RU2495930C1 - Method of production of glycosidase inhibitor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: fermentation of nutritional medium is carried out based on corn starch hydrolysates by actinomycete. Then ultrafiltration of the obtained filtrate of the culture liquid is carried out with an activity of 4250±250 IU/cm³ through membranes with cut-off of 5 kDa. The filtrate obtained is concentrated under vacuum of 0.082 mPa at a temperature of 25-30°C. It is clarified at the temperature of 30-40°C. Its ultrafiltration is carried out again through the membrane with cut-off of 1 kDa. The obtained glycosidase inhibitor is dried and ground.

EFFECT: invention enables to obtain the glycosidase inhibitor with increased activity.

1 tbl, 4 ex

Description

The invention relates to the food industry, namely food biotechnology, and relates to a method for producing a glycosidase inhibitor intended for use as a biologically active and special-purpose food additive in functional products of the dietary direction

A known method of producing a carbohydrate glycosidase inhibitor from a culture fluid with an activity of 3000 ± 200 IE / cm ³ or 6500 ± 500 IE / cm ³ obtained using actinomycetes (Streptomyces BKM Ac 1328-D and Streptomyces 839/64), including acidification of the culture fluid up to pH 3.6 ± 0.1 units, centrifugation, ultrafiltration through a UAM-300 cellulose acetate membrane (hollow fibers) at a pressure of 2.5-3.0 atm, depigmentation on anion exchange resin, lyophilization, which allows obtaining an inhibitor preparation with an activity of 130 ± 100 and 250 ± 200 IE / mg (Akulova N.Yu. Inhibitors of α-glucosidases from S treptomyces. Isolation and properties: abstract of dissertation ... Ph.D. in Biology: 03.00.04; AOOT NIITIAF. - St. Petersburg, 1993. - 20 p.).

However, the processes of preparation of ion exchangers and their regeneration, based on the use of, in particular, acids and alkalis, are laborious and are accompanied by a large amount of waste.

The closest to the invention is a method for producing a glycosidase inhibitor from a culture fluid filtrate obtained by fermentation of corn starch hydrolysates with actinomycetes (Streptomyces lucensis VKPM Ac-1734) and having an inhibitory activity of 3700 ± 180 IE / cm ³ , including purification of the culture fluid filtrate with a protein content of 5 , 0 ± 0.2 g / dm ³ , reducing sugars 6.0 ± 0.3 g / dm ³ , chroma 8.2 ± 0.4 e.p. by ultrafiltration on a VPU-15PA filtering apparatus equipped with a membrane with a cut-off of 15 kDa (hollow fibers) at a temperature of 20 ° C, concentrating the filtrate at a temperature of 90 ° C, clarification at a temperature of 25 ° C, drying at 55 ± 2 ° C and grinding (Khodkevich OA Development of technology for the biosynthesis of an α-glycosidase inhibitor by actinomycetes of the genus Streptomyces and the use of complex food additives based on it in bakery: abstract of dissertation ... candidate of technical sciences: 05.18.07; GU VNIIPAKK, GOU VPO SPb GU NiPT. ”- SPb., 2009. - 16 p.).

The known method allows to obtain a preparation of a glycosidase inhibitor with an activity of 700,000 ± 1000 IE / g However, ultrafiltration through a membrane with a cut-off of 15 kDa does not completely separate the ballast protein impurities, which at high temperature during denatured concentration denature and complicate the clarification process. In addition, as a result of concentration at high temperature, the color of the concentrate increases not only due to the concentration of colored impurities, but also due to the caramelization of reducing sugars, which also reduces the efficiency of clarification. The sequence of stages “concentration → clarification” specified in the method does not allow to reduce the protein content, which is an impurity and interferes with the inhibitory effect of the isolated preparation.

The technical result of the invention is to obtain a glycosidase inhibitor with increased activity.

The technical result of the invention is achieved by a method of producing a glycosidase inhibitor from a culture fluid filtrate obtained by fermenting a nutrient medium based on corn starch hydrolysates with actinomycetes, including ultrafiltration of the culture fluid filtrate, clarification and subsequent drying and grinding, in which according to the invention the culture fluid filtrate is used with an activity of 4250 ± 250 IE / cm ³ , ultrafiltration is carried out through membranes with a cut-off of 5 kDa, ultraviolet clarification Litrate is carried out immediately after ultrafiltration at a temperature of 30-40 ° C, then concentration under vacuum of 0.082 MPa at a temperature of 25-30 ° C and ultrafiltration through a membrane with a cut-off of 1 kDa is carried out.

Information confirming the possibility of achieving a technical result of the invention is presented in the examples.

In the examples, a culture fluid filtrate is used, which is obtained by fermenting a medium based on a corn starch hydrolyzate with actinomycete Streptomyces lucensis VKPM Ac-1734 or Streptomyces violaceus VKPM Ac-1743 and filtering the culture liquid.

The filtrate of the culture fluid is a liquid, the inhibitory activity of which is 4100-4500 IE / cm ³ , the protein content of 2-6 g / dm ³ , the content of reducing sugars 5-10 g / dm ^{3 the} optical density D at λ _max 360 nm is 8.1-11.3 e.o.p., characterizing the color of the solution.

Example 1

A filtrate of the culture fluid with an inhibitory activity of 4100 IE / cm ³ , a protein content of 2 g / dm ³ , a content of reducing sugars of 5 g / dm ³ , D at λ _max 360 nm is 8.1 e.p.

The filtrate of the culture fluid in an amount of 1000 cm ^{3 is} purified by ultrafiltration through a membrane with a cut-off of 5 kDa and a solution of 0.9 dm ^{3 is} obtained. The ultrafiltrate is clarified by mixing with it with a suspension of activated carbon at the rate of 0.5 g of coal per 1 dm ^{3 of} solution at a temperature of 40 ° C, and the resulting suspension is stirred for 30 minutes. The suspension is filtered and the clarified solution is concentrated under vacuum of 0.082 MPa (615 mm Hg) and a temperature of 25 ° C to a volume of 0.2 dm ³ . The resulting concentrate is purified by ultrafiltration through a 1 kDa cut-off membrane and a solution of 0.15 dm ^{3 is} obtained, which is dried at a temperature of 40 ° C and ground. In a dry inhibitor preparation, inhibitory activity is determined.

The results of the experiment are presented in table 1.

Example 2

Purification of the filtrate of the culture fluid, concentration, purification of the concentrate of the concentrate, drying and grinding is carried out according to example 1.

A filtrate of the culture fluid with inhibitory activity of 4300 IE / cm ³ , a protein content of 6 g / dm ³ , a content of reducing sugars of 10 g / dm ³ , D at λ _max 360 nm is 11.3 e.p.

Clarification of the ultrafiltrate is carried out with activated carbon in an amount of 1.0 g per 1 DM ³ solution at a temperature of 30 ° C for 60 minutes.

The experience data are presented in table 1.

Example 3

Purification of ultrafiltrates, clarification, drying and grinding is carried out according to example 1.

A filtrate of the culture fluid with an inhibitory activity of 4500 IE / cm ³ , a protein content of 4 g / dm ³ , a content of reducing sugars of 8 g / dm ³ , D at λ _max 360 nm is equal to 8.2 e.p.

Concentration is carried out at a temperature of 30 ° C.

The experience data are presented in table 1.

Example 4 (prototype)

A filtrate of a culture fluid with an inhibitory activity of 4100 IE / cm ³ , with a protein content of 5 g / dm ³ , a content of reducing sugars of 6 g / dm ³ , D at λ _max 360 nm is 10.5 e.p.

Purification of the filtrate of the culture fluid and clarification of the ultrafiltrate is carried out according to example 1. For ultrafiltration using a membrane with a cut-off of 15 kDa. Then the ultrafiltrate is concentrated at a temperature of 90 ° C to a volume of 0.1 dm ³ and clarified at a temperature of 25 ° C. The clarified solution is dried at a temperature of 55 ° C and ground. In a dry inhibitor preparation, inhibitory activity is determined.

The experience data are presented in table 1.

The results obtained in examples 1-4 by the proposed method and in example 5 by the prototype show that the sequence "ultrafiltration → clarification" and the use of membranes with a cut-off of 5 and 1 kDa in examples 1-4 can completely remove the ballast protein fraction and reduce color of solutions. Using the proposed method provides an improvement in the quality of the inhibitor preparation, namely, an increase in inhibitory activity of 8.6-11.4 times, compared with the prototype example and the complete removal of the protein that reduces the inhibitory effect.

Table 1 Indicators of the process of isolation of glycosidase inhibitors Example No. Name of intermediate or product Name of indicator Mass concentration, g / dm ³ , g / g * Color index, D at λ _max 360 nm, e.p. Inhibitory activity of IE / cm ³ ; IE / g * squirrel reducing sugars The culture fluid filtrate 2.0 5,0 8.10 4100 Ultrafiltrate (membrane clipping 5 kDa) 1,2 0.7 0.15 4100 one Clarified ultrafiltrate 0.8 0.7 0,03 4100 Concentrate 4.1 3.2 0.05 20,400 Ultrafiltrate (membrane clipping 1 kDa) absent 3.2 0.01 20,400 Dried ultrafiltrate absent 0.7 absent** 3000000 The culture fluid filtrate 6.0 10.0 11.3 4250 2 Ultrafiltrate (membrane clipping 5 kDa) 3,5 1,5 0.25 4250 Clarified ultrafiltrate 0.9 1,5 0,03 4250 Concentrate 4,5 7.5 0,07 21500 Ultrafiltrate (membrane clipping 1 kDa) absent 7.5 0.01 21500 Dried ultrafiltrate absent 0.8 absent** 3,500,000 3 The culture fluid filtrate 4.0 8.0 8.2 4500 Ultrafiltrate (membrane clipping 5 kDa) 2,4 1,2 0.20 4500

Clarified ultrafiltrate 0.8 1,2 0.02 4500 Concentrate 4.0 6.0 0.05 22500 Ultrafiltrate (membrane clipping 1 kDa) absent 6.0 0.01 22500 Dried ultrafiltrate absent 0.8 absent** 4,000,000 The culture fluid filtrate 5 6 10.5 4100 4 (prototype) Ultrafiltrate (membrane clipping 15 kDa) 3.2 3,5 0.65 4100 Concentrate 31.5 33.6 4.8 30600 Clarified ultrafiltrate 30.6 0.65 30600 10.6 Dried ultrafiltrate 0.5 0.4 1.3 ** 350,000 Note: * - for the dried preparation, ** - the color index is determined for the solution of dried ultrafiltrate with a mass fraction of 1%.

Claims (1)

A method of producing a glycosidase inhibitor from a culture fluid filtrate obtained by fermenting a nutrient medium based on corn starch hydrolysates with actinomycete, including ultrafiltration of the culture fluid filtrate, concentration of the ultrafiltrate, clarification, subsequent drying and grinding of the obtained inhibitor, characterized in that the culture fluid filtrate is used with an activity of 4250 ± 250 IU / cm ^3, ultrafiltration is carried out through a membrane with a cutoff of 5 kDa lightening ultrafiltrate osuschest lyayut directly after ultrafiltration at a temperature of 30-40 ° C, and then subjected to concentration under vacuum of 0.082 MPa at a temperature of 25-30 ° C and ultrafiltration through a membrane with cut-off 1 kD.