生鲜乳中优势嗜冷菌对UHT乳蛋白浓度的影响

doi:10.6048/j.issn.1001-4330.2023.09.028

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (9): 2314-2322.DOI: 10.6048/j.issn.1001-4330.2023.09.028

• 草业·畜牧兽医·食品工艺技术 • 上一篇下一篇

生鲜乳中优势嗜冷菌对UHT乳蛋白浓度的影响

王辉¹^,²(), 邵伟¹, 张少颖², 王富兰², 王帅², 武亚婷², 范雪², 赵艳坤²()

1.新疆农业大学动物科学学院/新疆肉乳用草食动物营养实验室,乌鲁木齐 830052
2.新疆农业科学院农业质量标准与检测技术研究所/新疆农产品质量安全实验室,乌鲁木齐 830091

收稿日期:2022-12-20 出版日期:2023-09-20 发布日期:2023-09-19
通信作者: 赵艳坤(1990-),女,新疆阿拉尔人,副研究员,博士,研究方向为乳品品质与安全,(E-mail) yankunzhao90@163.com
作者简介:王辉(1995-),男,辽宁锦州人,硕士研究生,研究方向为动物生产,(E-mail)1397571059@qq.com
基金资助:
新疆维吾尔自治区自然科学基金项目“hylB介导无乳链球菌对乳腺上皮细胞泌乳功能的调控机制研究”(2019D01B32);国家自然科学基金项目“二元信号系统CovS/CovR对牛源无乳链球菌毒力与耐药的分子调控机制研究”(32060797);新疆维吾尔自治区重大科技专项“乳品质量风险分析与预警技术研究应用”(2020A01001-3);新疆农业大学大学生创新项目(dxscx2021107)

Effect of dominant psychrophilic bacteria in fresh milk on the concentration of UHT Milk protein

WANG Hui¹^,²(), SHAO Wei¹, ZHANG Shaoying², WANG Fulan², WANG Shuai², WU Yating², FAN Xue², ZHAO Yankun²()

1. Xinjiang Key Laboratory of Meat Milk Production Herbivore Nutrition/College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, China
2. Key Laboratory of Agro-Products Quality and Safety of Xinjiang/ Institute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2022-12-20 Online:2023-09-20 Published:2023-09-19
Correspondence author: ZHAO Yankun(1990-),female, native place:Aral,Xinjiang. associate researcher, doctor's degree,research field: Dairy quality and safety.(E-mail) yankunzhao90@163.com
Supported by:
Natural Science Foundation of Xinjiang Uygur Autonomous Region "HyLB-mediated Regulation of Streptococcus Agalactiae on Lactation Function of Mammary Epithelial Cells"(2019D01B32);National Natural Science Foundation of China(32060797);Key Science and Technology Project of Xinjiang Uygur Autonomous Region "Research and Application of Dairy Quality Risk Analysis and Early Warning Technology"(2020A01001-3);Undergraduate Innovation Project of Xinjiang Agricultural University(dxscx2021107)

摘要/Abstract

摘要：

【目的】研究UHT乳在接入不同数量级的嗜冷菌时,不同储存温度下UHT乳中蛋白浓度的变化。【方法】(1)采用混培法,将原料乳从原液稀释至10^-5稀释梯度,每个梯度做2个平行,6.5℃培养10 d,分离纯化保留单个菌落;(2)运用嗜冷菌基因组DNA提取与PCR反应鉴定筛选出优势嗜冷菌;(3)选用乳平板可视鉴定法,筛选出产蛋白酶能力最强的菌株。(4)采用BCA蛋白浓度测定法,将UHT乳稀释8倍、12倍、20倍、24倍、28倍在磷酸盐缓冲液中,测定不同稀释倍数的UHT乳在蛋白浓度测定试验中线性关系最好的稀释倍数。(5)UHT乳同时接入10³、10⁴、10⁵ CFU/mL的优势嗜冷菌,3组试验组分别在4、7和21℃环境下储存,培养7 d,每24 h BCA蛋白浓度测定法检测一次蛋白浓度。【结果】(1)在33份生鲜乳样品中,共筛选出212株嗜冷菌。(2)假单胞菌为生鲜乳中优势嗜冷菌,分离率为5.19%。(3)99号菌株假单胞菌产蛋白酶能力最强,透明圈带直径为0.3 mm;(4)UHT乳在28倍稀释浓度下的蛋白定量线性系数为R²=0.998,线性关系最好。(5)4℃条件下时,假单胞菌添加量为10³、10⁴ 和10⁵ CFU/mL 3个试验组的蛋白浓度变化均不显著(P>0.05);7℃时,3组接菌量的蛋白浓度较对照组均表现差异显著(P<0.05); 21℃条件下,除了10⁴ CFU/mL试验组蛋白浓度下降差异显著(P<0.05),10³和10⁵ CFU/mL 2个试验组蛋白浓度下降极显著(P<0.01)。【结论】生鲜乳中嗜冷菌含量、储藏温度、储存时间对UHT乳蛋白浓度均有影响,温度越高、嗜冷菌含量越高,蛋白水解的程度越明显,影响UHT乳的品质。

关键词: 生鲜乳; 嗜冷菌; 蛋白酶; UHT乳; 蛋白浓度

Abstract:

【Objective】 To investigate the changes of protein concentration in UHT milk at different storage temperatures when UHT milk was exposed to different orders of cold-philic bacteria.【Methods】 (1) Mixed culture method was used to dilute the raw milk from the original liquid to a 10^-5 dilution gradient, and each gradient was parallel to each other.They were cultured at 6.5℃ for 10 days to isolate and purify and retain a single colony.(2) Genomic DNA extraction and PCR reaction were used to identify the dominant cold-philic bacteria.(3) Milk plate visual identification method was used to screen the strain with the strongest protease production ability.(4) BCA protein concentration determination method was applied, UHT milk was diluted 8 times, 12 times, 20 times, 24 times and 28 times dilution concentration in phosphate buffer, and the linear dilution ratio of UHT milk in protein concentration determination experiment was determined.(5) UHT milk was simultaneously inoculated with 10³, 10⁴ and 10⁵ CFU/mL dominant thermophilic bacteria.The three experimental groups were stored at 4, 7 and 21℃ for 7 days, respectively.Protein concentration was detected every 24hBCA.【Results】 (1) A total of 212 strains of thermophilic bacteria were screened out from 33 fresh milk samples.(2) Through PCR identification, pseudomonas was the dominant cold-philic bacteria in fresh milk, and the isolation rate was 5.19%; (3) Through the milk plate visual identification method, pseudomonas strain 99 showed the strongest protease production ability, and the diameter of the transparent ring was 0.3mm; (3) The linear coefficient of protein quantification in UHT milk at 28 times diluted concentration was R²=0.998, and the linear relationship was the best.(4) At 4℃, there was no significant change in the protein concentration of the three experimental groups supplemented with 10³CFU/mL, 10⁴ CFU/mL and 10⁵CFU/mL pseudomonas (P > 0.05); At 7℃, the protein concentration of the inoculated bacteria in the three groups was significantly different from that in the control group (P<0.05); At 21℃, except for 10⁴CFU/mL group, the protein concentration decreased significantly (P<0.05), the protein concentration in 10³CFU/mL and 10⁵ CFU/mL groups decreased significantly (P<0.01).【Conclusion】 The content of cryophilic bacteria in fresh milk, storage temperature and storage time all affect the protein concentration of UHT milk.The higher the temperature is, the higher the content of cryophilic bacteria is, and the more obvious the degree of protein hydrolysis is, thus affecting the quality of UHT milk.

Key words: fresh milk; psychrophilic bacterium; protease; UHT milk; protein concentration

中图分类号:

S879.1

王辉, 邵伟, 张少颖, 王富兰, 王帅, 武亚婷, 范雪, 赵艳坤. 生鲜乳中优势嗜冷菌对UHT乳蛋白浓度的影响[J]. 新疆农业科学, 2023, 60(9): 2314-2322.

WANG Hui, SHAO Wei, ZHANG Shaoying, WANG Fulan, WANG Shuai, WU Yating, FAN Xue, ZHAO Yankun. Effect of dominant psychrophilic bacteria in fresh milk on the concentration of UHT Milk protein[J]. Xinjiang Agricultural Sciences, 2023, 60(9): 2314-2322.

图/表 16

表1 标准曲线制作

Tab.1 Standard curve making

编号 Serial number	0	1	2	3	4	5	6
标准溶液 Standard solutio (μL)	0	0.5	2.5	5.0	10	15	20
PBS溶液 PBS solution (μL)	20	19.5	17.5	15	10	5	0
总体积 Total volume (μL)	20

图1 嗜冷菌PCR鉴定的凝胶电泳图谱

Fig.1 Gel electrophoresis of cold-philic bacteria identified by PCR

图2 乳平板鉴定结果

Fig.2 Identification results of milk agar plate

图3 进化树构建结果

Fig.3 Evolutionary tree construction results

表2 乳平板鉴定结果

Tab.2 Identification results of milk agar plate

菌株编号 Strain No.	测定结果 Measurement results(mm)
99号	0.4
93号	0.3
36号	0.15
46号	0
27号	0
19号	0
38号	0.1
48号	0
45号	0
32号	0
6号	0.1

图4 蛋白浓度测定标准曲线

Fig.4 Standard curve for protein concentration determination

图5 8倍稀释浓度标准曲线

Fig.5 8 times dilution concentration standard curv

图6 12倍稀释浓度标准曲线

Fig.6 12 times dilution concentration standard curve

图7 16倍稀释浓度标准曲线

Fig.7 16 times dilution concentration standard curve

图8 20倍稀释浓度标准曲线

Fig.8 20 times dilution concentration standard curve

图9 24倍稀释浓度标准曲线

Fig.9 24 times dilution concentration standard curve

图10 28倍稀释浓度标准曲线

Fig.10 28 times dilution concentration standard curve

图11 103CFU/mL接菌量下UHT乳蛋白浓度变化注:按蛋白浓度由高到低a、b、c、d、e(P<0.05),按顺序排序,字母相同为不显著(P>0.05),大写字母为极显著(P<0.01)。下同

Fig.11 103CFU/mL effect of bacterial addition on UHT milk protein concentration Note:From high to low protein concentration a、b、c、d、e(P<0.05),sort in order,same letters are not significant(P>0.05),capital letters are highly prominent(P<0.01),the same as below

图12 104CFU/mL接菌量下UHT乳蛋白浓度变化

Fig.12 104CFU/mL effect of bacterial addition on UHT milk protein concentration

图13 105CFU/mL接菌量下UHT乳蛋白浓度变化

Fig.13 105CFU/mL effect of bacterial addition on UHT milk protein concentration

图14 胞外蛋白酶导致的乳蛋白沉淀变化

Fig.14 The precipitation condition of extracellular protease hydrolysis of casein

参考文献 33

[1]	Crudden A, Patrick, Fox F, et al. Factors affecting the hydrolytic action of plasmin in milk[J]. International Dairy Journal, 2005, 15(4):305-313. DOI URL
[2]	楠丁呼思勤, 母智深, 陈芳, 等. 影响牛奶品质耐热酶的研究进展[J]. 食品工业科技, 2007,(4): 244-246.
	Nandinghusiqin, MU Zhishen, CHEN Fang, et al. Research progress of heat-resistant enzymes affecting milk quality[J]. Science and Technology of Food Industry, 2007,(4): 244-246.
[3]	A Matéos, M Guyard-Nicodème, FranoisBaglinière, et al. Proteolysis of Milk Proteins by Aprx, an Extracellular Protease Identified in Pseudomonas LBSA1 Isolated from Bulk Raw Milk, and Implications for the Stability of UHT Milk[J]. International Dairy Journal, 2015, 49: 78-88. DOI URL
[4]	Hu Z, Meng X C, Liu F. Isolation and Characterisation of Lytic Bacteriophages against Pseudomonas spp.a Novel Biological Intervention for Preventing Spoilage of Raw Milk[J]. International Dairy Journal, 2016, 55: 72-78. DOI URL
[5]	Baur C, Krewinkel M, Kutzli I, et al. Isolation and Characterisation of a Heat-Resistant Peptidase from Pseudomonas panacis Withstanding General UHT Processes[J]. International Dairy Journal, 2015, 49: 46-55. DOI URL
[6]	Ren T, Frank J, Christen G. Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles[J]. Journal Dairy Science, 1988, 71(6): 14328.
[7]	唐兵, 唐晓峰, 彭珍荣. 嗜冷菌研究进展[J]. 微生物学杂志, 2002, 22(1):51-53.
	TANG Bing, TANG Xiaofeng, PENG Zhenrong. Research progress of bacteriophilic bacteria[J]. Journal of microbiology, 2002, 22(1):51-53.
[8]	夏涵, 府伟灵. 快速提取细菌DNA方法的研究[J]. 现代预防医学, 2005, 32(5): 571-573.
	XIA Han, FU Weiling. Rapid extraction of bacterial DNA[J]. Modern Preventive Medicine, 2005, 32(5): 571-573.
[9]	辛亮. 原料乳中嗜冷菌多样性研究及 LAMP 快速检测方法的构建[D]. 哈尔滨: 哈尔滨工业大学, 2017.
	XIN Liang. Diversity of cold-philic bacteria in raw milk and construction of LAMP rapid detection method[D]. Harbin: Harbin Institute of Technology, 2017.
[10]	纪振杰, 郭德军, 王欣. 原料乳中嗜冷菌数的检测方法的比较[J]. 乳品科学与技术, 2007, 29(3): 131-132.
	JI Zhenjie, GUO Dejun, WANG Xin. Comparison of detection methods for the number of cold-philic bacteria in raw milk[J]. Dairy Science and Technology, 2007, 29(3): 131-132.
[11]	尤超, 赵大球, 梁乘榜, 等. PCR引物设计方法综述[J]. 现代农业科技, 2011, 9(17): 48-51.
	YOU Chao, ZHAO Daqiu, LIANG Chengbang, et al. A review of PCR primer design[J]. Modern Agricultural Science and Technology, 2011, 9(17): 48-51.
[12]	林少华, 罗红霞, 贾红亮, 等. 高通量测序技术结合传统方法筛选及鉴定奶酒中曲菌[J]. 中国奶牛, 2019, 6(6): 44-47.
	LIN Shaohua, LUO Hongxia, JIA Hongliang, et al. Screening and identification of Aspergillus in milk wine by high-throughput sequencing technology combined with traditional methods[J]. China Dairy Cows, 2019, 6(6): 44-47.
[13]	李德葆, 周雪平. 基因工程操作技术[M]. 上海: 上海科学技术出版社, 1996:35-42.
	LI Debao, ZHOU Xueping. Operation Technology of Genetic Engineering[M]. Shanghai: Shanghai Science and Technology Press, 1996: 35-42.
[14]	Sambrook J, Russell D. 分子克隆试验指南[M].黄培堂, 译. 3版. 北京: 科学出版社, 2002: 27-29.
	Sambrook J, Russell D. Experimental guide to molecular cloning (translated by HUANG Peitang, 3rd Ed.)[M]. Beijing: Science Press, 2002:27-29.
[15]	叶慧, 谢海华, 黄俊, 等. 基于16SrDNA序列分析和BIOLOG快速鉴定方法鉴定杭州地区原料乳中嗜冷菌[J]. 发酵科技通讯, 2015, 44(4): 24-29.
	YE Hui, XIE Haihua, HUANG Jun, et al. Identification of cryophilic bacteria in raw milk from Hangzhou region based on 16SrDNA sequence analysis and BIOLOG method[J]. Bulletin of Fermentation Science and Technology, 2015, 44(4): 24-29.
[16]	薛艳蓉, 王呈, 梁茂文, 等. 新疆乌鲁木齐传统酸奶中乳酸菌的多样性分析[J]. 中国奶牛, 2019,(4): 37-40.
	XUE Yanrong, WANG Cheng, LIANG Maowen, et al. Analysis on the diversity of lactic acid bacteria in traditional yogurt in Urumqi, Xinjiang[J]. China Dairy Cows, 2019,(4): 37-40.
[17]	吴序栎, 朱倩倩, 成小娟, 等. 牛乳β-乳球蛋白热稳定与免疫原性关系的光谱学研究[J]. 光谱学与光谱分析, 2011, 31 (8): 2205-2209.
	WU Xuli, ZHU Qianqian, CHENG Xiaojuan, et al. Study on the relationship between β -lactoglobulin thermal stability and immunogenicity in milk[J]. Spectroscopy and Spectral Analysis, 2011, 31 (8): 2205-2209. PMID
[18]	韩荣伟, 王加启, 郑楠. 热处理对牛乳成分的变化影响及热损标识物的选择[J]. 中国食物与营养, 2011, 17(7): 22-29.
	HAN Rongwei, WANG Jiaqi, ZHENG Nan. Effects of heat treatment on milk composition and selection of heat loss indicators[J]. Food and Nutrition in China, 2011, 17 (7): 22-29.
[19]	魏超, 周虹, 郭灵安, 等. 鸡蛋溶血巴斯德菌药敏试验及氨基酸代谢研究[J]. 西南农业学报, 2017, 30(6):1335-1339.
	WEI Chao, ZHOU Hong, GUO Lingan, et al. Drug sensitivity test and amino acid metabolism of Pasteurellahemolysticus in eggs[J]. Southwest China Journal of Agricultural Sciences, 2017, 30(6): 1335-1339.
[20]	Schokker E P, Singh H, Creamer L K. Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin[J]. International Dairy Journal, 2000, 10(12):843-853. DOI URL
[21]	岳喜庆, 王玥, 李鹏, 等. 原料乳中嗜冷菌低温蛋白酶的酶学性质研究[J]. 食品工业科技, 2009,(6): 174-176.
	YUE Xiqing, WANG Yue, LI Peng, et al. Study on the enzymatic properties of cryophosphorin in raw milk[J]. Science and Technology of Food Industry, 2009,(6): 174-176.
[22]	张云峰, 罗玉明, 王新风. 嗜热蛋白酶的研究与应用[J]. 淮阴师范学院学报 (自然科学版), 2003, (2): 245-249.
	ZHANG Yunfeng, LUO Yuming, WANG Xinfeng. Research and application of hyperthermophilicproteases[J]. Journal of Huaiyin Normal University (Natural Science Ed.), 2003, (2): 245-249.
[23]	王娇. 原料乳中嗜冷菌的变化和控制研究[D]. 哈尔滨: 黑龙江东方学院, 2014.
	WANG Jiao. Study on the change and control of cold-philic bacteria in raw milk[D]. Harbin: Heilongjiang Dongfang University, 2014.
[24]	Griffiths M W, Phillips J D, Muir D D. Rapid plate counting techniques for enumeration of bacteria in pasteurized double cream[J]. International Journal of Dairy Technology, 2007, 33 (1): 8-10. DOI URL
[25]	Solimar G, Machado, et al. Pseudomonasspp.And Serratia liquefaciensas Predominant Spoilers in Cold Raw Milk[J]. Journal of Food Science, 2015, 80(8):M1842-M1849. DOI URL
[26]	王辉, 吕加平, 刘鹭, 等. 耐热蛋白酶对UHT乳蛋白的水解作用[J]. 食品科学, 2010, 31(17): 228-231.
	WANG Hui, LYU Jiaping, LIU Lu et al. Hydrolysis of UHT milk protein by heat-resistant protease[J]. Food Science, 2010, 31 (17): 228-231.
[27]	韩秋红, 李琴, 李凯锋. 嗜冷菌对乳制品加工及贮存的影响和控制措施[J]. 现代食品, 2018, 19(15): 73-75.
	HAN Qiuhong, LI Qin, LI Kaifeng. Effects of cryophilic bacteria on processing and storage of dairy products and control measures[J]. Modern Food, 2018, 19(15): 73-75.
[28]	Chen T R, Wei Q K, Chen Y J. Pseudomonas spp.and Hafnia alvei growth in UHT milk at cold storage[J]. Food Control, 2011, 22(5):697-701. DOI URL
[29]	洪青, 李楠, 刘振民. 原料乳中嗜冷菌快速检测与控制技术研究进展[J]. 乳业科学与技术, 2019, 42(6): 41-45. DOI
	HONG Qing, LI Nan, LIU Zhenmin. Research progress in rapid detection and control of cold-philic bacteria in raw milk[J]. Dairy Science and Technology, 2019, 42(6): 41-45.
[30]	刘欣欣, 李发弟, 乐祥鹏. 羊奶成分和奶中主要蛋白的研究进展[J]. 中国畜牧杂志, 2016, 52(9): 87-91.
	LIU Xinxin, LI Fadi, LE Xiangpeng. Progress in the composition of sheep milk and major proteins in milk[J]. Chinese Journal of Animal Science, 2016, 52(9): 87-91.
[31]	Kelly A L, Mcsweeney P. Indigenous Proteinases in Milk[J]. Advanced Dairy Chemistry, 2003, 1: 495-544.
[32]	遇晓杰, 万丽葵. 原料奶中嗜冷菌和耐热芽孢菌的来源追溯研究[J]. 肉品卫生, 2005, (8): 29-30.
	YU Xiaojie, WAN Likui. Origin of thermophilic bacteria and thermophilic spores in raw milk[J]. Meat Hygiene, 2005,(8): 29-30.
[33]	Morita R Y. Psychrophilic bacteria[J]. Bacteriological Reviews, 1975, 39: 144-167. DOI PMID

生鲜乳中优势嗜冷菌对UHT乳蛋白浓度的影响

Effect of dominant psychrophilic bacteria in fresh milk on the concentration of UHT Milk protein

在线阅读

PDF下载

可视化

摘要/Abstract

引用本文

使用本文

图/表 16

参考文献 33

相关文章 3

编辑推荐

Metrics

[1]	傅力;章运;涂振东;叶凯. 米曲霉高产蛋白酶菌株的选育及在酱油酿造中的应用研究[J]. , 2009, 46(6): 1274-1278.
[2]	王玮;张志东;茆军;王涛;谢玉清;唐琦勇. 高温中性蛋白酶基因的克隆及毕赤酵母表达研究[J]. , 2009, 46(3): 635-638.
[3]	茆军;王玮;张志东;谢玉清;罗淑萍. 高温中性蛋白酶基因的克隆、表达及验证研究[J]. , 2008, 45(5): 956-959.