RU2393228C2 - Food fibre manufacturing technique - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method provides for depth cultivation of basidium fungi Pleurotus ostreatus on nutrient medium of the following composition (g/l): soya flour - 21, KH₂PO₄ - 3.5, MgSO₄ - 0.4, milk whey - 200 ml, water - up to 1 litre. Mycelium is exposed to deep freezing with further defrosting. Then its disintegration is carried out. Food fibres are cleaned by alternating treatment with cold and hot water, mix of 1 M sodium carbonate or sodium hydroxide with ethanol in the ratio of 1:2 and 0.5 M solution of citric acid. Yield of food fibres makes 16-18% of dry biomass, content of chitin - 16-19%, glucan - 40-50% of dry mass of food fibres.

EFFECT: food fibres with high content of chitin.

4 ex

Description

The invention relates to the field of biotechnology, in particular to a method for producing a biological agent - dietary fiber that improves the functioning of the digestive tract.

The beginning of the 21st century was marked by an increased interest in medicinal substances obtained from mycelial fungi and aimed at treating a number of serious and difficult to cure diseases, in particular, oncological, cardiovascular, wounds of various etiologies, etc. Successes in this direction led to the creation of a new branch of medicine , in which they began to use drugs with active principles derived from mushrooms.

Until the 80s of the last century, refined and purified from complex biological components products were introduced into human food. It was believed that such substances as pectin, collagen and cellulose are not absorbed by the body, and these ballast substances, as they began to call them, are unnecessary and even harmful. Later, these ballast biopolymers became known as dietary fibers (dietary fiber, PV). By the beginning of the XXI century, it was found that the absence of PV worsens the digestive tract and leads to diseases such as diabetes, obesity, hypertension, cholelithiasis. The ability to gel and change volume depending on conditions allows PV to influence intestinal motility and chyme evacuation processes. The presence of carboxyl groups in the PV of plant origin, which participate as a buffer in the processes of changing pH, has a great influence on the functioning of the gastrointestinal tract. The same groups provide the adsorption of ions, including multiply charged cations, which form stable chelate complexes with carboxyl groups. These data led to the creation of a new theory of nutrition, in which PV played a significant role, and over the past 10 years, none of the components of food has been devoted as much research as dietary fiber. Even the so-called "food pyramid" was created, which is very popular at the present time. The basis of the pyramid are those products that contain PV - bread, cereals, potatoes and other plant products. It is believed that an adult requires 25-30 grams of PV per day.

At present, abroad, PVs have long been widely used as food additives. In 1992 alone, over 300 new types of therapeutic and diagnostic nutritional supplements with PV appeared in the USA. PV is also widely used as additives to meat and vegetable products, when baking bread, making ice cream, chocolate, sweets, fish dishes. In recent years, high molecular weight polysaccharide biopolymers that are not digested and absorbed in the gastrointestinal tract and excreted have been the focus of attention of physicians and nutritionists. PV from mycelial fungi belonging to basidiomycetes and ascomycetes is represented by β-glucans, chitin, and heteropolysaccharides (polyuronides, hemicellulose, and pectin substances). These biopolymers are also found in the cell walls of fungi, and they are supposed to be used to cleanse the stomach of carcinogens and ions of heavy and radioactive metals. In addition, these biopolymers are able to enhance gastrointestinal motility (Wasser SP, Weis AL Medical properties of substances occurring in higher Basidiomycetes mushrooms: current perspectives (review) // Int. J. Med. Mushrooms. 1999. V.1. P .31-62).

For this purpose, so far only chitosan filaments obtained from the shells of crab and krill, which are manufactured by Vostok-Bor, are currently being used. For medical purposes, it is also intended to use the chitin-glucan complex (CHC) from higher fungi. The advantage of this complex obtained from mushrooms, in comparison with analogues of crustaceans, is that the HCG of the mushrooms:

1) has a higher sorption capacity and more actively sorb ions of heavy and radioactive metals. At the same time, the sorption properties of chitin and chitosan increase significantly in combination with glucans, in particular, with highly branched β- (1-3) -glucans with (1-6) side chains present only in fungi;

2) has a pronounced ability to absorb carcinogenic compounds and, in addition, can inhibit the development of cancer metastases. In particular, fungal glucans affect the suppressive activity of helper T cells, restoring the humoral immune system;

3) enhances the "laxative" effect, which doctors currently attach particular importance to, considering the "sluggish" stomach, as a cancer risk factor;

4) is much cheaper, not related to seasonality, production technologies are environmentally friendly and provide unlimited production. To obtain them, you can use the waste of other mushroom production, for example, obtaining citric acid and carotenoids.

Several methods are known for extracting and determining the content of dietary fiber, in particular from plants (Talmadge KW, Keegstra K., Bauer WD, Albersheim P. The structure of the plant cell wall // Plant. Physiol. 1973. V.51. No. 1. P.158-173). For this purpose, the material is homogenized, then a series of extractions is carried out, removing intracellular contents from the cells by washing with phosphate buffer (pH 6.0), ethanol, distilled water with acetone. The isolated cell walls, consisting mainly of cellulose, are washed 3 times with 80% ethanol, the initial buffer, centrifuged and starch is removed by treatment with α-amylase.

There are also known methods of producing PV of animal origin, which are used as necessary components for creating new and effective food products (Neklyudov A.D. Dietary fiber of animal origin. Collagen and its fractions as necessary components for new and effective food products // Prikl. Biochemistry and Microbiology. 2003. T. 19. No. 3. S.261-272). To produce collagen fibrils called tropocollagen, the skin of young animals is extracted with cold saline or diluted acids. There is also a method of producing PV from pulp - a waste from the processing of sugar beets. To this end, the pulp undergoes heat treatment, water extraction, pressing, drying and grinding. The result is a product containing pectin, which was called "beet fibers" (Kovaleva T. Medical dietary fiber // Informkonditer. 2005. S.1-3).

The closest in technical essence and the achieved effect is a method of obtaining PV from the mycelium of basidiomycetes of the genus Pleurotus (oyster mushrooms) (Chung P. Dietary fiber content and composition of cultivated edible mushroom fruiting bodies and mycelia // J. Agric. Food. Chem. 1996, V. 44. P. 468-471). According to this method, basidiomycetes, in particular, Pleurotus sajor-caju, are grown on a medium of the following composition (g / l): potato-dextrose broth - 24, KH ₂ PO ₄ - 0.4, K ₂ HPO ₄ - 1, MgSO ₄ - 0.5 and asparagine - 2. The fungal mycelium grown on this medium is separated from the culture liquid by filtration through a glass fiber filter, washed with distilled water and freeze-dried. A dry sample of mycelium is crushed in a mill, passed through a 0.5 mm sieve and extracted with petroleum ether with stirring. Next, the dry sample is treated with two amylases: resistant to heating with α-amylase for 15 minutes in a boiling water bath, and amyloglucosidase - 1 hour at 60 ° C to remove glycogen, then with a protease to solubilize proteins. After precipitation with 78% alcohol, the ethanol insoluble precipitate is separated by precipitation, it is weighed and the weight is adjusted with the weight of the ash and the residual protein content. The yield of chitin is 10.9%.

The disadvantage of this method is the use of expensive reagents, in particular, three enzyme preparations, the possible instability of their action in a scaled production environment, which can affect the quality of PV with a deviation from the standard. Confirmation of this can also be found in the work (Cherkasova E.I., Alekseeva M.F., Pastukhov M.O., Kirilenko Yu.K., Smirnova L.A. Enzymatic isolation of chitin complexes and chitin from oyster mushroom fruit bodies // Materials 7th International Conference. S.-Pb., Repino. 2003. S. 417-419), which shows that the use of enzymes to obtain HCG from oyster mushrooms leads to a decrease in chitin content.

The objective of the invention is to develop a more efficient, cheaper and less time-consuming method of obtaining PV, providing standardization of the final product when it is obtained by a biotechnological method in the factory. In this case, PV is obtained with a high content of chitin, which is the main component of the fungus for medical purposes.

The essence of the invention lies in the fact that the pre-frozen mycelium of the basidiomycetes of the genus Pleurotus ostreatus (oyster mushrooms) grown and separated by filtration is disintegrated, then they are extracted with cold and hot water, then with alkaline and acid solutions using a mixture of an aqueous solution of soda or sodium hydroxide with ethanol and an aqueous solution of citric acid, followed by separation of the precipitate by centrifugation. In this case, mushrooms are grown on a nutrient medium containing soy flour, milk whey, monosubstituted potassium phosphate and magnesium sulfate. The yield of PV is 17-18% of the dry mass of mycelium. Obtained by this method, PV contains 19% chitin and 50% β- (1-3) -glucan.

In the proposed method for the purification of PV from residual proteins and fats contained in the oyster mushroom mycelium, chemical treatment of biomass is used, however, taking into account the food purpose of the final product, weak acids and bases recommended for food production are used.

The invention is illustrated by the following examples.

Example 1

Basidiomycetes Pleurotus ostreatus strain INMI RAS No. 3 was grown on a medium of the following composition (g / l): soy flour - 21, KH ₂ PO ₄ - 3.5, MgSO ₄ - 0.4, milk whey - 200 ml, water - up to 1 liter The medium was sterilized at 1 atm. Fermentation was carried out in 2 L flasks with 300 ml of medium at a temperature of 26-28 ° C for 4 days. The biomass yield is 25 g / l. The specified strain is isolated from basidiospores of oyster mushrooms growing on aspen. Hat basidioma dark with a purple tint. The shape of the hats is basidioma linguistic. The size of the basidioma caps is 4 to 9 cm in diameter. The biomass obtained by separation from the culture fluid was washed with water and deep frozen for 8 hours and, after thawing, it was destroyed twice on a Panasonic MK-G58PR disintegrator. Next, the biomass was subjected to the following treatments:

Stage 1 - extraction with water at room temperature for 5 minutes with stirring,

Stage 2 - double water extraction at 98 ° C with stirring;

Stage 3 - double treatment with a 1 M solution of Na ₂ CO ₃ and ethanol in a ratio of 1: 2 for 15 minutes with stirring at room temperature;

Stage 4 - treatment with a 0.5 M citric acid solution for 5 minutes with stirring at room temperature.

The yield of PV is 16-18% of dry biomass. In PV, the content of chitin is 16-19%, glucan - 40-50%. The content of polysaccharides was determined as described (Tereshina V.M., Memorskaya A.S., Feofilova E.P. et al. Preparation of polysaccharide complexes from mycelial fungi and determination of their degree of deacetylation. Microbiology. 1997. V.66. No. 1. S.84-89). The glucosamine content is 14% by weight of PV.

Example 2

The method was performed as described in example 1, but as used basidiomycetes Pleurotus ostreatus strain INMI RAS №2, instead of Na ₂ CO ₃ using a 1M NaOH solution.

The indicated strain was isolated from basidiospores of oyster mushroom growing on a birch. The cap of the basidioma is ash-gray; by maturity it acquires a yellowish tint. The shape of the caps of the basidiomas is wide-chested. The size of the basidioma hats in diameter is from 3 to 10 cm.

The yield of PV is 15.8% of dry biomass, the content of PV of chitin is 15.9%, glucan is 40.3%.

Example 3

The method was carried out according to example 1, but the extraction with cold water was carried out for 10 minutes, the extraction with hot water for 20 minutes. Treatment with sodium bicarbonate and citric acid was carried out with heating - 80-90 ° C. The yield of PV is 16.4%, the content of chitin in them is 16.4, and glucan is 43.8%.

Example 4

The method was carried out according to example 1, but the extraction with cold water was carried out for 10 minutes, hot water for 20 minutes without stirring. Alkaline treatment was carried out with 1 M NaOH for 1 hour at 80 ° C, and citric acid for 20 minutes at 98 ° C. The yield of PV is - 12.1%, the content of PV of chitin - 17%, glucan - 52%.

Claims (2)

1. A method of producing dietary fiber, including the deep cultivation of mycelium of Pleurotus basidiomycetes on a nutrient medium containing carbon and nitrogen sources, followed by disintegration of the mycelium and purification of dietary fiber, characterized in that the species Pleurotus ostreatus is used as basidiomycete, before the disintegration of the mycelium is subjected deep freezing followed by thawing, and cleaning is carried out by sequential extraction with cold and hot water, a mixture of 1M sodium carbonate solution or sodium hydroxide I with ethanol in a ratio of 1: 2 and 0.5 M citric acid solution. 2. The method according to claim 1, characterized in that the cultivation is carried out on a nutrient medium of the following composition, g / l: soy flour - 21, KH ₂ PO ₄ - 3.5, MgSO ₄ - 0.4, whey - 200 ml , water - up to 1 liter.