Screening And Identification Of Potential Probiotics Yeasts From Raw Milk And The Influence On The Characteristics Of Camembert-type Cheese

Yeasts are main microorganisms in some fermented dairy products. The potential probiotics yeasts can be used in the Camembert cheese to enhance nutritional value and health protection of production. In the thesis, 129 yeasts strains isolated from raw milk in Beijing and Harbin were identified by physiological and molecular methods; and then, the experiments about probiotic selection were carried out in order to obtain some probiotics yeast strains; at last, the influence of 3 probiotic yeast strains, which had ability of cholesterol assimilation and antioxidant on the characteristics of Camembert- type cheese, were studied.The traditional physiology method and molecular method were used to classify the yeast strains isolated from raw milk in Beijing and Harbin. The results indicated that the main yeasts in the raw milk were Pichia guilliermondii, Galactomyces reessii, Trichosporon gracile, Rhodotorula mucilaginosa, Dipodascus capitatus, Issatchenkia orientalis, Yarrowia lipolytica and Geotrichum sp.The assays for probiotics screening and cholesterol assimilation in vitro showed that four strains, i.e., P. fermentans BY5, I. orientalis BY10, I. orientalis BY15 and Y. lipolytica HY4, were potential probiotic yeasts to assimilate cholesterol. The ability of lowering cholesterol was induced by the absorption of growing yeasts cells. The assays of scavenging DPPH free radical and inhibiting lipid peroxidation revealed that 10 yeast strains had antioxidant ability in both yeast cells and cell-free extracts, and 4 yeast strains including P. fermentans BY5 and I. orientalis BY10 were potential probiotics yeasts strains with antioxidant ability. At the same time, the incubation conditions such as initial pH, time and components in culture significantly influenced the antioxidant activity of cell-free extract from fermentans BY5 (P<0.05). The scavenging DPPH ability reached to the maximum value of 47%, 62% and 60% as initial pH 6, at the second day, as well as the culture with malt, respectively. Cell- free extracts from P. fermentans BY5 and I. orientalis BY10 could inhibit lipid peroxidation induced by 4-nitroquinoline 1-oxide (4-NQO). Compared with the control group, the MDA level was lower, the activity of some antioxidant enzyme as SOD, CAT and GSH were higher while the GPx dropped significantly (P<0.05). The genetic background evaluation for 3 protential probiotics yeasts strains revealed that the homology between BY5 and a P. fermentans strain originated from Xinjiang koumiss was 100%; the same as that of BY10 and an I. orientalis strain from the intestinal of a piglet. The genome of HY4 had a relatively close relationship with a Y. lipolytica from deep-sea eara with 98.6% homology. It meant that the genetics background of above strains were non- toxic. Furthermore, the acute toxicity test of the mouse proved that the milk fermented by selective yeasts were actually non-toxic.The growth curve of the strains of BY5, BY1 and HY4 under aerobic condition accorded with Monod model. The period of 0-16 h was their logarithmic growth phase and the growth of these strains was up to maximum at 64 h. At this time, the maximum growrh value of HY4 reached to 9.4 log cfu/ mL. All 3 yeast strains accelerated the growth of Lactococcus lactis Ll and Lactococcus lactis Lc. Symbiotic relationships were exhibited between the Lactococcus lactis Ll, Lactococcus lactis Lc and P. fermentans BY5 and I. orientalis BY10, respectively. The addition of BY5 and BY10 decreased the content of lactose and all 3 strains increased the concentrations of free amino acid (FAA) and free fatty acid (FFA) in the ultra high temperature (UHT) milk.The influence of 3 probiotics yeasts strains, i.e. P. fermentans BY5, I. orientalis BY10 and Y. lipolytica HY4, on the characeristics of Camembert-type cheese was discussed in this thesis. All selective yeast strains had inhibition on Pen. camemberti in cheese. The addition of yeasts did not change general characteristics of Camembert-type cheese such as the content of total solid and fat and the value of pH. However, the contents of total free amino acid and the individual amino acid in the experimental cheese were significantly different from the control (P<0.05). Moreover, BY10 made the content of non-protein nitrogen (NPN) and the NH3 in the cheese increase obviously. The results of PCA (principle component analysis) can not differentiate the experimental cheese and the control by analyzing the volatile compound, but by the mature period of the cheese which meant no significant effect on cheese for the selective yeasts. In addition, the P. fermentas BY5 increased the hardness and reduced the springing of products. For I. orientalis BY10, it softened the cheese while no change of textural properties was shown in the cheese with addition of Y. lipolytica HY4 contrasted to the control. As a result, the strains of BY5, BY10 and HY4 can be used as probiotics in cheese, and the antioxidant and cholesterol assimilation characteristics of the cheese deserve further analysis.