Elucidation of potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa

Dry-cured meat products are considerably popular around the world due to unique flavor.Proteolysis is one of the enzymatic reactions from which flavor substances are derived,which is affected by endogenous proteases.The purpose aimed to reveal the potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa in this paper.The dynamic changes of endogenous proteases activity,free amino acids,and volatiles during dry-cured pork coppa processing were characterized.The results showed that 5 kinds of free amino acids,Glu,Lys,Val,Ala,and Leu,were identified as significant contributors to taste.Meanwhile,key volatiles,such as hexanal,nonanal,octanal,benzaldehyde,3-methyl butanoic acid,2-methyl propanoic acid,and ethyl octanoate,greatly contributed to the flavor characteristics of dry-cured pork coppa.Further partial correlation analysis was performed to better elucidate the relationship among parameters.The results revealed that close relationship between endogenous proteases and key substances.RAP not only significantly affected the accumulation of key active-amino acids,but also affected the accumulation of ethyl octanoate,2,3-pentanedione,and 2,3-octanedione by regulating the accumulation of octanoic acid and Leu.In addition,cathepsin B and D,DPP II,DPP IV and RAP notably affected accumulation of hexanal.

Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder(Paralichthys olivaceus)

Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16 S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The Nterminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecu-lar-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

Effect of protease supplementation on apparent ileal crude protein and amino acid digestibility of over-processed soybean meals in broilers

Background: Nutritional value of proteins in feed ingredients can be negatively affected by hydrothermal processing, which causes large variation in the bioavailability of amino acids(AA) and negatively affects animal productive performance. Supplementation of exogenous proteases could increase the rate of digestion of damaged proteins, thereby increasing overall AA digestibility and bioavailability. The aim was to determine the effect of exogenous protease supplementation on the apparent ileal digestibility(AID) of crude protein(CP) and AA of soybean meals(SBM) with different degrees of hydrothermal processing in broilers.Methods: The experiment involved a 3 × 2 factorial arrangement, with SBM processing time(commercial SBM or autoclaved for 30 or 60 min at 120 ℃) and protease supplementation(not supplemented and supplemented) as factors. Protease was included at three times the recommended dose(0.06%) and the experimental diets were fed from 15 to 21 d.Results: The interaction between the effects of SBM processing and protease supplementation was significant for the AID of CP(P = 0.01), Trp(P = 0.01), Gly(P = 0.03) and Pro(P = 0.03), and also for the average daily gain(P = 0.01)and feed conversion ratio(P = 0.04). Increasing the processing time of SBM decreased(P < 0.0001) the AID of all amino acids, whilst the effect of protease supplementation was only significant for the AID of Phe(P = 0.02) and Tyr(P = 0.01).Conclusions: Exogenous protease supplementation at three times the commercial dose does not seem to offset the negative effects of hydrothermal processing of SBM on the apparent ileal digestibility of CP and amino acids or performance of broilers. Whilst positive numerical improvements of digestibility and performance(ADG and FCR)were noticed with protease supplementation at relatively mild processing levels, negative results were obtained with the harsh-processed meals.

Chlorogenic acid may be a potent inhibitor of dimeric SARS-CoV-2 main protease 3CLpro: an in silico study

Background:Since the emergence of coronavirus disease 2019 to date,there is no available approved drug or definitive treatment for coronavirus disease 2019 viral infection,and the identification of novel hits against therapeutic targets has become a global emergency.Echinacea purpurea is a traditional herb utilized to treat cough,fever,sore throat,respiratory tract infection,and so on as an immune stimulant.In this study,in silico molecular docking approach was used to screen phytocompounds from E.purpurea against severe acute respiratory syndrome coronavirus 2 main protease 3C-like protease(3CLpro)and severe acute respiratory syndrome coronavirus main peptidase(96%sequence similarity)to blunt the viral gene expression and viral replication.Methods:Initially,we screened phytocompounds for their druggability and ADMET property.Furthermore,x-ray crystallographic structures of main proteases 3CLpro and main peptidase having Protein Data Bank ID 6LU7 and 2GTB were used as protein targets for the identification of potential drug candidates.We performed docking using AutoDock Vina by PyRx 0.8 software.BIOVIA Discovery Studio Visualizer v2019 was used to analyze ligand-protein complex.The probable protein targets of the selected compound were predicted by BindingDB(P≥0.7).STRING and Kyoto Encyclopedia of Genes and Genomes pathways are utilized to identify the molecular pathways modulated by the predicted targets(FDR≤0.05),and the network interaction between compounds and protein pathways was constructed by Cytoscape 3.6.1.Results:Among all the compounds,chlorogenic acid showed druggable characteristics and scored the lowest binding energy with main protease and main peptidase via interacting with active site 1 domain amino acid residues.Interestingly,chlorogenic acid interacted with Phe140 main protease 3CLpro,which is potentially involved in the dimerization.Enrichment analysis identified chlorogenic acid to modulate insulin resistance,necroptosis,interleukin-17,tumor necrosis factor signaling pathway,legionellosis,T helper 17 cell differentiation,advanced glycation end products and receptor for advanced glycation end products,mitogen-activated protein kinase,Ras,estrogen,vascular endothelial growth factor,B-cell receptor,nuclear factor kappa B,Rap1,hypoxia inducible factor-1,phosphatidylinositide 3-kinase-Akt,insulin,mechanistic target of rapamycin,p53,retinoic acid inducible gene I like receptor,and ErbB signaling pathways.Conclusion:Chlorogenic acid may act as a potent main protease 3CLpro inhibitor and may also inhibit the severe acute respiratory syndrome coronavirus 2 dimerization,viral gene expression,and replication within the lung epithelium.Chlorogenic acid may go a long way in finding one of the multipronged solutions to tackle coronavirus disease 2019 viral infection in the future.

Comparative Effects of Coated Compound and Mono-component Proteases on Growth Performance and Nutritional Efficiency in Broiler Diets

Protease as feed additive is being used in poultry production as a partial replacement for protein sources for cost efficiency and reducing nitrogen excretion. However, diverse proteases may yield different responses under field conditions. A pellet diet study was conducted in Cobb broilers to assess the impact of coated compound （CC） and mono-component （MC） proteases with 5% replacement of digestible amino acids and 0.9% crude protein. Birds fed positive control diet had a better growth than those fed negative control diet, regardless of enzyme supplementation. However, CC protease had shown feed conversion ratio （FCR） like control in a reformulated diet, whereas negative control and MC protease missed to gain the feed conversion. In measures of nutritional efficiency, like energy efficiency, protein efficiency and amino acids efficiency （lysine and methionine）, the CC protease proved to be better than MC protease. In terms of European efficiency factor （EEF）, control and CC protease elicited a closer response, whereas the other two groups showed a drop. In this study, CC protease allowed partial substitution of digestible amino acids and crude protein, while maintaining feed efficiency and animal performance. It could be concluded that incorporating CC proteases is an efficient choice to maximize the utilization feed material resources and efficiency in animal protein production.

新型稀奶油-大豆分离蛋白香基的制备

为了丰富奶味香基蛋白源营养种类、降低成本并增强其风味强度,选用大豆分离蛋白部分替代稀奶油构建复合体系,采用两种不同的酶法结合乳酸菌发酵法,开发新型奶味香基,同时对两款香基的感官特性、主要代谢物和风味物质进行分析。结果表明,一步酶法结合发酵制备的香基具有较高水平的游离脂肪酸和中长链挥发性酸,并因此呈现出刺激性的酸败味等不良风味;而两步酶法结合发酵制备的香基积累了更高含量的多肽和氨基酸,短链挥发性酸以及3-羟基-2-丁酮、苯甲醛、糠醛、δ-十二内酯等重要挥发性风味物质,并获得更高的感官评价分数,更适于制备新型稀奶油-大豆分离蛋白复合风味香基。

超声协同嫩化剂改善淘汰蛋鸡肉嫩度的工艺优化及其对肉品质的影响

为筛选针对家禽养殖场大量淘汰蛋鸡的肉质嫩化处理方式,采用超声联合氯化钙、生物酶法对鸡胸肉进行嫩化,以剪切力为关键指标,筛选最佳超声参数,并重点分析了质构、保水性、蛋白分布、总巯基与氨基酸含量以及微观结构变化。结果显示,最佳超声参数为超声240 W、频率22 kHz、水浴温度35℃和处理时长10 min。相比于未处理组,超声联合蛋白酶处理后的鸡胸肉剪切力显著(P<0.05)降低64.02%。鸡胸肉的硬度、胶黏性和咀嚼性下降,其中咀嚼性显著(P<0.05)降低56.54%,降低效果明显优于氯化钙。同时,肉的蒸煮损失与离心损失程度也低于氯化钙。电泳结果显示,116 kDa附近大分子肌球蛋白重链降解,部分肌原纤维蛋白分解。其中,总巯基含量也不断降低,鸡胸肉蛋白发生氧化。当超声功率继续提升至300 W时,鸡胸肉中必需氨基酸苏氨酸、缬氨酸和赖氨酸含量显著(P<0.05)降低。微观结构观察显示,鸡胸肉的表观与截面结构发生崩解、深层肌纤维分离、分解与断裂。综上,超声联合蛋白酶处理后的鸡胸肉嫩度与品质得到显著改善。

产蛋白酶芽孢杆菌的筛选、耐酸性驯化及蛋白酶酶学性质分析

该研究利用透明圈法及蛋白酶活力测定从高温大曲中筛选高产蛋白酶的菌株,通过形态学观察和分子生物学技术对其进行菌种鉴定。进一步对筛选菌株的耐酸性进行驯化,并对其耐受性及所产蛋白酶酶学性质进行研究。结果表明,分离筛选到一株高产蛋白酶活力的菌株Cy3,经鉴定为蜡样芽孢杆菌(Bacillus cereus),其在最适生长pH 6条件下所产蛋白酶活性为505.0 U/m L。通过对菌株Cy3进行耐酸性驯化后,得到菌株Cy3-驯,成功将其生长p H驯化至5,培养15 h时OD600 nm值提高2.54倍。菌株Cy3-驯所产蛋白酶的最适反应温度从60℃提高至70℃,最适反应p H值为6,但活性及稳定性略有下降。菌株Cy3-驯具有较好的NaCl和葡萄糖耐受性,最高耐受含量分别为15%和60%,同时在乙醇体积分数8%以及乳酸体积分数3%时仍能生长。

麸醋醋醅中产酸、产蛋白酶葡萄球菌的筛选及产酶条件优化

为提高食醋风味,该研究采用透明圈法及高效液相色谱(HPLC)法从麸醋醋醅中分离筛选既产酸又产蛋白酶的菌株,采用形态学观察及分子生物学技术进行鉴定,对菌株安全性、最适生长条件及产蛋白酶条件进行考察,并将其应用于固态发酵食醋。结果表明,筛选出一株既产酸又产蛋白酶的菌株(编号为D-6),将其鉴定为表皮葡萄球菌(Staphylococcus epidermidis),无致病性;最适生长条件为培养温度37℃,pH 5.5,乙醇体积分数0.5%,葡萄糖含量3%;最佳产蛋白酶条件为:培养温度36℃,接种量4%,培养时间41 h。在此优化条件下,蛋白酶活性为36.12 U/g,与优化前相比提高了49.25%;将其应用于固态发酵食醋,总酸、氨基酸态氮、乙酸及乳酸含量均增加,其含量分别为0.95 mmol/10 g、0.21 g/100 g、182.2 mg/L、1472.6 mg/L。

哈茨木霉酸性蛋白酶基因的克隆表达及其水解应用

为了对哈茨木霉酸性蛋白酶(Acid Protease,Ap)的性质研究,采用RT-PCR克隆了哈茨木霉Ap基因,转化至毕赤酵母GS115菌株中获得高效表达,然后对该重组蛋白酶(Recombinant Acid Protease,rAp)的酶学性质和水解大豆分离蛋白的效果进行测定。结果表明,重组毕赤酵母在500 mL三角瓶中诱导表达时,发酵液中rAp酶活力达到21.50 U/mL。该rAp为天冬氨酸蛋白酶,最适温度为55℃,在40℃处理120 min仍具有较强的热稳定性;最适pH值为2.50,在不同pH缓冲液中处理24 h后,pH值2.00~5.00具有较强稳定性。Cu^(2+)、Ni^(2+)和Mn^(2+)能显著促进活性,相对酶活分别高达116.21%、113.79%和117.44%;而Fe^(2+)、Fe^(3+)、0.50%SDS和5.00%Trion X-100显著抑制活性,其相对酶活分别78.02%、79.26%、2.6%和13.19%。rAp和胃蛋白酶对大豆分离蛋白水解后,水解产物中蛋白相对含量分别为14.67%和3.64%,β-伴大豆球蛋白抗原性分别降低了30.01%和26.10%,球蛋白抗原性分别降低了22.37%和15.63%。从以上结果可知,rAp对大豆分离蛋白具有较强水解和降低抗原性的能力,具有潜在的应用开发价值。

响应面法优化混菌发酵膏状腐乳前发酵工艺

为满足市场对腐乳的多样化需求,采用多菌株混菌发酵制备膏状腐乳。以蛋白酶活力为评价指标,响应面法优化前发酵条件,并测定膏状腐乳理化指标、游离氨基酸组分及感官评分。结果表明,混菌发酵膏状腐乳前发酵的最优工艺条件为:少孢根霉(Rhizopus oligosporus):毛霉(Mucor)∶酵母∶植物乳植杆菌(Lactiplantibacillus plantarum)接菌比例为4∶2∶2∶2,接菌浓度1.0×10^(6)CFU/mL,发酵温度32℃,发酵时间60 h,在此条件下,混菌发酵膏状腐乳的蛋白酶活力为123.84μg/g,显著高于少孢根霉(90.07μg/g)和毛霉(102.14μg/g)单菌发酵腐乳(P<0.05)。混菌发酵膏状腐乳前发酵结束时游离氨基酸总量为5.77 mg/g,其中34.35%是必需氨基酸,含量为1.98 mg/g,高于单菌株少孢根霉(4.25 mg/g)和毛霉(3.66 mg/g)发酵腐乳的游离氨基酸总量。对比单菌和混菌发酵剂发酵膏状腐乳前发酵终点时的感官品评结果,混菌发酵膏状腐乳的综合评分最高,香气浓郁,且滋味丰富。综合来看,混菌发酵膏状腐乳在各方面均优于单菌发酵膏状腐乳。

参附注射液辅助治疗对急性心肌梗死大鼠NOD样受体蛋白3/半胱氨酸天冬氨酸特异性蛋白酶1介导的细胞焦亡信号通路以及炎性因子水平的影响

目的探讨参附注射液辅助治疗对急性心肌梗死(AMI)大鼠NOD样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)介导的细胞焦亡信号通路以及炎性因子水平的影响。方法40只大鼠随机分为假手术组、模型组、倍他乐克组(0.9 mg/kg)和联合组(倍他乐克0.9 mg/kg联合参附注射液6 mL/kg),每组10只,连续灌胃3周。检测造模前、造模后和治疗3周时大鼠血清肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)、白细胞介素(IL)-6、IL-1β和肿瘤坏死因子-α(TNF-α)水平。造模后和治疗3周时,比较各组大鼠左心室射血分数(LVEF)、左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)等心脏彩超参数以及心肌梗死面积。应用实时荧光定量聚合酶链式反应(qRT-PCR)及Western blot检测心肌梗死面积NLRP3、Caspase-1的mRNA及其蛋白表达水平。结果与假手术组相比,模型组造模后和治疗3周时cTnI、CK-MB、IL-6、IL-1β、TNF-α、心肌梗死面积、NLRP3 mRNA和Caspase-1 mRNA表达量均升高,差异有统计学意义(P<0.05);与模型组相比,倍他乐克组和联合组治疗3周时cTnI、CK-MB、IL-6、IL-1β、TNF-α、心肌梗死面积、NLRP3 mRNA和Caspase-1 mRNA表达量均降低,且联合组上述指标均低于倍他乐克组,差异有统计学意义(P<0.05)。结论参附注射液辅助治疗AMI能够进一步减轻心肌细胞损伤,缩小梗死面积,抑制炎症反应和细胞焦亡活性。

新型冠状病毒木瓜样蛋白酶抑制剂荧光共振能量转移高通量筛选模型的建立与应用

目的:以新型冠状病毒木瓜样蛋白酶(PLpro)为靶点,利用荧光共振能量转移(FRET)技术,建立用来筛选PLpro抑制剂的高通量筛选(HTS)模型。方法:通过大肠杆菌原核表达系统进行质粒构建、原核表达、分离纯化,获得高活性PLpro(以FRET检测PLpro生物活性),用的底物为两端被荧光基团Edans与淬灭基团Dabcyl修饰的多肽。通过优化实验条件,建立并评价PLpro抑制剂FRET高通量筛选模型,筛选苗头化合物。结果:利用大肠杆菌原核表达系统成功表达并分离出PLpro,其比活力>3000 U/mg。经过优化实验,确定模型筛选条件:设定HEPES缓冲液(pH 7.0)中NaCl浓度为50 mmol/L,孵育温度为25℃,孵育时间为30 min,PLpro浓度为18μmol/L,底物浓度为2μmol/L。测得Z′因子值为0.74,说明所建模型可用于高通量筛选。通过对天然产物化合物库进行筛选,发现十七烷一烯银杏酸(GA17∶1)对PLpro具有混合型抑制作用且IC50值为15.5μmol/L。结论:成功建立PLpro小分子抑制剂高通量筛选模型,初步确定GA17∶1是一种新型抗PLpro的苗头化合物,该筛选方法对于快速筛选新型冠状病毒PLpro的靶向抑制剂具有十分重要的意义。

生姜蛋白酶辅助金线鱼腐乳发酵工艺优化及理化性质研究

该试验以金线鱼鱼糜为原料,接种雅致放射性毛霉(Actinomucor elegans),辅助添加生姜蛋白酶制作金线鱼腐乳。采用单因素及响应面试验优化前发酵条件;在后发酵时期添加生姜蛋白酶(70 m L/500 g盐坯),检测后发酵时期腐乳理化指标的变化趋势,评价生姜蛋白酶效果。结果表明,前发酵过程中,金线鱼腐乳的氨基酸态氮含量逐渐上升,大分子蛋白降解为小分子多肽,最佳前发酵工艺为发酵时间78 h、发酵温度27℃、菌悬液接种量5.4%,优化得到的金线鱼腐乳毛坯的最大蛋白酶活为55.46 U/g。后发酵中添加了生姜蛋白酶的试验组的氨基酸态氮含量显著高于空白组,pH值比空白组稍低,可溶性固形物含量与空白组的变化规律相差无几,证明生姜蛋白酶酶解效果显著。

菜籽饼超微粉酶解制备抗氧化活性肽

为探究菜籽饼超微粉直接酶解制备抗氧化肽技术,该研究以菜籽饼超微粉为原料,利用碱性蛋白酶、风味蛋白酶对其进行酶解,通过测定2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)自由基清除能力、1,1-二苯基-2-苦基肼自由基清除能力和总抗氧化能力表征酶解产物体抗氧化活性,使用膜分离、Sephadex G-15凝胶层析对酶解产物进行分离纯化,利用高效液相色谱-串联质谱联用技术分析鉴定抗氧化活性组分的氨基酸序列。结果表明,风味蛋白酶酶解产物的肽含量较高,并以分子量小于1 kDa肽段为主,碱性蛋白酶解产物以5~10 kDa肽段为主,但同等浓度下(1 mg/mL)的碱性蛋白酶酶解产物抗氧化活性较高;膜分离后,同种酶解产物中低分子量(<1 kDa)组分具有更好的ABTS自由基清除能力和总抗氧化活性;通过凝胶层析分离纯化得到的高抗氧化活性组分J3经氨基酸序列鉴定,结合PeptideRanker生物活性预测筛选得到5个肽段,肽段的N-端均为苯丙氨酸且肽段中疏水性氨基酸的含量占比达50%以上。该结果可为深入开发菜籽饼蛋白资源提供理论参考。

补骨脂糖蛋白提取工艺优化及成分测定

为优化补骨脂糖蛋白(Psoralea corylifolia glycoprotein,PCG)提取工艺,并对PCG中成分进行分析,在单因素试验基础上,以PCG提取率为指标,对料液比、酶解时间、加酶量进行考察,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定PCG分子量,采用红外光谱仪、超高速全自动氨基酸测定分析仪、高效液相色谱仪测定PCG成分。结果表明,碱性蛋白酶对PCG提取率最高,优化后PCG的提取工艺为料液比为1∶30(g/mL)、酶解时间2.5h、加酶量3000U/g,提取得到的PCG分子量为17~75 kDa。PCG中含有氨基酸和糖类基团,氨基酸种类为16种,谷氨酸含量最高,为5.13%,单糖种类为5种,甘露糖含量最高,为44.43%。

蛋白酶对羊肚菌水解液滋味和风味物质的影响

以游离氨基酸总量为指标,研究不同蛋白酶对羊肚菌的酶解效果,筛选羊肚菌酶解增鲜工艺最适蛋白酶,并采用单因素试验以及L9(34)正交试验,优化最适蛋白酶的水解工艺,在此基础上采用氨基酸分析仪及气质谱联用(gas chromatography-mass spectrometry,GC-MS)技术对羊肚菌酶解前后氨基酸组成与挥发性风味物质进行分析评价。结果表明:中性蛋白酶酶解后游离氨基酸总量显著高于其他处理组,可以保持其鲜味和滋味的丰富性,因此选择中性蛋白酶对羊肚菌进行酶解处理。中性蛋白酶最佳酶解工艺条件为pH6.5、酶解温度45℃、中性蛋白酶添加量0.20%、酶解时间2.5 h。相较于未经酶解处理样品,该工艺条件下所得水解液(游离氨基酸总量220.54 mg/g)鲜味氨基酸、甜味氨基酸、苦味氨基酸浓度增加;醇类、酮类、醛类等挥发性风味物质相对含量增加,可以作为羊肚菌调味品加工基料。

红曲霉发酵龙虾壳产酸性蛋白酶分离纯化及酶学性质研究

小龙虾虾壳废弃物排放造成环境污染和资源浪费,而红曲霉可以转化利用虾壳蛋白质。以小龙虾虾壳为唯一氮源,烟灰色红曲霉BQ2液态发酵诱导其产胞外酸性蛋白酶。该酸性蛋白酶经硫酸铵盐析沉淀和DEAE-Sepharose CL-6B阴离子交换柱层析分离纯化,酶活为29.10 U/mL,纯化倍数为11.42,相对分子质量为65000。对其酶学性质进行研究,结果表明该酸性蛋白酶的最适反应pH为4.0,最适反应温度为40℃。该酶属于天冬氨酸蛋白酶,Pepstatin A能强烈抑制其活性,Cu^(2+)和Ca^(2+)能抑制该蛋白酶活性,而Mg^(+)、Zn^(2+)和Fe^(3+)对该蛋白酶的活性具有促进作用。