淡水鱼发酵对酸鱼品质影响的研究
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摘要
我国传统酸鱼不仅具有风味独特、酸香浓郁、鱼腥味低、回味醇厚、贮藏期长的产品特点，还能软化鱼体骨刺，带骨即食，是一种颇受消费者喜好，具有巨大开发潜力的优质发酵鱼制品。但该传统产品发酵周期长、含盐量不均、产品质量不稳定、随机性强、发酵条件难控制，因而制约了该产品的规模化、工业化生产。本论文依据传统酸鱼的微生物及生化特性，优选出适合工业化生产酸鱼的发酵剂，结合现代生物发酵技术，开发出了既保持传统酸鱼的风味特点，又能实现工业化生产的优质发酵鱼体。本研究对于探讨传统酸鱼的生化特性、微生物菌相变化及优势菌种特性，接种发酵酸鱼的理化、微生物变化、挥发性风味变化以及安全性具有重要的学术意义，为克服制约淡水鱼加工业的行业瓶颈，利用现代食品发酵技术建立工业化生产发酵鱼体技术提供了一定的理论依据，对促进我国淡水鱼加工业的发展具有重要的应用价值。
本文以我国6种不同产地的酸鱼作为研究对象，首先研究了这一传统发酵酸鱼的生化、微生物特性及安全性指标。结果表明：6种不同酸鱼中含有的肠道菌和假单胞菌属均在检测限以下，蛋白与水分含量分别为17.2%-22.9%,52.9%-58.1%，pH值在4.27-5.18之间，乳酸是酸鱼中的主要有机酸，SDS-PAGE分析显示重链蛋白和肌动蛋白发生剧烈水解。另外，酸鱼中测出较低含量的生物胺和TBARS。酸鱼的产品特性受原料、配辅料及发酵工艺条件等因素影响较大。
考察了我国传统酸鱼不同发酵期间的优势微生物及微生物菌相的变化及分布，重点对具有优良性状的21株植物乳杆菌、17株戊糖片球菌、19株木糖葡萄球菌和13株酿酒酵母菌的菌种特性进行了研究。对筛选的乳酸菌和葡萄球菌，主要探讨了的产酸速率、氨基酸脱羧酶活性、脂酶和蛋白酶活性、抗菌性等菌种特性。对酵母菌的菌种特性评价主要包括产气率、产酯力、耐盐性、耐酸性等。研究结果表明：乳酸菌和酵母菌在酸鱼发酵期间逐渐增加，发酵成熟后分别达8log cfu/g，6log cfu/g，葡萄球菌在发酵期间呈现先增后降的趋势，在发酵末期为4log cfu/g，肠道菌、假单胞菌属及芽孢菌在发酵期间逐渐降低至检测限以下。酸鱼中的乳酸菌主要包括植物乳杆菌、戊糖片球菌，接着是明串珠菌和食品乳杆菌，还有零星的肠球菌和干酪乳杆菌。葡萄球菌主要包括木糖葡萄球菌和腐生葡萄球菌。酵母菌主要包括酿酒酵母菌和异常汉逊酵母菌。酸鱼在发酵前期主要是明串珠菌和葡萄球菌，中后期是植物乳杆菌、戊糖片球菌、木糖葡萄球菌、腐生葡萄球菌、酿酒酵母菌和异常汉逊酵母菌，它们在酸鱼发酵过程中存在一个动态关系。肠杆菌等腐败菌主要集中在酸鱼发酵前期，随着pH值的降低及乳酸菌的大量繁殖，在酸鱼的发酵中后期没能测出肠杆菌等野生杂菌。21株乳酸菌在三种不同的温度条件下都能使pH值快速的降至4.5以下，大部分乳酸菌株显示对大肠杆菌、金黄色葡萄球菌、李斯特杆菌较强的抑菌效果，10株植物乳杆菌和4株戊糖片球菌能分泌细菌素，琼脂扩散法和SDS-PAGE分析5株植物乳杆菌有显示弱的蛋白酶活性，13株木糖葡萄球菌具有较强的蛋白酶活性，11株木糖葡萄球菌显示一定的脂肪酶活性。仅Lp-10具有酪氨酸脱羧酶活性，4株木糖葡萄球菌也测出具有氨酸脱羧酶活性，其中3株具有酪氨酸脱羧酶活性。通过对13株酿酒酵母菌的产香、耐乳酸、耐盐及蛋白酶试验，得出所有菌株都具有一定产酯能力和蛋白酶活性，Ss-3，Ss-8能耐受10%的食盐浓度。实验结果得出：Lp-15，Lp-21，Pp15，Sx-8，Ss-3，Ss-8显现出良好的菌种特性，具有开发成发酵鱼肉制品优良发酵剂的潜在价值。
采用从酸鱼中选育出的发酵剂，分别选用3组单菌种发酵剂(Lp-15, Lp21, Pp-15)模型和3组混合菌种发酵剂(S1:Lp-15,Sx-8,Ss8； S2:Lp21,Sx-8,Ss-3；S3:Pp-15,Sx-8,Ss-8)模型对鲤鱼鱼体进行发酵，使用传统发酵方法加工的酸鱼作为对照组。对接种发酵鱼制品发酵期间的品质变化进行了系统研究，主要包括发酵期间微生物、pH值、TA、蛋白降解、质构、挥发性风味及生物胺等的变化，结果表明：在各接种发酵剂酸鱼组发酵初期阶段，乳酸菌和酵母菌大量增殖，而葡萄球菌为发酵初期增殖较快，pH值快速降低，致病菌和腐败菌在检测限以下。混合菌种发酵剂组酸鱼发酵初期的葡萄球菌和酵母菌显示了更明显的增长速率，单菌种发酵剂组的TA值高于混合菌种发酵剂组。发酵成熟后，相较对照组，发酵剂酸鱼组的TBARS积累量降低，游离氨基酸和游离脂肪酸含量增加明显，SDS-PAGE分析显示肌原纤维蛋白和肌浆蛋白降解显著。产品感官评定表明：各发酵剂组样品的外形，风味，滋味及总体接受性显著高于对照组，各个产品的质购方面没有显著性差异，但接种混合菌种发酵剂组的酸鱼酸味更为柔和，因而，混合发酵剂制作的酸鱼更能满足消费者的需求，产品品质明显改善。GC/MS分析酸鱼组(对照组、发酵剂组、香辛料组)发酵过程中挥发性风味物质的变化显示，分别含有53、84、101种挥发性风味化合物。挥发性风味种类主要包括酸类、醛类、碳氢类、醇类、酮类、酯类、含氮化合物和呋喃类化合物，其中，碳氢类的萜类物质在香辛料组中含量最高，而乙醇和酯类物质在对照组中含量较低。发酵中形成的主要风味化合物为己醛、乙酸、乙醇、3-羟基-2-丁酮、2,3-丁二酮、3-甲基丁醛、乙酸乙酯、己酸乙酯、苯甲醛等挥发性物质。其中，乙醇和乙酸的含量最高，构成了发酵酸鱼独特的风味物质。推测这些挥发性的风味化合物主要是形成于发酵过程中的氨基酸的Strecker降解、脂肪水解氧化碳水化合物的分解代谢以及香辛料。接种混合发酵剂组酸鱼在发酵期间生物胺的变化表明，混合菌种发酵剂组酸鱼发酵期间较对照组挥发性盐基氮值明显降低，强烈抑制了肠道菌和假单胞菌的生长繁殖，发酵剂组酸鱼(S1，S3)的酪胺、尸胺、腐胺的积累受到显著抑制，但发酵剂组酸鱼(S2)的腐胺含量与对照组类似。各样品中的组胺和精胺均在检测限以下。此外，pH值、游离氨基酸与生物胺的产生没有发现具有直接的相关性。因而，混合发酵剂组(S1,S3)较S2具有更好的抑制酸鱼发酵过程中生物胺的产生，能更显著的改善发酵酸鱼品质，提高产品安全性。因而，使用从传统酸鱼中选育的菌种作为生产发酵鱼体的优良发酵剂，能够有效的改善酸鱼品质，从而为酸鱼实现工业化生产奠定坚实的理论基础。
Suan yu, which is a traditional fish product in China that maintains stable and retainsnutritional substances during storage without fishy odor and taste, can be eaten directly withsoftened fish bone spur. Therefore, freshwater fish can be potentially processed properly withconsiderably elevated product yield. However, this traditional product suffers from longfermentation cycle, uneven salt distribution, unstable product quality, random preparation anddifficultly NSlable fermentation condition. Therefore, the product has not been industrializedin large-scale manufacture. In our study, autochthonous microfloras were isolated from thetraditional Suan yu as starter cultures that were the most appropriate strains owing to theirtechnological and antimicrobial characteristics, armed with which will develop high qualityfermented fish that tastes uniquely and suits industrial production combining modern food andfermentation technology. This study is dedicated to understanding the changes of microbialphase and characteristics of selected starter cultures as well as chemical and microbialproperties, the origin of volatile flavor and the safety of Suan yu. A solid theoretical basis wasprovided using modern food fermentation technology to overcome the bottleneck, aiming topromote the development of freshwater fish processing industry eventually.
The chemical and microbial properties of Suan yu, a sanitary Chinese traditional low-saltfermented whole fish product which is highly nutritional, particularly flavored and long-termstorable, were studied. Six brands of Suan yu from different locations in China were selected.The characteristics of this traditional fermented fish, raw material and other spontaneouslyfermented fish differed significantly. The counts of Enterobacteria and Pseudomonads in thesix brands were below the detection limits. In addition, the products contained high proteinand low moisture contents ranging from17.2%to22.9%and52.9%to58.1%, respectively.The pH values ranged between4.27and5.18, and lactic acid was the major organic acid. Thesodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealsthe proteolysis of myosin heavy chain and actin chain. Moreover, lowly concentrated biogenicamine (BA) and thiobarbituric acid (TBARS) were found in all samples. The characteristicsof the six Suan yu brands were all influenced by raw materials, ingredients, and fermentationprocesses and conditions.
The variations of microbial phase and the distribution of dominant microorganism werestudied at different fermentation stages in naturally fermented Suan yu. Furthermore, thecharacteristics of21strains of Lactobacillus plantarum,17strains of Pediococcuspentosaceus,19strains of Staphylococcus xylosus, and13strains of Saccharomycescerevisiae were investigated. To select the most suitable candidates to be used as the startercultures for fermented fish products, the lactic acid bacteria (LAB) and Staphylococcus xylosus were screened for the acidifying rate, the amino-biogenic ability, and the proteolysis,lipolysis and antimicrobial activities. The Saccharomyces cerevisiae were screened for gasrelease rate, ester production ability, salt tolerance, acid tolerance and etc. The counts of LABand yeast gradually increased during fermentation and ripening to8log cfu/g and6log cfu/grespectively at the end of fermentation. The number of Staphylococci gradually increasedduring the first fermentation stage and then slowly decreased until the end of ripening (4logcfu/g). The counts of Enterobacteria, Pseudomonas and aerobic spore-formers whichdecreased during fermentation were lower than the detection limits ultimately. Most of theLAB isolated from Suan yu were Lactobacillus plantarum and Pediococcus pentosaceus,followed by leuconostoc and food lactobacillus, as well as sporadic enterococcus andlactobacillus casei. The predominant Staphylococcus isolated from Suan yu comprisedStaphylococcus saprophyticus and Staphylococcus xylosus. The yeast mainly consisted ofSaccharomyces cerevisiae and Hansenula anomala. The predominant microorganism in earlyfermentation included leuconostoc and staphylococcus aureus, and those detectedsubsequently included Lactobacillus plantarum, Pediococcus pentosaceus, staphylococcusxylose, Staphylococcus saprophyticus, Saccharomyces cerevisiae and Hansenula anomala.Enterobacteria and other spoilage organisms were mainly concentrated in the thresholdfermentation stage. The counts of Enterobacteria and other spoilage organisms were below thedetection limits with the growth of LAB and the decrease of pH during the later fermentation.All of these organisms were dynamically related in Suan yu during fermentation and ripening.Major LAB dramatically reduced the pH to lower than4.5at each temperature, which thussuppressed the growth of Listeria monocytogenes, Staphylococcus aureus and Escherichiacoli. Ten L. plantarum strains and four Pediococcus pentosaceus produced bacteriocins. Theagar plate assay and SDS-PAGE suggest that five L. plantarum strains mildly proteolyzedmyofibrillar proteins. None of LAB showed any lipolytic activity.13and11Staphylococcusxylosus strains were of intense proteolytic and lipase activities, respectively. Besides, LABwere mostly decarboxylase-negative, but the strain Lp-10was of L-tyrosine decarboxylaseactivity.4Staphylococcus xylosus strains exhibited decarboxylase activity, most of whichtargeted tyrosine. All the Saccharomyces cerevisiae were ester-production capable andprotease-active. Ss-3and Ss-8tolerated10%salt. Hence, Lp-15, Lp-21, Pp-15, Sx-8, Ss-3andSs-8displayed the optimal technological properties out of the isolated strains, which arefeasible to be applied as starter cultures in fermented fish products manufacture.
Three groups of single starter cultures (Lactobacillus plantarum15, Lactobacillusplantarum21,8and Pediococcus pentosaceus15) and three groups of mixed starter cultures(S1: Lactobacillus plantarum15, Staphylococcus xylosus8and Saccharomyces cerevisiae8;S2: Lactobacillus plantarum21, Staphylococcus xylosus8and Saccharomyces cerevisiae3;S3: Pediococcus pentosaceus15; Staphylococcus xylosus8and Saccharomyces cerevisiae8), isolated from Suan yu, were inoculated to produce the traditional fermented fish. Duringfermentation and ripening, the samples inoculated with different starter cultures weresubjected to rapid-growing LAB and yeast, declined pH, slow-growing spoilage bacteria andpathogens, and low TBARS accumulation. However, Staphylococcus grew more rapidly atthe beginning of fermentation. Staphylococcus and yeast inoculated mixed starter culturesalso grew more quickly initially. The TA values of the samples with single starter cultureswere higher than those with mixed starter cultures. The total free amino acids (FAA) of thesamples with starter cultures were higher compared to those of the NS (p<0.05). The proteinswere severely hydrolyzed during fermentation (SDS-PAGE). Moreover, the sensoryevaluation indicates the inoculator fermented fish was more acceptable than the NS was.Nevertheless, the samples inoculated with single starter cultures tasted slightly more acid thanthose inoculated with mixed starter cultures did. Therefore, the results suggest that beinginoculated with mixed starter cultures shortened the lag time that fermentation was initiatedand improved the quality of Suan yu. The alterations of the volatile flavor compounds in alltreated fermented Suan yu were identified by a GC/MS system during ripening, including NSbatch, sample with starter cultures batch and those with starter cultures and spices batch. Theresults show that the53,84and101volatile flavor compounds were detected in those samples,respectively. These compounds in Suan yu were clustered in acids, aldehydes, ketones,hydrocarbons, esters, alcohols and furans. Besides, the contents of terpenoids, one type ofhydrogens, were higher in the batch spices than those in other batches. The levels of estersand alcohols were higher in the batch spices and starter cultures compared to those in the NS.The main volatile flavor compounds included hexanal, acetic acid, ethanol,3-hydroxy-2-butanone,2,3-butanedione,3-methyl butyl aldehyde, ethyl acetate, ethylcaproate and benzaldehyde during ripening. The obviously increased quantities of acetic acidand ethanol during processing led to the distinct flavor. We infer that Strecker degradation,lipids hydrolysis oxidation, carbohydrate catabolism and spices mainly accounted for thegeneration of volatile compounds during ripening. Therefore, the most appropriate strainsisolated from the traditional Suan yu were used as the starter cultures, the flavor of Suan yuwas adjusted for public acceptance, and a solid theoretical basis was provided for the possibleindustrialization production. The effects of mixed starter cultures on the suppression of BAproduced during ripening were investigated. The results show that the mixed starter culturesrapidly decreased pH, and the accumulation of TVB-N were reduced compared to that of theNS. Moreover, the growth of contaminant microorganisms in the sample with mixed startercultures was inhibited. In addition, the contents of tyramine, cadaverine and putrescine in thesamples inoculated with starter cultures (S1and S3) were also restricted. The levels ofputrescine in the samples with S2were similar to those in the NS. Histamine and sperminewere not produced in any batch. Furthermore, pH and FAA and BA productions were not directly correlated. The study suggests that the samples inoculated with S1and S3inhibitedBA more significantly compared to those with S2did, thereby securing the product better.Thus, the quality of Suan yu can be substantially improved using the mixed starter culturesisolated from traditional Suan yu. The results herein are able to assist the commercializationof Suan yu theoretically.
本文以我国6种不同产地的酸鱼作为研究对象，首先研究了这一传统发酵酸鱼的生化、微生物特性及安全性指标。结果表明：6种不同酸鱼中含有的肠道菌和假单胞菌属均在检测限以下，蛋白与水分含量分别为17.2%-22.9%,52.9%-58.1%，pH值在4.27-5.18之间，乳酸是酸鱼中的主要有机酸，SDS-PAGE分析显示重链蛋白和肌动蛋白发生剧烈水解。另外，酸鱼中测出较低含量的生物胺和TBARS。酸鱼的产品特性受原料、配辅料及发酵工艺条件等因素影响较大。
考察了我国传统酸鱼不同发酵期间的优势微生物及微生物菌相的变化及分布，重点对具有优良性状的21株植物乳杆菌、17株戊糖片球菌、19株木糖葡萄球菌和13株酿酒酵母菌的菌种特性进行了研究。对筛选的乳酸菌和葡萄球菌，主要探讨了的产酸速率、氨基酸脱羧酶活性、脂酶和蛋白酶活性、抗菌性等菌种特性。对酵母菌的菌种特性评价主要包括产气率、产酯力、耐盐性、耐酸性等。研究结果表明：乳酸菌和酵母菌在酸鱼发酵期间逐渐增加，发酵成熟后分别达8log cfu/g，6log cfu/g，葡萄球菌在发酵期间呈现先增后降的趋势，在发酵末期为4log cfu/g，肠道菌、假单胞菌属及芽孢菌在发酵期间逐渐降低至检测限以下。酸鱼中的乳酸菌主要包括植物乳杆菌、戊糖片球菌，接着是明串珠菌和食品乳杆菌，还有零星的肠球菌和干酪乳杆菌。葡萄球菌主要包括木糖葡萄球菌和腐生葡萄球菌。酵母菌主要包括酿酒酵母菌和异常汉逊酵母菌。酸鱼在发酵前期主要是明串珠菌和葡萄球菌，中后期是植物乳杆菌、戊糖片球菌、木糖葡萄球菌、腐生葡萄球菌、酿酒酵母菌和异常汉逊酵母菌，它们在酸鱼发酵过程中存在一个动态关系。肠杆菌等腐败菌主要集中在酸鱼发酵前期，随着pH值的降低及乳酸菌的大量繁殖，在酸鱼的发酵中后期没能测出肠杆菌等野生杂菌。21株乳酸菌在三种不同的温度条件下都能使pH值快速的降至4.5以下，大部分乳酸菌株显示对大肠杆菌、金黄色葡萄球菌、李斯特杆菌较强的抑菌效果，10株植物乳杆菌和4株戊糖片球菌能分泌细菌素，琼脂扩散法和SDS-PAGE分析5株植物乳杆菌有显示弱的蛋白酶活性，13株木糖葡萄球菌具有较强的蛋白酶活性，11株木糖葡萄球菌显示一定的脂肪酶活性。仅Lp-10具有酪氨酸脱羧酶活性，4株木糖葡萄球菌也测出具有氨酸脱羧酶活性，其中3株具有酪氨酸脱羧酶活性。通过对13株酿酒酵母菌的产香、耐乳酸、耐盐及蛋白酶试验，得出所有菌株都具有一定产酯能力和蛋白酶活性，Ss-3，Ss-8能耐受10%的食盐浓度。实验结果得出：Lp-15，Lp-21，Pp15，Sx-8，Ss-3，Ss-8显现出良好的菌种特性，具有开发成发酵鱼肉制品优良发酵剂的潜在价值。
采用从酸鱼中选育出的发酵剂，分别选用3组单菌种发酵剂(Lp-15, Lp21, Pp-15)模型和3组混合菌种发酵剂(S1:Lp-15,Sx-8,Ss8； S2:Lp21,Sx-8,Ss-3；S3:Pp-15,Sx-8,Ss-8)模型对鲤鱼鱼体进行发酵，使用传统发酵方法加工的酸鱼作为对照组。对接种发酵鱼制品发酵期间的品质变化进行了系统研究，主要包括发酵期间微生物、pH值、TA、蛋白降解、质构、挥发性风味及生物胺等的变化，结果表明：在各接种发酵剂酸鱼组发酵初期阶段，乳酸菌和酵母菌大量增殖，而葡萄球菌为发酵初期增殖较快，pH值快速降低，致病菌和腐败菌在检测限以下。混合菌种发酵剂组酸鱼发酵初期的葡萄球菌和酵母菌显示了更明显的增长速率，单菌种发酵剂组的TA值高于混合菌种发酵剂组。发酵成熟后，相较对照组，发酵剂酸鱼组的TBARS积累量降低，游离氨基酸和游离脂肪酸含量增加明显，SDS-PAGE分析显示肌原纤维蛋白和肌浆蛋白降解显著。产品感官评定表明：各发酵剂组样品的外形，风味，滋味及总体接受性显著高于对照组，各个产品的质购方面没有显著性差异，但接种混合菌种发酵剂组的酸鱼酸味更为柔和，因而，混合发酵剂制作的酸鱼更能满足消费者的需求，产品品质明显改善。GC/MS分析酸鱼组(对照组、发酵剂组、香辛料组)发酵过程中挥发性风味物质的变化显示，分别含有53、84、101种挥发性风味化合物。挥发性风味种类主要包括酸类、醛类、碳氢类、醇类、酮类、酯类、含氮化合物和呋喃类化合物，其中，碳氢类的萜类物质在香辛料组中含量最高，而乙醇和酯类物质在对照组中含量较低。发酵中形成的主要风味化合物为己醛、乙酸、乙醇、3-羟基-2-丁酮、2,3-丁二酮、3-甲基丁醛、乙酸乙酯、己酸乙酯、苯甲醛等挥发性物质。其中，乙醇和乙酸的含量最高，构成了发酵酸鱼独特的风味物质。推测这些挥发性的风味化合物主要是形成于发酵过程中的氨基酸的Strecker降解、脂肪水解氧化碳水化合物的分解代谢以及香辛料。接种混合发酵剂组酸鱼在发酵期间生物胺的变化表明，混合菌种发酵剂组酸鱼发酵期间较对照组挥发性盐基氮值明显降低，强烈抑制了肠道菌和假单胞菌的生长繁殖，发酵剂组酸鱼(S1，S3)的酪胺、尸胺、腐胺的积累受到显著抑制，但发酵剂组酸鱼(S2)的腐胺含量与对照组类似。各样品中的组胺和精胺均在检测限以下。此外，pH值、游离氨基酸与生物胺的产生没有发现具有直接的相关性。因而，混合发酵剂组(S1,S3)较S2具有更好的抑制酸鱼发酵过程中生物胺的产生，能更显著的改善发酵酸鱼品质，提高产品安全性。因而，使用从传统酸鱼中选育的菌种作为生产发酵鱼体的优良发酵剂，能够有效的改善酸鱼品质，从而为酸鱼实现工业化生产奠定坚实的理论基础。
Suan yu, which is a traditional fish product in China that maintains stable and retainsnutritional substances during storage without fishy odor and taste, can be eaten directly withsoftened fish bone spur. Therefore, freshwater fish can be potentially processed properly withconsiderably elevated product yield. However, this traditional product suffers from longfermentation cycle, uneven salt distribution, unstable product quality, random preparation anddifficultly NSlable fermentation condition. Therefore, the product has not been industrializedin large-scale manufacture. In our study, autochthonous microfloras were isolated from thetraditional Suan yu as starter cultures that were the most appropriate strains owing to theirtechnological and antimicrobial characteristics, armed with which will develop high qualityfermented fish that tastes uniquely and suits industrial production combining modern food andfermentation technology. This study is dedicated to understanding the changes of microbialphase and characteristics of selected starter cultures as well as chemical and microbialproperties, the origin of volatile flavor and the safety of Suan yu. A solid theoretical basis wasprovided using modern food fermentation technology to overcome the bottleneck, aiming topromote the development of freshwater fish processing industry eventually.
The chemical and microbial properties of Suan yu, a sanitary Chinese traditional low-saltfermented whole fish product which is highly nutritional, particularly flavored and long-termstorable, were studied. Six brands of Suan yu from different locations in China were selected.The characteristics of this traditional fermented fish, raw material and other spontaneouslyfermented fish differed significantly. The counts of Enterobacteria and Pseudomonads in thesix brands were below the detection limits. In addition, the products contained high proteinand low moisture contents ranging from17.2%to22.9%and52.9%to58.1%, respectively.The pH values ranged between4.27and5.18, and lactic acid was the major organic acid. Thesodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealsthe proteolysis of myosin heavy chain and actin chain. Moreover, lowly concentrated biogenicamine (BA) and thiobarbituric acid (TBARS) were found in all samples. The characteristicsof the six Suan yu brands were all influenced by raw materials, ingredients, and fermentationprocesses and conditions.
The variations of microbial phase and the distribution of dominant microorganism werestudied at different fermentation stages in naturally fermented Suan yu. Furthermore, thecharacteristics of21strains of Lactobacillus plantarum,17strains of Pediococcuspentosaceus,19strains of Staphylococcus xylosus, and13strains of Saccharomycescerevisiae were investigated. To select the most suitable candidates to be used as the startercultures for fermented fish products, the lactic acid bacteria (LAB) and Staphylococcus xylosus were screened for the acidifying rate, the amino-biogenic ability, and the proteolysis,lipolysis and antimicrobial activities. The Saccharomyces cerevisiae were screened for gasrelease rate, ester production ability, salt tolerance, acid tolerance and etc. The counts of LABand yeast gradually increased during fermentation and ripening to8log cfu/g and6log cfu/grespectively at the end of fermentation. The number of Staphylococci gradually increasedduring the first fermentation stage and then slowly decreased until the end of ripening (4logcfu/g). The counts of Enterobacteria, Pseudomonas and aerobic spore-formers whichdecreased during fermentation were lower than the detection limits ultimately. Most of theLAB isolated from Suan yu were Lactobacillus plantarum and Pediococcus pentosaceus,followed by leuconostoc and food lactobacillus, as well as sporadic enterococcus andlactobacillus casei. The predominant Staphylococcus isolated from Suan yu comprisedStaphylococcus saprophyticus and Staphylococcus xylosus. The yeast mainly consisted ofSaccharomyces cerevisiae and Hansenula anomala. The predominant microorganism in earlyfermentation included leuconostoc and staphylococcus aureus, and those detectedsubsequently included Lactobacillus plantarum, Pediococcus pentosaceus, staphylococcusxylose, Staphylococcus saprophyticus, Saccharomyces cerevisiae and Hansenula anomala.Enterobacteria and other spoilage organisms were mainly concentrated in the thresholdfermentation stage. The counts of Enterobacteria and other spoilage organisms were below thedetection limits with the growth of LAB and the decrease of pH during the later fermentation.All of these organisms were dynamically related in Suan yu during fermentation and ripening.Major LAB dramatically reduced the pH to lower than4.5at each temperature, which thussuppressed the growth of Listeria monocytogenes, Staphylococcus aureus and Escherichiacoli. Ten L. plantarum strains and four Pediococcus pentosaceus produced bacteriocins. Theagar plate assay and SDS-PAGE suggest that five L. plantarum strains mildly proteolyzedmyofibrillar proteins. None of LAB showed any lipolytic activity.13and11Staphylococcusxylosus strains were of intense proteolytic and lipase activities, respectively. Besides, LABwere mostly decarboxylase-negative, but the strain Lp-10was of L-tyrosine decarboxylaseactivity.4Staphylococcus xylosus strains exhibited decarboxylase activity, most of whichtargeted tyrosine. All the Saccharomyces cerevisiae were ester-production capable andprotease-active. Ss-3and Ss-8tolerated10%salt. Hence, Lp-15, Lp-21, Pp-15, Sx-8, Ss-3andSs-8displayed the optimal technological properties out of the isolated strains, which arefeasible to be applied as starter cultures in fermented fish products manufacture.
Three groups of single starter cultures (Lactobacillus plantarum15, Lactobacillusplantarum21,8and Pediococcus pentosaceus15) and three groups of mixed starter cultures(S1: Lactobacillus plantarum15, Staphylococcus xylosus8and Saccharomyces cerevisiae8;S2: Lactobacillus plantarum21, Staphylococcus xylosus8and Saccharomyces cerevisiae3;S3: Pediococcus pentosaceus15; Staphylococcus xylosus8and Saccharomyces cerevisiae8), isolated from Suan yu, were inoculated to produce the traditional fermented fish. Duringfermentation and ripening, the samples inoculated with different starter cultures weresubjected to rapid-growing LAB and yeast, declined pH, slow-growing spoilage bacteria andpathogens, and low TBARS accumulation. However, Staphylococcus grew more rapidly atthe beginning of fermentation. Staphylococcus and yeast inoculated mixed starter culturesalso grew more quickly initially. The TA values of the samples with single starter cultureswere higher than those with mixed starter cultures. The total free amino acids (FAA) of thesamples with starter cultures were higher compared to those of the NS (p<0.05). The proteinswere severely hydrolyzed during fermentation (SDS-PAGE). Moreover, the sensoryevaluation indicates the inoculator fermented fish was more acceptable than the NS was.Nevertheless, the samples inoculated with single starter cultures tasted slightly more acid thanthose inoculated with mixed starter cultures did. Therefore, the results suggest that beinginoculated with mixed starter cultures shortened the lag time that fermentation was initiatedand improved the quality of Suan yu. The alterations of the volatile flavor compounds in alltreated fermented Suan yu were identified by a GC/MS system during ripening, including NSbatch, sample with starter cultures batch and those with starter cultures and spices batch. Theresults show that the53,84and101volatile flavor compounds were detected in those samples,respectively. These compounds in Suan yu were clustered in acids, aldehydes, ketones,hydrocarbons, esters, alcohols and furans. Besides, the contents of terpenoids, one type ofhydrogens, were higher in the batch spices than those in other batches. The levels of estersand alcohols were higher in the batch spices and starter cultures compared to those in the NS.The main volatile flavor compounds included hexanal, acetic acid, ethanol,3-hydroxy-2-butanone,2,3-butanedione,3-methyl butyl aldehyde, ethyl acetate, ethylcaproate and benzaldehyde during ripening. The obviously increased quantities of acetic acidand ethanol during processing led to the distinct flavor. We infer that Strecker degradation,lipids hydrolysis oxidation, carbohydrate catabolism and spices mainly accounted for thegeneration of volatile compounds during ripening. Therefore, the most appropriate strainsisolated from the traditional Suan yu were used as the starter cultures, the flavor of Suan yuwas adjusted for public acceptance, and a solid theoretical basis was provided for the possibleindustrialization production. The effects of mixed starter cultures on the suppression of BAproduced during ripening were investigated. The results show that the mixed starter culturesrapidly decreased pH, and the accumulation of TVB-N were reduced compared to that of theNS. Moreover, the growth of contaminant microorganisms in the sample with mixed startercultures was inhibited. In addition, the contents of tyramine, cadaverine and putrescine in thesamples inoculated with starter cultures (S1and S3) were also restricted. The levels ofputrescine in the samples with S2were similar to those in the NS. Histamine and sperminewere not produced in any batch. Furthermore, pH and FAA and BA productions were not directly correlated. The study suggests that the samples inoculated with S1and S3inhibitedBA more significantly compared to those with S2did, thereby securing the product better.Thus, the quality of Suan yu can be substantially improved using the mixed starter culturesisolated from traditional Suan yu. The results herein are able to assist the commercializationof Suan yu theoretically.
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