低盐乳酸菌法与传统法腌干鱼制品的风味比较

为了研究不同腌制方法对鱼肉风味的影响,为腌干鱼制品加工新技术的进一步优化和应用提供理论依据,采用固相微萃取-气相色谱-质谱(SPME-GC-MS)联用法分析,鉴定比较了鲜红牙原料采用低盐乳酸菌法和传统法腌干鱼制品的风味成分变化。结果表明:鲜红牙、低盐乳酸菌法腌干鱼和传统腌干鱼肉中分别检测出80、110、91种挥发性成分,醛、醇、酮类化合物是构成腌干鱼肉独特风味的主要成分。经低盐乳酸菌法腌干的鱼肉挥发性物质对风味贡献较大的醛、醇、酮类化合物总量达35种,而鲜鱼和传统腌干鱼肉中分别只有17和21种。低盐乳酸菌法腌干鱼肉风味物质中含有大量的醇、醛类物质,但不含有胺类物质,在保持传统腌制鱼肉风味的基础上增加了特有的花香味、水果香味及酒香味,提升了鱼肉感官品质。所以采用低盐结合复合乳酸菌法制备腌干鱼,不仅能缩短腌制时间,还能提升腌干鱼肉特有的风味,而且防止了胺类物质的产生,显著提高产品的品质和安全性。     

来源于腌干鱼的乳酸菌中抗氧化酶及胞外多糖研究

为研究从腌干鱼中获取的乳酸菌在细胞内外起抗氧化作用的主要活性物质,通过测定菌株胞内的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)活性以及提取胞外多糖(EPS)并测定其抗氧化性,利用超高效液相色谱法(UPLC)分析其结构。结果显示,9株菌的SOD、GSH-Px和CAT的最高活性分别为44.67、15.26和1.23 U/mg prot,对应菌株为L21、L4和L21;9株菌的EPS产量为(5.71±0.18)~(50.33±1.89)mg/L,L21的EPS最高纯度为56.16%±1.08%。L21中EPS的DPPH自由基清除能力和还原能力较高,综合抗氧化能力相对较强,结构分析发现其含有甘露糖(Man)、鼠李糖(Rha)、葡萄糖(Glu)和半乳糖(Gal)等4种单糖。研究表明,分离自腌干鱼的乳酸菌中,抗氧化能力越强的菌株细胞内表现出越高的抗氧化酶活性,同时细胞外EPS的产量和纯度也较高,并且EPS也具有较强的抗氧化能力。从中筛选出一株综合抗氧化能力最强的L21,可以进一步作为生物源性抗氧化剂。     

高产壳聚糖酶菌株的筛选及其产酶条件的优化

从虾塘底泥中分离到1株高产壳聚糖酶的菌株,将其命名为QY01.研究发现其最适培养基组分为胶体壳聚糖1.0%、葡萄糖0.1%、酵母粉0.3%、K2HPO4·3H2O 0.2%、MgSO4·7H2O 0.1%、NaCl 0.5%、(NH4)2SO4 0.5%、起始pH 6.最适培养条件为6%接种量,30℃、160 r/min培养72 h,在此条件下发酵液酶活可达10.87 U/mL,表明筛选到的菌株在降解壳聚糖方面具有较好的利用价值和开发前景.对QY01进行了16S rDNA形态特征及生理生化特性鉴定分析,经序列比对,该菌株与芽孢杆菌属的同源性高达99%;形态特征与生理生化鉴定结果与16S rDNA鉴定结果相符,初步鉴定为芽孢杆菌属(Bacillus sp.).     

高产壳聚糖酶菌株的筛选及其产酶条件的优化

从虾塘底泥中分离到1株高产壳聚糖酶的菌株,将其命名为QY01。研究发现其最适培养基组分为胶体壳聚糖1.0%、葡萄糖0.1%、酵母粉0.3%、K2HPO4·3H2O0.2%、MgSO4·7H2O0.1%、NaCl0.5%、(NH4)2SO40.5%、起始pH6。最适培养条件为6%接种量,30℃、160r/min培养72h,在此条件下发酵液酶活可达10.87U/mL,表明筛选到的菌株在降解壳聚糖方面具有较好的利用价值和开发前景。对QY01进行了16SrDNA形态特征及生理生化特性鉴定分析,经序列比对,该菌株与芽孢杆菌属的同源性高达99%;形态特征与生理生化鉴定结果与16SrDNA鉴定结果相符,初步鉴定为芽孢杆菌属(Bacillussp.)。     

饲用大豆肽生产菌株的初步筛选及发酵条件研究

通过发酵试验对三株芽孢杆菌进行筛选,并确定该菌株的最佳接种量、种龄和摇床速度。对其发酵条件进行优化,结果表明最适条件为:加水量94%、温度30℃、180r/min旋转摇床48h。发酵液的水解度达到15.9%,比优化前提高了1.92倍。     

响应面法优化鲅鱼抗氧化肽发酵条件

为减少加工废弃物蛋白所造成的环境污染,提高水产品的利用率,探索了回收利用鲅鱼(Spanish mackerel)加工后富含蛋白质的废弃物,以寻找制备鲅鱼抗氧化肽的最佳生产条件。借助Design-Expert数据处理软件对苏云金芽孢杆菌Hy-4发酵产抗氧化肽的条件进行优化;在单因素试验的基础上,利用PlackettBurman试验设计法对影响发酵制备鲅鱼抗氧化肽的培养条件进行筛选。确定了影响发酵液总抗氧化活性的3个主要影响因素(P0.5)为发酵温度、培养基初始p H和料液比;在此基础上,利用最陡爬坡实验逼近3个关键因素的最大响应区域,再利用Box-Behnken试验设计及响应面分析法进行回归分析,通过求解回归方程得到产抗氧化肽的最优条件为:发酵时间48 h,发酵温度30℃,培养基初始p H 6.8,接种量2%,料液比1.45 g/50 m L,菌龄24 h。经过验证,发酵液总抗氧化活性达到537.73 U,较优化前提高了57.2%,与回归方程的预测值570.11 U相比,相对误差为5.7%,说明模型能够较好地预测菌株Hy-4发酵产抗氧化肽发酵液的总抗氧化性。     

基于Illumina MiSeq技术比较二种多脂鱼在腌干过程中的菌相变化

通过比较研究多脂红肉鱼(蓝圆鲹)和白肉鱼(带鱼)腌干加工中菌相的变化规律,以探讨加工过程对菌相的影响并寻找具有抗氧化作用的优势菌。在腌干加工过程中采用Illumina平台的MiSeq技术比较分析了两种多脂鱼的菌相变化情况。结果显示,两种鱼的菌相主要分布在拟杆菌门、变形菌门;在科水平上,初始原料的蓝圆鲹和带鱼分别含7个和15个科的细菌,带鱼包括了蓝圆鲹的所有菌群,肠杆菌科作为共同的优势菌,在蓝圆鲹和带鱼中分别占47%和26%。从腌制开始,两种鱼的菌群数都大量减少,弧菌和芽孢杆菌科作为共同优势菌,前者平均占蓝圆鲹和带鱼的40.3%和42.2%,后者则平均占16.7%和13.3%。原料中,蓝圆鲹和带鱼都包含了肠杆菌科、假单胞菌、弧菌科和希瓦氏菌科这4种腐败菌,加工阶段,两种鱼的优势腐败菌都为弧菌科。乳酸菌包括链球菌科和乳杆菌科,仅出现在带鱼中。研究表明,在腌干加工中,带鱼的细菌减少程度大于蓝圆鲹,总体上均呈现下降趋势,两种鱼含共同的菌群和优势菌,却表现出明显的差异。腌干后两种鱼的腐败菌大大减少,说明腌干加工有利于降低鱼类腐败的可能性。可选择带鱼作乳酸菌的分离以进行后续的抗氧化研究。     

金枪鱼鱼骨胶原肽的制备及抗氧化活性研究

为制备金枪鱼鱼骨胶原肽,并对其抗氧化活性进行研究,利用酶解、超滤、凝胶色谱和反相高效液相色谱制备抗氧化胶原肽,采用氨基酸序列分析仪测定其氨基酸序列,利用质谱(ESIMS)确定其分子量,采用羟自由基、DPPH自由基、ABTS自由基和超氧阴离子自由基清除实验和脂质过氧化抑制实验对胶原肽抗氧化能力进行评价。结果显示,金枪鱼鱼骨胶原蛋白经胃蛋白酶和胰蛋白酶2步酶解和分离纯化得到1个十肽(TFCH-P2),经氨基酸序列分析和质谱(ESIMS)确定其氨基酸序列为Gly-Pro-Ala-Gly-Pro-Ala-Gly-Glu-Gln-Gly(GPAGPAGQEG),分子量为839.87 u([M+H]+840.68 u)。体外抗氧化实验结果表明,GPAGPAGQEG对羟自由基(EC500.18 mg/mL)、DPPH自由基(EC500.97 mg/mL)、ABTS自由基(EC500.52 mg/mL)和超氧阴离子自由基(EC500.38 mg/mL)具有良好的清除作用;GPAGPAGQEG亦显示出良好的脂质过氧化抑制作用。研究表明,胶原肽GPAGPAGQEG抗氧化活性良好,可以用于抗氧化相关的功能食品、药物或者食品添加剂。     

智利竹鱼渔场海洋微生物的物种多样性及其生物活性筛选

利用东海水产研究所开展的2012年东南太平洋智利竹鱼(Trachurus murphyi)渔场生态环境调查,对该海域75个站点进行了海洋微生物样品的采集;通过菌株的16S rRNA测序及GenBank数据库比对,分析了从所采样品中分离获得的海洋微生物菌株的物种多样性;通过菌株发酵、代谢产物提取及其抗菌滤纸扩散分析,对其发酵代谢产物粗提物进行了抗菌活性筛选;通过类胡萝卜素合成通路中八氢番茄红素合成酶(crtB)及脱氢酶(crtI)基因分析,对筛选到的抗菌活性菌株进行了基因筛选。结果表明,累计分离获得可培养海洋微生物菌株628株,其隶属12个属,其中的优势菌属包括亚硫酸盐杆菌属(Sulfitobacter)、弧菌属(Vibrios)及气单胞菌属(Aeromonas)。其中16株菌株的发酵代谢产物具有明显的抗菌活性,而其中12株活性菌株具有合成类胡萝卜素的crtB、crtI分子遗传基础。     

发酵鱼溶浆替代鱼粉对大菱鲆幼鱼生长、抗氧化能力、蛋白质代谢及相关基因表达的影响

为研究发酵鱼溶浆(FSW)替代鱼粉对大菱鲆(Scophthalmus maximus)幼鱼生长、抗氧化能力、蛋白质代谢及相关基因表达的影响,实验设正对照组(50%鱼粉),负对照组(30%鱼粉),在负对照组基础上分别以2%、4%、6%、8%的FSW替代鱼粉,分别命名为FSW2、FSW4、FSW6和FSW8组,饲喂初始体重为(30.00±0.03) g的大菱鲆幼鱼8周。结果显示,各组间幼鱼成活率均无显著差异(P>0.05),FSW2~FSW8组幼鱼增重率、蛋白质效率与正对照组无显著差异(P>0.05),但均显著高于负对照组(P<0.05)。FSW2~FSW8组全鱼和背肌粗蛋白含量与正对照组无显著差异,但显著高于负对照组(P<0.05);负对照组全鱼和背肌粗脂肪含量显著高于其他组(P<0.05)。负对照组血清谷丙转氨酶(ALT)、谷草转氨酶(AST)和甘油三脂(TG)均显著高于正对照组(P<0.05),负对照组、FSW2 ~FSW8组血清中总胆固醇(T-CHO)、ALT和AST呈先降低后升高的趋势,肝脏中ALT和AST含量则呈相反趋势。负对照组血清中高密度脂蛋白胆固醇(HDL-C)含量显著低于正对照组(P<0.05)。负对照组肝脏中蛋白激酶A(PKA)、蛋白激酶C(PKC)和乳酸脱氢酶(LDH)活性均显著低于正对照组(P<0.05)。与正对照组相比,负对照组肠道中氨基酸转运载体b0at1和小肽转运载体pept1表达量上调,氨基酸转运载体cat1、pat1表达量差异不显著,FSW2~FSW8组b0at1、cat1、pat1和pept1表达量均显著高于正对照组和负对照组(P<0.05)。综上所述,饲料中添加FSW显著改善了实验鱼对饲料蛋白质的利用率,缓解了植物蛋白造成的生长性能下降。以增重率为评价指标,添加FSW可使饲料中鱼粉的使用量降低至22%,且鱼体在生长和体组成上与50%鱼粉组无显著差异。     

GC—MS检测咸鱼中N-亚硝胺的条件优化

利用气相色谱-质谱联用（GC-MS）检测咸鱼中的N-亚硝胺,优化了样品前处理条件,比较了固相微萃取（SPME）和二氯甲烷超声萃取对N-亚硝胺的响应强度的影响,探讨了有机溶剂用量、萃取时间、萃取次数对测定的影响。采用选择离子法定性定量检测咸鱼中N-二甲基亚硝胺（NDMA）、N-二乙基亚硝胺（NDEA）、N-亚硝基吡咯烷（NPYR）和N-二丙基亚硝胺（NDPA）4种N-亚硝胺。结果显示,优化后的线性相关系数分别达到0.999 2、0.999 1、0.999 1和0.999 2;线性范围为0～10μg.mL-1;该方法重现性好,其相对标准偏差（RSD）均≤2.1%;空白加标回收率可达70%～80%;灵敏度高,检测限分别为0.038 6μg.kg-1、0.022 7μg.kg-1、0.031 6μg.kg-1和0.047 8μg.kg-1。     

Application of Native Lactic Acid Bacteria (LAB) for Fermentative Recovery of Lipids and Proteins from Fish Processing Wastes: Bioactivities of Fermentation Products

Fermentation using native lactic acid bacteria (LAB) was evaluated for its effectiveness in recovering lipids and proteins simultaneously from freshwater fish visceral waste (FVW). Five different LAB isolated from fish processing waste were employed in a fermentation process that involved 10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB. Cultures evaluated included four native isolates (Pediococcus acidilactici NCIM5368, Enterococcus faecalis NCIM5367, Pediococcus acidilactici FM37, and Pediococcus acidilactici MW2) from FVW with E. faecium NCIM5335 as the reference culture. Fermentation with native LAB resulted in recovery of > 90% oil present in the material as against no recovery in case of raw (unfermented) viscera and resulted in > 50% of degree of hydrolysis of proteins. The fatty acid profile of lipids was not affected by the fermentation process. The fermentation liquor, rich in hydrolyzed protein, exhibited antioxidant as well as antagonistic properties against several bacterial pathogens. The results clearly demonstrate the usefulness of fermentation using native isolates for simultaneous recovery of lipids and proteins from fish processing waste. It also asserts the value of fermentation as an eco-friendly method and aids in minimizing disposal/pollution problems associated with these solid wastes.     

Isolation,Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao Product

The aim of the study was to isolate, select, and identify proteolytic lactic acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by poured plate method. Proteolytic LAB were selected using agar skim milk media. Protease activity of LAB was determined based on the amount of tyrosine released in unit/mL. Proteolytic LAB were identified using API 50 CHL kit and 16S rRNA gene sequence analysis. The result showed that a total of 60 isolates were obtained from chao, and 57% of them were cocci-shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively. Identification by API 50 CHL kit showed that four rod-shape isolates were Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-shape isolates could not be identified as cocci bacterium. Rep-PCR results showed that there were two kinds of bands, namely thick and thin. Two isolates were selected from two types of bands that had the highest R for the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The results showed that Ags1-3 isolate was identified as Lactobacillus plantarum and Ags7-3 as Pediococcus acidilactici.     

Influence of glutamine and vitamin E on growth and antioxidant capacity of fish enterocytes

The present study explored the effect of glutamine and vitamin E on growth and anti-oxidation capacity of isolated fish enterocytes. Fish enterocytes were cultured with six medium, respectively, containing 0, 2.0, 4.7, 6.8, 8.1, 9.2 mmol L⁻¹ glutamine for 64 h. The results showed that glutamine could promote fish enterocytes proliferation and differentiation. Fish enterocytes were cultured with different medium containing 0, 2.5, 3.5, 4.5, 6.0, 7.0 μg mL⁻¹ vitamin E for 96 h. The results showed that cells proliferation and differentiation were not significantly enhanced, but anti-superoxide anion activity, anti-hydroxy radical activity, reduced glutathione concentration, the ratio between reduced and total glutathione in the cells were significantly enhanced, and the malondialdehyde concentration in the culture medium was significantly depressed with the vitamin E treatment. In the whole, the present results firstly indicated that glutamine could promote fish enterocytes growth, but vitamin E could not. Vitamin E could promote fish enterocytes antioxidant capacity and cellular structural integrity. These data would be instructive for glutamine and vitamin E supplement in aquaculture diets.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

Comparison of Lactic Acid Bacteria Fermentation with Acid Treatments for Chitosan Production from Shrimp Waste

The traditional procedure for chitosan production involves use of a strong acid (HCl) for demineralization of chitin. This study reports application of a mixed culture of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus lactis) fermentation in demineralization of chitin for chitosan production from shrimp waste. Chitosan produced from shrimp waste with lactic acid bacteria fermentation at 30°C for 72 h or 1 M lactic acid treatment at room temperature for 2 h followed by alkaline treatments (0.5 M NaOH at 25°C for 4 h followed by 12.5 M NaOH at 70°C for 10 h) contained very low protein (0.8–1.1%) and ash (<0.01%) contents with the same solubility (100%) as those of chitosan produced with hydrochloric acid treatment. Lactic acid bacteria fermentation and lactic acid treatment may be utilized in chitosan production from shrimp waste to reduce use of strong chemicals.     

Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish

Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55–95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe.