Fruit and vegetable consumption before and during pregnancy and developmental delays in offspring aged 2 years in Japan.

The association between fruit and vegetable consumption before and during pregnancy and offspring's physical growth has been well reported, but no study has focused on offspring's neurological development. We aimed to explore the association between maternal fruit and vegetable consumption before and during pregnancy and developmental delays in their offspring aged 2 years. Between July 2013 and March 2017, 23 406 women were recruited for the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study. Fruit and vegetable consumption was calculated using FFQ, and offspring's developmental delays were evaluated by the Ages & Stages Questionnaires, Third Edition (ASQ-3) for infants aged 2 years. Finally, 10 420 women and 10 543 infants were included in the analysis. Totally, 14·9 % of children had developmental delay when screened using the ASQ-3. Women in the highest quartile of vegetable consumption from pre-pregnancy to early pregnancy and from early to mid-pregnancy had lower odds of offspring's developmental delays (OR 0·74; 95 % CI 0·63, 0·89 and OR 0·70; 95 % CI 0·59, 0·84, respectively) than women in the lowest quartile. Women in the highest quartile of fruit consumption from early to mid-pregnancy had lower odds of offspring's developmental delays (OR 0·78; 95 % CI 0·66, 0·92) than women in the lowest quartile. In conclusion, high fruit and vegetable consumption before and during pregnancy was associated with a lower risk of developmental delays in offspring aged 2 years.

Dietary patterns before and during pregnancy and small for gestational age in Japan: a prospective birth cohort study.

BACKGROUND: Although small for gestational age (SGA) is a serious problem worldwide, the association of dietary patterns before and during pregnancy with SGA risk is unclear. We evaluated this association among Japanese pregnant women using three methods: reduced rank regression (RRR) and partial least squares (PLS), methods for extracting dietary patterns that can explain the variation of response variables, and principal component analysis (PCA), a method for extracting dietary patterns of the population. METHODS: Between July 2013 and March 2017, 22,493 pregnant women were recruited to the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study, a population-based prospective birth cohort study in Japan. Information on dietary intake was obtained using food frequency questionnaires, and dietary patterns were extracted using RRR, PLS, and PCA. Information on birth weight was obtained from obstetric records, and the birth weight SD score and SGA were defined by the method of the Japan Pediatric Society. The associations of dietary patterns with birth weight SD score and SGA risk were investigated using multiple linear regression and multiple logistic regression, respectively. RESULTS: A total of 17,728 mother-child pairs were included. The birth weight SD score was 0.15 ± 0.96, and the prevalence of SGA was 6.3%. The dietary patterns extracted by RRR and PLS were similar and characterized by a high intake of cereals and fruits and a low intake of alcoholic and non-alcoholic beverages in both pre- to early pregnancy and from early to mid-pregnancy. Higher adoption of the RRR and PLS patterns in both periods was associated with an increased birth weight SD score and lower risk of SGA. In contrast, the PCA1 pattern was not associated with birth weight SD score or SGA risk in either period. Although the PCA2 pattern was associated with increased birth weight SD score from early to mid-pregnancy, no other associations with birth weight SD score or SGA risk were observed. CONCLUSIONS: The dietary pattern with a high intake of cereals and fruits and a low intake of alcoholic and non-alcoholic beverages before and during pregnancy was associated with a decreased SGA risk in Japan.

Neoxanthin in young vegetable leaves prevents fat accumulation in differentiated adipocytes.

The present study aimed to investigate the effect of young leaves on fat accumulation in differentiated 3T3-L1 adipocytes. A potent preventive effect on fat accumulation was observed in fractions of young leaves of spinach, beet, and arugula extracted with a low-polarity solvent (hexane:acetone:ethanol:toluene = 10:6:7:6). This effect was seemingly associated with the leaf carotenoid content, including lutein, ß-carotene, and neoxanthin. Among these, only neoxanthin, with the characteristic structure of 5,6-monoepoxide and an allenic bond, significantly prevented fat accumulation in a dose-dependent manner. The preventive effect and carotenoid content, including neoxanthin, of these young leaves did not differ from those of the corresponding adult leaves. Therefore, our study demonstrated that young vegetable leaves, such as spinach, beet, and arugula leaves, contained neoxanthin, which prevented fat accumulation in adipocytes in vitro. In the future, the effectiveness of such young leaves and neoxanthin should be investigated in vivo.

Living environments long-term after the Great East Japan Earthquake and nutritional intake among recent mothers.

BACKGROUND AND OBJECTIVES: Although large-scale natural disasters and the resultant changes in living environments worsen dietary habits among adults immediately after the disasters, whether this association remains for a long period is unclear. This is particularly important for recent mothers because lactating women require additional nutrition for milk production. Thus, we investigated the association of living environments with dietary habits and nutritional intake of recent mothers between four and seven years after the Great East Japan Earthquake (11th March, 2011). METHODS AND STUDY DESIGN: We analyzed 8,551 mothers who participated to the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study. Living environments were characterized into four categories: "same home before the earthquake", "rental housing", "reconstructed home", and "acquaintance's home". Dietary habits and nutritional intake were evaluated using a food frequency questionnaire answered 12 months after their deliveries (the questionnaire was answered between March 2015 and July 2018). RESULTS: Multiple linear regression analyses demonstrated that mothers in 'rental housing' or 'reconstructed home' had a significantly lower intake of almost all nutrients or certain nutrients, respectively, compared with those residing in 'same home before the earthquake'. However, fewer significant differences were detected between the nutritional intake of the mothers lodging in an 'acquaintance's home' and that of those living in 'same home before the earthquake'. CONCLUSIONS: Our findings indicate that living environments long-term after largescale disasters are associated with dietary habits and nutritional intake among recent mothers.

Fruit and vegetable consumption before and during pregnancy and birth weight of new-borns in Japan: the Tohoku medical megabank project birth and three-generation cohort study.

BACKGROUND: Associations of fruit and vegetable consumption before and during pregnancy with birth weight of new-borns and the risk of low birth weight (LBW) remain unclear. METHODS: Between July 2013 and March 2017, we recruited 23,406 pregnant women in the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study). Fruit and vegetable consumption before and during pregnancy was calculated using food frequency questionnaires. Information regarding birth weight was obtained from medical records, and LBW was defined as < 2500 g. We used a multivariable linear regression model and a multivariate logistic regression model to assess associations between fruit and vegetable consumption and birth weight/risk of LBW. RESULTS: In total, 17,610 women were included in the analysis. Mean birth weight was 3061.8 ± 354.1 g, and 5.4% of the new-borns had LBW. Compared to women in the lowest quartile of fruit consumption between pre- and early pregnancy, women in the highest quartile had heavier new-borns (ß = 49.4; 95% CI: 34.1-64.7) and lower risk of LBW (OR: 0.79; 95% CI: 0.65-0.95). Women in the highest quartile of fruit consumption from early to mid-pregnancy also had heavier new-borns (ß = 32.3; 95% CI: 17.1-47.6), and they tended to have lower risk of LBW (OR: 0.83; 95% CI: 0.69-1.01). Results of analysing the association between changes in fruit consumption from pre- to mid-pregnancy and birth outcomes revealed that women with continuous high fruit consumption from pre- to mid-pregnancy had heavier new-borns (ß = 37.6; 95% CI: 25.0-50.3), but they did not have lower risk of LBW (OR: 0.90; 95% CI: 0.77-1.06). Associations involving vegetable consumption and birth weight/risk of LBW were not observed. CONCLUSIONS: Fruit consumption before and during pregnancy was positively associated with birth weight of new-borns and negatively associated with risk of LBW.

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu).

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

Growth characteristics of Lactobacillus brevis KB290 in the presence of bile.

Live Lactobacillus brevis KB290 have several probiotic activities, including immune stimulation and modulation of intestinal microbial balance. We investigated the adaptation of L. brevis KB290 to bile as a mechanism of intestinal survival. Strain KB290 was grown for 5 days at 37 °C in tryptone-yeast extract-glucose (TYG) broth supplemented with 0.5% sodium acetate (TYGA) containing 0.15%, 0.3%, or 0.5% bile. Growth was determined by absorbance at 620 nm or by dry weight. Growth was enhanced as the broth's bile concentration increased. Bile-enhanced growth was not observed in TYG broth or with xylose or fructose as the carbon source, although strain KB290 could assimilate these sugars. Compared with cells grown without bile, cells grown with bile had twice the cell yield (dry weight) and higher hydrophobicity, which may improve epithelial adhesion. Metabolite analysis revealed that bile induced more lactate production by glycolysis, thus enhancing growth efficiency. Scanning electron microscopy revealed that cells cultured without bile for 5 days in TYGA broth had a shortened rod shape and showed lysis and aggregation, unlike cells cultured for 1 day; cells grown with bile for 5 days had an intact rod shape and rarely appeared damaged. Cellular material leakage through autolysis was lower in the presence of bile than in its absence. Thus lysis of strain KB290 cells cultured for extended periods was suppressed in the presence of bile. This study provides new role of bile and sodium acetate for retaining an intact cell shape and enhancing cell yield, which are beneficial for intestinal survival.

Growth and bile tolerance of Lactobacillus brevis strains isolated from Japanese pickles in artificial digestive juices and contribution of cell-bound exopolysaccharide to cell aggregation.

Cell-bound exopolysaccharide (EPS) of the aggregable strain Lactobacillus brevis KB290 isolated from traditional Japanese pickles has been reported to protect against the effects of bile. However, there are no reports of bile tolerance mechanisms for other L. brevis strains that have aggregability. To elucidate the mechanism of bile tolerance of L. brevis KB290, we found 8 aggregable L. brevis strains out of 121 L. brevis strains isolated from traditional Japanese fermented pickles. We estimated their growth in artificial digestive juice and the amount of cell-bound EPS. We found 3 types of aggregation for these strains: filiform (<1 mm), medium floc (1-5 mm), or large floc (>5 mm). There was no significant difference in growth between nonaggregable and aggregable strains in the artificial digestive juice. The large floc strains selected from the aggregation strains showed significantly higher growth in the artificial digestive juice than nonaggregable strains. In medium and large floc strains, cell-bound EPS, mainly consisting of glucose, N-acetylglucosamine, and N-acetylmannosamine, were observed. The amount of EPS and each strain's growth index showed a positive correlation. We conclude that aggregable L. brevis strains were also protected by cell-bound EPS.

Cellular fatty acid composition and exopolysaccharide contribute to bile tolerance in Lactobacillus brevis strains isolated from fermented Japanese pickles.

Bile tolerance is a fundamental ability of probiotic bacteria. We examined this property in 56 Lactobacillus brevis strains isolated from Japanese pickles and also evaluated cellular fatty acid composition and cell-bound exopolysaccharide (EPS-b) production. The bile tolerance of these strains was significantly lower in modified de Man - Rogosa - Sharpe (MRS) medium (without Tween 80 or sodium acetate) than in standard MRS medium. Aggregating strains showed significantly higher bile tolerance than nonaggregating strains in MRS medium, but there was no significant difference in the modified MRS media. The relative octadecenoic acid (C18:1) content of the 3 most tolerant aggregating and nonaggregating strains was significantly higher when bile was added to MRS. In MRS without Tween 80, the relative C18:1 content was only marginally affected by addition of bile. In MRS without sodium acetate, only the 3 most tolerant nonaggregating strains increased their relative C18:1 content in the presence of bile. Meanwhile, culture in MRS without sodium acetate reduced EPS-b production in aggregating strains. In conclusion, both EPS-b and cellular fatty acid composition play important roles in bile tolerance of pickle-derived L. brevis.

Clusters in Infant Environmental Factors Influence School-Age Children's Vegetable Preferences in Japan.

It remains unclear how the various environmental factors are combined in practice to influence vegetable preferences in school-aged children. This study aimed to clarify the environmental factors during infancy and their association with vegetable preference in school-aged children. To find clusters of early childhood environmental factors, we conducted a factor analysis on 58 items related to early childhood environmental factors and a k-means cluster analysis using the factors obtained. The association of the extracted factors and clusters with vegetable preferences was assessed by multiple regression analysis. Twelve factors relating to vegetable eating, cooking and harvesting experience, and parental attitudes were extracted by factor analysis. Three clusters, "low awareness of experiences", "high awareness" and "low positive encouragement", were then extracted. In the multiple regression analysis, all 12 factors were found to be associated with vegetable preferences. Furthermore, it was found that the "high awareness" group had a significantly higher score for vegetable preference than the "low awareness of experiences" group (ß = 0.56, 95% CI: 0.37-0.74). Thus, the study found that environmental factors during infancy, in isolation and combination, influenced vegetable preferences in school-aged children. Assessing the combination of various environmental factors during infancy may contribute to a better understanding of future vegetable preferences.

Identification and Safety Assessment of Enterococcus casseliflavus KB1733 Isolated from Traditional Japanese Pickle Based on Whole-Genome Sequencing Analysis and Preclinical Toxicity Studies.

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required.

Cell-bound exopolysaccharides of Lactobacillus brevis KB290: protective role and monosaccharide composition.

We examined the survivability of Lactobacillus brevis KB290 and derivative strain KB392 in artificial digestive juices and bile salts. The strains have similar membrane fatty acids but different amounts of cell-bound exopolysaccharides (EPS). In artificial digestive juices, KB290 showed significantly higher survivability than KB392, and homogenization, which reduced the amount of EPS in KB290 but not in KB392, reduced the survivability only of KB290. In bile salts, KB290 showed significantly higher survivability than KB392, and cell-bound EPS extraction with EDTA reduced the survivability of only KB290. Transmission electron microscopy showed there to be a greater concentration of cell-bound EPS in KB290 than in either KB392 or EDTA-treated or homogenized KB290. We conclude that KB290's cell-bound EPS (which high performance liquid chromatography showed to be made up of glucose and N-acetylglucosamine) played an important role in bile salt tolerance.

Neoxanthin is undetectable in human blood after ingestion of fresh young spinach leaf.

In a previous study, we demonstrated that the carotenoid neoxanthin was contained in young leafy vegetables including spinach and showed a fat accumulation inhibitory effect in vitro. To evaluate the bioavailability of neoxanthin, a raw young spinach leaf (100 g day-1 for 4 weeks) intake test was performed on 14 participants (36.5 ± 8.0 years; male:female ratio = 9:5). Neoxanthin, neochrome, ß-carotene, and lutein concentration in the spinach and blood of participants (before and after the test) was measured using high performance liquid chromatography. Neither neoxanthin nor neochrome was detected in the blood samples, whereas ß-carotene and lutein concentration significantly increased (1.4- and 1.9-fold, respectively) during testing. Neoxanthin bioavailability in humans is low; thus, it is unlikely to have a fat accumulation inhibitory effect in vivo, contrary to the result in vitro. Ingesting the leafy vegetables raw can help maintain high neoxanthin levels, but it is not beneficial for neoxanthin bioavailability.

Definition of a Dietary Pattern Expressing the Intake of Vegetables and Fruits and Its Association with Intestinal Microbiota.

Daily dietary habits directly or indirectly influence the intestinal microbiota, and the resulting changes in its composition and metabolic activity alter the health conditions of the host. Although many studies have analyzed the association between individual nutrients/food items and intestinal microbiota, the assessment of the diet and intestinal microbiota from a macroscopic perspective has not yet been performed in Japan. Therefore, we focused on vegetables and fruits and aimed to identify dietary patterns of high intake of these foods and to examine their relationship with the intestinal microbiota. This cross-sectional study included 1019 healthy individuals aged ≥20 years in a rural area in northern Japan. Six dietary patterns were detected by factor analysis using the brief-type self-administered diet history questionnaire (BDHQ) data to identify the "vege pattern", which was the dietary pattern rich in vegetables and fruits. Permutational multivariate analysis of variance revealed changes in ß-diversity according to dietary patterns. In multivariable-adjusted models, the adherence to the vege pattern was positively correlated with α-diversity. This is the first study to reveal a correlation between intestinal microbiota and dietary habits rich in vegetables and fruits in a rural area of Japan.

Association of Plasma Lipopolysaccharide-Binding Protein Concentration with Dietary Factors, Gut Microbiota, and Health Status in the Japanese General Adult Population: A Cross-Sectional Study.

The influx of intestinal bacteria-derived lipopolysaccharide (LPS) into the blood has attracted attention as a cause of diseases. The aim of this study is investigating the associations between the influx of LPS, dietary factors, gut microbiota, and health status in the general adult population. Food/nutrient intake, gut microbiota, health status and plasma LPS-binding protein (LBP; LPS exposure indicator) were measured in 896 residents (58.1% female, mean age 54.7 years) of the rural Iwaki district of Japan, and each correlation was analyzed. As the results, plasma LBP concentration correlated with physical (right/left arms' muscle mass [ß = -0.02, -0.03]), renal (plasma renin activity [ß = 0.27], urine albumin creatinine ratio [ß = 0.50]), adrenal cortical (cortisol [ß = 0.14]), and thyroid function (free thyroxine [ß = 0.05]), iron metabolism (serum iron [ß = -0.14]), and markers of lifestyle-related diseases (all Qs < 0.20). Plasma LBP concentration were mainly negatively correlated with vegetables/their nutrients intake (all ßs ≤ -0.004, Qs < 0.20). Plasma LBP concentration was positively correlated with the proportion of Prevotella (ß = 0.32), Megamonas (ß = 0.56), and Streptococcus (ß = 0.65); and negatively correlated with Roseburia (ß = -0.57) (all Qs < 0.20). Dietary factors correlated with plasma LBP concentration correlated with positively (all ßs ≥ 0.07) or negatively (all ßs ≤ -0.07) the proportion of these bacteria (all Qs < 0.20). Our results suggested that plasma LBP concentration in the Japanese general adult population was associated with various health issues, and that dietary habit was associated with plasma LBP concentration in relation to the intestinal bacteria.

Relationships between fatty acid composition and bile tolerance in lactobacillus isolates from plants and from non-plant materials.

Twenty plant-derived and 18 non-plant-derived strains of Lactobacillus casei were compared for their growth in tryptone - yeast extract - glucose broth containing 0.3% bile by measuring absorbance at a wavelength of 620 nm after 24 h of incubation at 37 °C. Bile tolerance - a fundamental probiotic property - was calculated by dividing the experimental data by control values (growth without bile). We found that bile tolerance was strain specific but that the average bile tolerance of the plant-derived strains was significantly (P < 0.05) lower than that of the non-plant-derived strains tested. All tested strains could not deconjugate sodium taurocholate, indicating that the difference in bile tolerance was not due to the ability to deconjugate bile. The fatty acid compositions of the test strains with and without exposure to 0.3% bile were investigated, and a statistical correlation analysis between these compositions and their bile tolerance was conducted. The fatty acids correlated with bile tolerance differed between plant and non-plant lactobacilli. This is the first report to show that the origin (i.e., growth environment) of lactobacilli affects their fatty acid composition, which in turn, appears to be related to their bile tolerance.

Enterococcus casseliflavus KB1733 Isolated from a Traditional Japanese Pickle Induces Interferon-Lambda Production in Human Intestinal Epithelial Cells.

The association between lactic acid bacteria (LAB) and their immunostimulatory effects has attracted considerable attention; however, it remains unclear whether LAB can induce interferon-lambdas (IFN-λs) in human epithelial cells under conditions that do not mimic infection. In this study, we first employed a reporter assay to screen for a potential strain capable of inducing IFN-λ3 among 135 LAB strains derived from traditional Japanese pickles. Next, we assessed the strain's ability to induce the expression of IFN-λ genes and interferon-stimulated genes (ISGs), and to produce IFN-λs. As a result, we screened and isolated Enterococcus casseliflavus KB1733 (KB1733) as a potential strain capable of inducing IFN-λ3 expression. Furthermore, we clarified that KB1733 induced the expression of IFN-λ genes and ISGs related to antiviral functions, and that KB1733 induced IFN-λ1 and -λ3 expression in a dose-dependent manner up to 10 µg/mL. In addition, KB1733 significantly increased IFN-λ1 production compared to Enterococcus casseliflavus JCM8723T, which belongs to the same genera and species as KB1733. In conclusion, we isolated a unique LAB strain from traditional Japanese pickles that is capable of stimulating IFN-λ production, although further study is needed to investigate how KB1733 protects against viruses in mice and humans.

Grain consumption before and during pregnancy and birth weight in Japan: the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study.

BACKGROUND/OBJECTIVES: Studies have reported the effects of grain consumption on human health, but the association between maternal grain consumption before and during pregnancy and birth weight remains unclear. We evaluated the association between maternal grain consumption before and during pregnancy and birth weight/low birth weight (LBW). SUBJECTS/METHODS: Grain consumption was calculated using two semi-quantitative food frequency questionnaires (FFQs). The two FFQs evaluated consumption from pre- to early pregnancy and then from early to mid-pregnancy, respectively. Information concerning birth weight was obtained from birth records, and multivariable analyses for birth weight and LBW risk were conducted after adjusting for potential confounders. RESULTS: In total, 17,610 pregnant women (age, 31.8 ± 4.9 years; smoked during pregnancy, 16.1%; gestation period, 38.5 ± 2.5 weeks; first childbirth, 45.5%) and their singleton and term new-borns (birth weight, 3061.8 ± 354.1 g; LBW, 5.4%) were included in the analysis. Women in the highest quartile of grain consumption from pre- to early pregnancy had heavier new-borns (ß = 22.3; 95% confidence interval (CI): 5.8-38.9) but did not have a significantly lower LBW risk (odds ratio [OR]: 0.87; 95% CI: 0.71-1.07) than women in the lowest quartile. Women in the highest quartile of grain consumption from early to mid-pregnancy also had heavier new-borns (ß = 24.1; 95% CI: 7.1-41.1) but did not have a significantly lower LBW risk (OR: 0.85; 95% CI: 0.69-1.05) than women in the lowest quartile. CONCLUSIONS: Grain consumption before and during pregnancy was positively associated with birth weight.

Effects of Heat-Killed Levilactobacillus brevis KB290 in Combination with ß-Carotene on Influenza Virus Infection in Healthy Adults: A Randomized Controlled Trial.

Influenza, a seasonal acute respiratory disease caused primarily by the influenza virus A or B, manifests with severe symptoms leading to considerable morbidity and mortality and is a major concern worldwide. Therefore, effective preventive measures against it are required. The aim of this trial was to evaluate the preventive effects of heat-killed Levilactobacillus brevis KB290 (KB290) in combination with ß-carotene (ßC) on influenza virus infections in healthy Japanese subjects aged between 20 and 59 y throughout the winter season. We performed a randomized, double-blind, placebo-controlled, parallel-group trial from 16 December 2019 to 8 March 2020, comparing KB290 + ßC beverage with placebo beverage. The primary endpoint was the incidence of influenza based on a doctor's certificate. The incidence of influenza was not significantly different between the two groups. However, the subgroup analysis showed a significant difference between the two groups (influenza incidence: the KB290 + ßC group 1.9%, and the placebo group 3.9%) in the subgroup of subjects aged ˂40 y, but not in the subgroup of subjects aged ≥40 y. The results of this trial suggest that the combination of KB290 and ßC might be a possible candidate supplement for protection against the seasonal influenza virus infection in humans aged <40 y, although further clinical studies are needed to confirm the concrete preventive effect of this combination on influenza.