Effect of alcohols on lactose solubility.

In tests of effects of high (70 to 90% volume/volume) and low (2 and 5% volume/volume) alcohol concentrations, solubility of lactose decreased with increased alcohol concentration and decreased as alcohol chain length increased. Since lactose has lower solubility in alcohol, crystallization would be expected to be speeded by increased supersaturation. Composition of precipitates formed by the action of ethanol changed with time; alpha-lactose precipitated more rapidly at first, then beta-lactose. Time of crystallization was related directly to the percentage of beta-lactose (therefore, the relation was inverse for total alpha). However, the percentage of alpha-hydrate increased with time and with water content. Agitation during crystallization increased production of beta-lactose. Composition of the lactose precipitate varied greatly with concentration of alcohol (ethanol). When ethanol concentration was low, only alpha-hydrate was precipitated whereas at higher concentrations stable anhydrous alpha lactose also was precipitated, with the percentage of total alpha decreasing while the percentage of beta-lactose increased. Crystal shape changed from prisms initially to partially or fully developed tomahawks as time went by or as the percentage of ethanol decreased, and crystal color increased with crystallization time and as ethanol percentage decreased. Choosing long-chain alcohols and controlling suitable parameters enabled recovery of greater amounts and more desirable forms of lactose.

Selection of strain, growth conditions, and extraction procedures for optimum production of lactase from Kluyveromyces fragilis.

Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.