The Ultrastructure of Spirulina platensis— A New Source of Microbial Protein

The ultrastructure of the blue-green alga Spirulina platensis has been investigated. The appearance of photosynthetic thylakoids, polyphosphate and cyanophycin granules and nucleoplasm was studied. Cells of Spirulina have also been disintegrated and extracted with different concentrations of sodium hydroxide and the amount of extractable and isoelectrically precipitable nitrogen has been determined. High yields were obtained with about 60 % of the nitrogen precipitable after extraction of disintegrated cells.     

Reduction of the nucleic acid content of single-cell protein concentrates

Methods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.     

The influence of alkali and heat treatment on yeast protein

The soluble components in disintegrated cells of Saccharomyces cereivisiae have been characterized by means of extraction, centrifugation, dialysis, and gel filtration. The influence of alkali and heat treatment on the protein and RNA in the soluble fraction from disintegrated yeast cells and on functional properties of protein concentrates have been studied. After water extraction and centrifugation at 100000 g 42% of the nitrogen containing components of the disintegrated cells were recovered in the supernatant. By extraction at pH 11.5 an additional 31% of the nitrogen was solubilized. Half of the water-soluble nitrogen-containing components has a molecular weight lower than 5000. In the water- and alkali-soluble fractions about 80% of each amino acid was recovered The water-soluble protein was separated into 3 fractions by gel filtration on Sephadex G 200. The major portion of the protein had a molecular weight about 100,000. The amount of protein in this fraction was decreased after treatment at increasing pH and temperature. No degradation of protein to low molecular peptides occurred. The amount of RNA in the soluble fraction was only slightly influenced by alkali treatment and by heat treatment at pH 7.5 in the presence of 5% NaCl. RNA was not degraded to low molecular components of the treatments. The solubility of protein concentrates decreased after treatment at alkaline pH and after heat precipitation.     

Conditions for the extraction of DNA and RNA from tobacco pollen by sodium chloride and perchloric acid

Studying the influence of the pH of 10% NaCl solutions used for the extraction of RNA and DNA on the yield of both nucleic acids, the maxima of pH were found at which both types of nucleic acids pass into extracts better than at neutral pH and do not remain in residues of the experimental material.The perchloric acid extraction temperature was also studied for obtaining the hydrolysate of nucleic acids from the trichloroacetic acid precipitate of sodium chloride extracts differing by 5 °C within the range of 35 °C to 90 °C and it was found that in this wide range almost the same amount of RNA is extracted by the method used. However, at a lower temperature, some DNA remained in the extracted residue of the trichloroacetic acid precipitate of sodium chloride extracts.     

稀碱法提取食用蝇蛆蛋白

对蝇蛆粉充分脱脂后采用稀碱法提取蝇蛆蛋白。结果表明,液料比、浸提温度、碱浓度对提取得率有显著影响(P<0.01),提取时间对提取得率影响差异不显著。蛋白提取的最佳条件为:液料比20mL/g,浸提温度80℃,浸提时间2h,碱浓度1%(w/v)。在此条件下,所得蝇蛆蛋白粉蛋白氮含量为11.14%,蛋白得率为57.53%,提取效率为69.84%。对最优条件下得到的蝇蛆蛋白粉进行理化指标检测,并与速溶豆粉的企业标准以及脱脂乳粉的国家标准进行比较。结果表明,水分、灰分、脂肪、蛋白含量均已达到可食用级别的要求,重金属铅、汞、砷含量均低于限量要求,微生物检测不含致病菌和大肠杆菌,在最优提取条件下所得蝇蛆蛋白粉,综合指标已达可食用标准。     

Some methods for processing of single-cell protein

Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot-plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration. Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.     

Autolysis of disintegrated cells of the yeast saccharomyces cerevisiae

Optimum conditions for autolysis of disintegrated cells of S. cerevisiae are at 50–53°C and pH 5.5; the process is terminated after 6 h. In the presence of sodium chloride (3–5%) the autolysis is complete after 5 h. The yield of autolysis of disintegrated yeast cells is about 70% of autolytic product per yeast dry weight. The product obtained after centrifugation, filtration and drying has very good sensoric and physical properties.     

Conditions of Phenol Extraction of RNA from Rat Liver Subcellular Cytoplasmic Fractions I. Effect of a Precipitation of Microsomal RNA on its Purity

Purification of RNA extracted with phenol from rat liver microsomes by precipitation in sodium benzoate and phenol solutions resulted in the removal of a readily measurable amount of proteins and polysaccharides. The stabilization of RNA was observed by changes in the profiles of the centrifugation gradients. This effect was the same over a wide range of yields in RNA obtained with extraction in the presence of buffers with varying pH levels.     

Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae.

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50 degrees C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5-6 and temperatures between 50-60 degrees C. The main part of the RNase in the unincubated suspension was soluble also at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.     

Studies on apple protopectin. IV: Apple xyloglucans and influence of pectin extraction treatments on their solubility

Hemicelluloses were extracted from apple cell walls with 1 and 4 sodium hydroxide and 8 urea after depectinisation by a chelating agent, by a chelating agent and dilute sodium hydroxide or by a chelating agent and a pectin-lyase. The extracts were fractionated on Sephacryl S 500 and DEAE Sepharose CL-6B. The bulk of the hemicelluloses were solubilised by 4 sodium hydroxide. The main hemicellulose was a fucogalactoxyloglucan. Some low-molecular-weight mannans were also present. Part of the xyloglucans could be extracted by urea after pectin extraction by a chelating agent or by pectin-lyase but not after pectin extraction by dilute sodium hydroxide. Dilute sodium hydroxide probably insolubilised some of the pectins and hemicelluloses.     

Kinetic aspects and mechanism of liposome disintegration in polyoxyethylene lauryl ether and sodium cholate solutions

The disintegration behaviour of liposomes in polyoxyethylene lauryl ether (PLE) and sodium cholate solutions was studied by the turbidity disappearance method. In maximally solubilized systems of liposomes, the molar ratios (phosphatidylcholine/surfactant) were 0.43 and 1.8 for PLE and sodium cholate, respectively. The disintegration process of either unilamellar or multilamellar liposomes followed first-order kinetics. Based on a physical model in which liposomes heterogeneous in size were assumed to disintegrate from the outermost shell one by one, a mathematical expression of the turbidity disappearance rate was introduced and applied to explain the data thus obtained. Model calculations suggested that the number of disintegrated shells would not be so large, even if up to 50% reduction of the initial turbidity was observed. From the dependence of the pseudo-first-order rate constant (kobs) on the surfactant concentration for unilamellar liposomes, it was assumed in general that kobs consists of the contributions of the monomer and micellar fractions: for PLE, both fractions shared in the disintegration, but only the micellar fraction with sodium cholate. Furthermore, in the latter case, kobs depended on the initial liposome concentration. These results are likely to be consistent with the proposed modes of surfactant action classified as type A and type B (Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79).     

Disintegration of Microorganisms and Preparation of Yeast Cell Walls in a New Type of Disintegrator

Microbial cells were disintegrated in a new type of rotary disintegrator with a disc stirrer by a combination of shear force layers, collisions, and rolling of glass beads which were brought into motion by the stirrer. The rate of disintegration at a given dry bed volume of Ballotini beads and a given volume of cell suspension is proportional to the peripheral velocity of the stirrer up to 18 m/sec. Horizontal arrangement of the stirrer increases the effectiveness about five times; 100% disintegration of yeast cells was achieved under optimal conditions within 72 sec at a concentration of 3.5g (dry weight)/100 ml of suspension, and within 96 sec at a concentration of 10.5g (dry weight)/100ml. At 17.5 g (dry weight)/100 ml, the stirrer began to slip. The cell walls of yeast were obtained at the desired degree of crushing and the course of purification was determined by infrared spectral analysis.     

Determination of Chlorophenols in Soils by a Method Involving Alkaline Extraction and Solid-phase Preconcentration Prior to High-performance Liquid Chromatography

A well-reproducible method for determining five common chlorophenols in soils is described. Chlorophenols were extracted from a soil with sodium hydroxide and, after being acidified to pH 6.5, the extract was cleaned by partitioning between chloroform and sodium hydroxide. After adjusting the pH value to 5.5, the sample was concentrated in a C-18 minicolumn. The chlorophenols were desorbed with methanol, the extract was basified, the solvent volume was reduced to 100 μl, and the concentrated extract was acidified. An aliquot was analyzed by HPLC with UV detection (225 nm). Recovery experiments were performed at concentration levels of 1 ppm, 0.1 ppm, and 0.01 ppm. Recovery levels between 65% and 83% with a standard deviation of ±3.75 were achieved at the concentration level of 0.1 ppm. Detection limits between 2 ppb (2,4,6-trichlorophenol) and 2.5 ppb (4-chloro-3-methylphenol) were determined.     

Effects and model of alkaline waste activated sludge treatment

Due to lack of information about alkaline sludge treatment with high dose at ambient temperature, alkaline sludge treatment was investigated to know the effects of different doses (0-0.5 mol/L) of sodium hydroxide (NaOH) and calcium hydroxide [Ca(OH)2] at 0-40 degrees C. Results showed that NaOH was more suitable than Ca(OH)2 for sludge disintegration. For NaOH treatment, most efficient dose was about 0.05 mol/L (0.16 g/g dry solid), and 60-71% solubilization of organic matters was achieved in first 30 min during its treatment of 24h. The process is described by a transmutative power function model, and relation between reaction rate constant and temperature are according with Arrhenius equation. Low dose NaOH treatment (<0.2 mol/L) deteriorated sludge dewatering ability obviously, while the ability was recovered gradually with the increase of NaOH dose. For Ca(OH)2 treatment, the disintegrated floc fragments and soluble organic polymers can be re-flocculated with the help of calcium cations. Consequently sludge disintegration effect was counteracted and dewatering ability improved.     

Extraction of Wheat Flour Proteins with Sodium Dodecyl Sulfate and Their Molecular Weight Distribution

By extraction of wheat flour with sodium dodecyl sulfate (SDS) solution at pH 6.8, about 76% of the total flour nitrogen solubilized into clear supernatant. This solvent was more effective for extraction of wheat protein than 0.01 m acetic acid, aluminium lactate-lactic acid buffer (pH 3.1), AUC-solvent (0.1 m acetic acid, 3 m urea and 0.01 m cetyltrimethyl-ammomum bromide) and 3,5-diiodosalicylic acid lithium salt etc. The molecular weight distribution of the SDS-soluble proteins was studied by SDS-polyacrylamide gel electrophoresis and by molecular sieve chromatography on controlled pore glass (CPG–10–500) without prior reduction of disulfide linkages of the proteins. Most of the SDS-soluble proteins had molecular weight of less than 75,000, suggesting single-chained proteins. A small amount of relatively high molecular weight proteins which contained intermolecular disulfide linkages was also detected in the gel of electrophoresis, while high molecular weight protein which did not migrate into gel matrix during electrophoresis without prior reduction of disulfide linkages existed in trace amount in the SDS-soluble fraction.

The SDS-insoluble proteins were almost completely extracted by further extraction with SDS in combination with 2-mercaptoethanol or with mercuric chloride.     

Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with Triton X-100

Bull sperm that had been extracted with 0.2% Triton X-100 could be reactivated with ATP, and their movement closely resembled the motion of intact live sperm. Their motility required the presence of ATP, magnesium, and a medium of suitable salt concentration and pH. When Triton-extracted bull sperm were digested breifly with trypsin at pH 9.0, they appeared to reatin most of their normal structure, but subsequent exposure of the digested sperm to ATP caused a disintegration by light microscopy, using dark-field illumination, combined with an electron microscope study of preparations of the disintegrated sperm, demonstrated the presence of an active sliding mechanism of filament interaction in bull spermatozoa. Human sperm subjected to the same procedures showed similar patterns of reactivation and of disintegration.     

The glucan components of the cell wall of baker''s yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure

1. Commercial pressed baker's yeast, and cell walls prepared from it, were extracted in various ways and the products examined by a number of techniques, including infrared spectroscopy and electron microscopy. 2. The glucan components of the walls cannot be extracted from intact yeast cells by 3% (w/v) sodium hydroxide at 75 degrees , but at least one-third of the glucan of cell wall preparations is dissolved under these conditions, and more will dissolve after ultrasonic treatment. 3. If intact cells are given a preliminary treatment with acid the wall glucans dissolve in dilute aqueous alkali. 4. Acid conditions as mild as sodium acetate buffer, pH5.0, for 3hr. at 75 degrees are sufficient for this preliminary treatment; the glucan then dissolves in 3% sodium hydroxide at 75 degrees leaving a very small residue, which contains chitin and about 1% of the initial glucan of the wall. Dissolution is hindered by exclusion of air, or by a preliminary reduction with sodium borohydride, suggesting that some degradation of the glucan by alkali is taking place. 5. After treatment with 0.5m-acetic acid for 24hr. at 90 degrees the glucan dissolves slowly at room temperature in 3% sodium hydroxide, or in dimethyl sulphoxide. The extraction with acetic acid removes glycogen and a predominantly beta-(1-->6)-linked glucan (not hitherto recognized as a component of baker's yeast), but none of the beta-(1-->3)-glucan, which remains water-insoluble. 6. Without treatment with acid, the glucan is not significantly soluble in dimethyl sulphoxide, but can be induced to dissolve by ultrasonic treatment. 7. These results are interpreted by postulating the presence of an enclosing membrane, composed of chitin and glucan, that when intact acts as a semipermeable membrane preventing the escape of the alkali- and dimethyl sulphoxide-soluble fraction of the glucan. Mild acid treatments damage this membrane, and ultrasonic and ballistic disintegration disrupt it. 8. Some support for this hypothesis is given by the effects of certain enzyme preparations, which have been found to render a substantial part of the glucan extractable by dimethyl sulphoxide.     

The generation of valosin-like peptides from a precursor protein in vitro as an extraction artifact

Valosin is a 25 amino acid peptide recently isolated from the porcine gastrointestinal tract. The molecular forms of valosin-like immunoreactivity (VLIR) were examined following different tissue extraction procedures. Fractionation of tissue extracted with cold 0.1 M sodium hydroxide by Sephadex G50 gel permeation chromatography revealed a large form of VLIR (Kav = 0). Smaller forms of VLIR, Kav = 0.36 and 0.57 were obtained in tissue extracted by boiling in 0.5 M acetic acid. Acidification and boiling of the 0.1 M sodium hydroxide tissue extracts also generated smaller forms of VLIR of Kav = 0.36 and 0.57. Partially purified preparations of the large forms of VLIR extracted with sodium hydroxide could be disrupted into a smaller form of Kav = 0.57 by acidification and boiling. This smaller molecular form co-eluted with the synthetic 25 amino acid valosin standard. We conclude that valosin does not occur naturally but is an artifact generated by cleavage of a larger protein precursor upon acid extraction of tissues. Workers should be aware of the need to verify their extraction procedures when characterising novel peptides to avoid potential pitfalls such as acid/thermal cleavage of proteins.     

Factors Enhancing the Host-Cell Penetration of Toxoplasma gondii

The penetration into HeLa cells of Toxoplasma gondii was studied with a cell culture technique. The influence on the rate of penetration and the number of penetrating Toxoplasma parasites was tested by use of preparations of disintegrated parasites mixed with test parasites. These preparations were found to contain factors enhancing the penetrating rate of the parasites. This effect was demonstrable by use of untreated parasites as well as parasites lacking active motility owing to a previous exposure to Formalin. The preparations of disintegrated parasites contained, in addition, components inhibitory to the penetration-enhancing factors. These inhibitory components were able to reduce the penetrating capacity of normal Toxoplasma parasites, suggesting that the studied enhancing factors may play a role in the natural process of penetration. The efficacy of various techniques for disintegration of Toxoplasma parasites was investigated for release of penetration-enhancing factors from Toxoplasma parasites. The methods used resemble those used for liberation of lysosomal enzymes. Reduced osmotic pressure was obviously not adequate for release of enhancing factors, whereas the freezing and thawing procedure, sonic treatment, and irradiation produced high yields. It was difficult to evaluate the effect of incubation at acid pH on release of enhancing activity, because the penetration-promoting factors seemed unstable on both the acid and the alkaline sides of pH 7.6.     

葡萄皮中粗蛋白提取条件的研究

利用葡萄饮料厂生产的废料葡萄皮提取粗蛋白,并对影响粗蛋白提取率的几个因素进行了研究。这些因素包括提取溶剂的选择、提取剂浓度、提取温度、提取时间等,并设计正交实验选取最佳提取条件,即:以NaOH溶液为提取剂,NaOH浓度为0.8 mol·L-1,提取温度70℃,提取时间为80min。提取得到的粗蛋白中蛋白质质量分数为71.38％。