| Malolactic fermentation is mainly conducted by Oenococcus oeni which can reduce acidity,increase microbiological stability and improve flavor.Spontaneous MLF was carried out by a variety of microorganisms during the wine production process,which may result in problems like high level of volatile acid and biological amine.However,O.oeni starter is a good solution to these problems.Freeze-drying is usually used in the production of starter,but bacteria could be damaged and lead to bacterial death or reduced vitality in the process of freeze-drying,while the addition of lyoprotectant can effectively reduce bacterial damage and maintain viability of bacteria.The laboratory research found that the soluble extracellular polymeric substances secreted by O.oeni 28A-1 during growth and metabolism have significant lyoprotective activity during freeze-drying process.Studies have found that exopolysaccharides in bacterial extracellular polymeric substances play a dominant role in cryoprotection.We speculated that the lyoprotective activity of extracellular polymeric substances may be attributed to the exopolysaccharide in them.Therefore,this study investigated whether the exopolysaccharide in the extracellular polymeric substances of O.oeni dominated the freeze-drying resistance and conducted a preliminary study on the exopolysaccharide.This study explored whether the exopolysaccharide in the extracellular polymer substances of O.oeni dominated the freeze-drying resistance,and conducted preliminary studies on the physical and chemical properties,structure and biological activity of the exopolysaccharide.Further understanding of the O.oeni exopolysaccharide could provide information for the study of the structure-activity relationship,provide a reference for it as a natural lyoprotectant for Lactic acid bacteria,and provide a theoretical basis for the artificial synthesis of this exopolysaccharide in the future.In this paper,the exopolysaccharide of O.oeni 28A-1 were first extracted,and the lyoprotective activity performance was verified to lay the foundation for subsequent experiments.Then,the physical and chemical properties of exopolysaccharide were determined and their structures were preliminary characterized.Finally,the antioxidant activity was studied.The main results are as follows:(1)O.oeni increased exopolysaccharide secretion after a stable growth period.Under ATB medium conditions,the optimal fermentation time for O.oeni 28A-1 to produce sugar is 12 days.There are significant differences in the yield and lyoprotective activity of O.oeni's exopolysaccharide grown under different media conditions.The exopolysaccharide produced by O.oeni 28A-1 grown under the condition of ATB medium was significantly higher than that produced under SMD medium in terms of yield and lyoprotective activity.(2)The exopolysaccharide produced by O.oeni 28A-1 grown under the condition of ATB medium was significantly higher than the polysaccharides contained in the medium itself in terms of lyoprotective activity.Therefore,the excellent lyoprotective activity of EPS(EPS below refers to the exopolysaccharide produced by O.oeni 28A-1 grown under ATB medium)is mainly derived from the exopolysaccharides secreted by O.oeni,which eliminates the interference of polysaccharides in the culture medium.Adding lyoprotectant could significantly improve the freeze-drying survival rate of O.oeni.The best protection effect is 2.5%EPS,and its bacterial survival rate reached 81.43%±9.87%,which was significantly higher than other common lyoprotectant(trehalose,sucrose,monosodium glutamate),and also higher than the bacterial survival rate of O.oeni's extracellular polymer substances,so the exopolysaccharide of O.oeni extracellular polymer substances plays a leading role in lyoprotective activity,and O.oeni EPS has great potential as a natural lyoprotectant.(3)In the investigation of the physical and chemical properties of EPS,The EPS polysaccharide content was higher than that of the polysaccharide contained in the medium itself,which indirectly indicated that O.oeni 28A-1 produced exopolysaccharide and played a key role in lyoprotective activity.The protein content in EPS was 0.49%,the residual protein is mainly proteoglycan covalently bound to the polysaccharide.Ultraviolet spectrum full wavelength scanning revealed that EPS had weak absorption peaks near 260 nm and 280nm,indicating that EPS contained trace amounts of nucleic acids and proteins.The presence of a small amount of proteins and nucleic acids in EPS plays a vital role in its biological activity.The Mw of EPS measured by gel permeation chromatography was 7.73×10~4Da.The exopolysaccharide with higher Mw can better reduce the damage of strains caused by extreme low temperature.The absorption heat of EPS measured by differential scanning calorimetry was 116.67?.The relatively high endothermic value indicates that it has a strong water holding capacity,which can slow down the damage to cells caused by the loss of water during the freeze drying process and play a role in lyoprotective.The degradation temperature was 259.47?,which is consistent with the thermal performance report of polysaccharides,indicating that the main component of EPS is polysaccharides,and the higher degradation temperature indicates that EPS has good thermal stability.(4)In the structural characterization of EPS,the monosaccharide composition of EPS was analyzed by high performance liquid chromatography.The EPS is a heteropolysaccharide composed of mannose,rhamnose,glucose,galactose,arabinose,and galacturonic acid.Fourier transform infrared spectrophotometer was used to analyze the main functional groups of O.oeni 28A-1 EPS.The FT-IR spectrum of EPS showed various characteristic absorption peaks of polysaccharides.SEM and AFM were used to observe the microstructure of EPS in solid and liquid forms.SEM images showed that EPS solid had an irregular structure with a rough surface and a certain layered structure.AFM images showed that EPS had an irregular particle structure in the solution,the particle distribution was relatively uniform,and some areas were aggregated.The microstructure of EPS is similar to pectin hemicellulose polysaccharide,which is easy to embed and attach to the target sample,so it can be used as a protective agent or carrier in related research fields.(5)The analysis of EPS antioxidant activity shows that EPS has certain activity in removing ABTS,DPPH,hydroxyl and superoxide anion free radicals.The analysis of reducing ability shows that EPS achieves the purpose of anti-oxidation through various mechanisms.Therefore,EPS has good antioxidant activity.Compared with other lyoprotectant,EPS can not only improve the survival rate of bacteria,but also enhance the antioxidant activity of wine,broaden the scope of application and make it more valuable. |
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