Yeast strain preparation

Aspartase exhibits incredibly strict substrate specificity and thus is of little use in the preparation of L-aspartic acid analogues. However, a number of L-phenylalanine analogues have been prepared with various PAL enzymes from the yeast strains Rhodotorula graminis, Rhodotorula rubra, Rhodoturula glutinis, and several other sources that have been cloned into E. call.243 241 Future work in this area will likely include protein engineering to design new enzymes that offer a broader substrate specificity such that additional L-phenylalanine analogues could be prepared. 

Candida tropicalis PBR-2, a yeast strain isolated from soil, is capable of carrying out the enantioselective reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-l-propanamine 58 to (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-l-propanamine 59 (Fig. 18.18), a key intermediate in the synthesis of the chiral drug (S)-Duloxetine (Soni and Banerjee, 2005). The organism produced the enantiopure (S)-alcohol with a good yield (>80%) and almost absolute enan-tioselectivity, with an ee >99%. Parameters of the bioreduction reaction were optimized and the optimal temperature and pH for the reduction were found to be 30 °C and 7.0, respectively. The optimized substrate and the resting cell concentration were lg/1 and 250 g/1, respectively. The preparative-scale reaction using resting cells of C. tropicalis yielded the (S)-alcohol at 84-88% conversion and ee >99%. 

The culture medium for toxin production consists of 3 % nutrient broth (Difco Laboratories, Detroit, Ml, USA), 1.5% yeast extract (Difco Laboratories), 1 % ammonium sulfate, 1 % glucose, 0.4 % calcium carbonate, 0.4% soluble starch and 0.4% cysteine hydrochloride. Adjust the pH of the medium to 7.5 with 4 N NaOH and autoclave at 1 kg/cm for 15 min. After the temperature of the autoclave has fallen below 100 °C, cool the medium as soon as possible to 40-45 °C in tap water to avoid a decrease in reduction state of the medium, and then inoculate the culture of the toxin producing strain prepared as in Section 9.3.2. 

FIG. 6 Growth of Oenococcus oeni in Chardonnay wines prepared using yeast strains UCLM S325 ( , 50 mg/L total S02), Zymafluor VL1 ( , 33 mg/L total S02), CKS 102 (A, 18 mg/L total S02), EC1118 (X[CN4], 18 mg/L total S02), BKS 104 ( , 15 mg/L total S02), or Saint Georges SI01 (o, 0 mg/L total S02). Concentrations of total S02 in these wines was determined just before bacterial inoculation. (Adapted from Larsen et al., 2003 and with permission from the American Journal of Enology and Viticulture.)... 

Yeast strain differences and/or differences in culture medium result in a range of efficiencies of spheroplasting with the enzyme preparations we use. Pretreatment has allowed us to employ the conditions described below with different strain backgrounds grown in different media. Because we have made changes to our previously reported method for preparing yeast nuclei (Aris and Blobel, 1991), which serves as the starting material for the isolation of nucleoli, we present the entire method for nuclei here. 

The yeast responsible for alcoholic fermentation in winemaking is usually introduced into the must from the surface of the grapes, the surface of winery equipment, or from specifically prepared cultures. The fermentation process can occur either naturally, without inoculation, or by inoculating the must with selected starters. The use of locally selected yeast strains (usually belonging to the species Saccharomyces cereoisiae), with strain-specific metabolic characteristics can positively affect the final quality of the wine (Regodon et al., 1997 Romano et al., 2003). Several studies have clearly shown the effects of indigenous and inoculated yeast populations on the wine volatile composition (Mateo et al., 2001 Nurgel et al, 2003). 

Techniques for growing/expanding LAB starters vary somewhat but can generally be divided into either pure culture or coculture (LAB and wine yeast) methods. In the latter case, most utilize the same yeast strain that is used to carry out alcoholic fermentation. In either case, a properly prepared and expanded LAB starter should yield a stationary-phase inoculum of >10 CFU/mL. 

YPAD medium This is a general purpose, nutrient-rich medium for the routine propagation of yeast strains when specific selection conditions are not required. YPAD is prepared by combining 10 g yeast extract, 20 g peptone, 20 g dextrose, and 40 mg adenine sulfate in 1 L of distilled water, and autoclaving for 15 rain at 15 Ib/in. Adenine is included in the culturing medium for certain strains to inhibit reversion of adel and ade2 mutations. 

Enzyme preparations isolated from special yeast strains are used for saccharose (sucrose) inver-... 

Blondin and Vezhinet (1988), Edersen et al. (1988) and Dubourdieu and Frezier (1990) applied this technique to identify etiological yeast strains. Sample preparation is relatively easy. The yeasts are cultivated in a liquid medium, collected during the log phase, and then placed in suspension in a warm agarose solution that is poured into a partitioned mold to form small plugs. 

Inoculation of the juice is strongly recommended. The chosen yeast strain should be highly ethanol tolerant and should produce little volatile acidity in difficult fermentation conditions. The dry yeasts should not be introduced directly into the juice to start the fermentation for this type of winemaking. A yeast starter should be prepared in diluted must, supplemented with NH4+ and yeast hulls, then seeded with dried active yeast at a dose of 2.5 g/hl of the total volume to be inoculated. The starter is added to the must on the second day of fermentation, at a rate of 2% of the total volume. This increases the maximum yeast population, which controls fermentation rate and volatile acidity production (Section 2.3.4). In one experiment, adding yeast in this way reduced the final volatile acidity content by 20%. 

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