【作者】 胡永金；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学与工程， 2007， 博士

【摘要】 鱼糜具有蛋白质含量高、脂肪含量低、口感嫩爽等特点深受欢迎,但目前鱼糜生产原料多为海水鱼,淡水鱼应用较少。因淡水鱼多属难凝胶化鱼种,凝胶强度较低,易凝胶劣化。本论文根据微生物的发酵性能和鱼糜的基本制作工艺,结合现代食品与发酵技术,旨在研制出具有独特风味,高营养、高安全性、贮藏期长,符合中国人饮食心理等特点的优质发酵鱼糜。本研究对于了解鱼肉在发酵环境下化学成分的变化规律、蛋白质结构变化以及凝胶形成的机理具有重要的学术意义,本研究将为水产加工业建立生物发酵的新加工技术体系提供坚实的理论基础和科学依据,对于促进淡水鱼加工具有重要的应用价值。首先研究了7株乳酸杆菌(LC、LS、Lp-1、LP-5、Lp-8、Lp-10、Lp-15)、2株戊糖片球菌(PP和Pp-8)和1株木糖葡萄球菌(Sx-12)的生长、产酸、耐盐、产蛋白和脂肪分解能力、拮抗性、抑菌性等生理学特性。所有菌株在12-18h内都能进入对数生长阶段,具有较好的发酵活力;在24h内的产酸能力与发酵初期相比差异极显著。性能测定表明Lp-5, LC, Sx-12, PP, Lp-8, Lp-15显示出较好的发酵特性。分别以这六株较优的微生物为发酵剂,以鲢鱼肉糜为基质。分析在发酵剂作用下,鲢鱼肉糜理化和食用品质变化的规律和特征。结果表明,鱼肉糜经微生物作用后,pH值迅速由最初的6.6降至60 h时的4.05-4.46。而对照组,则升高到7.12。非蛋白质态氮和氨基态氮含量随发酵时间的增加而增加,但以接种木糖葡萄球菌增加最大,60 h时分别为8.32 mg/g和0.62 %。接种发酵剂的鲢鱼肉浆的TVBN值上升较慢,在发酵60 h后仍在新鲜标准内(15-25 mg/100g),也没有腐败臭味的产生。而未接种发酵剂的对照组在发酵到48 h时已呈现腐败现象。添加发酵剂可显著增加了鲢鱼肉糜的硬度、胶粘性、咀嚼性,提高鱼肉的白度,有效抑制Pseudomonas (假单胞菌属)、Enterobacteriaceae (肠道菌)等的生长繁殖(P<0.05)。除木糖葡萄球菌外,发酵剂的添加可有效抑制发酵过程中生物胺的产生。感官评定(风味、质地、外观、总体接受性)表明,添加发酵剂可显著改善鱼糜的品质,特别是菌株LC、PP、Lp-8显示出较高的感官特性。在研究了单一发酵剂的基础上,使用混合微生物发酵剂对鲢鱼肉糜进行发酵。在发酵过程中,乳酸菌大量生长增殖,成为优势菌种,pH值急速下降,48 h时降至4.21-4.36,混合发酵剂也可有效抑制肠道菌和假单胞菌的生长繁殖,降低挥发性盐基氮(TVBN)、TBA和生物胺的累积,并且可提高鲢鱼肉糜的凝胶强度和白度。SDS-PAGE、非蛋白质态氮、游离氨基酸和游离脂肪酸的结果显示接种混合发酵剂加快蛋白质和脂肪的降解,也显著提高了鲢鱼肉糜的感官特性。各混合发酵剂发酵样品在质地和外观方面没有显著性差异,但S-PXC(Lp-8,Lc和Sx-12)样品的风味和总体接受性显著高于S-PXP(Lp-8,Sx-12和PP)和S-XCP(Sx-12,Lc和Pp)样品。采用HS-SPME/GC-MS结合嗅觉监测器对S-PXC的挥发性风味成分进行分析,发酵24 h和48 h的鲢鱼肉鱼糜中分别检出42种和44种挥发性风味物质。对发酵鱼糜风味起贡献主要的挥发性风味物质为:乙酸、己醛、己醇、乙酸乙酯、2,3-戊二酮、2-戊烯-1-醇。鲢鱼糜在发酵过程中随发酵时间的增加其凝胶强度显著增加,为揭示凝胶形成机制,综合应用傅立叶变换红外、激光拉曼和圆二色性光谱等方法研究了鱼肉肌纤维蛋白结构的变化规律。在发酵过程中,活性巯基含量逐渐下降,而二硫键的含量和蛋白表面疏水性逐渐增加。FT-IR和Raman光谱分析表明氢键形成的分子间β-折叠结构在蛋白质-蛋白质相互作用形成中发挥着重要的作用。二硫键构象已由对照样品的全旁-旁-反式构象(gauche-gauche-trans)转为旁式(gauche)与反-旁-反式并存的构象。随发酵时间的增加,原来埋藏在蛋白质分子内部的酪氨酸残基逐渐暴露于分子的表面。肌纤维蛋白二级结构分析表明,β-折叠增加、无规卷曲和β-转角降低。发酵鱼糜热力学研究表明肌球蛋白的热变性温度和热焓随着发酵时间的增加而逐渐增加,发酵48 h热变性温度增加了3.28℃,热焓增加了0.176 J/g。而肌动蛋白则相反,48 h内热变性温度降低了1.43℃,热焓降低了0.015J/g。电镜照片揭示微生物发酵后的鱼糜结构从最初的溶胶状态变为凝胶状态,凝胶化后肌动球蛋白呈三维网状结构。鱼糜在发酵过程中凝胶强度的增加主要是S-S键、疏水键及氢键等相互作用的结果。鱼糜的发酵伴随着极为复杂的理化和微生物进程,采用不同发酵方法和发酵条件,发酵制品在发酵过程中所伴随着的生化进程将有所不同,导致发酵终产品食用品质各异。为此,在研究了单一因素的基础上,采用中心复合设计(Central Composite Design, CCD) 3×3响应曲面法优化混合发酵剂的发酵工艺条件。结果表明,各因素及其交互作用对响应值凝胶强度存在显著的相关性。在最优工艺条件下,最大凝胶强度达到1192.8 g.cm。研究了室温和冷藏(4℃)下,真空和非真空包装发酵鱼肉鱼糜品质的变化规律和特性。在贮藏过程中pH值、TBA、非蛋白氮随着贮藏时间的增加而增加,而白度值,水分含量和凝胶强度则降低,在冷藏条件下贮藏,可有效抑制肠道菌和假单胞菌的生长。通过微生物、理化卫生检测与感官评分进行相关分析,建立TVBN、TBA与贮藏时间、贮藏温度之间的动力学模型,预测鲢鱼肉发酵鱼糜在贮藏过程中货架寿命。试验表明TVBN变化和TBA的变化对一级化学反应和阿仑尼乌斯(Arrhenius)方程具有很高的拟和精度。并求出了TVBN变化反应的En和KA分别为52.433 kJ/mol和1.344×1011,TBA的生成反应的En和KA分别为32.6239 kJ/mol和4.785×108。更多还原

【Abstract】 Surimi, a high protein content, low fat content, and taste tender, are very popular in the world. But the raw materials of surimi are mainly used marine fish and freshwater fish is seldom used for difficult to form gel, and has lower gel strength and easy gel deterioration. Application of bio-fermentation method to maintain and improve the quality of products or inhibit the growth of bacteria is a good food processing and preservation method which has been widely used in the livestock meat processing, such as fermented ham and sausage. This study, according to the microbial fermentation performance and surimi production of the basic technology and combined with modern food and fermentation technology, will develop fermented surimi with a unique flavor, high-nutrition, high-security, long-term storage. This study has an academic significance for understanding structural changes of fish protein in the context of fermentation and the formation mechnisms of gelation, and establishs a new bio-fermentation processing technology system to provide a solid theoretical and a scientific basis for the aquatic processing industries.Determination of the seven Lactobacillus (LC, LS, Lp-1, LP-5, Lp-8, Lp-10. Lp-15), two Pediococcus pentosaceus (PP and Pp-8) and a Staphylococcus xylosus (Sx-12) growth, acid, salt, protein and fat production capacity of decomposition, antagonism, such as antibacterial physiology. The results showed that these strains have good fermentation and growth properties. The acid production for 9 Lactobacillus species in 24 hours was significantly different compared with the 0 h. During 24 h fermentation, the pH of samples with Lp-8 decreased rapidly from around 6.12 to 3.95; pH for Sx-12 decline slowly, and at 48 h pH was 5.0. LS and Lp-1 had more poor salt tolerance than the other bacterial. Performances of a preliminary indication for Lp-5, Lc, Sx, Pp, Lp-8, Lp-15 showed good fermentation characteristics and used them as the starter cultures to make fermented surimi. As fermentation time increased, silver carp sausages inoculated with starter (Lp-5, Lc, Sx-12, PP, Lp-8, Lp-15) showed a lower pH than the control Non-protein nitrogen and aminopeptide nitrogen content increased with fermentation time, Staphylococcus xylosus showed the largest contend during 60 h fermentation. TVBN value of the samples inoculated starter is still fresh standard (15-25 mg/100 g) after 60 h fermentation. In contrast, the control has been corruption after 48 h. Starter cultures exhibited higher texture profiles (hardness, gumminess, springiness and chewiness) and whiteness than the control (P < 0.05), and inhibit the growth and reproduction of Pseudomonas and Enterobacteriaceae(p <0.05). Apart from Sx-12, adding starter effectively suppressed the accumulation of histamine, cadaverine, putrescine, tryptamine and tyramine during fermentation. Sensory evaluation (flavor, texture, appearance and overall acceptability) showed that adding starter significantly improve the quality of surimi,especially strains LC, PP, Lp-8 showed higher sensory characteristics.On the basis of the of a single starter, the mixed starter cultures were used in the manufacture of fermented surimi. During the 72 h fermentation at 30°C, silver carp surimi inoculated with mixed starter cultures resulted in a rapid pH decrease, suppression in the growth of Enterobacteriaceae, Pseudomonas, yeasts and molds, and exhibited higher texture profiles (hardness, gumminess, springiness and chewiness) and whiteness than the control (P < 0.05). The changes in non-protein nitrogen (NPN), free amino acid and SDS-PAGE indicated severe hydrolysis of muscle protein occurred during fermentation. Polyunsaturated fatty acids were higher in quantity in sausages with cultures compare to the control. No significant differences for taste, texture, and appearance were found among batches with mixed starters. The sausage inoculated with the combination of lactobacillus plantarum-15, Staphylococcus xylosus-12 and Lactobacillus casei subsp casei-1.001 (S-PXC) gained highest scores for flavor and overall acceptability. Application headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) and olfactory monitors was identified for the determination of flavor-contributing volatiles for silver carp sausages fermented with mixed starter cultures. Fourty-two and fourty-four compounds were identified from the silver carp sausages fermented at 24 h and 48 h, respectively. Acetic acid, hecanal, hexanol, ethyl acetate and 2,3-pentanedione, 2-pentene-1-ol were the main flavor compounds of fermented silver carp sausages inoculated S-PXC. The gel strength of silver carp surimi inoculated with mixed starter cultures increased with the time. To reveal the gel formation mechanism, the changes of the structure of myofibrillar proteins was determined by using fourier transform infrared (FT-IR), laser Raman and circular dichroism spectroscopy methods. During the process of fermentation, activity sulfhydryl decreased with the time, and the content of disulfide bonds and hydrophobic surface protein with fermentation time increased. FT-IR and Raman spectroscopy showed the formation of hydrogen bonds between the molecular-folded structure of the protein-protein interaction formation played an important role. Disulfide conformation samples from the control of the whole next-next-trans conformation (gauche-gauche-tran s) adjacent to (the gauche) and anti-next-trans conformations coexist. With the fermentation time, the original buried tyrosine residues gradually exposed to the surface. the two structures of the myofibrillar proteins further analysis showed that fermentation resulted in a decreased proportion of coil structures and increased the proportion ofβ-sheet structures. The thermal denaturation temperature and enthalpy of myosin gradually increased with the fermentation time. During fermentation 48 h, thermal denaturation temperature and enthalpy increased 3.28°C and 0.176J/g than the fresh muscle, respectively. In contrast, the actin decreased 1.43°C for heat denaturation temperature and 0.015J/g for enthalpy. The lower pH significantly affects the muscle fiber protein denaturation temperature and enthalpy. Microstructural study revealed that fermented silver carp sausage had a three-dimensional network. A more void and open structure correlated well with fermentation time and was associated with higher acids. The gel strength of silver carp surimi during fermentation increased with the time mainly was the interactions of S-S bond, hydrogen bonding and hydrophobic bond.Surimi during the fermentation accompanied by an extremely complex physico-chemical and microbiological changes. Fermentation, using different methods and fermentation conditions, accompanied by a different biochemical process, which resulted in fermented end-products varying quality. Therefore, on the basis of single factors study, a three-level Central Composite Design (CCD) factorial design was employed combining with response surface methodology (RSM) to optimize the fermentation condition for the production of fermented silver carp surimi. Response surface analysis showed that and the interaction of the factors of response values gel strength and overall acceptability were significantly correlated. The greatest strength gel is 1192.8 g.cm at the optimizing conditions.Comparative analysis at room temperature and refrigerated (4℃), vacuum and vacuum-packed meat fermentation characteristics of fermented surimi biochemical changes. The results showed pH, TBA, and the non-protein nitrogen increased with storage time, and the whiteness, moisture content, and the gel strength decrease during storage. Cold storage can effectively inhibit the growth of Enterobacteriaceae and Pseudomonas, but the samples storage at room temperature, these microorganisms increased. The changes of TVBN and TBA of fermented silver carp surimi at different storage temperature of 20℃、25℃、30℃were studied. The kinetics models of TVBN and TBA with respect to storage time and temperature was established so as to predict the quality change and shelf life of fermented silver carp surimi during storage. The changes of TVBN and TBA value with time and temperature showed precision of the first order chemical reaction and Arrhenius equation. EA and K0 of TVBN and TBA change reaction were obtained. These were 52.433 kJ/mol, 1.344×1011 and 32.6239 kJ/mol, 4.785×108, respectively. The kinetics model could provide an important reference for the predication the changes of fermented silver carp surimi during storage. The shelf life of fermented silver carp surimi at different storage temperature was calculated based on the kinetics models.更多还原