含Nisin脱脂乳粉制备及应用的研究
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摘要
本课题对含Nisin脱脂乳粉的制备及应用进行了系统的研究，主要研究结果如下：
以乳酸乳球菌PCM2379为出发菌株，经紫外线及氯化铯复合诱变使其产Nisin能力提高了32％。以脱脂乳为基础培养基，通过添加酵母粉，及在发酵过程中调控pH值，使产Nisin能力提高了3倍，达到1800IU／ml。
在杀菌后，将含Nisin的脱脂乳真空冷冻制成粉。脱脂乳粉中Nisin的热变性属于1.8级反应。在70℃、80℃、90℃、100℃条件下其D值分别为184.91min、77.18min、21.53min、9.37min，在此温度范围内Z值为22.52℃，表观活化能为122.033KJ／mol。采用LTLT(63℃ 30min)和HTST(85℃15s)巴氏杀菌，其效价分别下降到86.82％、98.2％，采用UHT(115℃ 10min、130℃2s)灭菌其效价分别下降到3.43％、80.58％。
将含Nisin的脱脂乳粉分别添加到原料乳、鲜乳、酸奶中，观察其在乳制品中的应用：添加30-70IU／ml的Nisin可使原料乳中的细菌、芽孢菌的生长明显受到抑制，酸度变化明显缓慢，但低温菌生长受影响不大；添加35IU／mlNisin可明显延长鲜乳的保质期2-3倍；即使添加少量Nisin(5IU／ml)也会影响酸奶的发酵过程。
This research subject is to prepare skimed milk powder containing Nisin and to
applicate it in dairy products. Research results are as follow:
A mutant strain with hight yield of Nisin was obtained from lactococcus lactis 2379 by many mutagenetic treatment with ultraviolet ray and CsCL. Compared with primitive strain , the Nisin potency of the mutant strain was increased by over 32%. Through neutralize test to control pH changes during fermentation, the strain produced at least 3-fold greater bacteriocin in skimed milk culture supplemented with yeast extract} and the Nisin potency was 1800IU/ml. After pasteurized, the skimed milk powder containing Nisin was prepared by using lyophilization. Thermodynamic analysis indicated that the number of reaction degree of heat denaturation of Nisin in skimed milk powder was n=1.8. At temperatures of 70℃, 80℃ ,90℃, 100℃, D values for Nisin denaturation were 184.91min, 77.18min, 21.53min,and 9.37min respectively. With this temperature range,Z value was 22.52, and the apparent active energy was 122.033KJ/mol. LTLT(63 ℃ ,30min)and HTST(85℃ ,15s)pasteurization made the Nisin potency decrease to 86.82%and 98.2%,UHT(
115℃ 10min, 130℃2s )sterilization made the Nisin potency decrease to 3.43% and 80.58%. Concentration of 30-70IU/ml Nisin in raw milk was able to inhibit the growth of bacteria and spores-forming bacteria, but the effect of Nisin on the growth of psychrotrophic bacteria was weak; Concentration of 35IU/ml Nisin was able to extend the shelf-life of sterilized milk up to 30 days at 4℃, three times longer than the control group; during fermentation, concentration of 5IU/ml Nisin was able to inhilit the growth of lactic bacteria.
以乳酸乳球菌PCM2379为出发菌株，经紫外线及氯化铯复合诱变使其产Nisin能力提高了32％。以脱脂乳为基础培养基，通过添加酵母粉，及在发酵过程中调控pH值，使产Nisin能力提高了3倍，达到1800IU／ml。
在杀菌后，将含Nisin的脱脂乳真空冷冻制成粉。脱脂乳粉中Nisin的热变性属于1.8级反应。在70℃、80℃、90℃、100℃条件下其D值分别为184.91min、77.18min、21.53min、9.37min，在此温度范围内Z值为22.52℃，表观活化能为122.033KJ／mol。采用LTLT(63℃ 30min)和HTST(85℃15s)巴氏杀菌，其效价分别下降到86.82％、98.2％，采用UHT(115℃ 10min、130℃2s)灭菌其效价分别下降到3.43％、80.58％。
将含Nisin的脱脂乳粉分别添加到原料乳、鲜乳、酸奶中，观察其在乳制品中的应用：添加30-70IU／ml的Nisin可使原料乳中的细菌、芽孢菌的生长明显受到抑制，酸度变化明显缓慢，但低温菌生长受影响不大；添加35IU／mlNisin可明显延长鲜乳的保质期2-3倍；即使添加少量Nisin(5IU／ml)也会影响酸奶的发酵过程。
This research subject is to prepare skimed milk powder containing Nisin and to
applicate it in dairy products. Research results are as follow:
A mutant strain with hight yield of Nisin was obtained from lactococcus lactis 2379 by many mutagenetic treatment with ultraviolet ray and CsCL. Compared with primitive strain , the Nisin potency of the mutant strain was increased by over 32%. Through neutralize test to control pH changes during fermentation, the strain produced at least 3-fold greater bacteriocin in skimed milk culture supplemented with yeast extract} and the Nisin potency was 1800IU/ml. After pasteurized, the skimed milk powder containing Nisin was prepared by using lyophilization. Thermodynamic analysis indicated that the number of reaction degree of heat denaturation of Nisin in skimed milk powder was n=1.8. At temperatures of 70℃, 80℃ ,90℃, 100℃, D values for Nisin denaturation were 184.91min, 77.18min, 21.53min,and 9.37min respectively. With this temperature range,Z value was 22.52, and the apparent active energy was 122.033KJ/mol. LTLT(63 ℃ ,30min)and HTST(85℃ ,15s)pasteurization made the Nisin potency decrease to 86.82%and 98.2%,UHT(
115℃ 10min, 130℃2s )sterilization made the Nisin potency decrease to 3.43% and 80.58%. Concentration of 30-70IU/ml Nisin in raw milk was able to inhibit the growth of bacteria and spores-forming bacteria, but the effect of Nisin on the growth of psychrotrophic bacteria was weak; Concentration of 35IU/ml Nisin was able to extend the shelf-life of sterilized milk up to 30 days at 4℃, three times longer than the control group; during fermentation, concentration of 5IU/ml Nisin was able to inhilit the growth of lactic bacteria.
引文
1 马希汉，尉芹．微生物素在食品保藏中的应用．食品科学，1994，7：13～14
2 李平兰等．乳球菌肽(Nisin)的特性及应用．中国乳品工业，1994，25(4)：18～20
3 Rogers L A.The inhibiting effect of streptococcus on lactobacillus bulgayicus.Journal of Bacteriology, 1928, 16: 321-325
4 Whitehead. A substance inhibiting bacterial growth,produced by certain strains of lactic streptococci.Biochemistry, 1933,27:1993～1800
5 李东，孙健，彭沈军．天然生物食品防腐剂乳酸链球菌素的研究发展．食品与发酵工业，1995，4：58～61
6 Mattick A T R.A powerful inhibitory substance produced by group N streptococci.Natu-r e, 1994,154:551
7 Hirsch A,Grinsted E.A note on the inhibition of an anaerobic sporeformer in Swiss type cheese by a Nisin producing streptococcus.Journal of Dairy Research, 1951, 18:198～204
8 Gowans,J.L.Some properties of Nisin.Brit.J.pharmacol,1952,7:438～445
9 J.Delves-Broughton.Nisin.and Its Uses as a Food preservative.Food Technology, 1990, 11:100～117
10 Hurst A.Nisin Advance in Applied Microbiology,1981,27:85～123
11 Gross,E.and morel,J.L.The structure of Nisin.J.Am.Chem Soc, 1971,93:4634
12 李绍昌，梁同春．乳酸链球菌素的开发及市场前景．化工科技市场，2001，3：15～16
13 马立如，张占路，李宏．新型食品防腐剂—NISIN．河北省科学院学报，2001，18(3)：179～181
14 李平兰等，乳酸链球菌素研究进展．微生物学通报，1998，25(5)：295～298
15 那淑敏，还连栋．羊毛硫细菌素及其应用．微生物学通报，1999，26(6)：30～433
16 Suarez.M.ANA.et al. One-step Purification of NisinA by Immunoaffinity Chromatography. Microbiology, 1997,63(12):4990-4992
17 Willem M D et al. Properties of NisinZ and distribution of its gene NhinZ in Iactococcus lactis. Applied and Environmental Microbiology, 1993,59(1):213～215
18 Hurst,A.Nisin.Adv.Appl..Microbiol,1981,27:85～93
19 Tramer,J.The inhibitory action of Nisin on bacillces stearothermophilus.J.Sci food Agric, 1964,15(8):522～528
20 Heinemann,b.,voris,Letal. Vse of Nisin in processing food products.Food Technology, 1965,19:592
21 江芸，周光宏等．周产Nisin部分特性的研究．食品科学，2001，22(4)：18～21
22 Scott, V.H.Effect of Nisin on the outgrowth of clostridium botulinum spores.J.Food Sci;1981,46:117～124
23 Somers,E.B.Further studies on the antibotulinal effectirenss of Nisin in acidic media.J.Food Sci,1981,46:1972～1980
24 胡国平，叶嗣颖．乳链菌肽(Nisin)的研究进展．中国微生态学杂志，2001，13(2)：115～117
25 Morris,S.L.,Walsh,R.C.,and Hansen,J.N.Identification and characterization of some bacterial membrane sulphydryl groups which am targets of bacteriostatic and antibiotic action.J.Biol.Chem,1984,259:13590～13595
26 Christense,D.,et al. Collapse of the proton motive force in listeria monoacytogenes caus-e d by a bacteriocin produced by pediococeus acidilactici,Appl.Environ.Microbiol, 1992, 58
(10):3312～3315
27 Abee,T. Jakko.,et al.Bacteriocins:modes of action and potentials in food preservation and control of food poisoning.J.Food Microbiol,1995,28:169～185
28 Koen venema, Gerard Veneema and Jankok.Lactococcal bacteriocins:mode of action and immunity.Trends in Microbidogy,1995,3(8):299～304
29 Jack,Ralph, W.,John R.Tagg and Bibek Ray,Bacteriocins of grampositive bacteria, Microbiol.Rev., 1995,59:171～200
30 许扬，孙红斌．乳酸菌细菌素作用方式及其的活性的影响因素．中国畜产与食品，1998，5(3)：131～133
31 Siegers K, Entian K D.Genes involved in immunity to the lantibiotic Nisin produced by lactococcecs Lactis.Appl.Environ.Microbiol,1995,61:1082～1089
32 陈秀珠，那淑敏等．乳链菌肽在乳制品防腐保鲜中应用的研究．中国乳品工业，1994，27(2)：3～5
33 Claypool,L.,Stambo,C.R.Residence time of Nisin in the oral cavity following consumption of chocolate milk containing Nisin.J.Dairr Sci, 1966,49:314～317
34 崔健超，张柏林等．Nisin的研究现状．河北农业大学学报，2001，24(4)：104～109
35 Thomas,M.a.The processed cheese industry.Dept of Agricuiture,New Sacth Wales,Australist, 1977
36 Somers,E.B.Antibomlinal effectiveness of Nisin in pasteurized processed cheese spreads.J.Food protect,1987,50:842～847
37 钱平，吴德元．乳酸菌肽在蔬菜罐头中的应用．食品科学，1998，19(3)：28～29
38 沈颜新，姚日标等．乳酸链球菌素在食品工业中的应用．食品科学，1998，19(7)：35～37
39 还连栋，陶勇等．乳链菌肽高产菌株的选育及其基因定位．微生物学报，1995，35(5)：364～367
40 杨博，王永华等．利用菌株的乳酸菌肽免疫性筛选菌株．食品与发酵工业，2000，26(5)：19～21
41 王效军．中国乳业发展趋势．中国乳品工业，2001，29(1)：39～40
42 谢俊杰，佘世望，许杨，孙红斌．微生物天然防腐剂．食品科学，1999，1：13～14
43 Betty E,et al. Improved Medium for Lactic streptococci and Their Bacteriophages.Applied Microbiology,1975,29(6):807～813
44 刘稳，马桂荣．乳酸菌肽发酵条件的研究．食品与发酵工业，1996，3：37～40
45 江芸，周光宏等．国产Nisin部分特性的研究．食品科学，2001，22(4)：18～21．
46 内村泰，冈田早苗著．乳酸菌实验手册．内蒙古农业大学：内蒙古乳制品研究培训中心译制，1999．13～15
47 陈天寿主编．微生物培养基的制造与应用．北京：中国农业出版社，1995．138
48 金世琳主编．乳品工业手册．北京：轻工业出版社，1987．896～897
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52 Chan-lck cheigh ,Hak-Jong Choi et al. Influence of growth conditions on the production of a
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60 潘利华，夏云梯等．Nisin对酸奶品质的影响研究．合肥工业大学学报，2002，25(4)：620～623
61 杨洁彬等编．乳酸菌—生物学基础及应用．北京：中国轻工业出版社，1999．98～109
62 Citti,J.E.,W.E.Sandine.Comparison of slow and fast acid—producing streptococcus lactis. J.Dairy Sci,1965,48:14～18
63 陈秀珠，何松等．乳链菌肽高产菌株AL2的发酵条件研究．微生物学通报，1995，22(4)：215～217
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67 武若飞，李艳华，吴平等．UHT奶的质量与原料奶的关系．中国乳品工业，1997，25(2)：34～35
68 郭清泉，张兰威等．Nisin在酸奶制品中的应用．食品与机械，2001，84(4)：30～31
2 李平兰等．乳球菌肽(Nisin)的特性及应用．中国乳品工业，1994，25(4)：18～20
3 Rogers L A.The inhibiting effect of streptococcus on lactobacillus bulgayicus.Journal of Bacteriology, 1928, 16: 321-325
4 Whitehead. A substance inhibiting bacterial growth,produced by certain strains of lactic streptococci.Biochemistry, 1933,27:1993～1800
5 李东，孙健，彭沈军．天然生物食品防腐剂乳酸链球菌素的研究发展．食品与发酵工业，1995，4：58～61
6 Mattick A T R.A powerful inhibitory substance produced by group N streptococci.Natu-r e, 1994,154:551
7 Hirsch A,Grinsted E.A note on the inhibition of an anaerobic sporeformer in Swiss type cheese by a Nisin producing streptococcus.Journal of Dairy Research, 1951, 18:198～204
8 Gowans,J.L.Some properties of Nisin.Brit.J.pharmacol,1952,7:438～445
9 J.Delves-Broughton.Nisin.and Its Uses as a Food preservative.Food Technology, 1990, 11:100～117
10 Hurst A.Nisin Advance in Applied Microbiology,1981,27:85～123
11 Gross,E.and morel,J.L.The structure of Nisin.J.Am.Chem Soc, 1971,93:4634
12 李绍昌，梁同春．乳酸链球菌素的开发及市场前景．化工科技市场，2001，3：15～16
13 马立如，张占路，李宏．新型食品防腐剂—NISIN．河北省科学院学报，2001，18(3)：179～181
14 李平兰等，乳酸链球菌素研究进展．微生物学通报，1998，25(5)：295～298
15 那淑敏，还连栋．羊毛硫细菌素及其应用．微生物学通报，1999，26(6)：30～433
16 Suarez.M.ANA.et al. One-step Purification of NisinA by Immunoaffinity Chromatography. Microbiology, 1997,63(12):4990-4992
17 Willem M D et al. Properties of NisinZ and distribution of its gene NhinZ in Iactococcus lactis. Applied and Environmental Microbiology, 1993,59(1):213～215
18 Hurst,A.Nisin.Adv.Appl..Microbiol,1981,27:85～93
19 Tramer,J.The inhibitory action of Nisin on bacillces stearothermophilus.J.Sci food Agric, 1964,15(8):522～528
20 Heinemann,b.,voris,Letal. Vse of Nisin in processing food products.Food Technology, 1965,19:592
21 江芸，周光宏等．周产Nisin部分特性的研究．食品科学，2001，22(4)：18～21
22 Scott, V.H.Effect of Nisin on the outgrowth of clostridium botulinum spores.J.Food Sci;1981,46:117～124
23 Somers,E.B.Further studies on the antibotulinal effectirenss of Nisin in acidic media.J.Food Sci,1981,46:1972～1980
24 胡国平，叶嗣颖．乳链菌肽(Nisin)的研究进展．中国微生态学杂志，2001，13(2)：115～117
25 Morris,S.L.,Walsh,R.C.,and Hansen,J.N.Identification and characterization of some bacterial membrane sulphydryl groups which am targets of bacteriostatic and antibiotic action.J.Biol.Chem,1984,259:13590～13595
26 Christense,D.,et al. Collapse of the proton motive force in listeria monoacytogenes caus-e d by a bacteriocin produced by pediococeus acidilactici,Appl.Environ.Microbiol, 1992, 58
(10):3312～3315
27 Abee,T. Jakko.,et al.Bacteriocins:modes of action and potentials in food preservation and control of food poisoning.J.Food Microbiol,1995,28:169～185
28 Koen venema, Gerard Veneema and Jankok.Lactococcal bacteriocins:mode of action and immunity.Trends in Microbidogy,1995,3(8):299～304
29 Jack,Ralph, W.,John R.Tagg and Bibek Ray,Bacteriocins of grampositive bacteria, Microbiol.Rev., 1995,59:171～200
30 许扬，孙红斌．乳酸菌细菌素作用方式及其的活性的影响因素．中国畜产与食品，1998，5(3)：131～133
31 Siegers K, Entian K D.Genes involved in immunity to the lantibiotic Nisin produced by lactococcecs Lactis.Appl.Environ.Microbiol,1995,61:1082～1089
32 陈秀珠，那淑敏等．乳链菌肽在乳制品防腐保鲜中应用的研究．中国乳品工业，1994，27(2)：3～5
33 Claypool,L.,Stambo,C.R.Residence time of Nisin in the oral cavity following consumption of chocolate milk containing Nisin.J.Dairr Sci, 1966,49:314～317
34 崔健超，张柏林等．Nisin的研究现状．河北农业大学学报，2001，24(4)：104～109
35 Thomas,M.a.The processed cheese industry.Dept of Agricuiture,New Sacth Wales,Australist, 1977
36 Somers,E.B.Antibomlinal effectiveness of Nisin in pasteurized processed cheese spreads.J.Food protect,1987,50:842～847
37 钱平，吴德元．乳酸菌肽在蔬菜罐头中的应用．食品科学，1998，19(3)：28～29
38 沈颜新，姚日标等．乳酸链球菌素在食品工业中的应用．食品科学，1998，19(7)：35～37
39 还连栋，陶勇等．乳链菌肽高产菌株的选育及其基因定位．微生物学报，1995，35(5)：364～367
40 杨博，王永华等．利用菌株的乳酸菌肽免疫性筛选菌株．食品与发酵工业，2000，26(5)：19～21
41 王效军．中国乳业发展趋势．中国乳品工业，2001，29(1)：39～40
42 谢俊杰，佘世望，许杨，孙红斌．微生物天然防腐剂．食品科学，1999，1：13～14
43 Betty E,et al. Improved Medium for Lactic streptococci and Their Bacteriophages.Applied Microbiology,1975,29(6):807～813
44 刘稳，马桂荣．乳酸菌肽发酵条件的研究．食品与发酵工业，1996，3：37～40
45 江芸，周光宏等．国产Nisin部分特性的研究．食品科学，2001，22(4)：18～21．
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