| Casein is one of the main protein in cow's milk,which is digested to produce many bioactive peptides,such as opioid peptides,angiotensin converting enzyme inhibitory peptides,immunomodulating peptides,antibacterial peptides,antithrombotic peptides,mineral binding peptides,in addition to this besides,in recent years investigators also found that anti-inflammatory peptide,and those bioactive peptides are beneficial to people's health.However,casein is one of the major allergens of cow's milk allergy in infants,the main clinical manifestation are skin symptoms,such as urticaria,angioedema,eczema,and the next characteristics are gastrointestinal manifestations.In addition,casein is a complex of several phosphorus containing proteins,existing in milk with micellar state,presenting as a rough spherical particle,which largely leads to the indigestibility of milk protein in the infant's stomach.Therefore,it is of great significance to reduce the allergic reaction and promote the hydrolysis of casein.High hydrostatic pressure(HHP)technology is a new type of non thermal sterilization technology,which can disrupt protein secondary,tertiary and quaternary structures,alter their conformation,improve the digestibility of protein and reduce the allergic reaction.The purpose of this study is to use high hydrostatic pressure processing technology,and casein from bovine milk were pretreated at different pressure level,temperature,pressure dwell time and pressure cycle,following by digested at the imitative condition of adult and infant gastrointestinal environment,respectively.In this paper,the effects of casein on digestion and allergic reaction under different conditions were studied and orthogonal array design was used to optimize the operating conditions.The Caco-2 cell monolayer model was established to evaluate the bioavailability of peptide derived from the enzymatic digestion of casein after high hydrostatic pressure.Objective1.The effects of high hydrostatic pressure(HHP)treatment on elimination of allergens in casein from bovine milk were studied by imitative gastrointestinal digestion models and orthogonal array design was used to optimize the HHP operating conditions.2.The effects of high hydrostatic pressure(HHP)treatment on digestion in casein from bovine milk were studied by gastrointestinal digestion models and orthogonal array design was used to optimize the HHP operating conditions.3.The Caco-2 cell monolayer model was established to evaluate the bioavailability of peptide derived from the enzymatic digestion of casein after high hydrostatic pressure.Methods1.Casein from bovine milk were pretreated at different pressure level,temperature,pressure dwell time and pressure cycle,following by digested at the imitative condition of adult and infant gastrointestinal environment,respectively.The eliminating effectiveness of allergens was detected by indirect competitive enzyme-linked immunosorbent assay.Orthogonal array design was used to optimize the HHP operating conditions.2.Casein from bovine milk were pretreated at different pressure level,temperature,pressure dwell time and pressure cycle,following by digested at the imitative condition of adult and infant gastrointestinal environment,respectively.The hydrolysis degree of casein from bovine milk was detected by trinitro-benzene-sulfonic acid method(TNBS).Orthogonal array design was used to optimize the HHP operating conditions.3.MTT assay was used to evaluate the cytotoxic effects of six casein peptide on Caco-2 cells.4.The success of Caco-2 cell monolayer model was estimated by the measurement of the TEER value,the transportation of sodium fluorescein,and activity of alkaline phosphatase.The Caco-2 cell monolayer model was established to evaluate the bioavailability of peptide derived from the enzymatic digestion of casein after high hydrostatic pressure.5.The bioavailability of casein peptide derived from the enzymatic digestion of casein after high hydrostatic pressure was evaluated by detecting the absorption of peptide nitrogen in Caco-2 cell monolayer model.Results1.After high hydrostatic pressure treatment,the bovine casein was digested in an imitative adult and infant gastrointestinal environment,respetively.The results showed that casein allergen was eliminated after enzymatic digestion with imitative adult and infant gastrointestinal digestive models.The optimal HHP combined with adult gastrointestinal digestivemodelstreatment was performed at the condition of 350 MPa.30?,15 min,3 times cycle,and the inhibition rate was 22.67%.The optimal HHP combined with infant gastrointestinal digestive models treatment was performed at the condition of 350 MPa,30 0C?20 min,l times cycle,and the inhibition rate was 39.95%.2.After the same HHP treatment,the bovine casein was digested in an imitative adult and infant gastrointestinal environment,respetively.The results showed that the degree of hydrolysis of casein was improved after enzymatic digestion with imitative adult and infant gastrointestinal digestive models.The optimal HHP combined with adult gastrointestinal digestive models treatment was performed at the condition of 350 MPa?35?,30 min,2 times cycle,and the degree of hydrolysis was 12.68%.The optimal HHP combined with infant gastrointestinal digestive models treatment was performed at the condition of 300 MPa,30 ?,35 min,2 times cycle,and degree of hydrolysis was 11.52%.3.The MTT assay results showed that in the range of experimental doses(1.0.2.0?3.0?4.0?5.0mg/mL),6 groups of casein peptides have no obvious cytotoxic effect on Caco-2 cells.4.Day 21 after Caco-2 cells planted on transwell plate,TEER value was greater than 500 Q · cm2.Day 19 and 21 after Caco-2 cells planted on transwell plate,the fluorescein sodium apparent permeability coefficient(Papp)were distributed in the range of 0.1-0.7x10-6cm/s.In day 21,AP/BL is 5.32.Those results indicated that the Caco-2 cell monolayer model was dense and complete,and can be used for the transport experiments of peptide derived from the enzymatic digestion of casein.5.In the imitative adult gastrointestinal environment,the absorption rate of casein peptides in the control group was 7.26%,compared with the control group,the absorption rate of casein peptides(9.44%)in the optimal hydrolysis group was the highest(P<0.05),and the absorption rate of casein peptides in the optimal allergen reduction group was 7.88%(P>0.05).In the imitative infant gastrointestinal environment,the absorption rate of casein peptides in the control group was 5.76%,the absorption rate of casein peptides in optimal hydrolysis group and allergen reduction group is significantly higher than that of the control group(P<0.05),the absorption rate of casein peptides(6.94%)in the hydrolysis group was the highest.ConclusionOrthogonal array design was used to optimize the high hydrostatic pressure operating conditions to eliminate allergens and improve enzymolysis of casein from bovine milk.After high hydrostatic pressure treatment,the bovine casein was digested in an imitative adult and infant gastrointestinal environment,respetively,and obtaining casein peptides.Caco-2 cell monolayer model was successfully established and was used to evaluate the bioavailability of casein peptides.The bioavailability of the casein peptide derived from HHP combinating with the imitative adult and infant gastrointestinal digestive models was higher than that of the untreated casein peptide. |
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