Processing yeast to reduce its nucleic acid content. Induction of intracellular RNase action by a simple heat-shock procedure,and an efficient chemical method based on extraction of RNA by salt solutions at low pH

Autolytic breakdown of the RNA of Saccharomyces cerevisiae can be induced by subjecting a suspension of yeast in NaCl solution at pH 6 to heat shock, produced by bringing the suspension to 50°C and then adding enough water at 100°C to increase the temperature momentarily to 68°C, followed after 1 min by the addition of cold water to reduce it to 50°C. During continued incubation of the suspension (60 g yeast solids and 0.5 mol NaCl/litre) at 50°C, the rate of degradation of yeast RNA is as rapid as when autolysis is triggered by treatment of the yeast with organic solvents, or by drying and rehydration. The procedure is compared with an efficient chemical method for reduction of RNA content, by which SCP can be produced with a protein/nucleic acid ratio of over 100. This is based on heating a suspension of yeast in 0.5 M-NaCl at pH 1.4 to 70°C for a period of 2 h. This technique can be applied to yeast from which low-molecular weight constituents have first been removed by extraction with water at 70-100°C, with a considerable reduction in nuisance due to odours.     

Chemical methods for the reduction of the purine content of baker's yeast, a form of single-cell protein.

Chemical procedures, suggested by methods formerly used to prepare yeast RNA, or in the analysis of nucleic acids, were applied to the preparation of nucleic acid-reduced baker's yeast. Water at 80–100°C extracted nucleotides but no nucleic acid from the cell, with a loss of 20% total solids and 16% total nitrogen. Water at 120°C removed part of the nucleic acid, and nearly all was extracted by 5% NaCl solution at 120°C. At room temperature, 0.5 N -HC1 removed RNA from a suspension of heatkilled yeast. NaOH at a pH of about 12.5 extracted RNA from suspensions of fresh or of heat-killed yeast. In both cases the cells lost 25–35% solids and a similar proportion of crude protein (Kjeldahl N). The RNA-reduced yeast preparations may require further treatment, such as extraction with organic solvents, to improve appearance or palatability.     

Autolytic methods for the reduction of the purine content of baker's yeast,a form of single-cell protein

When a suspension of baker's yeast was incubated at 50°C, the cells became permeable to H⁺, a process completed in about 4 h. Cellular constituents of low molecular weight then diffused into the medium, but only after a long lag period were cell nucleic acids and proteins degraded by intracellular enzymes. The lag period was shorter at higher temperatures, when RNase and proteinases suffered some thermal denaturation. It was abolished if, before autolysis at 50°C, yeast was plasmolysed by ethyl acetate, or heated to 70°C for 1 min, treatments which, it is thought, disorganised the membrane of the yeast cell vacuole, within which the enzymes are located. Heat treatment, when effective, was sufficiently severe to disorganise the yeast cell plasma-lemma, with the consequence that a single adjustment of pH immediately following heat treatment served to maintain a constant pH, without addition of buffer, during the subsequent incubation at 50°C. Autolysis was not found to be an entirely suitable procedure for the reduction of the nucleic acid content of baker's yeast, because of the concomitant and severe loss of protein.     

EFFECT OF ZINC AND COBALT ON YEAST GROWTH AND FERMENTATION

Yeast grown at 30° C. in a synthetic medium containing protein yields a greater crop when zinc at 50 μg./ml. is added, but the yeast is more prone to autolysis. In a medium lacking protein, there is a similar stimulatory effect but the zinc has little effect on autolysis. If the yeast is grown at 35° C. in the synthetic medium containing protein, the addition of zinc at 50 μg./ml. has little effect. In similar experiments at 30° C. with cobalt in the range 5–50 μg./ml., protein utilization by the yeast is stimulated but growth is retarded.     

Optimization of Rice Bran Fermentation Conditions Enhanced by Baker’s Yeast for Extraction of Protein Concentrate

The rice bran fermentation conditions for extraction of protein concentrate was enhanced by the use of baker’s yeast at optimized conditions using response surface methodology (RSM). A central composite design with three independent variables: fermentation temperature (25 to 35°C), yeast concentration (1 to 5%) and fermentation time (10 to 24 h) was used to study the response variable (protein yield). Results indicated that the generated regression model represented the relationship between the independent variables and the responses. Also, all linear terms, two quadratic terms (fermentation temperature and time) and all interactive terms had significant (p < 0.05) effect on the protein yield. The optimum conditions for yeast pretreatment of rice bran protein extraction were achieved at 30°C for 17 h using 3% yeast concentration to obtain a protein yield of 23.37%, which showed no significant difference (p > 0.05) from the response surface methodology predicted protein yield (23.02%). The use of baker’s yeast in the fermentation of rice bran for extraction of protein concentrate can be more effectively used to improve the extraction yield compared to natural fermented (15.43%) and untreated rice bran (10.16%).     

酵母抽提物生产工艺的优化

对以新鲜面包酵母为原料生产酵母抽提物的工艺进行了研究和探讨，在充分考虑影响酵母抽提物的质量指标以及酵母自溶过程中糖类物质、蛋白质和RNA的降解过程的基础上，得出了优化的酵母抽提物生产工艺。     

COMPARISON OF METHODS FOR THE DETERMINATION OF CELL VIABILITY IN STORED BAKER'S YEAST

The classical plate method for discriminating between viable and nonviable yeast cells in stored baker's yeast was compared with dead cell staining techniques using methylene blue and three fluorochrome stains. The increase of dead yeast cells during storage of baker's yeast for up to 16 days at 5°C, 20°C and 35°C was determined. During prolonged storage, especially at 35°C, the death rate increased rapidly and the yeast cake began to soften because of autolysis. In these conditions the choice of method for determining the proportion of dead cells proved to be of great importance. Useful results for yeast stored for some time at 35°C could be obtained only by the fluorochrome technique using primuline, acridine orange or acriflavine as fluorochromes.     

Effect of procedures for the reduction of the nucleic acid content of SCP on the DNA content of Saccharomyces cerevisiae.

Autolysis of a suspension of baker's yeast, triggered by heat shock, organic solvents or drying/rehydration, had previously been found to degrade yeast RNA to diffusible products, leaving a resistant, cell-bound fraction characterised by a high adenine: guanine ratio, which it was suggested might be DNA. Analysis for DNA by the diphenylamine reaction now shows that even when autolysis (following heat shock) is prolonged to the point at which the cells lose 42% of dry matter and half their protein content, no DNA leaves the cells. Overnight treatment of freeze-dried yeast preparations with In perchloric acid at room temperature extracts the purines of DNA in the free form, and estimations of DNA content based on this agree with the results of the diphenylamine method in the case of autolysed or alkaline salt-extracted yeast preparations, which have a low RNA content. With untreated yeast, which contains 20 times as much RNA as it does DNA, the picture is complicated by a slow, but significant depurination of RNA by 1N perchloric acid. The implications of this, and other findings, are considered in respect of analytical procedures for yeast DNA and RNA content.     

Changes in dry matter concentration gradients in stored apples

Longitudinal and transverse dry matter concentration gradients were measured in Cox's Orange Pippin apples at harvest and during storage. Dry matter concentration was higher at the calyx end of the fruit at harvest than in the central zone and at the stalk end. This difference was maintained as dry matter concentration in the whole fruit declined during cold storage. Dry matter concentration decreased from the peel to the core in median transverse slices at harvest but this gradient diminished during storage at 4°C in either air or 1·25%v/v O₂. After 11 weeks' storage dry matter concentrations became generally lower in all zones of the air-stored fruit than those in apples stored in 1·25% v/v O₂, but transverse distribution patterns of dry matter were similar in apples in either regime throughout storage. Proportions of dry matter in the outer zones declined whilst those in the inner cortex and core increased. The relevance of these changes to the quality of stored apples is discussed.     

Application of Enzymes in Soy Milk Production to Improve Yield

Soy milk was produced from full fat flour by preparing a slurry in water at a water to flour ratio of 10 to 1. The slurry was heated to 50°C, and different enzymes were evaluated in order to optimize protein and solids yields in the milk. The reaction period was 1 hr and after inactivation of the enzymes by boiling for 15 min the mixtures were centrifuged at 1500 ×g and the protein and dry matter solubility indices (PSI and DSI) were calculated together with protein and dry matter yields in the obtained milk. The best results were obtained with a neutral proteinase, increasing the protein and solids yields from 33% and 42%, respectively, to 73% and 66%, respectively, at 0.5% dosage level compared to the control sample with no enzyme treatment.     

Mass Production Method for Rice Protein Isolate and Nutritional Evaluation

We developed a method for mass production of rice protein isolate (RPI) and evaluated its nutritional quality in rats. To obtain thick slurry, rice flour was mixed with a 0.6% Termamyl 120L-solution (1:2, w/v) at room temperature (～23°C). The slurry was heated at 97°C for 2 hr with stirring. Gelatinization and liquefaction occurred simultaneously. RPI obtained by filtration and washing with boiling water, was more than 90% pure protein (dry matter basis). It also contained 6.4% dietary fiber, 1.3% ash, and 1.1% carbohydrate. RPI diets (40–50%) allowed the maximum growth in rats comparable to that with 25% casein diet.     

Detoxification of commercial united kingdom rapeseed meal by glucosinolate hydrolysis with exogenous myrosinase and the extractability of the aglucones by aqueous industrial methylated spirits

The aims of this study were to investigate the detoxification of United Kingdom commercial rapeseed meal by alcoholic extraction of aglucones released after treatment with mustard seed myrosinase. Aglucones released from the commercial rapeseed meal were compared with those resulting from autolysis of laboratory-defatted rapeseed. 3-Butenyl isothioc yanate, 5-vinyl-oxazolidene-2-thione (VOT) and 1-cyano-2-hydroxy-3-butene were detected in commercial rapeseed meal after glucosinolate hydrolysis. In comparison, autolysis of rapeseed at 5°C gave VOT, 1-cyano-2-hydroxy-3-butene and epithionitriles, but at 60°C epithionitrile release was significantly reduced. In addition, 1-cyano-3,4-epithiobutane was detected in the autolysed samples. Aglucones released at 40°C from commercial rapeseed meal by mustard seed myrosinase were 85% extractable in 60–90% (v/v) aqueous industrial methylated spirits. Extraction was significantly higher than previously reported for the intact glucosinolate. Double extraction of the myrosinase-treated meal with 80 % (v/v) aqueous industrial methylated spirits gave a meal with no detectable intact glucosinolate or aglucone content.     

Tea extraction methods in relation to control of epimerization of tea catechins

Effect of water temperature and ethanol concentration on epimerization and extractability of tea catechins was investigated. The results showed that epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) were partially epimerized into gallocatechin gallate (GCG) and catechin gallate (CG), respectively, when tea catechins extract was heated in water solution at 100 °C for 2 h or dry tea was extracted in water at 100 °C. The epimerization of the catechins was inhibited if the tea catechins extract was heated as solid powder and the dry tea was extracted in 50% (v/v) ethanol or in water at 80 °C or below. When the dry tea was extracted in water, the extractability of catechins increased with the increase of extraction temperature up to 100 °C, but there was no statistically significant difference in total catechins between 80 °C and 100 °C. When teas were extracted using ethanol solutions, the highest extractability of total catechins was obtained in 50% (v/v) ethanol for dry tea and in 75% (v/v) ethanol for fresh tea leaf. In order to reveal the real profiles of tea catechins in teas to be tested, dry tea should be extracted in 50% (v/v) ethanol for 10 min, while fresh tea leaf should be extracted in 75% (v/v) ethanol for 10 min. For commercial extraction, temperature should be controlled at 80 °C if water is used as the solvent. Copyright © 2007 Society of Chemical Industry     

Effects of developmental stage,cold acclimation and diet on the cold tolerance of three species of Cryptolestes (Coleoptera: Laemophloeidae)

Flat grain beetles (Coleoptera: Laemophloeidae) are common stored-product insect pests in Canada, infesting cereals in grain bins, equipment and end products in flour mills. We studied the cold tolerance of the three most common flat grain beetles: Cryptolestes ferrugineus, Cryptolestes turcicus and Cryptolestes pusillus, by measuring the survival at −10 °C and supercooling point (SCP) for different life stages (egg, young larva, old larva, pupa and adult) reared on flour mixed with brewer’s yeast. Probit analysis was used to estimate the lethal time for 50 and 95% mortality. This was done with non-acclimated individuals (only held at 30 °C) or cold-acclimated individuals (held at 18, 10 and 5 °C, for 1 week/temperature). In general, adults were the most cold-hardy stage for each of the species. Acclimated insects were anywhere from no increase in cold tolerance to 14-fold more cold-tolerant than the corresponding non-acclimated stage and species. Cryptolestes ferrugineus was most cold-tolerant species (58 d at −10 °C to reach 95% mortality for acclimated adult), C. turcicus was the next most cold-tolerant, (39 d) and C. pusillus was the least cold-tolerant (11 d). The cold tolerance of adults reared on three diets was measured both for acclimated and non-acclimated insects. The adults reared on grain diet (whole wheat kernels, cracked wheat kernels and wheat germ (90:5:5 mass ratio) were the most cold-tolerant, adults reared on white-wheat flour and brewer’s yeast diet (95:5 mass ratio) had the next highest cold tolerance followed by the adults reared on 100% white-wheat flour. Supercooling point (SCP) of insects ranged from −20.6 to −26.7 °C. In general, acclimated insects had slightly lower SCP than non-acclimated insects.     

Effect of 1-Monoglycerides on Viscoelastic Properties of Processed Cheese

This article deals with the influence of selected 1-monoglycerides (1-monocaprylin, 1-monocaprin, 1-monolaurin, 1-monopalmitin, and 1-monostearin) in a concentration of 0.25% w/w on viscoelastic and sensory properties of the processed cheese with 45 and 50% fat in dry matter after 14 days of storage at temperature of 6 ± 2°C. With increasing number of carbons in the molecule of fatty acid, which is ester-bound in 1-monoglycerides, both the storage G′, and the loss G″ moduli increased in whole frequency range. This enhancement was relatively smaller in the group of processed cheeses with 45% fat in dry matter compared to the group of processed cheeses with 50% fat in dry matter. The additions of 1-monocaprylin, 1-monocaprin, and 1-monolaurin were not sensorial acceptable due to the off-flavour. The effect of addition of 1-monopalmitin and 1-monostearin (from 0.1 to 0.5% w/w) has also been presented.     

Water uptake by cocoa seeds during fermentation-like incubation

During anaerobic incubation in citric acid/NaOH (35 mmol; pH 5.5) at 40°C the fresh weight of ripe cocoa seeds (without testae) increases considerably (17–27% within 40 h) by water absorption. This is similar to water uptake during germination at 25–30°C. At 50°C in the same medium, weight increase (3.5–7.3% within 40 h) as well as net water uptake is low. Subcellular compartmentation is destroyed at 50 but not at 40°C. Since water uptake is greatly reduced or is impaired at 40°C in the presence of osmotically active substances like sucrose, it is concluded that intact vacuoles in the cotyledons are responsible for high water absorption. In the presence of acetic acid (200 mmol; 50°C) weight increase is low but loss of dry matter is increased by exudation. These findings are discussed with respect to protein vacuole swelling and proteolysis during fermentation and germination.     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

Identification by GeLC‐MS/MS of Trypsin Inhibitor in Sarcoplasmic Proteins of Three Tropical Fish and Characterization of Their Inhibitory Properties

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB‐SP), bigeye snapper (BS‐SP), and yellow croaker (YC‐SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB‐SP and YC‐SP. Alpha‐1‐antitrypsin with molecular mass of 45 to 50 kDa was identified in YC‐SP by gel‐based liquid chromatography‐tandem mass spectrometry (GeLC‐MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde‐3‐phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB‐SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB‐SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB‐SP could be a promising protein ingredient for enhancing surimi gel texture.     

Production of a high concentration of ethanol from potato tuber by high gravity fermentation

To produce a high concentration of ethanol from viscous potato tuber mash, potato tuber mash containing high contents of solids (28%) was prepared by grinding the potato tuber without the addition of water. The viscosity of the potato mash was reduced by using Viscozyme (0.1%) at 50°C for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90°C for 30 min and optimal conditions for the simultaneous saccharification and fermentation (SSF) of the potato mash for ethanol production were investigated using statistical methods. Using 2⁴ factorial design, saccharifying-enzyme and incubation temperature were found to be important factors. Using response surface methodology, the optimal saccharifying-enzyme dosage and incubation temperature were determined to be 1.45 AGU/g dry matter and 31.3°C, respectively. Under these optimal conditions for SSF, 14.92%(v/v) ethanol with 91.0% of theoretical yield was produced after 60 h, and all the starch was completely used up.     

Quantification of Factors Which Influence Nisin's Inhibition of CIostridium botulinum 56A in a Model Food System

We modeled nisin's anticlostridial activity and assessed the antagonistic or potentiating influences of food ingredients. The model systems contained yeast extract, proteose peptone, and glucose; were supplemented with protein (0.075, 0.75, 7.5% w/v), phospholipid (0.075, 0.75, 7.5% w/v), or soluble starch (5, 17.5, 30% w/v); and were adjusted to pH 5.5, 6.0, or 6.5. Samples inoculated with 10⁴/mL spores were incubated at 15, 25, or 35°C. Statistical analysis developed an equation (r²= 0.76) that modeled the response and identified temperature as the most significant (α 0.001) variable. Nisin lost effectiveness with increasing temperature. Nisin concentration had significant positive and phospholipid negative, linear effects. Many interactive effects were significant (α 0.20). Nisin inhibited C. botulinum until its residual level dropped below a threshold, which decreased from 154 IU/mL at 35°C to 12 IU/mL at 15°C.