Production method of glucan-chitosan complex from yeast biomass of brewing waste

FIELD: biotechnologies.

SUBSTANCE: production method of glucan-chitosan complex from yeast biomass of brewing waste involves mechanical and ultrasonic treatment of yeast biomass, destruction of proteins by treatment of the obtained suspension using alkali reagents with further extraction of a target product. As biomass, Saccharomyces living yeast is used. First, yeast is frozen to -15°C during 24 hours. After mechanical destruction, biomass is treated for 15 minutes at 20°C in an ultrasonic bath with frequency of an emitter of 35 kHz and voltage of 285 W. Biomass is acidified with chlorhydric acid till pH=5.5 and treated with ferment preparation in the amount of one pellet containing lipase - 3500 units of Ph.Eur., amylase - 4200 units of Ph.Eur. and protease - 250 units of Ph.Eur. per kilogramme of biomass in terms of dry substance; then, lipid components of yeast are removed. Fermentation is performed at t=20-29°C during 30-60 minutes. Destruction of proteins is performed at 55°C by means of a water bath during 60 minutes by treatment using 4% water solution of caustic soda at the ratio of yeast biomass and alkali, which is equal to 1:4. The medium is neutralised and hydrosol of glucan-chitosan complex is deposited by centrifugation during 10 minutes. The deposit is dried at t=55°C during 48 hours.

EFFECT: invention allows improving the quality of the obtained complex and its biological activity.

3 ex

 

The invention relates to the field of biotechnology, and in particular to methods of allocation glucan-chitosan complex, and can be used in laboratory and industrial practice to get glucan-chitosan complex from yeast waste food brewing industry, for further use in medicine as a component of dietary fiber, Microbiology and in the production of polymeric materials.

Known "Method of production of glucan-chitosan complex, including the state of deproteinization mycelium chitin-containing microscopic fungus treatment with dilute sodium hydroxide solution and deacetylation concentrated sodium hydroxide solution at elevated temperature, using mycelium of the fungi selected from the group including Aspergillus niger, Blakeslia trispora, Actinomyces roseum, P.chrysogenum, and deproteinization perform two to four treatments at 60-90°C, after which the reaction mass is treated with a dilute solution of hydrochloric, nitric or phosphoric acid and washed from fats and lipids in organic solvent, and the deacetylation spend 20-29%sodium hydroxide solution at 110-125°C for 30-120 minutes

RF patent for the invention №2043995, IPC: SW 37/08, publ. 1995.09.20.

The well-known "Method of obtaining chittangong complex, including the cultivation of the fungus Aspergillus nier with subsequent treatment of the mycelium diluted and concentrated solutions of NaOH, the hillshade from alkali and freeze drying of the target product. Thus obtained after culturing mycelium pre-exposed to 2 N. hydrochloric acid, then treated with a mixture containing 0.1% detergent, 0.1 ethanol(96%) on the basis of 2% NaOH solution, at a ratio of complex reagents and mycelium 3:1, as a concentrated solution of NaOH used it 20-25%solution and the treatment is carried out for 1-2 hours.

RF patent for the invention №2121505, IPC: SR 19/04, publ. 1998.11.10.

The disadvantage of these methods is the use of waste products of the antibiotics and citric acid, represented by microscopic fungi mycelium, with the amounts of waste (mycelial biomass) is disproportionately small compared to the waste from the brewing industry, which on average brewing companies in Russia up to 20-25 tons per day. Also the disadvantages include the complexity of technology pretreatment of biomass, as well as a multi-stage and multiple processing, resulting in additional financial costs. The need for the use of concentrated solutions of alkali, more than 20% at a temperature of over 100°C, furthermore, multiple flushing alcohol solutions ethanol entails additional costs.

The closest analogue to the method proposed in the invention the technical solution is the Way to obtain beta-glucans in cell walls of yeast", characterized by the fact that yeast cells destroy the mechanical method, a suspension of the washed cell walls treated with ultrasound at a frequency of 18 to 30 kHz. Preferably 18-22 kHz, related polysaccharides destroy by treatment with alkali or enzyme and precipitation produce the target product.

RF patent for the invention №2216595, IPC: C02F 3/34, publ. 2007.12.10.

The technical result consists in increasing the quality glucan-chitosan complex and its biological activity by obtaining it from yeast waste food brewing industry.

The technical result is achieved in that the Method of obtaining glucan-chitosan complex of the yeast biomass waste brewing production" includes mechanical and ultrasonic treatment yeast biomass, the destruction of proteins by treating the resulting suspension with an alkaline reagent, followed by separation of the target product. And as yeast biomass using waste from the brewing industry in the form of live yeast "Saccharomyces cerevisiae". When this yeast pre-slowly frozen to a temperature of -15°C for 24 hours. After mechanical destruction of the biomass is placed in an ultrasonic bath with frequency oscillator 35 kHz and a power of 285 watts and process for 15 minutes at 20°C. the resulting biomass of popcicle the t hydrochloric acid to pH=5.5 and treated with the enzyme preparation in the number one tablet, containing the lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur. and protease - 250iu Ph.Eur. 1 kg of yeast biomass in terms of dry substance, then remove the lipid components of the yeast. When the fermentation process is carried out at t=20-29°C for 30-60 minutes Further destruction of proteins is carried out using alkaline components at a temperature of 55°C in a water bath for 60 minutes with 4%aqueous sodium hydroxide solution at a ratio of biomass-based yeast and alkali equal to 1:4. Then neutralize the environment and precipitate the Hydrosol glucan-chitosan complex by centrifugation for 10 min. the precipitate in the form of chitin-glucan complex is dried at t=55°C within 48 hours.

The main product containing the lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur. and protease - 250iu Ph.Eur., is Mezim® Forte, one of the drugs for the correction of exocrine pancreatic insufficiency.

The method is as follows: waste brewing production, containing a mixture of different strains of Saccharomyces cerevisiae in the form of biomass (30-35% in terms of dry substance) slowly freeze within 24 hours at the temperature of 15°C, this results in the preferential growth of large ice crystals, which contribute to the destruction of the yeast cells. Then the frozen biomass split into not the large chunks of size (1×1×1 cm). Were placed in a conical flask with a small amount of cold water (t=4°C). The flask is still frozen chunks of yeast prevented in an ultrasonic bath with frequency oscillator 35 kHz, 285 watts and were treated by Ultrazvuk for 15 minutes at a water temperature in the bath of 20°C. this procedure was complete destruction of the yeast cells, since under the action of ultrasonic vibrations created cavitation effect. Cavitation bubbles in the liquid, pulsing and zahlebyvayas near cellular structures, led to destruction, and yet still not melted ice crystals (acting as "explosive submunitions") inside and outside cells resulted in more greater destructive effect.

At the first station of deproteinization and removal of lipid components obtained biomass was acidified with hydrochloric acid to pH=5.5 and added enzyme preparation 1 tab., for example Mezim® Forte, one of the drugs for the correction of exocrine pancreatic insufficiency, it contains: lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur., protease - 250iu Ph.Eur.) 1 kg of yeast biomass (in terms of dry substance). The fermentation process was carried out in a water bath at t=25°C for 30 minutes

Then, the resulting biomass-based yeast was mixed with NaOH solution. The final destruction of proteins (deproteinization) and removal of lipid substances carried and in 4%aqueous caustic soda solution at a ratio of biomass-based yeast and alkali 1:4, at temperature t=55°C on a water bath for 60 minutes At this at the end of the process in the flask are separated liquid into 2 fractions. The top layer is transparent, oily liquid (light brown liquid) and the lower layer of colorless liquid (in the form of a slightly transparent suspension). The solution was cooled. Shared by dekotirovaniem two fluids. Later used the bottom layer of the liquid.

Then the resulting solution was brought to neutral reaction with hydrochloric acid. This Hydrosol yeast chitin-chitosan complex was settled during the day, because the particles of chitin-glucan complex is very small. The process of sedimentation of particles of chitin-glucan complex was precipitated by centrifugation for 10 min at 3000g. After centrifugation formed a dense precipitate is light gray. Then the obtained chitin-glucan complex was dried in a dry-heat Cabinet at t=55°C within 48 hours.

Examples of specific performance of the method for production of chitin-glucan complex.

Example 1. Live biomass of yeast 1 kg slowly froze in the refrigerator up to t=-15°C within 24 hours. Then the frozen biomass was split into small pieces of size (1×1×1 cm). Were placed in a conical flask with a small amount of cold water (t=4°C). The flask is still frozen chunks of yeast was placed is in an ultrasonic bath frequency oscillator 35 kHz, 285 watts (peak power 300 W) and treated with ultrasound for 15 minutes at a water temperature in the bath of 20°C. the Obtained biomass was acidified with hydrochloric acid to pH=5.5, the addition of the enzyme preparation 1 table. (ingredients: Lipase - 3500 IU Ph.Ew., amylase - 4200 IU Ph.Eur., protease - 250iu Ph.Eur.) 1 kg of yeast biomass (in terms of dry substance). Fermentation at t=25°C for 30 minutes the resulting mass of yeast was mixed with 4%aqueous NaOH solution at a ratio of yeast and alkali 1:4 at a temperature of t=55°C on a water bath for 60 minutes and Then the solution was cooled and brought to a neutral reaction with hydrochloric acid. This Hydrosol yeast chitin-chitosan complex was settled during the day, because the particles of chitin-glucan complex is very small. After a day of liquid above the sludge decantation, pasty residue is light gray in color was placed in a dry-heat Cabinet. Then the chitin-glucan complex was dried in a dry-heat Cabinet at t=55°C for 72 hours. The mass of chitin-glucan complex was 80 g, i.e., the output amounted to 8% by weight of the original yeast biomass.

Example 2. Live biomass of yeast 1 kg slowly froze in the refrigerator up to t=-15°C within 24 hours. Then the frozen biomass was split into small pieces of size (1×1×1 cm). Were placed in a conical flask with a small amount of cold in the water (t=4°C). The flask is still frozen chunks of yeast was placed in an ultrasonic bath frequency oscillator 30 kHz, 285 watts (peak power 300 W) and were treated by Ultrazvuk for 25 min when the water temperature in the bath of 20°C. the Obtained biomass was acidified with hydrochloric acid to pH=5.5, the addition of the enzyme preparation 1 table. (ingredients: Lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur., protease - 250iu Ph.Eur.) 1 kg of yeast biomass (in terms of dry substance). Fermentation at t=29°C for 30 minutes the resulting mass of yeast was mixed with 4%aqueous NaOH solution at a ratio of yeast and alkali 1:4 at a temperature of t=50°C water bath for 90 minutes and Then the solution was cooled and brought to a neutral reaction with hydrochloric acid. The process of sedimentation of particles of chitin-glucan complex was precipitated by centrifugation for 10 min at 3000g. After centrifugation were formed dense pasty precipitate light gray in color, was placed in a dry-heat Cabinet. Then the chitin-glucan complex was dried in a dry-heat Cabinet at t=55°C for 48 hours. The mass of chitin-glucan complex was 10 g, i.e., the output amounted to 10% by weight of the original yeast biomass.

Example 3. Live biomass of yeast 1 kg slowly froze in the refrigerator up to t=-20°C within 24 hours. Then the frozen biomass was split into small piece and size (0,5×0,5×0,5 cm). Were placed in a conical flask with a small amount of cold water (t=4°C). The flask is still frozen chunks of yeast was placed in an ultrasonic bath frequency oscillator 25 kHz, 285 watts (peak power 300 W) and were treated by Ultrazvuk for 30 min at the temperature of the water in the bath of 20°C. the Obtained biomass was acidified with hydrochloric acid to pH=5.5, the addition of the enzyme preparation 1 table. (ingredients: Lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur., protease - 250iu Ph.Eur.) 1 kg of yeast biomass (in terms of dry substance). Fermentation at t=20°C for 60 minutes resulting mass of yeast was mixed with 4%aqueous NaOH solution at a ratio of yeast and alkali 1:4 at a temperature of t=60°C water bath for 45 minutes and Then the solution was cooled and brought to a neutral reaction with hydrochloric acid. The process of sedimentation of particles of chitin-glucan complex was precipitated by centrifugation for 10 minutes at 3000g. After centrifugation were formed dense pasty precipitate a light grey color. The sediment contained in the tube after centrifugation was mixed with ethyl alcohol 96% in the ratio of 1:1 (to remove residual lipophilic substances), then put the tube in a horizontal shaker and shake for 30 minutes, Re-centrifuged using centrifugation for 10 min at 3000g. Received OS the dock was dried at 25°C for 8 hours. Then the chitin-glucan complex was dried in a dry-heat Cabinet at t=50°C for 24 hours. While the resulting product was lighter in comparison with products 1 and 2 options. The mass of chitin-glucan complex was 10 g, i.e., the output amounted to 10% by weight of the original yeast biomass.

Offered as an invention of "a method of obtaining a glucan-chitosan complex allows the use of cheap waste brewing production, containing a mixture of different strains of Saccharomyces cerevisiae. This, ultimately, contributes to a significant reduction of the obtained product and increase profitability glucan-chitosan complex.

The method of obtaining glucan-chitosan complex of the yeast biomass waste Breweries, including mechanical and ultrasonic treatment yeast biomass, the destruction of proteins by treating the resulting suspension with an alkaline reagent, followed by separation of the target product, characterized in that as the yeast biomass using waste from the brewing industry in the form of live yeast Saccharomyces cerevisiae, the yeast pre-slowly frozen to a temperature of -15°C for 24 h, and after mechanical destruction of the biomass is placed in an ultrasonic bath with frequency oscillator 35 kHz and modestly W and treated for 15 min at 20°C, the resulting biomass is acidified with hydrochloric acid to pH=5.5 and treated with the enzyme preparation in the number one tablet, containing the lipase - 3500 IU Ph.Eur., amylase - 4200 IU Ph.Eur. and protease - 250iu Ph.Eur. 1 kg of yeast biomass in terms of dry substance, then remove the lipid components of yeast, the fermentation process is carried out at t=20-29°C for 30-60 min, and the further destruction of proteins is carried out using alkaline components at a temperature of 55°C in a water bath for 60 min by treatment with 4%aqueous sodium hydroxide solution at a ratio of biomass-based yeast and alkali equal to 1:4, then neutralize the environment and precipitate the Hydrosol glucan-chitosan complex by centrifugation for 10 min, the precipitate in the form of chitin-glucan complex is dried at t=55°C for 48 hours



 

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