开菲尔粒增殖工艺及其发酵乳制品生理功能特殊性的研究
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摘要
本文通过对比研究、正交实验、营养强化和动物实验等方法对开菲尔粒的复活传代、
清洗传代、增殖条件、开菲尔酸奶生产的最佳工艺条件以及开菲尔的功能特性进行了广
泛的研究。结果表明：
粒状开菲尔粒的活力在连续培养到第六代时达到最高活力（pH值为3.72）。在此之
后随着传代次数的增加活力开始下降，经过第七代培养后从第八代开始，开菲尔粒的活
力始终稳定在4.20左右。而片状开菲尔粒的活力最高只能达到pH值3.94，利用其得到
的开菲尔发酵乳酸度也明显不如粒状开菲尔粒得到的发酵乳（前者pH值4.74，后者pH
值4.52），而且刺激性酸味较大，不柔和。通过对比在以后的课题研究中采用了粒状开
菲尔粒。
清洗对开菲尔粒的影响极大，既使采用隔代清洗开菲尔粒的活力也需连续培养十六
代才能达到所需活力（pH值3.75），在工艺条件完全相同的情况下得到这一结论，显然
采用不清洗连续传代更具有实际意义。
对开菲尔粒增殖速度影响最大的是乳的浓度。在控制最佳的培养条件下（乳的浓度
12％，培养温度22℃，开菲尔粒接种量5％），不清洗连续培养十一代后，质量即可增殖一
倍，增殖率达到104.73％；在其它的营养强化条件中，无机盐对开菲尔粒的增殖有显著
的效果，连续培养十代后增殖率最高可达到167.85％；糖和氨基酸对开菲尔粒的增殖影
响相对较小，在培养第二代时增殖效果明显减弱，而进入到培养的第三代时不但无增殖
作用，相反抑制了开菲尔粒的增殖。
利用制备的开菲尔工作发酵剂进行乳的发酵条件要求极高，只有在高温和高接种量
（28℃，接种量为12％）长时间发酵培养的条件下才能获得品质较好的产品。此时，开
菲尔发酵乳中的微生物从菌相的平衡关系到其活力都发生了极大的变化，若利用得到的
发酵乳做工作发酵剂进行乳的发酵，条件要求非常苛刻，所得产品的酸度也相对较低（pH
值4.63）。
对于开菲尔的功能性研究，本课题通过小白鼠的动物实验，通过测定血清中甘油三
酯、胆固醇和血糖浓度验证了开菲尔酸奶具有极好的降血脂和降血糖功能（P＜0.01）。
In this article, we make an extensive study on resurgent subculture, purging subculture, proliferation condition, and the optimum technological condition of Kefir yoghurt using the methods such as comparison research, orthogonal experiments, nutrition enrichment, and the animal experiments. Here are the results:
The vril of the grainy Kefir grains can reach the highest point under the continuative culture at its 6th generation (pH3.72). Afterwards, the vril begins to decrease as the times of subculture increases, and from the 8~ generation on, after the 7~ generation culture, the vril is stabilized at about 4.2. But the highest vril of flaky Kefir grains can only reach its pH value at 3.94. The acidity of the fermented milk made of the flaky Kefir grains is lower the that of the fermented milk made of the grainy Kefir grains. (pH of the former is 4.74, while pH of the latter is only 4.52). Besides, the fermented milk made of the flaky Kefir grain has the strong irritant sour, the flavor is not mild. After the comparison, we choose the grainy Kefir grain as the object on which we make our study.
Purging influences the Kefir grains greatly, the vril of the Kefir grains can only be the vril we need under the continuative culture for 16 generations (pH3.75) even though we purge it every other generation. Under the same technological condition, we draw such a conclusion, so it is of more practical significance for us to use the method of non-purging continuative subculture.
The factor which can influence the proliferation of Kefir grains most is the density of milk. Under the optimum culture condition (the density of milk: 12%, culture temperature: 22 ~C, inoculation quantity of Kefir grains: 5%) using non-purging continuative culture for 11 generations, the quality can be doubled, its proliferation rate is 104.73%. In other nutrition enrichment conditions, inorganic salts can have an distinguished effect on the proliferation of Kefir grains, after continuative culture for 10 generations , its proliferation rate can reach 167.85%; sugar and amino acid have a comparative small effect on its proliferation, the proliferation effect obviously decreases during the culture of 2nd generation, as for the 3rd generation , the proliferation is not intensified but withheld.
The milk ferment technological condition using cultured Kefir as working ferment is very strict. Only under such conditions as high temperature, large inoculation quantity
(temperature: 28~C, inoculation quantity: 12%) and long fermented culturing time, can we obtain the product of high quality. At this time, there are great changes taking place in microorganism colony of Kefir fermented milk from the balance relationship to its vril, if we use this fermented milk, the technological condition will be very strict, and the acidity of the product is comparative low (pH=4.63).
In order to study on Kefir functions, we do the animal experiments on rats, measure triglyceride, cholesterol and blood sugar, so this proves that Kefir yoghurt has the admirable function of lowering blood fat and blood sugar (p
清洗传代、增殖条件、开菲尔酸奶生产的最佳工艺条件以及开菲尔的功能特性进行了广
泛的研究。结果表明：
粒状开菲尔粒的活力在连续培养到第六代时达到最高活力（pH值为3.72）。在此之
后随着传代次数的增加活力开始下降，经过第七代培养后从第八代开始，开菲尔粒的活
力始终稳定在4.20左右。而片状开菲尔粒的活力最高只能达到pH值3.94，利用其得到
的开菲尔发酵乳酸度也明显不如粒状开菲尔粒得到的发酵乳（前者pH值4.74，后者pH
值4.52），而且刺激性酸味较大，不柔和。通过对比在以后的课题研究中采用了粒状开
菲尔粒。
清洗对开菲尔粒的影响极大，既使采用隔代清洗开菲尔粒的活力也需连续培养十六
代才能达到所需活力（pH值3.75），在工艺条件完全相同的情况下得到这一结论，显然
采用不清洗连续传代更具有实际意义。
对开菲尔粒增殖速度影响最大的是乳的浓度。在控制最佳的培养条件下（乳的浓度
12％，培养温度22℃，开菲尔粒接种量5％），不清洗连续培养十一代后，质量即可增殖一
倍，增殖率达到104.73％；在其它的营养强化条件中，无机盐对开菲尔粒的增殖有显著
的效果，连续培养十代后增殖率最高可达到167.85％；糖和氨基酸对开菲尔粒的增殖影
响相对较小，在培养第二代时增殖效果明显减弱，而进入到培养的第三代时不但无增殖
作用，相反抑制了开菲尔粒的增殖。
利用制备的开菲尔工作发酵剂进行乳的发酵条件要求极高，只有在高温和高接种量
（28℃，接种量为12％）长时间发酵培养的条件下才能获得品质较好的产品。此时，开
菲尔发酵乳中的微生物从菌相的平衡关系到其活力都发生了极大的变化，若利用得到的
发酵乳做工作发酵剂进行乳的发酵，条件要求非常苛刻，所得产品的酸度也相对较低（pH
值4.63）。
对于开菲尔的功能性研究，本课题通过小白鼠的动物实验，通过测定血清中甘油三
酯、胆固醇和血糖浓度验证了开菲尔酸奶具有极好的降血脂和降血糖功能（P＜0.01）。
In this article, we make an extensive study on resurgent subculture, purging subculture, proliferation condition, and the optimum technological condition of Kefir yoghurt using the methods such as comparison research, orthogonal experiments, nutrition enrichment, and the animal experiments. Here are the results:
The vril of the grainy Kefir grains can reach the highest point under the continuative culture at its 6th generation (pH3.72). Afterwards, the vril begins to decrease as the times of subculture increases, and from the 8~ generation on, after the 7~ generation culture, the vril is stabilized at about 4.2. But the highest vril of flaky Kefir grains can only reach its pH value at 3.94. The acidity of the fermented milk made of the flaky Kefir grains is lower the that of the fermented milk made of the grainy Kefir grains. (pH of the former is 4.74, while pH of the latter is only 4.52). Besides, the fermented milk made of the flaky Kefir grain has the strong irritant sour, the flavor is not mild. After the comparison, we choose the grainy Kefir grain as the object on which we make our study.
Purging influences the Kefir grains greatly, the vril of the Kefir grains can only be the vril we need under the continuative culture for 16 generations (pH3.75) even though we purge it every other generation. Under the same technological condition, we draw such a conclusion, so it is of more practical significance for us to use the method of non-purging continuative subculture.
The factor which can influence the proliferation of Kefir grains most is the density of milk. Under the optimum culture condition (the density of milk: 12%, culture temperature: 22 ~C, inoculation quantity of Kefir grains: 5%) using non-purging continuative culture for 11 generations, the quality can be doubled, its proliferation rate is 104.73%. In other nutrition enrichment conditions, inorganic salts can have an distinguished effect on the proliferation of Kefir grains, after continuative culture for 10 generations , its proliferation rate can reach 167.85%; sugar and amino acid have a comparative small effect on its proliferation, the proliferation effect obviously decreases during the culture of 2nd generation, as for the 3rd generation , the proliferation is not intensified but withheld.
The milk ferment technological condition using cultured Kefir as working ferment is very strict. Only under such conditions as high temperature, large inoculation quantity
(temperature: 28~C, inoculation quantity: 12%) and long fermented culturing time, can we obtain the product of high quality. At this time, there are great changes taking place in microorganism colony of Kefir fermented milk from the balance relationship to its vril, if we use this fermented milk, the technological condition will be very strict, and the acidity of the product is comparative low (pH=4.63).
In order to study on Kefir functions, we do the animal experiments on rats, measure triglyceride, cholesterol and blood sugar, so this proves that Kefir yoghurt has the admirable function of lowering blood fat and blood sugar (p
引文
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2． Koroleva,N．S．Starters for fermented milks．Section 4 kefir and kumys starters． International Dairy Federation Bulletin．1988，227：35~40
3． Robinson RK．Studies on the multiplication of lactic acid bacteria present in the composition of kefir grains and on their intracellular structure as determined in the electron microscope．Dairy microbiology．1985． 272
4． Yokoi，H．；Watanabe，T．，Toba,T． Some taxonomical characteristics of encapsulated Lactobacillus sp．KPB-167B Isolated from kefir grains and characterization of its extracellular polysaccharide．International Journal of Food Microbiology 1991，13： 257~264
5． K ooiman P .The chemical structure of kefiran．the water-soluble polysaccharide of the kefir grain．Carbohydrate research．1968，7：200~211
6． La．Riviere．Kefiran．a novel polysaccharide produced in the kefir grain by Lactobacillus brevis．Archir fUr milkrobiologie．1967,59：269~278
7． Kandler,O．&Kuanth．P. Lactobacillis kefir sp．nov．，a component of the microflora of kefir． Systematic and Applied Microbiology．1983，4：286~294
8． Rosi，J．&Rossi，J．The kefir micrl-organisms：The lactic acid bacteria．Scienza e Tecnica Lattiero-Casearia 1978，29：291~305
9． Obera,N．Microbiological studies of kefir grains．Diss．Tokyo Univ．Agric．，Jpn．1980
10． 崛俊民昭和62年度日本农芸化学大会讲演要旨集．1987． 145
11． Toba T. A medium for the isolation of capsular bacteria from kefir grains．Agri．Biol．Chem． 1986，50：2673~2674．
12． Fojisawa,T．，S．Adachi，T. Toba,K．Arihara,and T．Mitsuoka．Lactobacillus kefiranofaciens sp．nov．isolated from kefir grains．International Journal of System Bacteriology, 1988． 38：12~14
13． Haruhido yodoi．Isolation and Characterization of Polysaccharide-Producing Bactfia from Kefir Grains J．Dairy Sci．1990． 73：1 684~1689
14． AY Tamime．Fermented milks and their future trends．Part Ⅱ．Technological aspects．Journal of Dairy Research 1988，55：281~307
15． Marshall V．M．&Cole，W．M． Methods for making kefir and fermented milks based on kefir．Journal of Dairy Research，1985． 52：451~456
16． Ohara,N；Suzuki，M;Okada,S．Identification of lactic acid bacteria isolated from freeze-dried kefir grains．Japanese Journal of Food Microbiology 1997，13(4) ：165～171．
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