RU2108381C1 - Base for nutrient medium being used for cultivation of microorganism - Google Patents

Abstract

FIELD: cultivation of microorganism; microbiological synthesis of irreplaceable amino acids, antibiotics and vitamins. SUBSTANCE: hydrolyzate of concentrated whey is proposed as base for nutrient medium. Said hydrolyzate is prepared by deep acid hydrolysis of whey, the process is carried out at 90-125 C within 12-1 h in the presence of 2.0-5.0 N mineral acid (2.0-4.0 N sulfuric acid being preferred). Summary amount of glucose and galactose in said hydrolyzate is at least 50 %, summary amount of amino acid being not more than 13500 mg as calculated for dry residue. EFFECT: increased assimilability all of whey nutrients. 2 cl, 6 tbl

Description

The invention relates to a nutrient medium derived from whey. Such a basis can be used in the technology of microbiological synthesis:
- predominantly essential amino acids (especially L-lysine, as well as threonine, tryptophan, leucine, valine, proline),
- antibiotics (for example: oxytetracycline and tetracycline) and
- vitamins (e.g. riboflavin)
as well as in the production of preferably baker's yeast.

 The need for these microbiological synthesis products is very high. Therefore, raw materials for the implementation of the technological processes of their production are presented with a complex of systematically tightening hardly compatible requirements.

Indeed, it is highly desirable that the raw materials used be:
- is substantially balanced in terms of the qualitative and quantitative composition of the nutrients necessary and sufficient for the efficient cultivation of microorganisms and therefore most fully assimilated in the processes of microbiological synthesis;
- available for use on an industrial scale;
- practically non-toxic not only for cultivated microorganisms, but also for production personnel and consumers of the final products, if the remains of nutrient media are included in the target products as an integral part;
- thermostable and, accordingly, allowing thermal sterilization;
- as homogeneous as possible.

 Separate fulfillment of these requirements or some of their partial combinations does not present significant difficulties.

 Indeed, it is widely known to use protein-vitamin concentrate (BVK, or paprika) as a complete nutrient medium base for the cultivation of microorganisms producing amino acids, vitamins, and proteolytic enzymes (see, for example, 1. Beker V.F. ., Becker ME, Lysine of microbial synthesis - Riga: Zinatne, 1974, p. 37-38; 2. Pankova, T. A. Application of BVK acid hydrolyzate in the production of lysine // Microbiological industry, 1983, N 3, p. 9 )

 However, BVK obtained by microbiological synthesis using Candia fungi cultivated on oil paraffins, under conditions of a systematic increase in energy prices, is becoming less and less accessible for industrial microbiological synthesis of amino acids. In addition, BVK is very dangerous as an allergen for industrial personnel, is fire and explosive during transportation and storage, and, finally, serves as a source of hard-to-remove ballast impurities in target products such as amino acid-containing feed additives.

 If we take into account that the synthesis of BVK in itself is extremely environmentally hazardous, it is clear that materials from safer and easier renewable sources should be used as nutrient media for the cultivation of microorganisms.

 An example of such a material is corn extract obtained from key waters during the processing of corn grain into starch (see, for example, the already mentioned book by V.F. Becker and M.E. Becker, p. 36). This extract, as well as BVK, can serve as a complete nutrient-rich complex of growth substances (and richer in vitamins) for the cultivation of microorganisms producing amino acids, vitamins and proteolytic enzymes.

 However, it is usually abundantly infected with vulgar microflora (see, for example, Novakovskaya S.S., Shishatsky Yu.I. Production of baking yeast. Handbook - M .: Agropromizdat, 1990, p. 58) and contains large ones (up to 3 mm across) suspended particles, which makes sterilization difficult. In addition, the proportion of corn extract in the total production of starch and syrup industry is small and its production is not able to cover the needs of the microbiological and food industries.

 Therefore, the problem of reliable provision of microbiological production with raw materials remains quite acute.

 One of the directions of its solution may be the use of milk production waste in the form of whey obtained in the manufacture of cottage cheese and cheeses. This serum contains up to 6% by weight of solids, including up to 4% of disaccharide (lactose), from 0.6 to 1% of proteins (milk proteins: albumin, casein) and a complex of mineral salts and vitamins characteristic of milk (see, for example, Rostros N.K. Handbook of whole milk production - M .: Food industry, 1976).

 However, in its original form, this serum contains up to 94% water, which makes it inappropriate to transport and direct use as a source of nutrients for the cultivation of microorganisms. In addition, the lactose and proteins contained in it in its original form can be absorbed only by some industrial strains of microorganisms.

 Therefore, it is usually subjected to additional processing.

 One of the options for such processing involves the isolation of milk protein (“albumin milk”) from the original whey, partial hydrolysis of albumin using 3% hydrochloric acid or 30% acetic acid, or a proteolytic enzyme (namely, Protosubtilin GZh) and the use of a hydrolyzate as the basis of nutrient media for the cultivation of microorganisms producing amino acids (see Mezhinya G.R., Yakovich V.T., Duntse M.E. and Liepins G.K. Use of albumin milk in the biosynthesis of amino acids // Producers a Mineral Acids and Enzymes - Riga: Zinatne, 1978, pp. 80-85).

 However, the hydrolyzate described in comparison with the initial whey is significantly depleted in nutrients such as sugars, vitamins, mineral salts and trace elements, which subsequently forces them to significantly enrich nutrient media from other sources. In addition, acid hydrolysis of the protein gives a low yield of free amino acids, and when using acetic acid, the target product is oversaturated with the products of its neutralization. Enzymatic hydrolysis by protosubtilin GZh, which is a Bacillus subtilis drug, which is not purified from spores, leads to the pollution of nutrient media based on "albumin milk" with heat-resistant microflora.

 Therefore, in the production of basic nutrient media for the cultivation of microorganisms, it is preferable to use whey concentrates, for example, containing 40-60% solids (see, in particular: TU 49-195-72) or having a residual moisture content of not more than 5% (see TU 49-189-72).

 Of the known bases of this type, the basis of nutrient media for the cultivation of microorganisms - with the goal of microbiological synthesis of L-lysine) according to French patent 2526808 is the closest to the one proposed by technical essence.

 This base is a partial hydrolyzate of concentrated whey, which is obtained using enzymes acting on hydrocarbons (for example, beta-galactosidase).

 Such enzymes convert the disaccharide (lactose) into a mixture of monosaccharides (glucose and galactose), present in the target product and against the background of unchanged proteins, mineral salts and vitamins.

 Accordingly, during the cultivation of microorganisms in liquid nutrient media prepared on the basis of this hydrolyzate, only the absorption of carbon from carbohydrate sources significantly increases. Assimilation of protein nutrients is possible insofar as cultured strains are able to assimilate them independently. It should be noted that most industrial strains producing amino acids and vitamins and some strains producing antibiotics do not contain proteolytic enzymes and therefore only free amino acids and oligopeptides are utilized, the concentration of which in this hydrolyzate is negligible.

 In connection with the foregoing, the invention is based on the task of improving the concentrated whey hydrolyzate to create a basis for a culture medium for the cultivation of microorganisms that would be most fully assimilated in the processes of microbiological synthesis.

The problem is solved in that the basis of the nutrient medium for the cultivation of microorganisms, which is a hydrolyzate of concentrated whey, according to the invention is a hydrolyzate, which is obtained by deep acid hydrolysis of this serum in the presence of 2.0-5.0N mineral acid at a temperature of 90-125 ^o C for 12 to 1 hour

 This hydrolyzate contains essentially a mixture of amino acids and monosaccharides with an admixture of vitamins and salts of mineral and organic acids customary for the specified raw materials. Thus, the digestibility of almost all nutrients of whey by cultivated microorganisms is significantly increased.

 The first additional difference is that the hydrolyzate is obtained in the presence of 2.0-4.0 N sulfuric acid, which is most preferable both in technological and in consumer aspects. Indeed, sulfuric acid can be introduced into these raw materials in a highly concentrated (up to 96%) form, which makes it possible to obtain a target product more saturated with nutrients. Further, the low volatility of sulfuric acid virtually eliminates the need for trapping its vapor in the hydrolyzate process. And finally, salts (especially ammonium sulfate) obtained by neutralizing sulfuric acid are usually more easily absorbed by most cultivated microorganisms.

Further, the invention is illustrated:
- specific data on the qualitative and quantitative composition of the proposed basis of a nutrient medium for the cultivation of microorganisms.

- a detailed description of the method for its preparation with specific examples of its implementation and
- the test results of the basis of nutrient media according to the invention in microbiological synthesis processes.

 Averaged data on the qualitative and quantitative composition of the proposed framework are given in table. 1 in comparison with the data for the most complete in terms of the complex of nutrients similar basics of nutrient media for the cultivation of microorganisms.

 As can be seen from the table. 1, the proposed framework, inferior to the BVK hydrolyzate in the total concentration of amino acids and in the concentration of most individual amino acids, significantly exceeds the BVK hydrolyzate in the presence of monosaccharides that are easily assimilated by microorganisms-producers. Moreover, the apparent advantage of the BVK hydrolyzate in comparison with the hydrolyzate according to the invention in terms of amino acid content is “neutralized” by almost twice as much ammonium sulfate content. Therefore, the hydrolyzate of BVK, when introduced into culture media for the cultivation of microorganisms, has to be substantially diluted.

 If we take into account that BVK itself is an expensive product of microbiological synthesis and that such a synthesis of BVK is environmentally very dangerous, then it is clear that the utilization of (also environmentally hazardous in its pure form) whey provides significant advantages.

 In the General case, the method of manufacturing the proposed basis of a nutrient medium for the cultivation of microorganisms is as follows.

 In the initial whey concentrate, on the basis of passport data or by the results of additional laboratory analysis, the dry residue is determined and, if necessary, adjusted to about 40% by weight (since it is impossible to hydrolyze the dry whey concentrate, and concentrates with a greater than 40% dry residue technologically inconvenient due to low fluidity).

 The indicated possible dilution of the raw materials can be carried out both before preparation and during the preparation of acidic working solutions for hydrolysis.

 Further, in compliance with well-known safety rules, the aforementioned acidic working solutions are prepared on the basis of a strong mineral (preferably hydrochloric or, most preferably, sulfuric) acid so that the normality is in the range of 2.0-5.0 N (and for sulfuric acid - in narrower range 2.0-4.0N).

Then the complex of milk proteins and galactose contained in the prepared acidic working solutions are subjected to deep hydrolysis at a temperature of 90-125 ^o C for 12 to 1 h, that is, the faster the higher the temperature.

 The described method differs significantly from the known (see, for example, Gaurovits F. Chemistry and protein functions. - M .: MIR, 1965, p. 36-40) of the method for the complete hydrolysis of protein-containing materials in 6N hydrochloric acid by boiling samples in sealed glass ampoules for 72 hours for analytical needs. These differences relate primarily to hydrolysis conditions (temperature, lack of oxygen, acidity of the medium, duration, use of HCl only as a catalyst) and, accordingly, the quality of the resulting product. Indeed, the neutralization of the known hydrolyzate before introduction into the culture medium will lead to an excessive concentration of chlorides.

 To justify the intervals of the modes of execution of the described method, a series of experiments were conducted.

 The experiments of the 1st series were aimed at establishing the permissible and preferred ranges of acidity of working solutions, the formulations of which corresponded to those indicated in table. 2.

 Working solutions were prepared in 250 ml flasks. In options 1-5, water was first poured into the flask, and in option 6 - 40% whey concentrate. Then, with continuous stirring, sulfuric acid was supplied through a dropper.

Flasks with solutions were placed in an autoclave and kept at a temperature of 125 ^° C for 2 hours. The depth of hydrolysis of carbohydrates (lactose) was estimated by the glucose content, and the depth of protein hydrolysis by the total nitrogen content in the hydrolyzate.

 Analyzes of hydrolysates showed that the glucose content in all cases was 10.5%, which indicates the complete hydrolysis of lactose, regardless of the acidity of the working solutions.

The final concentration of amine nitrogen in the hydrolysates is presented in column 6 of the table. 2. An analysis of these data indicates that:
- an acidity of less than 2N is insufficient for the effective hydrolysis of milk proteins;
- acidity of more than 5N does not increase the efficiency of hydrolysis of milk proteins;
- acidity at the 4N level is quite sufficient for the effective hydrolysis of milk proteins and is more acceptable from the point of view of the subsequent utilization of ammonium salts in the processes of cultivation of microorganisms.

 The experiments of the 2nd series were aimed at establishing the permissible temperature range of hydrolysis.

 Working solutions, as in the 1st series, were prepared in 250 ml flasks according to the recipe of the 3rd option from the table. 2. The hydrolysis temperature was set as indicated in the table. 3. During hydrolysis, the flasks in options 3.1, 3.2 and 3.3 were equipped with a reflux condenser and heated in an oil bath, and in options 3.3, 3.5 and 3.6 they were autoclaved. Hydrolysis was carried out for 12 hours

The final results of the analyzes of hydrolysates are presented in columns 3 and 4 of the table. 3 confirm that hydrolysis temperatures below 90 ^° C and above 125 ^° C are ineffective.

 The experiments of the 3rd series were aimed at establishing an acceptable interval for the duration of hydrolysis. For this, two groups of experiments were carried out at different hydrolysis temperatures.

Working solutions, as in the 1st series, were prepared in 250 ml flasks according to the recipe of the 3rd option from the table. 2. The hydrolysis temperature was set at levels corresponding to the numbers of options from the table. 3 (that is, for examples containing in the numbers a combination of the numbers "3.5" - 125 ^o C and "3.2" - 90 ^o C). Accordingly, the same table. 3, hydrolysis used reflux refrigerators or autoclaves.

 The results of the analysis of the finished hydrolysates are given in table. 4.

As can be seen from the table. 4, the duration of hydrolysis of less than 1 h leads to a sharp decrease in the depth of hydrolysis of proteins even at a temperature of 125 ^o C and to a no less sharp decrease in the efficiency of hydrolysis of lactose at a temperature of 90 ^o C. Further, hydrolysis for more than 12 hours at 90 ^o C does not increase amino acids and monosaccharides .

 The suitability of the concentrated whey hydrolyzate as the basis of nutrient media for the cultivation of microorganisms was carried out for the synthesis of different amino acids using different producer strains.

 Microbiological synthesis of L-lysine.

Microbiological synthesis was performed in 750 ml Erlenmeyer flasks. In the experiments used: 2 ml of washout with physiological solution of a 24-hour culture of Brevibacterium sp. VNIIgenetika-90 grown on jambs of meat and peptone agar - as a seed; 20 ml of culture media that were prepared:
a) using the proposed base in the form of a concentrated whey hydrolyzate (hereinafter referred to as KMS hydrolyzate), neutralized with ammonia before mixing with other conventional sources of nutrients (this medium is designated below as “experimental” with the addition of, if necessary, digital indices to indicate compositional options ) and
b) using, as the most effective of the known bases, the already mentioned BVK hydrolyzate (such a medium is designated below as “control”).

Flasks with nutrient media were sterilized for 30 min at a temperature of 121 ^° C, adjusted to pH 7.2 by dropwise addition of a 25% sterile ammonia solution, and seed was introduced.

Fermentation was carried out at a temperature of 30 ^° C. for 72 hours on a shaker at 250 rpm with an amplitude of 50 mm.

 In the prepared culture fluid, the lysine concentration (in g / l) and the lysine yield (as a fraction of the consumed sugar) were determined.

 The composition of these media (in% by weight) and the results of the analysis of the culture fluid are given in table. 5.

 Similarly to the above, microbiological synthesis of lysine was carried out with the producer strain Brevibacterium lactofermentum NITIA-88 on nutrient medium Experimental-1. The concentration of lysine in the culture fluid was 51 g / l with a yield of 0.44 for the control and 56.4 g / l and a yield of 0.45 for the experimental nutrient medium.

 As can be seen from the table. 5 and non-tabular data, in all these cases, the proposed basis of nutrient media is more effective and allows the rejection of a deficient corn extract as an ingredient in nutrient media.

 Microbiological synthesis of tryptophan.

 The inoculum was the inoculum of Bacillus subtilis VNII-genetics-15. As a control, a nutrient medium was used in accordance with the industry regulations of the USSR Ministry of Health and Biotechnology ODA N04688648-001-93, and as an experimental medium, an environment based on KMS hydrolyzate.

 The composition of the media (indicating the concentration of ingredients in g / l) and the results of the analysis of the culture fluid are given in table. 6.

In preparing the culture media, the urea solution was sterilized by filtration through a membrane, and the solution of the remaining components was autoclaved at a temperature of 121 ^° C for 30 minutes. 30 ml of culture medium and 2 ml of inoculum were added to 750 ml Erlenmeyer flasks. Fermentation was carried out at a temperature of 37 ^° C for 48 hours on a shaker at 250 rpm and an amplitude of 50 mm.

 As can be seen from the table. 6, in the microbiological synthesis of tryptophan, the hydrolyzate KMS according to the invention is completely equivalent to the corn extract as the basis of the nutrient medium.

 Microbiological synthesis of chlortetracycline and vitamin B12.

For the synthesis used a nutrient medium based on the hydrolyzate KMS according to the invention, which had a composition, wt.%:
Vegetable starch - 2.0
Hydrolyzate KMS - 2.0
Ammonium Nitrate - 0.7
Sodium Chloride - 0.2
Calcium carbonate (chalk) - 0.4
Cobalt Chloride - 0.00005
Sunflower oil - 0.1
The indicated medium in an amount of 50 ml was introduced into 750 ml Erlenmeyer flasks and sterilized at 121 ^° C for 30 minutes. Then, benzyl thiocyanate was added to the flasks at the rate of 1.5 μg / ml of medium and 2.5 ml of mycelium of the Actinomyces aureofaciens chlortetracycline producer strain were sown.

Fermentation was carried out for 56 hours at a temperature of 28 ^° C. on a shaker at 250 rpm and an amplitude of 50 mm.

 Analysis in the culture fluid revealed 6000 units / ml of chlortetracycline and 1.5 μg / ml of vitamin B12.

 Industrial applicability of the invention is justified by the above examples. In addition, it should be noted the environmental effect of recycling dairy industry waste, which often fell into industrial wastewater and polluted surface water.

Claims (2)

1. The basis of the nutrient medium for the cultivation of microorganisms, which is a concentrated whey hydrolyzate, characterized in that the hydrolyzate is obtained by deep acid hydrolysis of the specified serum in the presence of 2.0 - 5.0 N mineral acid at 90 - 125 ^o C for 12 to 1 h and contains at least 50% glucose and galactose and a total of at least 13500 mg% amino acids calculated on the dry residue.  2. The base according to claim 1, characterized in that the hydrolyzate is obtained in the presence of 2.0 - 4.0 N sulfuric acid.

Images