| Ochratoxin A(OTA) is a common mycotoxin in wine, and Aspergillus carbonarius is considered to be the main cause of OTA contamination in grapes and its products. Inhibiting the growth and toxin production of OTA-producing A.carbonarius is an important measure to control OTA contamination in wine. This paper used A.carbonarius that was isolated from grape surface and had the highest OTA yield as material. Through artificial and natural fermentation methods, the growth and toxin-producing regularity of A.carbonarius and other molds were tracked during the process of winemaking. Main factors affected growth and toxin production of OTA-producing fungi were studied and the influences and mechanisms of A.carbonarius on wine quality were analyzed. The antifungal substances produced by Bacillus subtilis were purified and identified and then applied in winemaking process for inhibiting the contamination of molds and OTA. The main results obtained are listed as follows:(1) Growth and toxin production regularity of molds and OTA-producing fungi during winemaking process. During the natural winemaking process, molds contaminated in must including Aspergillus section of Nigri(9 strains), Penicillium sp.(11 strains), Cladosporium sp.(2 strains), Alternaria sp.(one strain), Aspergillus sp.(one strain) and unknown species(5strains). When A.carbonarius was artificial added to the winemaking process, Aspergillus sp.and other molds contamination were increased rapidly after destemming and crushing treatment(at 3h), and reached the maximum at 8-24 h, but began to decrease at 48 h when yeast grow rapidly, and then dropped to undetected level at 72 h. OTA content was increasing during the early stage of winemaking and reached a maximum value at 8 h or 48 h and then decreased sharply. With the increase of the added amount of A. carbonarius, OTA content in wine increased. Most OTA was removed from the liquid portion and remained in the pomaces after the pressing operation and the OTA content in the fermented liquid dropped dramatically.(2) Main factors affected growth and toxin production of OTA-producing fungi were analyzed. The trends of fungal contamination and OTA content in five types of wine grapes were consistent with addition of OTA-producing fungi during the natural winemaking process.However, the overall contamination in different grape varieties were significantly different,and Shiraz grape had the highest content of fungal and OTA contamination. Addition of SO2 could inhibit the growth and OTA production of OTA-producing fungi during winemaking.When the SO2 and ethanol concentrations at 500 mg/L and 2-4%, respectively, growth and OTA production of A. carbonarius were completely inhibited. SO2 and alcohol content were the main factors inhibiting the growth and OTA production of OTA-producing fungi during the fermentation process.(3) Influences and mechanisms of OTA-producing fungi on wine quality were investigated. The artificial adding OTA producing fungi had no significantly effects on alcohol, soluble solids and reducing sugar content during fermentation. However, the contents of ethanol, 2, 3-butanediol and the total content of ester were decreased. Meanwhile, the contents of higher alcohols, total acid, alkane compounds, phenols, aldehydes, ketones and other types of volatile compounds in wine were increased, especially for the glycerol content.The reason were that the A. carbonarius spores could release some substances from its internal or surface when it was mixed cultivated with the S.cerevisiae and these substances could promote the synthesis of glycerol in S.cerevisiae. These substances could increase the genes expression related with glycerol metabolism and gene of STL1 and GPP1 had the biggest increase, suggesting that this promoting effect might be associated with oxidative balance pathways of glycerol metabolism. In converse, S.cerevisiae had certain inhibitory effect on toxin production of OTA-producing fungi.(4) Identification of lipopeptides and their antifungal properties to inhibit OTA-producing fungi under the condition of winemaking were investigated. Antifungal substances that stable to the winemaking conditions could be extracted by salting out method combined with pH adjustment(3.0) and high temperature(121 ℃, 30min) treatments to remove impurity. The main components of obtained antifungal substances were identified as lipopeptides based on their properties and molecule weight. It found that the lipopeptides had higher antifungal activity to OTA-producing fungi than to yeast. Lipopeptides could inhibit more than 95% of growth and OTA production of OTA-producing fungi and could inhibit 29 different types of fungi isolated from natural winemaking process. The minimum inhibitory concentrations of antifungal lipopeptides for OTA-producing fungi in the PDA, SNM solid medium and SNM liquid culture were 200 μg/m L, 50 μg/mL and 400 μg/m L, respectively,which were much lower than the minimum inhibitory concentrations of SO2, that were 4 g/L in SNM solid medium and 2 g/L in SNM liquid culture, respectively. By scanning electron microscopy analysis, it was found that antifungal lipopeptides could make the mycelia surface roughness and vesicle expanded and become smooth, but could not form metulae and phialide.Further studies found that antifungal lipopeptides in concentrations more than 100μg/mL could make the spores wall ruptured and restrain the spore germination, whereas in concentrations less than 50 μg/m L, mainly inhibit the generation of metulae and phialide on the top of vesicle and then restrain the formation of sporogenous structure and spores.(5) Antifungal effect of the lipopeptides during the winemaking process and its impact on product quality were investigated. Lipopeptides were applied in winemaking process, and the results showed that they could significantly reduce the contamination of molds and OTA-producing fungi and the inhibitory effect was better than the addition of sulfur dioxide alone. Lipopeptides addition amount at 200 μg/mL did not affect the changes of soluble solids,sugar, alcohol, total acid, glucose and fructose, but reduced higher alcohols, ketones and total phenols, and improved the types and content of esters, fatty acids and hydrocarbons in wine.In contrast, the aroma feelings were best in an addition concentration of 200 μg/mL, which had dense apple, pear and other fruit flavor, and also some fancy floral and spice flavor. When the addition concentration was 400 μg/mL, or 200 μg/mL of antifungal lipopeptides added with SO2 together, the wines had better taste, balance, coordination, strong flavor and ductility.The innovations of this dissertation are as following: growth and toxin-producing regularity of OTA-producing fungi during winemaking process are revealed; and the influence of OTA-producing fungi on winemaking process and wine quality and its mechanism are clarified. Lipopeptides which have effectively inhibitory effects on contamination of molds and OTA-producing fungi during winemaking process are obtained, providing new research ideas and methods for controlling the mold and OTA contamination and improving the sulfur dioxide alternative brewing technology during winemaking. |
PDF Full Text Request