Glycinin和β-Conglycinin对不同食性鱼类生长及肠道组织的影响

【目的】研究大豆球蛋白(Glycinin)和β-伴大豆球蛋白(β-Conglycinin)对不同食性鱼类生长及肠道组织的影响。【方法】以健康的鲤、埃及胡子鲇和草鱼为试验对象,以添加鱼粉饲料为对照组,另设置添加Glycinin 60.0mg/g和β-Conglycinin 40.0 mg/g的2个试验组,各配制成3种等氮(粗蛋白含量分别为鲤360.2 g/kg,埃及胡子鲇400.5 g/kg,草鱼300.6 g/kg)、等能(鲤15.26 MJ/kg,埃及胡子鲇15.86 MJ/kg,草鱼15.6 MJ/kg)的半精制饲料,在室内控温单循环养殖系统中进行为期6周的饲养试验,比较分析Glycinin和β-Conglycinin对不同食性鱼类生长、饲料利用及肠道组织影响的差异。【结果】Glycinin和β-Conglycinin对不同食性鱼类生长及肠道组织影响不同。鲤Glycinin组和β-Conglycinin组的特定生长率、饲料效率和蛋白质效率均下降,其中β-Conglycinin组与对照组差异不显著(P>0.05);埃及胡子鲇Glycinin组和β-Conglycinin组的特定生长率、饲料效率和蛋白质效率均较对照组显著下降(P<0.05);草鱼Glycinin组和β-Conglycinin组的特定生长率、饲料效率和蛋白质效率均极显著低于对照组(P<0.01)。Glycinin组、β-Conglycinin组鲤和埃及胡子鲇中肠、后肠及草鱼前肠、中肠、后肠的皱襞高度均极显著低于对照组(P<0.01);观察鲤、埃及胡子鲇、草鱼前肠、中肠、后肠的组织切片发现,不同食性鱼类的Glycinin组和β-Congly-cinin组肠道组织结构完整性出现了不同程度的损伤,Glycinin组鲤前肠、后肠和埃及胡子鲇后肠及草鱼前肠绒毛均有不同程度破损,固有层组织较疏松而且断裂;β-Conglycinin组埃及胡子鲇后肠和草鱼前肠的绒毛有不同程度的断裂或破损。【结论】在本试验条件下,从生长性能、饲料利用和肠道生长指标来看,大豆主要抗原蛋白对草鱼的影响最大,其次是埃及胡子鲇,对鲤的影响较小。

致仔猪过敏性大豆抗原蛋白β-conglycinin单克隆抗体的制备与鉴定

研究以人工合成的对应于β-conglycinin分子α'亚基上的一段抗原表位肽(RPQHPER,78-84α')作为半抗原,与载体蛋白(OVA)交联后,作为免疫原制备了致仔猪过敏性大豆抗原蛋白β-conglycinin的单克隆抗体(Mab 6G4),并应用酶联免疫吸附等方法进行鉴定。结果表明,制备的单抗Mab 6G4属于IgG1;该抗体能与大豆β-conglycinin特异性结合,IC50为4.7 ng/mL,并对β-conglycinin表现出较强的亲和力,亲和常数达6.9×109 L/mol。因此,制备的单克隆抗体Mab 6G4为分析大豆制品中β-conglycinin的含量及探讨β-conglycinin对仔猪的致敏机理提供了一个潜在的分析工具。

β-Conglycinin在不同食性鱼类肠道组织中的分布规律

【目的】研究β-伴大豆球蛋白(β-Conglycinin)在不同食性鱼类肠道组织中的分布规律。【方法】以健康的鲤鱼、埃及胡子鲇和草鱼为研究对象,以鱼粉为对照组,以添加40mg/g提纯的β-Conglycinin组为试验组,各配制成2种等蛋白(粗蛋白质量分数分别为鲤鱼360.2g/kg,埃及胡子鲇400.5g/kg,草鱼300.6g/kg)、等能(鲤鱼15.2MJ/kg,埃及胡子鲇15.8MJ/kg,草鱼15.6MJ/kg)的半精制饲料,在室内控温单循环养殖系统中进行为期6周的饲养试验,饲养试验结束后,采用免疫组织化学方法探讨β-Conglycinin在不同食性鱼类肠道中分布规律的差异。【结果】β-Conglycinin在鲤鱼和草鱼前、中、后肠的积累光密度呈上升趋势,鲤鱼后肠与前肠差异极显著(P<0.01),草鱼中、后肠的积累光密度与前肠差异极显著(P<0.01);埃及胡子鲇前肠、中肠、后肠的积累光密度差异不显著(P>0.05)。【结论】在本试验条件下,β-Conglycinin主要分布在鲤鱼后肠,草鱼的中、后肠,埃及胡子鲇的前肠、中肠和后肠。

β-Conglycinin酶解肽对仔猪空肠上皮细胞通透性及紧密连接蛋白表达的影响

本研究通过测定大豆抗原β-Conglycinin酶解肽(52ku)对仔猪空肠上皮细胞(IPEC)通透性及紧密连接蛋白Occludin与ZO-1基因mRNA表达的影响,为探索其对肠道损伤的相关机制提供依据。本试验分别采用不同浓度(0、0.125、0.25、0.5、1、2mg·mL-1)52ku酶解肽处理IPEC 2、4、6、8、12、24h后,通过测定细胞存活力(MTT值)、跨上皮细胞电阻值(TEER)以及细胞培养液中碱性磷酸酶(AP)的活性,检测52ku酶解肽对细胞膜通透性的影响。并选取0.5mg·mL-1 52ku酶解肽处理IPEC 24h后,用实时荧光定量PCR方法检测Occludin与ZO-1基因mRNA表达量的变化情况。结果表明,52ku酶解肽处理仔猪空肠上皮细胞后,MTT值和TEER呈时间-剂量依赖性降低(P<0.05)。24h处理后,AP活性呈显著升高趋势(P<0.05)。与对照组相比,52ku酶解肽可使Occludin和ZO-1基因mRNA相对表达量均呈显著下降趋势(P<0.05)。总之,52ku酶解肽使仔猪空肠上皮细胞通透性增加,并降低Occludin或ZO-1的mRNA表达。

Protective Effect of Bacillus subtilis Peptidoglycan (PG) on β-conglycinin-induced Intestinal Epithelial Cells Damage of Juvenile Carp (Cyprinus carpio)

[Objective] The paper was to investigate the protective effects of different concentrations of Bacillus subtilis peptidoglycan(PG) on β-conglycinin-induced inflammatory injury in intestinal epithelial cells of juvenile carp(Cyprinus carpio).[Method] In 24-cell microplates, the intestinal epithelial cells(IECs) of juvenile carp were primarily cultured for 72 h at 26°C and 6% CO2, and then the IECs were randomly divided into6 groups with 4 replicates per group. One of the six groups was set as negative control group, and the other groups were all supplemented with 1.0 mg/mL β-conglycinin in culture medium to establish inflammatory injury. At 24 h post induction, the culture media were changed into B. subtilis PG culture media with the concentrations of 0(positive control group), 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively. The samples were collected to measure the antioxidant and anti-inflammatory indices at 12, 24 and 36 h post culture.[Result]β-conglycinin exposure significantly decreased the activity of ASA, AHR, SOD, CAT, GPx, and increased the PC content and the mRNA expression of inflammatory cytokines(IL-1β, IL-8, TNF-α1,IL-10 and TGF-β). At 12, 24 and 36 h post PG treatment, the activities of ASA, AHR, SOD, CAT, GPx and the content of PC in cells decreased in a dose-dependent manner;the mRNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and those of IL-10 and TGF-β were up-regulated.[Conclusion] Different concentrations of B. subtilis PG could protect IECs oxidative damage induced by β-conglycinin and improve the antioxidant capacity of IECs. High concentration of PG could improve the anti-inflammatory ability of IECs by inhibiting inflammatory factors and promoting the gene expression of anti-inflammatory cytokines.

Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybeanβ-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and theβ-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters ofβ-conglycinin was optimized by single factor and L_(9)(3^(4))orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity ofβ-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity ofβ-conglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration causedmonotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions of protein concentration 30mg/mL,enzymedosage0.7%,hydrolysis time40min,temperature 55°C and pH8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Effect of extrusion on the structure and antigenicity of soybean β-conglycinin

β-Conglycinin,the main component of 7S globulin in soybean protein,is also a key soybean antigen protein that causes allergic reactions.Extrusion technologies have received considerable attention as amethod for modifying soybean protein allergens.This study investigated the changes inβ-conglycinin structure and antigenicity upon extrusion.Isoelectric precipitation,ammoniumsulfate precipitation,and sepharose CL-6B gel filtration were used to isolate and purifyβ-conglycinin from soybean powder,and single-factor and orthogonal tests were used to study the effects of water content,extrusion temperature,screw rotation speed,and feeding speed on the antigenicity ofβ-conglycinin after extrusion.Fourier transforminfrared spectrometry(FTIR)was then employed to analyze the structure ofβ-conglycinin after extrusion under the optimal conditions determined by the orthogonal test.The results showed that extrusion significantly reduced the antigenicity ofβ-conglycinin(P<0.05),and the degree of influence of the factors studied may be ordered as extrusion temperature>feeding speed>screw rotation speed>water content.The optimal parameters were temperature at 130°C,screwrotation speed at 140 r/min,and feeding speed at 35 g/min.Under these conditions,the contents ofα-helix,β-pleated sheet,andβ-turn structures inβ-conglycinin were significantly reduced(P<0.05),while the contents of random coils were significantly increased(P<0.05).The peak absorption intensity of amides I,II,and III also decreased.Taken together,the findings suggest that extrusion could be an effective method for reducing the antigenicity ofβ-conglycinin.

An innovative method used for the identification of N-glycans on soybean allergenβ-conglycinins

β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.

Enzymatic kinetics ofβ-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and analysis of antigenicity of hydrolyzed peptide

β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.

Cytoprotection and Cytothetic Effects of GLP-2 on Enterocytes from a Weaned Piglet Injured by β-conglycinin in Vitro

The experiment was conducted to study the effects and possible mechanism of GLP-2 on proliferation,metabolism and apoptosis of cultured enterocytes from a 28-d weaned piglet injured by exposure to β-conglycinin.A cell damage model was established to investigate cell proliferation, metabolism and apoptosis by exposing primary cell cultures of intestinal epithelial cells(IEC) to 1.2 and 2.4 mg/mL β-conglycinin.A 2×3 factorial experiment was then used to study the effect of different GLP-2 concentrations of(1×10-9,1×10-8 and 1×10-7mol/L),in combination with the two concentrations ofβ-conglycinin.Cells exposed to the allergenβ-conglycinin had decreased(P【0.05) MTT OD;decreased (P【0.01) protein retention and total protein content of cells;increased(P【0.01) LDH and caspase-3 activities and decreased(P【0.05) Na+,K+-ATPase activity.When GLP-2 was used in combination withβ-conglycinin,MTT OD,protein retention,total protein content and Na+,K+-ATPase activity significantly increased(P【0.05);LDH activity gradually decreased(P【0.05 or P【0.01) and Caspase-3 activity significantly decreased(P【0.01) with increasing concentrations of GLP-2.The results indicated thatβ-conglycinin had adverse effects on proliferation and integrity of IEC in vitro.GLP-2 relieved or prevented the adverse effects ofβ-conglycinin on proliferation and integrity of IEC by regulating Na+,K+- ATPase and Caspase-3 activities,and consequently affecting cell metabolism.

大豆β-Conglycinin对鱼类健康的影响及分离方法

大豆抗原蛋白是限制大豆蛋白在水产饲料中广泛应用的主要因素。β-伴大豆球蛋白(β-Conglycinin)是免疫原性最强的大豆抗原蛋白,饲料中β-Conglycinin含量过高会导致鱼类生长速度下降、消化酶活力下降、肠道组织发生损伤,甚至死亡。本文概述β-Conglycinin的理化性质、对鱼类生长性能、肠道组织、消化酶活力、非特异性免疫指标和抗氧化能力的影响、分离提纯方法及去除工艺,为进一步研究大豆抗原蛋白的致敏机理提供参考,同时为合理开发利用大豆蛋白源提供依据。

Effect of lipid peroxidation on the allergenicity and functional properties of soybeanβ-conglycinin(7S)and glycinin(11S)

This study aimed to analyze the effect of lipid peroxidation on the allergenicity and functional properties of soybeanβ-conglycinin(7 S)and glycinin(11 S).Oxidation complexes were determined using the lipid peroxidation method.Functional properties were analyzed based on emulsifying and foaming properties.The potential allergenicity was evaluated by in vitro and in vivo methods.The results found that oxidation altered structures of the proteins and resulted in the formation of cross-linked protein polymers.The emulsion and foaming properties of the proteins were improved after oxidation.The IgE-binding capacity of 7 S and11 S reduced after oxidation.KU812 cell assays showed that both histamine and IL-4 release decreased after oxidation treatment.A mouse model showed that oxidation reduced the IgE,IgG,and IgG1 levels,as well as reduced histamine and mMCP-1 release in serum,which might suppress the allergic reaction.In conclusion,the lipid peroxidation treatment likely causes changes to the functional properties of soybean,decreasing the potential allergenicity of 7 S and 11 S.

Identification of Co-dominant SSR Markers Associated with Genes Controlling α'- and cr-subunit-null β-conglycinin Phenotypes in Soybean (Glycine max (L.) Merr.)

Studies have shown that the three subunits of β-conglycinin are the main potential allergens of soybean sensitive patients.And β-conglycinin has adverse effects on nutrition and food processing.So solation and production of lines with lowerβ-conglycinin content has been the focus of recent soybean breeding projects.Soybean lines with deficiency in one or all subunits of β-congIycinin have been obtained.An effective and rapid system to identify such mutations will facilitate genetic manipulation of the β-conglycinin subunit composition.Here,two segregating F2 populations were developed from crosses between Cgy-1/cgy-1(CC),anα'-lacking line(△α'),and DongNong 47(DN47),a wild-type(Wt)Chinese soybean cultivar with normal globulin components,and Cgy-2/cgy-2(CB),an a-lacking line(△α),and DN47.These populations were used to estimate linkage among the egy-1(conferring α'-null)and cgy-2(α-null)loci and simple sequence repeat(SSR)markers.Seven SSR markers(Sat_038,Satt243,Sat_307,Sat_109,Sat_231,Sat_108 and Sat_190)were determined to co-scgregate with cgy-1,and six SSR markers(Satt650,Satt671,Sat_418,Sat_170,Satt292 and Sat_324)co-segregated with cgy-2.Linkage maps being composed of seven SSR markers and egy-1 locus,and six SSR markers and the cgy-2 locus were then constructed.It assigned that the egy-1 gene to chromosome 10 at a position between Sat_307 and Sat_231,and the cgy-2 gene to chromosome 20 at a position between Satt650 and Satt671.These markers should enable map-based cloning of the egy-1 and cgy-2 genes.For different subunit-deficiency types[α'-null,α-null and(α'+α)-null types],the two sets of SSR markers could also detect of polymorphism between three normal cultivars and seven related mutant lines.The identification of these markers is great significance to the molecular marker-assisted breeding of soybean/9-conglycinin subunits.

Effect of Allelic Variation of β-conglycinin α-subunit Locus cgy-2 on Soybean(Glycine max) Quality Ⅰ. Evaluation of Free Amino Acid Content

The effects of the deficiency of the allergenicα-subunit on soybean amino acid(AA)composition were studied using the cultivar Dongnong 47(DN47)as a genetic background.The near-isogenic line(NIL)NIL-DN47-Δα of DN47,with an introgression of theα-null trait allele from the high protein donor parent RiB,was created by marker assisted background selection and used to investigate the AA content and nutritional quality.The contents of crude protein,the total AAs,the total essential amino acids(EAAs)and sulfur-containing(Met and Cys)AAs increased by 4.11%,4.16%,5.20% and 11.96%,respectively in NIL-DN47-Δα compared with DN47.Analyses of the total EAAs(TEAAs)and the EAA index(EAAI)revealed that both parameters in NIL-DN47-Δα were higher than those in DN47.The null-allele of theα-subunit positively affected the AA scores.The quantitative changes in free AAs(FAAs)in the developing seeds of NIL-DN47-Δα and DN47 were compared as of 15 days after flowering(DAF)until maturity.The results showed that the total FAA content in NIL-DN47-Δα was significantly higher than that in the DN47 throughout the late maturation stage(40-60 DAF)of seeds.The high concentration of the FAAs in cgy-2 mutant seeds was a consequence of the high rates of synthesis and/or accumulation of individual FAAs during seed maturation where 25 DAF was an important turning point in the accumulation of the FAAs.The FAA contents of single soybeanα-null,double(α+α′)and triple(α+α′+group I)-null mutant combination lines were investigated.In all of these combinations,introduction of the cgy-2 gene invariably raised the FAA content of mature seeds above that of the DN47.In summary,the enhanced protein quality in cgy-2 mutants resulted from several factors.(1)There was a general increase in the contents of most AAs and FAAs in NIL-DN47-Δα.(2)The induced synthesis of free Arg contributed effectively to the high FAAs of various storage-protein-deficiency mutants.For example,in the S2(null α,group I),the free Arg content was seven times as much as that of DN47,accounting for more than half of the total FAA content in the seed.(3)The increase of sulfur-containing AAs in theα-null type NIL mainly resulted from elevated Met content.These data suggested that the cgy-2 mutation might improve the protein quality of soybean seeds and that lacked of the allergenicα-subunit resulted in increased the FAA content.

Identification of lncRNAs Associated withβ-conglycininα-null Allele Based on a Genome-wide Transcriptome Analysis ofα-null-type Hypoallergenic Soybean(Glycine max)Near-isogenic Lines

Soybean mutants withα-nullβ-conglycinin are associated with high nutritional value and low allergenic risk.Although long noncoding RNAs(lncRNAs)are increasingly recognized as functional regulatory components affecting eukaryotic gene expression,little is known about lnc RNA profiles inα-null-type hypoallergenic soybeans.In this study,a genome-wide integrative analysis of lncRNAs,m RNAs and epigenomic data in the soybean cgy-2(confirmedα-null)near-isogenic line(NIL)and its recurrent parent Dongnong47(DN47)was conducted.Nineteen novel lncRNAs that were differentially expressed(DE)only in the NIL at 18 days after flowering(i.e.,α-null-associated DE lncRNAs)were delected.Sixteen putative soybean stress-responsive lncRNAs were identified,and observed to regulate 257 stress-related genes DE in the NIL.This result indicated that theα-null allele might represent an intrinsic defect stress that altered the expression of various stress-related genes inα-null-type hypoallergenic soybean.Additionally,25 epigenetic-related lncRNAs regulated 831 DE epigenetic-related genes and simultaneously initiated multiple epigenetic activities,including ubiquitination,methylation and acetylation.Kyoto encyclopedia of genes and genomes(KEGG)analysis indicated that the biosynthesis of amino acids pathway was enriched with 83 DE genes regulated by nine DE lncRNAs.Changes in the expression of these lncRNAs and genes might be the reason for the altered amino acid composition in the NIL.Among all detected DE lncRNAs,MSTRG.12518 was the most conspicuousα-null-specific cis/trans-lnc RNA that played an efficient,versatile and vital role in the NIL.The data indicated that the lnc RNA profile differed between the NIL and DN47.Variations in lncRNAs,gene expression levels and DNA methylation states likely contributed to the intrinsic defect stress response mechanism inα-null-type hypoallergenic soybeans.

β-Conglycinin对不同发育时期鲤鱼消化酶活力的影响

【目的】研究β-伴大豆球蛋白(β-Conglycinin)对鲤幼鱼、稚鱼蛋白酶和淀粉酶活力的影响。【方法】以初始体质量为(10.06±0.14)g/尾的鲤稚鱼和(110.23±0.23)g/尾的鲤幼鱼为研究对象,以鱼粉为动物蛋白源,面粉、糊精为糖源,混合油脂(m(鱼油)∶m(玉米油)=1∶1)为脂肪源,分别配制5种等氮(鲤幼鱼和稚鱼粗蛋白质量分数分别为36%和40%)、等能(鲤幼鱼和稚鱼总能分别是15.2和16.9 MJ/kg)的半精制饲料,其β-Conglycinin的添加量(质量分数)分别为0(CK),2.0%,4.0%,6.0%和8.0%,每组饲料设3个重复,在控温单循环养殖系统中进行为期8周的饲养试验,试验结束后,取鲤幼鱼、稚鱼的前、中、后肠道和肝胰脏,分别用福林-酚试剂法和淀粉酶试剂盒法,测定肠道和肝胰脏蛋白酶及淀粉酶的活力。【结果】鲤幼鱼肝胰脏蛋白酶活力各组之间差异不显著(P>0.05);β-Conglycinin添加量为6.0%和8.0%组的前肠、中肠蛋白酶活力显著低于对照组(P<0.05);而β-Conglycinin添加量为8.0%组后肠蛋白酶活力显著低于对照组(P<0.05)。在鲤稚鱼肝胰脏和后肠,β-Conglycinin添加量为8.0%组的蛋白酶活力显著低于对照组(P<0.05);前肠蛋白酶活力则以2.0%,4.0%,6.0%和8.0%添加组显著低于对照组(P<0.05);中肠蛋白酶活力为4.0%,6.0%和8.0%添加组显著低于对照组(P<0.05)。β-Conglycinin对鲤幼鱼和稚鱼肝胰脏、前肠、中肠及后肠淀粉酶活力均无显著影响(P>0.05)。【结论】鲤幼鱼配合饲料中β-Conglycinin的添加量不应超过6.0%;鲤稚鱼配合饲料中β-Conglycinin的添加量不应超过2.0%。

大豆抗原蛋白β-conglycinin线性表位及其不同种属动物过敏血清结合能力比较

试验通过生物信息学方法预测β-conglycinin线性表位,综合已发表文献筛选出四段具有代表性氨基酸序列合成短肽,采用ELISA和点杂交法检测表位肽段与仔猪、犊牛、兔三种不同种属动物过敏血清结合程度差异。结果表明:通过生物信息学方法预测合成的肽段均为β-conglycinin表位肽;三种动物过敏血清与四段表位肽结合能力存在显著差异,其中四段表位肽与仔猪、犊牛过敏血清结合能力显著高于兔过敏血清(P<0.05);同一动物与四段不同肽段结合敏感程度差异显著(P<0.05),兔过敏血清与肽段β1结合能力最强,仔猪过敏血清与α1肽段结合能力最强,犊牛过敏血清与肽段β2肽段结合能力最强。结果为寻找β-conglycinin最佳优势表位提供理论支持,同时为揭示大豆抗原蛋白种属间致敏差异机理奠定基础。

Glycinin和β-Conglycinin对不同生长阶段猪生长性能和粗蛋白消化率的影响

选取仔猪(7.06±0.18)kg、生长猪(44.54±9.04)kg和肥育猪(78.93±7.37)kg军牧一号去势公猪各15头,用于3个生长阶段的动物试验,每一阶段分为A、B、C 3组,其中A组为对照组,B、C以Glycinin或β-Conglycinin的提纯产物作为日粮唯一大豆蛋白来源,每组5个重复。仔猪在28日龄断奶,试验分组之前,猪均不接触任何豆科日粮。7 d试验期过后,收集消化道各段食糜,测定干物质和粗蛋白表观消化率。结果表明:Glycinin或β-Conglycinin降低仔猪或生长猪的生产性能以及干物质、粗蛋白在消化道各段的表观消化率(P<0.001),最终导致回肠养分表观消化率的绝对值与对照组相比减少3%～6%。2个生长阶段猪的粗蛋白质表观消化率只在胃中受抗原蛋白的影响有显著差异(Glycinin,P=0.005;β-Conglycinin,P=0.001),仔猪的下降程度大于生长猪。

Soybeanβ-conglycinin and glycinin reduced growth performance and the intestinal immune defense and altered microbiome in juvenile pearl gentian groupers Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂

The utilization efficiency of soy protein is affected by its 2 anti-nutritional substancesdthe antigens bconglycinin and glycinin.This study investigated their effects on the growth performance,intestinal immune defense,andmicrobiome in juvenile pearl gentian groupers(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂).Three isonitrogenous and isolipidic diets were formulated containing fishmeal supplemented with 70 g/kgβ-conglycinin or 100 g/kg glycinin,or no supplementation(control).Each experimental diet was fed to quadruplicate groups with 30 fish in each tank for 8 weeks.Dietary inclusion of eitherβ-conglycinin or glycinin significantly reduced weight gain and specific growth rates,and cell proliferation of the distal intestine.Histological evaluation of the intestine tract revealed the inflammation signs,characterized by reducing of plica height and width as well as the number of the goblet cells,and widening of the lamina propria.The group fed theβ-conglycinin diet had reduced lysozyme activity,contents of immunoglobulin M and complements 3 and 4.Increased activities of caspase-3 and-9 were observed in the group fed the bconglycinin diet compared to the other 2 groups.In the intestinal microbiota,the relative abundances of the potentially pathogenic genera Photobacterium and Vibrio were significantly higher in the glycinin group than those in others.Therefore,the existence of soybean antigens(β-conglycinin or glycinin)could damage the structural integrity of the intestine,reduce immune defense,reshape the intestinal microbiome and,ultimately,impair growth in fish.

In vitro digestibility of soybean β-conglycinin hydrolysates

The in vitro digestibility of alcalase enzymatic hydrolysates of β-conglycinin was studied. The results showed that the zeta potentials of β-conglycinin hydrolysates decreased and their electronegativity increased when digested with pepsin and trypsin. Furthermore, the content of peptides with molecular weight from 10 kDa to 20 kDa remained stable, while those with higher molecular weight （〉20 kDa） decreased, and those with lower molecular weight （〈10 kDa） increased. The proportion of highly hydrophobic peptides decreased in the process of the in vitro digestion, but no significant change in the surface hydrophobicity indices of digestion products was observed （P 〈 0.05）. These results indicate that the β-conglycinin hydrolysates were degraded through in vitro digestion, but the degree of degradation was relatively low. Peptides with molecular weight from 10 kDa to 20 kDa in the β-conglycinin hydrolysates resisted the digestion by pepsin and trypsin and they remained stable during the in vitro digestion processes.