Effects Of Milk Supplementation And Genetic Variants On The Gut Microbiota And Metabolic Health In Subjects With And Without Lactose Malabsorption

Milk has a complete and well-proportioned nutritional composition,and also is an optimal source of dietary calcium.Higher dairy intake not only was associated with lower risk of osteoporosis,hypertension,cardiovascular disease,but also contributed to lower all-caused mortality.But the current dairy consumption was merely 24.7 g/d in Chinese adults,which would be much lower than the recommendation amount（300 g/day）of“Chinese dietary guidelines”.Lactase deficiency is the main cause of lactose malabsorption.Lactose malabsorbers can not fully digested lactose in the small intestine,and undigested lactose will be fermented by colonic bacteria into gases（H₂,CO₂ and CH₄）and short chain fatty acids.Some lactose malabsorbers occur abdominal bloating,abdominal pain,diarrhea,and other uncomfortable symptoms,which is called“lactose intolerance”.The prevalence rate of lactose malabsorption in Chinese population is up to 86%.This is supposed to restrict the dairy intake and exert a long-term and adverse influence on the nutrition and health status in Chinese population.Accordingly,to explore the potentially different effects of milk on gut microbiota and metabolic health is of great significance in drafting personalized recommendation on milk consumption,and improving nutrition and health status of Chinese population.Section 1:Effects of Milk Supplementation on the Metabolic Health in Subjects with and without Lactose MalabsorptionObjective:To investigate the effect of milk intake on the anthropometric indexes,blood pressure and plasma biochemical indicators in subjects with and without lactose malabsorption.Methods:This study was a dietary intervention study in subjects with and without lactose malabsorption.Initially,all the volunteers conducted the hydrogen breath test to examine the lactose malabsorption status.According to these results,lactose absorbers and malabsorbers were 1:1 matched according to sex,age,body mass index（BMI）as well as baseline dairy intake.In a 4-week intervention period,all the participants were instructed to include one box of 250mL whole milk in their routine diet.In the beginning and ending of the study,all the participants were instructed to complete a 3-day food record,measure anthropometry indexes and collect blood samples together.Anthropometry indexes and blood pressure was measured according to standard methods,plasma glucose control indicators,lipids profile,inflammatory factors and markers of oxidative stress were measured using commercial kits,plasma indicators of liver and kidney function were measured using automatic biochemical analyzer.Results:227 subjects completed the hydrogen breath test.32 was lactose absorbers（14.1%）,while the other 195 was lactose malabsorbers（85.9%）.Finally,31 pairs of lactose malabsorbers and lactose absorbers were recruited into the intervention study,including 44men and 18 women,with age ranging from 20 to 31 years,BMI ranging from 16.3 to 25.9kg/m².All the participants finished the study.There were no differences in height,weight,BMI,waist circumference,hip circumferences,blood pressure,glucose control indicators,lipids profile,inflammatory factors and oxidative stress parameters between two groups.During the study,all the participants had good compliance,dairy biomarkers（plasma pentadecanoic acid）significantly increased after the study（P<0.001）.After 4-week supplementation of milk,dietary energy and other nutrients remained stable.Although body weight,BMI and blood pressure remain unchanged,body fat mass（P=0.034 for LM,P=0.015 for LA）and body fat percentage（P=0.018 for LM,P=0.022 for LA）significantly decreased with milk intake in subjects with and without lactose malabsorption,but no difference was observed between two groups.No significant effect of milk on indicators of glucose control,lipids profile,inflammation,oxidative stress,liver and kidney function in all the participants.Conclusion:A supplementation of 250 mL milk per day has no significantly differential metabolic effects on subjects with and without lactose malabsorption.Adequate milk consumption would not lead to adverse effect on the metabolic health in lactose malabsorbers.Section 2:Effects of Milk Supplementation on the Gut Microbiota and Its Metabolites in Subjects with and without Lactose MalabsorptionObjective:To investigate the effect of milk intake on the gut microbiota and its metabolites in subjects with and without lactose malabsorption,to clusters the enterotype of all the participants,and to evaluate effects of enterotype on the responses of lactose absorbers and malabsorbers to milk supplementation.Methods:In the three days before and after the intervention study,all the participants randomly collected a portion of fecal samples.Gut microbiota composition was measured using 16S rRNA gene sequencing and the levels of short chain fatty acids in stool and plasma were measured with gas chromatography-mass spectrometry.Enterotyping was clustered based on the relative abundance of gut microbiota composition at the genus level.Results:The 4-week supplementation of milk had no effect on gut microbiota structure in subjects with and without lactose malabsorption,but selectively altered gut microbiota composition of lactose malabsorbers.After milk intervention,the relative abundances of Actinobacteria（P<0.001,FDR=0.001）,Bifidobacterium（P<0.001,FDR<0.001）and two bacteria related to production of short chain fatty acids,including Anaerostipes（P<0.001,FDR<0.001）and Blautia（P=0.037,FDR=0.111）dramatically increased.Furthermore,changes in above taxa in both groups were significantly different between two groups（Actinobacteria:P=0.018;Bifidobacterium:P=0.029;Anaerostipes:P=0.016;Blautia:P=0.003）.In addition,supplementation of milk did not change the concentration of short chain fatty acids in stool and plasma in lactose malabsorbers as well as lactose absorbers.The relative abundance of Actinobacteria（P=0.016）and Bifidobacterium（P=0.004）merely significantly increased in the lactose malabsorbers with bacteriodes enterotype,but not lactose malabsorbers with Prevotella enterotype and all the lactose absorbers.Similarly,body fat mass（P<0.001for LM,P=0.020 for LA）and body fat percentage（P<0.001 for LM,P=0.025 for LA）significantly decreased in lactose malabosorbers and absorbers with bacteriodes enterotype,but not those with Prevotella enterotypeConclusion:A supplementation of 250 mL milk per day has bifidogenic effects on lactose malabsorbers,and could selectively alter the relative abundance of beneficial bacteria.Furthermore,individuals with different enterotype differently respond to the supplementation.Section 3:Interaction Effects of Milk Supplementation and Genetic Variants on the Gut Microbiota and Metabolic Health in Subjects with and without Lactose MalabsorptionObjective:To explore the genetic variants specially associated with lactose in Chinese population,to assess the prediction performance of several genetic variants potentially associated with lactose malabsorption,to discuss the interaction effects of milk supplementation and genetic variants on the gut microbiota and metabolic health.Methods:Genetic variants specially associated with lactose malabsorption in Chinese people was detected with Tag SNP,genotyping was conducted with microarray,the prediction performance was evaluated using weighted or unweighted genetic risk score（GRS）.Results:Two genetic variants well associated with lactose malabsorption in European population were not associated with lactose absorption in Chinese population.Among 24 Tag SNP selected from LCT and MCM6,rs11903319（P=0.010）and rs72972158（P=0.020）were significantly associated with lactose malabsorption.Weighted GRS has a better prediction performance of lactose malaborbers（OR=27,95%CI:3²85,P=0.006）.Milk consumption decreased body fat mass（Low GRS:P=0.015;Medium GRS:P=0.027;High GRS:P=0.038）and body fat percentage（Low GRS:P=0.020;Medium GRS:P=0.035;High GRS:P=0.044）in individuals with different genetic risk of lactose malabsorption,but did not significantly influenced the indicators of body weight,BMI,glucose control,lipids profile,inflammatory factors and oxidative stress in subjects with different genetic risk scores.There was no interaction effects of milk supplementation and genetic variants in gut microbiota structure,but significant interaction effects were observed in Actinobacteria（P=0.010）and Bifidobacterium（P=0.036）.Conclusion:A supplementation of 250 mL milk per day has bifidogenic effects on individuals with high genetic risk of lactose malabsorption,but has no adverse metabolic effect on them.Accordingly,it is acceptable to include milk as one part of the routine diet.It is of great significant in personalized milk recommendation and improving the nutrition and health status of our population,especially for improving dairy calcium intake.