Isolation And Identification Of Bacteria From Kefir And Study On Compound Fermentation Milk Of Fine Strains

Kefir, which contains a small amount of alcohol, is one of the most popular fermented milk. It originates from Caucasus which has one of the highest life expetency in the world. According to reports, kefir possesses lots of physiological functions such as gastrointestinal system improvement, enhancement of immunity, inhibition of tumor cell, to mention a few. At present, kefir can only be produced with kefir grains, and kefir grian is a natural fermentation culture, it can be easily changed by the cultured environment and the geographical origin. In the industrial production of kefir, the further development of kefir is restricted by the limitation, the volatility and unknown microorganisms in kefir grains. In order to solve these shortcomings, this study mainly focuses on the following three aspects, the production of kefir grains, the isolation, purification and identification of the microorganisms in different kefir grains, and the development of pure culture starters producing fermented milk. Finally, fermented milk with stable taste and flavor was developed. The main conclusions of the researchs are as follows.At the start of the study, the proliferation culture medium and conditions of kefir grains were optimized. The results showed that skim milk is the best medium compared with raw milk and low- fat milk. And the optimal concentration was 40%. Adding carbon source, nitrogen source and inorganic salt had no significant effect on the proliferation of kefir grains, and its growth was hindered extently. Taking economic factor and the effect of experiment into account, the later experiment selected 20% of the skimmed milk as the initial culture medium. The results of optimization on the proliferation conditions showed that the processeswith not cleaning and smaller size promoted the proliferation of kefir grains. The single factor experiment results showed that the the kefir grains proliferation rate increased with the increase of temperature, speed, time, inoculation amount, p H, but after reaching a certain degree, decreased with the rise of them. Response surface experiment showed that optimal culture temperature was 30.4 ℃, the inoculation amount was 2.12%, the rotation speed was 99 r/min. Verification test showed that the average growth rate of kefir grains is 14.68%, the ratio of the initial conditions of 11.12 % increased by 32.01%.Then, six kefir grains from different origins were isolated, purified, identified, respectively. The results showed that there were 12 strains in kefir grains N, 3 strains of Kazachstania unispora, 1 strain Pichia kudriavzevii, 1 strain Dekkera anomala, 2 strains of Leuconostoc mesenteroides, 2 strains of Lactobacillus kefiri, 1 strain Acetobacter tropicalis, 1 strain of Acetobacter syzygii, and 1 strain Acetobacter cibinongensis. There were 10 strains in kefir grains X, 2 strains of Kazachstania unispora, 1 strain Kluyveromyces marxianus, 1 strain Pichia kudriavzevii, 1 strain Leuconostoc mesenteroides, 3 strains of Lactobacillus kefiri, 1 strain Acetobacter lovaniensis, and 1 strain Acetobacter syzygii. There were 7 strains in kefir grains SK, 1 strain Kazachstania unispora, 1 strain Pichia kudriavzevii, 4 strains of Lactobacillus kefiri, and 1 strain Acetobacter syzygii. There were 13 strains in ke fir grains SX, 1 strain Kazachstania unispora, 1 strain Kluyveromyces marxianus, 1 strain Pichia occidentalis, 1 strain Candida stellimalicola, 5 strains of Lactobacillus kefiri, 1 strain Acetobacter lovaniensis, 2 strains of Acetobacter okinawensis, and 1 strain Acetobacter syzygii. There were 6 strains in kefir grains TX, 1 strain Kluyveromyces marxianus, 4 strains of Lactobacillus kefiri, and 1 strain Acetobacter syzygii. There were 7 strains in kefir grains FX, 1 strain Kluyveromyces marxianus, 1 strain Pichia kudriavzevii, 4 strains of Lactobacillus kefiri, and 1 strain Acetobacter cibinongensis.At last, the 6 kinds of kefir grains were evaluated on sensory and flavor. Kefir X showed the best flavor and sensory among them. And it got the highest score of 89 in sensory experiments. The volatile components analysis showed that the content of acetoine is very high in kefir X. It matched to the result of sensory experiments. The ratio of Leuconostoc mesenteroides(XR1) and Lactobacillus kefiri(XR2, XR3, XR4) was determined by the counts in same palte. Then according to the proportion of LAB stains(XR1:XR2:XR3:XR4 was 5:1:2:3) in kefir grains X, fermented milk was developed. It had good flavor,and the fermentation time was short. And the flavor of the developed milk was close to the kefir X. It demonstrated a kind of novel fermented milk was successfully developed. Compared with kefir X, the novel milk showed the comparable contents in crude polysaccharide and protein. The acidity and water holding capacity of them were very close. Nutrition index determination results showed that the fermented milk has better nutritional function. It demonstrated the experiment was successful.