Extraction of immunoglobulin-G from colostral whey by reverse micelles

Separation of immunoglobulin G (IgG) from the other colostral whey proteins was carried out by reversed micellar extraction. The colostral whey was diluted to 5 times its original volume with 50 mM phosphate buffer at pH 6.35 containing 100 mM of sodium chloride. The aqueous solution was then mixed with an equal volume of isooctane containing 50 mM bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), and shaken at 200 rpm and 25 degrees C for 10 min. After extraction, the mixture was separated to the aqueous phase and the reversed micellar phase by centrifugation. This procedure extracted most of the non-IgG proteins to the reversed micellar phase and recovered more than 90% of the IgG in the aqueous phase. The IgG in the aqueous phase had a purity of 90%, and still possessed immunological activity. AOT was not detectable in the aqueous phase.     

One-step separation of lysozyme by reverse micelles formed by the cationic surfactant,cetyldimethylammonium bromide

Lysozyme was selectively extracted from reconstituted freeze-dried egg-white, using reverse micelles formed by the cationic surfactant, cetyldimethylammonium bromide (CDAB). The major egg-white proteins, including ovalbumin and ovotansferrin, were solubilized into the organic phase while lysozyme was recovered in the aqueous phase. The solubilization behaviours of proteins were manipulated by processing parameters, including pH and salt concentration in the aqueous phase and concentration of surfactant in the organic phase. The optimum extraction was achieved with sodium borate buffer (50 mM, pH 9, no added KCl) and organic phase containing 50 mM CDAB. After the forward extraction, 96% of total lysozyme activity was recovered. Lysozyme was efficiently purified, more than 30-fold with only a single forward extraction. The suggested extraction procedure has advantages in terms of time and cost compared to traditional reverse micellar extraction which requires both forward and backward extraction steps.     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

Optimizing Assay and Extraction of Lipoxygenase in Wheat Germ

Using three-level seven-factor response surface methodology, wheat germ lipoxygenase (LPO) assay conditions were standardized. The important parameters were concentration of the substrate (linoleic acid), and surfactant (Tween 20), pH and temperature. The standardized LPO assay conditions for the 1 mL reaction volume were : 450 μM linoleic acid, 129 μM Tween 20, 175 mM ethanol, 1 mM EDTA, pH 6.2 (phosphate buffer), ionic strength 100 mM and 40°C. LPO extraction conditions were standardized by sequential variation of parameters. Optimum conditions were a single extraction of defatted wheat germ flour at 2–5°C with magnetic stirring of an extractant acetate buffer pH 4.5, ionic strength 100 mM, at buffer-to-solid ratio 10:l.     

THE ROLE OF AMINO GROUPS IN THE BIOLOGICAL AND ANTIGENIC ACTIVITIES OF CLOSTRIDIUM PERFRINGENS TYPE A ENTEROTOXIN

Clostridium perfingens type A enterotoxin has 8 amino groups which are readily reactive with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) in 20 mM phosphate buffer, pH 6.8, and 10 amino groups which react with TNBS only after denaturation with 6.5 M guanidine · HCl ≤ 25°C. The one sulfhydryl group of enterotoxin is not reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the native molecule (phosphate buffer, pH 6.8), but is reactive in the presence of 0.2% sodium dodecyl sulfate at 25°C.
i     

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

Forty-one strains of Kluyveromyces fragilis (J?rgensen) 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.     

POST-MORTEM GL YCOL YSIS IN OX SKELETAL MUSCLE: EFFECT OF ADDING NICOTINAMIDE-ADENINE DINUCLEOTIDE TO DILUTED MINCE PREPARATIONS

ABSTRACT— Samples of mince, prepared from ox sternomandibularis muscle about 75 min postmortem, were diluted with one volume of 0.16M potassium chloride containing 0, 50 or 100 mM potassium phosphate with or without 1 mg of nicotinamide-adenine dinucleotide (NAD)/ml. Changes in pH and in the concentrations of glycolytic intermediates and the principal nucleotides were followed for about 6 hr after dilution. NAD was lost rapidly, its adenine moiety being converted to inosine nucleotide by the enzymes present. When the diluent did not contain inorganic phosphate (P_i) inclusion of NAD resulted in a somewhat faster loss of adenosine triphosphate (ATP), earlier cessation of glycolysis and higher ultimate pH. Inclusion of NAD in the preparations containing added P_i led to a reduction in both phosphorylase and phosphofructokinase activity. It also prevented the accumulation of fructose diphosphate. This was the result of lowering the phosphofructokinase activity rather than of increasing the glyceraldehyde-3-phosphate dehydrogenase activity. Adenosine triphosphatase (ATPase) activity, after an initial period during which it was unaffected by inclusion of NAD, was greater in the preparations containing added P_i and NAD than in the corresponding preparations without added NAD.     

IMMOBILIZATION OF PAPAIN ON AN ANION EXCHANGE RESIN BY PHYSICAL ADSORPTION FOLLOWED BY CROSS LINKING WITH GLUTARALDEHYDE

A practical method involving physical adsorption by vacuum drying of papain on Dowex MWA-1 (mesh 20–50), followed by intermolecular cross linking with 1.0% glutaraldehyde under neutral pH conditions was developed. Potassium phosphate buffer (0.1 M, pH 12.5) was better for immobilization of papain than was acetate buffer (0.1 M, pH 4.0). Nitrogen recovery was increased by increasing the reaction time with glutaraldehyde from 0.5 to 15 min and enzyme concentration from 1 to 80 mg/0.3 mL buffer/0.2 g resin. Recovery of absolute and proteolytic activities, using casein as a substrate, was increased when reaction time was increased from 0.5 to 5 min. These activities were increased in a papain concentration range of 5 mg/0.2 mL buffer/0.2 g resin to 5 mg/0.3 mL buffer/0.2 g resin and then decreased with greater volumes of buffer, ranging from 11.2 to 8.6%. Relative activity recovery was decreased with an increase in reaction time and enzyme concentration, ranging from 40.5 to 7.5% and 47.5 to 1.5%, respectively. Cysteine (0.08 M) and EDTA (2 mM) enabled more than 95% of the original activity of immobilized papain to be maintained for 2 months at 5°C; NaHSO₃ and ascorbic acid inactivated immobilized papain.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

Microbial Safety in Radio-frequency Processing of Packaged Foods

ABSTRACT: Thermal resistance of Clostridium sporogenes (PA 3679) was determined at 115.6 °C, 118.3 °C, and 121.1 °C (240 °F, 245 °F, and 250 °F, respectively) in phosphate buffer (pH 7.0) and mashed potatoes (pH 6.3) using aluminum thermal-death-time (TDT) tubes developed at Washington State Univ. D-values were 1.8, 1.1, and 0.62 min in phosphate buffer and 2.2, 1.1, and 0.61 min in mashed potatoes at 115.6 °C, 118.3 °C, and 121.1 °C, respectively. Z-values were 12 °C and 10 °C in phosphate buffer and mashed potatoes, respectively. The thermal inactivation kinetic results were then used to validate a novel thermal process based on 27.12 MHz radio frequency (RF) energy. Trays of mashed potatoes inoculated with PA 3679 were subjected to 3 processing levels: target process (F₀∼4.3), under-target process (F₀∼2.4), and over-target process (F₀∼7.3). The microbial challenge test data showed that microbial destruction from the RF process agreed with the calculated sterilization values. This study suggests that thermal processes based on RF energy can produce safe and shelf-stable packaged foods.     

Reduction of Escherichia coli O157:H7 and Salmonella spp. on laboratory-inoculated mung bean seed by chlorine treatmentt

Three U.S. outbreaks of foodborne illness due to consumption of contaminated raw mung bean sprouts occurred in the past 2 years and were caused by Salmonella Enteritidis. The original source of the pathogens is thought to have been the seed. The aim of this study was to determine whether treatment with aqueous chlorine would eliminate the pathogens from mung bean seed inoculated in the laboratory with four-strain cocktails of Escherichia coli O157:H7 and Salmonella spp. Treatments (for 5, 10, or 15 min) with buffered (500 mM potassium phosphate, pH 6.8) or unbuffered solutions containing 0.3 or 3.0% (wt/vol) Ca(OCl)2 were tested. In order to mimic common commercial practice, seed was rinsed before and after treatment with sterile tap water. Treatment for 15 min with buffer (500 mM potassium phosphate, pH 6.8) or sterile water in combination with the seed rinses resulted in maximum reductions of approximately 3 log10 CFU/g. The largest reductions (4 to 5 log10 CFU/g) for the chlorine treatments in combination with the rinses were obtained after treatment with buffered 3.0% (wt/vol) Ca(OCl)2 for 15 min. Treatment of mung bean seed for 15 min with unbuffered or buffered 3.0% (wt/vol) Ca(OCl)2 did not adversely affect germination. Even though treatments with 3% (wt/vol) Ca(OCl)2 in combination with the water rinses were effective in greatly reducing the populations of both bacterial pathogens, these treatments did not result in the elimination of the pathogens from laboratory-inoculated seed.     

High pressure assisted extraction of proteins from wet biomass of Arthrospira platensis (spirulina) – A kinetic approach

The application of high-pressure technology (100–600 MPa, 20 °C for up to 20 min) for cell disruption and consequent extraction of proteins -including C-phycocyanin- from the cyanobacteria Arthrospira platensis (spirulina), was investigated. Wet spirulina biomass was suspended in three different aqueous systems (deionized water, phosphate buffer, 10% sodium chloride solution). During a-24 h post processing storage period at 20 °C, the concentration of total soluble proteins and C-phycocyanin content and purity were measured. Color-spectrum and chroma analyses were also performed. The use of deionized water and phosphate buffer as processing/extraction media favoured the extraction process compared to the NaCl solution. Proteins extraction was significantly assisted by pressure. Equal/higher intensity than 400 MPa led to lower C-phycocyanin extraction yields, probably due to denaturation of proteins. High pressure conditions at 300 MPa for 10 min -using deionized water or phosphate buffer as medium- were selected as optimum, leading to higher extraction yields and purity extracts within ~2 h after processing.     

FACTORS AFFECTING THE SODIUM CHLORIDE EXTRACTABILITY OF MUSCLE PROTEINS FROM CHICKEN BREAST, TROUT WHITE AND LOBSTER TAIL MUSCLES

The amount of protein extracted from chicken breast muscle at low salt (0–50 mM NaCl) increased as the salt concentration of the extracting solutions increased. The addition of 10 mM sodium phosphate buffer pH 7 (P_i) caused a marked increase in protein extractability at all salt concentrations. A particular polypeptide chain of about 150,000 daltons appeared to be particularly sensitive to the extraction conditions. At high salt (0.6M NaCl, 50 mM sodium phosphate buffer pH 7.0) a second extraction still contained significant amounts of protein. The amount of protein extracted was maximized at a 1/20 dilution. On the other hand, the protein extract-ability of trout white muscle, showed a smaller P_i effect and very little dependence on low salt concentration. The protein extractability of lobster flexor muscle showed little change with either increased salt or P_i. For all three muscles extraction over time with either high or low salt remained essentially constant after the first day with the most protein being extracted from lobster muscle and the least from chicken muscle.     

Recovery of proteins from beef bone and the functionality of these proteins in sausage batters

Four solutions [4% sodium chloride (control), 4% sodium chloride with 0.3% sodium tripolyphosphate (STP), 0.3% tetrasodium pyrophosphate (TTP) or 0.05 M sodium hydroxide (NaOH)] were used to extract proteins from beef bones. Three bone solution ratios (1:1, 1:4 or 1:10), three bone types [vertebra (lumbar), rib (4-7) and leg (femur)] and two methods of protein recovery from the extraction slurries [dialysis against 0.03 M potassium phosphate buffer (pH 5.3) and acid precipitation] were evaluated. Solutions containing phosphates or NaOH were more effective in extracting protein than sodium chloride alone. Total protein recovery was highest from vertebra bones while extraction of proteins from leg bones resulted in the lowest recovery. A solution to bone ratio of 1 to 10 recovered more total protein from vertebra or rib bones than leg bones. Dialysis recovered more total protein from extraction solutions when the protein concentration was low. Acid precipitation, however, worked best if the protein concentration in the extraction solution was high. Extraction procedures resulted in some myosin degradation. Proteins extracted from beef bone materials and recovered by dialysis, performed equally as well as other commercially available proteins when added to a finely comminuted sausage product.     

Determination of Spiramycin and Josamycin in Milk By HPLC and Fluorescence Detection

ABSTRACT: A reversed-phase high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of spiramycin and josamycin in milk. The extract obtained from milk, using liquid-liquid extraction, was treated with cyclohexa-1,3-dione in ammonium acetate buffer (pH 7.0) for 60 min at 90°C. The derivative was chromatographed on an octadecylsilane (ODS) column at 45°C. Separation was performed by using a mobile phase consisting of an acetonitrile-methanol-phosphate buffer (pH 6.0) mixture. Fluorescence detection was achieved at 375 nm for the excitation wavelength and 450 nm for the emission wavelength. Linearity was demonstrated between 50 and 500 μg/L. Recoveries were about 90% and the detection limits were 8 and 13 μg/L for spiramycin and josamycin, respectively.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

PRODUCTION AND PROPERTIES OF PENCILLIUM ROQUEFORTI LIPASE

SUMMARY: A Penicillium roqueforti strain produced maximal amounts of lipase when grown in 0.5% casitone-1% Proflo broth, pH 5.5, at 27°C. Addition of butteroil, com oil or olive oil to the growth medium inhibited the lipase production. Under pH stasis the partially purified lipase of P. roqueforti had an optimum pH of 8.0 and an optimum temperature of 37°C. Maximum lipolytic activity occurred with 5% butteroil emulsion as the substrate. Manganese chloride and magnesium chloride stimulated the enzyme activity. Calcium, sodium and potassium sale had no appreciable effect on lipolysis; silver, mercury and zinc salts were inhibitory. The lipase was thermolabile, being inactivated completely within 10 min at 50°C. The lipase hydrolyzed tributyrin, tricaprylin, tricaprin, tripropionin and triolein in decreasing order.     

SQUID PROTEIN ISOLATE: EFFECT OF PROCESSING CONDITIONS ON RECOVERY YIELDS

Loigo species of squid was investigated as a potential source of protein isolate. The various process parameters which influence extraction of protein (particle size, time, extraction pH, salt concentration, relative amount of solvent to squid tissue and temperature) were investigated. From this study the following parameters were chosen to optimize extraction: pH 11 (sodium hydroxide) or 4% salt concentration (sodium chloride, sodium hexametaphosphate in aqueous extractant); temperature = 22°C; time = 45 min; particle diameter = 2–3 mm; solvent-to-squid ratio = 10:1. Under these conditions, about 85% of the squid protein can be extracted. 65% of the extracted nitrogen is recovered as protein isolate by isoelectric precipitation at pH 5.     

混合反胶团提取α-淀粉酶

以十六烷基三甲基溴化铵(CTAB)和脱水山梨醇单硬脂酸酯聚氧乙烯醚(Tween-60)为混合表面活性剂溶于正丁醇-异辛烷中构成反胶团系统,萃取纯化α-淀粉酶。研究不同萃取条件下,α-淀粉酶的萃取率。其中反胶团相组成为：ρ(CTAB+Tween-60)＝4g/L;n(CTAB):n(Tween-60)＝2.0:1.0;V(正丁醇):V(异辛烷)＝1.0:1.0。水相组成为：α-淀粉酶配制的粗酶液,此时c(NaCl)＝0.04mol/L,水相pH11.04。结果表明：萃取温度40℃、V(有机相):V(水相) ＝2.0:1.0、振荡时间10min时,α-淀粉酶萃取率可达91%;反萃取水相组成为c(NaCl)＝2.5mol/L、pH4.5、V(水相):V(有机相)＝1.0:2.0,反萃取振荡时间10min、温度50℃时,α-淀粉酶反萃取率可达65%。反胶团相可重复使用,当V(水相):V(有机相)＝1.0:2.75时,反胶团第2次α-淀粉酶萃取率达到71%。     

Preservation of minced pelagic fish by combined methods

The stability of unwashed and washed mince from Spanish sardines ( Sardinops sagax ) treated by mild heating, low pH (5.7–6.0), potassium sorbate (0.05–0.2%), and sodium chloride (2.0–6.0%) was studied. Addition of 6% salt and 0.2% sorbate at pH 5.7 was the best factor combination and when accompanied by heat treatment (10min, 80°C) produced a partly cooked product, microbiologically stable at 15°C for at least 15 d. Washing and mild heat treatment (2min, 80°C) helped to reduce the initial microbial load of the mince by a factor of 100 and, in combination with the above formulation, the product had a storage life of over 15 d at 15°C, compared with less than 3d for the original unwashed mince.