Method for producing mycelial culture biomass from truffle mushroom microbiome

Abstract

The invention relates to biotechnology. What is proposed is a method for producing a biomass of mycelial cultures that contain unsaturated fatty acids using a truffle mushroom microbiome. The biomass is produced by artificial in vitro cultivation in laboratory or factory conditions. The method is described using the examples of cultures derived from the fruiting body microbiomes of three different truffle mushrooms: Tuber magnatum (Piedmont white truffle), Tuber melanosporum (Perigord black truffle) and Choiromyces venosus (Troitsky white truffle). A high concentration of unsaturated fatty acids is achieved by growing the mycelial biomass under conditions of restricted aeration that significantly reduce oxidation of the biochemical products. The invention makes it possible to simplify the production process and to reduce the time taken to obtain a biomass having a high content of biologically active substances (unsaturated fatty acids and antioxidants).

Description

[0001] A METHOD FOR OBTAINING BIOMASS OF MYCELIAL CULTURES FROM THE MICROBIOME OF TRUFFLE MUSHROOMS

[0002] The invention relates to biotechnology, namely to the process of obtaining mycelial biomass of fungi isolated from the microbiome of truffle fruiting bodies.

[0003] Methods for producing mycelial forms of fungal biomass are widely known, aimed at using the resulting biomass in food or medical biotechnology. In these cases, the target products in the biomass are either protein and flavor components (food biotechnology) or a specific biochemical compound (medical biotechnology). Furthermore, the resulting biomass may contain unsaturated fatty acids, which act as antioxidants.

[0004] Similar methods for producing fungal mycelial biomass "in vitro" are known (here and below, "in vitro" means not on soil plantations, not in natural conditions, but as cultures grown in laboratory or industrial settings). Examples of such similar patents include the following: [1] [2]. Both of these methods are focused on the targeted production of protein biomass, rather than the production of unsaturated fatty acids (antioxidants). Both of these methods require aeration of the medium during cultivation.

[0005] Mycelial biomass is cultivated in a similar manner in cases where the applicants do not indicate the purpose of obtaining protein. For example, patents [3], [4].

[0006] The aeration regime during cultivation in these cases is not the subject of patenting and is described indirectly, for example, as the free flow of air from the atmosphere into Petri dishes with the culture: “The mycelium of white truffles (e.g., Tuber borchii or Tuber magnatum) or black truffles (e.g., Tuber melanosporum) is first grown under sterile conditions and in Petri dishes in a culture medium favorable for its growth (e.g., in a solidified medium containing agar). (10 g / l) with the addition of malt extract (1%) or potato dextrose agar (15 g / l) with the pH adjusted to 6-5-7.0.” (US Patent US 9277760 B2).

[0007] Methods for obtaining biomass of pure cultures of truffle and other mushrooms "in vitro" by submerged cultivation are also known. This method involves growing biomass in a liquid medium accompanied by aeration (bubbling). During submerged cultivation, fungal biomass grows in a liquid medium under dynamic conditions, where the nutrient medium is continuously bubbling with air. It is generally accepted that constant aeration energetically (due to the supply of an oxidizing agent—oxygen) ensures enhanced biomass growth. Examples of these methods are given in the following sources: [5], [6], [7], [8], [9],

[0010] ,

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[0008] All of the listed methods for culturing mushrooms to obtain mycelial biomass in vitro do not consider the selection of mushroom cultures as a selection from the truffle microbiome based on the production of unsaturated fatty acids; also, all of the listed methods for culturing mushrooms to obtain mycelial biomass in vitro do not consider the production of unsaturated fatty acids (antioxidants) as a result of specified cultivation conditions for aeration and do not aim to reduce the content of the oxidant (oxygen).

[0009] In fact, none of the methods considers establishing reduced oxidizer levels based on dissolved oxygen concentrations as a patentable method. Aeration in the cited analogous methods is considered a means of increasing fungal biomass, not the content of target biochemical products (unsaturated fatty acids) in the biomass.

[0010] The closest to the proposed method in terms of the set of essential features and therefore chosen by the authors as a prototype is the method according to patent US 9277,760 "Production of natural truffle flavors from truffle mycelium" [3]. In the known method, the mycelium is grown "in vitro". In accordance with the description of this method, it provides information on the resulting composition of truffle biomass during cultivation without any limitation of access of atmospheric oxygen: "The mycelium of white truffles (e.g., Tuber borchii or Tuber magnatum) or black truffles (e.g., Tuber melanosporum) is first grown under sterile conditions and in Petri dishes in a culture medium favorable for its growth (e.g., in a solidified medium containing agar). (10 g / l) with the addition of malt extract (1%) or potato dextrose agar (15 g / l) with pH adjusted to 6-5-7.0.

[0011] The disadvantages of this prototype method in comparison with the patented method are: 1. The lack of preliminary selection of crops for the ability to rapidly grow and form unsaturated fatty acids, which leads to the lack of guaranteed production of unsaturated fatty acids in significant quantities.

[0012] 2. The absence of aeration restrictions during cultivation, which leads to proportionally greater oxidation of biochemical products of the biomass, that is, a correspondingly lower content of under-oxidized products (antioxidants, unsaturated and polyunsaturated fatty acids).

[0013] The objective of the proposed invention is to create a method for obtaining mycelial biomass of fungi isolated from the microbiome of truffle fruiting bodies that provides an increased content of unsaturated fatty acids in the target biomass.

[0014] The technical result of the proposed invention is the production of mycelial biomass of cultures with an increased content of unsaturated fatty acids isolated from the microbiome of truffle fruiting bodies.

[0015] The technical result is achieved in that in the Method for obtaining biomass of mycelial cultures from the microbiome of truffle mushrooms, including growing "in vitro" under sterile conditions on a culture medium of mycelial culture biomass, the cultures are preliminarily selected during successive passages-reseedings according to the ability to rapidly grow and according to the formation by the mycelial cultures of fatty acids exceeding 10% of the weight of dry mycelial biomass, with unsaturated fatty acids exceeding 30% of the total amount of fatty acids; and the biomass is grown under conditions of reduced oxygen content from 2 to 10%, at a temperature of 22-28°C and a humidity of 60-80% for 14-30 days; and a mixture of organic compounds containing proteins is used as a culture medium.

[0016] An additional distinguishing feature of this method is that the fruiting body of Tuber magnatum (Piedmont white truffle), Choiromyces venosus (Trinity white truffle), or Tuber melanosporum (Périgord black truffle) is used as the truffle mushroom microbiome. Examples are provided below that illustrate the invention's implementation, confirming the technical result achieved using a culture medium consisting of a mixture of organic compounds containing proteins.

[0017] In both examples, three protein nutrient media were used to cultivate mycelium isolated from the truffle microbiome in order to compare the fatty acid composition (weight % of the total analyzed) of the mycelial biomass grown under microaerobic conditions in vitro on different media with the biomass of natural fruiting bodies of the mushrooms Choiromyces venosus and Tuber magnatum.

[0018] Media with protein components:

[0019] 1) Medium T is a modification of the "semi-synthetic cheese medium" of Hansen-Nielsen (Hansen BV / , Nielsen PV 1997. Development of a semisynthetic cheese medium for fungi using chemometric methods. J. Dairy Sci. V. 80 (7): 1237-1245. doi: 10.3168 / jds.S0022-0302(97)76052-l). Our modification consists in the fact that one component (agar) is removed from the medium. Accordingly, the composition of medium T (modified Hansen-Nielsen medium), in grams per 1 liter of solution (distilled water): casein - 100, 90% lactate - 8.3, lactose - 7.9, CaCl2 х 2H2O - 7.3, MgSO4 x 7H2O - 2.6, NaCl - 26.0, FeSO4 x 7H2O - 0.025. Protein component - casein, 100 g / l.

[0020] 2) the classic Sabouraud medium for the counting of filamentous fungi is a ready-made medium accepted in practice, available commercially as a product of various companies. Composition according to (Ьдп8: / / 8Ьор.1аЬргер.ги / ргобисС / среда-сабуро-агар-биотехновация / #:~:1ех1=Состав) in grams per 1 liter of solution (distilled water): dry enzymatic peptone - 7.0, enzymatic soy flour hydrolysate - 3.0, crystalline hydrated glucose - 40.0, clarified autolyzed yeast extract - 4.0, microbiological agar - 12.0. Protein component - peptone and partially hydrolysates of soy flour and yeast - up to 14 g / l.

[0021] 3) "Nutrient medium for growing edible mushroom mycelium" in accordance with RU2013931C1 (reissuance of Soviet copyright certificate SU4941177). The composition of the medium, in accordance with the formula of this invention, in grams per 1 liter of solution (distilled water): wheat grain - 240, natural zeolite - 604, gypsum - 20, chalk - 8. Protein component - grain protein, quantity unknown.

[0022] Example 1.

[0023] Laboratory glassware (glasses used as culture chambers) were filled with a loose protein medium with a humidity of up to 30%.

[0024] Beakers containing protein medium were sterilized for 30 minutes at an elevated pressure of 0.5 atm and a temperature of 105°C. The sterilized media were inoculated with a pure culture previously obtained by microbiological isolation from the fruiting bodies of the truffle fungus Choiromyces venosus (also Choiromyces meanderingris, the Trinity white truffle).

[0025] The culture-inoculated beakers containing the media were incubated at 24°C under microaerobic conditions. Microaerobic conditions were achieved by partially sealing the culture chambers without compromising the authenticity of the inoculated cultures (i.e., without contamination by other microorganisms).

[0026] Mushroom growth was monitored visually. After 14 and 28 days of growth, samples were collected for fatty acid analysis.

[0027] For analysis, the fungal biomass was lyophilized (dehydrated at low temperatures to a dry state) and methylated.

[0028] The biomass of fruiting bodies of mushrooms grown in natural conditions was prepared in a similar manner: the fruiting bodies were crushed, lyophilized (dehydrated at a low temperature to a dry state) and methylated.

[0029] Fatty acid analysis was performed using established methods using an Agilent 5977B GC / MSD gas chromatograph with a 7890B gas chromatograph and software, using an HP-5MS column (5% phenylmethylsilicone, 30 m x 0.25 mm x 0.25 μm).

[0030] In accordance with the capabilities of the method and instrumentation:

[0031] 1) the analyzed fatty acids were identified;

[0032] 2) the ratio (weight%) in the analyzed fatty acids was determined;

[0033] 3) Taking into account the weights of the analyzed samples and the amount of fatty acids in the compared standards, the weight content of fatty acids per unit of dry matter of the fungal biomass was determined. The fatty acid analysis results for the culture grown under microaerobic conditions in vitro on protein media were compared with the fatty acid analysis results for the fruiting bodies of Ch. venosus truffle mushrooms grown in natural conditions.

[0034] The results of analyses of fatty acids identification and determination of their ratios in the biomass of Ch. venosus (Table 1, 2, 3) showed the following:

[0035] 1) the biomass of fungal cultures grown under microaerobic conditions “in vitro” contained the same and even richer spectrum of unsaturated fatty acids as the biomass of fruiting bodies grown under natural conditions;

[0036] 2) with the time of in vitro cultivation, the ratio between different fatty acids in the grown mycelial biomass changed.

[0037] Tables 1, 2, and 3 present a comparison of the fatty acid composition (weight percent of the total analyzed) of mycelial biomass grown under microaerobic conditions in vitro with the biomass of natural fruiting bodies of Ch. venosus mushrooms. Unsaturated fatty acids are highlighted in bold.

[0038] Table 1 Table 2

[0039] Table 3

[0040] The analysis of the weight content of fatty acids per unit of dry matter of Ch. venosus fungal biomass in the fungal biomass cultivated in vitro and in natural fruiting bodies (Table 4) showed the following: in general, the content of fatty acids per unit of dry biomass cultivated in vitro increased over 28 days of growth and exceeded the content in natural fruiting bodies.

[0041] Table 4 presents data comparing the fatty acid content (weight % of dry biomass) of the mycelial biomass grown under microaerobic conditions in vitro with the biomass of natural fruiting bodies of Ch. venosus mushrooms depending on the cultivation time and the type of in vitro cultivation medium.

[0042] Table 4

[0043] Example 2.

[0044] Example 2 differs from Example 1 in the object, namely, the species of truffle mushroom. In Example 2, sterilized media were inoculated with a pure culture previously obtained by microbiological isolation from the truffle mushroom Tuber magnatum (Piedmont white truffle).

[0045] All conditions and procedures for cultivation and analysis in Example 2 are the same as in Example 1.

[0046] The results of the research and analysis for Example 2 are presented in Tables 5, 6 and 7.

[0047] Table 8 presents a comparison of the fatty acid composition (weight percent of the total analyzed) of mycelial biomass grown under microaerobic conditions in vitro with the biomass of natural fruiting bodies of Tuber magnatum mushrooms. Unsaturated fatty acids are highlighted in bold. Table 5

[0048] Table 6 Table 7

[0049] Table 8 presents data comparing the fatty acid content (wt.% of dry biomass) of mycelial biomass grown under microaerobic conditions in vitro with the biomass of natural fruiting bodies of Tuber magnatum mushrooms depending on the cultivation time and the type of in vitro cultivation medium.

[0050] Table 8

[0051] The results of the analyses of the identification of fatty acids and the determination of their ratios in the biomass of Tuber magnatum (Table 5, 6, 7) showed the same as in Example 1, namely: 1) the biomass of fungal cultures grown under microaerobic conditions “in vitro” contained the same and even richer spectrum of unsaturated fatty acids (including omega-unsaturated fatty acids) as the biomass of fruiting bodies grown in natural conditions;

[0052] 2) with the time of in vitro cultivation, the ratio between various fatty acids, including omega-unsaturated fatty acids, changed in the grown biomass.

[0053] The examination of the weight content of fatty acids per unit of dry matter of fungal biomass in the fungal biomass cultivated in vitro and in natural fruiting bodies (Table 8) showed the same as in Example 1, namely: in general, the content of fatty acids per unit of dry biomass, including omega-unsaturated fatty acids in the biomass cultivated in vitro, increased over 28 days of cultivation and, taking into account the weight fraction, exceeded the content in natural fruiting bodies.

[0054] Conclusion on examples 1-2

[0055] The use of the proposed method for in vitro cultivation of mycelial biomass of cultures isolated from the microbiome of truffle fruiting bodies, compared to the prototype method, allows for obtaining a higher content of biologically active unsaturated fatty acids (antioxidants) in the biomass.

[0056] The truffle mushroom species Tuber magnatum and Choiromyces venosus belong to different genera of the family Tuberaceae (Truffleaceae). Therefore, the demonstrated applicability of the method to the mycelium of the microbiome of two different genera of the truffle family demonstrates its applicability to various species within this group. As an additional test, the proposed method was successfully tested by the applicants, yielding similarly positive results, by isolating the target mycelial culture, which produces unsaturated fatty acids, from the fruiting body of the Tuber melamosporum (Périgord black truffle).

[0057] The proposed method can be used to produce fungal biomass with a high content of unsaturated fatty acids (antioxidants). The grown biomass and / or antioxidants obtained from it can be used as raw materials for cosmetic products, as well as feed or food additives. References:

[0058] [1] “Method for obtaining protein biomass of a fungus” Russian Federation Patent RU2189395 C2. Published: 20.09.2002. Bulletin No. 26.

[0059] [2] Method for producing fungal protein biomass. Russian Federation Patent RU 2511041 C1. Published: 10.04.2014. Bulletin No. 10.

[0060] [3] Production of natural truffle flavors from truffle mycelium. US Patent US 9277760 B2, priority date 04 / 26 / 2012, priority date outside the US 07 / 03 / 2009: WO 2011 / 001349 A1.

[0061] [4] Method for producing mushrooms. Russian Patent RU2689951, patent validity date 30.09.2016, conventional priority: 01.10.2015 IT 102015000057159.

[0062] [5] Bukhalo A.S. Higher edible basidiomycetes in pure culture. Kyiv: Naukova Dumka, 1988, 144 p.;

[0063] [6] Liu QN, Liu RS, Wang YH, Mi ZY, Li DS, Zhong JJ, Tang YJ Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides. Bioresour. Technol. 2009. V. 100(14): 3644-3649;

[0064] [7] Dedyukhina E.G., Chistyakova T.I., Weinstein M.B. Biosynthesis of arachidonic acid by micromycetes (review). Applied Biochemistry and Microbiology, 2011, v. 47 (2), pp. 125-134;

[0065] [8] Method for obtaining fungal protein biomass. Russian Patent RU 2511041, priority date 10.09.2012;

[0066] [9] Dedyukhina EG, Chistyakova TI, Kamzolova SV, Vinter MV, Vainshtein MB Arachidonic acid synthesis by glycerol-grown Mortierella alpina. European Journal of Lipid Science and Technology. 2012. V. 114: 833-841;

[0067]

[0010] Dedyukhina EG, Chistyakova TI, Mironov AA, Kamzolova SV, Morgunov IG, Vainshtein MB Arachidonic acid synthesis from biodiesel-derived glycerol by Mortierella alpina. European Journal of Lipid Science and Technology. 2014. V. 116 (4): 429-437;

[0068] [I] Dedyukhina E.G., Chistyakova T.P., Mironov A.A., Kamzolova S.V., Minkevich I.G., Weinstein M.B. Effect of pH, aeration and temperature on the synthesis of arachidonic acid by Mortierella alpine. Applied biochemistry and microbiology. 2015. Vol. 51 (2). P. 243-250;

[0069]

[0012] Method for obtaining protein biomass of the basidiomycete fungus Pleurotus pulmonarius. Russian Patent RU2588474, priority date 11.03.2015;

[0070]

[0013] Tang YJ, Liu RS, Li NM. Current progress on truffle submerged fermentation: a promising alternative to its fruiting bodies. Appl. Microbiol. Biotechnol. 2015. V. 99: 2041-2053.

Claims

Invention formula 1. A method for obtaining biomass of mycelial cultures from the microbiome of truffle mushrooms, including growing "in vitro" under sterile conditions on a culture medium of mycelial culture biomass, characterized in that the cultures are preliminarily selected during successive passages-reseedings for the ability to rapidly grow and for the formation by the mycelial cultures of fatty acids greater than 10% of the weight of dry mycelial biomass, with unsaturated fatty acids greater than 30% of the total amount of fatty acids, and the biomass is grown under conditions of reduced oxygen content from 2 to 10% at a temperature of 22-28°C and a humidity of 60-80% for 14-30 days; and a mixture of organic compounds containing proteins is used as a culture medium.

2. The method according to claim 1, characterized in that the fruiting body of Tuber magnatum (white Piedmont truffle) or Choiromyces venosus (white Trinity truffle), or Tuber melanosporum (black Perigord truffle) is used as the microbiome of truffle mushrooms.

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