CS271503B1 - Soya cultivating medium and method of its production - Google Patents

Abstract

New soya nutrient medium falls into the branch of industrial microbiology. This medium consists of oligopeptides and amino acids in the form of a transparent sterile yellow brown solution. This solution consists of (calculated as dry basis) 52.0 to 57.0 weight % of nitrogen substances, of which 15.0 to 17.0 weight % are represented by peptidic or free amino acids; of 16.5 to 19.0 weight % of sodium chloride, of 16.4 - 16.6 weight % of phosphatic buffer, of 2.2 to 2.5 weight % of reducing sugars, off 1.0 to 1.25 weight % of glucose, and of 3.65 to 11.9 weight % of non-nitrogenous substances of extract fatty acids, glycerol and mineral substances. The solution is reached by a combined proteolytic effect of phylogenetically different enzymes: papain and pancreatin. Introducing papain into enzymatic hydrolysis of soya proteins beside pancreatin, the pancreatic proteases break by papain prepared higher and middle-molecular peptides to oligopeptides and amino acids, while the optimum reaction conditions for papain are in pH = 7.0 regulated by means of sodium hydroxide and optimum reaction conditions for pancreatin are in pH = 7.5 - 8.2.

Description

The new soy nutrient medium falls within the field of industrial microbiology. This medium consists of oligopeptides and amino acids in the form of a clear sterile yellow-brown solution, characterized in that it consists, calculated as dry matter, of 52,0 to 57,0% by weight of nitrogen substances, of which 15,0 to 17,0% by weight are peptic or free amino acids, from 16.5 to 19.0 wt% sodium chloride, from 16.4 to 16.6 wt% phosphate buffer, from 2.2 to 2.5 wt% or from reducing sugars, from 1.0 to 1, 25% by weight of glucose and from 3.65 to 11.9% by weight of nitrogen-free extracts, fatty acids, glycerol and minerals.

The solution is achieved by the combined proteolytic effect of phylogenetically differing enzymes pajalm and pancreatin. By incorporating papain du enzymatic hydrolysis of soy protein to pancreatin, the pancreatic proteases cleave papain-prepared above and medium-molecular peptides up to oligopeptides and amino acids, with optimal reaction conditions for papain at pH = 7.0 adjusted with sodium hydroxide and optimal reaction conditions for pancreatin pH value - 7.5 - 8.2.

(13) B1 Int. Cl. ⁵

C 12 N 1/20

CS 271 503 B1

The invention relates to a soy nutrient medium based on oligopeptides and amino acids in the form of a sterile yellow-brown solution and to a process for its preparation.

Solved problems belong to the field of production of veterinary bacterial vaccines.

Current methods of enzymatic hydrolysis of protein substrates relate to application! only animal, plant, or microbial enzymes in mass production solo. Such as pepsin digestion of plant proteins (peptic peptone broth for VF, peptic algal peptone), trypsin štiqjerle vegetable protein (tryptic soy peptone) _f tryptic digestion of animal protein (meat peptone bouillon bases casein tryptic hydrolyzate). In the above examples, it is a multiplication of one species or one enzyme system per substrate. However, it is more difficult to enter the same hydrolytic process with two enzymes or enzyme systems.

The above drawbacks using one enzyme system have been solved by preparing a new soy nutrient medium from oligopeptides and amino acids in the form of a clear, sterile yellow-brown solution, calculated as dry matter, from 52 to 57 percent by weight of nitrogen, of which 15 to 17 percent by weight peptide or free amino acids, from 16.5 to 19.0% by weight of sodium chloride, from 16.4 to 19.0% by weight of sodium chloride;

16.6% by weight of phosphate buffer, 2.2 to 2.5% by weight of reducing sugars, 1.0 to 1.25% by weight of glucose and 3.65 to 11.9% by weight of nitrogenous, non-fatty acids, glycerol and minerals.

A novel process for the production of soy broth is characterized in that the starting substrate soy protein and large molecule peptides are cleaved by papain endopeptidase in the neutral pH range, producing medium molecular peptides which are further hydrolyzed to oligopeptides and amino acids by pancreatic peptidases, lipases are released from starch and fat by glucose and glycerol with fatty acids, and after consumption of a certain amount of 5 mol / l sodium hydroxide to maintain optimal pH in the hydrolyzate, it is further adjusted by adjusting the pH, clearing by sedimentation and filtration and sterilized by filtration and autoclaving.

It is an object of the invention to change the physical and biochemical parameters of the technological process for the production of soy nutrient medium. The decisive change is the use of plant proteases in the form of a commercial paprika preparation and an animal multienzyme pancreatin preparation, which are biochemically suitable for the enzymatic hydrolysis of aqueous soybean delipidized flour extract. This is similar to the biochemical interaction that exists between pepsin and pancreatic enzymes, but in the case of papain, a low pH can be avoided, which not only complicates the process of elongation, but also reduces the quality of the nutrient medium compared to papine.

Several advantages are achieved according to the invention. In particular, a significant increase in the number of Erystipelothrix rhusiopathiae - strain WR - 2 in 1 ml soy nutrient medium is achieved by substantially increasing its nutritional value from the original mines. 1. 10 ⁷ cfu in 1 ml per min.

. 10 cfu in 1 ml soy nutrient medium. Using papain in tandem with pancreatin in the destruction of soy protein, it has been shown that peptide papain grafts as endopeptidase W are pancreatin carboxypeptidases more accessible spatially and quantitatively than exopeptidases. Similar to the pancreatin serine proteases are the phylogenetically more accessible peptide steps, although with a higher molecular weight than the uncleaved polypeptide (protein). Of the declared values, an increase in the total nitrogen content from at least 2,400 mg is remarkable. l ” ¹ per min. 3000 mg. l ¹ and amino nitrogen content from min. 500 mg / ^l for min. 700 mg. As a test strain, E. r. - WR - 2 selected because it has the highest difficulty in both surface and submerged cultures because of its high sensitivity to bacterial growth inhibitors.

CS 271 503 Bl. EXAMPLE 1 An aqueous suspension of soybean delipidized flour is adjusted to pH = 8.0 to initiate cold alkaline extraction. After completion, the protein substrate is pasteurized at 70 ° C for 30 minutes. Shortly thereafter, the temperature is stabilized at 60 ° C and adjusted to a starting pH of 7.0. At the same time protease stimulator - CaCl ₂ = 0.01% is added. The papain suspension is added at a concentration of 0.7 - 1.0% depending on the enzymatic activity. Papain hydrolysis lasts 1 hour and the pH is adjusted with C (NaOH) = 5 mol. l ” ¹ . Upon completion, the temperature is lowered to 44 ° C and the pancreatin suspension is added at a concentration of 2.0-2.5% Abbot depending on the enzymatic activity. Pancreatin hydrolysis lasts 2 hours and the pH is maintained in the range of 7.5-8.2 opiates with C (NaOH) = 5 mol.

<After the hydrolysis, the nutrient medium is stabilized by the addition of phosphate buffer before finalization. Finalization includes sedimentation, supernatant separation, filtration, sterilization, incubation, final control and storage.

The invention offers new cultivation possibilities, especially in the cultivation of newly isolated bacteria using both in veterinary medicine and in the food or feed industry (fermentation food products, silage preservation by microbial fortification and the like).

Claims (2)

Example 1 An aqueous suspension of soybean delipidized flour is adjusted to pH = 8.0 to produce alkaline extraction. Upon completion, the protein substrate is pasteurized at 70 ° C for 30 minutes. Shortly thereafter, the temperature is stabilized at 60 ° C and the pH is adjusted to 7.0. At the same time, a protease stimulator - CaCl 2 = 0.01% - is added. The papain suspension is added at a concentration of 0.7-1.0% depending on the enzymatic activity. The papain hydrolysis lasts 1 hour and the pH is adjusted with C (NaOH) = 5 mol. The temperature was lowered to 44 ° C and the pancreatin suspension was added at a concentration of 2.0-2.5%, depending on the enzymatic activity. Pancreatin hydrolysis lasts 2 hours and the pH is maintained at 7.5-8.2 with a C (NaOH) = 5 mol. 1. * After the hydrolysis is complete, the nutrient medium is stabilized by addition of phosphate buffer prior to finalization. Finalization includes sedimentation, supernatant separation, filtration, sterilization, incubation, exit control and storage. The invention offers new cultivation possibilities especially in the cultivation of newly isolated bacteria using both in veterinary medicine and in the food or feed industry (fermentation food products, preservation of silages by microbial fortification, etc.). SUBJECT OF THE INVENTION 1. Oligopeptide and amino acid-based soybean nutrient medium in the form of a clear sterile yellow-brown solution, consisting of, calculated as dry matter, from 52 to 57% by weight of nitrogenous substances, of which 15 to 17% are peptide or free amino acids , from 16.5 to 19.0% by weight of sodium chloride, from 16.4 to 16.6% by weight of phosphate buffer, from 2.2 to 2.5% by weight of reducing sugars, from 1.0 to 1.25% by weight of glucose and 3.65 to 11.9% by weight of nitrogen-free extract, fatty acids, glycerol and minerals. 2. A method for producing a soy nutrient medium according to claim 1, wherein the starting substrate soy protein and large molecule peptides are cleaved by a papain endopeptidase in the neutral pH region, wherein the resulting mid-molecular peptides are further hydrolyzed to oligopeptides by pan-creatine peptidases. amino acids, while gluconic and glycerol with fatty acids are released from starch and fat by the action of pancreatic amylase and lipase, whereby after consumption of a certain amount of sodium hydroxide to maintain the optimum pH in the hydrolyzate, it is further adjusted by adjusting the pH; by filtration and filtration and sterilized by filtration and autoclaving. AND