Milk allergy is one of the most common food allergies,affecting 6%of young children,andβ-lactoglobulin(β-LG)is the main milk allergen.Clostridium tyrobutyricum Z816 was selected for the degradation ofβ-LG,which was...Milk allergy is one of the most common food allergies,affecting 6%of young children,andβ-lactoglobulin(β-LG)is the main milk allergen.Clostridium tyrobutyricum Z816 was selected for the degradation ofβ-LG,which was successfully reduced by about 90%using permeabilized bacteria under the optimized conditions.The hydrolyzed peptides were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and analyzed by molecular modeling,which indicated that C.tyrobutyricum Z816 could effectively degrade the antigenic epitopes ofβ-LG.Finally,the concentration and digestibility ofβ-LG in actual samples was quantified using enzyme-linked immunosorbent assay(ELISA)and gastrointestinal digestion simulation experiments.The results showed more than 92%ofβ-LG in actual samples was hydrolyzed,and the gastric and total digestibility of whey protein isolate(WPI)was improved by 85.96%and 64.51%,respectively.Therefore,C.tyrobutyricum Z816 offers an effective method to degradeβ-LG and reduce the occurrence of milk allergies,which has great significance for the development of hypoallergenic dairy products.展开更多
Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L anti...Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.展开更多
Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent o...Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the展开更多
Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteris...Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.展开更多
In this study,a label-free,portable and reproducible immunochip based on quartz crystal microbalance(QCM)was developed for the qualitative detection ofβ-lactoglobulin(β-LG),an allergen,in milk products.Experimental ...In this study,a label-free,portable and reproducible immunochip based on quartz crystal microbalance(QCM)was developed for the qualitative detection ofβ-lactoglobulin(β-LG),an allergen,in milk products.Experimental parameters in the fabrication and regeneration procedure such as pH of the coupling microenvironment,amount of anti-β-LG antibody and regeneration reagent were optimized in detail.Under optimal conditions,the proposed QCM immunochip exhibited good recognition of β-LG,with a calibration curve of ΔF=12.877 C_(β-LG)^(0.4809)(R^(2)=0.9982)and limit of detection of 0.04μg/mL.Additionally,this portable QCM immunochip had good stability,high specificity,and no obvious cross-reaction to three other milk proteins(α-casein,α-lactalbumin,and lactoferrin).It could compete a qualitative measurement within5 min,and could be reused at least ten times.In the β-LG analysis of actual milk samples,the developed QCM immunochip yielded reliable and accurate results,which correlated strongly with those from the standard HPLC method(R^(2)=0.9969).Thus,the portable,stable,and reproducible QCM immunochip developed in this study allowed the rapid,cost-effectively and sensitively measure theβ-LG in milk products.展开更多
The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often t...The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.展开更多
Lipid-based nanocarriers have staged a remarkable comeback in the oral delivery of proteins and peptides, but delivery efficiency is compromised by lipolysis. β-Lactoglobulin(β-lg) stabilized lipid nanoparticles, in...Lipid-based nanocarriers have staged a remarkable comeback in the oral delivery of proteins and peptides, but delivery efficiency is compromised by lipolysis. β-Lactoglobulin(β-lg) stabilized lipid nanoparticles, including nanoemulsions(NE@β-lg) and nanocapsules(NC@β-lg), were developed to enhance the oral absorption of insulin by slowing down lipolysis due to the protection from β-lg. Cremophor EL stabilized nanoemulsions(NE@Cre-EL) were prepared and set as a control. The lipid nanoparticles produced mild and sustained hypoglycemic effects, amounting to oral bioavailability of 3.0% ± 0.3%, 7.0% ± 1.1%, and7.7% ± 0.8% for NE@Cre-EL, NE@β-lg, and NC@β-lg, respectively. Aggregation-caused quenching(ACQ)probes enabled the identification of intact nanoparticles, which were used to investigate the in vivo and intracellular fates of the lipid nanoparticles. In vitro digestion/lipolysis and ex vivo imaging confirmed delayed lipolysis from β-lg stabilized lipid nanoparticles. NC@β-lg was more resistant to intestinal lipolysis than NE@β-lg due to the Ca^(2+)-induced crosslinking. Live imaging revealed the transepithelial transport of intact nanoparticles and their accumulation in the liver. Cellular studies confirmed the uptake of intact nanoparticles. Slowing down lipolysis via food proteins represents a good strategy to enhance the oral absorption of lipid nanoparticles and thus co-formulated biomacromolecules.展开更多
基金supported by the National Key R&D Program of China(2017YFC1600404)the National Natural Science Foundation of China(31922070,22008114)the Natural Science Foundation of Jiangsu Province(BK20180038,BK20200684)。
文摘Milk allergy is one of the most common food allergies,affecting 6%of young children,andβ-lactoglobulin(β-LG)is the main milk allergen.Clostridium tyrobutyricum Z816 was selected for the degradation ofβ-LG,which was successfully reduced by about 90%using permeabilized bacteria under the optimized conditions.The hydrolyzed peptides were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and analyzed by molecular modeling,which indicated that C.tyrobutyricum Z816 could effectively degrade the antigenic epitopes ofβ-LG.Finally,the concentration and digestibility ofβ-LG in actual samples was quantified using enzyme-linked immunosorbent assay(ELISA)and gastrointestinal digestion simulation experiments.The results showed more than 92%ofβ-LG in actual samples was hydrolyzed,and the gastric and total digestibility of whey protein isolate(WPI)was improved by 85.96%and 64.51%,respectively.Therefore,C.tyrobutyricum Z816 offers an effective method to degradeβ-LG and reduce the occurrence of milk allergies,which has great significance for the development of hypoallergenic dairy products.
基金Sponsored by the Young Scholar Scientific Research Foundation of China CDC[2015A202]:The establishment of testing platform of quantitatively detecting main protein of cow milk by using protein chip technique
文摘Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.
基金supported by the Natural Science Foundation of China (30871752)the High-tech Industrialization Funding of Guangdong Province (2009B011300010)
文摘Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the
基金Supported by the Project of Science & Technology Plans in Heilongjiang Province in the 11th Five-year Period (GB07B407)
文摘Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.
基金supported by the National Natural Science Foundation of China(31972147)Project of Tianjin Science and Technology Plan(19PTSYJC00050)+1 种基金the Open Project Program of State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science and Technology(SKLFNS-KF-202011)Project program of Key Laboratory of Food Nutrition and Safety,Ministry of Education,Tianjin Key Laboratory of Food Nutrition and Safety,China(JYB202002)。
文摘In this study,a label-free,portable and reproducible immunochip based on quartz crystal microbalance(QCM)was developed for the qualitative detection ofβ-lactoglobulin(β-LG),an allergen,in milk products.Experimental parameters in the fabrication and regeneration procedure such as pH of the coupling microenvironment,amount of anti-β-LG antibody and regeneration reagent were optimized in detail.Under optimal conditions,the proposed QCM immunochip exhibited good recognition of β-LG,with a calibration curve of ΔF=12.877 C_(β-LG)^(0.4809)(R^(2)=0.9982)and limit of detection of 0.04μg/mL.Additionally,this portable QCM immunochip had good stability,high specificity,and no obvious cross-reaction to three other milk proteins(α-casein,α-lactalbumin,and lactoferrin).It could compete a qualitative measurement within5 min,and could be reused at least ten times.In the β-LG analysis of actual milk samples,the developed QCM immunochip yielded reliable and accurate results,which correlated strongly with those from the standard HPLC method(R^(2)=0.9969).Thus,the portable,stable,and reproducible QCM immunochip developed in this study allowed the rapid,cost-effectively and sensitively measure theβ-LG in milk products.
文摘The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.
基金funded by the Science and Technology Committee of Shanghai Municipality (Nos.19430741400, 23S11901500,23ZR1413100, and 21430760800)the National Natural Science Foundation of China (Nos.81973247 and 82030107)。
文摘Lipid-based nanocarriers have staged a remarkable comeback in the oral delivery of proteins and peptides, but delivery efficiency is compromised by lipolysis. β-Lactoglobulin(β-lg) stabilized lipid nanoparticles, including nanoemulsions(NE@β-lg) and nanocapsules(NC@β-lg), were developed to enhance the oral absorption of insulin by slowing down lipolysis due to the protection from β-lg. Cremophor EL stabilized nanoemulsions(NE@Cre-EL) were prepared and set as a control. The lipid nanoparticles produced mild and sustained hypoglycemic effects, amounting to oral bioavailability of 3.0% ± 0.3%, 7.0% ± 1.1%, and7.7% ± 0.8% for NE@Cre-EL, NE@β-lg, and NC@β-lg, respectively. Aggregation-caused quenching(ACQ)probes enabled the identification of intact nanoparticles, which were used to investigate the in vivo and intracellular fates of the lipid nanoparticles. In vitro digestion/lipolysis and ex vivo imaging confirmed delayed lipolysis from β-lg stabilized lipid nanoparticles. NC@β-lg was more resistant to intestinal lipolysis than NE@β-lg due to the Ca^(2+)-induced crosslinking. Live imaging revealed the transepithelial transport of intact nanoparticles and their accumulation in the liver. Cellular studies confirmed the uptake of intact nanoparticles. Slowing down lipolysis via food proteins represents a good strategy to enhance the oral absorption of lipid nanoparticles and thus co-formulated biomacromolecules.
