| Bovine milk contains plenty of nutrients and active substances.It plays an important role in promoting growth and development,ensuring health and preventing diseases.Due to the potential occurrence of pathogenic microorganisms in raw milk,and in order to extend its shelf life,raw milk needs to undergo thermal processing procedures.Current thermal processing procedures primarily include pasteurization,ultra-high temperature(UHT)sterilization,and in-container sterilization.Different processes have different heating intensities,which causes different degrees of physicochemical changes of milk contents.In order to evaluate the biological impact of these changes on the intestine,intestinal cells were selected as in vitro cell models,and cell viability and cellular genes expressions were tested,respectively.Furthermore,lactoferrin,one of the milk functional active proteins,was selected as a subject,and we studied its anti-colon cancer effect in vitro and in vivo.The effect of heat treatment on the activity of lactoferrin against colon cancer was also analyzed.The experiment results are summarized as follows:Firstly,CCK-8 test showed that the activities of human colon cancer cell Caco-2 and human normal colon cell NCM460 were significantly reduced when cultured in in-container sterilized milk compared with raw milk,pasteurized milk,and UHT milk groups(p <0.05).This was partly due to the reduction in lysine content in the in-container sterilized milk,as well as changes in the structure of individual amino acid(the D-type proportion of protein-bound amino acids: tryptophan,aspartic acid,and the free amino acid: cysteine,increased significantly).More importantly,the in-container sterilized milk caused a strong cellular oxidative stress characterized by suppressed glutathione peroxidase activity,reduced intracellular GSH content,and diminished total antioxidant capacity.Secondly,in this study,we established a model for the cultivation of intestinal cells in vitro using pre-digested milk juice.Based on this model,transcriptome analysis was performed on differentiated Caco-2 cells cultured with different thermal processed milk.The results showed that raw milk,pasteurized milk and UHT milk had similar gene expression regulation patterns for intestinal cells(2 and 17 DEGs in pasteurized milk group and UHT milk group compared with raw milk group),while the cell gene expression in the in-container sterilized milk group was significantly different from the former three groups.The results of KEGG analysis showed that DEGs of the in-container sterilized milk culturing cells were mainly enriched in the metabolic process related pathways(especially peroxisomes metabolism pathway),and the expressions of DNA damage inducing genes,antioxidant genes and aldosterone reductase genes increased significantly compared with other three groups(p<0.05).Thirdly,lactoferrin significantly inhibited colon cancer cell line HT-29 in terms of its growth,migration and invasion(p<0.05).Lactoferrin significantly arrested the HT-29 cell cycle in the G1 phase and inhibited the expressions of angiogenesis pathway-related protein factors in HT-29 cells.The HT-29 tumor-bearing mouse test showed that oral administration of 100 mg/kg lactoferrin significantly inhibited the growth rate of tumor mass(p<0.05).In addition,the heat treatment decreased the inhibitory activity of lactoferrin to a certain extent,and the lactoferrin substantially lost its relevant inhibitory ability at 100 °C.The above experiments revealed that high temperature heating,especially over-heating,might produce potential toxic by-products in milk and could cause damage to intestinal cells.For the first time,the biological effects of different thermal processed milk on intestinal cells were studied in a holistic level.The activity of lactoferrin in inhibiting colon cancer tumors was gradually lost in a temperature dependent manner.The results of this study shed light on the importance of controlling thermal processing temperature during dairy production. |
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