The method of producing lysozyme from egg white
(57) Abstract:The invention relates to a process for the production of lysozyme from egg white. The invention consists in the obtaining of lysozyme undiluted egg protein by column chromatographic process on the cation exchanger, providing processing for 1 cycle of large quantities of protein by a small number of the cation, the receipt of the eluate of high purity with a high concentration of lysozyme. For that egg protein is passed through a column of strongly acidic, macroporous cation exchange resin, then the cation exchanger is washed with water or sodium chloride solution with a concentration of not higher than 0.2 M, then elute lysozyme, skipping alkaline solutions of inorganic salts with a pH of 8.5-13,2. The composition of the eluent may include soda Na2CO3or potash (K2CO3, chlorides, sulfates or phosphates of sodium or potassium in a concentration of 0.05-2 M, and ethyl alcohol at a concentration of 10-40%. 3 C.p. f-crystals, 1 table. The invention relates to techniques for the production of egg white lysozyme, used as a preservative additive in veterinary medicine, medicine, food industry.A method of obtaining lysozyme, in which egg protein is treated with solutions of the om-known method is the low activity of the obtained lysozyme, the complexity and duration of the process. Obtained in this method the saline waste egg whites and the products are of very limited use.A method of obtaining lysozyme from egg white by processing undiluted egg protein ion-exchange sorbents with subsequent desorption of lysozyme aqueous solutions of salts. As the ion exchange sorbent use macroporous weak acid cation exchangers, for example, "Duolith C-464"  According to this method, native, undiluted with water or buffer solutions of egg protein is brought into contact with a weak acid cation exchange resin in the reactor, then the cation exchanger is washed from the ballast proteins salt solution with a salt concentration of no higher than 0.2 M or water and then elute lysozyme aqueous salt solution, for example, 0.1 M nutrifaster buffer (pH 7.5 to 8.3) containing 0.15 M sodium chloride. Later in the collected eluate add inorganic salt crystallization or vysalivaniya lysozyme, or the eluate provide for subsequent drying.The use of weak acid cation exchangers type "Duolith" for the first time allowed to obtain the lysozyme of raw materials were extracted only the target product, and the consumer properties of egg white, the confectionery industry, in the formulation of powder products, which resulted in a wide spread of this method. The method, however, is not without disadvantages.The most important disadvantages of this method include the following:
all weak acid cation exchangers, including "Duolith", strong (3-5 times) change its volume when changing the pH or ionic strength balancing their solutions, which makes it almost impossible to carry out a chromatographic process in the column. The implementation of such a process on an industrial scale is possible only in the reactor, which is technically difficult and is associated also with a strong deterioration of the cation;
only a relatively small amount of egg protein can be processed within 1 cycle a certain amount of weak acid cation exchanger (3-4 volume protein 1 by volume of cation exchange resin);
after processing of the cation salt solutions with the purpose of desorption of lysozyme latter is recovered in the form of a highly diluted solution (1-3 mg/ml), which may contain large (up to 50%) of the amount of ballast proteins.In General, these shortcomings lead to the increasing complexity of technology and the rise of lysozyme.The present invention assessory process to process large quantities of raw materials at low cost sorbents and to obtain a sufficiently pure eluate with a high concentration of lysozyme.The invention consists in the following.Egg protein is passed through a column of strongly acidic (pKa 1-2) macroporous cation exchange resin, in particular KU-2S, and desorption of lysozyme are eluent with a pH of 8.5-13,2. The composition of the eluent may include soda or potash at a concentration of 0.05-2 M Eluent may contain additional or other inorganic salts chlorides or sulfates or phosphates of sodium or potassium in concentrations less than or equal to the concentration of soda. The composition of the eluent can also include ethyl alcohol at a concentration of 10-40%
Use nanabhai macroporous strongly acidic cation exchange resin provides the possibility of lysozyme in a column process, which allows to increase the performance of the method and reduce the cost of obtaining lysozyme.The substance used soda, potash, neutral salts and ethanol are equivalent from the point of view of their debilitating effect on the electrostatic interaction of polyelectrolytes in water. The specified range of pH values is due to the fact that at pH below 8.5 does not occur desorption of Lisa what is fact, when the concentration is below 0.05 M not going desorption of lysozyme, and at concentrations above 2 M there is a "salting out" of lysozyme on the cation exchanger, which reduces the degree of desorption. These concentrations of ethanol are determined by the same circumstances.Specific values of the concentrations of salts within the specified range is chosen based on the nature of the subsequent procedures. So, if after desorption conduct desalting of the eluate with subsequent drying, it is advisable to use an eluent with a low salt concentrations (e.g., 0.1 M). If after desorption conduct salting out of lysozyme from the eluate, it is advisable to use an eluent with high concentrations of salts (for example, 0.4 M). Acceptable from the point of view of Economics of the process are the concentration (total) salts of 0.4 M, ethanol 20% In General, the higher the concentration of salts and ethanol eluent, the more concentrated (but not less than 10 mg/mg) is the target solution of lysozyme (eluate).The method is as follows.The original egg white pass with the speed of 15-45 ml/h/cm2through a column of cation exchange resin KU-2P 7-15 volume of the cation exchanger. After that, the sorption stop eluent until while the lysozyme is not desorbed, with speeds up to 45 ml/h/cm2. Thereafter, the eluate can be obessolivanie and dry or to carry out the crystallization of lysozyme by known methods.Examples of implementation of the method.Example 1. Through the column with a volume of 8 ml (1.8 to 2.1 cm) with a cation exchange resin KU-2P missed a 200 ml egg whites with a speed of 6 ml/hour at a temperature of 20oC, then distilled water 30 ml, then 0,11 NaOH (pH 13,2) in an amount of 100 ml of the cation exchanger was servirovaci 70% of the original amount (activity) of lysozyme, desorbitados 40% of the adsorbed lysozyme.Examples 2 to 15, are shown in table illustrate the efficiency of eluting solutions with different salt composition, different pH and different ethanol content.Example 16. Through the column with a volume of 0.8 l (1010,2 cm) with a cation exchange resin KU-2P missed 8 l egg whites with a speed of 420 ml/HR, then 3 liters of 0.2 M NaCl, and then 2 l of eluent composition 0.2 M NaCl, 0.2 M Na2CO3and 30% ethanol at a speed of 250 ml/hour. As a result, the cation exchanger was servirovaci 83% of the original amount of lysozyme was desorbitados 80% of the adsorbed amount of lysozyme. Next, the eluate was absoluely known manner and subjected to lifelinebeta. 1. The method of producing lysozyme from egg white by processing undiluted egg protein ion-exchange sorbents with subsequent desorption of lysozyme eluted salts, characterized in that the ion exchange sorbent using a strongly acidic cation exchangers and desorption exercise eluted salts with a pH of 8.5 to 13.2.2. The method according to p. 1, characterized in that the composition of the eluting solution is injected soda or potash at a concentration of 0.05 to 2 M3. The method according to p. 1 or 2, characterized in that the composition of the eluting solution is further added chlorides, or sulfates, or phosphates of sodium or potassium in a concentration of 0.05 to 2M.4. The method according to p. 1, or 2, or 3, characterized in that the composition of the eluting solution is further added ethyl alcohol at a concentration of 10 - 40%
FIELD: biotechnology, biochemistry.
SUBSTANCE: invention relates to producing the biologically active complex eliciting antioxidant and immunomodulating activity and used in medicine, cosmetics, veterinary science and food industry. The biologically active complex preparing by enzymatic hydrolysis of muscle tissue represents complex of biologically active compounds involving carnosine and anserine in the amount 85-97 wt.-% of the native content of these components in poultry muscle tissue, 1-7 weight parts of amino acids, 0.5-12 weight parts of oligopeptides of molecular mass 10 kDa, not above, and 0.1-15 weight parts of cyclic and polycyclic phenolic compounds as measured for 1 weight part of carnosine and anserine in the complex. This complex is prepared by enzymatic hydrolysis of milled and homogenized water muscle tissue in preferable dilution homogenate with water in the range 0.2-0.6 and with using proteolytic enzymes in the amount 2-5 wt.-% of the protein content and working at pH 4.5-8.5 and at enhanced temperature being preferably at 45-65°C. Product is isolated as extract or powder prepared in drying the extract. Invention provides enhancing effectiveness of the claimed complex.
EFFECT: improved method for preparing, valuable properties of complex.
7 cl, 6 tbl, 6 ex
SUBSTANCE: the present innovation deals with treating vestibular and auditory disorders, trigeminal nerve's neuralgia and peripheral paresis of facial nerve of herpetic etiology. The method deals with introduction of an antiherpetic preparation followed by immunomodelling therapy with the use of polyoxidonium. Moreover, after treating with antiherpetic preparation before introducing polyoxidonium it is necessary to conduct additional successive therapy: with preparations of trophic action as milgamma or neuromultivit at simultaneous introduction of antioxidant, then comes intratissue injection of cerebrolysine being behind the top of mastoid process.
EFFECT: the innovation enables to decrease the quantity of relapses due to stopping the development of herpes simplex virus, restore conductivity along nervous fiber and improve endoneural circulation.
3 cl, 3 ex
SUBSTANCE: method involves applying antiherpetic therapy using Phamcyclovir. Then, Trental is sequentially introduced during 5 days with 2-5 ml of cerebrolysin being concurrently intratissularly introduced behind mastoid process tip. Mexidol is administered for 10 days. The treatment is finished by introducing Polyoxydonium.
EFFECT: combined microcirculation improvement; high neurotrophic and antioxidant activity; secondary immune deficiency adjustment; eliminated labyrinth hydrops.
FIELD: medicine, neurology, in particular treatment of disseminated sclerosis.
SUBSTANCE: complex therapy includes plasma exchange, interferonotherapy, administration of copaxon, cytostatics, symptomatic and bracing agents, and cyclosporin A. Administration of steroids is excluded. Additionally ceruloplasmine intravenously drop-by-drop in dose of 100 mg and cerebrolysate intramuscularly in dose of 10 ml for 10 days are administered. Claimed method provides stable remission up to 12 months for 89.2 % of patients.
EFFECT: decreased invalidisation due to reconstitution of regulatory relationship between nervous and immune systems.
1 ex, 1 tbl
FIELD: medicine, neurology, pediatrics.
SUBSTANCE: method involves administration of aminolone in the dose 0.25-0.5 g, 2-3 times per a day for 6-8 months, cerebrolysine in the dose 1.0-1.2 ml by intramuscular route, course 15-20 injections, mydocalm in the dose 50-75 mg, 2 times per a day for 1-1.5 month, vitamin B12 in the dose 150-300 mcg, course 15-20 injections wherein injections of vitamin B12 and cerebrolysine are alternated in each other day; in 1.5 month after onset of this treatment prefizone in the dose 1 ml by intramuscular route is administrated every day, course 15-20 injections, pyrogenal in the dose 100-200 MTD is administrated by course 15-20 injections and curative physical culture is carried out with heat procedure in sauna (dry bath) for 3 months, and after termination of medicinal therapy hyppotherapy is carried out for 3-5 months for each other day. Method allows straightening the kyphoscoliotic carriage and to improve joins mobility due to increase of the motions volume in them. Invention can be used in treatment of spastic diplegia in infantile cerebral paralysis.
EFFECT: improved treatment method.
FIELD: medicine, ophthalmology.
SUBSTANCE: after termination of surgery operation as to strabismus the method involves catheterization of Tenon's space by using the irrigative system for the successive fractional administration of preparations of riboflavin, cerebrolysin, dicynone, caffeine, emoxipine and taufon in the dose 0.3 ml for each drug. Also, method involves carrying out the computer training by designation of the complex "Shooting range/gallery" and "Pursuit" wherein the patient combines color symbols that change colors and sizes in cyclic change of positions, 1 séance for each type of exercise every day. Administration of drugs and computer trainings are carried out for 10 days. Such performance of the method provides the effective treatment of strabismus and recovery of binocular vision based on the complex effect on all links of coordination activity of sensor and motor eye apparatus. Invention can be used in treatment of strabismus.
EFFECT: improved and effective method of treatment.
FIELD: veterinary, in particular agents for prophylaxis and treatment of gastric diseases in calves.
SUBSTANCE: the fist foremilk of calved in summer cows is collected, filtered, bottled in sterile 1 l vessels and frozen in freezing apparatus at 20-22°C. In winter-spring period foremilk is defrosted and conserved with potassium sorbate in ratio of 2.5 ml of 40 % potassium sorbate solution per 1 l of foremilk.
EFFECT: environment friendly method for stimulation of calves immunity.
FIELD: medicine, biochemistry, pharmacy, biotechnology.
SUBSTANCE: invention relates to a method for preparing polyelectrolyte microparticles containing the end substance and showing sensitivity to alteration of the environment composition. Method involves preparing oppositely charged polyelectrolytes on microaggregates containing an encapsulated substance. These polyelectrolyte microparticles can be used both in medicine as systems used in delivery drugs and providing pH-sensitive release of encapsulated substance and in biotechnology as biocatalysts stabilized with respect to unfavorable conditions. Invention provides preparing polyelectrolyte microparticles characterizing by the high content of active substance - up to 90% of microparticles mass. Proposed method is sample and involves lesser amounts of steps.
EFFECT: improved preparing method.
9 cl, 3 tbl, 2 dwg, 61 ex
SUBSTANCE: method involves applying mono therapy with Cerebrolysin intramuscularly introduced once a day in the morning at a daily dose of 0.1 ml/kg of weight as 20 days long course or Encephabol to be taken 3 times a day in 30 min after having taken meals. Encephabol is given at a dose of 50 mg in 2-5 years old children cases or at a dose of 100 mg in 6-10 years old children cases. Male children take Encephabol in not longer than 10 days long course. Female children take Encephabol in a course being longer than 10 days.
EFFECT: enhanced effectiveness of genome instability adjustment.