果味奶生产技术及果味物质在牛体内代谢机制的初步研究
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摘要
本论文采用瘤胃瘘管、动静脉插管和乳腺培养技术等先进技术手段,系统地研究了果香味剂在奶牛瘤胃、血液和乳腺中的代谢变化,并采用固相微萃取、吹扫捕集结合气质联用技术对果香味剂、瘤胃液、血液、乳腺培养液和牛奶中的风味物质进行了检测,以更全面地深入地探讨果香味剂在奶牛体内的代谢机制。本研究分为三部分:第一部分为果香味剂中果味物质测定方法的建立
选择不同的萃取头、电解质水平、萃取温度和萃取时间,通过正交实验设计,筛选出SPME最佳萃取参数。结果表明:①.采用SPME对果香味剂分析其最佳萃取头、NaCl用量、萃取温度和萃取时间分别为PDMS、0.32g/mL、50℃和30min。②.通过SPME-GCMS分析果香味剂中共有70种化合物,主要成分为酯类、醇类、醛类、酸类、酮类、酚类等。
第二部分为果香味剂最适添加剂量的筛选及对奶牛生产性能和牛奶风味的影响选择体重、泌乳期(71±5d)、胎次、产奶量相近,且健康无疾病的奶牛30头,共分5组,每组6头,日粮中果香味剂添加剂量分别为0、5000、6500、7500和9000g/t(精料DM基础)。当这些奶牛处于泌乳期152±4d时对其进行相同分组,添加相同剂量的果香味剂,试验期间观察奶牛生产性能和牛奶风味的变化。结果表明:①.果香味剂可改善乳风味和提高乳品质,其最适添加剂量为7500g/t。②.果香味剂可提高产奶高峰期奶牛的产奶量,对于产奶高峰期后奶牛的产奶量具有一定减缓作用,经济效益显著。
第三部分为果香味剂在瘤胃、血液和乳腺中的代谢变化研究试验1.果香味剂在奶牛瘤胃中的代谢及对瘤胃内容物和风味的影响选择2只体况良好,体重相近,安装有永久性瘤胃瘘管的奶牛,采用体内和体外培养相结合的方法研究果香味剂对瘤胃内容物及其风味的影响。结果表明:①.果味剂可显著提高瘤胃液中的干物质降解率(P < 0.05),说明果香味剂确实可以提高动物消化力,进而提高饲料转化率。②.体外培养表明果香味剂对瘤胃液产气量、pH值、NH3-N水平、乙酸、丙酸、丁酸、总VFA和乙/丙酸比影响不显著(P > 0.05),而体内试验则可显著提高瘤胃液中乙酸、丙酸、丁酸和总VFA含量(P < 0.05),显著降低瘤胃液NH3-N水平和乙/丙酸比(除0h外)(P < 0.05)。③.根据体外试验结果综合评定,得出其最适添加剂量为7500g/t,与上述试验结果相一致。④.通过采用SPME/GC-MS对瘤胃液进行分析,表明试验组中均含有果味剂中的一些风味物质成分。
试验2.果香味剂在奶牛乳腺动静脉差中的代谢变化选择2只体况良好,体重相近,处于泌乳前中期的荷斯坦奶牛,安装有乳静脉和阴外动脉插管供采集血液用。结果表明:果香味剂可进入到奶牛血液中,且阴外动脉血中风味物质含量将近是乳静脉血中风味物质含量的2～3倍。奶牛乳腺中风味物质摄取量随着采食时间的延长呈先上升后下降的变化趋势,其乳腺摄取/牛奶产出比均很高,说明奶牛采食后果香味剂中的风味物质被乳腺大量摄取,但仅有少量的物质进入到牛奶中改善牛奶风味。
试验3.果香味剂对乳腺细胞生长、细胞周期、乳成分和培养液风味的影响
选用自制的奶牛乳腺上皮细胞,果香味剂添加剂量分别为0、5、1、15、20和30μg/mL。结果表明:①.当添加剂量为15μg/mL时,细胞数量、培养液乳成分含量及进入S期和G2/M期细胞比例均达到最高(P < 0.05),之后开始下降;当浓度达30μg/mL时,开始抑制细胞生长和细胞周期的增殖。②.运用PT-GCMS对乳腺培养液中风味成分分析表明试验组中均含有果味剂中的一些风味物质成分,且当添加剂量为15μg/mL时,各物质成分含量为最高(P < 0.05),之后呈下降趋势。
综合上述研究结果表明:通过奶牛瘤胃、血液和乳腺代谢变化和初步的机制的研究表明果味剂中的一些物质成分能通过牛体血液进入到乳中,进而改善乳风味和提高乳品质,且体内外试验均证实其最适添加剂量为7500g/t(精料DM基础)。本研究还进一步得出日粮中添加果香味剂在进入到奶牛体内的不同部位均发生了较大的代谢变化,部分果味物质比例升高、部分果味物质数量减少、部分果味物质消失,所以真正进入到牛体内的果味物质组合发生了变化。尽管都改善了牛奶的风味,但进入到牛奶中的成分发生了较大的变化。本研究结果为果香味剂在奶牛养殖应用中提供了实验依据。
The metabolic changes of the mixed fruit flavor meal in the rumen, blood and mammary gland of the lactating cow by rumen cannulas, arterio-venous catheter, and mammary gland culture technique were studied in this paper. The results were as follows:
Part1 The development of measuring method for the mixed fruit flavor meal The best extraction parameter of SPME (Solid Phase Microextraction), the different fiber, electrolyte, extraction temperature and time were screened out in this trial. The results showed:①The fiber, electrolyte, extraction temperature and time of the SPME was PDMS, NaCl (0.32g/mL), 50℃and 30min, respectively.②There were 70 compounds in the mixed fruit flavor meal, including ester, mellow, aldehyde, ketone and hydroxybenzene, and so on.
Part2 Studies on the optimal supplemental dosage of the mixed fruit flavor meal and effects on the cow performance and milk flavor
30 lactating cows with the same weight, lactation(71±5d) and milk yield were divided into five groups to investigate the effects of the mixed fruit flavor meal on the cow performance and milk flavor in peak milk, the trail was continuously taken out in lactation(152±4d). During the period, the cows were fed a basal diet adding different dosage mixed fruit flavor meal, i.e 0, 5000, 6500, 7500 and 9000g/t (based on DM of concentration), respectively. The results showed:①the mixed fruit flavor meal could improve the milk quality and flavor, and the optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t.②the milk yield in peak milk was increased and the decline rate of milk yield in the mid and late lactation was decreased. The total economic benefits were significant.
Part3 Studies on the metabolic changes of the mixed fruit flavor meal in the rumen, blood and mammary gland
⑴The metabolic changes of the mixed fruit flavor meal in the rumen
Two cows with permantent ruminal cannulas were fed a basal diet in early lactation and used to investigate the effects of the mixed fruit flavor meal on the fermentation index and flavor in the rumen. The results showed:①The mixed fruit flavor meal could increase significantly the DM disappearance rate(P < 0.05), which resulted in increasing the dietary digestion and the feed coefficiency.②The effects of the mixed fruit flavor meal on the gas production, pH, NH3-N concentration, acetate, propionate, butyrate, TVFA and A/P ratio were not significant in the batch culture in vitro (P > 0.05), but could significantly increase the ruminal content of acetate, propionate, butyrate and TVFA (P < 0.05), and so did and the NH3-N concentration and A/P ratio (expect 0h) in vivo (P < 0.05).③According to avaluation of Multiple-Factors Associative Effects Index (MFAEI), the optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t, the same as the result of the breeding trail.④Some flavor compounds of the mixed fruit flavor meal were found in the rumen liquid by SPME/GC-MS, including Acetic acid, 2-methylpropyl ester; 1-Butanol,3-methyl-, acetate; Butanoic acid, 2-methylpropyl este; Butanoic acid, ethyl ester; 1-Butanol,3-methyl-,propanoat; Propanoic acid, pentyl ester; Butanoic acid, 3-methylbutyl ester; and so on.
⑵The metabolic changes of the mixed fruit flavor meal measured by the arterio-venous difference cross the mammary gland
Two cows with the permanent catheters at the external pudic artery and subcutaneous abdominal vein were fed a basal diet and used to investigate the metabolic changes of the mixed fruit flavor meal in the arterio-venous difference. The results showed that some flavor compounds of the mixed fruit flavor meal were measured in the external pudic artery and subcutaneous abdominal vein blood by PT-GCMS, and the flavor compounds content of the mixed fruit flavor meal in the external pudic artery were nearly 2～3 times in subcutaneous abdominal vein. The mammary uptake of flavor compounds was higher than the output from milk.
⑶The effects of the mixed fruit flavor meal on the cell growth and cycle in the mammary epithelium cell culture and milk composition
The mammary epithelial cells in the culture were home made, the dosage of the mixed fruit flavor meal 0, 5, 10, 15, 20 and 30μg/mL was added in the culture trail. The results showed:①The cell number and the cell proportion in S and G2/M period were the highest when the mixed fruit flavor meal dosage was 15μg/mL, but cell growth and cycle proliferation were stopped when the dosage was 30μg/mL.②These compounds from the mixed fruit flavor meal were found in the mammary culture liquid by PT-GCMS, and their levels were the highest when the dosage was 15μg/mL (P < 0.05).
In conclusion, the above results indicated that the flavor compounds could improve the milk flavor and quality. The optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t (DM basal of concentrate). The fruit flavor compounds in the added in the mixed fruit flavor meal changed in the different site of the lactating cow’s body. some of the fruit flavor compounds were increased in ratio, some of compounds were decreased in qualitity, maybe some were disappeared in the animal body. Although the mixed fruit flavor meal could improve the milk flavor, the kind and ratio of the fruit flavor compounds in milk were changed.
选择不同的萃取头、电解质水平、萃取温度和萃取时间,通过正交实验设计,筛选出SPME最佳萃取参数。结果表明:①.采用SPME对果香味剂分析其最佳萃取头、NaCl用量、萃取温度和萃取时间分别为PDMS、0.32g/mL、50℃和30min。②.通过SPME-GCMS分析果香味剂中共有70种化合物,主要成分为酯类、醇类、醛类、酸类、酮类、酚类等。
第二部分为果香味剂最适添加剂量的筛选及对奶牛生产性能和牛奶风味的影响选择体重、泌乳期(71±5d)、胎次、产奶量相近,且健康无疾病的奶牛30头,共分5组,每组6头,日粮中果香味剂添加剂量分别为0、5000、6500、7500和9000g/t(精料DM基础)。当这些奶牛处于泌乳期152±4d时对其进行相同分组,添加相同剂量的果香味剂,试验期间观察奶牛生产性能和牛奶风味的变化。结果表明:①.果香味剂可改善乳风味和提高乳品质,其最适添加剂量为7500g/t。②.果香味剂可提高产奶高峰期奶牛的产奶量,对于产奶高峰期后奶牛的产奶量具有一定减缓作用,经济效益显著。
第三部分为果香味剂在瘤胃、血液和乳腺中的代谢变化研究试验1.果香味剂在奶牛瘤胃中的代谢及对瘤胃内容物和风味的影响选择2只体况良好,体重相近,安装有永久性瘤胃瘘管的奶牛,采用体内和体外培养相结合的方法研究果香味剂对瘤胃内容物及其风味的影响。结果表明:①.果味剂可显著提高瘤胃液中的干物质降解率(P < 0.05),说明果香味剂确实可以提高动物消化力,进而提高饲料转化率。②.体外培养表明果香味剂对瘤胃液产气量、pH值、NH3-N水平、乙酸、丙酸、丁酸、总VFA和乙/丙酸比影响不显著(P > 0.05),而体内试验则可显著提高瘤胃液中乙酸、丙酸、丁酸和总VFA含量(P < 0.05),显著降低瘤胃液NH3-N水平和乙/丙酸比(除0h外)(P < 0.05)。③.根据体外试验结果综合评定,得出其最适添加剂量为7500g/t,与上述试验结果相一致。④.通过采用SPME/GC-MS对瘤胃液进行分析,表明试验组中均含有果味剂中的一些风味物质成分。
试验2.果香味剂在奶牛乳腺动静脉差中的代谢变化选择2只体况良好,体重相近,处于泌乳前中期的荷斯坦奶牛,安装有乳静脉和阴外动脉插管供采集血液用。结果表明:果香味剂可进入到奶牛血液中,且阴外动脉血中风味物质含量将近是乳静脉血中风味物质含量的2～3倍。奶牛乳腺中风味物质摄取量随着采食时间的延长呈先上升后下降的变化趋势,其乳腺摄取/牛奶产出比均很高,说明奶牛采食后果香味剂中的风味物质被乳腺大量摄取,但仅有少量的物质进入到牛奶中改善牛奶风味。
试验3.果香味剂对乳腺细胞生长、细胞周期、乳成分和培养液风味的影响
选用自制的奶牛乳腺上皮细胞,果香味剂添加剂量分别为0、5、1、15、20和30μg/mL。结果表明:①.当添加剂量为15μg/mL时,细胞数量、培养液乳成分含量及进入S期和G2/M期细胞比例均达到最高(P < 0.05),之后开始下降;当浓度达30μg/mL时,开始抑制细胞生长和细胞周期的增殖。②.运用PT-GCMS对乳腺培养液中风味成分分析表明试验组中均含有果味剂中的一些风味物质成分,且当添加剂量为15μg/mL时,各物质成分含量为最高(P < 0.05),之后呈下降趋势。
综合上述研究结果表明:通过奶牛瘤胃、血液和乳腺代谢变化和初步的机制的研究表明果味剂中的一些物质成分能通过牛体血液进入到乳中,进而改善乳风味和提高乳品质,且体内外试验均证实其最适添加剂量为7500g/t(精料DM基础)。本研究还进一步得出日粮中添加果香味剂在进入到奶牛体内的不同部位均发生了较大的代谢变化,部分果味物质比例升高、部分果味物质数量减少、部分果味物质消失,所以真正进入到牛体内的果味物质组合发生了变化。尽管都改善了牛奶的风味,但进入到牛奶中的成分发生了较大的变化。本研究结果为果香味剂在奶牛养殖应用中提供了实验依据。
The metabolic changes of the mixed fruit flavor meal in the rumen, blood and mammary gland of the lactating cow by rumen cannulas, arterio-venous catheter, and mammary gland culture technique were studied in this paper. The results were as follows:
Part1 The development of measuring method for the mixed fruit flavor meal The best extraction parameter of SPME (Solid Phase Microextraction), the different fiber, electrolyte, extraction temperature and time were screened out in this trial. The results showed:①The fiber, electrolyte, extraction temperature and time of the SPME was PDMS, NaCl (0.32g/mL), 50℃and 30min, respectively.②There were 70 compounds in the mixed fruit flavor meal, including ester, mellow, aldehyde, ketone and hydroxybenzene, and so on.
Part2 Studies on the optimal supplemental dosage of the mixed fruit flavor meal and effects on the cow performance and milk flavor
30 lactating cows with the same weight, lactation(71±5d) and milk yield were divided into five groups to investigate the effects of the mixed fruit flavor meal on the cow performance and milk flavor in peak milk, the trail was continuously taken out in lactation(152±4d). During the period, the cows were fed a basal diet adding different dosage mixed fruit flavor meal, i.e 0, 5000, 6500, 7500 and 9000g/t (based on DM of concentration), respectively. The results showed:①the mixed fruit flavor meal could improve the milk quality and flavor, and the optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t.②the milk yield in peak milk was increased and the decline rate of milk yield in the mid and late lactation was decreased. The total economic benefits were significant.
Part3 Studies on the metabolic changes of the mixed fruit flavor meal in the rumen, blood and mammary gland
⑴The metabolic changes of the mixed fruit flavor meal in the rumen
Two cows with permantent ruminal cannulas were fed a basal diet in early lactation and used to investigate the effects of the mixed fruit flavor meal on the fermentation index and flavor in the rumen. The results showed:①The mixed fruit flavor meal could increase significantly the DM disappearance rate(P < 0.05), which resulted in increasing the dietary digestion and the feed coefficiency.②The effects of the mixed fruit flavor meal on the gas production, pH, NH3-N concentration, acetate, propionate, butyrate, TVFA and A/P ratio were not significant in the batch culture in vitro (P > 0.05), but could significantly increase the ruminal content of acetate, propionate, butyrate and TVFA (P < 0.05), and so did and the NH3-N concentration and A/P ratio (expect 0h) in vivo (P < 0.05).③According to avaluation of Multiple-Factors Associative Effects Index (MFAEI), the optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t, the same as the result of the breeding trail.④Some flavor compounds of the mixed fruit flavor meal were found in the rumen liquid by SPME/GC-MS, including Acetic acid, 2-methylpropyl ester; 1-Butanol,3-methyl-, acetate; Butanoic acid, 2-methylpropyl este; Butanoic acid, ethyl ester; 1-Butanol,3-methyl-,propanoat; Propanoic acid, pentyl ester; Butanoic acid, 3-methylbutyl ester; and so on.
⑵The metabolic changes of the mixed fruit flavor meal measured by the arterio-venous difference cross the mammary gland
Two cows with the permanent catheters at the external pudic artery and subcutaneous abdominal vein were fed a basal diet and used to investigate the metabolic changes of the mixed fruit flavor meal in the arterio-venous difference. The results showed that some flavor compounds of the mixed fruit flavor meal were measured in the external pudic artery and subcutaneous abdominal vein blood by PT-GCMS, and the flavor compounds content of the mixed fruit flavor meal in the external pudic artery were nearly 2～3 times in subcutaneous abdominal vein. The mammary uptake of flavor compounds was higher than the output from milk.
⑶The effects of the mixed fruit flavor meal on the cell growth and cycle in the mammary epithelium cell culture and milk composition
The mammary epithelial cells in the culture were home made, the dosage of the mixed fruit flavor meal 0, 5, 10, 15, 20 and 30μg/mL was added in the culture trail. The results showed:①The cell number and the cell proportion in S and G2/M period were the highest when the mixed fruit flavor meal dosage was 15μg/mL, but cell growth and cycle proliferation were stopped when the dosage was 30μg/mL.②These compounds from the mixed fruit flavor meal were found in the mammary culture liquid by PT-GCMS, and their levels were the highest when the dosage was 15μg/mL (P < 0.05).
In conclusion, the above results indicated that the flavor compounds could improve the milk flavor and quality. The optimal supplemental dosage of the mixed fruit flavor meal was 7500g/t (DM basal of concentrate). The fruit flavor compounds in the added in the mixed fruit flavor meal changed in the different site of the lactating cow’s body. some of the fruit flavor compounds were increased in ratio, some of compounds were decreased in qualitity, maybe some were disappeared in the animal body. Although the mixed fruit flavor meal could improve the milk flavor, the kind and ratio of the fruit flavor compounds in milk were changed.
引文
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22.马燕芬,卢德勋,罗婷等.固相微萃取GC-MS技术分析果味香味剂成分的研究.中国乳品工业,2009,8
23. Schlichtherle-Cerny H., Oberholzer D. Comparison of aroma compounds of mild and traditional acidic yogurts using SPME-GC-O-MS [J]. 5th NIZO Dairy Conference Prospects for Flavour Formation and Perception Papendal (NL), June 13-15, 2007
24.马永昆,陈计峦.SDE和SPME两种不同方法对库尔勒香梨芳香成分的分析[J].国际饮料工业大会,2002.
25.曾庆亮.浅论饲料中添加香味剂的必然性和重要性.中国饲料,1993,(8):37-39
26.刘俊奇.饲料香味剂及其应用.饲料研究,1995,(3):31-32
27. Kland K.,王放银.调味剂的应用现状及其发展展望.国外畜牧学,1994,(6):26-28
28.宋强华.饲料增味剂及其功效[J].粮食与饲料工业,1993,(1):23-25
29.陈一夫.兔用调味剂的开发应用[J].中国养兔杂志,1994,(5):32-34
30.王成章,王志祥,汪雪芳.芝麻饼加香味素可替代豆粕育肥蛋公鸡[J].中国饲料,1996,(4):27-28
31.郭芳彬.动物饲料调味剂的研究与应用[J].饲料世界,1991,(6):16-19
32.林圭,柏宝山,杨加杰.添加乳猪香对乳猪采食和增重的影响[J].饲料研究,1993,(12):5-6
33.徐公义,娄秀山.香味素饲喂断奶仔猪试验[J].饲料工业,1994,15(9):26-27
34.高剑蓉,何国声.猪用香味剂[J].饲料工业,1994,15(5):17-18
35.佟建明,萨仁娜.仔猪饲喂香味素的效果试验[J].中国饲料1993,(6):32
36.沈利明,吴国华,绊金忠.猪用饲料风味剂的研究[J].饲料工业,1994,15(8):29-30
37. Arave C. W. D., Purcell and M. Engstrom, Effects of feed flavors on improving choice for a ten percent meat and bone meal dairy concentrate [J]. J Dairy Sci, 1989(72): 563
38.甘长涛.牛饲料调味剂的作用[J].饲料工业, 1997,(3): 39～40
39.张栓林,黄应强.调味剂在养牛生产中的应用[J].饲料与畜牧, 1998,(6): 23～24
40.吕继蓉,赵华修,喻麟,李小兵,曾凡坤.添加不同香味剂对奶牛生产性能的影响.饲料研究, 2004,7:33-35
41. BramLey A J, Murdough P A and Pankey J W. In vitro culture of bovine teat duct explants [J]. J Dairy Sci, 1996, 79(Suppl. 1) 214
42. Erdmann B, and Breter H. Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium [J]. CeZZ Tissue Res, 1993, 272: 383-389
43. Cifrian E, Guidry A J, O’Brien C N, Keys J E and Marquardt W W. Bovine mammary teat and ductal epithelial cell cultures [J]. Am J Vet Res, 1994, 55: 239-246
44. Ebner K. E., Hoover C.R., Hageman E. C., Larson B. L. Cultivation and properties of bovine mammary cell culture [J]. Exp Cell Res’, 1961, 23: 373-385.
45. Goodman G. T., Akers R.M., Friderici K.H., Tucker H. A. Hormonal regulation of alpha-lactalbumin secretion from bovine mammary tissue cultured in vitro [J]. Endocrinology, 1983, 112(4): 1324-1330.
46. McGrath M. F. A novel system for mammary epithelial cell culture [J]. J Dairy Sci, 1987, 70(9): 1967-1980.
47. Schmid E., Franke W.W., Grund C., Schiller D.L., Kolb H., Paweletz N. An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays [J]. Exp Cell Res’, 1983, 146(2): 309-328.
48. Boris Z,M arilyn V D,W aren S, et al. Establishment and characterization of bovine mammary epithelial cell line unique properties in vitro cell [J]. Dev Biol,1996,32: 138- 148.
49.欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].中国兽医学报,1999;19(6)
50.欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].西北农林科技大学学报,2003;31(3)
51. Thordarson G., Ogren L. Day J.R., Bowens K., Fielder P., Talamantes F. Mammary gland development and alpha-lactalbumin production in hypophysectomized pregnant mice [J]. Biol Reprod, 1989, 40(3): 517-524.
52. Zheng Y. M., Peng X. R., Xu Y. P., Shi Y. Q., Zhang Y. Study on appearance of goat mammary epithelial cells which cultured in vitro [J]. J Northwest Sci-tech Univ Agricul For (Nature Science), 2004, 32(3): 37-41.
53.崔立莉.奶牛乳腺上皮细胞体外培养体系的建立及其应用[D].新疆农业大学硕士学位论文,2006,20-28.
54.李震,王英,刘慧丽等.牛乳腺上皮细胞的分离培养[J].上海农业学报,2000,16(3): 25-28.
55.多曙光,吴应积,罗奋华,旭日干.牛乳腺上皮细胞的分离培养及其生物学特性[J].动物学研究, 2006, 27(3): 299-305
56.王治国.奶牛乳腺上皮细胞的体外培养及应用[D].中国农业科学院硕士学位论文, 2007,23-30.
57.郑宇才等,水牛乳腺上皮细胞的体外培养[J].西南民族学院学报,1994,20(1):42-44
58.林桂娟等,机械破碎法分离奶牛乳腺上皮细胞的体外培养研究[J],畜牧与兽医, 2004,36(7): 4-6.
59. Allen J S K. Flow cytometry: assaying experimental polyploid fish and shellfish[J] . A quaculture, 1983,33:317-328.
60. Zhang J B. Practical Methods and Techniques of Cell Biology[M]. Beijing: United Press of Beijing Medicine University and Peking Union Medical College ,1995. 2092213.
61. Zhai Z H ,Wang X Z and Ding M X. Cell-Biology[M]. Beijing: Higher Education Press, 2000,357-360.
62. Bazemore R, Goodner K L, Rouseff R L. Food Chemtoxicol[M]. 1999, 64:800
63.刘杨岷,王利平,袁身淑.固相微萃取气质联用分析白兰花的香气成分[J].无锡轻工大学学报, 2001, 4: 32-36
64.赵军.原料奶中挥发性风味物质的分析评论及其保护技术体系的研究[D].内蒙古农业大学硕士学位论文,2009
65.冯宗慈,高民.通过比色法测定瘤胃液氨氮含量方法的改进[J].内蒙古畜牧与饲料科学,1993,(4):40-41
66.张吉鹍,李龙瑞,邹庆华.饲料间的组合效应及其在粗饲料科学搭配上的应用[J].饲料广角,2003,21:26-30.
67. Delamaire, E and Guinard-Flament J. Increase milking intervals decreases the mammary blood flow and mammary uptake of nutrients in dairy cows [J]. J Dairy Sci, 2006,89:3439-3446
68.白英.微量元素锌(Zn)对山羊β-酪蛋白合成及分泌影响的研究[D].内蒙古农业大学博士学位论文, 2007
69. Nicoletti I, Migliorati G, Psgliacci M,C , et al. A rapid and simple method for mearsuring thymocyte apoptosis by propidium iodide staining and flow cytometry [J]. J Immunol Methods, 1991,139∶271.
70. Tounekti O, Belehradek J, Mir Jr , et al. Relationships between DNA fragmentation , chromatin condensation , and changes in flow cytometry profiles detected during apoptosis [J]. Exp Cell Res, 1995,217∶506.
71. Steffen A, Pawliszyn J. Analysis of Flavor Volatiles Using Head Space Solid-phase Microextraction[J]. J Agric Food Chem, 1996, 44: 2187-2193.
72. Verhoeven H, Beuerle T, Schwab W. Solid-phase Microextraction: Artifact Formation and Its Avoidance[J]. Chromatographia, 1997,46(1-2): 63-66.
73. Fisher C, Fisher U. Analysis of Corktaintin Wine and Corkmateria at Olfactory SubThreshold Lever by Solid-phase Microextraction[J]. J Agric Food Chem, 1997, 45: 1995-1997.
74. Alexandra S, Jnusez P. Analysis of Flavor Volatile Using Headspace Solid Phase Microextraction [J]. J Agric Food Chem, 1996, 44: 2187-2193.
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18. Bezman Y., Mayer F., Takeoka G. R. Differential efffets of tomato (Lycopersicon esculentum mill) Matrix on the volatility of important aroma compounds [J]. J Agric Food Chem, 2003, 51(3): 722-726.
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20. Jordan M. J. Using HS-SPME to determine the effects of reducing insoluble solids on aromatic composition of orange juice [J]. Lebensm-Wissu-Technol, 2001, (34):244-250.
21.陈计峦,吴继红,冯作山.新疆库尔勒香梨果酒的择发性成分的分析[J].酿酒科技, 2005,(3): 87-89.
22.马燕芬,卢德勋,罗婷等.固相微萃取GC-MS技术分析果味香味剂成分的研究.中国乳品工业,2009,8
23. Schlichtherle-Cerny H., Oberholzer D. Comparison of aroma compounds of mild and traditional acidic yogurts using SPME-GC-O-MS [J]. 5th NIZO Dairy Conference Prospects for Flavour Formation and Perception Papendal (NL), June 13-15, 2007
24.马永昆,陈计峦.SDE和SPME两种不同方法对库尔勒香梨芳香成分的分析[J].国际饮料工业大会,2002.
25.曾庆亮.浅论饲料中添加香味剂的必然性和重要性.中国饲料,1993,(8):37-39
26.刘俊奇.饲料香味剂及其应用.饲料研究,1995,(3):31-32
27. Kland K.,王放银.调味剂的应用现状及其发展展望.国外畜牧学,1994,(6):26-28
28.宋强华.饲料增味剂及其功效[J].粮食与饲料工业,1993,(1):23-25
29.陈一夫.兔用调味剂的开发应用[J].中国养兔杂志,1994,(5):32-34
30.王成章,王志祥,汪雪芳.芝麻饼加香味素可替代豆粕育肥蛋公鸡[J].中国饲料,1996,(4):27-28
31.郭芳彬.动物饲料调味剂的研究与应用[J].饲料世界,1991,(6):16-19
32.林圭,柏宝山,杨加杰.添加乳猪香对乳猪采食和增重的影响[J].饲料研究,1993,(12):5-6
33.徐公义,娄秀山.香味素饲喂断奶仔猪试验[J].饲料工业,1994,15(9):26-27
34.高剑蓉,何国声.猪用香味剂[J].饲料工业,1994,15(5):17-18
35.佟建明,萨仁娜.仔猪饲喂香味素的效果试验[J].中国饲料1993,(6):32
36.沈利明,吴国华,绊金忠.猪用饲料风味剂的研究[J].饲料工业,1994,15(8):29-30
37. Arave C. W. D., Purcell and M. Engstrom, Effects of feed flavors on improving choice for a ten percent meat and bone meal dairy concentrate [J]. J Dairy Sci, 1989(72): 563
38.甘长涛.牛饲料调味剂的作用[J].饲料工业, 1997,(3): 39～40
39.张栓林,黄应强.调味剂在养牛生产中的应用[J].饲料与畜牧, 1998,(6): 23～24
40.吕继蓉,赵华修,喻麟,李小兵,曾凡坤.添加不同香味剂对奶牛生产性能的影响.饲料研究, 2004,7:33-35
41. BramLey A J, Murdough P A and Pankey J W. In vitro culture of bovine teat duct explants [J]. J Dairy Sci, 1996, 79(Suppl. 1) 214
42. Erdmann B, and Breter H. Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium [J]. CeZZ Tissue Res, 1993, 272: 383-389
43. Cifrian E, Guidry A J, O’Brien C N, Keys J E and Marquardt W W. Bovine mammary teat and ductal epithelial cell cultures [J]. Am J Vet Res, 1994, 55: 239-246
44. Ebner K. E., Hoover C.R., Hageman E. C., Larson B. L. Cultivation and properties of bovine mammary cell culture [J]. Exp Cell Res’, 1961, 23: 373-385.
45. Goodman G. T., Akers R.M., Friderici K.H., Tucker H. A. Hormonal regulation of alpha-lactalbumin secretion from bovine mammary tissue cultured in vitro [J]. Endocrinology, 1983, 112(4): 1324-1330.
46. McGrath M. F. A novel system for mammary epithelial cell culture [J]. J Dairy Sci, 1987, 70(9): 1967-1980.
47. Schmid E., Franke W.W., Grund C., Schiller D.L., Kolb H., Paweletz N. An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays [J]. Exp Cell Res’, 1983, 146(2): 309-328.
48. Boris Z,M arilyn V D,W aren S, et al. Establishment and characterization of bovine mammary epithelial cell line unique properties in vitro cell [J]. Dev Biol,1996,32: 138- 148.
49.欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].中国兽医学报,1999;19(6)
50.欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].西北农林科技大学学报,2003;31(3)
51. Thordarson G., Ogren L. Day J.R., Bowens K., Fielder P., Talamantes F. Mammary gland development and alpha-lactalbumin production in hypophysectomized pregnant mice [J]. Biol Reprod, 1989, 40(3): 517-524.
52. Zheng Y. M., Peng X. R., Xu Y. P., Shi Y. Q., Zhang Y. Study on appearance of goat mammary epithelial cells which cultured in vitro [J]. J Northwest Sci-tech Univ Agricul For (Nature Science), 2004, 32(3): 37-41.
53.崔立莉.奶牛乳腺上皮细胞体外培养体系的建立及其应用[D].新疆农业大学硕士学位论文,2006,20-28.
54.李震,王英,刘慧丽等.牛乳腺上皮细胞的分离培养[J].上海农业学报,2000,16(3): 25-28.
55.多曙光,吴应积,罗奋华,旭日干.牛乳腺上皮细胞的分离培养及其生物学特性[J].动物学研究, 2006, 27(3): 299-305
56.王治国.奶牛乳腺上皮细胞的体外培养及应用[D].中国农业科学院硕士学位论文, 2007,23-30.
57.郑宇才等,水牛乳腺上皮细胞的体外培养[J].西南民族学院学报,1994,20(1):42-44
58.林桂娟等,机械破碎法分离奶牛乳腺上皮细胞的体外培养研究[J],畜牧与兽医, 2004,36(7): 4-6.
59. Allen J S K. Flow cytometry: assaying experimental polyploid fish and shellfish[J] . A quaculture, 1983,33:317-328.
60. Zhang J B. Practical Methods and Techniques of Cell Biology[M]. Beijing: United Press of Beijing Medicine University and Peking Union Medical College ,1995. 2092213.
61. Zhai Z H ,Wang X Z and Ding M X. Cell-Biology[M]. Beijing: Higher Education Press, 2000,357-360.
62. Bazemore R, Goodner K L, Rouseff R L. Food Chemtoxicol[M]. 1999, 64:800
63.刘杨岷,王利平,袁身淑.固相微萃取气质联用分析白兰花的香气成分[J].无锡轻工大学学报, 2001, 4: 32-36
64.赵军.原料奶中挥发性风味物质的分析评论及其保护技术体系的研究[D].内蒙古农业大学硕士学位论文,2009
65.冯宗慈,高民.通过比色法测定瘤胃液氨氮含量方法的改进[J].内蒙古畜牧与饲料科学,1993,(4):40-41
66.张吉鹍,李龙瑞,邹庆华.饲料间的组合效应及其在粗饲料科学搭配上的应用[J].饲料广角,2003,21:26-30.
67. Delamaire, E and Guinard-Flament J. Increase milking intervals decreases the mammary blood flow and mammary uptake of nutrients in dairy cows [J]. J Dairy Sci, 2006,89:3439-3446
68.白英.微量元素锌(Zn)对山羊β-酪蛋白合成及分泌影响的研究[D].内蒙古农业大学博士学位论文, 2007
69. Nicoletti I, Migliorati G, Psgliacci M,C , et al. A rapid and simple method for mearsuring thymocyte apoptosis by propidium iodide staining and flow cytometry [J]. J Immunol Methods, 1991,139∶271.
70. Tounekti O, Belehradek J, Mir Jr , et al. Relationships between DNA fragmentation , chromatin condensation , and changes in flow cytometry profiles detected during apoptosis [J]. Exp Cell Res, 1995,217∶506.
71. Steffen A, Pawliszyn J. Analysis of Flavor Volatiles Using Head Space Solid-phase Microextraction[J]. J Agric Food Chem, 1996, 44: 2187-2193.
72. Verhoeven H, Beuerle T, Schwab W. Solid-phase Microextraction: Artifact Formation and Its Avoidance[J]. Chromatographia, 1997,46(1-2): 63-66.
73. Fisher C, Fisher U. Analysis of Corktaintin Wine and Corkmateria at Olfactory SubThreshold Lever by Solid-phase Microextraction[J]. J Agric Food Chem, 1997, 45: 1995-1997.
74. Alexandra S, Jnusez P. Analysis of Flavor Volatile Using Headspace Solid Phase Microextraction [J]. J Agric Food Chem, 1996, 44: 2187-2193.