Study On The Loss And Control Of Low-abundance Proteins In Bovine Milk Powder Processing

Bovine milk is the most popular dairy product among consumers all over the world,and its related commercial products,such as WPC,MPC are the main raw materials of infant formula products.Protein is the third most component of bovine milk,which is of huge significance for the development and health of neonates and infants.In addition,the native structure would be seriously modified by the thermal processing and lose its bioactivity in vivo.Generally,the protein in cow milk could be categorized into three groups,that is casein,whey,and milk fat globule membrane(MFGM)proteins.The molecular characteristics of these proteins vary with the different structures.For example,caseins mainly exist in the form of micelles,and the molecule contains more proline,resulting in the lack of stable higher structures and better molecular stability;whey protein exists in mainly globular form,with more cysteine and disulfide bonds in the molecule,would readily get denatured and aggregated upon heating.Besides well-known ?-LA and ?-LG,plenty of low-abundance proteins are present in the milk serum.MFGM proteins are mainly glycosylated proteins,and there are relatively few studies on their molecular characteristics.Recently,as the proteomic rapidly developed,more and more MFGM proteins were identified in milk.Studies have shown that milk serum and MFGM proteins are rich in bioactive components which have health-promoting benefits for newborns.Therefore,a systematic study of these proteins during industrial processing and their corresponding biological activities is of great significance for improving milk quality.In this study,we firstly collected common domestic and foreign infant formula products and their main ingredients WPC,and determined the bioactive proteins in them,such as immunoglobulin and lactoferrin.The results showed that almost no bioactive proteins could be found in these products.In order to reveal the key point which results in the loss of these bioactive substances,this thesis mainly conducts researches through the following aspects:Firstly,this paper used LC-MS/MS-based proteomics and enzyme-linked immunoassay(ELISA)method to track and analyze the loss of active whey protein in the entire milk powder production process by simulating the milk powder processing,including heat sterilization,concentration,spray drying,etc.Besides,bioinformatics methods were used to study the physiological functions of these heat-sensitive proteins.Results showed that there is almost no immunoglobulin and lactoferrin in the finally obtained milk powder products;in total,a number of 391 proteins were identified in milk serum and 89 proteins were shared.In the raw milk,161 proteins were identified while only 128 proteins were identified in the sterilized milk.Further,the abundance of other milk serum protein is markedly lowered,and a large amount of protein loss mainly occurs in the sterilization stage,while the concentration and spray drying process has little effect on these bioactive proteins in milk.Next,the paper compared the influences of several common thermal processes on protein quality by simulating common dairy processing technology with raw milk(R)as control,including low-temperature long time or Hold pasteurization(LTLT/Hop),high-temperature short time(HTST)pasteurized milk,extended shelf-life milk(ESL),ultra-high temperature instant sterilized milk(UHT)and spray dried milk powder(S).The study used label-free proteomics to study the changes in low-abundance proteins in milk serum and the lactose glycosylation of ?-LA and ?-LG.Results showed that the abundance and types of low-abundant proteins in ESL,UHT,and spray-dried milk decreased significantly,and a certain degree of lactose glycosylation occurred.In addition,the content of bioactive proteins in milk after different treatments was determined by ELISA,including milk immunoglobulins(Ig G,Ig A,Ig M),lactoferrin(LTF),and xanthine oxidase(XO).After pasteurization treatment,the bioactive protein can retain 35-70%,while these proteins are almost undetectable after other treatments,and HTST can retain more LTF and LTLT can retain more Ig G and XO.Finally,the author compared the results of ELISA with those identified by mass spectrometry and found a good similarity.Using ELISA,the feasibility of Label-free proteomics in studying milk protein changes during thermal processing was further verified.In view of the thermal damage caused by high-intensity heat treatment to whey protein,this paper further compared the effects of non-thermal sterilization(UV radiation and ultrasonic treatment)and different intensities of pasteurization treatment on bovine whey proteome for the first time.The results showed that ultraviolet radiation(4500 J/L)and ultrasound treatment(60W,6 min)can greatly reduce the number of microorganisms in milk,reaching a reduction of 5log10.The proteomic results show that UV treatment can retain all the milk serum proteins in milk,while low-intensity pasteurization and ultrasonic treatment can cause a certain degree of thermal damage to the protein;and a large amount of immunologically active whey protein was damaged in high-temperature pasteurization,such as lactoferrin,peroxidase,complement protein,immunoglobulin,etc.GO(Gene ontology)function enrichment indicated that those damaged proteins are mainly located on the cell membranes and intercellular substances,and related to cells,metabolism,immune response,and catalytic functions.Finally,the proteomic data were again verified by matching the contents of LTF,Ig G,and LPO by ELISA.Homogenization is an important operation dairy processing,which would make the milk more stable.Shear homogenization would not only efficiently reduce the size distribution of milk fat globules(MGF)but also may change the MFGM proteins and lipid-derived volatile components.Ultrasonication would reduce the bacterial load as well as reduce the size distribution of MFG.This paper next compared the effects of shear homogenization and ultrasonic homogenization on MFGM and volatile components in milk.The data indicated that homogenization performance of ultrasonication was elevated as the ultrasonic intensity(treatment power and time)increased.Furthermore,the homogenization efficiency at 40? is better than 25?.Ultrasound treatment with proper conditions(35 k J/L)can achieve a similar homogenization effect as conventional shear homogenization(the size distribution of milk fat globules is about 1 ?m).Under the similar homogenization effect,ultrasonic homogenization can better retain milk fat globular proteins(the types and abundance of MFGM proteins after ultrasonication are both higher than those of shear homogenization).Both homogenization methods increased the specific surface area(SSA)of MFG,and caseins were the dominant components that adsorbed on the interface of newly-formed MGF membranes.Both shear homogenization and ultrasonic homogenization change the volatile components in milk,among which the content of free short-chain fatty acids is higher,such as butyric acid,hexanoic acid,and caprylic acid,while the content of chloroform decreased after shear homogenization while the contents of limonene and ethyl butyrate increased after ultrasonic homogenization.Based on these obtained results,it was found that the critical control point that resulted in the loss of bioactive proteins in milk is the thermal processing,but not concentration or spraydrying,and non-thermal treatments were able to realize better retention of these proteins as well as a satisfied microbiological quality.Thus,we investigated the effects of non-thermal treatments on the bio-active proteins during milk powder processing relative to common thermal processing.Results showed that ultrasonication and UV-C treatments were able to realize a similar bactericidal effect compared with thermal processing.Ultrasonication increased the solubility and brightness of spray-dried milk powder compared with thermal and UV-C treatments,and this is mostly attributed to the strong homogenization of ultrasonication.Moreover,ultrasonication and UV-C treatments both increased the retentions of immunoglobulins,lactoferrin,and XO activity of milk powder as expected.UV-C did not result in a significant protein oxidization and lipids peroxidation in milk as thermal processing did.GC-MS results showed that the main volatile components in milk are acids,ketones,and aldehydes,and non-thermal treatments increased the contents of acids and aldehydes significantly.Ultrasonication further increased the acids in milk,and this is potentially attributed to the fact that ultrasonication facilitated the hydrolysis of triglyceride by lipase.