RU2059417C1 - Method for sterilization of nutrient media - Google Patents

Abstract

FIELD: microbiological, food and other industries. SUBSTANCE: method for sterilization of nutrient media includes heat treatment of media with preliminary acidification of media by introduction of mineral or organic acids or their combinations, neutralization after heat treatment with alkali solutions. Acids and respective alkalis are selected on the assumption of full or partial replacement of that or any other salt contained in the treated medium. EFFECT: higher efficiency. 8 cl, 3 tbl

Description

 The invention relates to the microbiological industry, and in particular to methods of sterilization of culture media intended for the cultivation of microbial and other producers.

 The proposed method can be used to sterilize nutrient media of various compositions used in microbiological and other industries, as well as for the disinfection of organic waste from livestock, poultry farms, waste from microbiological industries, sewage, soil, etc. In addition, the method can be used in the food industry to sterilize fruit and vegetable juices, mashed potatoes, wines, etc.

 It is well known that organic and inorganic components of nutrient media are largely seeded with extraneous microflora. In particular, molasses, starch and starch-containing raw materials (flour of various origin), yeast autolysates, BVK, corn extract, acidic and enzymatic protein hydrolysates, etc. are highly seeded.

 For microbiological industries, microbial contamination is of particular danger, i.e. the presence in the environment of vegetative cells and spores (especially heat-resistant spores). Viral contamination may be of slightly less importance. As for the organic waste of agricultural farms, wastewater, soil, the cells of pathogenic microorganisms, viruses, Protozoa cells, helminth eggs, and also germ (seeds) of weeds are the most dangerous.

Existing methods for the sterilization of nutrient media are based mainly on the use of high temperatures (autoclaving at 120-160 ° ^C. The disadvantages of this method are the high costs for heat sterilization and partial destruction of the organic constituents of culture media, especially sugars, running at high temperatures. Thus, during sterilization by autoclaving at ¹²⁰ ° C and above reactions are caramelization of the sugars (melaninoobrazovaniya) as well as the reaction of sugars with amino acids (melanoidins). Visually this it is manifested in the fact that the medium after autoclaving changes color (turns brown). As a result of the destruction of sugars, the yield of the final fermentation products decreases.

 Multiple attempts have been made to develop and introduce alternative sterilization methods into microbiological production. In particular, methods for the so-called chemical sterilization of culture media are described. As biocides (or decontaminants), i.e. chemical agents with the ability to kill vegetative cells and spores of microorganisms as well as viruses, the most known oxidizing agents (hydrogen peroxide, ozone, ethylene oxide, propylene oxide), halogen-containing compounds, aldehydes (formaldehyde, glutaraldehyde), sodium azide, beta-propiolactone, hydroxylamine, hydrazine , Quaternary ammonium compounds, etc. The listed compounds are widely used both individually and in the form of mixtures as disinfectants.

 As shown in the experiment, bacterial spores are 100-1000 times more resistant to heat and to chemical agents than vegetative cells. Under the action of ethylene oxide, this difference is not observed. Ethylene oxide is a very active decontaminant. Using ethylene oxide, you can sterilize agar, peptone, starch, various sugars, dextrin, glycogen (Matveev V.E. Smirnov E.V. Thermal sterilization of sugar solutions. ONTITE. Glavmikrobioprom, 1976). Beta propiolactone is also a very effective decontaminant. This substance is 25 times more effective than formaldehyde and 4000 times more ethylene oxide (cited by Matveev, 1981).

 The above bactericides are currently not used for sterilization of culture media in microbiological industries. The main reasons for this are the high toxicity of these compounds for humans and animals, the difficulties associated with the complete inactivation of biocides after sterilization (which implies the introduction of additional reagents inactivating biocides into the environment), as well as the fact that biocides interact with the components of the environment and change its composition (aldehydes, for example, interact with the amino groups of proteins and peptides, oxidizing agents change the redox potential of the environment, etc.).

 Organic and inorganic acids are also known to have bactericidal properties. In particular, it was noted that at acidic pH values, sterilization of culture media is more effective. Unlike many other biocides, acids are not toxic compounds, when they are neutralized with alkali, harmless salts are formed.

 Szepessy at all. 1983 (US Pat. No. 4,817,333) describes an acid-base seed disinfection process. In order to combat nematodes and pathogens that attack rice plants, the seeds of the latter are treated at room temperature with a solution of mineral or organic acids at a concentration of 1-10 g / l for several hours (usually 3 hours). The acid is then neutralized with alkali powder or carbonate of alkaline or alkaline earth elements. With this treatment, both nematodes and pathogenic microorganisms completely die. Plant seeds do not lose their germination.

 Ueno et all. 1983 (US Patent No. 4647458) for the sterilization of certain food products (ham, sausages, chickens, etc.), as well as machines and equipment for the food industry, offer a bactericide, consisting of a synergistic mixture of ethyl alcohol and organic plus phosphoric acid. In the proposed mixtures, the concentration of ethanol varies from 1 to 14% organic acid from 0.3 to 13% phosphoric acid from 0.03 to 0.7%. Bactericidal mixtures are quite effective and completely kill the used test cultures (E. coli cells) during 30 sec It is important to note that in the proposed mixtures, the concentration of acids should provide a solution pH below 4.0. At pH greater than 4.0, the effectiveness of the bactericidal mixture decreases. In the cited work, the authors, however, did not investigate the activity of the proposed synergistic mixtures with respect to bacterial spores. Despite the fact that the literature has repeatedly noted an improvement in the efficiency of sterilization of media during acidification, however, this issue has not been deeply and systematically studied. Sterilization methods using acidification of the media to mitigate the temperature conditions of heat treatment are not known.

Thus, despite the large number of studies devoted to the development of methods of chemical (thermochemical) sterilization, not one of the proposed methods has currently found application in microbiological production. The sterilization of culture media in microbiological plants is still carried out by heating a neutral (in the sense of pH) medium under excess pressure, i.e. at a temperature exceeding 120 ° ^C.

 The purpose of the invention is the development of a sterilization method that makes it possible to reduce the temperature of heat treatment of nutrient media and ensures absolute sterility of the media with almost complete preservation of nutrient components.

 The above goal can be achieved by acidifying the medium to pH values from 1 to 3.5.

 The method of thermochemical sterilization is as follows.

In combination with the temperature factor, organic and inorganic acids, along with bactericidal, have sporocidal activity. In this case, sporocidal activity at a given temperature is proportional to the concentration of hydrogen ions, i.e., increases with decreasing pH. Thus, at pH 3.0, complete sterilization of culture media of various compositions achieved at ⁹⁰ ° C for 40 min. With subsequent neutralization of the acid with sterile alkali, media suitable for the cultivation of microorganisms can be obtained. Thus, by acidification of culture media with mineral or organic acids can be reduced sterilization temperature of 30-40 ^{° C,} compared with sterilization at neutral pH, under the same heat treatment time.

 The proposed method of soft sterilization is inherently a type of thermochemical sterilization, i.e., it combines two methods of thermal and chemical sterilization.

Acceptable acidity, in which heat treatment is sufficient for sterilization and does not affect fermentation, depends on the nature of the producer strain, the temperature of the heat treatment and the selected acid (alkali). pH environments subjected to sterilization, may vary generally from 1 to 4.0 and a temperature ^of from 70 ^to 100 ° C at a residence time of from 30 minutes to 2 hours. The optimum pH value sufficient for complete sterilization media at 90-100 ^about C for 40-60 minutes, located in the region from 2.7 to 3.5. The consumption of acids to achieve the indicated pH depends on the composition of the nutrient media and varies from 0.1 to 2-3%. In the above pH range, the fermentation equipment of microbiological production has sufficient corrosion resistance.

Sterilization at acidic pH values within the permissible acidity range is not accompanied by the destruction of organic components of the medium (in particular, sugars and amino acids) and does not (which is very important) upon subsequent neutralization of the acid with alkali, to slow the growth rate of the producer culture or to reduce the yield of final fermentation products . Environment Dominated acid sterilization at 70-100 ^{° C,} does not visually change their pigmentation. Indeed, according to published data, sterilization of aqueous solutions of glucose as well as fructose, maltose, arabinose at acidic pH values (at pH 3-4) contributes to greater sugar conservation than at a neutral pH value (Matveev, 1981). As for sucrose, this sugar during heat treatment is most stable at pH 8.5. However, even if partial sucrose hydrolysis occurs at pH 3.0, the products of this hydrolysis of fructose and glucose are resistant to degradation and, along with sucrose, are equally well utilized by various producers. At pH values of 2.7-3.5 and at a temperature below 100 ^{° C} and no noticeable destruction of amino acids. Indeed, for example, alanine and glycine shown that the rate of decomposition of amino acids during the heat treatment (130 ° ^C) maximum at pH 5-7 and at acidic and alkaline pH values sharply decreased (VE Matveev Scientific bases microbiological techniques. M. Agroproduct, 1958). The acid sterilization regimen is also gentle on vitamins.

 An important point in the invention is the choice of acid and the appropriate base for sterilization. During soft (acid) sterilization, in many cases the mineral composition of the medium does not change, since the selection of the appropriate acids (and alkalis) for sterilization is made on the basis of the composition of a particular medium, namely, partially or completely replacing one or another salt entering the medium with appropriate acid and base. The following acids can be used for acid sterilization: hydrochloric, sulfuric, phosphoric, nitric, formic, acetic, lactic, propionic and other acids, as well as their combinations. In the event that there are no salts in the recipe of the nutrient medium, the choice of acid for mild sterilization is carried out experimentally, i.e. those acids are taken whose salts, at appropriate concentrations, do not inhibit the growth of the producer culture and do not negatively affect the yield of fermentation products.

Acid neutralization after heat treatment is carried out with sterile solutions of alkalis: NaOH, KOH, NH ₄ OH, Ca (OH) ₂ , Mg (OH) ₂ , or with carbonates of alkaline or alkaline earth elements: Na ₂ CO ₃ , K ₂ CO ₃ , CaCO ₃ , MgCO ₃ , (NH ₄ ) ₂ CO ₃ and others. The pH of the medium is adjusted to the desired optimal value for the development of a particular producer. First, a calculated amount of alkali, equivalent to the amount of previously added acid, is added, then a fine adjustment of the pH is carried out using color indicators or a pH meter.

According to the data obtained, at pH values at least above 2.5, and at room temperature, the spores of many microorganisms may not lose their viability for a very long time. Vegetative cells at acidic pH values (below 3.5), on the contrary, die quickly enough. Media with acidic pH values, thus, self-cleaning from the cells of many non-spore-forming microbes. This property of hydrogen ions can be used for short-term (for several days) storage of still non-sterile nutrient media in microbiological plants. A sufficiently rapid death of spores in the pH range of 2.5-3.5 occurs only at 70-100 ^about C.

The sporocidal activity of acids is associated with the ability of H ⁺ ions to cause DNA apurinization. This process, as is known from the literature, is effective at a pH below 4.0. The absence of the sporocidal effect of dilute acids at room temperature is apparently due to the fact that H ⁺ ions cannot penetrate through the dehydrated protein shell of spores at this temperature. At 70-100 ^C occurs hydration shell spores and H ⁺ ions reach the DNA. If the mechanism of action of acids is just this, then this means that the biocidal activity of acids is extremely high and spreads to viruses. Protozoa, helminth eggs, weed seeds, etc.

 PRI me R 1. Soft sterilization of the medium for the producer of lysine.

In experiments on the fermentation of the producer of lysine Сorynebacterium glutamicum strain VNIIgenetika 95-10, the following composition was used:
Beet molasses 26 wt.

 Corn hydrolyzate 6 vol.

K ₂ NRA ₄ 0.2%
MgSO ₄ · 7H ₂ O 0.15%
(NH ₄ ) ₂ · SO ₄ 2%
NH ₄ Cl 1.5%
CaCO ₃ 3%
The pH of the medium is 6.9 7.1, adjusted to the desired value with 40% NaOH before sterilization. The accepted sterilization mode is 0.8 atm, 30 min. During sterilization by autoclaving, molasses is diluted with tap water 1.5 times and sterilized with CaCO ₃ . The remaining components are sterilized together.

For a fermentation experiment in flasks, three 200 ml portions of medium were prepared. One portion of the medium was sterilized in an autoclave (control). The other two portions were sterilized at ⁹⁰ ° C in the presence of hydrochloric acid. For this, all components of the medium (with the exception of CaCO ₃ , which was sterilized separately) were combined in one flask, the volume was brought up to 195 ml with tap water. The amount of NH ₄ Cl introduced into the medium was reduced taking into account the amount of HCl used to sterilize the medium. Various amounts of conc. HCl (33%) to pH 2.7, in one embodiment, and pH 3.0 in another embodiment. The pH of the medium was monitored using a pH meter. Consumption conc. Hcl was about 2 vol.

After acidification and check pH solutions carefully transferred into sterile vials (500 ml) was cotton-plugged and heated in water bath at ⁹⁰ ° C for 40 min. (not taking into account the time required to heat the contents of the bottle to 90 ^° C, which is 15-20 minutes). Periodically, the vials with the medium were mixed. After temperature treatment, the medium was cooled, then the acid was neutralized with a sterile ammonia solution (25%). The pH of the medium was adjusted to 6.9-7.0 using a pH meter. The sterility test was carried out by plating the neutralized medium on plates with nutrient agar (Hottinger agar was usually used). The plates were incubated at ³⁷ ° C for 3 days. In all cases, sterility was complete.

The sterile medium was poured into 20 ml into sterile rocking flasks with previously added CaCO ₃ . The culture of the producer strain was introduced into flasks with medium. Cultivation of cells was performed on a shaker at ³⁰ ° C for 96-hours. The concentration of lysine in the medium was determined chromatographically. In the experiments performed on media subjected to mild sterilization, the lysine yield was at the control level and amounted to about 70 g / l.

 PRI me R 2. Soft sterilization of a nutrient medium for lactic acid bacteria.

To cultivate the cells of lactic acid bacteria Streptococcus faecium and Bifidum bacterium, a medium of the following composition was used,
Corn Extract 0.9 (V / V)
Beet molasses 1.5 (V / V)
K ₂ HPO ₄ 0.02 (W / V)
KH ₂ PO ₄ 0.02
NaNO ₃ 0.3
Trace elements:
MnSO ₄ , ZnSO ₄ , CoSO ₄ at 0.0005 each
CaCO ₃ 0.6
Whey up to 100; pH of the medium is 6.6-6.9.

 The sterilization mode of the neutral medium at an excess pressure of 0.8 atm for 30 minutes During sterilization, all components of the medium can be together. The pH of the medium is adjusted to the desired value before autoclaving.

For the experiment, two 300 ml portions of medium were prepared. One portion containing all the components was poured into 50 ml vials with cotton plugs (per 200 ml) and sterilized in an autoclave (control). A second portion containing all components except CaCO ₃ was sterilized in the presence of hydrochloric acid. To do this, using conc. Hcl pH of the medium was adjusted to a value of 2.7 in one case, and 3.0 in another case. The acidified medium was poured into sterile bottles (200 ml) with cotton plugs, 50 ml in each bottle. The vials were heated in water bath at ⁹⁰ ° C for 60 min. (20 minutes heating to 90 ^{° C} and incubated 40 min at this temperature). Periodically, the vials with the medium were mixed. After incubation at 90 ° ^C the medium was cooled, neutralized acid sterile solution NH ₄ OH (25%). pH was adjusted to the desired value by the indicator bromtimol blue and tested for sterility by plating aliquots on agar plates Hottingepa and by prolonged incubation media at 37 ° ^C. In all cases, sterility was complete. Prepared sterile medium for 3 days were incubated at ³⁷ ° C and germinated.

Neutralized sterile media was poured into sterile 50 ml vials, sterile chalk (CaCO ₃ ) was added and then incubated with a fresh culture of lactic acid bacteria. Cultivation of cells produced stationary (without shaking) at 37 ^{° C} for 18 hours. The cell titer was determined by plating aliquots of appropriate dilutions on plates with a meat-peptone agar (MPA) and incubating the plates at ³⁷ ° C for 18-24 hours. In the experiments performed in liquid media subjected to mild sterilization, the cell titer was: B. bifidum (1.09 1.30) x 10 ⁹ ; Str. faecium (4.12 5.95) x 10 ⁹ . Close values of cell titer were obtained in the control, i.e. on autoclaved media. Differences in the rate of crop development were also not observed.

 PRI me R 3. Mild sterilization of the medium for the producer of riboxin.

The cultivation of Bacillus subtilis cells strain VNIIgenetics 265-76 producer of riboxin was performed on a medium of the following composition,
Glucose 12
BVK 2.5
NH ₄ NO ₃ 2.0
MgCl ₂ 0.5
CaCO ₃ 2.0 pH 6.8-7.0, is added by adding 40% KOH before autoclaving. The accepted autoclaving mode is 0.8 atm. 30 minutes. When sterilizing by autoclaving, all components of the medium, except for CaCO ₃ , can be together. CaCO _{3 is} sterilized separately.

For the experiment, 1000 ml of medium was prepared. Of this volume, 200 ml of autoclave sterilization (control). In this case, the pH of the medium was previously adjusted to the desired value. The remaining 800 ml of medium was divided into 8 portions of 100 ml. To acidify the media used conc. Hcl and conc. H ₂ SO ₄ . The amount of acid added and the steady acidity of the media (pH) are presented in table. one.

After acidification, the medium was transferred into sterile bottles (200 ml) with cotton plugs. The vials were heated in water bath at ⁹⁰ ° C for 60 minutes (20 minutes heating to 90 ^{° C} and 40 min. Hold at this temperature). Periodically, the contents of the vials were mixed by shaking. After the temperature treatment of the medium, the acid was cooled and neutralized with sterile 40% KOH. The pH was adjusted to 6.8-7.0. After adjusting the pH of the medium was checked for sterility. In all cases, sterility was complete. The media could be at room temperature for at least 2 weeks and not germinate.

For fermentation, sterile CaCO ₃ and 25 ml of sterile medium were added to the rocking flasks. The media were then inoculated with a fresh producer culture. The cell culture was performed with shaking at 34 ^{° C} for 6 days. The result of the experiment is presented in table. one.

 As can be seen from the table, on media subjected to mild sterilization, the yield of riboxin increases by 3-5% compared with the control (in the control, the yield of riboxin was 35 g / l). Apparently this is due to the fact that with soft sterilization, the components of the medium, in particular glucose, are not destroyed.

Experience soft sterilization medium for producing riboksina was repeated with the difference that the heat treatment is conducted in a boiling water bath (at 98 ° ^C), with the same processing time. The result of fermentation was the same yield of riboxin was 3-5% higher than the control
PRI me R 4. Mild sterilization of the medium for the producer of inosinic acid.

The producer of inosinic acid of the Brevibacterium ammonoagenes cell strain 79-5 (VNIIgenetika) was grown on a medium of the following composition,
Glucose 12
Corn Extract 5
KN ₂ RO ₄ 3.0
MgSO ₄ 1.0
Urea 0.72
Adopted autoclaving sterilization mode at 0.8 atm 30 min. Urea is sterilized separately at 0.5 atm. 30 minutes.

 The pH of the medium is supplied after autoclaving using sterile 40% KOH to 7.0.

For the experiment, 300 ml of medium was prepared (all components without urea). One portion of the medium with a volume of 100 ml was autoclaved (control). A second portion (200 ml) was sterilized by the addition of hydrochloric acid. To this end, conc. Hcl at the rate of 1 ml of acid per 100 ml of medium. The pH of the medium was 2.33. The acidified mixture was transferred into sterile vials with cotton stoppers and heated for 1 hour in water bath at 90 ^C. After incubation temperature vials were cooled and the acid neutralized with a sterile solution of 40% KOH. Sterile urea was added to the neutralized mixture (pH about 6.0), and only then the pH of the medium was adjusted with alkali to 7.0.

 The resulting media were absolutely sterile and did not germinate upon storage for two weeks at room temperature.

Inosinic acid producer cells were cultured in rocking tubes. 10 ml of medium was added to the tube and inoculated with the producer cell culture. Cultivation was performed on a shaker with intensive aeration at 34 ^{° C} for 6 days.

 Determination of the concentration of inosinic acid in QOL was carried out chromatographically. As it turned out, the yield of inosinic acid on media subjected to mild sterilization was 3-5% higher than the level of inosinic acid in the control.

 PRI me R 5. Mild sterilization of the environment for the producer of riboflavin.

Producer of riboflavin strain Bacillus subtilis VNIIgenetika 1-304 / pMX45. In experiments on fermentation in rocking flasks, a medium of the following composition was used, BVK 2 Sucrose 10 MgSO ₄ 0.05 Urea 0.6
The pH of the medium is 6.5-7.0, adjusted with 40% KOH. The sterilization mode of the neutral medium is 0.8 atm. 30 minutes. Urea (60% solution) is sterilized separately.

For the experiment, 1000 ml of medium containing all components except urea was prepared. One portion of the medium (200 ml) was autoclaved (control). The remainder was mildly sterilized. To this end, various amounts of conc. Hcl or conc. H ₃ PO ₄ . The amount of introduced acid and the steady-state pH values are presented in table. 2.

Acidified solutions were transferred into sterile cotton plug vials. The vials with the medium was heated for 1 hour on a boiling water bath (ie. E. At 98 ^C). After heat treatment, the vials with the medium were left at room temperature overnight. Then, the acid was neutralized with 40% KOH. Before neutralization, sterile urea was introduced into the acidified medium (1 ml of a 60% solution per 100 ml of medium). The pH was adjusted to the desired value using the indicator bromothiol blue. The prepared medium was checked for sterility and then poured into rocking flasks (750 ml) 25 ml into each flask. Inoculum was added to the flasks. Culturing of the producer cells was performed at ³⁷ ° C for three days.

 The concentration of riboflavin in QOL was determined spectrophotometrically.

As it turned out (see Table 2), the yield of riboflavin on a medium subjected to mild sterilization exceeded that on a medium subjected to autoclaving by 3-4%
In another experiment, cells of the strain of the producer of riboflavin were cultured on a laboratory liter fermenter (Hitachi) with a volume of 1 l. In this case, the main medium contained, BVK 2 MgSO ₄ 0,05 (NH ₄ ) ₂ SO ₄ 0,2 Defoamer 0,1
The feeding medium contained: 1. Sugar 50 2. Corn extract 6
For the experiment, we prepared two portions of the basic medium of 700 ml and two portions of the recharge of 250 ml. In one case, 0.45 ml of conc. H ₂ SO ₄ to a pH of 2.30; in another case, 0.4 ml conc. H ₂ SO ₄ to a pH of 2.95. To 250 ml of recharge was also added conc. H ₂ SO ₄ , in one case, 0.45 ml to a pH of 2.3, in the other case of 0.35 ml to a pH of 2.65.

 After acidification, the medium was transferred into sterile vials (1.5 and 0.5 l) with cotton plugs. Vials of media were heated for 60 minutes in a boiling water bath. After the temperature treatment, the bottles were cooled at room temperature and the acidic media were checked for sterility (0.2-0.3 ml of medium was plated on plates with Hottinger agar).

 In all cases, sterility was complete.

To neutralize the acid, the calculated amount of a sterile solution of 40% NaOH was added to the vials. Fine adjustment of the pH of the fermentation medium (basic) was carried out in a fermenter using an automatic supply of a 7% solution of NH ₄ OH.

 Fermentation was carried out for 3 days with makeup and automatic subtraction to pH 7.0 with intensive aeration and stirring. In the control experiment, fermentation was carried out on a medium subjected to autoclaving.

 During the fermentations, the yield of riboflavin on media with mild sterilization was slightly higher (3%) compared with the control.

 PRI me R 6. Mild sterilization of a nutrient medium for the producer of chlortetracycline.

The fermentation experiments of the strain Streptomyces aureofaciens 737 (VNIIgenetika) were carried out on a medium of the following composition, Corn flour 8 Corn extract 0.8 Kormobakterin 0.25 NH ₄ Cl 0,4 NH ₄ NO ₃ 0,6 (NH ₄ ) ₂ HPO ₄ 0,035 Fat (fish) 3.0 CaCO ₃ 0.8 pH 6.7-6.9, added using a NaOH solution before sterilization. Sterilization mode of a neutral medium autoclaving at 0.8 atm. 30 minutes. Wednesday sealed before sterilization at 95-100 ^{° C} for 5 minutes, then to reduce the viscosity in amilorizin medium is added at a concentration of 0.001% by autoclaving all ingredients except for CaCO ₃ are present together.

For the experiment, 5 portions of medium of 100 ml each were prepared. One portion containing all the components, but without chalk, was poured into 25 ml rocking flasks and sterilized in an autoclave. (Chalk was sterilized separately). The remaining portions did not contain NH ₄ Cl and CaCO ₃ , they were sterilized by a mild method. (Chalk was introduced into rocking flasks and autoclaved). For sterilization to separate portions of the medium were added various amounts of concentrated HCl at the rate of 1.5, 1.0, 0.75 and 0.5% in mixed thoroughly, transferred to sterile vials with cotton-plugged and incubated in water bath at ⁹⁰ ° C. within 60 minutes After heat treatment, the medium was cooled to room temperature and the acid was neutralized by adding sterile 25% ammonia. (When neutralized, NH ₄ Cl is formed, which is a component of the medium). Sterility was determined by plating 0.4 ml of medium on Hottinger agar plates and in parallel by introducing 0.4 ml of the medium in the test tubes with Hottinger broth and subsequent incubation tubes at ³⁷ ° C for 3 days. In all cases, sterility was complete. The sterile medium was then poured (25 ml each) into rocking flasks containing CaCO ₃ .

Cultivation was performed at 32 ^{° C} on a shaker at 240 rev / min for 144 hours. Determination chlortetracycline concentrations produced after 2, 3-day incubation and at the end of fermentation.

 As it turned out, at a concentration of Hcl equal to 1.5 and 1% there is a slowdown in the sporulation rate compared to the control. The antibiotic yield was also reduced. At a concentration of HCl equal to 0.75 and 0.5%, the development of culture and its sporulation proceeded normally. The yield of chlortetracycline also corresponded to the control and amounted to 1500 μg / ml.

 PRI me R 7. Soft sterilization of the environment for the culture of ginseng cells.

The ginseng cell culture medium included the following components, mg / L: Casein hydrolyzate 500 Sucrose 30 Mesoinositol 80 NH ₄ NO ₃ 820 KNO ₃ 950 CaCl ₂ (anhydrous) 220 MgSO4 185 KH ₂ PO ₄ 85
Set of trace elements:
Fe chelate (FeSO ₄ · 7H ₂ O
plus Trilon B) Thiamine chloride 0.4 Alpha-naphthylacetic acid 2 Kinetin 2
The initial pH of the medium is 7.15; after sterilization, 6.6-6.7.

 The accepted sterilization mode is autoclaving at 0.8 atm for 30 minutes.

 Preparation of a nutrient medium.

 Separately, they are prepared and sterilized 1) a solution of salts (components 4-8), 2) a solution of trace elements, 3) a solution of Fe-chelate, 4) a solution of thiamine chloride, 5) a solution of naphthylacetic acid, 6) a solution of kinetin. Before sterilization, the components are mixed, the volume is adjusted to 1 liter with distilled water, the mixture is poured into 100 ml flasks and sterilized in an autoclave.

 For the experiment, 1000 ml of medium was prepared. Of this amount, 400 ml of the medium was poured into 100 ml flasks and sterilized by autoclaving (control). The remaining 600 ml of medium was mildly sterilized. For this, individual portions of the medium in 100 ml each were acidified using concentrated hydrochloric, sulfuric or phosphoric acids or a mixture of these acids. The number of acids added to the medium and pH of the medium are presented in table. 3.

 The acidified media were transferred into sterile cotton-plug vials and heated in a boiling water bath for 60 minutes. After this, the medium bottles were cooled at room temperature and the acid was neutralized with a sterile solution of 10% NaOH. The pH of the media was monitored by the bromothymol blue indicator.

Ready media were stored at room temperature for 2 weeks. Sterility was not completely germinated. Sterile media were then used to grow ginseng cell culture. Cultivation was carried out in shake flasks with 100 ml medium with vigorous shaking for 7 days at 30 ° ^C. Cell growth on culture media subjected to mild sterilization did not differ from control.

The above examples show that the proposed method ensures absolute sterility nutrient media at temperatures below 100 ° ^C at a heat treatment time of 40-60 min. The minimum pH value sufficient to achieve complete sterility in the above heat treatment mode is below 3.5. Increasing the concentration of ions [H +] (lowering of pH to 3.0 and below) can reduce heating medium temperature to 90 ^{° C} and even lower at the same time (40-60 min) of heat treatment.

 It is important to emphasize that for some media there are pH limits below which acidification of media should not be done, as this can adversely affect the producer growth rate and the yield of the final fermentation products.

The most optimal pH values for mild sterilization, at which the consumption of acids and corresponding bases is minimal, the effect (corrosion) on the fermentor equipment is minimal, the minimal destruction of the medium components and the medium composition does not change significantly. Typically this pH 2.7-3.5 (with the heating temperature of 90-100 ^{° C} and time of incubation for 40-60 min). The claimed pH of the environments for acid sterilization is 1-5.0.

At pH higher than 3.5 (in the region 3.5-5.0) heat treatment of the medium should be conducted at a temperature of above 100 ^{° C,} namely in the range of ^{100 °} C to 110-115 ^{° C} (with the same heat treatment time equal to 40-60 minutes.).

 In many cases, 0.1-0.3% (V / V) of mineral acid is sufficient to acidify the media and achieve the desired pH (from 2.7 to 3.5). In the case of molasses as well as flour media, characterized by a large buffer capacity, up to 1-2% of acid is required. However, even in these cases, the use of acid will be cost-effective and will give some savings already for the reason that the cost of acid and alkali is always lower than the cost of the corresponding salt formed during their interaction.

Thus, the proposed method due to the acidification of the medium makes it possible to reduce the sterilization temperature of 30-40 ^{° C,} which should result in significant savings in heat energy that goes to the sterilization.

 The proposed method is universal, i.e. can be used to sterilize media of the most diverse composition, suitable for the cultivation of both bacterial and fungal producers, and for the cultivation of plant and animal cell cultures. Moreover, the method is suitable for sterilization of both liquid and solid (agarized) media. In the latter case, the neutralization of the acid should be carried out at a temperature above the pour point of the agar.

 The proposed method is environmentally friendly, since the reagents used (acids and alkalis) in a concentrated form are non-toxic (although they require careful handling) and are used in the microbiological industry. In the form of dilute solutions (i.e., at pH 2.5-3.5), acids are completely harmless to the human body. The products of the neutralization of acids and alkalis (i.e. salts), on the other hand, are components of media and are used by growing cells.

The proposed method is easily scalable and can be used in the microbiological industry. Indeed, at pH 2.7-3.5 and 90-100 ^{° C} fermenternoe equipment resistant to corrosion, and the presence in the fermentors the pH sensors and devices for mixing the medium is very convenient to neutralize the acid and bring the pH to the desired value.

The soft (acid) sterilization method is suitable for use in sterilizers of various designs, including devices with ONS system for continuous sterilization. In this case, in order to reduce the time of heat treatment of the media in the incubator to 7-15 minutes, the temperature of the acidified media can be raised to 110-115 ^about C.

Mild acid sterilization practically does not destroy the components of the medium, in particular sugars, amino acids, and this makes it possible to increase the yield of fermentation products by 3-5%, which can be significant savings on the scale of microbiological production. Regarding polysaccharides (starch, dextran, etc.), Then at acidic pH and 90-100 ^{° C,} they undergo some hydrolysis to form glucose. During the heat treatment (mild sterilization) of starch-containing media, some dilution is observed (viscosity drop). The appearance of free glucose can adversely affect the productivity of some fungal producers, in particular those that are sensitive to trace amounts of glucose. To avoid this, mild sterilization should be carried out at a pH above 3.0. For other producers, the presence of glucose is indifferent.

 The high efficiency of the proposed method of soft sterilization, among other things, is associated with the ability of acids to dissolve small particles of soil that are present in many components of the medium, for example, molasses, and are not amenable to dissolution and sterilization at neutral pH values.

 An important aspect of low-temperature acid sterilization is that it can be used for the neutralization (disinfection) of waste from microbiological industries (e.g., biomass and sewage), organic waste from livestock and poultry farms, feces, soil, etc. In this case, it is desirable to use acids such as sulfuric, phosphoric, hydrochloric, or mixtures of these acids, and neutralize with ammonia so that the organic solution is enriched with mineral salts necessary for the development of beneficial microflora and cultivation of plants. When disinfecting organic waste in many cases, complete sterilization is not necessary; it is enough to kill pathogenic microflora, viruses, helminth eggs, weed embryos. Therefore, in these cases, it is possible to lower the temperature of the heat treatment by increasing the acidity of the medium and the exposure time.

The proposed method, in addition, can be used in the food industry to sterilize fruit and vegetable juices, mashed potatoes, wines, etc. In this case, it is most expedient to use hydrochloric acid from mineral acids for acidification, and to neutralize NaOH or Na ₂ CO ₃ . Since the buffer capacity of juices is very low, the consumption of acid and, accordingly, alkali will be insignificant, so that the salt (NaCl) formed after neutralization does not affect the taste of the drink.

Claims (8)

 1. METHOD OF NUTRITION STERILIZATION, including thermal treatment of the medium, characterized in that before the thermal treatment, the medium is acidified with mineral or organic acids or a combination thereof, and after thermal treatment they are neutralized with alkaline solutions to a pH value optimal for subsequent use, while acids and the corresponding alkalis are selected based on the condition of complete or partial pinching of one or another salt that is part of the medium.  2. The method according to p. 1, characterized in that the medium is acidified to a pH of 1 to 5.  3. The method according to p. 1, characterized in that the medium is acidified to a pH of 2.7 to 3.5. 4. The method according to p. 1, characterized in that the heat treatment of the acidified medium is carried out at 70 120 ^o C. 5. The method according to p. 1, characterized in that the heat treatment is carried out at 90 to 100 ^o C.  6. The method according to PP. 1, 4 and 5, characterized in that the heat treatment is carried out for 30 to 120 minutes  7. The method according to PP. 1, 4 and 5, characterized in that the heat treatment is carried out for 40 to 60 minutes  8. The method according to p. 1, characterized in that for neutralization using sterile solutions of hydroxides, carbonates and bicarbonates of alkali and alkaline earth metals and ammonia.

Images