Degradation of naturally occurring and engineered antimicrobial peptides by proteases

We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.

The Technology Research of Anti-oxidation Peptide Preparation by Alkaline Protease Hydrolyzing Wheat Germ Meal

Wheat germ meal is the by production of oil extracting, and a great quantity of it has been wasted, thus the quantity of lost protein is great. In order to use wheat germ meal proteins adequately, wheat germ proteins were hydrolyzed to anti-oxidation peptides by using alkaline protease. Through the single factor analysis and regression analysis, the optimized experiment conditions of hydrolysising wheat germ meal to wheat germ peptides were enzymatic quantity 0.8%（w/w）, material to liquid ratio 1∶12.3, enzymolysis time 2.1 h. Under these conditions, the scavenging effect was 49.78%,the DH was 22% and peptides content in enzymatic hydrolysate was 1.9%（w/w）.By SDS-PAGE electrophoresis,the molecular weight range of wheat germ peptides were below 10 ku and most were between 4.54 and 5.63 ku.The wheat germ proteins could be used ful y and grain resources would be saved.

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.

Investigating the effects of peptide-based,MOS and protease feed additives on the growth performance and fecal microbial composition of weaned pigs

Background:Digestive disorders in weaning pigs remain a major challenge for swine producers.Different types of commercial feed additives have been developed to promote gut health and development in young pigs,but their effects on resident gut microbial communities remain largely unexplored.The aim of this study was to investigate the impact of a peptide-based product(Peptiva)in combination with mannose oligosaccharides(MOS)and an exogenous protease on the performance and fecal microbiome of nursery pigs.Methods:A total of 1097 weaned pigs were divided into 44 pens(24-26 pigs/pen)with each pen randomly assigned to one of four experimental diets as part of Phase Ⅱ and Phase Ⅲ of a standard nursery phase feeding program.Fecal samples collected from representative control and treatment pigs were used to investigate bacterial composition profiles by high throughput sequencing of PCR-generated amplicons targeting the V1-V3 region of the 16S rRNA gene.Results:Higher gain:feed was observed for pigs fed Peptiva and MOS compared to Controls during the period when experimental diets were fed,but the benefits of supplementation were not maintained after pigs were transitioned to a non-supplemented diet.Three candidate bacterial species,identified as Operational Taxonomic Units(OTUs),were found to have significantly different abundances between control samples and treatment samples during the same phase.In Phase Ⅲ samples,SD_Ssd-00039,predicted to be a strain of Streptococcus alactolyticus based on nucleotide sequence identity,was the most highly represented of these OTUs with an average abundance in pigs fed Peptiva,MOS and protease that was 3.9 times higher than in Controls.The report also presents evidence of microbial succession that occurred during the trial,with 16 of the 32 most abundant OTUs found to vary between Phase Ⅱ and Phase Ⅲ samples for the same dietary treatment.Conclusions:Dietary supplementation with a combination of a peptide-based product,MOS,and protease increased the growth performance of weaned pigs compared to control animals during the nursery phase,but these benefits were no longer observed within 2 weeks after all animals were transitioned to a non-supplemented diet.Supplementation with these feed additives was found to modulate the composition of the swine gut microbiome during this period.

The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains （HN3.11, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 was found to have the highest antioxidant activity （61.4%） and the highest angiotensin I converting enzyme （ACE）-inhibitory activities （68.4%）. The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

Novel insight into the formation and inhibition mechanism of dipeptidyl peptidase-Ⅳ inhibitory peptides from fermented mandarin fish(Chouguiyu)

Fermented foods are a potential source to produce novel dipeptidyl peptidase-IV inhibitory peptides(D4IPs).In this study,the fermented mandarin fish(Chouguiyu)was used to screen D4IPs and their formation mechanism was studied by metagenomics and peptidomics.A total of 400 D4IPs with DPP-IV inhibition structure and high hydrophobicity were identified.The correlation network map showed that Lactococcus,Bacillus,Lysobacter,Pelagivirga,Kocuria,Escherichia,Streptococcus,and Peptostreptococcus were significantly correlated with the most D4IPs.Four stable D4IPs,including KAGARALTDAETAT,GEKVDFDDIQK,VVDADEMYLKGK,and GQKDSYVGDEAQ were respectively from the precursor proteins parvalbumin,troponin,myosin,and actin,and were mainly formed by the hydrolysis of subtilisin(EC 3.4.21.62),aspartic proteinase(EC 3.4.23.1),thermolysin(EC 3.4.24.27),oligopeptidase B(EC 3.4.21.83),and proteinase P1(EC 3.4.21.96)from Bacillus,Kocuria,Lysobacter,Lactococcus,and Peptostreptococcus.The inhibition mainly resulted from the hydrogen bond and salt bridge between D4IPs and DPP-IV enzyme.This study provides important information on the proteases and related microbial strains to directionally prepare D4IPs in Chouguiyu.

Antioxidant Activity of Peptides Obtained from Cotton Ground Oil-Cake Proteins

Antioxidant activity of the peptides derived from proteins of defatted cottonseed kernels and cotton ground oil-cake by their enzymatic hydrolysis with acidic （Asp. niger） and neutral proteinases （Bac. amyloliquefaciens） was studied. Antioxidant activity of the derived peptides depended on the used proteins and enzymes. The peptides derived by using of neutral proteinase possessed higher antioxidant activity, in comparison with the peptides derived by acidic proteinases.

Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction

AIM:To determine the molecular mechanisms of Shugan decoction(SGD) in the regulation of colonic motility and visceral hyperalgesia(VHL) in irritable bowel syndrome(IBS).METHODS:The chemical compounds contained in SGD were measured by high-performance liquid chromatography.A rat model of IBS was induced by chronic water avoidance stress(WAS).The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension.Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining.The contents of tumor necrosis factor(TNF)-αin colonic tissue and calcitonin-gene-related peptide(CGRP)in serum were measured by ELISA.The protein expression of serotonin[5-hydroxytryptamide(5-HT)],serotonin transporter(SERT),chromogranin A(Cg A)and CGRP incolon tissue was measured by immunohistochemistry.RESULTS:SGD inhibited colonic motility dysfunction and VHL in rats with IBS.Blockers of transient receptor potential(TRP)vanilloid 1(TRPV1)(Ruthenium Red)and TRP ankyrin-1(TRPA1)(HC-030031)and activator of protease-activated receptor(PAR)4 increased the pain pressure threshold,whereas activators of PAR2and TRPV4 decreased the pain pressure threshold in rats with IBS.The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1(capsaicin),TRPV4(RN1747),TRPA1(Polygodial)and PAR2(AC55541).In addition,CGRP levels in serum and colonic tissue were both increased in these rats.TNF-αlevel in colonic tissue was also significantly upregulated.However,the levels of 5-HT,SERT and Cg A in colonic tissue were decreased.All these pathological changes in rats with IBS were attenuated by SGD.CONCLUSION:SGD alleviated VHL and attenuated colon motility in IBS,partly by regulating TRPV1,TRPV4,TRPA1,PAR2,5-HT,Cg A and SERT,and reducing CGRP and TNF-αlevel.

Systematic functional analysis and potential application of a serine protease from cold-adapted Planococcus bacterium

In this study, a gene encoding serine protease(PmSpr288)from cold-adapted bacterium, namely Planococcus maritimus XJ11, was cloned and overexpressed in Escherichia coli BL21(DE3). Bioinformatics analysis revealed that PmSpr288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49, His86 and Ser251. Moreover, PmSpr288 was found to be active over broad alkaline pH and low-moderate temperature, and exhibited wide range of protein substrate specificity. In addition, PmSpr288 was able to hydrolyze the meat proteins actin and myosin, and molecular docking results suggested that the crucial interaction between PmSpr288 and actin/myosin complexes was mainly occupied by hydrogen bonds. The muscle protein hydrolysates of silver carp prepared by PmSpr288 was shown to have antioxidant activity via DPPH radical scavenging assay, which presented an IC_(50) valve of 1.309 mg/mL. In conclusion, these characteristics imply that PmSpr288 has potential biotechnological application prospect for the production of bioactive peptides.

发酵棉籽粕对育肥猪生长性能、蛋白酶活性及肠道小肽转运载体的影响

文章旨在研究发酵棉籽粕对育肥猪生长性能,蛋白酶活性及肠道小肽转运载体的影响。试验将200头体重(36.49±1.34)kg的育肥猪分为4组,每组5个重复,每个重复10只。各组分别使用0%、1%、2%和4%的发酵棉籽粕替换豆粕。试验为期100d,预饲期10d,正试期90d。在初重无显著差异的基础上(P>0.05)添加2%和4%的发酵棉籽组肥猪的FBW显著降低3.12%和3.83%(P<0.05);添加2%和4%的发酵棉籽组肥猪的ADG显著降低4.44%和5.57%(P<0.05);添加2%和4%的发酵棉籽组肥猪的料重比显著提高5.51%和5.57%(P<0.05)。添加2%和4%的发酵棉籽组肥猪的十二指肠胃蛋白酶活性显著降低(P<0.05);添加4%的发酵棉籽组肥猪的空肠胃蛋白酶和胰蛋白酶活性均显著降低(P<0.05)。添加2%和4%的发酵棉籽组肥猪的十二指肠CAT1和PepT1基因表达显著降低(P<0.05);添加1%~4%的发酵棉籽组肥猪的空肠CAT1和PepT1基因表达显著降低(P<0.05);添加1%~4%的发酵棉籽组肥猪的回肠CAT1基因表达显著降低(P<0.05)。总之,发酵棉籽可能通过下调肥猪肠道蛋白酶活性和小肽转运载体mRNA表达影响肠道蛋白和氨基酸消化吸收,从而抑制育肥猪生长。

棉籽肽制备工艺的初步研究

本文以水解度和DPPH·清除能力作为评价指标,采用单因素试验和响应面法优化棉籽肽制备工艺。结果表明,最佳工艺条件为酶解温度53.5℃,液料比18,酶解时间4 h。对最佳工艺进行验证,棉籽蛋白的水解度为22.7%,质量浓度为1.23 mg/mL的棉籽肽DPPH·清除能力为85.12%。

碱性蛋白酶酶解蛋白制备活性肽的质控研究

以胶原蛋白和大豆分离蛋白为主要研究对象,应用体积排阻色谱(SEC)结合多角度激光光散射(MALLS)及示差折光(RI)联用技术对碱性蛋白酶酶解蛋白制备肽段的过程进行了详细分析。在酶解过程中,不同来源的胶原蛋白(牛、鸡)和大豆分离蛋白在分子量分布、体系的分散度及粒径变化规律上表现出了典型的差异。结果表明,牛胶原蛋白在酶解开始时就迅速被降解产生了大量小分子肽段,鸡胶原蛋白相对于牛胶原蛋白在相同的酶解条件下更难产生小分子肽段,大豆分离蛋白随着酶的加入则是表现出了先聚集再降解的特性。SEC-MALLS技术可以有效把控蛋白酶解的过程以及体系中肽段分子量分布,该技术可以为活性肽产品的开发及品质检测提供依据。

沙丁鱼鱼鳞胶原蛋白肽的制备及其抗氧化活性研究

目的优化沙丁鱼(Sardinapilchardus)鱼鳞胶原蛋白肽的制备工艺和评价不同分子量大小胶原蛋白肽的抗氧化活性。方法以沙丁鱼鱼鳞为原料,通过酶工程结合单因素与响应面实验优化胶原蛋白肽制备工艺,采用超滤膜分级分离鱼鳞胶原蛋白肽,并通过体外自由基化学体系分析对比不同分子量大小(10、5、1 kDa)的胶原蛋白肽的抗氧化活性。结果采用碱性蛋白酶进行酶解鱼鳞,胶原蛋白肽的得率最高;最优酶解工艺参数为:碱性蛋白酶加酶量7526 U/g、料液比1:20(g/mL)、pH 10.5、酶解温度67℃、酶解时间4 h,此条件下鱼鳞胶原蛋白肽得率最高,达43.44%±1.59%。通过超滤获得的分子量小于1k Da的鱼鳞胶原蛋白肽对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和羟基自由基(hydroxyl radical,·OH)的清除能力最强,其半抑制浓度(half maximal inhibitory concentration,IC_(50))分别为0.72 mg/mL和1.76 mg/mL;分子量为1~5 kDa的鱼鳞胶原蛋白肽对超氧阴离子自由基(superoxide radical,O^(2-)·)的清除效果最好,其IC50为1.45 mg/mL。结论碱性蛋白酶能够有效制备鱼鳞胶原蛋白肽,且小于1 kDa的鱼鳞胶原蛋白肽抗氧化活性最好。

酶解法制备阿胶蛋白肽工艺条件优化及抗氧化活性分析

目的:研究酶解法制备阿胶蛋白肽的工艺条件及阿胶蛋白肽的抗氧化活性。方法:以阿胶为原料,通过酶解实验优化碱性蛋白酶Alcalase 2.4L制备阿胶蛋白肽的酶解工艺条件。以酶解pH、酶与底物比、酶解温度作为考察因素,以阿胶的水解度为指标,在单因素实验的基础上,通过正交试验确定最佳工艺条件;利用高效凝胶色谱法测定酶解前后阿胶溶液的分子量分布情况,并通过检测对ABTS+和DPPH自由基的清除率,考察酶解前后阿胶溶液的抗氧化活性,从而综合分析酶解效果;采用4种不同大小孔径的超滤膜分离阿胶蛋白肽液,研究经超滤膜分离后不同分子量阿胶蛋白肽的抗氧化活性。结果:阿胶溶液的最佳酶解条件为:pH10.5、酶与底物比9%、酶解温度63℃,此条件下测得阿胶酶解液的水解度为15.93%±1.32%;酶解后阿胶溶液的分子量大部分分布在5000 Da以下;当浓度为10 mg/mL时,未酶解和酶解后的阿胶蛋白肽溶液ABTS+自由基清除率分别为75.83%±0.32%和93.16%±0.21%,DPPH自由基清除率为16.93%±2.41%和39.95%±1.27%;经超滤处理获得五组不同分子量的阿胶蛋白肽(>30 kDa、10~30 kDa、3~10 kDa、1~3 kDa和<1 kDa),分析比较发现低分子量的阿胶蛋白肽抗氧化效果较好。结论:碱性蛋白酶Alcalase 2.4L能够有效制备阿胶蛋白肽,且阿胶蛋白肽具有良好的抗氧化活性。试验结果为后续开展阿胶蛋白肽的抗氧化机制研究提供科学依据。

复合酶酶解法制备中华草龟皮胶原蛋白肽的工艺优化

该研究探讨了不同种类蛋白酶对中华草龟皮胶原蛋白酶解液的水解度和DPPH自由基清除率的影响,并进一步研究了复合蛋白酶对胶原蛋白的酶解效果。此外,通过正交试验优化了复合酶酶解条件。结果表明,由胰蛋白酶和碱性蛋白酶按1∶1组成的复合酶对龟皮胶原蛋白进行酶解,可显著提高酶解液的水解度和DPPH自由基的清除率(P<0.05);优化的复合酶酶解工艺为复合酶添加量5000 U/g、酶解pH值7.5、酶解温度50℃、酶解时间3 h,在该酶解工艺条件下,水解度达(51.19±2.45)%,DPPH自由基清除率达(51.78±2.21)%。影响复合酶酶解效果的主次顺序为酶添加量>酶解pH值>酶解时间>酶解温度。

嗜碱碱性卤杆菌蛋白酶在枯草芽孢杆菌中的分泌表达和优化

该研究通过信号肽筛选和启动子优化构建了一种在枯草芽孢杆菌中高效分泌嗜碱碱性卤杆菌蛋白酶的表达系统。结果表明,信号肽筛选和启动子优化的组合策略显著提高了该蛋白酶在枯草芽孢杆菌中的胞外表达量。在173种信号肽中,SP_(Ydjm)介导该蛋白酶分泌的效率最高,是原始信号肽SP_(aprE)所介导酶活力的13.8倍。而串联启动子P_(HapII)-P_(ylb)对表达的促进作用最为显著,使酶活力进一步提升1.7倍。经发酵培养基适配,该表达系统在摇瓶中产生的蛋白酶活力为18600 U/mL。进一步使用5 L发酵罐发酵后,酶活力达到34050 U/mL。该表达体系有效的提升了嗜碱碱性卤杆菌蛋白酶的分泌量,为该酶的生产与应用提供了良好的基础。

酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺优化

为获得优质的核桃蛋白血管紧张素转化酶(ACE)抑制肽的制备原料及优化其制备工艺,采用连续提取法从脱脂核桃粕中依次分离出清蛋白、球蛋白、醇溶蛋白、谷蛋白-1和谷蛋白-25种组分蛋白,测定5种组分蛋白的占比及ACE抑制率,以ACE抑制率最大的组分蛋白作为原料采用酶解法制备ACE抑制肽,在筛选出最适酶解用酶基础上,采用单因素试验与响应面试验优化酶解制备核桃蛋白ACE抑制肽的工艺。结果表明:5种组分蛋白中谷蛋白-1占比仅次于谷蛋白-2,且其ACE抑制率最高,以核桃谷蛋白-1为原料,在筛选出胃蛋白酶作为酶解用酶基础上,经工艺优化得到最优的酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺条件为酶解温度46℃、酶解时间6 h、酶用量4.2%(以底物质量计)、酶解pH 1.6,在该条件下所得核桃谷蛋白-1酶解液的ACE抑制率为(50.08±2.34)%。因此,核桃谷蛋白-1经胃蛋白酶酶解可生产ACE抑制活性较高的核桃多肽。

传统中药材全蝎热稳定性多肽的消化酶抗性研究

目的:研究传统中药材全蝎三种热稳定性多肽BmKcug2、BmKcug2-P1和Martentoxin-P2的消化酶抗性。方法:结合重组表达、消化酶耐受实验、高效液相色谱技术及结构分析,评价多肽BmKcug2、BmKcug2-P1和Martentoxin-P2的肠胃蛋白酶稳定性。结果:多肽BmKcug2对胃蛋白酶、胰蛋白酶、α-胰凝乳蛋白酶都有很好的稳定性;多肽BmKcug2-P1对胃蛋白酶稳定性较好,对胰蛋白酶也具有一定的抗性,但对α-胰凝乳蛋白酶稳定较差;多肽Martentoxin-P2的酶稳定性谱与BmKcug2-P1类似。结论:本研究发现了全蝎毒腺中存在多肽能够耐受肠胃蛋白酶的消化作用,阐明了全蝎毒腺中多肽的消化酶稳定性,为深入研究全蝎中有效药用成分提供了思路,为系统研究全蝎多肽活性成分和口服给药途径之间的关系奠定了基础。

Production,purification and activity evaluation of three novel antioxidant peptides obtained from grass carp(Ctenopharyngodon idella)scale waste by microbial protease BaApr1 hydrolysis

In this study,an alkaline protease BaApr1 from the Bacillus altitudinis W3 was chosen to hydrolysis grass carp(Ctenopharyngodon idella)scales.The hydrolysate of alkaline protease BaApr1 exhibited the best antioxidant activity compared to other protease hydrolysates.The optimal hydrolysis conditions for BaApr1 were an enzyme dosage of 1250 U/g,a hydrolysis time of 7 h,a pH of 9.5 and a temperature of 50℃.Three novel peptides were purified using ultrafiltration,anion exchange chromatography,gel filtration chromatography and ultra-performance liquid chromatography,and their sequences were identified as Tyr-Val-Gln-Ala-Gly-Ala-Ala-Gly-Ala-Ala-Ala-His(SHP2),Val-Lys-Leu-Tyr-Val-Leu-Leu-Val-Pro(SHP4),and Val-Gln-Val-Leu-Ala-Gly-Pro-Val-Val-Lys-Leu-Tyr(SHP5)with molecular weights of 1086.53 Da,1043.69 Da and 1285.79 Da,respectively.Among them,SHP2 exhibited the highest scavenging activity on DPPH;(EC_(50)4.08 mg/mL),ABTS+;(EC_(50)0.23 mg/mL)and HO;(EC_(50)2.78 mg/mL),and the strongest reducing power.Additionally,SHP5 can significantly inhibit lipid peroxidation in the linoleic acid system.In conclusion,three peptides isolated from scales of hydrolysate of grass carp showed great antioxidant activity and might be used as potential food ingredients and pharmaceuticals.

风味蛋白酶酶解桑葚制备抗氧化肽的工艺研究

利用风味蛋白酶酶解桑葚粉制备抗氧化肽。以多肽得率和DPPH·清除率为指标,在单因素试验的基础上通过响应面法优化酶解工艺。结果表明:最佳酶解条件为酶解温度54℃、pH 6.95、酶解时间4.25 h、酶添加量8%(以桑葚蛋白质量计)、料液比30∶1(g/L),在此条件下桑葚多肽得率为16.76%±0.58%、DPPH·清除率为62.45%±0.93%,制得的桑葚抗氧化肽具有较好的抗氧化活性。