RU2511427C1 - Strain fusarium sambucinum - producent of fungal protein biomass - Google Patents

Abstract

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.

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Description

Technical field

The invention relates to biotechnology and pharmacology and relates to the production of a new strain-producer of edible fungal protein biomass. Most effectively, the present invention can be used in the production of mushroom protein biomass by liquid-phase deep cultivation in the food and medical industries.

State of the art

Among the producers of fungal protein biomass obtained by liquid phase deep cultivation, strains of the genera Agaricus are known [Kyoungju Kirn et al. (2011). Bioproduction of mushroom mycelium of Agaricus bisporus by commercial submerged fermentation of the production of meat analogue.// J Sci Food Agric, Vol. 91, pp. 1561-1568], Pleurotus [Jin-Zhong Wu. Studies on submerged fermentation of Pleurotus tuber-regium (Fr.) Singer - Part 1: physical and chemical factors affecting the rate of mycelial growth and bioconversion efficiency./ Jin-Zhong Wu, Peter C.K. Cheung, Ka-Hing Wong, Nian-Lai Huang // Food chemistry. - 2003. - V.81. - pp. 389-393], Panus [RU 2186851, C12P 21/00, C12N 1/14, C12N 1/14, C12R 1: 645, publ. 08/10/2002], 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. 02/26/1985; US 4466988, A23J 1/18, publ. 08/21/1984; Weibe, 2004. Myco-protein from Fusarium venenatum: a well-established product for human consumption.// Appi Microbiol Biotechnol, vol. 58, pp. 421-427], Neurospora [US 4938972, A23L 1/00; publ. 07/03/1990; Murray Moo-Young, Yusuf Chisti, Dagmar Vlach (1993). Fermentation of cellulosic materials to mycoprotein foods.// Biotech Adv., Vol.11, pp. 469-479], strains of the Mukorov order: families of Chonaephoraceae (Blakeslea trispora, Gilbertella persicaria), Cunninghamellaceae (Absidia pseudocylindrospora), Mortiereere) and Mucoraceae (Rhizopus stolonifer, Rhizopus miehei, Rhizopus pusillus, Rhizopus oligosporus, Rhizopus oryzae; Mucor heimalis, Mucor rouxii; Rhizomucor meihei), as well as Phycomyces blakesleeanus 1/3, 1/20 AU 1/3, US 70/3 1/18, 1/20, 1/23 US18 3/23 B23, 1/23. 05/16/2006] and others.

Known producer strains of protein biomass Pleurotus ostreatus VKPM F-697 [RU 2092548, C12N 1/14, C12P 21/00, C12N 1/14, C12R 1: 645, publ. 10.10.1997] and Pleurotus ostreatus VKPM F-720 [RU 2126831, C12N 1/14, C12P 21/00, C12N 1/14, C12R 1: 645, publ. 02/27/1999], cultivated in deep conditions on various liquid media (medium with peptone, molasses, soy-starch, corn-starch, potato-glucose and mineral medium with glucose). The mass fraction of crude protein in the biomass of the proposed strains is from 40 to 50% AFM. The disadvantage of these strains is the relatively low biomass productivity - from 0.21 to 0.37 g · l · h ^-1 , as well as biomass deficiency in such irreplaceable amino acids as cystine and valine. The presence of a wide range of unconventional aromas of biomass can also be a disadvantage, since it becomes necessary to vary the composition of the nutrient medium depending on the further use of biomass.

Known producer strains of protein biomass Pleurotus ostreatus 2-204 VKPM F-811 [RU 2189395, C12P 21/00, C12N 1/14, C12N 1/14, C12R1: 645, publ. September 20, 2002] and Panus tigrinus 3-204 VKPM F-810 [RU 2186851, С12Р 21/00, C12N 1/14, C12N 1/14, C12R 1: 645, publ. 08/10/2002], cultivated in deep conditions on various liquid nutrient media. The disadvantages of these strains are a fairly low content of crude protein in the biomass (from 28 to 40% AFM), as well as a relatively poor fatty acid composition (the relative content of unsaturated fatty acids in the biomass is: oleic (C _{18: 1} ) - 19.5 and 23, 2%; linoleic (C _{18: 2} ) - 7.1 and 13.6%, respectively; linolenic (C _{18: 3} ) - 1.3%).

Known use as producers of protein biomass of strains of the genus Penicillium: Penicillium nonatum and Penicillium chrysogenum [US 3912825, A23J 1/00, publ. 10/14/1975]. The deep cultivation of the strains is carried out on various raw materials of plant origin, for example feed wheat, potato hydrolysates, molasses, bagasse and / or citrus waste. The disadvantage of the proposed strains is the relatively low content of crude protein in the biomass - from 42 to 46% AFM (total Kjeldahl nitrogen - from 6.81 to 7.38%, conversion factor - 6.25).

Known use as producers of protein biomass of strains of the genus Fusarium: Fusarium solani, Fusarium oxysporum and Fusarium graminearum [US 4466988, A23J 1/18, publ. 08.21.1984]. Closest to the proposed strain is a strain of Fusarium graminearum Schwabe ATCC 20334 [US 4347, AH 15/00, publ. 12.12.1978], currently 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 deep conditions on various carbon substrates (glucose, maltose, reed molasses, starch-containing substrates and their hydrolysates ) The maximum growth rate of the producer strain in deep culture is 0.28 h ^-1 [Anthony PJ Trinci, 1992. Myco-protein: A twenty-year overnight success story.// Mycol. Res. - 1992 .-- Volume 96 (1). - pp.1-13]. The mass fraction of crude protein in biomass is from 45 to 54% (total Kjeldahl nitrogen - from 7.2 to 8.6%, conversion factor - 6.25). The disadvantage of this strain is the relatively high content of palmitic acid biomass in the lipid fraction (13 g / kg AFM) [Mycoprotein GRAS Notification, 2001], which limits the amount of product consumed in the diet, since palmitic acid increases low-density cholesterol in the blood and subsequently causes such diseases like vascular thrombosis, atherosclerosis, diseases of the cardiovascular system [Nicolosi RJ 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 RJ, Wilson TA, Rogers EJ, Kritcheysky D // The journal of lipid research. - 1998 .-- Volume 39 (10). - pp. 1972-1980]. The relative content of valuable unsaturated fatty acids in the biomass is relatively low and is: oleic (C _{18: 1} ) - 14%; linoleic (C _{18: 2} ) - 43%; linolenic (C _{18: 3} ) - 9%, and the total amount of unsaturated fatty acids is 78% of the total amount of fatty acids. Another disadvantage of the proposed strain is the increased content of RNA in the biomass (from 7 to 12% AFM), which significantly reduces the amount of biomass allowed for human consumption (not more than 2 g per day) and requires the stage of its denucleination.

Thus, at present, the producer protein biomass producing strain is not known, capable of high-protein biomass accumulation with a high growth rate with an increased amount of valuable unsaturated fatty acids and a relatively low content of nucleic acids.

Disclosure of invention

The new strain Fusarium sambucinum D-104 is characterized by a high maximum specific growth rate under liquid-phase deep cultivation (from 0.28 to 0.32 h ^-1 ), a significant content of protein substances in biomass (from 50 to 63% AFM) and an increased amount of valuable unsaturated fatty acids (from 83 to 87% of the total fatty acids). The strain provides a low content of nucleic acids in the biomass (from 4 to 5% AFM).

Strain Fusarium sambucinum Fuck. var. ossicolum (Berk. et Curt.) D-104, deposited in the VKPM collection under the number F-1161, was isolated on the basis of productivity in the process of deep cultivation on starch-containing media of strain D-002, obtained in 1981 as strain VSB-917, deposited in VKPM collection 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 (VKPM F-1161) is closely related to the non-pathogenic non-toxin-forming strain of Fusarhim sambucinum VSB-917 (VKPM F-169), which is confirmed by the data of PCR fingerprint.

Cultural-morphological and physiological-biochemical characteristics of the strain. On an agarized beer wort (7 ° Ball), the colony at the beginning of growth is grayish-white in color with a well-developed flocculent-felt air mycelium. Over time, the light becomes pinkish. The edge of the colony is less dense, slightly pressed. The size of the colonies on agarized wort at a temperature of 28 ° C after 4 days is 68-70 mm. The aerial mycelium is well developed, grayish-white in color, then pinkish. With aging, it acquires a light pinkish-brown color, then ocher shades to light brown. Sporodochia and pionotta does not form. The substrate mycelium is not stained. Reverse unchanged. It does not secrete pigments on Wednesday; it does not form exudate.

The strain grows on glucose peptone agar, glucose ammonium agar, meat peptone agar, Saburo medium. On meat and peptone agar, the mycelium is substrate, the aerial mycelium is weak, pressed, the edge of the colony is pressed. On diagnostic media (rice, potato slices) purple, lilac, and raspberry shades are absent. It forms a salmon-colored mycelium on rice, which acquires ocher hues to dark brown during aging, and the rice stains in brown tones. On the slices of potato it forms pinkish, with aging olive-ocher to dark brown mycelium.

On an agarized wort, hyphal aerial mycelium hyphae are 2.5–5.0 μm in diameter. In the growing zone of the colony, hyphae in the early age phase are even, long, with a homogeneous or fine-grained cytoplasm, with a small number of vacuoles, with distinctly visible cell walls. Branching at an acute angle. The tips of the gif are rounded. In the aging part of the colony, hyphae in the late age phase are inlaid with drops of oil, then with differentiation of the cytoplasm in the form of large structures (vacuoles, lipid inclusions). In the late phases, hyphae of different diameters, varying 2-4 or more times (from 2.5-3.5 microns to 12 microns). There are swellings on hyphae and chlamydospores. Chlamydospores apical and intercalary in glomeruli, knots and chains, ocher-brown, roundish, smooth, 8-12 microns.

Conidial sporulation in the form of macroconidia and microconidia on aerial mycelium, sometimes in deep culture. Macroconidia are hyaline fusiform and sickle-shaped (slightly curved), often with 3-5 septa, without constrictions. Dimensions 30-50 × 5 microns. Sickle-shaped spores with a pronounced pedicle, the upper cell gradually uniformly narrowed, slightly bent, elongated. Microconidia are unicellular or with 1-3 septa, elongated or fusiform. Sclerotia does not form.

Under conditions of deep cultivation on a nutrient medium with molasses, hyphae in the exponential growth phase are young, long, with a homogeneous cytoplasm and distinctly visible cell walls. At a low mixing speed, the mycelium is represented by free hyphae and hyphal agglomerates. GIF endings are rounded, spear-shaped or swollen. Then there is a differentiation of the cytoplasm (lipid inclusions, vacuoles), which increases with age. Large vacuoles appear. In an aging culture, hyphae are convoluted, uneven, the differentiation of cellular contents is strongly pronounced, thickening of hyphae and signs of accumulation of metabolites in the culture medium, metabolic plaques on hyphae, inlaid with metabolites appear. With prolonged storage, the formation of chlamydospores in chains occurs. GIF endings are thinned. Empty cells are present in the visual field.

Physiological and biochemical characteristics. Aerobe. Actively growing in the temperature range from 22 to 34 ° C, in the range of pH 3.5-7.0. The optimum growing temperature is (27 ± 1) ° C. It utilizes glucose, fructose, sucrose, xylose, lactose, galactose, maltose, raffinose, as well as mannitol, xylitol, ethanol, glycerin. Starch hydrolyzes. It uses peptone, urea, ammonium salts, nitrates for nitrogen nutrition. For growth does not need amino acids. It has a high growth rate in deep culture on media with products of various types of grain (wheat, rye, triticale), potatoes, sugar beets and cane, juices of root crops and green part of Jerusalem artichoke, turnip, table beet and other species, as well as their hydrolysates.

The applicability of this strain is illustrated by examples.

Example 1

The culture of the strain Fusarium sambucinwn D-104 was stored on beveled agarized wort in test tubes at a temperature of + 4 ° C with periodic reseeding every 6 months. The seed was grown under aseptic conditions at a temperature of 26 to 28 ° C in 250 ml rocking flasks containing 100 ml of the following medium: 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 , tap water - the rest, pH 5.8, by reseeding the culture from a solid nutrient medium (jamb) at the rate of 1 jamb to 1 rocking flask - the first passage. Duration of cultivation of seed: the first passage - 96 hours, the second - 48 hours, the third and subsequent - 24 hours

Deep cultivation was carried out batchwise in a 30 L fermentation apparatus with a working volume of 20 L at a temperature of 26 to 28 ° C, an initial pH of 5.8, an overpressure of 0.4 atm, and aeration with air under sterile conditions of 1.0 L / L / min with mechanical stirring 400 rpm turbine-type mixer on a nutrient medium of the above composition. The medium was inoculated with mycelial suspension (second and subsequent passages) in an amount of 10% by volume.

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

The chemical composition of biomass,% of AFM:

Total protein - 56.5;

Lipids - 7.8;

Nucleic acids - 4.1.

The content of unsaturated fatty acids was 83.5% of the total FA.

Example 2

The cultivation of seed was carried out under aseptic conditions at a temperature of 26 to 28 ° C in 250 ml rocking flasks containing 100 ml of a nutrient medium of the following composition: beet molasses - 40.0 g / l (2% by RW), ammonium nitrate - 3 , 0 g / l, monosubstituted potassium phosphate - 1.2 g / l, tap water - the rest, pH 5.8, by reseeding the culture from a solid nutrient medium (jamb) at the rate of 1 jamb to 1 rocking flask - the first passage. Duration of cultivation of seed: the first passage - 96 hours, the second - 48 hours, the third and subsequent - 24 hours

Depth cultivation was carried out batchwise in a 30 L fermentation apparatus with a working volume of 20 L at a temperature of 26 to 28 ° C, at an initial pH of 5.8 (gauge pressure 0.4 atm, air aeration under sterile conditions of 1.0 L / l / min with mechanical stirring 400 rpm with a turbine-type mixer) on a nutrient medium of the following composition: beet molasses - 40.0 g / l (2% by RV); NH ₄ NO ₃ - 3.0 g / l, KH ₂ PO ₄ - 1.2 g / l, propinol B-400 - 0.4 g / l. The medium was inoculated with mycelial suspension (second and subsequent passages) in an amount of 10% by volume.

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

The chemical composition of biomass,% of AFM:

Total protein - 60.5;

Lipids - 6.9;

Nucleic acids - 4.5.

The content of unsaturated fatty acids was 84.7% of the total FA.

Example 3

The seed was grown under aseptic conditions at temperatures from 26 to 28 ° C in 250 ml rocking flasks containing 100 ml of the following medium: wheat starch - 30 g / l, NH ₄ NO ₃ - 4.5 g / l, KH ₂ PO ₄ - 1.8 g / l, condensed corn extract - 10 g / l, tap water - the rest, pH 5.8, by reseeding the culture from a solid nutrient medium (jamb) at the rate of 1 jamb to 1 rocking flask - first passage. Duration of cultivation of seed: the first passage - 96 hours, the second - 48 hours, the third and subsequent - 24 hours

The deep cultivation was carried out in a periodic mode in a 30 L fermentation apparatus with a working volume of 20 L at a temperature of 26 to 28 ° C, in the pH-stat mode at a value of 4.0 (gauge pressure 0.4 atm, air aeration under sterile conditions 1, 0 l / l / min with mechanical stirring 400 rpm with a turbine type mixer) on a nutrient medium of the following composition: wheat starch - 30.0 g / l; ammonium nitrate - 4.5 g / l; potassium phosphate monosubstituted - 1.8 g / l; condensed corn extract - 10.0 g / l, propinol B-400 - 0.4 g / l. The starch was subjected to preliminary hydrolysis with the enzyme preparation amylosubtilin at the rate of 0.024% by volume of the nutrient medium (4.8 g / l). The medium was inoculated with mycelial suspension (second and subsequent passages) in an amount of 10% by volume.

The growth rate of the culture of the producer strain was 0.32 h ^-1 .

The chemical composition of biomass,% of AFM:

Total protein - 63.0;

Lipids - 6.2;

Nucleic acids - 5.0.

The content of unsaturated fatty acids was 87.0% of the total FA.

The amino acid composition of the biomass of the producer strain is close to the ideal FAO / WHO protein (Table 1). There is a slight limitation on sulfur-containing amino acids (methionine, cystine), while the biomass of the strain is enriched in lysine, which allows it to be combined with grain components that are deficient in this amino acid to obtain a product with a balanced amino acid composition.

Table 1 Essential Amino Acids The amino acid content of an ideal FAO / WHO protein, in% of the total amino acid The amino acid content in the biomass of the strain F.sambucinum, in% of the sum of amino acids Leucine 7.0 7.0 Isoleucine 4.0 4.0 Lysine 5.5 8.0 Methionine + cystine 3,5 3.0 Phenylalanine + tyrosine 6.0 8.0 Tryptophan 1,0 1,0 Threonine 4.0 6.0

Valine 5,0 6.0

The lipid fraction of the mycelium of the strain Fusarium sambucinum D-104 contains a significant amount of phospholipids (up to 33% of the total lipid), which indicates its nutritional value. The total amount of polyunsaturated fatty acids is from 83 to 87% of the total amount of fatty acids in the biomass, which exceeds this indicator for the analogue strain (the content of polyunsaturated fatty acids in the strain Fusarium graminearum Schwabe ATCC 20334 is 78% of the total amount of fatty acids). The composition of unsaturated fatty acids brings the lipid complex of biomass closer to the most valuable vegetable oils, in particular soybean oil and wheat germ oil (Table 2).

table 2 Soybean oil lipid fraction,% of total FA The lipid fraction of wheat germ oil,% of total FA The lipid fraction of the biomass of the strain F. Sambucinum,% of the total FA Palmitoleic - - 0.8-1.1 Oleic 24.0 21.0 21.0-23.0 Linoleic 52.0 52.0 51.0-52.0 Linolenic 9.0 10.0 8.0-9.0 Arachidonic - - 1.5-1.7 Isoarachidonic - - 0.8-1.0

Claims (1)

The strain Fusarium sambucinum VKPM F-1161 is a producer of protein food biomass.