| Tibetan kefir is a type of kefir grain that originated in Tibet, China. Similar to kefir, Tibetan kefir consists of gelatinous, irregular-shaped grains formed by a symbiotic combination of yeasts, lactic acid bacteria and acetic acid bacteria. Tibetan kefir has good fermentation characteristics, and the fermented milk has many biological functions. Compared with traditional fermented milk, Tibetan kefir fermented milk has unique alcohol flavor produced by yeasts. However, the components of Tibetan kefir microorganism were very complex, thus using traditional menthods to starter propagation easily led to flora imbalance and flavor changes, which limited its industrial production. Because of such problems, in this dissertation, a comprehensive research was made based on Tibetan kefir from some aspects including the antioxidant activities, rheological properties and structural features of exopolysaccharide(EPS) produced by Tibetan kefir, and the generation mechanism of Tibetan kefir grains. This study systematically studied on those problems and got the following results and conclusions:(1) Characteristics and antioxidant activity of EPS produced by Tibetan kefir. The EPS was purified by ethanol precipitation, deproteinization and ethanol precipitation. The EPS did not contain uronic acid and was composed of glucose and galactose in a ratio of 1: 1.88 in Fourier transform infrared spectroscopy analysis and monosaccharide composition analysis. Scanning electron microscopy images of the EPS showed smooth and compact surfaces without pores. The EPS had a melting point of 131.46 ℃ and had an endothermic enthalpy change of 209.6 J/g, indicating good stability on heating analysed using a differential scanning calorimeter. The EPS could effectively remove DPPH and ABTS free radicals, reduce Fe3+ ion and protect protein from oxidative damage produced by AAPH and Cu2+/H2O2, exhibiting high antioxidant activities.(2) Rheological and emulsifying properties of EPS produced by Tibetan kefir. The rheological properties of the aqueous EPS solution were detected by rheometer, and the influence of concentration, pH, temperature and salt ions on the rheological properties of EPS was analysed. The results showed as follows: The aqueous EPS solution was a typical non-Newtonian pseudoplasric fluid and had good thickening peropertity. The viscosity at concentrations of 10 mg/mL had little change with good temperature resistance at 10 ℃ to 85 ℃. The viscosity increased after being frozen(-20 ℃), showing good freeze-thaw stability. The viscosity obviously decreased when the pH changed from 7.0 to 4.0 or 10.0, characterized by acidic and alkaline instability. The addition of Na+ significantly increased the viscosity of EPS solutions, while the visvosity of the EPS solutions greatly decreased when Ca2+ was added. The emulsifying activity and stability of the EPS increased as the concentration increased, and were not effected by pH and NaCl.(3) Component analysis and structure determination of of EPS produced by Tibetan kefir. the EPS was applied to a DEAE-Sepharose Fast Flow anion exchange column, and TKEP1, TKEP2 and TKEP3 were obtained by eluting with water, 0.25 and 0.5 mol/L NaCl, respectively. The fraction TKEP1 was further applied on a Sephadex G-100 gel column, and eluted with water and fractionated into three fractions(TKEP1-A, TKEP1-B, TKEP1-C). TKEP1-A and TKEP1-B were combined with a small amount of protein, only TKEP1-C did not include protein. Further analysis showed that, The carbohydrate content of TKEP1-C was 98.7 %(Phenol-sulfuric acid method), and the molecular mass of the EPS was 2.065×103 Da(High performance gel permeation chromatography). TKEP1-C was composed of glucose and galactose in a molar ratio of 1.17: 1(Gas chromatographic method), with possible presence of β-glycosidic bond and α-glycosidic bond(Fourier infrared spectrum method).The resluts of methylation analysis revealed that TKEP1-C was a linear heteropolysaccharide and the main connection modes were 1, 4-Gal, 1, 4-Glc, 1, 3-Gal, 1, 6-Gal and 1, 6-Glc, with 1, 4-Glu(31.03 %) and 1, 6-Gal(23.08 %) accounted for larger proportion, and T-glc was connected to the non-reducing end.(4) EPS-producing strains isolated form Tibetan kefir and characteristics of the EPSs. Fifteen organisems were isolated from Tibetan kefir and identified via molecular biology method. There were fifteen species of microorganisms: three strains of Leuconostoc pseudomesenteroides, two strains of Lactococcus lactis subsp. Lactis, four strains of Acetobacter okinawensis, one strain of Saccharomyces cerevisiae and five strains of Kazachstania unispora.Six of them were EPS-producing strains in skim milk medium, which included RJ-1, RJ-2 and RJ-5(Leuconostoc pseudomesenteroides), RJ-4(Acetobacter okinawensis), JM-5 and JM-7(Kazachstania unispora). Monosaccharide composition and surface microstructure analysis showed that the features of EPSs produced by single strain were different from those of EPS produced by Tibetan kefir. The EPSs produced by single strain could protect protein from oxidative damage induced by AAPH, similar to the EPS produced by Tibetan kefir.(5) Microbiological basis of the formation of Tibetan kefir grains. The auto-aggregation and co-aggregation abilities, biofilm formation and species interaction were taken to expore the formation mechanism of Tibetan kefir grains. The results showed that JM-7 and RJ-4 showed faster and better auto-aggregation and co-aggregation abilities and RJ-5 showed the best biofilm formation abilit, indicating the vital function of the three strains in the process of Tibetan kefir grains formation. Small irregular-shaped grains were obtained via mixed cultivation of the fifteen strains isolated from Tibetan kefir. The new grains were poor adhesion and breakable and could not continue to adhere and largen in the process of transfer. The microstructure and surface morphology of the new grains and the EPS produced by mixed cultivation were simiar to those of Tibetan kefir. The determination of volatile constituent demonstrated that the fermented milk produced by mixed cultivation hold the unique flavor similar to Tibetan kefir. |
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