发酵香肠中葡萄球菌、微球菌的分离筛选及应用
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摘要
本研究从自然发酵香肠中分离筛选葡萄球菌和微球菌菌株,评估木糖葡萄球菌的发酵和益生特性,目的是筛选出适用于发酵香肠的优势菌株。并结合乳酸菌制成混合发酵剂用于发酵香肠生产,评估发酵剂对发酵香肠中的微生物、物理化学和感官特性变化的影响。主要结果和结论如下:
1.本研究从成熟阶段的发酵香肠中筛选出138株菌,菌株经过生化鉴定显示微球菌58株,其中克氏微球菌38株(65.5%)。葡萄球菌80株,73株(91%)凝固酶阴性和脱氧核糖核酸酶阴性,其中有60株属于木糖葡萄球菌(82.2%)。微球菌全部产色素,不符合发酵剂特点。菌种筛选主要从60株木糖葡萄球菌中选择。
2. 60株木糖葡萄球菌经过硝酸盐还原酶等发酵特性实验初筛和生物胺检测、乙偶姻检测、蛋白酶活性和脂肪酶活性测试实验复筛,筛选出符合条件的4株菌。
3.研究所筛选出的4株木糖葡萄球菌的益生特性,发现能够抵抗较强的酸性环境和较高浓度的胆盐,2株菌C1S23、C3R10产生细菌素。葡萄球菌与乳酸菌的拮抗试验表明,木糖葡萄球菌C1S23、C3R10和乳酸菌11没有拮抗性。
4.测试不同pH值、温度、NaCl浓度、亚硝酸盐浓度对木糖葡萄球菌C1S23、C3R10菌株生长的影响,结果显示适合发酵香肠环境。
5.优选出的菌株木糖葡萄球菌C1S23,C3R10和乳酸菌11(酸化菌株)作为发酵剂生产发酵香肠,主要研究发酵剂对微生物、理化和感官指标变化的影响。
6.微生物分析结果显示各组发酵和成熟期间乳酸菌始终处于支配地位。对照组微球菌的生长明显受酸度或不能竞争过其它菌群的影响,生长受到了抑制,添加发酵剂各组微球菌生长也受到一定的抑制,但由于接种量大,基本成缓慢增长趋势。添加发酵剂各组肠杆菌菌落总数显著下降。
7.理化分析结果显示添加发酵剂各组pH值显著低于对照,发酵三天达到最低值,以后稍有回升。发酵剂的加入对水分活度、灰分含量的变化没有影响,使非蛋白氮含量增加。发酵剂的加入对发酵香肠的质构有着显著的影响,在硬度、咀嚼性和胶粘性方面要显著高于对照组。色泽差异显著,S1和S2组亮度、红度要显著高于对照组。结合感观评价,两组都可作为肉品发酵剂用于发酵香肠的生产。
The aims of this study are to isolated staphylococci and micrococci from traditional sausages and to determine the metabolic and probiotic properties of selected Staphylococcus xylosus, in order to select potential starter cultures for fermented sausages. And employ the mixed starters combined with Lactobacillus in the manufacture of fermented sausages, in order to evaluate the effect of the starter cultures on the physico-chemical, microbiological and sensory properties of typical fermented sausages. The main results and conclusion are as follows:
1. One hundred and thirty-eight strains isolated during the ripening of a traditional fermented sausage were characterized. Fifty-eight isolated belonged to Micrococcus,with thirty-eight strains(sixty-five point five percent of Micrococcus)belonged to Micrococcus kristinae. Eighty strains belonged to Staphylococcus species, and senventy-three strains(ninty-one percent of Staphylococci)were coagulase- negative and nuclease-negative, and in them there are sixty strains belonged to Staphylococcus xylosus. All of the Micrococcus have pigment, did not apt for using as starters. Starter cultures main separeted from sixty strains of s.xylosus.
2. In order to select strains apt for used in fermented sausages, screening first passed technological properties, for example: nitrate reductase. And second passed detection of biogenic amine production, acetoin production, assessment of proteolytic activity and lipolytic activity. At last, from sixty strains of s. xylosus, we isolated four strains. 3. Studyed probiotic of the four strains. We found that they can resistant to strong acidic environment and higher concentrations of bile salt, and two strains C1S23 and C3R10 have bacteriocin. Antibiosis test between lactic bacteria and s. xylosus showed that: s.xylosus C1S23 and C3R10 and LAB11 had no antibiosis.
4. The strains of s.xylosus C1S23 and C3R10 were also tested for growth ability at different temperatures, pH and NaCl and nitrite concentrations. The results showed that they were suitable to make starter cultures.
5. Two starters formulation were developed combining the proteolytic but not lipolytic s.xylosus C1S23 with the Lactic Acid Bacteria 11 (starter S1) and the s.xylosus C3R10 with the same strain of LAB 11 (starter S2). We employed them in the manufacture of fermented sausages, in order to evaluate the effect of the starters on physico-chemical, microbiological and sensory properties of fermented sausages.
6. In all the samples LAB dominated the microflora from the beginning of the fermentation and ripening; Growth of Micrococcaceae in contral sample was inhibited, appeared to be significantly affected by acidification or by the environment of sausage. And the growth of Micrococcaceae in samples with starter S1 and S2 was also inhibited, but because of the great inoculation volume, the growth was basically increased slowly; Samples S1 and S2 showed a reduction tide in Enterobacteriaceae over the same period of time.
7. pH value of sample S1 and S2 was significantly lower than the control, after three days reaching the lowest level, and later there was a gradual increased. Inoculated with starter cultures had no effect on the water activity and ash, and made the content of NPN increased. Adding of starter cultures had significantly influenced in Texture. It was significantly increased in hardness, Chewiness and gumminess compared with the control sample. Both S1 and S2 exhibited significant increases in Lightness and redness compared with the control sample. From sensory analysis, it was seen that both starter cultures can be suitable to be used in fermented sausages.
1.本研究从成熟阶段的发酵香肠中筛选出138株菌,菌株经过生化鉴定显示微球菌58株,其中克氏微球菌38株(65.5%)。葡萄球菌80株,73株(91%)凝固酶阴性和脱氧核糖核酸酶阴性,其中有60株属于木糖葡萄球菌(82.2%)。微球菌全部产色素,不符合发酵剂特点。菌种筛选主要从60株木糖葡萄球菌中选择。
2. 60株木糖葡萄球菌经过硝酸盐还原酶等发酵特性实验初筛和生物胺检测、乙偶姻检测、蛋白酶活性和脂肪酶活性测试实验复筛,筛选出符合条件的4株菌。
3.研究所筛选出的4株木糖葡萄球菌的益生特性,发现能够抵抗较强的酸性环境和较高浓度的胆盐,2株菌C1S23、C3R10产生细菌素。葡萄球菌与乳酸菌的拮抗试验表明,木糖葡萄球菌C1S23、C3R10和乳酸菌11没有拮抗性。
4.测试不同pH值、温度、NaCl浓度、亚硝酸盐浓度对木糖葡萄球菌C1S23、C3R10菌株生长的影响,结果显示适合发酵香肠环境。
5.优选出的菌株木糖葡萄球菌C1S23,C3R10和乳酸菌11(酸化菌株)作为发酵剂生产发酵香肠,主要研究发酵剂对微生物、理化和感官指标变化的影响。
6.微生物分析结果显示各组发酵和成熟期间乳酸菌始终处于支配地位。对照组微球菌的生长明显受酸度或不能竞争过其它菌群的影响,生长受到了抑制,添加发酵剂各组微球菌生长也受到一定的抑制,但由于接种量大,基本成缓慢增长趋势。添加发酵剂各组肠杆菌菌落总数显著下降。
7.理化分析结果显示添加发酵剂各组pH值显著低于对照,发酵三天达到最低值,以后稍有回升。发酵剂的加入对水分活度、灰分含量的变化没有影响,使非蛋白氮含量增加。发酵剂的加入对发酵香肠的质构有着显著的影响,在硬度、咀嚼性和胶粘性方面要显著高于对照组。色泽差异显著,S1和S2组亮度、红度要显著高于对照组。结合感观评价,两组都可作为肉品发酵剂用于发酵香肠的生产。
The aims of this study are to isolated staphylococci and micrococci from traditional sausages and to determine the metabolic and probiotic properties of selected Staphylococcus xylosus, in order to select potential starter cultures for fermented sausages. And employ the mixed starters combined with Lactobacillus in the manufacture of fermented sausages, in order to evaluate the effect of the starter cultures on the physico-chemical, microbiological and sensory properties of typical fermented sausages. The main results and conclusion are as follows:
1. One hundred and thirty-eight strains isolated during the ripening of a traditional fermented sausage were characterized. Fifty-eight isolated belonged to Micrococcus,with thirty-eight strains(sixty-five point five percent of Micrococcus)belonged to Micrococcus kristinae. Eighty strains belonged to Staphylococcus species, and senventy-three strains(ninty-one percent of Staphylococci)were coagulase- negative and nuclease-negative, and in them there are sixty strains belonged to Staphylococcus xylosus. All of the Micrococcus have pigment, did not apt for using as starters. Starter cultures main separeted from sixty strains of s.xylosus.
2. In order to select strains apt for used in fermented sausages, screening first passed technological properties, for example: nitrate reductase. And second passed detection of biogenic amine production, acetoin production, assessment of proteolytic activity and lipolytic activity. At last, from sixty strains of s. xylosus, we isolated four strains. 3. Studyed probiotic of the four strains. We found that they can resistant to strong acidic environment and higher concentrations of bile salt, and two strains C1S23 and C3R10 have bacteriocin. Antibiosis test between lactic bacteria and s. xylosus showed that: s.xylosus C1S23 and C3R10 and LAB11 had no antibiosis.
4. The strains of s.xylosus C1S23 and C3R10 were also tested for growth ability at different temperatures, pH and NaCl and nitrite concentrations. The results showed that they were suitable to make starter cultures.
5. Two starters formulation were developed combining the proteolytic but not lipolytic s.xylosus C1S23 with the Lactic Acid Bacteria 11 (starter S1) and the s.xylosus C3R10 with the same strain of LAB 11 (starter S2). We employed them in the manufacture of fermented sausages, in order to evaluate the effect of the starters on physico-chemical, microbiological and sensory properties of fermented sausages.
6. In all the samples LAB dominated the microflora from the beginning of the fermentation and ripening; Growth of Micrococcaceae in contral sample was inhibited, appeared to be significantly affected by acidification or by the environment of sausage. And the growth of Micrococcaceae in samples with starter S1 and S2 was also inhibited, but because of the great inoculation volume, the growth was basically increased slowly; Samples S1 and S2 showed a reduction tide in Enterobacteriaceae over the same period of time.
7. pH value of sample S1 and S2 was significantly lower than the control, after three days reaching the lowest level, and later there was a gradual increased. Inoculated with starter cultures had no effect on the water activity and ash, and made the content of NPN increased. Adding of starter cultures had significantly influenced in Texture. It was significantly increased in hardness, Chewiness and gumminess compared with the control sample. Both S1 and S2 exhibited significant increases in Lightness and redness compared with the control sample. From sensory analysis, it was seen that both starter cultures can be suitable to be used in fermented sausages.
引文
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[2] Acton J. C., Dick R. L. Composition of some commercial dry sausages [J]. Food Sci., 1976, V41:971-972
[3] 王艳梅,马俐珍.发酵肉制品的研究现状现状[J].肉类工业,2004(6)
[4] 郭锡铎.我国发酵肉制品研究进展与未来[J].肉类工业,2004(5)
[5] Lucke F. K., Hechelmann H. Starter cultures for dry sausages and ham composition and effect [J]. Fleischwirtsch, 1987, V69: 1597-1598
[6] Hayes P. R. Food Microbiology and Hygiene [M]. London and New York: Elsevier Applied Science Publishers, 1985
[7] Shimokomaki M., et al. Charqui meat are hurdle technology meat products [J]. Food Rev., 1998, V 14(4): 339-349
[8] Paleari M.A., Beretta G., Colombo F. Buffalo meat as a sated and cured product [J]. Meat Science, 2000, V54: 565-567
[9] Geisen R., Lvcke F.K., Krockel L. Starter and protective cultures for meat and products [J]. Fleischwirtschaft, 1992, V 72(6): 894- 898
[10] Hammes W.P., Knauf H. J. Starters in the processing of meat products [J]. Meat Science, 1994, V36: 155 -168
[11] 张元生,译.微生物在肉制品加工中的应用[M].北京:轻工业出版社,1991
[12] Hammes W. P. Starter culture in meat production. Chem. Mikrobiol. Technol [J]. Lebensm, 1986, V9: 131-143
[13] Liepe H. U. Starter cultures in meat production. In Biotechnology. Basel [J]. Verlag Chemie, 1982, V5:400-424
[14] 施正学.肉品发酵剂[J].食品与发酵工业,1996, 6:50-53
[15] Nychas G. J., Arkoudelos J. S. Staphylococci aheir role in fermented sausages applied Bacteriology [J]. Symposium Supplement, 1990, V32:167-188
[16] 王海燕,张春江.发酵香肠中微生物产生的酶及其作用[J].肉类研究,2001,1:15-17
[17] Stahnke L.H. Dried sausages fermented with Staphylococcus xylosus at different temperatures and with different ingridient levels. Part II. Volatile components [J].Meat Science, 1995, V41:211 –223
[18] Berdague J. L., Monteil P. Effects of srarter cultures on the formation of flavourcompounds in dry sausages [J]. Meat Science, 1993, V35:229-240
[19] Stahnke L.H., Holck A., Jensen A. Maturity Acceleration of Italian Dried sausage by Staphylococcus carnosus-Relationship between Maturity and Flavor Compounds [J]. Journal of Food Science, 2002, V 67 (5): 1914-1921
[20] 夏文水.肉制品加工原理与技术[M].北京:化学工业出版社,2002,12
[21] 罗欣.发酵剂微生物及其代谢与发酵香肠的工艺控制[J].食品与发酵工业,2002, 28(3):67-71
[22] Montel M.C., Reitz J., Talon R. Biochemical activities of Micrococcaceae and their effects on the aromatic profiles and odours of a dry sausage model [J].Food Microbiology, 1996, V3: 489-499
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