一株产蛋白酶芽孢杆菌紫外诱变育种的初步研究

水产品加工废弃物容易造成环境污染,但其富含蛋白质,如果加以充分利用可以减少环境污染和提高经济效益。目前,抗氧化肽已经得到了广泛的应用,已经筛选了1株解淀粉芽孢杆菌Hy-7,以其发酵水产加工废弃物得到了较高的抗氧化肽产率。以此为出发菌,采用紫外线诱变的方法筛选出1株蛋白酶活力较高的菌株Hy-743,以鲅鱼加工后的废弃物为原料,接种该菌株进行发酵。该突变株较未经紫外线诱变的出发菌蛋白酶活力提高了40.1%,酶解物总抗氧化活性提高了10.2%。经遗传稳定性测定,该突变株在传代8次之后其遗传稳定性仍然良好。对菌株Hy-743进行响应面法优化发酵条件,得到最佳发酵条件为发酵温度30℃、发酵时间48 h、培养基初始p H6.6、菌龄26 h,接种量2%、料液比1.7 g/50 m L。筛选出的菌株酶活力较强、遗传稳定性好,可以用于发酵水产品及其加工废弃物制取抗氧化肽。     

苏云金芽孢杆菌酵解大西洋鲑加工废弃物制备抗氧化酵解液研究

为减少因大西洋鲑加工废弃物中的蛋白成分而造成的环境污染和资源浪费,同时提高其综合利用价值,探索以大西洋鲑加工废弃物制成的干粉为原料,使用一株苏云金芽孢杆菌,通过控制料液比、接种量、发酵时间、温度、pH和菌龄等发酵条件来酵解鱼粉,以制取抗氧化酵解液。首先以总抗氧化活性和α-氨基氮含量为指标,找到最佳单因素条件;然后在单因素试验的基础上,以水解度和总抗氧化活性为指标,使用正交实验L_(25)(5~6)对发酵条件进行优化,确定最佳发酵条件为:料液比2.0 g/50 m L,接种量3%,发酵时间72 h,温度30℃,pH 6.6,菌龄48 h,该条件下酵解得到的发酵液总抗氧化活性为503.59 U,鱼粉水解度为17.29%。研究表明,采用苏云金芽胞杆菌酵解大西洋鲑加工废弃物以制取抗氧化酵解液,具有一定的可行性。     

利用大西洋鲑内源酶酶解加工废弃物制取抗氧化肽的研究

大西洋鲑(Salmo salar)加工废弃物中含有丰富的蛋白质,具有较高的开发利用价值,直接遗弃会造成资源浪费和环境污染,探索利用自身内源蛋白酶酶解制取抗氧化肽具有重要的意义。对大西洋鲑内脏所含内源蛋白酶的性质进行了初步研究,测得主要含有3种内源蛋白酶,最适p H为2.0、6.0、9.0,p H 9.0的碱性蛋白酶具有最高的酶比活力。以水解度和总抗氧化活性为指标,通过单因素试验确定大西洋鲑内源蛋白酶处理鲑鱼的加工废弃物干粉料的最适条件,再用正交实验L16(45)对其酶解条件进行优化,确定影响酶解液总抗氧化活性的主要影响因素(P﹤0.05)为温度、p H和酶解时间,比较后得到最佳酶解条件为:温度35℃、加酶量0.7 m L/50 m L、料液比0.7 g/50 m L、p H 9.0、酶解时间7 h,该条件下酶解液的总抗氧化活性为258.29 U,大西洋鲑加工废弃物干粉料水解度为15.00%。研究表明,采用大西洋鲑内源蛋白酶酶解鲑鱼加工下脚料并制取抗氧化肽,具有一定的可行性。     

水产加工废弃物发酵法制备抗氧化肽的研究

以水产加工废弃物小黄鱼头与鱼骨、日本鼓虾虾皮为原料,接种蛋白酶产生菌ZS2发酵后,以紫外分光光度法和邻苯三酚自氧化法测定不同发酵时间发酵液抗氧化活性及其肽含量。结果表明,两种原料发酵一段时间后,肽的含量趋于稳定,15g左右的样品发酵后肽的含量维持在1600μg／ml左右;发酵液对超氧阴离子的清除效果在开始发酵30小时内处于较低水平,到发酵后期,随着时间的延长,发酵液对超氧阴离子的清除能力显著增强,鱼头鱼骨发酵液的抑制率超过50％;日本鼓虾发酵液对超氧阴离子的清除效果更为显著,抑制率在发酵102小时时超过80％。     

梅童鱼内源蛋白酶自水解工艺的优化

为提高海捕低值鱼加工利用率,合理利用鱼糜及鱼糜制品加工中的副产物,充分利用鱼类自身内源酶,寻求低值鱼精深加工的低碳高效利用,以梅童鱼(Collichthys lucidus)为研究对象,利用鱼类自身的内源蛋白酶将鱼体内的蛋白质酶解,在自然p H条件下,观察了液料比、自溶温度、时间对梅童鱼蛋白水解的影响,以可溶性蛋白/总蛋白的值为响应值,采用响应面分析优化自溶条件。通过求解回归方程得到的最优自溶条件为:液料比3.4,温度53℃,时间267 min。经过验证,可溶性蛋白/总蛋白的值为18.35%,与回归方程的预测值18.55%相比,相对误差1.1%,说明模型能较好地预测自溶过程中可溶性蛋白/总蛋白的值。在此条件下,测得水解度为15.84%,过0.45μm微滤膜蛋白质量比为9.83%。研究表明,该水解工艺可为生产鱼露、活性肽、氨基酸等提供技术依据。     

海水小球藻与养殖固废混合水解产酸过程研究

为有效提高海洋微藻两相发酵产沼气效率和产气过程的稳定性,开展了海水小球藻与养殖固废混合水解产酸特性的研究。探讨了水解料液的初始p H、料液浓度、接种量、藻液与养殖固废质量比(藻-废质量比)对海水小球藻水解产酸的影响。单因素试验结果表明,当初始料液p H为7.00时,更有利于料液中挥发性脂肪酸(VFA)的积累;料液总固体(TS)浓度增大,可有效提高料液VFA产率;当接种量为8%~12%时,料液中VFA浓度可提高90%左右。此外,控制合理的藻-废质量比,能够缩短水解产酸的启动时间,获得较高VFA浓度和系统稳定性。综合因子试验结果表明,各因素对料液VFA浓度的影响次序为:料液浓度接种量藻-废质量比,且当料液浓度为7%、接种量为8%、藻-废质量比为2∶1时,料液中VFA浓度最高,为4 760 mg/L。     

鲣鳔蛋白抗氧化酶解物制备工艺

为有效提高鲣鳔蛋白的附加值,研究以DPPH自由基清除率为抗氧化活性评价指标,采用蛋白酶酶解制备活性多肽的工艺,选用菠萝蛋白酶、复合蛋白酶、碱性蛋白酶、木瓜蛋白酶、胃蛋白酶、胰蛋白酶、中性蛋白酶7种酶在各自最适的条件下酶解,筛选出复合蛋白酶为最适用酶,通过单因素实验分别研究加酶量、溶液初始p H、酶解温度和时间对酶解物抗氧化活性的影响,在此基础上,根据响应面法优化鲣鳔抗氧化酶解物的制备工艺。结果显示,最佳酶解工艺条件为加酶量8.53 U/mg,p H 5.54,温度50.03°C,时间5.07 h。此外,利用超滤法对最佳条件下制备的酶解物进行初步分级,得到分子质量分别为大于10 000 u、3000~10 000 u和小于3000 u的3段组分,且这3段组分对DPPH自由基的半抑制浓度IC50值分别为0.64、0.52和0.37 mg/m L。研究表明,最优条件下制备的酶解物的DPPH清除率达72.00%,与模型预测值71.60%接近,且其中小于3000 u的组分具有较强的DPPH自由基清除活性。     

响应面优化沙蚕抗氧化物的超高压提取及其抗氧化活性研究

为了探讨超高压提取条件对沙蚕提取物中总酚含量的影响,采用超高压提取技术对双齿围沙蚕的抗氧化活性物质进行提取,比较不同提取条件(料液比、提取压力、提取时间及提取温度)对沙蚕提取物中总酚含量的影响,在单因素试验的基础上,采用3因素3水平的2次响应面法优化超高压提取工艺参数。结果表明,超高压提取沙蚕总酚的最佳工艺参数为:料液比1∶40(m/v,g/m L),提取压力378.72 MPa,提取时间28 min,提取温度20℃,在此提取条件下沙蚕提取物的总酚含量为8.39μg/mg干基,其清除超氧阴离子自由基、羟自由基及DPPH自由基的IC50(半数抑制浓度)分别为40.13、212.41及2.00 mg/mL。沙蚕超高压提取物具有很强的抗氧化活性。     

嗜水气单胞菌拮抗菌甲基营养型芽孢杆菌WM-1发酵条件优化

为优化嗜水气单胞菌拮抗菌——甲基营养型芽孢杆菌WM-1的发酵条件,获得抑菌物质产生的最佳培养基配方和培养条件,本实验以嗜水气单胞菌为指示菌,以发酵上清液的抑菌活性为检测指标,采用单因素和正交实验设计,对菌株WM-1的发酵条件进行优化。结果显示,菌株WM-1最适发酵培养基为可溶性淀粉1.0%、牛肉膏1.5%、硫酸镁0.07%、磷酸氢二钾0.05%;最佳发酵条件为初始pH 7.5、培养温度30°C、装液量20%、摇床转速210 r/min、培养时间48 h。在最适培养基和最佳发酵条件下,菌株WM-1发酵液抑菌圈直径达(22.5±0.50)mm,抑菌活性比优化前提高了40.6%。本研究为嗜水气单胞菌的防治提供了新的材料、为甲基营养型芽孢杆菌应用于嗜水气单胞菌的生物防控提供了理论基础。     

基于美拉德反应的狭鳕鱼排复配酶解工艺优化

为提高水产品加工副产物中蛋白质资源的综合利用率,采用复合蛋白酶和风味蛋白酶对阿拉斯加狭鳕鱼排进行复配酶解。以水解度为指标,考察初始p H、温度、料液比、加酶量、复配酶比例、时间对酶解液水解度的影响。在单因素试验基础上,采用Box-Behnken中心组合设计和响应面分析法,确定最佳的酶解工艺条件为:初始p H 7.0,温度47℃,料液比1∶2,加酶量3.0%,复合蛋白酶与风味蛋白酶比例1∶1,酶解时间3 h,此时酶解液水解度可达到38.74%。进一步的美拉德反应试验研究表明,在持续酶解9 h后,酶解液水解度、游离氨基酸含量增加趋势均趋于平缓,同时对美拉德反应产物的风味评价差异不显著,因此最终确定酶解时间为9 h。该研究可为利用狭鳕鱼排酶解液进行美拉德反应制备不同肉风味香料提供理论依据。     

Antioxidant Activity of Protein Isolate Obtained by the pH Shift Technique from Hydrolyzed and Unhydrolyzed Shrimp Processing Discards

Protein isolates from hydrolyzed and unhydrolyzed shrimp waste were prepared using the pH shift method. The conditions for protein isolation from unhydrolyzed shrimp waste by the pH shift method were standardized with respect to waste to distilled water ratio (1:2.5, 1:5, 1:7.5, and 1:10 w/v) and the alkaline pH (9.0, 10.0, and 11.0) to which the waste was to be adjusted before protein precipitation for higher yield of protein isolate. The yield of precipitate with waste to distilled water ratio of 1:2.5 (17.3 ± 0.9) was lower (p < 0.05) compared to the other three dilutions. The pH to which the waste is adjusted before precipitating the proteins at pH 4.5 had no effect (p > 0.05) on the yield. The antioxidant activity of the protein isolate obtained by the pH shift method from unhydrolyzed waste was compared with that obtained from the waste hydrolyzed with Alcalase®. The DPPH scavenging activity of protein isolate after enzyme hydrolysis was significantly higher than that of protein isolate from unhydrolyzed waste. With respect to recovery of total antioxidant activity, protein isolation from unhydrolyzed waste was superior to hydrolyzed waste due to higher yield.     

Protein Hydrolysates from Shrimp (Metapenaeus dobsoni) Head Waste: Optimization of Extraction Conditions by Response Surface Methodology

Utilization of shrimp head waste from the processing industry offers a solution to environmental pollution and also produces valuable bioactive peptides. The present study was aimed to optimize the alcalase assisted extraction conditions by response surface methodology (RSM) to obtain protein hydrolysates from shrimp (Metapenaeus dobsoni) head waste with maximum antioxidant activity. Based on the models derived by RSM, the optimized conditions for maximum degree of hydrolysis (DH) were pH 8.2, temperature of 45.4°C, and enzyme/substrate (E/S) of 1.8%. Meanwhile, the optimized conditions for maximum antioxidant activity of protein hydrolysates as measured by 2, 2-diphenyl-1-picrylhydrozyl (DPPH) and ferric reducing antioxidant power (FRAP) assays were pH 8.2 and 8.1, temperatures of 49.7 and 59.4°C, and E/S of 2.1 and 2.7%, respectively. The maximum predicted values for DH, DPPH, and FRAP were 42.44%, 39.64% inhibition, and 8.3 μM Fe (II)/g wet wt. of sample, respectively. The extraction of protein hydrolysates at optimal conditions derived by these models resulted in DH, DPPH, and FRAPs of 40.31%, 38.93%, and 8.21 μM Fe (II)/g of sample, respectively, demonstrating the fitness of the models. The present study demonstrates the potential of Metapenaeus dobsoni head waste as a source of protein hydrolysates with prospective antioxidant activity.     

响应面法优化中性蛋白酶酶解海湾扇贝副产物制备抗氧化酶解物研究

采用响应面法优化中性蛋白酶酶解海湾扇贝(Argopecten irradias)副产物蛋白制备抗氧化酶解物工艺。以DPPH自由基清除率和还原能力为响应值,在底物浓度、温度、时间、酶与底物比4个单因素实验基础上,优化确定3个显著性因素。获得实验条件下最佳制备工艺为:底物浓度25 mg·mL^-1、温度50℃、时间3.8 h、酶与底物比1.5%。在此条件下,实验验证酶解海湾扇贝副产物蛋白制备抗氧化酶解物的DPPH清除率为81.93%(预测值为83.19%),还原能力为58.12%(预测值为58.92%),DPPH自由基清除率和还原能力的IC 50值分别为0.264 mg·mL^-1和3.136 mg·mL^-1,且表现出较强的抗氧化活性。研究表明,优化后的回归模型用于预测中性蛋白酶酶解海湾扇贝副产物蛋白制备抗氧化酶解物是可行的。     

In Vitro Comparative Evaluation of Antioxidant Activity of Hydrophobic and Hydrophilic Extracts from Algicolous Fungi

The screening of potential antioxidant activities of hydrophobic (ethyl acetate) and hydrophilic (polysaccharide) extracts from both mycelia and fermentation media of eleven algicolous fungi was performed using three antioxidant assays. Algicolous fungi were able to ferment low-cost nutrients composed of potato infusion, glucose, and natural seawater and produce hydrophobic and hydrophilic metabolites with antioxidant properties. Signi?cant differences were observed in antioxidant activities of extracts from the various species evaluated using total antioxidant activity (TAA), hydroxyl radical scavenging activity (HRSA), and ferric reducing antioxidant power (FRAP), as well as their total sugar contents (TSC) of hydrophilic extracts and total phenolic contents (TPC) of hydrophobic extracts. TSC of endopolysaccharides was higher than exopolysaccharides and showed a significant correlation with both TAA and FRAP. Most of mycelial extracts were richer in phenolics than fermentation media, and TPC of hydrophobic extracts was significantly correlated with their FRAP. Mycelial extracts for most of algicolous fungi showed higher antioxidant activities than fermentation media. Both hydrophobic and hydrophilic extracts of algicolous fungi showed good antioxidant properties, especially as hydrogen-donating antioxidants, which could be considered for future applications in medicine, food production, or cosmetic industry.     

水产加工副产物源抗氧化肽的研究现状与展望

我国是水产资源大国,近年来的水产加工业发展较快,产生了大量水产品加工副产物。其中许多副产物的蛋白质含量较高,可通过酶解得到不同功能性活性肽,进而实现高值化的利用。提高对水产品加工副产物的利用不仅可以减少资源的浪费,提高资源利用率,而且对降低企业成本,增加企业经济效益,减少环境污染也具有重要现实意义。本文主要对水产品加工副产物制备抗氧化肽的来源、抗氧化肽分离纯化方法、抗氧化检测评价方法以及抗氧化肽作用机制的研究进展进行综述。     

Optimization of Antioxidant Activity and Phenolic Compound Extraction Conditions from Red Seaweed (Laurencia obtuse)

ABSTRACT

Response surface methodology was used to investigate the effect of ultrasound-assisted extraction conditions including solvent:seaweed ratio (10:1–30:1), extraction temperature (30–50°C), and extraction time (30–60 min) on the total phenolic compounds and antioxidant activity of red seaweed (Laurencia obtuse) extracts. The optimum extraction parameters for maximum phenolic content were as follows: solvent:seaweed ratio, 30:1; extraction temperature, 50°C; and extraction time, 42.8 min. The experiment results showed that the solvent:seaweed ratio was the most significant parameter for the extraction. The optimum extraction conditions for maximum antioxidant activity were as follows: solvent:seaweed ratio, 24.3:1; extraction temperature, 45.3°C; and extraction time, 58 min. Under the above-mentioned conditions, the experimental total phenolic content and antioxidant activity value were 26.23 ± 0.75 mg GAE/g seaweed and 120.89 ± 0.81 TEAC, respectively, of seaweed tested, which are well compatible with the predicted contents.     

Influence of Degree of Hydrolysis on Chemical Composition,Functional Properties,and Antioxidant Activities of Chinese Sturgeon (Acipenser sinensis) Hydrolysates Obtained by Using Alcalase 2.4L

Protein hydrolysates from Chinese sturgeon were prepared using Alcalase 2.4L enzyme. Under the optimum conditions (enzyme–substrate ratio of 3.5%, pH of 8.5, and temperature of 55°C), the degree of hydrolysis (DH) was 13.8%, 16.7%, and 19.1% after 1, 3, and 6 h, respectively. The contents of crude protein and amino acid increased at DH of 19.1% to 86.97% and 78.29%, respectively. There was an obvious increase in the low-molecular-weight peptides, which could enhance the hydrolysate’s functional properties such as solubility, representing more than 90% at different pH levels. The obtained protein hydrolysates revealed good emulsification properties and high oil absorption. Furthermore, good antioxidant activities such as 1, 1-diphenyl-2-picrylhydrazyl (DPPH) inhibition, ferric reducing power, and ferrous ion (Fe²⁺⁾ chelating ability were attained depending on the solution concentration. The findings indicate that the functional and antioxidant properties of protein hydrolysates could be useful in many applications of the food industry.     

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability,isolated from freshwater aquaculture pond

Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca²⁺ 0.8, Mg²⁺ 0.2, Mn²⁺ 0.4, Fe²⁺ 0.6, Al³⁺ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics.     

Shelf-Life of Spanish Mackerel (Scomberomorus commerson) from Northern Australian Waters

Abstract

The shelf-life of Spanish mackerel caught by line fishing in Northern Australian waters was evaluated. Spiked fish were stored in ice either headed and gutted or left uncut and subjected to sensory, nucleotide, pH, histamine and microbial analysts and the rigor pattern recorded. There was little difference between the two product forms. Rigor in uncut fish lasted for 14 ± 9.8 hours and in headed and gutted fish for 19.6 ± 16.3 hours. Spanish mackerel retained good sensory quality for up to 14 days. The bacterial load reached 105 cfu/g by 18 days. K values were low for most of the storage time, < 40%. Histamine levels did not develop more than 10 mg/kg during storage. The pH of Spanish mackerel did not change much even though bacterial levels become high.