Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Osteopontin Levels in Human Milk Vary Across Countries and Within Lactation Period: Data From a Multicenter Study.

OBJECTIVES: Osteopontin (OPN) is a multifunctional protein expressed in many cell types, tissues and body fluids with the highest concentrations found in milk; significantly higher in human than in bovine milk. Intervention studies have indicated beneficial effects of supplementing infant formula with bovine OPN. In this multicenter study, we determined the OPN content in human milk samples from 629 Chinese, Danish, Japanese and Korean mothers. METHODS: At each study site, milk samples were collected and analyzed for OPN and protein concentration using ELISA and infrared spectroscopy, respectively. RESULTS: A total of 829 milk samples from 629 women were included. When delivering the first sample, mean maternal age was 31.4 years (SD 4.0), and median infant age was 13.4 weeks (interquartile range 4.6-17.9). The median OPN concentration varied across sites; from 99.7 mg/L in Danish, 185.0 mg/L in Japanese, 216.2 mg/L in Korean to 266.2 mg/L in Chinese mothers (P < 0.001), corresponding to 1.3%, 2.4%, 1.8% and 2.7% of the total protein content (OPN/protein%) (P < 0.05), respectively. Based on 75 Chinese and 33 Japanese mothers delivering more than 1 sample, multilevel (mixed model) linear regression analysis showed a decrease in OPN concentration with infant age (ß = (-11.3), 95% confidence interval (CI) = (-13.9) to (-8.8) and ß = (-2.1), 95% CI = (-3.2) to (-0.9), respectively). CONCLUSIONS: In this large multicenter study, we observed statistically significant differences in the OPN concentration and the OPN/protein% in human milk samples between countries. Based on mothers delivering more than 1 sample, a significant decrease within the lactation period was observed.

T-cell epitope-containing hypoallergenic ß-lactoglobulin for oral immunotherapy in milk allergy.

BACKGROUND: Optimally hydrolyzed ß-Lactoglobulin (ßLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic ßLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of ßLg when preparing ßLg hydrolysates using food-grade trypsin. METHODS: We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of ßLg (ßLg102-124 ) and prepared an edible ßLg hydrolysate containing ßLg102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-ßLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum. RESULTS: In ßLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of ßLg102-124 with an increase in hydrolysis time in a dose-dependent manner. ßLg102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible ßLg hydrolysate retained ßLg102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE. CONCLUSIONS: Chymotrypsin activity inhibited the formation of ßLg102-124 in the trypsin hydrolysate of ßLg. This ßLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.

Effects of a Bovine Lactoferrin Formulation from Cow's Milk on Menstrual Distress in Volunteers: A Randomized, Crossover Study.

Dysmenorrhea is a highly prevalent complaint and highly undiagnosed gynecologic condition. Dairy products have a potential in the management of menstrual distress, and bovine lactoferrin can help the subjective dysphoria associated with dysmenorrhea. In the present study, we aimed to investigate the effects of a lactoferrin formulation isolated from cow's milk on menstrual symptoms in volunteers. A double-blind, randomized, placebo-controlled, crossover study of the iron-lactoferrin complex (FeLf) was performed in thirty-five healthy Japanese women. Participants received the 150 mg FeLf (per day) or placebo from day ten of the luteal phase to day four of the follicular phase. The Moos Menstrual Distress Questionnaire (MDQ) was measured for menstrual distress, and heart rate variability was measured as an index of autonomic nerve balance during menses. A visual analog scale for menstrual pain, and a verbal rating scale for quality of life during the first three days of menstruation were measured. The MDQ score for the automatic nervous system subscale was lower and the parasympathetic nervous system activity was greater in FeLf than in placebo for intention-to-treat or per-protocol populations. The other variables were not different between the groups. No treatment-related side effects were observed during the study. The results indicate that FeLf can provide a beneficial effect on the psychological symptoms in women affected by menstrual distress.

Dairy consumption, bone turnover biomarkers, and osteo sono assessment index in Japanese adults: A cross-sectional analysis of data from the Iwaki Health Promotion Project.

Purpose: Dairy foods are nutritional sources of calcium, phosphorus, protein, and other nutrients that improve bone health. However, the effects of dairy consumption on bone biomarkers in the Japanese population remain unclear. This study explored the association between dairy consumption and bone biomarkers in Japanese adults. Methods: This cross-sectional study was conducted as part of the Iwaki Health Promotion Project in Aomori, Japan. In total, 1063 adults were included in the analysis. Bone turnover marker levels were measured in local citizens during their annual medical checkups. The calcaneus osteo sono assessment index (OSI) was calculated using a quantitative ultrasound technique. The dietary intake of foods and nutrients was estimated using a food frequency questionnaire. Linear regression models were established using dairy consumption and bone biomarkers with adjustments. Statistic significance was considered by P < 0.05. Results: In multivariate models, the tartrate-resistant acid phosphatase 5b and parathyroid hormone concentrations were inversely associated with dietary dairy consumption after adjusting for age and sex. The undercarboxylated osteocalcin, a procollagen type I N-terminal peptide to bone alkaline phosphatase ratio, and OSI were the directly associated with dairy consumption in multivariate models with adjustment. Conclusions: Dairy consumption is partially associated with bone turnover biomarkers and OSI in adult Japanese participants. Habitual consumption of dairy foods may contribute to the nutritional supplementation for maintaining bone health, including turnover and structure. Clinical trial registry number and website where it was obtained: The Japanese Clinical Trials Registry (UMIN000040459), https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000046175.

Dietary Patterns Associated with General Health of Breastfeeding Women 1-2 Months Postpartum: Data from the Japanese Human Milk Study Cohort.

Background: The effects of dietary patterns on health outcome of lactating women remain unclear. Objectives: To describe the dietary patterns of lactating Japanese women and explore the association between dietary patterns and their general health. Methods: This study included 1096 lactating women from the Japanese Human Milk Study Cohort. The maternal diet during lactation 1-2 mo postpartum was determined using a FFQ. Dietary patterns were identified using a factor analysis based on the energy-adjusted intake of 42 food items. Trend associations between maternal and infant variables and quartiles of dietary pattern scores were tested, and logistic regression was performed to estimate the OR and 95% CI of maternal self-reporting anemia, constipation, rough skin, sensitivity to cold, and mastitis. Results: Four dietary patterns were identified in this study. The versatile vegetable diet, characterized by a high intake of vegetables, mushrooms, seaweeds, and tofu, was associated with maternal age, BMI prepregnancy and during the lactation periods, education, household income, and anemia. The plain Japanese diet contained a high intake of typical Japanese foods such as rice and miso soup and a low intake of bread and some confectioneries and was associated with maternal BMI during both periods. The salad vegetable diet, characterized by a high intake of raw vegetables and tomatoes with mayonnaise or dressing, was associated with parity and season in which data collection was conducted. The seafood diet, characterized by a high intake of fish, squid, octopus, shrimp, and shellfish, was associated with days postpartum and sensitivity to cold. Conclusions: Four dietary patterns were identified and were independently associated with socioeconomic factors. The versatile vegetables diet and seafood diet were associated with anemia and sensitivity to cold, respectively, among the participants. This trial was registered at the Japanese Clinical Trials Registry (https://center6.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000017649) as UMIN000015494.

Comparison of 30 Cytokines in Human Breast Milk between 1989 and 2013 in Japan.

Milk cytokines play a vital role in mucosal immunity during infancy by supporting immune development and functions. Although the maternal background characteristics influence milk cytokines, changes in cytokine levels across generations remain unclear. Colostrum (C, n = 48) and mature milk (MM, n = 49) samples were collected from lactating Japanese women in 1989 (2727 samples) and 2013 (1408 samples). Milk cytokines were comprehensively measured using a suspension array and immunosorbent assays. The positive rates and cytokine concentrations were compared between the two generations using logistic and multiple regression analyses. Twenty-eight cytokines tested positive in all sample groups (1989-C, 1989-MM, 2013-C, and 2013-MM). The median osteopontin (OPN) level was significantly higher in the 1989-C group than in the 2013-C group (318.1 vs. 137.5 µg/mL; p = 0.0016) but did not differ between the MM groups. The median TGF-ß1 level was significantly lower in the 1989-MM group than in the 2013-MM group (1056.2 vs. 1330.8 pg/mL; p = 0.008) but did not differ between the C groups. Most cytokines were comparable between generations, except for potential variation in the C-OPN and TGF-ß1 levels. Milk cytokine secretion may reflect temporal changes in maternal background characteristics; however, the results from the analysis of 30-year-old samples may have influenced the milk cytokine levels. Further studies are needed with a larger number of milk samples collected from the same individuals at multiple time points over a wide lactation period, with detailed data on the maternal and infant background characteristics and diets.

Ramen Consumption and Gut Microbiota Diversity in Japanese Women: Cross-Sectional Data from the NEXIS Cohort Study.

A cross-sectional study involving 224 healthy Japanese adult females explored the relationship between ramen intake, gut microbiota diversity, and blood biochemistry. Using a stepwise regression model, ramen intake was inversely associated with gut microbiome alpha diversity after adjusting for related factors, including diets, Age, BMI, and stool habits (ß = -0.018; r = -0.15 for Shannon index). The intake group of ramen was inversely associated with dietary nutrients and dietary fiber compared with the no-intake group of ramen. Sugar intake, Dorea as a short-chain fatty acid (SCFA)-producing gut microbiota, and γ-glutamyl transferase as a liver function marker were directly associated with ramen intake after adjustment for related factors including diets, gut microbiota, and blood chemistry using a stepwise logistic regression model, whereas Dorea is inconsistently less abundant in the ramen group. In conclusion, the increased ramen was associated with decreased gut bacterial diversity accompanying a perturbation of Dorea through the dietary nutrients, gut microbiota, and blood chemistry, while the methodological limitations existed in a cross-sectional study. People with frequent ramen eating habits need to take measures to consume various nutrients to maintain and improve their health, and dietary management can be applied to the dietary feature in ramen consumption.

Xanthophylls in Human Milk and Maternal Diet: A Cross-sectional Analysis of Data from the Japanese Human Milk Study Cohort.

Background: Maternal diet and sociodemographic factors influence xanthophyll concentration and composition in human milk. However, the importance of dietary patterns regarding the intake of fruits, vegetables, and xanthophylls remains unclear. Objective: The aim was to determine the composition of xanthophylls in the human milk of Japanese mothers and explore associations of xanthophylls with dietary and sociodemographic factors. Methods: This cross-sectional study was conducted in the early phase of the Japanese Human Milk Study. Xanthophyll content was measured using liquid chromatography at 30-36 d postpartum. Maternal intake of foods, nutrients, and dietary supplements was estimated using a food-frequency questionnaire. Linear regression models were established using xanthophylls, maternal diet, and sociodemographic factors. Results: Xanthophyll concentrations were measured in human milk from 118 mothers. The xanthophyll concentration varied among individuals. The median (IQR) concentrations of lutein, zeaxanthin, and ß-cryptoxanthin were 65.6 ng/mL (51.6-103.4 ng/mL), 18.6 ng/mL (12.9-25.8 ng/mL), and 15.6 ng/mL (9.0-26.0 ng/mL), respectively. In multivariate models, the lutein concentration was associated independently with dietary green vegetables, exclusive breastfeeding, and education (r 2 = 0.153 for the model; ß ± SE: 0.468 ± 0.198, 25.048 ± 10.222, and 13.460 ± 6.774; standardized ß = 0.210, 0.217, and 0.175; P = 0.019, 0.016, and 0.049 for dietary green vegetables, exclusive breastfeeding, and education, respectively). For zeaxanthin, exclusive breastfeeding was the most appropriate predictor (r 2 = 0.085; ß ± SE: 7.811 ± 3.300; standardized ß = 0.218; P = 0.020). The highest predictive power for human milk ß-cryptoxanthin was obtained with dietary ß-cryptoxanthin (r 2 = 0.258; ß ± SE: 0.089 ± 0.015; standardized ß = 0.468; P < 0.001), attributed to maternal citrus intake. Conclusions: ß-Cryptoxanthin in human milk was the xanthophyll most influenced by the maternal diet in Japanese women. The ß-cryptoxanthin concentration in human milk was reflected by the maternal ß-cryptoxanthin intake, mainly attributed to Japanese citrus consumption. This trial was registered in the Japanese Clinical Trials Registry (https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000017649) as UMIN000015494.

Milk Phospholipid Profiling Among Japanese Women with Differing Docosahexaenoic Acid Levels.

To characterize the milk phospholipids (PLs) profile using phosphorus-31 nuclear magnetic resonance (31P-NMR) and to investigate the effect of elevated milk docosahexaenoic acid (DHA) levels on PLs profiles in Japanese mothers. Methods: Milk samples from eligible patients with high and low DHA from a former cross-sectional study (n = 20; n = 10 for each group) were included. Fifteen milk PLs were analyzed using 31P-NMR, and the profiles were compared group-wise using Mann-Whitney U-test. The P value of <0.05 was considered statistically significant. Results: The median DHA content in milk was 1.13% and 0.29% for the high and low milk DHA groups, respectively. Twelve PLs, excluding lysophosphatidylserine, cardiolipin, and phosphatidylglycerol, were detected in all participants with 100% positive results. The median concentrations and proportions of total PLs, sphingophospholipids, and glycerophospholipids were comparable between groups. The proportions of choline-containing glycerophospholipid were significantly higher in the high milk DHA group than that in the low milk DHA group (24.09% [median, interquartile range: 23.08%-26.38%] and 21.41% [20.74%-22.84%], P = 0.019). Although the proportions of phosphatidylinositol were significantly lower in the high milk DHA group than that in the low milk DHA group (6.62% [5.75%-6.72%] versus 7.63% [7.11%-8.16%], P = 0.002), while that of phosphatidylcholine (21.90% [18.51%-23.22%] versus 19.78% [18.17%-20.26%], P = 0.059) and alkyl-acyl phosphatidylcholine (0.60% [0.40%-0.74%] versus 0.33% [0.14%-0.51%], P = 0.059) were higher in the former than that in the latter. Conclusions: Our results were comparable to that of the previous literature. Large variations in the milk DHA might affect the composition of choline-containing glycerophospholipids in Japanese mothers. However, possible confounders were not excluded in the study populations.

Probiotic Supplementation and Human Milk Cytokine Profiles in Japanese Women: A Retrospective Study from an Open-Label Pilot Study.

The benefits of probiotic supplementation to lactating mothers on human milk cytokines are inconclusive. Thus, we performed a comprehensive open-label pilot trial analysis of 27 human milk cytokines in lactating women with allergies (one to three months postpartum) to determine the effect of supplementation with a mixture of new probiotic strains. Participants voluntarily joined the probiotic (n = 41) or no supplementation control (n = 19) groups. The probiotic group took three probiotic tablets (Lactobacillus casei LC5, Bifidobacterium longum BG7, and Bacillus coagulans SANK70258) daily for one to three months postpartum. Milk samples were collected at one, two, and three months postpartum, and cytokine levels were measured using multiplex assays. The effects were analyzed using multivariate regression models. Eleven cytokines showed a positive rate of over 50% in the milk samples throughout testing in both groups. The positive rates of IL-1 receptor antagonist and IL-7 changed significantly with lactation progression in logistic regression models after adjusting for time and supplementation, whereas rates of other cytokines showed no significant differences. The lactational change patterns of IL-10 concentrations differed significantly between the two groups. A short-term supplementation of probiotics affects human milk cytokine levels in lactating women with a possible placebo effect still existing. Future placebo-controlled studies are needed to support these results, based on the estimated sample sizes in this study.

Cohort profile: Japanese human milk study, a prospective birth cohort: baseline data for lactating women, infants and human milk macronutrients.

PURPOSE: The Japanese Human Milk Study, a longitudinal prospective cohort study, was set up to clarify how maternal health, nutritional status, lifestyle and sociodemographic and economic factors affect breastfeeding practices and human milk composition. This would eventually determine factors affecting the growth and development of infants and children. PARTICIPANTS: A total of 1210 Japanese lactating women who satisfied the inclusion criteria, were invited across the country at various participating sites, between 2014 and 2019. Finally a total of 1122 women were enrolled in this study. FINDINGS TO DATE: Among 1122 eligible participants, mean age at delivery was 31.2 (SD 4.4) years and mean prepregnancy BMI was 20.8 (SD 2.7). Among these women, 35% were previously nulliparous and 77.7% had college, university or higher education. The mean gestational period was 39.0 (SD 1.3) weeks. Caesarean section was reported among 11.9%; mean infant birth weight was 3082 (SD 360) g. Of the infants, 53.7% were male. Overall, our participants appeared to be healthier than the general population in Japan. Analyses of the 1079 eligible human milk samples obtained at the first and second months postpartum showed the following composition: carbohydrate, 8.13 (SD 0.32) g/100 mL; fat, 3.77 (SD 1.29) g/100 mL; and crude protein, 1.20 (SD 0.23) g/100 mL. We also analysed osteopontin, fatty acid, vitamin D and phospholipid levels in limited subcohorts of the samples. FUTURE PLANS: Follow-up surveys will be conducted to obtain milk samples every 2 months for 12 months and to investigate mother and child health until the children reach 5 years of age. These will be completed in 2024. We plan to longitudinally analyse the composition of macronutrients and various bioactive factors in human milk and investigate the lifestyle and environmental factors that influence breastfeeding practices, maternal and child health, and child development. TRIAL REGISTRATION NUMBER: UMIN000015494; pre-results.

Association of DHA Concentration in Human Breast Milk with Maternal Diet and Use of Supplements: A Cross-Sectional Analysis of Data from the Japanese Human Milk Study Cohort.

BACKGROUND: DHA (22:6n-3) is essential for neurodevelopment in children, and its concentration in human breast milk is historically high in Japan. Dietary patterns in Japan might affect the fatty acid (FA) composition among lactating mothers. OBJECTIVES: This study aimed to characterize the composition of milk FAs and to identify any dietary and sociodemographic factors associated with the variability of DHA concentration in breast milk in the Japanese population. METHODS: This cross-sectional study was performed as part of the Japanese Human Milk Study. Milk FAs were analyzed by GC at 1-6 mo postpartum, and maternal diet was estimated using an FFQ, including 11 types and cooking methods of seafoods, and the use of DHA supplements. The association of milk DHA with maternal diet and sociodemographic factors was investigated. RESULTS: Milk FA concentrations were measured in 78 mothers, including 24 who routinely used DHA supplements. The DHA concentration in milk (overall median: 0.62%; IQR: 0.47%-0.78%) was higher in women who took DHA supplements than in women who had never used DHA supplements (0.74%compared with 0.55%; P = 0.011). A linear regression model showed the association of milk DHA concentration with maternal dietary intake of grilled fish (ß ± SE: 0.006 ± 0.003; standardized ß: 0.234; r 2 = 0.232, P = 0.036) after adjustment for DHA supplementation status, maternal and infant age, maternal BMI, and infant birth weight. Other FA concentrations were consistent, whereas caproic acid (6:0), undecylic acid (11:0), pentadecylic acid (15:0), palmitoleic acid (16:1n-7), and vaccenic acid (18:1n-7) varied by DHA supplementation status. CONCLUSIONS: The DHA concentration in human milk may be influenced by maternal grilled fish consumption and frequent DHA supplementation in lactating Japanese women. Milk DHA concentrations may reflect a dietary habit in Japanese mothers.This trial was registered at www.umin.ac.jp/ctr as UMIN000015494.

Maternal Health and Nutrition Status, Human Milk Composition, and Growth and Development of Infants and Children: A Prospective Japanese Human Milk Study Protocol.

It is unknown whether maternal health and nutrition are related to human milk composition or growth and development of infants and children. Here, we describe a protocol for a prospective five-year cohort study to clarify (i) how maternal health and nutrition, socioeconomic factors, and lifestyles affect human milk composition, and (ii) whether these are associated with growth and development of infants and children. In our study, we recruited 1210 Japanese mothers with singleton pregnancies from 73 obstetrics clinics and hospitals across Japan, between 2014 and 2019. We will measure the following: health information regarding maternal-child dyads using a self-administered questionnaire, maternal nutrition during breastfeeding using a Brief self-administrated Diet History Questionnaire, the development of infants and children using the Kinder Infant Development Scale, and the stress related to child rearing using the Mother's Child Care Stress Scale. Simultaneously, we will collect human milk every 2 months during the first year after birth to measure its composition and levels of macronutrients. This study will generate useful data to investigate whether health status, nutritional status, lifestyle, and socioeconomic factors affect human milk composition and the growth and development of infants and children.

Effects of Probiotic Supplementation on TGF-ß1, TGF-ß2, and IgA Levels in the Milk of Japanese Women: An Open-Label Pilot Study.

Background: Dietary probiotics supplementation in lactating mothers may help prevent allergic disease in infants. However, owing to a lack of consistency in nutritional and safety outcomes associated with probiotics, this topic remains controversial. Methods: In this open-label pilot trial conducted between April 2013 and December 2013, we evaluated the safety of probiotic supplementation with 5 × 109 CFU of Lactobacillus casei LC5, 5 × 109 CFU of Bifidobacterium longum BG7, and 2 × 108 CFU of Bacillus coagulans SANK70258 in lactating women who exhibited allergies for 2 months (1-3 months postpartum); we also evaluated the effects of probiotic supplementation on transforming growth factor-ß (TGF-ß) and immunoglobulin A (IgA) levels in human milk. Participants self-selected to join the probiotic (n = 41; age [median (interquartile range [IQR]), y] 33 [27-39], body mass index [BMI] [median (IQR), kg/m2] 21.8 [19.5-22.8]) or no supplementation control group (n = 19; age [median (IQR), y] 33 [23-43], BMI [median (IQR), kg/m2) 19.6 [18.4-22.1]). Probiotics (three tablets) received were taken as daily supplements. Milk samples were collected at 1, 2, and 3 months postpartum, and TGF-ß1, TGF-ß2, and IgA levels were measured. Results: No adverse effects were observed in the probiotic group, according to the self-recorded diary during the study period. Milk IgA decreased with increasing postpartum months in both groups. In contrast, TGF- ß1 and ß2 were not affected by lactation periods, and showed different patterns over time between the two groups. TGF-ß1, TGF-ß1, and IgA levels were significantly correlated at baseline (respectively p < 0.05). However, the correlation between TGF-ß1 and IgA became non-significant by the end of the intervention (p = 0.063). Conclusion: Altogether, probiotic supplementation was tolerated with respect to no dropout and 91.5% adherence. Although probiotic supplementation might affect human milk TGF-ß levels, a positive effect of probiotic supplementation was not entirely supported. Future placebo-controlled studies are needed to further support the efficacy and safety of probiotic supplementation. Clinical Trial Registration: www.umin.ac.jp/ctr/, identifier: UMIN000036059.

Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.

Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of ß-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of ß-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag.

Serum albumin forms a lactoferrin-like soluble iron-binding complex in presence of hydrogen carbonate ions.

The iron-lactoferrin complex is a common food ingredient because of its iron-solubilizing capability in the presence of hydrogen carbonate ions. However, it is unclear whether the formation of a stable iron-binding complex is limited to lactoferrin. In this study, we investigated the effects of bovine serum albumin (BSA) on iron solubility and iron-catalyzed lipid oxidation in the presence of hydrogen carbonate ions. BSA could solubilize >100-fold molar equivalents of iron at neutral pH, exceeding the specific metal-binding property of BSA. This iron-solubilizing capability of BSA was impaired by thermally denaturing BSA at ≥ 70 °C for 10 min at pH 8.5. The resulting iron-BSA complex inhibited iron-catalyzed oxidation of soybean oil in a water-in-oil emulsion measured using the Rancimat test. Our study is the first to show that BSA, like lactoferrin, forms a soluble iron-binding complex in the presence of hydrogen carbonate ions.

Iron-lactoferrin complex reduces iron-catalyzed off-flavor formation in powdered milk with added fish oil.

UNLABELLED: The iron-lactoferrin complex (FeLf) is useful for dietary iron supplementation. However, the effects of FeLf on iron-catalyzed off-flavors in lipid-containing food products have not been reported. In this study, we investigated the effects of FeLf on off-flavors development during the production and storage of powdered milk with added fish oil. Powdered milk samples were formulated with FeLf or iron (II) sulfate, then stored at 37 °C for 5 mo. A sensory evaluation revealed that FeLf delayed the development of oxidized flavor and reduced metallic taste in the powdered milk compared with iron (II) sulfate. Headspace gas chromatography-mass spectrometry analysis showed that oxidized volatile compounds, such as pentanal, hexanal, heptanal, octanal, 1-penten-3-one, (Z)-4-heptenal, (E, E)-2,4-heptadienal, and (E)-2-octenal, were less developed in the powdered milk containing FeLf than in that containing iron (II) sulfate. Iron and lactoferrin (Lf) were retained in the high-molecular-weight (>10000 Da) fraction of the reconstituted FeLf-containing powdered milk after its manufacture and storage, whereas the antigenicity of Lf was lost after ultrahigh-temperature processing at 120 °C for 5 s. These results suggest that FeLf reduces the iron-catalyzed off-flavors that develop during the production and storage of powdered milk. The stable iron-holding property of FeLf contributes to the inhibition of oxidized and metallic volatile formation, although the loss of Lf antigenicity did not affect the stability of FeLf and the iron-catalyzed formation of off-flavors in the powdered milk. Consequently, FeLf is a suitable candidate for the simultaneous supplementation of a single food with iron and fish oil. PRACTICAL APPLICATION: The supplementation of food products with iron and fish oil is a useful approach to redressing their inadequate intake in many populations. The iron-lactoferrin complex can protect food products against the off-flavors caused by iron-catalyzed lipid oxidation. Our results show that the iron-lactoferrin complex is useful for the simultaneous fortification of foods and nutraceuticals with iron and fish oil because this complex also reduces the formation of iron-catalyzed off-flavors in powdered milk enriched with fish oil.