Strain fusarium sambucinum - producent of fungal protein biomass. RU patent 2511427.

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is strain of Fusarium sambucinum, deposited in VKPM collection under number F-1161. Claimed strain is producent of protein food biomass.

EFFECT: invention makes it possible to accumulate biomass with high protein content with higher quantity of valuable unsaturated fatty acids, complete in composition.

2 tbl, 3 ex

The technical field

The invention relates to biotechnology and pharmacology and the receipt of a new strain-producer of edible mushroom protein biomass. Most effectively present invention can be used in the production of mushroom protein biomass by the method of liquid-phase submerged culture in the food and medical industry.

The level of technology

Among the producers of mushroom protein biomass obtained by the liquid-phase submerged culture, known strains of birth Agaricus [Kyoungju Kirn et al. (2011). Bioproduction of mushroom mycelium of Agaricus bisporus by commercial submerged fermentation of the production of meat analogue.// J Food Sci Agric, Vol.91, pp.1561-1568], Pleurotus [Jin-Wu Zhong. Studies on submerged fermentation ofPleurotus tuber-regium (Fr.) Singer - Part 1: physical and chemical factors affecting the rate of mycelial growth and bioconversion efficiency./ Jin-Wu Zhong, Peter C.K. Cheung, Ka-Hing Wong, Nian-Lai Huang// Food chemistry. - 2003. - V.81. - pp.389-393], Panus [EN 2186851, C12P 21/00, C12N 1/14, C12N 1/14, C12R 1:645, publ. 10.08.2002], Pholiota [El-Makhzangy A. Propagation of three mushrooms genera in submerged culture of mango stone infusion./ A. El-Makhzangy// Alexandria journal of food science & technology. - 2004. - V.1. - No.2. - pp.23-29], Fusarium [US 4501765, A23J 3/00, publ. 26.02.1985; US 4466988, A23J 1/18, publ. 21.08.1984; Weibe, 2004. Myco-protein from Fusarium venenatum: a well-established product for human consumption.// Appi Environ Biotechnol, vol. 58, pp.421-427], Neurospora [US 4938972, A23L 1/00; publ. 03.07.1990; Murray Moo-Young, Yusuf Chisti, Dagmar Vlach (1993). Fermentation of cellulosic materials to mycoprotein foods.// Biotech Adv., Vol.11, pp.469-479], strains of procedure (:family Chonaephoraceae (can trispora, Gilbertella persicaria), Cunninghamellaceae (Absidia pseudocylindrospora), Mortierelaceae (Mortierella alpina) and tuberculariaceae, genus Fusarium (Rhizopus stolonifer, Rhizopus miehei, Rhizopus pusillus, Rhizopus oligosporus, Rhizopus oryzae; Mucor heimalis, Mucor rouxii; Rhizomucor meihei)and Phycomyces blakesleeanus [US 7045160 B1, A23J 1/18, A23J 3/20, publ. 16.05.2006] and other

Known strains-producers of protein biomass Pleurotus ostreatus ACIM F-697 [EN 2092548, C12N 1/14, C12P 21/00, C12N 1/14, C12R 1:645, publ. 10.10.1997] and Pleurotus ostreatus ACIM F-720 [EN 2126831, C12N 1/14, SR 21/00, C12N 1/14, C12R 1:645, publ. 27.02.1999], cultivated in the underlying conditions for the various liquid media (Wednesday with peptone, molasses, soya-starch, corn starch, potato-glucose and mineral environment with glucose). Mass fraction of protein in biomass proposed strains is from 40 to 50% of the AFM. The disadvantage of these strains is relatively low biomass productivity - from 0,21 to 0.37 g·l·h -1 , and the scarcity of biomass such indispensable amino acids, as cystine and valine. The wide range of non-traditional flavors of biomass can also be a disadvantage, as it is necessary for the variation of the composition of the nutrient medium depending on the further use of biomass.

Known strains-producers of protein biomass Pleurotus ostreatus 2-204 ACIM F-811 [RU 2189395, SR 21/00, C12N 1/14, C12N 1/14, C12R1:645, publ. 20.09.2002] and Panus tigrinus 3-204 ACIM F-810 [EN 2186851, SR 21/00, C12N 1/14, C12N 1/14, C12R 1:645, publ. 10.08.2002], cultivated in the underlying conditions for the various liquid nutrient mediums. The disadvantages of these strains are quite low content of crude protein in biomass (from 28 to 40% AFM), and relatively poor fatty acid composition (relative content of unsaturated fatty acids in biomass is: oleic (18:1 ) - 19,5 and 23.2%; linoleic (18:2 ) - 7,1 and 13.6%, respectively; linolenic (18:3 ) - 1,3%).

Known to use as producers of protein biomass strains of Penicillium genus: Penicillium nonatum and Penicillium chrysogenum [US 3912825, A23J 1/00, publ. 14.10.1975]. Deep cultivation strains carried out on a variety of raw materials of plant origin, such as feed wheat, hydrolysates potato, sugar beet molasses, bagasse and/or citrus waste. The disadvantage of the proposed strains is relatively low content in biomass crude protein - from 42 to 46% of ACM (total nitrogen on Kjeldal from for 6.81 to 7.38%, the conversion rate - 6,25).

Known to use as producers of protein biomass strains of the genus Fusarium: Fusarium solani, Fusarium oxysporum and Fusarium frost [US 4466988, A23J 1/18, publ. 21.08.1984]. The closest to the proposed strain is a strain of Fusarium frost Schwabe ATCC 20334 [US 4347, AN 15/00, publ. 12.12.1978], now identified as Fusarium venenatum ATCC PTA-2684 [Wendy T. Yoder and Lynne M. Christianson, 1998. Species-Specific Primers Resolve Members of Fusarium section Fusarium. Taxonomic Status of the edible "Quom" Fungus Reevaluated.// Fungus genetics and biology, Vol.23, pp.68-80], cultivated in the underlying conditions for the various carbon substrates (glucose, maltose, cane molasses, starch-containing substrates and their hydrolysates). The maximum growth rate of producer strain in the deep culture is 0,28 h -1 [Anthony P.J. Trinci, 1992. Myco-protein: A twenty-year overnight success story.// Mycol. Res. - 1992. - Volume 96(1). - pp.1-13]. Mass fraction of protein in biomass is 45 to 54% (of total nitrogen on Kjeldal - from 7.2 to 8.6%, the conversion rate - 6,25). The disadvantage of this strain is relatively high content in the lipid fraction of biomass palmitic acid (13 g/kg AFM) [Mycoprotein GRAS Notification, 2001], which limits the number of product consumed in the diet, as palmitic acid contributes to the increase in blood cholesterol of low density and subsequently causes such diseases as vascular thrombosis, atherosclerosis, diseases of the cardiovascular system [Nicolosi R.J. Effects of specific fatty acids (8:0, 14:0, cis-18:1, trans-18:1) on plasma lipoproteins, early atherogenic potential, and LDL oxidative properties in the hamster. /Nicolosi R.J., T.A. Wilson, Rogers E.J., Kritcheysky D// The journal of lipid research. - 1998. - Volume 39(10). - pp.1972-1980]. The relative content of valuable unsaturated fatty acids in biomass is relatively low and amounts: oleic (18:1 ) - 14%; linoleic (18:2 ) - 43%; linolenic (18:3 ) - 9%, and the total number of unsaturated fatty acids is 78% of the total fatty acids. Another disadvantage of the proposed strain is high content of RNA in biomass (from 7 to 12% AFM), which significantly reduces acceptable for human consumption the amount of biomass (not more than 2 g / day) and requires stage of denuklearizatsii.

Thus, at the present time is not known for producing strains of food protein biomass, capable of high-speed growth to accumulate high protein biomass with a high number of valuable unsaturated fatty acids and relatively low content of nucleic acids.

Disclosure of the invention

A new strain of Fusarium sambucinum D-104 characterized the maximum specific growth rate in conditions of liquid-phase submerged cultivation (from 0.28 to 0.32 h -1 ), significant content of proteins in biomass (from 50% to 63% AFM) and the increased number of valuable unsaturated fatty acids (from 83 to 87% of the amount of fatty acids). Strain provides a low content of nucleic acids in biomass (4 to 5% AFM).

Strain Fusarium sambucinum Fuck. var. ossicolum (Berk. et Curt.) D-104, deposited in the collection of the PMBC number F-1161, selected on the basis of productivity in the process of deep cultivation on starch-containing environments strain D-002 obtained in 1981 as a strain VSB-917, deposited in the collection of the PMBC number F-169 as Polyporus squamosus and subsequently identified as Fusarium sambucinum Fuck. var. ossicolum (Berk. et Curt.) Bilai. Thus, the strain F. sambucinum D-104 (ACIM F-1161) is closely related non-pathogenic methoxynaphthalene strain Fusarhim sambucinum VSB-917 (ACIM F-169), which is confirmed by the data of PCR fingerprint.

Cultural morphological and physiological-biochemical peculiarities of the strain. On agar wort (7th Ball) of the colony in the early growth grayish-white in color with a well-developed flocculent-felt aerial mycelium. With time in the light acquire a pinkish hue. The territory of the colony less dense, slightly pressed. The size of colonies on agar wort at a temperature of 28 degrees C over 4 days is 68-70 mm Aerial mycelium is well developed, grayish-white in color, then rozovyi. During aging becomes light pinkish-brown color, then okhranye shades until light brown. Sporodochia and pinotti does not form. Substrate mycelium not painted. Reversum unchanged. Pigments in the environment does not emit, exudation does not form.

Strain grows on glycopeptides agar, glukozamina agar, mesopatamia agar, environment Saburo. On mesopatamia agar substrate mycelium, aerial mycelium weak, pressed, the edge of the colony pressed. On diagnostic environments (rice, potato), purple, violet, crimson shades are missing. On the rice forms mycelium salmon color that during aging acquires okhranye shades to dark brown, rice colors in brown tones. On the slices of potato forms pinkish, with aging olive-ohrany to dark brown mycelium.

Physiological-biochemical signs. Aerobe. Actively growing in the temperature range from 22 to 34 C, in the range of pH 3.5-7,0. Optimum temperature of cultivation (27 of + / -1)OC S. Disposes of glucose, fructose, sucrose, xylose, lactose, galactose, maltose, raffinose, and mannitol, xylitol, ethanol, glycerin. Starch hydrolyzes. For nitrogen nutrition uses peptone, urea, ammonium salts, nitrates. For growth does not need amino acids. Has a high rate of growth in the deep culture in products with products of processing of various types of grain (wheat, rye, triticale), potatoes, sugar beet and cane juice roots and green part of Jerusalem artichoke, turnips, beetroot, and other species and their hydrolysates.

The applicability of this strain is illustrated by examples.

Example 1

Culture of Fusarium strains sambucinwn D-104 kept on stubble agar wort in test tubes at a temperature of +4oC with periodic re-seeding every 6 months. The cultivation of seed were carried out in aseptic conditions with temperature of 26 to 28 degrees Celsius in katalozni flasks with a volume of 250 ml, containing 100 ml of culture medium of the following composition: sucrose - 30,0 g/l, corn extract - 10.0 g/l, NH 4 NO 3 - 3.0 g/l, KH 2 PO 4 - 1,0 g/l, MgSO 4 ·H 2 O - 0.1 g/l, ZnSO 4 ·H 2 O - 0.01 g/l, water-pipe water - the rest, pH 5,8, through the preservation of culture with solid nutrient media (jamb) at the rate of 1 jamb 1 catalogno flask - the first passage. The duration of the growing seed: the first passage - 96 h, the second - 48 h, the third and subsequent 24 hours

Deep cultivation was carried out in the periodic mode in a fermentation apparatus 30 litres displacement of 20 l at a temperature of 26 to 28 C, the initial pH value of 5.8, the excessive pressure 0,4 MPa, aeration in sterile conditions 1.0 l/l/min with mechanical agitation 400 rpm mixer turbine type on a nutrient medium the above composition. Wednesday was inocularea mycelial suspension (the second and the subsequent passages) in the amount of 10% by volume.

The growth rate of culture of strain-producer was 0.28 h -1 .

The chemical composition of the biomass, % to the AFM:

Total protein - 56,5;

Lipids - 7,8;

Nucleic acids of 4.1.

The content of unsaturated fatty acids amounted to 83.5% of the amount of the LCD.

Example 2

The cultivation of seed were carried out in aseptic conditions at temperature from 26 to 28 degrees Celsius in katalozni flasks with a volume of 250 ml, containing 100 ml of culture medium of the following composition: molasses sugar beet - 40,0 g/l (2% PB), ammonium nitrate - 3.0 g/l, potassium phosphate one-deputizing - 1,2 g/l, water-pipe water - the rest, pH 5,8, through the preservation of culture with solid nutrient media (jamb) at the rate of 1 jamb 1 catalogno flask - the first passage. The duration of the growing seed: the first passage - 96 h, the second - 48 h, the third and subsequent 24 hours

Deep cultivation was carried out in periodic mode in a fermentation apparatus 30 litres displacement of 20 l at a temperature of 26 to 28 C, with the initial pH value of 5.8 (the excessive pressure 0,4 MPa, aeration in sterile conditions 1.0 l/l/min with mechanical agitation 400 rpm turbine agitator type) on a nutrient medium of the following composition: molasses sugar beet - 40,0 g/l (2% PB); NH 4 NO 3 - 3.0 g/l, KH 2 PO 4 - 1,2 g/l, propanol B-400 - 0.4 g/L. Wednesday was inocularea mycelial suspension (second and subsequent passages) in the amount of 10% by volume.

The growth rate of culture of strain-producer was 0.28 h -1 .

The chemical composition of the biomass, % to the AFM:

Total protein - 60,5;

Lipids - 6,9;

Nucleic acid - 4,5.

The content of unsaturated fatty acids amounted to 84.7 per cent of the amount of the LCD.

Example 3

The cultivation of seed were carried out in aseptic conditions at temperature from 26 to 28 degrees Celsius in katalozni flasks with a volume of 250 ml, containing 100 ml of culture medium of the following composition: wheat starch - 30 g/l, NH 4 NO 3 - 4.5 g/l, KH 2 PO 4 - 1.8 g/l, corn extract condensed - 10 g/l, water-pipe water - the rest, pH 5,8, through the preservation of culture with solid nutrient media (jamb) at the rate of 1 jamb 1 catalogno flask - the first passage. The duration of the growing seed: the first passage - 96 h, the second - 48 h, the third and subsequent 24 hours

Deep cultivation was carried out in periodic mode in a fermentation apparatus 30 litres displacement of 20 l at a temperature of 26 to 28 C, the pH mode-when statorvane the value of 4.0 (gauge pressure 0,4 MPa, aeration in sterile conditions 1.0 l/l/min with mechanical agitation 400 rpm turbine agitator type) on a nutrient medium of the following composition: wheat starch - 30,0 g/litre; the ammonium nitrate - 4.5 g/l; potassium phosphate one-deputizing - 1,8 g/l; corn extract condensed - 10.0 g/l, propanol B-400 - 0.4 g/L. the Starch was exposed to the primary enzyme hydrolysis drug amilosubtilin in the calculation of 0,024% to volume of a medium (4.8 g/l). Wednesday was inocularea mycelial suspension (second and subsequent passages) in the amount of 10% by volume.

The growth rate of culture of strain-producer was 0,32 h -1 .

The chemical composition of the biomass, % to the AFM:

Total protein - 63,0;

Lipids - 6,2;

Nucleic acid - 5,0.

The content of unsaturated fatty acids was 87,0% of the amount of the LCD.

Essential amino acids

The content of amino acids in the ideal protein FAO/who % of the amount of amino acids

The content of amino acids in biomass strain F.sambucinum, in % of the amount of amino acids

Isoleucine

Methionine+cystine

Phenylalanine+tyrosine

Tryptophan

Threonine

Lipid fraction mycelium strain Fusarium sambucinum D-104 contains a large number of phospholipids (up 33% from the amount of lipids), which indicates its food-balanced. The total number of polyunsaturated fatty acids is from 83 to 87% of the total amount of fatty acids in the biomass, which is greater than that in strain-analog (content of polyunsaturated fatty acids in strain Fusarium frost Schwabe ATCC 20334 is 78% of total fatty acids). The structure of unsaturated fatty acids brings lipid complex of biomass to the most valuable vegetable oils, particularly soya oil and wheat germ oil (table 2).

Lipid fraction soybean oil, % of the amount LCD

Lipid fraction of oil and wheat germ % of the amount LCD

Lipid fraction of biomass strain F.Sambucinum, % of the amount LCD

Palmitoleic

21,0-23,0

Linoleic

51,0-52,0

Linolenic

Arachidonic

Sourahindaby

Strain Fusarium sambucinum ACIM F-1161 producer of protein food biomass.