Study On Composition Of Human Milk Under Cold Storage

In recent years,more and more breast-feeding women choose to pump human milk out and under cold storage first before feeding their babies.They prefer pre-stored human milk to feed babies instead of direct feeding.Cold storage of human milk has gradually become a trend.However,one of the major issues in cold storage of human milk is to investigate the quality change during storage since there is no universally accepted standard presently.In this study,the effects of storage temperature(4,-0.5,-3,and-18℃)and storage duration(2,4,7,14 d)on the quality of fresh human milk were studied.The total aerobic bacterial counts and nutritional composition in human milk,the microstructure of human milk fatty globule(HMFG),the protein composition and flavor of human milk fatty globule membrane(MFGM)were studied under different storage conditions.This work aimed to provide data support for the cold storage of human milk and the development of refrigerator designed for maternal and baby in the future.The main findings were as follows:(1)The effect of cold storage conditions on the basic physical and chemical properties of human milk was studied.The total aerobic bacterial counts increased significantly when stored at 4°C for 7 days compared with the fresh human milk,exceeding the standard of1×10~5cfu/m L established by the National Milk Bank Association,and decreased significantly at-0.5℃and-3℃for 7 days.No significant changes were observed within 14 days of storage at-18℃.The p H value of human milk gradually decreased with the extension of storage duration and the increase of storage temperature,and significantly decreased when stored at4℃for 2 days.The protein content of human milk insignificantly changed during the entire storage period.The fat content of human milk decreased significantly when stored at 4℃for7 days,while no significant changes were observed under other storage conditions.The lactose content in human milk decreased significantly when stored at 4℃for 2 days with continuous decrease subsequently,and it decreased significantly when stored at-0.5℃for 7days.No significant changes was observed at-18℃and-3℃during the entire storage.The SOD activity of human milk decreased significantly when stored at 4℃for 4 days with continuous decrease subsequently,however,there was insignificant difference in SOD activity between varied temperatures at the same storage duration.The non-enzymatic antioxidant Vc in human milk was sensitive to both storage temperature and storage duration.The degradation of Vc was inhibited lower storage temperature and shorter storage duration.The content of Ig G in human milk decreased significantly when stored at 4℃for 2 days,and changed insignificantly within 7 days of storage at-0.5℃and-3℃.It decreased significantly when stored at-18℃for 4 days,and then remained stable.The Ig A content in human milk decreased significantly when stored at 4℃for 7 days,and insignificant changes were observed under other storage conditions.The activity of human milk lysozyme decreased significantly under cold storage for 2 days,and it continued to decrease,especially at 4℃.(2)The effect of cold storage condition on human milk lipids was investigated.The changes in human milk lipid were studied and the related parameters during storage were recorded,including non-esterified fatty acid content,fatty acid composition and content,particle size,potential and microstructure of HMFG.The non-esterified fatty acid content in human milk gradually increased with the extension of storage duration,and more increment in non-esterified fatty acid content was observed under higher temperature.The proportion of saturated fatty acid,monounsaturated fatty acids and polyunsaturated fatty acids in human milk changed insignificantly when stored under different conditions,however,the corresponding content increased to a certain extent,consistent with the change trend of non-esterified fatty acid content.The particle size distribution of HMFG changed insignificantly during the entire storage period of 4,-0.5,and-3℃.A broader particle size distribution was observed when stored at-18℃for 2 days,and it continued to broaden subsequently.The morphology of HMFG began to change when stored at-18℃for 4 d,and the breakage of MFGM edges was observed.The accumulation and coalescence of HMFG were observed at the 7th day of storage,and more remarkable changes were observed at the14th day.However,no similar changes were observed under other temperatures,which also indicated that the accumulation and fusion of HMFG resulted in the increase of particle size distribution of human milk after storage at-18℃.(3)The effect of cold storage conditions on MFGM protein was investigated.The changes of MFGM protein in fresh human milk and stored for 4 days under different temperatures were characterized by SDS-PAGE and LC-MS/MS proteomics.Comparing the reduction and non-reduction electrophoresis patterns of SDS-PAGE,it is found that most of the MFGM proteins were in the form of disulfide bonds rather than monomeric proteins.Visualized changes of MFGM proteins could be observed under different temperatures when stored at a low temperature for 4 days.LC-MS/MS detected 1432,1004,998,1144 and 1199quantifiable proteins when stored at 0 d,4,-0.5,-3 and-18℃for 4 d,respectively.PCA and hierarchical cluster analysis showed that the overall MFGM protein information stored at 4℃and-0.5℃was similar,while that of-3℃and-18℃storage was similar and clustered into the same category as that of fresh sample(0 d).Compared with the fresh sample(0 d),202kinds of proteins significantly changed at all 4 storage temperatures.According to the GO functional annotations,more than 80%of the differential proteins had molecular functions of catalytic activity and binding effect,and the differential proteins involved in cell and metabolic processes accounted for the majority(56%)of cellular processes.According to the cell components classification,nearly half(46%)of the proteins belonged to the cellular anatomical entity,and 43%belonged to the intracellular.(4)The influence of cold storage conditions on the flavor of human milk was studied.Electronic nose,GC-MS and electronic tongue were used to analyze the changes in flavor(i.e.,odor and taste)of human milk during storage.The PCA results of qualitative analysis of electronic nose showed that the odor of human milk at different storage temperatures began to differ under cold storage for 4 days compared with the fresh human milk.The PCA results of qualitative analysis of GC-MS showed that the odor of human milk stored at 4℃and-0.5℃was different from that of fresh human milk at the 4th days.The difference was mainly caused by lauric acid,decanoic acid and caproic acid.According to the experimental results,there was insignificant difference in overall odor between the samples under cold storage and the fresh sample,if stored at 4℃and-0.5℃for less than 2 days,or stored at-3℃and-18℃for less than 7 days.PCA analysis was performed on the taste signals detected by the electronic tongue.The changes in signal intensity of astringent,astringent aftertaste and bitter aftertaste in human milk suggested the taste of human milk stored at different temperatures began to differ after 4 days under cold storage,and then the difference gradually increased.This observation was similar to the PCA analysis of volatile flavor substances detected by electronic nose and GC-MS.In conclusion,if the particle size and microstructure of HMFG are not considered,-18℃is more conducive to maintaining the stability of fresh human milk,followed by-3,-0.5 and4℃.The storage duration of human milk could be prolonged at-3℃compared with 4℃and-0.5℃.The structure of MFGM is more stable at-3℃compared with-18℃,and the overall physical and chemical properties are closer to those of-18℃.Therefore,if the human milk is under cold storage for short-term(<7 days),a better storage performance is observed when stored at-3℃could compared with that stored at 4℃,-0.5℃and-18℃.