RU2409019C2 - Method of bacillar thermoanaerobic preparation of high-quality straw substrate for intense non-sterile cultivation of oyster mushroom - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to agriculture and biotechnology, namely, to mushroom growing, based on culture of xylotroph oyster mushroom, cultivated using industrial intensive European technology. Method of bacillar thermoanaerobic preparation of high-quality straw substrate for intense non-sterile cultivation of oyster mushroom includes preliminary fermentation of substrate by soaking it in reservoirs and using to do pasteurisation and main thermal fermentation of thermal chambers using microorganisms of Bacillus type. For this purpose bacillar microorganisms are used of Bacillus type, namely, thermophil culture B.cereus.

EFFECT: method makes it possible to produce high-quality straw substrate to grow oyster mushrooms, to simplify process of preliminary fermentation and to reduce time for preparation of substrate of high selectivity and efficiency.

6 cl, 4 tbl, 2 ex

Description

The present invention relates to agriculture and biotechnology, namely to mushroom farming, based on the culture of xylotroph mushroom oyster mushroom ordinary, grown according to industrial intensive European technology.

Xylosaprotroph - oyster mushroom (Pleurotus ostreatus) - is one of the most valuable objects of world and domestic mushroom growing. It is the most economically profitable among other cultivated species of fungi [1, 2].

There are two methods for its artificial cultivation: extensive (in the summer on chunks of aspen, poplar, in three-year revolutions) and intensive (from the end of summer to the beginning of next summer, in two or three “wave” two-month mushroom rotations) under controlled indoor conditions, on straw or sawdust substrates.

The whole variety of existing methods and techniques for intensive cultivation of oyster mushrooms are two different technologies. The first is industrial semi-sterile Euro technology, where mushrooms grow on large blocks or perforated plastic bags filled with pre-prepared (inoculated cereal seed mycelium) and an accessible straw straw substrate (cereal straw, floor, corn kernels, sunflower husks, etc.) pre-prepared (inoculated with grain inoculum mycelium). . The second is Asian, absolutely sterile technology, with the fruiting of mushrooms from the openings of small vessels (ceramic, glass, polypropylene) or thermoplastic bags enriched with nutritious additives (bran, cereal, dust, flour, etc.) with a sterile sawdust substrate (in Russia practically not used for commercial production of mushrooms [3, 4]). European technology is characterized by lower costs of manual labor and potential reserves for its further modernization, mechanization and automation, effective use in large industrial mushroom production.

After the development and implementation of Euro-technology (40 years ago), thorough heat treatment (autoclaving, long-term steaming or boiling) was used to prepare the substrates [5]. The substrate prepared for inoculation (usually straw) was actually sterile, but did not have selectivity. Therefore, after inoculating it with seed mycelium, especially in the absence of a “clean” zone (that is, if strict asepsis rules are not respected), a rapid development of competing microorganisms contaminating spores was observed, which often led to a significant decrease, and in some cases to a complete loss of yield . This was confirmed in early studies (Table 1) of the authors of this technical solution when developing the scientific and technological foundations of intensive cultivation of oyster mushrooms in the Amur region.

Among the most harmful [6] are the so-called “weed” molds (micromycetes from the genera Trichoderma, Aspergillus, Penicillium, Rhizomukor, etc.). In the process of metabolism, most of them release strong toxins (antimycotics), to which the not yet developed oyster mushroom mycelium is not resistant, it stops growing and dies. A strong negative effect on the development of oyster mushroom mycelium toxins of micromycetes, which do not deteriorate even after many hours of exposure to high temperatures, was found during the production of grain planting mycelium in the GNU-DV of the Order of TKZ NIISH Agricultural Academy. In order to obtain a high-quality nutrient substrate for growing oyster mushrooms according to the European technology, with any method of preparation, careful selection of lots of high-quality cereal straw, which should be dry, golden in color, have no foci of dampness and dampness, is important. Moreover, high-quality cereal straw is not sterile and contains a certain composition of resting forms of microorganisms represented by bacteria, actinomycetes and microscopic fungi [9]. When moistening such a straw raw material, the activity of microflora begins to increase rapidly. The art of preparing a high-quality selective substrate is to preserve the beneficial (thermophilic) microflora and to sufficiently increase its number, at the same time to inactivate or destroy harmful competitive organisms.

Currently, the aerobic fermentation method is widely known, which allows one to create a selective substrate with good oyster mushroom yield [5, 6, 9, 10]. Fermentation of the substrate differs from simple heat treatment in that when the temperature rises to 60-62 ° C (more than 10 hours), only partial pasteurization of the substrate occurs (mesophilic vegetative forms of a wide spectrum are destroyed, but the thermophilic spore-forming bacteria are preserved), and a constant supply of fresh air (in tunnels) creates favorable conditions for the development of beneficial aerobic microflora, which allows to obtain the highest quality straw nutrient medium for mycelia oyster mushroom (Pleurotus ostreatus). This method (aerobic fermentation and preservation of thermophilic spore-forming bacteria) is closest to the claimed [9, 10].

However, for such aerobic fermentation, complex and expensive special capital structures, the so-called tunnels, are necessary. The process of classical aerobic fermentation of straw with preliminary long-term moistening in heaps is long (up to 10 days) and problematic for regions with harsh winters.

There is a need to modernize the process, simplify it and reduce the cost by combining technological operations for soaking fragmented straw and conducting preliminary anaerobic low-temperature fermentation, but with increased efficiency - due to the use of specially selected and cultivated effective microorganisms - optional anaerobes for filling the aqueous suspension.

The objective of the present invention was to develop a method of using an effective pure bacillary culture in the anaerobic preparation of high-quality straw substrates for growing oyster mushrooms, simplifying the preliminary fermentation process and reducing the time for preparing the substrate with economically sufficient selectivity and fruitfulness.

The problem is solved by using anaerobic fermentation as a bacillary microorganism of the thermophilic culture of B. cereus from the genus Bacillus.

It was assumed that from thermophilic microorganisms they increase the selectivity of the substrate for growing oyster mushrooms of common actinomycetes and bacilli [5, 9], which have high antagonistic activity against many types of microorganisms [12]. It was experimentally found (Table 2) that the greatest effectiveness in protecting against competitors and in stimulating the productivity of oyster mushrooms growing on “living” substrates is inherent in the species of the genus Bacillus taken for experiment. It was determined that to improve the quality of the substrate and productivity in oyster mushrooms, bacillary microorganisms are most suitable. A patent search showed that the bulk of the spore-forming bacteria of the genus Bacillus are mesophiles with an optimum growth of up to 40-45 ° C, and only a few representatives are thermophilic, i.e. grow at temperatures up to 65 ° C [13]. Thermophilic species of bacilli are divided into groups. The first group combines bacteria with a bacillary (rod-shaped) type of sporulation, in which two subgroups are distinguished: B. subtilis (aerobes) and B. cereus (optional anaerobes). B.subtilis (formerly called the “hay” stick), like another well-known representative of B.mesentericus (Vas.subtilis var. Mesentericus), or “potato” stick, use carbohydrates only under aerobic conditions. Bacteria of the subgroup B.subtilis are soil bacteria of the southern type, most common in arable lands of the main grain-producing regions [14]. In Russia and abroad [15] a number of B.subtilis strains have been patented that inhibit the growth of phytopathogenic fungi.

For bacteria of the B.cereus subgroup, a distinctive feature is the ability to use sugars both aerobically and anaerobically, i.e. in the absence of atmospheric oxygen [13], including and the second representative of you. cereus var. micodes (according to modern classification). These are northern species that are dominant in ecosystems, where the processes of transformation of organic matter weakly occur, i.e. in wet hayfields and fields with heavy, periodically waterlogged soils. Vas.cereus is a producer of thermostable broad-spectrum antibiotic zwittermicin [17].

The bacilli of the first group (the bacillary type of sporulation) are the most thermophilic, their vegetative forms are able to maintain vital activity under long-term exposure to temperatures of 60-62 ° C. Their matured resting spores, having a multilayer shell, acquire ultra-high thermal stability, which is due to the accumulation of calcium ions in them, associated with picolinic acid [18]. They withstand dry heat at 100 ° C for 3 hours (or boiling for 30 minutes). The resistance of the spores to temperature is significantly affected by the acidity of the medium. With decreasing pH, the stability of spores usually decreases [18]. Bacteria of the second group B.maserans and B.polymyxa are also used to give selectivity to substrates during the cultivation of oyster mushroom [10]. Like the bacteria of the first group, they are able to ferment carbohydrates; when grown under anaerobic conditions, these cultures have the ability to fix nitrogen, which is important when preparing a high-quality substrate, but they are less heat-resistant and therefore are able to grow and develop only at temperatures below 58 ° C [6]. In addition, they have not yet discovered active metabolites that could not only inhibit growth, but also radically inhibit competitive microscopic fungi, as the bacilli of the first group do.

The proposed method is as follows. High-quality strawy agricultural waste (straw, floor, dust) that has passed through combine threshing, dry, golden, without foci of dampness and dampness is selected, soaked in hot water with a weak wiring of B.cereus bacillary thermophilic culture, in enameled containers (vats) with lids and left for subsequent slow cooling - a simplified diagram. Or tanks filled with hot water with B.cereus wiring are placed in thermostats for 12-hour heating (pasteurization) at 60 ° C and subsequent 1.5-2-day cooling to 30 ° C - a classic scheme. The culture of B. cereus is used "pure", grown on a nutrient medium KGA, young age (up to 15 days). B. cereus bacilli are introduced into the prepared substrate in a moderate amount - at the rate of one test tube in a 50-75-liter capacity or one lawn of an ordinary Petri dish for a 1.5-2-ton capacity. This applies to both schemes.

This allows you to have in the moisture of the prepared substrate, at the time of its inoculation with oyster mushroom, the content of live bacilli is about 10 CFU / cm (g).

Example 1. In the spring of 2007, a large-scale experience was laid (Table 3) for experimental verification of the proposed method of using “pure” B.cereus cultures to create high-quality selective substrates according to aerobic fermentation schemes (in tunnels known in the country and abroad [5]) , which were previously developed for the production of compost for growing champignons), but with the transfer to the anaerobic path, i.e. pre-fermentation by 2.5-day soaking of the substrate in containers and using thermal rooms (thermostats) for pasteurization and basic thermo-fermentation, which corresponds to the existing technological level of commodity production of oyster mushrooms in most regions of the country. It was found (Table 3) that, when soaking and preparing a straw substrate, a weak aqueous distribution of “young” (fresh) B.subtilis or B.cereus cultures is used, the productivity of the cultivated fungus significantly increases relative to the control. The highest productivity of oyster mushroom in the experiment on anaerobically fermented oat sex was observed in variants with B.cereus. It was found (option 4) that it is impossible to use a mixture of these most important bacillary microorganisms for soaking the substrates, since in the nutrient medium they compete and inhibit each other's development, while the quality of the finished substrate decreases. The selectivity of anaerobically fermented substrates in all variants of the experiment was high. This allows for inoculation (especially when using B.cereus) without fear of the formation of foci of competitors (that is, outside the “clean” zone) and reduce the consumption of expensive grain inoculum mycelium to 3-4%.

It is noted in the literature [9] that, when preparing a substrate, sometimes bacilli can secrete antimycotics in amounts that severely limit the development of competitive molds, but sometimes oyster mushrooms. This was also observed by the authors when treating the substrate with boiling water, when, due to the lack of freshly sown bacillary cultures, old, long-stored cultures that had already dried up were used when they exceeded the norm and brought two B.cereus tubes into the tank with a huge amount of mature (resting) spores and, on the contrary, not pure straw was used as a substrate, but crushed dried rods of selection corn with a low initial titer of natural (including thermophilic) microflora, since the harvesting of ears was manual I was carried out in wrappers with their preventive drying.

Example 2. To understand the mechanisms of the observed phenomena and verify the modified accelerated (simplified) method of anaerobic fermentation (using B.cereus culture), an experiment was carried out that included two options. In each variant, there was a sac uncontaminated with oyster mushroom mycelium for control. Nothing grew on the control bag of the first variant for 15 days, which was almost 2 times the period (8 days) before the accumulation of weed micromycetes biomass in the control bag of the second variant. On the inoculated two-kilogram bags in the first variant, growth retardation was observed not only for competitors, but also for oyster mushrooms, which negatively affected the formation of the first wave of mushrooms. But the productivity of the second wave was lower than in the second option.

When pouring the substrate with boiling water, vegetative forms of a wide range of harmful and beneficial microorganisms die or get very stressful, including thermotolerant and thermophilic bacteria, but at the same time the germination and rapid development of their resting spores is activated, there is a progressive excessive production of active metabolites, to which there is virtually no counteraction in the first embodiment. Therefore, in order to improve the quality of the substrates obtained by the simplified scheme of anaerobic fermentation, the type and quality of the feedstock, its microbiological condition, and the volume of the B.cereus culture introduced are of importance, as in the classical schemes. The modernized scheme is the soaking of the substrate with a weak wiring of B.cereus with hot water in enameled containers (tanks) with lids, placing them in thermostats for 12-hour heating (pasteurization) at 60 ° C and subsequent 1.5-2-day cooling to temperature 30 ° С.

The proposed method convincingly proves that with anaerobic methods of substrate fermentation (in heat chambers and vats), when there are changes in conditions from aerobic to anaerobic, it is more effective for high-quality straw preparation (against the background of its initial microflora) to use a “young” culture of optional anaerobic B. cereus, in contrast to the B.subtilis aeroba previously recommended in the literature.

The use of a method of anaerobic increase in the selectivity and quality of nutrient substrates, modified and adapted for conditions similar to those characteristic of the Amur Region, helps to stabilize the commodity production of common oyster mushrooms, which is conducted according to industrial non-sterile European technology.

Literature

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Claims (6)

1. A method of bacillary thermal anaerobic preparation of a high-quality straw substrate for intensive non-sterile cultivation of common oyster mushroom, including preliminary fermentation of the substrate by soaking it in containers and using heat transfer chambers for pasteurization and main thermofermentation using microorganisms of the genus Bacillus, characterized in that microorganisms use bacillus Bacillus, namely the thermophilic culture of B.cereus. 2. The method according to claim 1, characterized in that they use a "pure" B.cereus culture grown on the KGA nutrient medium, young age up to 15 days. 3. The method according to claim 1, characterized in that B.cereus bacilli are introduced into the prepared substrate at the rate of one test tube for a 50-75-liter capacity or one lawn of an ordinary Petri dish for a 1.5-2-ton capacity. 4. The method according to claim 1, characterized in that they use straw agricultural waste - straw, floor, dust, passed through the combine threshing. 5. The method according to claims 1 to 4, characterized in that the straw agricultural waste is soaked in hot water, with the introduction of a B.cereus bacillary culture, in enameled containers with lids, followed by slow cooling. 6. The method according to claims 1 to 4, characterized in that the containers filled with hot water with the added B.cereus bacillary culture are placed in heat chambers for 12-hour heating - pasteurization at 60 ° C, followed by 1.5-2-day cooling to 30 ° C.