Effects Of Different Processing Treatments On MiRNA And Protein Of Exosomes In Goat Milk Products

As one of the most important sources of human daily foods,milk is rich in nutrients and bioactive substances.Cow and goat milk are major dairy foods in China.At present,the market share of goat milk is much lower than that of cow milk.However,with the increase of research on goat milk,there is growing interest in goat milk.Like other milk,goat milk dairy products drank by consumers are processed from raw goat milk,undergoing homogenization,pasteurization,concentration,and spray drying.The nutrients and bioactive substances in goat milk may be changed during the processing.Extracellular vesicles（EVs）are membranous nanoparticles secreted by all cells and present in all biological fluids,including exosomes,microvesicles and apoptotic bodies.As a major subtype of EVs,exosomes were also found in milk.Because of the encapsulation of exosomes,a large number of bioactive contents such as nucleic acids,proteins would avoid degradation in digestive tract,and could be taken up by intestinal cells,which may play an important regulatory role in human body.The effect of processing on exosomes in goat milk and its products still remains unclear.Therefore,in this study,the exosomes were isolated from different goat dairy products,including raw goat milk,pasteurized goat milk and goat milk formula.Then,the miRNA and proteome in three kinds of exosomes were analyzed using a combination of Next-generation sequencing and mass spectrometry.First of all,in order to minimize the contaminant of exosomes with goat milk components,especially high-abundance casein,on the isolation of exosomes,we optimized the pretreatment of samples and found that rennin could remove the high-abundance proteins in milk.Then,the process of milk exosome isolation was established:After defatted and rennin pretreated,the milk derived exosomes were obtained by sucrose cushion ultracentrifugation,followed by q EV chromatography.Exosomes obtained by the above isolation process were identified and compared.The exosomes isolated from different samples were identified by Western blot,Nanoparticle Tracking Analysis and Transmission Electron Microscope.We found that the sizes of three kinds of exosomes（raw goat milk exosomes:g-s EV,pasteurized goat milk exosomes:pg-s EV,milk formula exosomes:p-s EV）were decreased during processing,with the average size of g-s EV,pg-s EV and p-s EV were 195.7 nm,168.8nm and 143 nm,respectively.And the particles number of per m L milk of g-s EV and pg-s EV was 1.18×10¹¹ and 2.2×10¹¹,respectively,while the number of p-s EV is1.53×10⁸,which is significantly lower than the others.Then miRNAs were extracted from the exosomes of three kinds of exosomes and detected by next-generation sequencing.The results showed that miRNAs were all enriched in the exosomes.A total of 39 differential expression miRNAs between g-s EV and pg-s EV were found,of which 38 miRNAs were down-regulated in pg-s EV.Goat milk formular was produced from goat milk homogenization,pasteurization,concentration,and spray drying undergoing.There are 46 differential expression miRNAs were screened between g-s EV and pg-s EV,including 36 down-regulated miRNAs in p-s EV,and 32 differential expression miRNAs were identified between pg-s EV and p-s EV,including 4 down-regulated miRNAs in pg-s EV.Some of the down-regulated miRNAs after processing associated with immune regulation and tumors.The results of Gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis were showed as follow:Compared with g-s EV,the target genes of top 3 miRNAs that significantly down-regulated in pg-s EV were mainly involved in negative regulation of transcription from RNA polymerase II promoter and axon guidance pathway.Compared with g-s EV,the target genes of top 3 miRNAs that significantly down-regulated in p-s EV were mainly involved in striatum development and Erb B signaling pathway,and the target genes of top 3 miRNAs that significantly up-regulated in p-s EV were mainly involved in positive regulation of transcription from RNA polymerase II promoter and focal adhesion pathway.Compared with pg-s EV,the target genes of top 3 miRNAs that significantly down-regulated in p-s EV were mainly involved in regulation of transcription,DNA-templated and herpes simplex virus type 1 infection pathway,and the top 3miRNAs that significantly up-regulated in p-s EV were mainly involved in regulation of cell morphogenesis and Gn RH signaling pathway.Finally,exosomal proteins were extracted and performed for proteomic analysis using data independent acquisition（DIA）technology.The results showed that 910,1174 and 798 proteins were identified in g-s EV,pg-s EV and p-s EV,respectively.There were 339 differential expression proteins between g-s EV and pg-s EV,of which209 proteins were down-regulated.There were 425 differential expression proteins between g-s EV and p-s EV,of which 293 proteins were down-regulated.As for pg-s EV and p-s EV,436 differential expression proteins were detected,of which 236proteins were down regulated.Proteins lost in processing were mainly related to immune responses,transcriptional regulation and binding,etc.The results of GO and KEGG analysis were showed as follow:The differential expression proteins between g-s EV and pg-s EV were mostly involved in positive regulation of DNA binding and the complement and coagulation cascades pathway,etc.The differential expression proteins between g-s EV and p-s EV were mostly involved in Arp2/3 complex mediated actin nucleation and endocytosis pathway,etc.As for pg-s EV and p-s EV,the differential proteins were mainly involved in intercellular protein transport and endocytosis pathways,etc.In conclusion,we found that the particle size of goat milk exosomes decreases with processing such as homogenization,pasteurization,concentration,and spray drying.It was also found that pasteurization had little effect on the numbers of exosomes,but processing such as spray drying would greatly reduce the numbers exosomes.Moreover,miRNAs and proteins in milk exosomes changed with processing,which were down-regulated mainly,part of them involved in immunity and tumor.