RO116205B1 - PROCEDURE FOR OBTAINING AN ACTIVE PRODUCT, FOR HUMAN USE, FOR ADJUSTMENT OF INTESTINAL FLOUR - Google Patents

Abstract

The invention relates to a process for the production of an active product for human use for the regulation of intestinal flora by biosynthesis with the aid of the microorganism Bacillus subtilis, comprising the steps of: obtaining the inoculum by cultivating the ICCF-77A strain on a medium containing corn extract and mineral salts at 30 ... 32 ° C for 18 ... 20 hours, bioprocess itself by vegetative development on an identical medium and under the same conditions with a maximum sporulation of more than 90% of the bacterial population, between 22-26 hours, decanting and centrifugation at 3000 rpm for 20-30 minutes, cooling, drying the lymphophilized sporulated biomass to give a powder of 1.7. 10 ... 2. 10spori / gram, crude protein with 8 ... 15% dry substance and free amino acids: aspartic acid, valine, leucine, gluconic acid, alpha-alanine and heavy metals 0,001%.

Description

RO 116205 B

The present invention relates to a process for obtaining a product for human use, for regulating intestinal flora.

The microorganism Bacillus subtilis is used therapeutically to regulate the intestinal flora, due to its ability to sporulate and release, at the moment of germination, enzymes, in a fairly large quantity. Ingested in sporulated form, Bacillus subtilis crosses the stomach and duodenum without being altered by gastric HCl, pepsins and pancreatic enzymes. Germination is maximal at the ileocecal region and continues along the colon.

During this journey, Bacillus subtilis exerts its specific properties, positively influencing the intestinal microflora.

Bacillus subtilis spores, during germination, release two types of enzymes: paradigestive enzymes and bacteriolytic enzymes.

Paradigestive enzymes, synthesized by Bacillus subtilis during germination, help break down carbohydrates, lipids, and proteins in the body.

Bacteriolytic enzymes directly lyse pathogenic microorganisms [Ex: Proteus, pathogenic Staphylococci, etc.) by delipidating the external wall.

The highly polymorphic enzymatic equipment explains the effectiveness of the microorganism in regulating intestinal disorders and intestinal meteorism, the most common symptoms of fermentative diarrhea.

Worldwide, there are known processes for obtaining products active in regulating intestinal flora, using the Bacillus IP 5832 strain. These products have the disadvantage that the Bacillus IP 5832 strain is sensitive to chemotherapeutic agents, such as those from the quinolone class (nalidixic acid, norfloxacin) and some antibiotics such as, for example, from the tetracycline class - doxycycline.

The process for obtaining an active product for human use, for regulating the intestinal flora through biosynthesis with the help of the microorganism Bacillus subtilis, according to the invention, consists in that it comprises the phases of: obtaining the inoculum by cultivating the Bacillus subtilis ICCF-77A strain on a medium containing corn extract and mineral salts, at 3O...32°C, for 18...20 h, the bioprocess itself by vegetative development on an identical medium and under the same conditions, with maximum sporulation in a proportion of over 90% of the bacterial population between 22...26 h, decantation and centrifugation at 3000 rpm, for 20...30 min, under cooling, drying the sporulated biomass by lyophilization, obtaining a powder with 1.7x1O ^1o ...2.1O ¹² spores/gram, crude protein with 8...15% dry matter and free amino acids: aspartic acid, valine, leucine, gluconic acid, a-alanine and heavy metals below 0.001%.

The process for making the active product, according to the invention, has the following advantages:

- Bacillus subtilis ICCF-77A spores achieve maximum germination (over 99%) in a 3 h shorter time interval compared to known products;

- increased resistance to chemotherapeutic agents (such as nalidixic acid, norfloxacin);

- widening the range of resistance to tetracyclines (doxycycline).

According to the invention, a product for human use is obtained through biosynthesis, useful and active in normalizing the acidophilic and anaerobic intestinal flora, and the neutrophilic microflora.

An example of an embodiment of the invention is given below.

Claims (1)

RO 116205 B Prepare an inoculum medium composed of the following ingredients: corn extract 1 g (50% d.u.); ammonium sulfate 0.1 g; calcium chloride sicc 0.01 g; manganese sulfate 0.001 g; magnesium sulfate 0.05 g and tap water ad. 100 ml, pH 6.5.,,6.7 corrected with sol. NaOH 40%. Sterilization of the inoculum medium is done at 120°C for 30 min. 50 The sterilized inoculum medium is inoculated with 1% v/v suspension obtained from 5 ml of sterile distilled water, in which the vegetative culture of Bacillus subtilis ICCF77A from a maintenance culture tube is resumed. The development of the seeded inoculum medium takes place at 30...32°C for 18...20 h. 55 Finally, the developed inoculum appears as a gray liquid, with a characteristic odor, non-foaming, with a faint ring of sporulated film on the walls of the bottle. Microscopically, the inoculum appears as a pure culture containing vegetative forms and a small number of released spores. In general, these spores tend to agglomerate in large, refrigerating clumps. ⁶⁰ With this inoculum, the bioprocess medium consisting of: corn extract 3.0 g (50% d.u.) is seeded in a ratio of 0.5...1.0% v/v; ammonium sulfate 0.1 g; calcium chloride dry 0.01 g; manganese sulfate 0.001 g; magnesium sulfate 0.05 g; tap water ad. 100 ml; pH 6.5...6.7. Sterilization 30 min at 125°C. The bioprocess takes place in a reactor equipped with a turbine-type stirrer, with 65 air bubblers, at the following parameters: temperature 32...33°C, air 1 1/1 medium/min, pressure 0.5 atm. During the bioprocess, the following are monitored: pH evolution; bacterial biomass concentration; dissolved oxygen consumption; degree of sporulation and purity of the culture. After an interval of 22...26 h, the fermentation medium presents the following 70 characteristics; pH 8.2...8.5: spores over 90% and rare vegetative forms. The number of spores is approximately 1. 1O ¹⁰ cfu/ml. The resulting fermentation medium is processed by decantation, centrifugation at 3000 rpm for 20...30 min and drying by lyophilization. The final lyophilized product is presented in the form of a light white-gray powder, non-hygroscopic, with a characteristic taste and odor. The product contains a concentration of 1.7. 10 ¹⁰ ...2.10 ¹⁰ spores/g crude protein 8...15% on dry matter, free amino acids: aspartic acid, valine, leucine, gluconic acid, α-alanine and heavy metals below 0.001%, permissible limit for human use drugs. Claim Process for obtaining an active product for human use, for regulating the intestinal flora through biosynthesis with the help of the microorganism Bacillus subtilis, characterized in that it comprises the phases of: obtaining the inoculum by cultivating the ICCF-85 77A strain on a medium containing corn extract and mineral salts, at 30...32°C, for 18...20 h, bioprocess itself by vegetative development on an identical medium and under the same conditions, with maximum sporulation in a proportion of over 90% of the population RO 116205 B bacterial between 22 and 26 h, decantation and centrifugation at 3000 rpm, for 20...30 90 min, under cooling, drying of the sporulated biomass by lyophilization, obtaining a powder with 1.7x1O ^1o ...2. 10 ¹² spores/gram, crude protein with 8...15% dry matter and free amino acids: aspartic acid, valine, leucine, gluconic acid, α-alanine and heavy metals 0.001%. President of the examination committee: chemist Hăulică Mariela Examiner: biochemist Crețu Adina Computerized editing and proofreading - OSIM Printed at: State Office for Inventions and Trademarks