Bacillus coagulans and application thereof

Abstract

The invention belongs to the technical field of functional microorganism screening and application, and particularly relates to bacillus coagulans and application thereof. The bacillus coagulans is separated from an excrement sample of a healthy pig and is preserved in China Center for Type Culture Collection in Wuhan University, Wuhan, China on December 26, 2024, and the preservation number is CCTCC NO: M20242912. The strain does not generate hemolysin, cannot dissolve blood cells, is sensitive to various antibiotics, is good in safety, has excellent heat resistance, gastric acid resistance and cholate resistance, is high in organic acid yield, can efficiently remove zearalenone toxin, can be used as a feed additive or a mycotoxin detoxification agent to be widely applied to livestock and poultry breeding, and has wide application prospects. The feed additive can significantly promote the growth of bred animals, improve the immune performance and oxidation resistance of the animals, improve the intestinal flora structure and maintain intestinal health, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganism screening and application, and particularly relates to Bacillus coagulans and application thereof. Background Art

[0002] In modern animal husbandry, the long-term and irrational use of antibiotics not only threatens animal health but also poses a potential risk to human public health and safety. Consequently, global restrictions on the use of antibiotics in feed are becoming increasingly stringent, creating an urgent need to find alternatives to antibiotics.

[0003] Furthermore, in today's feed industry and livestock production, the problem of mycotoxin contamination of feed raw materials is becoming increasingly serious. Feed is highly susceptible to mold contamination during cultivation, storage, and processing, resulting in the production of a variety of mycotoxins. Zearalenone is a common and particularly harmful mycotoxin in feed. In livestock production, when animals ingest feed contaminated with zearalenone, not only does it severely damage the quality and nutritional value of the feed, but it also poses potential risks to the animals' growth performance, reproductive function, and human health. Traditional methods for removing zearalenone from feed primarily include physical adsorption and chemical treatment. However, physical adsorption has limited adsorption effects, while chemical treatment may introduce new chemical residues, posing a potential threat to animal health and food safety. Furthermore, these methods often require complex equipment and process conditions, resulting in high costs and difficulties in large-scale application in actual production.

[0004] Currently, biological detoxification is the most common method for removing zearalenone toxins. This method primarily detoxifies mycotoxins through the action of microbial enzymes or by adsorbing them onto microbial cell walls. This method offers advantages such as high efficiency, environmental friendliness, and low cost. It can be widely applied in the food, feed, and agricultural sectors, significantly reducing the harmful effects of mycotoxins on humans and animals. Therefore, the identification of microbial strains that are highly effective in degrading mycotoxins has been a research hotspot in recent years. Summary of the Invention

[0005] The present invention aims to provide a Bacillus coagulans and its application. The Bacillus coagulans has excellent heat resistance, gastric acid resistance, and bile salt resistance, high organic acid production, and the ability to efficiently degrade zearalenone toxin, thus having broad application prospects.

[0006] On the one hand, the present invention provides a Bacillus coagulans strain, named WL-64 (Bacillus coagulans), which was deposited in the China Center for Type Culture Collection of Wuhan University, Wuhan, China on December 26, 2024, with a deposit number of CCTCC NO: M20242912.

[0007] The 16s rDNA sequence of the Bacillus coagulans WL-64 strain is SEQ ID NO: 1.

[0008] The present invention also provides the use of the Bacillus coagulans WL-64 in feed or food production.

[0009] The present invention also provides the use of the Bacillus coagulans WL-64 in the production of a mycotoxin detoxifying agent.

[0010] The mycotoxins are any one or more of zearalenone, aflatoxin, fumonisin, ochratoxin, T-2 toxin, and vomitoxin.

[0011] The present invention also provides a microbial preparation comprising the Bacillus coagulans WL-64.

[0012] The present invention also provides application of the microbial preparation in feed or food production.

[0013] The Bacillus coagulans WL-64 provided by the present invention is non-hemolytic, sensitive to kanamycin, gentamicin, ampicillin, erythromycin, azithromycin and tetracycline, and has high safety; it also has extremely strong heat resistance, gastric acid resistance and bile salt resistance.

[0014] Bacillus coagulans WL-64 can metabolize and produce high-yield organic acids. After 24 hours of fermentation, the production of lactic acid, formic acid, acetic acid and propionic acid in its supernatant reached 15.04g / L, 0.9g / L, 9.2g / L and 10.3g / L, respectively.

[0015] Bacillus coagulans WL-64 exhibited significant inhibitory effects against Escherichia coli K88, Staphylococcus aureus, Salmonella pullorum, Salmonella suis, Clostridium perfringens from chicken, Clostridium perfringens from pigs, Haemophilus parasuis, and Streptococcus suis SS-2. Among them, Bacillus coagulans WL-64 had the strongest inhibitory effect against Salmonella pullorum, with an inhibition zone diameter of 23.5 mm.

[0016] Bacillus coagulans WL-64 has a strong antioxidant capacity. The scavenging rates of its fermentation broth on DPPH free radicals and hydroxyl free radicals are 85.3% and 58.3%, respectively. The scavenging rates of its bacterial suspension on DPPH free radicals and hydroxyl free radicals are 62.9% and 50.7%, respectively, achieving unexpected technical results.

[0017] Bacillus coagulans WL-64 is highly effective in removing zearalenone from liquid culture media, achieving a removal rate of 89.7%. This strain can be directly used to detoxify feed ingredients. After 24 hours of treatment, zearalenone concentrations in corn flour, soybean meal, bran, and cottonseed meal decreased by 90.0%, 73.2%, 68%, and 86.3%, respectively, demonstrating significant detoxification efficacy.

[0018] Bacillus coagulans WL-64 can be widely used in food or feed production as an additive or mycotoxin detoxifier. It can significantly improve the body's immune and antioxidant properties, improve the structure of intestinal flora, maintain intestinal health, promote growth, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the colony morphology of Bacillus coagulans WL-64;

[0020] Figure 2 This is a microscopic morphological picture of Bacillus coagulans WL-64;

[0021] Figure 3 This is a diagram showing the inhibitory effect of Bacillus coagulans WL-64 on Salmonella pullorum. DETAILED DESCRIPTION

[0022] The following are specific embodiments of the present invention, which describe the technical solutions of the present invention in detail but are not intended to limit the scope of protection of the present invention.

[0023] Example 1 Screening of Bacillus coagulans WL-64

[0024] A 10g fecal sample from healthy pigs at a Qingdao pig farm was added to a 250ml Erlenmeyer flask containing glass beads. 90mL of physiological saline was added and mixed thoroughly. The sample was incubated at 80°C for 10 minutes to kill non-spore-forming bacteria. A gradient dilution was performed, and 100μL of the appropriate gradient dilution was plated onto an MRS solid plate. The plate was then incubated inverted at 45°C for 48 hours. Single colonies isolated by multiple streaking were inoculated into MRS liquid medium and incubated at 45°C for 24 hours. The bacterial and spore morphology was observed microscopically. Five strains, WL-3, WL-8, WL-12, WL-38, and WL-64, were Gram-positive with terminal spores. The pH of the fermentation broth of each of the five strains was further measured to identify strains with high acid production.

[0025] The results showed that the WL-64 strain had the strongest acid production ability, and the pH dropped to 4.0 after being cultured at 45℃ for 24 hours.

[0026] Example 2 Identification of Bacillus coagulans WL-64

[0027] 1. Morphological identification:

[0028] The WL-64 strain grew well in MRS solid medium. The colonies were moist and white with neat edges and smooth surface. Figure 1 ). Observation under a microscope revealed that the bacteria were rod-shaped, single or in pairs, with spores at the end and oval in shape ( Figure 2 ).

[0029] 2. Physiological and biochemical identification:

[0030] The strain was subjected to physiological and biochemical identification, and the results are shown in Table 1. The strain was consistent with the physiological and biochemical characteristics of Bacillus coagulans and was preliminarily identified as Bacillus coagulans.

[0031] Table 1 Physiological and biochemical identification of WL-64 strain

[0032]

[0033] Molecular identification: The genome of strain WL-64 was extracted and used as a template for PCR amplification using the universal bacterial 16s rDNA primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Amplification conditions were: 98°C for 3 min, followed by 30 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 90 s, and finally 72°C for 10 min. Positive amplified fragments were purified and sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0034] The 16S rDNA sequence of the WL-64 strain is shown in SEQ ID NO: 1. The details are as follows:

[0035]

[0036] Homology comparison analysis of SEQ ID NO: 1 with related sequences in NCBI showed that the WL-64 strain was Bacillus coagulans, named Bacillus coagulans WL-64, and deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on December 26, 2024, with the deposit number CCTCC NO: M20242912.

[0037] Example 3 Characteristic Analysis of Bacillus coagulans WL-64

[0038] Bacillus coagulans WL-64 strain stored at -80°C was streaked onto MRS solid medium and incubated at 45°C for 48 hours. A single colony was selected and inoculated into MRS liquid medium and incubated at 45°C for 17 hours. A 10% inoculum of Bacillus coagulans WL-64 strain was transferred to fresh MRS liquid medium and incubated at 45°C with shaking for 24 hours. The cells were centrifuged at 4000 rpm for 5 minutes to obtain bacterial cells. The cells were washed twice with saline and resuspended in saline to obtain a bacterial suspension.

[0039] 3.1 Hemolytic test:

[0040] Prepare TBS basal medium, sterilize it by autoclave at 121°C for 15 minutes, wait for the medium to cool to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into a plate; pick a single colony of Bacillus coagulans WL-64 and streak it onto the prepared blood cell plate, incubate it at 37°C for 24 hours, and observe whether the test bacteria show hemolysis.

[0041] The results showed that no lysis zone appeared, that is, Bacillus coagulans WL-64 did not produce hemolysin and could not dissolve blood cells, and was safe.

[0042] 3.2 Antibiotic sensitivity test:

[0043] The agar diffusion paper method was used to determine the sensitivity of the strain to antibiotics (sensitive: inhibition zone greater than 20 mm), and the sensitivity level of the strain to antibiotics was determined by the size of the inhibition zone.

[0044] Table 2 Antibiotic sensitivity test of Bacillus coagulans WL-64

[0045]

[0046] The results are shown in Table 2. Bacillus coagulans WL-64 is sensitive to kanamycin, gentamicin, ampicillin, erythromycin, azithromycin, and tetracycline, and has good biosafety.

[0047] 3.3 Artificial gastric juice and artificial intestinal juice tolerance:

[0048] The bacterial suspension was added to simulated artificial gastric juice (pH = 1.5, 2.0) and simulated artificial intestinal juice (0.3% bile salt, 0.5% bile salt) for 2 hours, and samples were taken for plate colony count. The number of live bacteria without adding gastric juice or bile salt was used as a control to calculate the survival rate of the strain.

[0049] The results showed that the survival rate of Bacillus coagulans WL-64 was above 90% after being treated with artificial gastric juice and artificial intestinal juice, indicating that the strain had extremely strong tolerance.

[0050] 3.4 Heat resistance:

[0051] The bacterial suspension of Bacillus coagulans WL-64 was treated at different temperatures of 70℃, 80℃, 85℃ and 90℃ for 10 minutes, and the survival rate was above 90%, which shows that the strain has strong heat resistance.

[0052] 3.5 Determination of organic acid production:

[0053] After Bacillus coagulans WL-64 was cultured in MRS liquid medium at 45°C with shaking for 24 h, the supernatant was collected for determination of organic acid content.

[0054] The results showed that the content of organic acids produced by the metabolism of Bacillus coagulans WL-64 was relatively high, among which the production of lactic acid, formic acid, acetic acid and propionic acid reached 15.04g / L, 0.9g / L, 9.2g / L and 10.3g / L, respectively.

[0055] Example 4 Analysis of the antibacterial ability of Bacillus coagulans WL-64

[0056] The inhibitory effect of Bacillus coagulans WL-64 on common animal pathogens was detected by the Oxford cup method.

[0057] First, according to the type and requirements of pathogens, select appropriate culture medium to prepare 10 6 CFU / mL of pathogenic bacteria solution, add 0.1mL 10 6 Then place a 6mm Oxford cup in the center of the plate and add 50μL of a 10 6 A suspension of Bacillus coagulans WL-64 at 500 CFU / mL was incubated at 37°C for 24 hours, and the diameter of the inhibition zone was measured. A 50 μL MRS liquid medium was inoculated as a control. Each experiment was repeated three times.

[0058] Table 3 In vitro antibacterial test of Bacillus coagulans WL-64

[0059]

[0060] From the results in Table 3, it can be seen that Bacillus coagulans WL-64 has a significant inhibitory effect on Escherichia coli K88, Staphylococcus aureus, Salmonella pullorum, Salmonella suis, Clostridium perfringens from chicken, Clostridium perfringens from pig, Haemophilus parasuis, and Streptococcus suis SS-2. Among them, Bacillus coagulans WL-64 has the best inhibitory effect on Salmonella pullorum, with an inhibition zone diameter of 23.5 mm ( Figure 3 ).

[0061] Example 5 Determination of the in vitro antioxidant capacity of Bacillus coagulans WL-64

[0062] (1) DPPH free radical scavenging ability

[0063] Take 1mL of the fermentation broth and bacterial suspension of Bacillus coagulans WL-64 and mix them thoroughly with 1mL of DPPH solution (0.4mmol / L) respectively, and react at room temperature in the dark for 30min. After the reaction is completed, measure the sample absorbance A at 517nm. 样品 The sample was replaced with an equal volume of normal saline as a control, and the absorbance of the control group was recorded as A 对照 .

[0064] The DPPH radical scavenging rate of the strain was calculated as follows:

[0065] Clearance (%) = (1 – A 样品 / A 对照 )×100%.

[0066] (2) Hydroxyl radical scavenging ability

[0067] 200 μL of bacterial suspension and fermentation broth were mixed thoroughly with 200 μL of 2.5 mmol / L 1,10-phenanthroline, 200 μL of 2.5 mmol / L FeSO4 solution, 200 μL of 0.02 mmol / L PBS, and 200 μL of 20 mmol / L H2O2, respectively. The absorbance A of the sample was measured at a wavelength of 536 nm in a 45 ° C water bath for 1 h. a .

[0068] The hydroxyl radical scavenging rate was calculated as follows:

[0069] Hydroxyl radical scavenging rate = (A a -A b ) / (A c -A b )×100%.

[0070] Among them, A b To replace the sample with an equal volume of deionized water, A c The sample and H2O2 were replaced by equal volumes of deionized water.

[0071] The results showed that Bacillus coagulans WL-64 has a strong antioxidant capacity. The scavenging rates of its fermentation broth on DPPH radicals and hydroxyl radicals were 85.3% and 58.3%, respectively, and the scavenging rates of the bacterial suspension on DPPH radicals and hydroxyl radicals were 62.9% and 50.7%, respectively, achieving unexpected technical results.

[0072] Example 6 Preparation of Bacillus coagulans WL-64 bacterial powder

[0073] 1. Preparation of Bacillus coagulans WL-64 fermentation broth

[0074] A single colony of Bacillus coagulans WL-64 was picked and inoculated into MRS liquid culture medium and cultured at 45°C for 17 h. Bacillus coagulans WL-64 was transferred to fresh MRS liquid culture medium at a 10% inoculum volume and cultured with shaking at 45°C for 24 h as seed liquid.

[0075] The seed liquid of Bacillus coagulans WL-64 was transferred to the fermentation medium at a volume ratio of 5%. The fermentation formula was as follows: 10.0 g / L soybean meal, 10.0 g / L corn steep liquor, 10.0 g / L corn starch, 2.0 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, and 0.25 g / L manganese sulfate; the culture was carried out at 45°C and 200 rpm for 20-24 hours. The fermentation was terminated when the morphology of Bacillus coagulans WL-64 was normal, without foreign bacteria, and the spore rate was 90% or above. After the fermentation was completed, the temperature was raised to 60°C and maintained for 10 minutes to obtain a Bacillus coagulans WL-64 fermentation broth. The number of Bacillus coagulans WL-64 spores in the fermentation broth was 9.0×10 9 CFU / mL, and the spore rate is 90% or above.

[0076] 2. Preparation of Bacillus coagulans WL-64 powder

[0077] The fermentation liquid of Bacillus coagulans WL-64 was thoroughly mixed with a protective agent (10% corn starch, 5% sucrose, 2% gum arabic, 1% vitamin C and 0.5% sodium L-glutamate) to obtain a spray mixture. A centrifugal spray dryer was used for spray drying, with the inlet air temperature controlled at 150°C and the outlet air temperature at 70°C. The bacterial powder yield reached 85% or more, and the number of Bacillus coagulans spores in the bacterial powder was 10 11 CFU / g.

[0078] Example 7 Degradation of Zearalenone Toxin by Bacillus coagulans WL-64

[0079] 1. Detoxification effect of Bacillus coagulans WL-64 in liquid culture medium

[0080] Take 1g of bacteria as 10 11Bacillus coagulans WL-64 powder was inoculated into 100 mL of MRS liquid medium. Zearalenone was added to a final concentration of 5 μg / mL and cultured at 45°C and 200 rpm for 6, 12, 18, 24, and 32 hours. The residual zearalenone concentration in the medium was determined using a zearalenone ELISA kit. MRS liquid medium supplemented with zearalenone alone, without inoculation, served as a control.

[0081] Zearalenone removal rate (%) = (zearalenone content in the control group - zearalenone content in the experimental group) / zearalenone content in the control group × 100%.

[0082] The results showed that within 6-24 hours of culture, as the culture time prolonged, the removal rate of zearalenone by Bacillus coagulans WL-64 gradually increased. The removal effect of zearalenone was the strongest at the 24th hour, with a removal rate of 89.7%, achieving unexpected technical results.

[0083] 2. Detoxification effect of Bacillus coagulans WL-64 on feed raw materials corn flour, soybean meal, bran, and cottonseed meal

[0084] Take 1g of bacteria as 10 11 Bacillus coagulans WL-64 powder at a concentration of 100 CFU / g was inoculated into 100g of corn meal, soybean meal, bran, and cottonseed meal, all feed ingredients contaminated with zearalenone. The mixture was thoroughly mixed and incubated at 45°C for 24 hours. Feed ingredients not inoculated with Bacillus coagulans WL-64 served as a control.

[0085] 1g of each experimental and control feed ingredient was ground and passed through a 20-mesh sieve. The ground feed was placed in a conical flask, and 25mL of a 6:4 mixture of methanol and water was added. The mixture was shaken for 15 minutes, filtered, and the filtrate was collected and appropriately diluted to serve as the test solution. The zearalenone toxin content in the test solution was determined using a zearalenone enzyme-linked immunosorbent assay kit.

[0086] The results showed that after 24 hours of fermentation of Bacillus coagulans WL-64 in corn flour, soybean meal, bran and cottonseed meal contaminated with zearalenone, the concentrations of zearalenone decreased by 90.0%, 73.2%, 68% and 86.3%, respectively, indicating that Bacillus coagulans WL-64 has a significant removal effect on zearalenone.

[0087] Example 8 Application of Bacillus coagulans WL-64 in broiler farming

[0088] The feeding experiment was conducted in a chicken farm in Qingdao, Shandong Province. 300 7-day-old yellow-feathered broilers were randomly divided into two groups, with 5 replicates in each group and 30 chickens in each replicate. 8 The control group was fed a basal diet. The experimental period was 45 days, with free access to food and water, and immunization was performed according to the conventional immunization procedure.

[0089] 1. Growth performance determination

[0090] During the experiment, the daily feed intake was recorded for each replicate. The animals were weighed 12 hours before and after the experiment, and the average daily feed intake, average daily weight gain, and feed-to-gain ratio were calculated.

[0091] Average daily feed intake = total feed consumption / number of experimental days.

[0092] Average daily weight gain = (final weight - initial weight) / test day.

[0093] Feed-to-weight ratio = average daily feed intake / average daily weight gain.

[0094] The results showed that compared with the control group, the average daily weight gain and average daily feed intake of broilers in the experimental group increased by 35.5% and 17.7% respectively, and the feed-to-weight ratio decreased by 13%.

[0095] 2. Immune organ index

[0096] When broiler chickens were 52 days old, after fasting for 12 hours, one chicken with a body weight close to the average was selected from each replicate for slaughter. The spleen, bursa of Fabricius and thymus were dissected and weighed, and the indexes of each immune organ were calculated.

[0097] Immune organ index (%) = (immune organ weight / live weight) × 100%.

[0098] The results showed that compared with the control group, the spleen index, bursa index and thymus index of the experimental group broilers increased by 50.2%, 35.9% and 64.2%, respectively, indicating that the Bacillus coagulans WL-64 provided by the present invention can significantly improve the immune function of broilers.

[0099] 3. Immune performance

[0100] After the experiment, blood was collected from 52-day-old broilers under the wing vein and centrifuged at 4000 rpm for 10 min to obtain serum. The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum were determined using ELISA kits from Nanjing Jiancheng Bioengineering Research Institute.

[0101] The results showed that compared with the control group, the levels of IgA and IgM in broiler serum increased by 32.5% and 29.7% respectively, and the IgG content did not change significantly, indicating that Bacillus coagulans WL-64 can improve the immunity of yellow-feathered broilers to a certain extent and promote their growth and development.

[0102] 4. Antioxidant function

[0103] The levels of glutathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in broiler serum were determined using kits from Nanjing Jiancheng Bioengineering Institute.

[0104] The results showed that compared with the control group, the serum T-SOD, GSH-Px, and T-AOC activities of broiler chickens fed Bacillus coagulans WL-64 increased by 32.0%, 25.7%, and 28.9%, respectively, and the serum MDA content decreased by 23.8%. This shows that Bacillus coagulans WL-64 can significantly improve the antioxidant capacity of broiler chickens.

[0105] 5. Effects of Bacillus coagulans WL-64 on cecal flora

[0106] The cecum of the broilers in the experimental group and the control group were collected respectively. 1 g of cecum was taken, added into 9 ml of sterile saline and shaken, and diluted 10 times step by step. The appropriate gradient dilutions were inoculated into MacConkey medium and MRS solid medium respectively. After culturing at 37°C for 24 h and 48 h respectively, Escherichia coli and lactic acid bacteria were counted.

[0107] The results showed that compared with the control group, the number of Escherichia coli in the cecum of broilers fed Bacillus coagulans WL-64 decreased by 32.1%, while the number of lactic acid bacteria increased by 25.7%. This shows that Bacillus coagulans WL-64 can significantly improve the microbial community structure in the cecum of yellow-feathered broilers, which is beneficial for maintaining intestinal health.

[0108] In summary, the Bacillus coagulans WL-64 provided by the present invention has high biosafety and strong resistance to heat, gastric acid, and bile salts. This strain can metabolize and produce high levels of organic acids, exhibiting significant inhibitory effects against various pathogens, including Escherichia coli K88 and Staphylococcus aureus. It can also efficiently remove zearalenone, significantly enhance immune and antioxidant properties, improve intestinal flora structure, maintain intestinal health, and promote growth. It has broad application prospects as an additive or mycotoxin detoxifier in food and feed production.

Claims

1. A Bacillus coagulans, characterized in that The deposit number of the Bacillus coagulans is CCTCC NO: M20242912.

2. The Bacillus coagulans according to claim 1, wherein The 16s rDNA sequence of the Bacillus coagulans is SEQ ID NO:

1.

3. Use of the Bacillus coagulans according to claim 1 in food production.

4. Use of the Bacillus coagulans according to claim 1 in feed production.

5. Use of the Bacillus coagulans according to claim 1 in the production of a mycotoxin detoxifying agent.

6. The use according to claim 5, characterized in that The mycotoxins are any one or more of zearalenone, aflatoxin, fumonisin, ochratoxin, T-2 toxin, and vomitoxin.

7. A microbial preparation, characterized in that The microbial preparation comprises the Bacillus coagulans according to claim 1.

8. The microbial preparation according to claim 7, wherein The amount of live Bacillus coagulans in the microbial preparation is not less than 10 9 U / g.

9. Use of the microbial preparation according to claim 7 or 8 in feed production.

10. Use of the microbial preparation according to claim 7 or 8 in the production of a mycotoxin detoxifying agent.

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