Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes.

OBJECTIVE: To examine the results of a 5-year trial using cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle. DESIGN: Retrospective clinical evaluation of pregnancy rates (PRs) per cycle after freezing pronuclear stage human oocytes. SETTING: Tertiary care academic center. PATIENTS: Six hundred seventeen patients treated in 776 IVF-ET cycles from January 1987 to December 1991 (less oocyte donation cycles). MAIN OUTCOME MEASURE: Pregnancy rate per cycle after transfer of pre-embryos developed from thawed pronuclear stage oocytes. RESULTS: Three thousand seven hundred thirty-one oocytes were frozen. Of these, 2,039 were thawed. One thousand three hundred seventy-seven survived thawing (68%), and 1,370 were transferred after passing through syngamy to at least the first cleavage (68%). Of patients with thawing, 359 of 401 (90%) (449 of 505 cycles [89%]) received intrauterine transfer. One hundred thirty-three separate clinical pregnancies were established from 128 different cycles (128/449; 29%); 5 cycles had two thaws, each of which resulted in pregnancy. This PR is less than the overall fresh PR observed in patients who had excess pronucleate oocytes frozen (279/776; 36%) but is remarkably similar when adjusted for the number of pre-embryos transferred per cycle. The age of the patient at the time of cryopreservation and the number of quality of pre-embryos ultimately available for transfer were important factors in the establishment of pregnancy. The mode of ovarian stimulation and duration of cryostorage did not prove meaningful. CONCLUSIONS: Cryopreserved pronucleate oocytes that survive freezing, thawing, and progress through syngamy demonstrate a similar potential for implantation and pregnancy when compared with fresh conceptuses, the cumulative effect of which is an enhanced total PR per cycle.

Kinetics and mechanisms of reactions catalysed by immobilized lipases.

This review focuses on the kinetics and mechanisms of reactions catalysed by immobilized lipases. The effects of pH, temperature, and various substances on the catalytic properties of immobilized lipases and on the processes by which they are deactivated are reviewed and discussed.

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: IV. Effects of temperature.

A lipase from Aspergillus niger, immobilized by adsorption on microporous polypropylene hollow fibers, was used to effect the hydrolysis of the glycerides of melted butterfat at pH. 7.0 at 40, 50, 55, and 60 degrees C. Mcllvane buffer was pumped upward through the lumen, and melted butterfat was pumped upward through the shell side of a hollow fiber reactor. Nonlinear regression methods were employed to determine the kinetic parameters of models based on combinations of three nested rate expressions for the hydrolysis reaction with three nested rate expressions for thermal deactivation of the enzyme. A rate expression containing four lumped parameters is sufficient to model the release of free fatty acids as a function of reactor space time and time elapsed after immobilization. Nonlinear regression methods were also employed in global fits of the data to rate expressions containing an explicit dependence on temperature. For the reaction conditions used in this research, a 14-parameter rate expression is necessary to accurately model the overall release of free fatty acids as a continuous function of the absolute temperature, initial substrate concentrations, reactor space time, and time elapsed after immobilization of the lipase.

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part II. Uniresponse kinetic studies.

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40 degrees C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped cocurrently through the shell side of a shell-and-tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four-parameter rate expression (which includes a two-parameter deactivation rate expression and a two-parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn-1,3 positions of the triglycerides can be released in the hollow-fiber reactor.

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part III. Multiresponse kinetic studies.

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the continuous hydrolysis of the glycerides of butter oil at 40 degrees C and pH 7.0. The effluent concentrations of 10 different free fatty acid products were measured by high-performance liquid chromatography (HPLC). Multiresponse nonlinear regression methods were used to fit the data to a multisubstrate rate expression derived from a Ping Pong Bi Bi mechanism in which the rate-controlling step is deacylation of the lipase. Thermal deactivation of the enzyme was also included in the mathematical model of reactor performance. A postulated normal distribution of v(max) with respect to the chain length of the fatty acid (with an additive correction for the degree of unsaturation) was tested for statistical significance. The model is useful for predicting the free fatty acid profile of the lipolyzed butteroil product over a wide range of flow rates.

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part I. lipase adsorption studies.

Adsorption of proteins from a crude preparation containing a lipase from Aspergillus niger on microporous polypropylene hollow fibers was studied at six different temperatures. Langmuir isotherms accurately describe the overall adsorption equilibria. Lipase is selectively adsorbed relative to the other proteins in the crude preparation. Hence, immobilization also provides further purification of the lipase. The predictions of the Langmuir model for the change in the specific activity of lipase upon adsorption are consistent with experimental results. The loading capacity of the hollow fibers decreases and the adsorption constant increases as temperature is increased. This effect is more significant in the case of lipolytic activity than it is for the total amount of adsorbed protein. Small, positive enthalpy changes are associated with the adsorption of lipase on these hydrophobic membranes.

Hydrolysis of lactose in skim milk by immobilized beta-galactosidase (bacillus circulans).

A novel chemical reactor, consisting of beta-galactosidase from Bacillus circulans immobilized onto a ribbed membrane made from polyvinylchloride and silica, was used to hydrolyze the lactose constituent of skim milk. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of rate expressions based on a proposed enzymatic mechanism that includes the formation of oligosaccharides. High-performance liquid chromatography (HPLC) methods were employed to monitor the concentrations of all species present in the effluent stream. For the experimental conditions used in this research, rate expressions which include the formation of trisaccharides, the inhibition effects of both the alpha and beta anomers of galactose, and the corresponding mutarotation reaction are sufficient to model the reaction network.

Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil.

A lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat-sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35 degrees C. For the reaction conditions used in this research, a pseudo-zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis-Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell-shaped (or Gamma-type) distribution. Data taken at five different pH values were fit assuming a Dixon-Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first-order kinetics with a half-life of 19.9 days at 35 degrees C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped-substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.

Use of novel immobilized beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk.

beta-galactosidase from Aspergillus oryzae immobilized in an axial-annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30 degrees C, 40 degrees C, and 50 degrees C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three-parameter rate expression which includes the separate competitive inhibition effects of alpha- and beta-galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the beta anomer of galactose (E(A) = 10.3 kcal/mol). At 40 degrees C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial-annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.

Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor.

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.

Lactose hydrolysis by immobilized beta-galactosidase in capillary bed reactor.

The hydrolysis of lactose by immobilized beta-galactosidase was studied in a continuous-flow capillary bed reactor operating at 30 degrees C. Solutions containing 50, 100, and 150 g lactose and 0.5 g sodium acetate/L were fed to the reactor. Lactose conversions ranging from 24% to greater than 99% were achieved at reactor space times ranging from 0.06 to 6.3 min. These conversion data were successfully modeled in terms of a plug flow reactor model and a form of Michaelis-Menten kinetics which included competitive inhibition by both the alpha and beta forms of galactose.

Hydrolysis of lactose in skim milk by immobilized beta-galactosidase in a spiral flow reactor.

beta-galactosidase from Aspergillus Oryzae immobilized in a spiral flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Residence time distribution measurements were used to assess the amount of longitudinal dispersion occurring as a consequence of the spiral flow pattern and the semiporous nature of the polymeric material used to construct the spiral. It was possible to model the flow conditions as tubular flow with a Peclet number that was a linear function of the reactor space time. Nonlinear regression methods were used to determine the kinetic parameters of three proposed enzymatic rate expressions. The best fit of the data was obtained using a rate expression containing separate terms for competitive inhibition of the reaction by both the a and beta anomers of galactose. This kinetic model also incorporates the kinetics of the mutarotation between these forms. At 30 degrees C and a space time of 7 minutes, 80% of the lactose present in skim milk can be converted to glucose and galactose.

Effects of dietary tryptophan levels on growth, feed/gain, carcass composition and liver glutamate dehydrogenase activity in rainbow trout (Salmo gairdneri).

1. The tryptophan requirement of rainbow trout (initial body wt, 13 g) was estimated by feeding diets containing varied levels of tryptophan from 0.06 to 0.5% of diet for 6 weeks. 2. The estimated tryptophan requirement was 0.20-0.25 (0.57-0.71)% of diet (dietary proteins). 3. Nitrogen retention increased and feed/gain decreased with dietary tryptophan levels up to 0.14%, but no further effect was observed at levels above 0.14%. 4. Carcass protein content gradually increased and lipid and ash contents decreased with increasing dietary tryptophan levels. 5. Dietary tryptophan levels did not affect hepatosomatic index or liver glutamate dehydrogenase activity.

Protein conversion efficiencies of four test diets based on milk, two milk and tea treatments, and casein.

Milk consumption in 80 households in western Kenya averaged .25 to 1.00 L per family per day. Most of this milk was processed into a milk and tea beverage. Polyphenols, the major determinants of tea flavor and character, form soluble complexes with casein when black tea infusions are mixed with milk. Test diets were prepared from the following treatments: a) casein reference diet; b) milk, no tea leaves; c) milk added after heat treatment; and d) traditional Kenyan-style tea (i.e., milk and tea leaves heated together). Four groups of 10 male 21-day-old Sprague Dawley weanling rats were assembled and assigned to the test diets. Weight gain and protein intake (g/g) during a 28-d test period for the four diets were 1.94, 2.99, 2.93, and 2.77. The least significant difference test showed weight gain for protein intake was greater for rats on the milk-based diet and the diet with milk added after heating than for rats on the traditionally prepared milk and tea drink. Rats did most poorly when assigned to the casein reference diet. Modification of traditional Kenyan tea preparation practices (i.e., adding milk after tea leaves have been removed) might improve the nutritional value of the most popular milk product in western Kenya.

Comparison of rat hepatic cholesterol biosynthesis during skim milk versus whey permeate ingestion.

Whey permeate is an ultrafiltrate of whey that is devoid of protein but contains lactose, salts, and other soluble low molecular weight compounds. These experiments compared cholesterol concentrations of blood plasma, hepatic lipids, and hepatic cholesterol biosynthesis of rats ingesting skim milk powder versus whey permeate powder. Groups of young male rats weighing 90 to 92 g were fed a casein-based diet into which skim milk powder or whey permeate powder was incorporated isocalorically. No effects of skim milk or whey permeate on plasma cholesterol concentrations were observed at any time during 5-wk of feeding. However, 3-hydroxy-3-methylglutaryl co-enzyme A reductase activity was increased by either skim milk or whey permeate feeding. Hepatic cholesterol, triglyceride, and phospholipid concentrations at wk 5 were unchanged. Plasma and hepatic cholesterol responses of rats to whey permeate ingestion are similar to those that occur with skim milk consumption, and plasma and hepatic cholesterol concentrations do not reflect necessarily an increase in hepatic cholesterol biosynthesis.

Variability in Cholesterolemic Response of Rats Consuming Skim Milk.

The hypocholesterolemic property of skim milk powder (SMP) was investigated using rats in a series of six separate experiments. SMP was incorporated at a level of 25% by weight into stock diets or semipurified diets with casein or soy as the protein source. Ingestion of SMP with casein-based diets for periods of 5 to 12 weeks produced a transient hypocholesterolemia only when diets were introduced within 1 week of weaning. In two different experiments, when weanling rats were fed stock diets with or without SMP for periods of 8 and 16 weeks, SMP ingestion led to a transient decrease in plasma cholesterol (CH) levels in only one experiment. Finally, SMP was not hypocholesterolemic when fed to weanling rats with a soy-based diet for 8 weeks. Together, these results reveal that consumption of SMP does not consistently elicit hypocholesterolemia in rats. We found that to observe significant differences in plasma CH levels between the control and SMP groups, semipurified diets containing SMP had to be fed to rats within the first week of weaning; in addition, the diets had to be free of, or reduced in other components with cholesterol-lowering properties. Finally, the SMP-induced hypocholesterolemia that we observed appeared to facilitate the normal rate of decline in plasma CH levels during the post-weaning period, producing only a transient difference between control and SMP groups.

Plasma and hepatic cholesterol and hepatic HMG CoA reductase levels in rats fed fluid or powdered skim milk.

The effects of skim milk powder (SMP) and fluid skim milk (FSM) on plasma cholesterol (CH) and hepatic liquid concentrations, and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase activity of rats of different ages were compared. Groups of young (23 days old) and older (45 days old) rats were fed a casein -based diet and provided tap water; the casein-based diet and FSM as fluid source; or tap water and the casein diet into which SMP (25% by wt.) had been isocalorically incorporated. Plasma CH concentrations were determined at 0, 1.5, 3 and 5 wk, hepatic total lipid, triglyceride and CH at 5 wk. Half of each group were killed at wk 3 and the other half at wk 5 for determinations of HMG CoA reductase activity. Both FSM and SMP decreased plasma CH levels at 1.5 and 3 wk of feeding in the young rats; plasma CH concentrations of the older rats were not altered by either FSM or SMP. Both milk derivatives increased HMG CoA reductase activity at wk 3 and wk 5 in both ages of rat, whereas hepatic lipid levels were unchanged. In these experiments the effects of feeding FSM of SMP along with a casein-based diet were comparable and included an increase in HMB CoA reductase activity, no change in hepatic lipid levels, and a decrease in plasma CH; the latter response depended on the initial age of the rat.

Absence of fatty livers in rhesus monkeys fed orotic acid.

Pairs of rhesus monkeys were fed for 10 wk a basal diet containing 1% orotic acid or 10% nonfat milk powders. Amounts of total lipids in the liver and hepatic morphology were normal after 10 wk indicating that orotic acid in the diet did not induce fatty livers in rhesus monkeys.

Heat resistance of salmonellae in concentrated milk.

The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level.