RU2075312C1 - Method of preparing hydrolyzate for virological media - Google Patents

Abstract

FIELD: virology, nutrient media. SUBSTANCE: meat-bone raw is placed into the closed volume and treated with live steam under pressure 5 atm for 2.5 h, not less. Volume is sealed and product is kept for 4 h (not less) up to the final temperature 100 C (not below). Broth is filtered, dried and the obtained powder is diluted in deionized water at ratio powder : water = 1:10 (not less) followed by addition of pancreas extract and preserving agent. Hydrolysis is carried out at 37-39 C for 48 h (not less) followed by the repeated heat treatment of preparation and its filtration. Then product can be dried. Method can be used for preparing nutrient media used in veterinary science. EFFECT: improved method of preparing. 7 cl

Description

 The invention relates to methods for preparing the basics of nutrient media used, for example, in veterinary medicine and other fields.

A known method of obtaining the basis of a nutrient medium for tissue cultures from amino acids of muscle tissue [1]
Since meat proteins contain no more than 18 amino acids in their composition, there are no more in the resulting composition, which is in most cases insufficient. In addition, meat as a raw material must meet the highest requirements, which leads to high cost.

 Also known is a method of producing a hydrolyzate from an amino acid base obtained from dairy products [2] In this case, the nutrient medium obtained by many hours of hydrolysis of milk in liquid or powder form contains 16 18 amino acids, and the dry matter content does not exceed 5 - 7% which in most cases is not valuable enough.

Closest to the claimed method is a method involving heat treatment in a closed volume with hot steam of meat and bone raw materials in water after deboning carcasses, mainly cattle, at a pressure in this volume of 1.5 to 5 atm. and separating the resulting broth, drying it, dissolving in deionized water in a ratio of at least 1 10, heat treating the resulting solution at a temperature of at least 70 degrees Celsius, subsequent cooling of the solution, adding pancreatic extract and preservative to the resulting solution, followed by hydrolysis at 37 39 degrees Celsius for at least 8 hours, repeated heat treatment and filtration of the resulting hydrolate [3]
It is known that after deboning, 5 8% of cartilage remains in the bones and up to 20% of the bone weight of the muscle tissue. Cartilage tissue contains up to 20% protein and up to 2.2% minerals, and collagen is up to 34% by weight of dry skim bone. Under the influence of temperature, collagen is welded, which is accompanied by the breaking of hydrogen bonds holding polypeptide chains in the three-dimensional structure of collagen. As a result, these chains bend and twist, collagen structure loosens. With further heating, collagen peptization occurs with the formation of glutin and its hydrolysis. The final product is free amino acids.

 The broth from meat and bone raw materials contains (in the feedstock) absolutely dry matter up to 14, protein up to 10, phosphorus up to 0.2, calcium up to 0.06, sodium chloride up to 0.09.

 In this case, the product is used in liquid and dry form, before drying, filtration and its separation are carried out.

The composition of this product contains 19 amino acids (in relation to protein):
1. Cystine and 2. Cystathionine 12.84 + 0.39 3. Lysine 7.53 + 0.54 4. Arginine 10.85 + 1.15 5. Glycine and 6. Serine 24.0 + 0.84 7. Aspartic acid and 8. Hydroxyproline 1.66 + 0.15 9. Threonine and 10. Glutamic acid 13.24 + 0.38 11. Alanine 5.7 + 0.28 12. Proline 3.07 + 0.26 13. Tyrosine 3.44 + 0.66 14. Valine and 15. Methionine 3.77 + 0.33 16. Phenylalanine 3.87 + 0.25 17. Leucine 4.35 + 0.33 18. Ornithine 5.72 + 0 9 19. Tripotophan 2.8 + 0.1.

 In the known method, the ratio of the protein product to water is essential.

 The solution was boiled for about 30 minutes, cooled to 40 degrees Celsius, an enzymatic preparation of the pancreas and chloroform were added. The hydrolysis was carried out at a temperature of 37–39 degrees for at least 2 days, after which the hydrolyzate was re-boiled for 50 minutes, deionized water was added to its original volume, cooled, filtered and sterilized in an autoclave. The resulting nutrient medium, although it has strong properties, however, has insufficient efficiency and range of applications.

 The aim of the proposed method is to obtain from the specified raw materials a product of higher nutritional value and standard composition.

 This goal is achieved by the fact that heat treatment with hot steam is carried out for at least 2.5 hours at a steam condensation temperature corresponding to the aforementioned pressure value, after which the closed processing volume is sealed and the raw materials are kept in the resulting broth for at least 4 hours until the final temperature of the broth is less than 100 degrees Celsius, while, as an extract of the pancreas, use toluene extract or toluene alcohol extract or toluene or ethyl alcohol.

 In this case, before heat treatment, meat and bone raw materials are crushed, and the ratio of the mass of raw materials to the mass of water is at least 3 1, and the repeated heat treatment of the hydrolyzate is carried out at a temperature of at least 100 degrees Celsius, after repeated heat treatment, the hydrolyzate is diluted with deionized water to the original volume. Along with this, chloroform is used as a preservative, and the final product is dried to a powder state.

 A search conducted in patent and technological literature showed that the claimed combination is unknown, i.e. it meets the condition of patentability “novelty”.

 Since there is a need to obtain a product in this way, the claimed meets the condition of "industrial applicability".

 And since the product is obtained with higher properties and obtaining a product with such properties and quality is not obvious to the average specialist, the claimed meets the condition of "inventive step".

 The method is as follows. The meat and bone raw materials after deboning carcasses, mainly cattle, are loaded into a container, for example, a tank for trapping fat from bone, and fill it with water. In this case, the ratio of raw materials to water is chosen at least 3 1. It should be noted that, if necessary, the raw materials are crushed before loading, after which the container is closed, the safety valve is adjusted to a pressure in the range from 1.5 to 5 atm. and sharp steam is supplied to the vessel under pressure, the condensation temperature in the processing vessel of which corresponds to the aforementioned pressure value, i.e. significantly exceeds 100 degrees Celsius and is determined by the known dependence of temperature on the pressure of the phase transition of water from a liquid to a gaseous state.

 The specified treatment is carried out for less than 2.5 hours, while the fat released from the bone collecting on the surface of the liquid can be removed periodically. After the end of the heat treatment time, the steam supply is stopped and the treatment volume is sealed, for example, all inlet and outlet pipes are closed, and the safety pipe sufficiently seals the steam pipe.

 After sealing, the raw materials are kept in the resulting broth for at least 4 hours until the final temperature of the broth is at least 100 degrees Celsius, this is achieved by the quality of the insulation of the processing vessel. Then the resulting broth is removed, if necessary, separating it from the fat layer, filtered, separated and dried, for example, to a powder state.

The resulting protein product, an intermediate raw material for the hydrolyzate, contains (in absolutely dry matter) at least 94.5 protein, 0.3 phosphorus, 0.6 calcium, 0.9 sodium chloride, 0.5 ash, 0.6 fat-like substances , which in some positions is 2 10 times higher than the known protein product (according to the prototype) and contains 27 amino acids, which significantly exceeds the known amino acid composition. The resulting protein product has the following amino acid composition (mg / 100 mg):
1.2 cystine and cystathionine 0.454; 3 lysine 2.086; 4 arginine 4,538; 5 - glycine 5.996; 6 serine 2,337; 7 asparagine 3,678; 8 aspartic acid; 9 glutamine; 4.135; 10 glutamic acid; 11 hydroxyproline 6.695; 12 - threonine 1.219; 13 alanine 4.869; 14 proline 9.667; 15 tyrosine 1,096; 16 - valine 0.546; 17 methionine 0.689; 18 phenylalanine 1.522; 19 leucine 2.470; 20 ornithine 0.287; 21 isoleucine 0.509; 22 histidine 0.802; 23 ethanolamine 0.209; 24 tirphotophan 0.095; 25-A-aminobutyric acid 0.320; 26-B-aminobutyric acid 0.155; 27-Gamma-aminobutyric acid 0.6148.

 Thus, starting from the 21st position of the amino acid composition of the protein product, amino acids are listed that are not previously found in the known protein products obtained by heat treatment of meat and bone raw materials and which are the basis for the hydrolyzate.

 It should be noted that the raw materials of cattle are most effective in this case, since pork rye gives a significant amount of fat, which is difficult to separate from the resulting broth. A similar drawback, although to a lesser extent inherent in raw materials obtained after lamb carcass deboning, although it should be noted that in all cases the intermediate protein product obtained from pork and mutton carcasses is incomparably more effective than the known intermediate protein product.

 The longer thermal effect of betting at temperatures up to 130 degrees Celsius and the subsequent exposure of the raw materials to the broth allows for more efficient process of bone collagen peptization and collagen hydrolysis, which leads to a wider range of amino acids in the resulting broth and greater broth saturation with chemical compounds.

 After receiving the protein product in the form of a broth, it is separated and filtered and then dried to a powder state. Then diluted in water at a ratio of at least 1 10 at a temperature of about 70 degrees Celsius and above until complete dissolution. Next, the resulting solution is cooled and toluene extract of the pancreas is added, and as a preservative, chloroform and at a temperature of 37–39 degrees Celsius carry out hydrolysis for more than 2 days. Next, the hydrolyzate is boiled, diluted with deionized water to the original volume, cooled, filtered and sterilized. The resulting base can be used in liquid or powder form.

 An example of a specific implementation of the method. Meat and bone raw materials after deboning carcasses from the cutting shop are fed for grinding, where raw bones, tendons, cartilage, etc. grind into fractions of a length of not more than 5 cm, after which the crushed raw material is loaded into a tank for bone fat melting, in which it is most simple to implement the heat treatment process. Set the safety valve to 2 atm. start supplying hot steam to the container. The temperature in the tank rises to 120 degrees Celsius, excessive pressure in the tank is released through the safety valve. Under the influence of the ambient temperature, bone fat is released from the bone and collected in the upper part of the vessel 7. After 1 2 hours, the formed layer of fat can be drained from the upper part of the tube, but fat can be removed at the end of processing. The thermal effect on the raw materials leads to the formation of glutin polypeptide chains, which dissolve in water, and at a temperature of about 100 degrees Celsius, the collagen is hydrolyzed, the product of which is the free amino acids found in broth 7. The yield of the broth in this case is 40% by weight of the raw material.

 After exposure to hot steam, after 2.5 hours, all nozzles are closed, thereby sealing the container and holding the raw material in the broth for 4 hours. During this exposure, the broth is also saturated with free amino acids, i.e. collagen fibers are almost completely utilized from the bone and a wider set of amino acids is determined in the resulting broth, which is then filtered and dried.

 When using pressure of 1.5 atm. the concentration of solids is achieved in broth 7 8% while the use of pressure of 5 atm. allows you to get up to 20% solids in the broth.

 Studies on the influence of the heat exposure time on the concentration of solids in the broth showed that 7% is the minimum value, less than which there will not be formed a sufficient number of non-substituted amino acids in the required amount. A study of the exposure time of raw materials after treatment with hot steam showed that the hydrolysis of the product of peptization of collagen bone fibers at an acceptable (up to 80%) level is completed in 4 hours, which fully meets the degree of readiness of the resulting protein product at a final broth temperature of at least 100 degrees Celsius.

 It should be noted that only the totality of all stages of the thermal effect on meat and bone raw materials allows not only a very high degree of destruction of collagen bone fibers, but also to complete the process of collagen hydrolysis. It is impossible to achieve such a degree of destruction by simple thermal exposure, similarly to other influences with subsequent exposure of meat and bone raw materials in the broth, it is impossible to obtain such a degree of hydrolysis and, therefore, the indicated spectrum of amino acids in the composition of a new protein product that was previously unknown.

 The composition of the obtained protein product, consisting of at least not 95% (in dry form) of protein, determines its widespread use as a basis for the production of nutritious hydrolyzate and is a more effective protein base than meat water, enzymatic meat hydrolysates (Hottinger's meat digests) casein, etc.

 Next, the resulting product was dissolved in a volume of deionized water at a concentration of the specified powder, for example, a 10 12% solution was boiled for 30 minutes, after which 20% of the enzymatic toluene preparation of the pancreas and 0.5% chloroform were added to the product at a temperature of 40 degrees Celsius. In this case, the enzymatic toluene preparation is made from the native pancreas of cattle (cattle), which is cleaned of fat, films, blood vessels, thoroughly washed with distilled water and sterile saline. The washed pancreas is squeezed from the water, thoroughly crushed, one and a half quantities are added to its weight, the amount of distilled water and 17% to the total weight of chemically pure toluene. The suspension is placed in a glass bottle, closed with a rubber stopper and shaken vigorously for 30 minutes until a homogeneous white mass is obtained. Then the suspension of the gland is left for 1 to 2 days at a temperature of 1 to 4 degrees Celsius, after which the drug becomes ready for use. Immediately prior to enzymatic hydrolysis, the toluene preparation is filtered through two layers of gauze, carefully squeezing by hand.

 Hydrolysis is carried out at a temperature of 37 degrees Celsius for at least 48 hours with mechanical stirring. After that, the hydrolyzate is boiled, distilled water is added to the original volume, cooled and autoclaved at 110 degrees Celsius for at least 20 minutes. After that, the hydrolyzate can be used directly, but can also be dried for subsequent preservation. If necessary, a dry hydrolyzate is used to obtain a solution.

 Further, after receiving the hydrolyzate for preparing a culture medium for cell cultures, a 0.5% hydrolyzate solution is prepared on Hanks medium, vitamins, bovine serum, antibiotics are added to it according to official prescriptions.

 The growth medium of digested and primarily trypsinized cell cultures 18, monkey kidneys, guinea pig kidneys, human embryo fibroblasts, mouse embryos, and others were tested on a nutrient medium from this hydrolyzate.

 The control was 199 medium and a lactoalbumin hydrolyzate. The biological value of the cultures was evaluated according to cell growth, monolayer formation time, viability of the grown cultures and the nature of cell growth.

As a result of cultivation of the primary cell lines on a medium with the given hydrolyzate, it was revealed that the monolayer forms up to 4 days, the cell growth was on average 7 9 times. When sowing primary trypsinized seeds, a sowing dose of 200,500 thousand cells in 1 ml of medium. Starting from the first day of growth to the formation of a monolayer, there is a constant increase in the number of cells by an average of 4 to 5 times, on the first day they are glued 25 40%
For primary trypsinized cell cultures of guinea pig kidney and white mouse kidney, the proliferation index was 0.6 0.8 and 1.5 1.6, respectively, which corresponds to the same parameters of medium 199 with lactoalbumin hydrolyzate.

 Since, in comparison with meat, a powdered protein product has an incomparably more standard composition, differing 4 times in the high content of protein, peptones, amino acids, bacteriological media based on the powder of this protein product are 5 times more economical than other media. Obtained in the process of implementing the method, the protein product is almost one and a half times more effective than the previously known product from meat and bone raw materials. Comparison of the culture media obtained from this product for tissue cultures with culture media from protein-containing raw materials of blood, fish, dairy products, etc. as well as their mixtures shows the incomparable effectiveness of the new nutrient medium in all applications.

 Practice has shown that the high standard composition of the resulting protein product from meat and bone raw materials is an effective basis for obtaining microbiological culture media, being an extremely beneficial substitute for meat and other protein products, and, in some cases, the resulting protein product is the only possible basis for a number of virological media, this multiplicity of replacing this product with known others reaches 10 15. The cost effectiveness of the resulting nutrient medium is at least 2 30 r of greater than when receiving the culture medium of the above standard protein products or mixtures thereof. The availability of meat and bone raw materials and the possibility of using standard equipment for the extraction of bone fat to obtain a protein product in its liquid phase, and in the powder phase as the most intermediate protein product of standard vacuum spray dryers, allows for the shortest possible time to establish a new nutrient medium with the widest range described above actions.

Sources of information
1. Babich M. A. Nutrient media. Laboratory research methods, M. Higher School, 1954.

 2. Kozakov A.M. Preparation of culture media from meat and bone meal, g. Nutrition Issues, 1938, 8, 112.

 3. Karysheva A.F. Dry protein concentrate and its use in the preparation of culture media for microorganisms, diss. for the Ph.D. Minsk, 1972.

Claims (7)

1. A method of producing a hydrolyzate for virological environments, including heat treatment in a closed volume with hot steam of meat and bone raw materials in water after deboning carcasses of mainly cattle at a pressure in the closed volume of 1.5 5.0 atm and separating the resulting broth, filtering it, separating , drying, diluting the obtained powder in deionized water with a ratio of the mass of the latter to the mass of water of at least 1 10, heat treatment of the resulting solution at a temperature of at least 70 ^o C, subsequent cooling solution, the addition of pancreatic extract and preservative, hydrolysis at a temperature of 37 ^° C for at least 48 hours, repeated heat treatment and filtration of the obtained hydrolyzate, characterized in that the heat treatment of the feedstock with hot steam is carried out for at least 2.5 hours at a steam temperature corresponding to the aforementioned pressure value, after which the closed treatment volume is sealed and the raw materials are held in the resulting broth for at least 4 hours to a final broth temperature of at least 100 ^o C, while as an extract the pancreas uses toluene extract, or ethyl alcohol extract, or toluene, or ethyl alcohol.  2. The method according to claim 1, characterized in that before heat treatment, the meat and bone raw materials are ground.  3. The method according to p. 1, characterized in that the ratio of the mass of meat and bone raw materials to the mass of water in the closed processing volume before heat treatment is at least 3 1. 4. The method according to p. 1, characterized in that the repeated heat treatment of the hydrolyzate is carried out at a temperature of not less than 100 ^o C.  5. The method according to p. 1, characterized in that after repeated heat treatment, the hydrolyzate is diluted with deionized water to the original volume.  6. The method according to p. 1, characterized in that chloroform is used as a preservative.  7. The method according to p. 1, characterized in that they carry out the drying of the hydrolyzate to a powder state.