of a heat-treated Bifidobacterium bifidum OLB6378 concentrate.

Several bacterial strains, including probiotic strains, have undergone evaluations for their safety and potential beneficial health effects. Some of these strains have been introduced into various markets, including that for infant products. However, certain probiotic strains have been linked to serious infections in infants, such as septicemia and meningitis. Given this, it is crucial to assess the safety of each probiotic strain, including those of Bifidobacterium, which is a common genus of probiotics. One such strain, Bifidobacterium bifidum OLB6378 (NITE BP-31), referred to as OLB6378 hereafter, has been selected for use in infants. To determine its genotoxicity and general toxicity potential, a heat-treated OLB6378 concentrate was subjected to various tests, including the bacterial reverse mutation test, in vitro chromosome aberration test, in vivo micronucleus test, and single- and 90-day oral gavage toxicity studies in rats. No significant differences were observed compared with negative controls in any of genotoxicity tests. The single-dose toxicity study employed dose levels of 560, 1,693, and 5,092 mg/kg, representing the total solid contents of culture concentrates containing OLB6378 (equivalent to 8.1 × 1011, 2.4 × 1012, and 7.4 × 1012 cells/kg of Bifidobacterium, respectively). In the 90-day toxicity study, dose levels of 280, 853, and 2,546 mg/kg/day were used (equivalent to 4.0 × 1011, 1.2 × 1012, and 3.7 × 1012 cells/kg/day, respectively). Importantly, the heat-treated OLB6378 concentrate did not induce any signs of toxicity in any of the conducted toxicity studies. In conclusion, the heat-treated OLB6378 concentrate exhibited no genotoxicity potential, and the no-observed-adverse-effect level in the 90-day toxicity study was determined to be 2,546 mg/kg/day (equivalent to 3.7 × 1012 cells/kg/day). This suggests that heat-treated OLB6378 can be safely utilized as a food source.

Highest concentration of breast-milk-derived exosomes in colostrum.

BACKGROUND: Exosomes are nanosized extracellular vesicles, that play important roles in intercellular immune regulation. They have potential therapeutic utility for neonatal diseases including necrotizing enterocolitis. Breast-milk-derived exosomes have recently shown beneficial effects on intestinal damage in vitro and in vivo. However, the chronological change in breast-milk-derived exosome concentrations after delivery are unclear. METHODS: In this prospective study, we enrolled 17 mothers who delivered premature infants admitted to a neonatal intensive care unit in Japan. We measured the consecutive concentrations of breast-milk-derived exosomes in the mothers for 48 weeks after delivery. RESULTS: The median concentration of breast-milk-derived exosomes was 1.62 × 108 particles/ml in colostrum, showing a significant decrease after 2 weeks (P < 0.01). There was no association between the exosome concentration in colostrum and maternal perinatal factors including parity, mode of delivery, maternal age, and gestational age at delivery. CONCLUSIONS: We concluded that breast-milk-derived exosomes were the richest in colostrum. Our basic data regarding breast-milk-derived exosomes are expected to aid in the clinical application of exosomes for treating neonatal diseases.

Chronological changes of serum exosome in preterm infants: A prospective study.

BACKGROUND: Exosomes, which are observed in all human fluid, including serum, are nanosized extracellular vesicles with a mechanism of intercellular communication. Potential clinical applications of exosomes in neonatal diseases have recently been discussed. However, the characteristics of exosomes in serum during early infancy is unclear. METHODS: In this prospective study, we evaluated the chronological changes in the concentration of serum-derived exosomes of 20 infants for 12 months after birth. RESULTS: The average concentration of serum-derived exosomes was 4.6 × 1010 particles/mL at birth and increased significantly until the age of 48 weeks. There was a moderate correlation between the gestational age and the concentration of serum-derived exosomes both at birth (r = 0.54, P = 0.01) and during the 8 weeks after birth (r = 0.48, P < 0.001). A multivariable analysis showed that gestational age at birth was associated with the concentration of serum-derived exosomes at birth (partial regression coefficient, 0.86; 95% confidence interval, 0.37-1.37; P = 0.002). CONCLUSIONS: The concentration of serum-derived exosomes in preterm infants increased both chronologically and by gestational age after birth. These basic data may help to further understand physiology of exosomes in preterm infants.

Effects of the intake of non-live Bifidobacterium bifidum on the faecal IgA of full-term infants: a double-blind, randomised, placebo-controlled study.

Bifidobacterium bifidum OLB6378 (OLB6378) was selected as a strain that enhances the production of secretory immunoglobulin A (IgA) in vitro. This ability of non-live OLB6378 has been shown by a clinical trial in preterm infants. In the present study, we examined whether non-live OLB6378 also enhances the production of secretory IgA, even in full-term infants. One hundred full-term infants were allocated to receive formula with (BbF group, 49 infants) or without non-live OLB6378 (PF group, 51 infants). Breastfeeding was prioritised, so infant formula was used for infants with breastfeeding difficulties. The intervention was initiated by five days of age. The faecal IgA concentration and OLB6378 level were determined at one, two, four, and eight weeks of age. Faecal IgA in the BbF group (1.04 ± 0.47 mg/g of faeces, n=45) was significantly higher than that in the PF group (0.85 ± 0.42 mg/g of faeces, n=49) at four weeks of age (p=0.047). OLB6378 was not detected in faeces at any age. This indicated that production of secretory IgA in full-term infants may also be enhanced by non-live OLB6378 intake.

Poor Bifidobacterial Colonization Is Associated with Late Provision of Colostrum and Improved with Probiotic Supplementation in Low Birth Weight Infants.

This study aimed to evaluate the association between bifidobacterial colonization in low birth weight infants and perinatal factors, including the timing of initial colostrum and the effect of probiotics on this colonization. In this non-randomized controlled trial, we enrolled 98 low-birth-weight infants from a neonatal intensive care unit (NICU) in Japan. Infants were divided into three groups: group N (no intervention), group H (received non-live bifidobacteria), and group L (received live bifidobacteria). The number of bifidobacteria in the infants' stools at 1 month of age was measured using real-time polymerase chain reaction (PCR). We divided infants into "rich bifidobacteria" (≥104.8 cells/g feces) and "poor bifidobacteria" (<104.8 cells/g feces) subgroups. The ratio of "rich bifidobacteria" infants was 20/31, 34/36, and 30/30 in groups N, H, and L, respectively. In group N, the "rich bifidobacteria" group received first colostrum significantly earlier than the "poor bifidobacteria" group (1 day vs. 4 days, P < 0.05). Compared with the N group, both groups H and L had a significantly high proportion of "rich bifidobacteria" infants (P < 0.05). Bifidobacterial colonization was poor in premature infants at 1 month compared with term infants, and the level of colonization was associated with the timing of initial provision of colostrum. Providing probiotics to premature infants can improve bifidobacterial colonization.

Probiotics and the development of very low birthweight infants: follow-up study of a randomised trial.

OBJECTIVE: To investigate the effect of Bifidobacterium bifidum OLB6378 on the development of very low birthweight (VLBW) infants at 18 months of corrected age. DESIGN: Long-term follow-up study of a cluster-randomised, placebo-controlled trial. PATIENTS: VLBW infants (birth weight <1500 g) born between January 2010 and March 2011 and managed at 19 neonatal intensive care unit facilities assigned to two groups to account for the effect of probiotic cross-contamination within facilities. INTERVENTIONS: For VLBW infants, administration of OLB6378 as a probiotic was started within 48 hours of birth and continued until the body weight reached 2000 g. MAIN OUTCOME MEASURES: At 18 months of corrected age, physical status and developmental quotient (DQ18) were assessed. The distribution of DQ18 scores was categorised into four levels of development: <70, significant developmental delay; 70-84, moderate developmental delay; 85-99, without developmental delay; ≥100, average development or better. RESULTS: Among 153 infants assigned to the OLB6378 administration group and 130 assigned to the placebo administration group, 102 and 105 infants, respectively, underwent the 18-month medical examination. The distribution of developmental levels (DQ18 scores <70, 70-84, 85-99 and ≥100) was significantly more favourable for OLB6378 administration (12, 12, 25 and 40 infants, respectively) than for placebo administration (15, 17, 23 and 24 infants, respectively) (ordered logistic regression analysis: partial correlation coefficient, 0.589; P value, 0.038). CONCLUSIONS: Although limited by assessment rates, result suggests that OLB6378 may have a beneficial effect on the psychological development in VLBW infants. CLINICAL TRIAL REGISTRATION: UMIN000002543.

Bifidobacterium and enteral feeding in preterm infants: cluster-randomized trial.

BACKGROUND: This study evaluated the benefit of Bifidobacterium bifidum OLB6378 (B. bifidum) in very low-birthweight (VLBW) infants (birthweight <1500 g) for the acceleration of enteral feeding. METHODS: A cluster-randomized, double-blind, placebo-controlled trial was conducted in 19 hospitals, divided into two groups: the B group (n = 10 hospitals; B. bifidum given to infants within 48 h of birth) and the P group (n = 9 hospitals; infants received a placebo). The primary outcome was establishment of enteral feeding after birth, defined as the postnatal day at which enteral feeding exceeded 100 mL/(kg/day). Secondary outcomes were defined as incidence of morbidity and somatic growth before discharge. RESULTS: Overall, 283 VLBW infants were enrolled in the study: B group, n = 153; and P group, n = 130. Enteral feeding was established within 21 days after birth in 233 infants, of whom 119 received B. bifidum and 114 received placebo until their bodyweight reached 2000 g. Enteral feeding was established significantly earlier in the B group, at 11.0 ± 3.6 days versus 12.1 ± 3.8 days in P group (P < 0.05). Infant growth during the stay in the neonatal intensive care unit was not different between groups, but the incidence of late-onset sepsis among all enrolled infants was significantly lower in the B group (3.9%, 6/153) than in the P group (10.0%, 13/130; P < 0.05). No differences were observed in the incidence of other adverse outcomes including mortality. CONCLUSIONS: B. bifidum in VLBW infants accelerated the establishment of enteral feeding after birth without increasing the incidence of adverse effects.

Strain-specific identification of Bifidobacterium bifidum OLB6378 by PCR.

The aim of the present study was to develop a strain-specific polymerase chain reaction (PCR) primer set for the detection of Bifidobacterium bifidum OLB6378 (OLB6378) that can serve as suitable probiotics for infants. The random amplified polymorphic DNA (RAPD)-PCR technique was used to obtain OLB6378-specific PCR products. One OLB6378-specific RAPD-PCR product was obtained after testing 97 RAPD primers, and was sequenced. Thirteen PCR primer sets were designed from the sequence. One PCR primer set was found to amplify one PCR product when genomic DNA of OLB6378 was used as template. The primer set did not amplify any PCR product when the other genomic DNA was used as template. The primer set was tested with 47 strains of B. bifidum and 20 strains of the other Bifidobacterium species. As a result, we developed an OLB6378-specific primer set, one that should be useful not only for the detection of OLB6378 but also for the quantification of OLB6378.

Safety evaluation of Propionibacterium freudenreichii ET-3 culture.

Propionibacterium freudenreichii ET-3 culture, a cell-free product of whey fermentation using P. freudenreichii ET-3 (7025), has been shown to promote the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA), and therefore, has potential use in the food and supplement industries. Although currently used as a food ingredient in Japan, the safety of this novel ingredient has not been previously evaluated through traditional toxicity testing. Therefore, here we report the results of standard toxicological testing performed on P. freudenreichii ET-3 culture. In a 4-week oral toxicity study, administration of 6000mg/kg body weight/day P. freudenreichii ET-3 culture was without compound-related adverse effects on clinical signs, body weights, food consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and gross and microscopic findings in male and female Sprague-Dawley rats. Furthermore, in vitro mutagenicity testing demonstrated that P. freudenreichii ET-3 culture was non-mutagenic in the bacterial reverse mutation assay using a standard battery of bacterial strains (Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA) and non-clastogenic in Chinese hamster lung cells in the mammalian chromosome aberration test. Together, the results of these studies support the safety of P. freudenreichii ET-3 culture for use in foods for human consumption.

Reduced thymic size and numbers of splenic CD4+ and CD8+ cells in artificially reared mouse pups.

The effect of early nutrition on the development of the immune tissue and T cells of mouse pups was examined. Newborn mice were divided into three experimental groups: mother-reared (MR) pups, pups that were fed on a milk substitute from the first day (AR-0), and the third day (AR-2), using a hand-feeding system. The average thymic size of the AR-2 pups was respectively significantly larger and smaller than that of the AR-0 and MR pups. In contrast, the splenic sizes of the AR-0 and AR-2 pups were greater than that of the MR pups. The numbers of CD4+CD8- and CD4-CD8+ cells in the spleen of the MR pups were significantly higher than those in the AR-0 pups. These results indicate that early nutrition affected the sizes of the thymus and spleen and the composition of CD4+CD8- or CD4-CD8+ T cells in the spleen.