High-temperature-resistant candida intermedia bzd-4 and application thereof in soybean meal fermentation

Abstract

The application discloses high-temperature-resistant Candida intermedia BZD-4 and application of the Candida intermedia BZD-4 in soybean meal fermentation, and has the classification number Candida intermedia CCTCC NO: M 2021828 and is preserved in the China Center for Type Culture Collection on July 8, 2021. The high-temperature-resistant Candida intermedia BZD-4 can be applied to soybean meal fermentation, can inhibit the growth of low-temperature miscellaneous bacteria, reduces interference, and achieves better fermentation effect. The Candida intermedia BZD-4 provided by the application has the characteristics of high-temperature resistance, can still maintain a good growth state at 37 DEG C to 40 DEG C, and is an effective strain suitable for high-temperature fermentation process. The high-temperature fermentation process can effectively shorten the soybean meal fermentation period, improve production efficiency, is suitable for industrialization popularization, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of microbial and fermentation engineering technology, specifically to a thermotolerant interstitial Candida bacillus BZD-4 and its application in soybean meal fermentation. Background Technology

[0002] Soybean meal is a byproduct of soybean oil extraction. It contains various anti-nutritional factors, which not only destroy the nutritional value and bioavailability of the feed (Wang Wei, Sun Shijun et al., 2013), but also significantly reduce the production performance of poultry. Bio-fermentation is a widely used method for treating anti-nutritional factors in soybean meal (Ma Jing, 2016). During microbial fermentation, microorganisms produce hydrolytic enzymes to eliminate anti-nutritional factors in soybean meal and increase the polypeptide content, thereby improving animal digestibility and absorption. Furthermore, the probiotics produced by microbial fermentation have significant effects on improving animal gut health and reducing antibiotic use. Therefore, the microbial fermentation of soybean meal to prepare fermented soybean meal has become a research hotspot in the feed industry in recent years, with solid-state fermentation being the main method for producing fermented soybean meal. In actual production, the prepared fermenting agent is generally mixed with fermentation materials, culture medium, water, and other auxiliary materials using a conventional feed mixing device, maintaining the necessary fermentation conditions (Yang Jianying, Li Yuanxiao, 2012). Modern solid-state fermentation is generally carried out at medium and low temperatures, with fermentation temperatures typically around 36℃. However, summers in Hubei are characterized by high temperatures, with indoor temperatures reaching over 37℃. Within this temperature range, unwanted microorganisms in the environment can grow, affecting the overall fermentation effect. Therefore, high-temperature fermentation strains and processes are key to solving this problem.

[0003] *Candida intermedia*, also known as *Candida mesenteroides* (Barnett, 1991; Editorial Group of *Common and Common Fungi*, 1978), belongs to the genus *Candida*. *Candida*, also called Candida, is a common fungus widely distributed in nature, commonly found in soil and on plants. There are over 200 species within the genus *Candida*, including pathogenic strains and economically valuable strains (such as glycerol-producing *Candida*). *Candida intermedia* has been widely used in antibacterial applications in various crops, inhibiting diseases such as Penicillium and Solanum mold through antagonistic effects. *Candida intermedia* possesses a unique xylose transport system that inhibits glucose and promotes xylose diffusion, exhibiting high xylose affinity. Therefore, it can utilize xylose for physiological activities, showing great potential in degrading hemicellulose and cellulose. Therefore, developing a heat-resistant *Candida intermedia* and applying it to high-temperature soybean meal fermentation to shorten the fermentation cycle and utilize various sugars in soybean meal to increase protein content is an important aspect of this invention. Summary of the Invention

[0004] The purpose of this invention is to address the problems of poor fermentation results caused by the growth of miscellaneous bacteria in modern solid-state fermentation, which is carried out at medium and low temperatures. This invention provides a heat-resistant Candida albicans strain BZD-4 and its application in soybean meal fermentation. The Candida albicans strain BZD-4 of this invention has heat resistance, maintaining good growth at 37℃-40℃, making it an effective strain suitable for high-temperature fermentation processes. High-temperature fermentation can effectively shorten the soybean meal fermentation cycle, improve production efficiency, and is suitable for industrial-scale promotion.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] The present invention relates to a heat-resistant intermediate Candida BZD-4, which is classified as Candida intermedia, has the accession number CCTCC NO: M 2021828, and was deposited at the China Center for Type Culture Collection on July 8, 2021.

[0007] The screening process for Candida intercalaris BZD-4 of the present invention is as follows:

[0008] (1) Preparation of culture medium:

[0009] Screening medium: xylose 50g / L, yeast extract 3g / L, fermented soybean meal extract 1000mL (fermented soybean meal 100g, water 1000mL, boiled, filtered through gauze, and brought to 1000mL), agar powder 20g / L, sterilized at 115℃ for 20min.

[0010] Fermentation medium: soybean meal; water = 1:0.5, unsterilized.

[0011] (2) Initial screening

[0012] Orchard soil was placed in a porcelain dish, and chopped cooked corn cobs and soybean meal were mixed evenly with the soil at a mass ratio of 1:4, with the cooked corn cobs and soybean meal accounting for 5-10% of the total mass. The mixture was in situ enriched at 37℃ for 14 days before sampling. The corn cobs contain a large amount of cellulose and hemicellulose, which, on the one hand, provide oxygen for yeast growth through aeration, and on the other hand, are slowly decomposed into sugars by microorganisms. These sugars, along with the raffinose and stachyose in the soybean meal, provide a carbon source for the yeast. Simultaneously, the soybean meal contains a relatively high amount of nitrogen, which can be used to screen for microorganisms that effectively utilize soybean meal as a substrate for growth.

[0013] Take 10g of sample and place it in a 250mL Erlenmeyer flask containing 90mL of culture medium. Shake at 120rpm for 30min. Spread 0.2mL of the sample onto a petri dish. Perform three parallel samples simultaneously. Select typical colonies for isolation and purification. Based on the morphology of the colonies on the plates and microscopic observation of the water immersion slides, preserve strains with typical yeast morphology for subsequent fermentation screening.

[0014] The isolated strains were activated and cultured for fermentation experiments. After initial screening, 20 strains with strong ability to remove raffinose and stachyose from soybean meal were obtained. The content of raffinose and stachyose after degradation is shown in Table 1. Among them, strain 04 performed well, with the content of raffinose and stachyose after degradation both below 0.1%.

[0015] Table 1. Degradation of raffinose and stachyose in soybean meal by the initial screening strains.

[0016]

[0017]

[0018] (3) Second screening

[0019] Twenty strains with high removal capabilities of raffinose and stachyose were re-screened under the same conditions, resulting in a strain with the highest and most stable removal capability (strain No. 04). ITS sequencing confirmed that it shared 98.31% of the Max indentation of *Candida intermedia*. The sequencing results are shown in the attached sequence listing. This strain was identified as *Candida intermedia* BZD-4, and was deposited at the China Center for Type Culture Collection on July 8, 2021, with the classification and naming: *Candida intermedia* BZD-4.

[0020] The morphological and physiological-biochemical characteristics of *Candida intercalaris* BZD-4 obtained in this invention are as follows: (1) On YPD medium, white to creamy-colored colonies, see [reference needed]. Figure 1 (2) Microscopic examination of bacterial cells showed that they were oval-shaped. See [reference needed] Figure 2 (3) The main physiological characteristics are: it can ferment with pentose, it can ferment with glucose and xylose, it has high tolerance to osmotic pressure and is tolerant to ethanol, glucose and NaCl.

[0021] Since *Candida metasporum* plays a dominant role in soybean meal fermentation, and the fermentation environment has a significant impact on its growth and metabolism, the conditions during soybean meal fermentation may affect its function. Therefore, we conducted a series of tolerance tests on BZD-4, as detailed below.

[0022] (1) pH tolerance

[0023] Experimental Procedure: YPD liquid culture medium was prepared and the pH was adjusted to 3.88, 4.87, 6.00, 6.85 (natural pH), 7.64, and 8.44, respectively. 5 mL of each medium was dispensed into PA bottles and sterilized at 115°C for 20 min. BZD-4 yeast was inoculated and activated twice in the natural pH medium to prepare seed culture. This seed culture was then inoculated at a 1% inoculation rate into YPD media at different pH values ​​and cultured at 30°C for 3 days. After 2 days, the absorbance (OD) at 600 nm was measured using a microplate reader. 600 Calculation and initial OD 600 The difference ΔOD 600 The experimental results are attached. Figure 4 As shown, the *Candida metamorphosus* BZD-4 of this invention can grow at pH values ​​between 3.88 and 8.44.

[0024] (2) Oxygen dependence

[0025] Experimental procedure: YPD liquid culture base was prepared in Erlenmeyer flasks, and 1 mL of Candida metamorphosum BZD-4 seed culture was inoculated. The culture was carried out for 36 h under shaking (aerobic, 30℃, 200 rpm) and static (anaerobic, 30℃) conditions, respectively, and the growth of colonies was observed.

[0026] Experimental results are as follows Figure 5 As shown, compared with the initial inoculation medium at 0h (Figure A), colonies grew under both aerobic (Figure B) and static (Figure C) conditions. Among them, the growth of Candida albicans BZD-4 was better under aerobic conditions and worse under anaerobic conditions, indicating that the Candida albicans of the present invention can grow under both aerobic and anaerobic conditions, but the presence of oxygen can promote its large-scale reproduction.

[0027] (3) Temperature tolerance

[0028] Experimental Procedure: YPD liquid culture medium (natural pH) was prepared and dispensed into sterile PA bottles at 115℃ for 20 min. Seed culture was prepared by inoculating *Candida albicans* BZD-4 (the present invention) with *Saccharomyces cerevisiae* (common *Saccharomyces cerevisiae*) and *Saccharomyces cerevisiae* (thermotolerant *Saccharomyces cerevisiae*) after two activations. This seed culture was inoculated into PA bottles at a 1% inoculation rate and cultured at set temperatures of 30℃, 37℃, 40℃, 45℃, and 50℃ for 2 days. The absorbance (OD) at 600 nm was measured using a microplate reader. 600 Calculation and initial OD 600 The difference ΔOD 600 .

[0029] Experimental results are as follows Figure 6 As shown, Candida metamorphosum BZD-4 grows well at 40℃ and grows better than ordinary Saccharomyces cerevisiae and thermotolerant Saccharomyces cerevisiae, indicating that Candida metamorphosum BZD-4 of the present invention can adapt to the high temperature environment of 40℃ and grow well.

[0030] (4) Tolerance to NaCl concentration

[0031] Experimental procedure: Culture media containing NaCl solutions of 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, and 24% by mass were dispensed into 18 mL aliquots into 250 mL Erlenmeyer flasks. Each flask was inoculated with 2 mL of bacterial suspension, with an initial concentration of *Candida* yeast of 4.4 × 10⁻⁶. 8 CFU / mL, static incubation at 28℃, shaking the Erlenmeyer flask every 4 hours, followed by microscopic counting after 72 hours. Tolerance is defined as the concentration of the substance that causes an order-of-magnitude decrease in bacterial count.

[0032] Experimental results are as follows Figure 7 As shown, the number of cells decreased after adding a certain mass fraction of NaCl solution, indicating that osmotic pressure affects the activity of this strain. The number of cells decreased by an order of magnitude when the NaCl concentration was 12%, indicating that the tolerance of the interstitial Candida BZD-4 strain to NaCl was 12%, that is, the interstitial Candida BZD-4 strain of this invention has good growth at a NaCl mass fraction of 8-10%.

[0033] (5) Tolerance to ethanol concentration

[0034] Experimental procedure: Culture media containing ethanol solutions of 2%, 4%, 6%, 8%, 10%, 12%, 14%, and 16% by mass were dispensed into 18 mL aliquots into 250 mL Erlenmeyer flasks. Each flask was inoculated with 2 mL of bacterial suspension, with an initial concentration of *Candida* yeast of 4.4 × 10⁻⁶. 8 CFU / mL, static incubation at 28℃, shaking the Erlenmeyer flask every 4 hours, followed by microscopic counting after 72 hours. Tolerance is defined as the concentration of the substance that causes an order-of-magnitude decrease in bacterial count.

[0035] Experimental results are as follows Figure 8 As shown, ethanol is a metabolite of the strain of this invention. Therefore, the concentration of ethanol affects the degree of feedback inhibition. In the range of 2%-4%, Candida mesenteroides showed good growth. Under the condition of 6%, the number of strains showed a significant order of magnitude decrease, indicating that the 6% ethanol concentration had a strong feedback inhibition effect on the strains. Therefore, the tolerance to ethanol is 6%, and the strains can grow well in the range of 2%-4%.

[0036] (6) Glucose concentration tolerance

[0037] Experimental procedure: Culture media containing glucose solutions with mass fractions of 55%, 58%, 61%, 64%, 67%, 70%, 73%, 76%, 79%, and 82% were dispensed into 18 mL aliquots into 250 mL Erlenmeyer flasks. Each flask was inoculated with 2 mL of bacterial suspension, with an initial concentration of *Candida* yeast of 4.4 × 10⁻⁶. 8CFU / mL, static incubation at 28℃, shaking the Erlenmeyer flask every 4 hours, followed by microscopic counting after 72 hours. Tolerance is defined as the concentration of the substance that causes an order-of-magnitude decrease in bacterial count.

[0038] Experimental results are as follows Figure 9 As shown, excessively high glucose concentrations affect the osmotic pressure of the environment and also stimulate yeast to undergo anaerobic metabolism. Figure 9 It can be seen that the order of magnitude of Candida metasporum decreases when the glucose concentration is 58%, indicating that this strain can tolerate a glucose concentration of 58%.

[0039] This invention also provides an application of thermostable Candida albicans BZD-4 in soybean meal fermentation, comprising the following steps:

[0040] (1) Use soybean meal as raw material and control the moisture content of the raw material to be between 40% and 50%;

[0041] (2) Add neutral protease and proteinase K with an enzyme activity ratio of (1-2):1 to the raw materials. The inoculum size of Candida albicans BZD-4 is 0.1 × 10⁻⁶. 7 CFU ~ 0.5 × 10 7 The inoculation amount of *Lactobacillus paracasei* per gram of dry substrate is 0.2 × 10⁻⁶ CFU / gram of dry substrate. 8 CFU-0.5×10 8 CFU;

[0042] (3) Mix the above raw materials evenly, pile them up to a height of 80cm to 100cm, and ferment at a temperature of 37℃ to 40℃ for 12h to 24h;

[0043] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has a crude protein content of 50% to 55%, a moisture content of 10% to 12%, a relative content of acid-soluble protein of more than 15%, raffinose and stachyose degraded to less than 0.02%, and a miscellaneous bacteria rate of less than 0.1%.

[0044] The addition of complex proteases (neutral protease and proteinase K) in this invention can degrade crude protein in soybean meal into smaller protein molecules, thus allowing yeast and lactic acid bacteria to be further utilized, promoting microbial growth and the utilization of other components.

[0045] This invention utilizes Candida metasperidium during soybean meal fermentation to degrade large-molecule proteins (crude protein) in soybean meal. Simultaneously, the yeast's growth process has a "concentration effect" on protein feed, increasing the protein level in the feed. Yeast itself is also an excellent source of microbial protein, and its cell protein can enhance the protein quality of fermented soybean meal. Furthermore, yeast can effectively utilize oligosaccharides (raffinose and stachyose) in soybean meal, reducing the content of undesirable oligosaccharides and minimizing their impact on animals.

[0046] The high-temperature fermentation process in this invention can inhibit the growth of medium- and low-temperature bacteria, reduce interference, and achieve better fermentation results. The *Candida metamorphosus* BZD-4 provided by this invention has high-temperature resistance, maintaining good growth at 37℃-40℃, making it an effective strain suitable for high-temperature fermentation processes. High-temperature fermentation can effectively shorten the soybean meal fermentation cycle, improve production efficiency, and is suitable for industrial-scale promotion. It is a key technology for soybean meal fermentation and has significant application value. Attached Figure Description

[0047] Figure 1 The colony morphology of *Candida intercalaris* BZD-4 of this invention on YPD medium;

[0048] Figure 2 The morphology of Candida albicans BZD-4 of this invention under a microscope;

[0049] Figure 3 This is the liquid fermentation growth curve of *Candida albicans* BZD-4 of the present invention;

[0050] Figure 4 This is a pH tolerance experiment diagram of *Candida intercalaris* BZD-4 according to the present invention;

[0051] Figure 5 This is an experimental diagram of the oxygen tolerance of Candida albicans BZD-4 according to the present invention, where A represents 0h of inoculation; B represents aerobic conditions on a shaker; and C represents static culture conditions.

[0052] Figure 6 This is a temperature tolerance test diagram of the *Candida intercalaris* BZD-4 according to the present invention;

[0053] Figure 7 This is a diagram of the experimental tolerance of the interstitial Candida albicans BZD-4NaCl according to the present invention;

[0054] Figure 8 This is a diagram of the ethanol tolerance test of Candida albicans BZD-4 according to the present invention;

[0055] Figure 9 This is a diagram of the glucose tolerance experiment of Candida albicans BZD-4 according to the present invention. Detailed Implementation

[0056] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0057] Example 1

[0058] The thermotolerant intertype Candida BZD-4 of this embodiment is classified as Candida intermedia, with accession number CCTCC NO: M 2021828, and was deposited at the China Center for Type Culture Collection on July 8, 2021.

[0059] Example 2

[0060] This embodiment describes the application of a thermoresistant Candida albicans BZD-4 in soybean meal fermentation. In the laboratory setting, the soybean meal fermentation process was as follows:

[0061] In this embodiment, the optimal culture conditions for Candida intercalaris BZD-4 were shake flask culture at 40°C, with a shaker speed of 120 rpm, using YPD liquid medium (10 g / L glucose, 10 g / L peptone, and 5 g / L yeast extract) to obtain the Candida intercalaris BZD-4 seed culture.

[0062] Soybean meal fermentation experiments were conducted using *Candida metasporum*. The experimental conditions were: soybean meal loading 120g, material-to-water ratio 1:0.5 (w / v), compound protease (including 100U / g neutral protease and 50U / g proteinase K) added at 150U / g dry material, fermentation temperature 40℃, fermentation time 48h, and *Candida metasporum* BZD-4 seed culture inoculation amount 5%. The final fermented soybean meal contained 50.28±0.395% crude protein, 0.01% raffinose, 0.01% stachyose, and 15.008±0.390% relative acid-soluble protein. In this embodiment, the compound protease can degrade the crude protein in soybean meal into smaller protein molecules, thus facilitating further utilization by *Candida metasporum* BZD-4, thereby promoting microbial growth and the utilization of other components.

[0063] Example 3

[0064] This embodiment describes the application of a heat-resistant Candida albicans BZD-4 in soybean meal fermentation. The soybean meal fermentation process in large-scale production is as follows:

[0065] The moisture content of soybean meal was controlled at 50%, and the inoculum size of *Candida metaplasia* was 0.5 × 10⁻⁶. 7 CFU / g dry material was mixed evenly, piled up to a height of 100cm, and fermented at 40℃ for 24 hours. After fermentation, the product was dried at low temperature, crushed and packaged. The final product contained 51.83% crude protein, 10.23% moisture, 10.52% acid-soluble protein, 0.005% raffinose, 0.02% stachyose, and 0.03% miscellaneous bacteria.

[0066] Example 4

[0067] Because *Lactobacillus paracasei* produces organic acids such as lactic acid and acetic acid, which can lower the pH of the fermented soybean meal system, a lower pH is also the optimal growth environment for yeast. At the same time, acetic acid and lactic acid are excellent antibacterial substances; the presence of lactic acid and acetic acid in feed can increase the sour aroma of the feed during feeding, thereby increasing animal feed intake. Therefore, adding *Lactobacillus paracasei* for fermentation can improve feed quality and promote the synergistic fermentation process of bacteria and enzymes. The *Lactobacillus paracasei* used in this invention is *Lactobacillus paracasei* BZD-3, with accession number CCTCC NO:M2021827, classified as *Candida intermedia*, and deposited at the China Center for Type Culture Collection on July 23, 2021.

[0068] This embodiment describes the application of a thermotolerant Candida albicans BZD-4 combined with Lactobacillus paracasei in high-temperature fermented soybean meal, including the following steps:

[0069] (1) Soybean meal was used as raw material (soybean meal was purchased from COFCO Group), and the moisture content was controlled at 50% without any other treatment;

[0070] (2) Add neutral protease and proteinase K with an enzyme activity ratio of 2:1 to soybean meal, with an enzyme activity of 150 U / g dry material. The inoculum size of Candida albicans BZD-4 is 0.3 × 10⁻⁶. 7 The comparison results of 16S rDNA sequencing of the *Lactobacillus paracasei* strain with CFU / g dry material showed that it was only a subtype of *Lactobacillus paracasei*, with an inoculum size of 0.2 × 10⁻⁶. 8 CFU / g dry matter.

[0071] (3) After mixing evenly, pile up to a height of 100cm, control the fermentation temperature at 40-43℃, and ferment for 16h.

[0072] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 53.45% crude protein, 10.12% moisture, 15.85% acid-soluble protein, 0.01% raffinose, 0.01% stachyose and 0.05% miscellaneous bacteria.

[0073] Example 5

[0074] This embodiment describes the application of a thermotolerant Candida albicans BZD-4 combined with Lactobacillus paracasei in high-temperature fermented soybean meal, including the following steps:

[0075] (1) Use soybean meal as raw material and control the moisture content to 45%.

[0076] (2) Add neutral protease and proteinase K in an enzyme activity ratio of 1:1 to soybean meal, with an enzyme activity of 150 U / g dry material. The inoculum size of Candida albicans BZD-4 is 0.4 × 10⁻⁶.7 The inoculum size of *Lactobacillus paracasei* was 0.4 × 10⁻⁶ CFU / g dry material. 8 CFU / g dry matter.

[0077] (3) After mixing evenly, pile up to a height of 100cm, control the fermentation temperature at 37-40℃, and ferment for 24 hours.

[0078] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 53.83% crude protein, 10.42% moisture, 15.98% acid-soluble protein, 0.02% raffinose, 0.02% stachyose and 0.05% miscellaneous bacteria.

[0079] Example 6

[0080] This embodiment describes the application of a thermotolerant Candida albicans BZD-4 combined with Lactobacillus paracasei in high-temperature fermented soybean meal, including the following steps:

[0081] (1) Use soybean meal as raw material and control the moisture content to 50%.

[0082] (2) Add neutral protease and proteinase K in a 1:1 ratio to soybean meal, with an enzyme activity of 200 U / g dry material. The inoculum size of Candida albicans BZD-4 is 0.5 × 10⁻⁶. 7 The inoculum size of *Lactobacillus paracasei* was 0.5 × 10⁻⁶ CFU / g dry material. 8 CFU / g dry matter.

[0083] (3) After mixing evenly, pile up to a height of 80cm, control the fermentation temperature at 42-40℃, and ferment for 12 hours.

[0084] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 54.32% crude protein, 10.51% moisture, 16.68% acid-soluble protein, 0.02% raffinose, 0.01% stachyose and 0.01% miscellaneous bacteria rate.

[0085] Example 7

[0086] To compare the fermentation performance of the *Candida metasporum* BZD-4 of this invention with that of common high-temperature brewing yeast, a comparison was made with Example 4. The high-temperature brewing yeast in this example was purchased from Angel Yeast, purified by plate separation, and obtained through high-temperature screening. Its optimal growth temperature was 34-36℃, its acid tolerance was pH 2.5, its ethanol tolerance was 13%, and its glucose tolerance was 60%. The high-temperature brewing yeast was used for high-temperature soybean meal fermentation, including the following steps:

[0087] (1) Use soybean meal as raw material and control the moisture content to 50%.

[0088] (2) Add neutral protease and proteinase K in an enzyme activity ratio of 1:1 to soybean meal, with an enzyme activity of 150 U / g dry material. The inoculum size of high-temperature brewing yeast is 0.3 × 10⁻⁶. 7 The inoculum size of *Lactobacillus paracasei* was 0.2 × 10⁻⁶ CFU / g dry material. 8 CFU / g dry matter.

[0089] (3) After mixing evenly, pile up to a height of 100cm, control the fermentation temperature at 40-43℃, and ferment for 16h.

[0090] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 53.35% crude protein, 10.22% moisture, 13.41% acid-soluble protein, 0.12% raffinose, 0.55% stachyose and 0.5% miscellaneous bacteria.

[0091] Example 8

[0092] To compare the fermentation performance of the *Candida metasporum* BZD-4 of this invention with that of common high-temperature brewing yeast, a comparison was made with Example 5. The high-temperature brewing yeast in this example was purchased from Angel Yeast, purified by plate separation, and obtained through high-temperature screening. Its optimal growth temperature was 34-36℃, its acid tolerance was pH 2.5, its ethanol tolerance was 13%, and its glucose tolerance was 60%. The high-temperature brewing yeast was used for high-temperature soybean meal fermentation, including the following steps:

[0093] (1) Use soybean meal as raw material and control the moisture content to 45%.

[0094] (2) Add neutral protease and proteinase K in an enzyme activity ratio of 1:1 to soybean meal, with an enzyme activity of 150 U / g dry material. The inoculum size of high-temperature brewing yeast is 0.4 × 10⁻⁶. 7 The inoculum size of *Lactobacillus paracasei* was 0.4 × 10⁻⁶ CFU / g dry material. 8 CFU / g dry matter.

[0095] (3) After mixing evenly, pile up to a height of 100cm, control the fermentation temperature at 37-40℃, and ferment for 24 hours.

[0096] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 53.68% crude protein, 10.30% moisture, 13.78% acid-soluble protein, 0.26% raffinose, 0.64% stachyose and 0.6% miscellaneous bacteria.

[0097] Example 9

[0098] To compare the fermentation performance of the *Candida metasporum* BZD-4 of this invention with that of common high-temperature brewing yeast, a comparison was made with Example 6. The high-temperature brewing yeast in this example was purchased from Angel Yeast, purified by plate separation, and obtained through high-temperature screening. Its optimal growth temperature was 34-36℃, its pH tolerance was 2.5, its ethanol tolerance was 13%, and its glucose tolerance was 60%. The high-temperature brewing yeast was then used for high-temperature soybean meal fermentation, comprising the following steps:

[0099] (1) Use soybean meal as raw material and control the moisture content to 50%.

[0100] (2) Add neutral protease and proteinase K to soybean meal at a ratio of 1:1, with an enzyme activity of 200 U / g dry material. The inoculum size of high-temperature brewing yeast is 0.5 × 10⁻⁶. 7 The inoculum size of *Lactobacillus paracasei* was 0.5 × 10⁻⁶ CFU / g dry material. 8 CFU / g dry matter.

[0101] (3) After mixing evenly, pile the mixture to a height of 80cm, control the fermentation temperature at 42-45℃, and ferment for 12 hours.

[0102] (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has 54.35% crude protein, 10.35% moisture, 14.31% acid-soluble protein, 0.11% raffinose, 0.25% stachyose and 0.3% miscellaneous bacteria.

[0103] By comparing Examples 4 and 7, Examples 5 and 8, and Examples 6 and 9, it can be seen that when ordinary high-temperature yeast is used to replace Candida intermedia in the soybean meal fermentation process, the crude protein content is comparable, but the degradation degree of raffinose and stachyose, the relative content of acid-soluble protein, and the rate of miscellaneous bacteria are all worse than those of Candida intermedia.

[0104] Example 10

[0105] To verify the effects of three different yeasts (Candida intercalaris BZD-4, thermotolerant yeast, and common room-temperature yeast) and fermentation time on the high-temperature fermentation of soybean meal, this example investigated the effects of the three yeasts and fermentation times of 24 h and 12 h on crude protein, acidity, raffinose, stachyose, acid-soluble protein, and contamination rate. The fermentation steps were as follows:

[0106] (1) Soybean meal was used as raw material (soybean meal was purchased from COFCO Group), and the moisture content was controlled at 50% without any other treatment;

[0107] (2) Add neutral protease (100 U / g) and proteinase K (50 U / g) at an enzyme activity ratio of 2:1 to soybean meal, resulting in an enzyme activity of 150 U / g dry material. Inoculate with *Candida metasporum* BZD-4, thermotolerant yeast, and common room-temperature yeast, respectively, at an inoculation amount of 0.4 × 10⁻⁶. 7 CFU / g dry matter.

[0108] (3) Mix the materials of Candida intercalaris BZD-4, thermotolerant yeast and ordinary room temperature yeast evenly, pile them up to a height of 100cm, control the fermentation temperature at 40℃, and ferment for 12 and 24 hours respectively.

[0109] (4) After fermentation for 12 h and 24 h, the products were dried at low temperature, pulverized, and packaged. The crude protein, moisture, relative content of acid-soluble protein, raffinose, stachyose, and contamination rate of the products inoculated with Candida intercalaris BZD-4, thermotolerant yeast, and ordinary room temperature yeast were tested at 12 h and 24 h, respectively. The test results are shown in Table 2 below.

[0110] Table 2. Fermentation product indicators of Candida albicans BZD-4, thermotolerant yeast, and common room-temperature yeast.

[0111]

[0112] As shown in Table 2 above, the interstitial Candida BZD-4 of the present invention exhibits the best fermentation indicators under the above fermentation conditions, with low raffinose and stachyose content and low contamination rate. Furthermore, the indicators after 12 hours of fermentation are comparable to those after 24 hours of fermentation, both of which are superior to those of thermotolerant yeasts and ordinary room-temperature yeasts after 24 hours of fermentation. Therefore, the selected interstitial Candida BZD-4 can effectively shorten the fermentation time when applied to high-temperature fermented soybean meal.

Claims

1. A heat-resistant Candida metatype ( Candida intermedia BZD-4, characterized in that: The accession number is CCTCC NO: M 2021828.

2. The application of the heat-resistant interstitial Candida BZD-4 as described in claim 1 in soybean meal fermentation.

3. The application of the heat-resistant Candida albicans BZD-4 as described in claim 2 in soybean meal fermentation, characterized in that, Includes the following steps: (1) Using soybean meal as raw material, the moisture content of the raw material should be controlled at 40% to 50%; (2) Add neutral protease and proteinase K with an enzyme activity ratio of (1-2):1 to the raw materials. The inoculum size of Candida albicans BZD-4 is 0.1×10⁻⁶. 7 CFU ~ 0.5 × 10 7 The inoculation amount of *Lactobacillus paracasei* per gram of dry substrate is 0.2 × 10⁻⁶ CFU / gram of dry substrate. 8 CFU-0.5×10 8 CFU; (3) Mix the above raw materials evenly, pile them up to a height of 80cm to 100cm, and ferment at a temperature of 37℃ to 40℃ for 12h to 24h; (4) After fermentation, the product is dried at low temperature, crushed and packaged. The final product has a crude protein content of 50% to 55%, a moisture content of 10% to 12%, a relative content of acid-soluble protein of more than 15%, raffinose and stachyose degraded to less than 0.02%, and a miscellaneous bacteria rate of less than 0.1%.

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