Dvorstia and method for fermenting corn steep liquor using the same

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a Dvorst's bacteria and a method for fermenting corn steep liquor by using the same.The present application provides a Dvorst's bacteria capable of efficiently degrading vomitoxin in corn steep liquor, and corn steep liquor is fermented by using the Dvorst's bacteria and Candida krusei in cooperation, so that the pH of the corn steep liquor can be increased, the content of vomitoxin and anti-nutritional factors in the corn steep liquor can be reduced, and the content of choline in the corn steep liquor can be significantly increased, the quality of the corn steep liquor is obviously improved, the corn steep liquor after the cooperative fermentation can be better applied in the field of animal feed, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Dvorstia strain and a method for fermenting corn syrup using the same. BACKGROUND

[0002] Feed and its feed raw materials are extremely susceptible to contamination by mycotoxins during growth and storage, and mycotoxins enter the food chain, posing a threat to the health of animals and seriously restricting the development of animal husbandry. Vomitoxin (DON) is one of the most widely contaminated mycotoxins, which is mainly produced by fusarium and widely exists in grains such as corn, wheat and barley. Vomitoxin mainly affects protein synthesis by affecting the synthesis of DNA and RNA and blocking translation initiation, and this toxin can cause reproduction performance disorders such as loss of appetite, growth inhibition, metabolic disorder, immune disorder, infertility in female animals, abortion in pregnant animals, fetal abnormalities, and serious damage to animal husbandry.

[0003] Corn syrup is a concentrate of corn soaking water in the process of wet extraction of corn starch. Corn syrup contains rich protein, soluble sugar and short chain starch, and also contains rich amino acids, minerals and trace elements, etc., and is a high-quality animal feed raw material. However, corn syrup contains a certain amount of sulfite, which can cause sulfite residues in livestock and poultry if fed for a long time; and the pH of corn syrup is relatively low, and long-term feeding of corn syrup can also affect the digestive system of animals and cause impacts on their health and production performance; in addition, corn syrup is also contaminated by vomitoxin, which seriously restricts its application.

[0004] In the prior art, CN103387950 discloses a Dvorstia strain, which can degrade vomitoxin in grains. CN118406618A also discloses a Dvorstia strain and its application, which still has the ability to degrade vomitoxin after being cultured in a low pH environment, and has good tolerance to low pH environment. However, these prior arts do not focus on the problems of increasing the pH of corn syrup and degrading sulfite.

[0005] Therefore, how to increase the pH of corn syrup while reducing the content of vomitoxin and sulfite in corn syrup has become a technical problem to be solved in the field. SUMMARY

[0006] Firstly, the present application provides a Dvorstia strain (Dvorstia sp. Devosia sp. ), which is Dvorstia sp. C2-8, and its preservation number is CGMCC No. 32030.

[0007] The strain can efficiently degrade vomitoxin in corn syrup, and can completely degrade 100 µg / mL of DON within 10 min, thereby solving some of the problems existing in the prior art.

[0008] Furthermore, the present invention provides a microbial inoculum containing the aforementioned Dvora bacteria C2-8.

[0009] In some embodiments, the microbial agent is a freeze-dried fermentation broth of Dvora bacteria C2-8.

[0010] In some embodiments, the freeze-dried fermentation broth powder also includes a freeze-drying protectant.

[0011] Furthermore, the present invention provides a product containing the Dvora bacteria C2-8 or the microbial agent.

[0012] Furthermore, the present invention provides the application of the Dvora bacteria C2-8 or the microbial agent in the preparation of products.

[0013] In some implementations, the product is feed or feed additive.

[0014] Furthermore, the present invention also provides the application of the Dvora bacteria C2-8, the microbial agent, or the product in the degradation of vomitoxin.

[0015] In some embodiments, the present invention provides the use of the Dvora bacteria C2-8, the microbial agent, or the product in the degradation of vomitoxin in neutral corn steep liquor.

[0016] Furthermore, the present invention also provides a method for fermenting corn steep liquor, comprising: fermenting *Candida cruzi* (… Candida krusei )109 was inoculated into corn steep liquor for fermentation, and sterilized after the pH reached above 6.5. Then, it was inoculated with Dvora bacteria C2-8 for secondary fermentation; the Candida crus-galli ( Candida krusei The accession number for 109 is CGMCC No.32031.

[0017] By using Candida croceta 109 and Dvora C2-8 to co-ferment corn steep liquor, the pH of the corn steep liquor can be increased, the content of vomitoxin and antinutritional factors in the corn steep liquor can be reduced, and the choline content in the corn steep liquor can be increased.

[0018] Preferably, the anti-nutritional factor is phytic acid and sulfite ions.

[0019] The *Candida cruzi* 109 strain in this invention was isolated and screened from fermented foods. This strain was deposited on September 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification is *Candida cruzi*. Candida krusei The accession number is CGMCC No.32031.

[0020] The Devosia C2-8 in the application is isolated and screened from wheat, and the strain is preserved in the China General Microbiological Culture Collection Center on September 20, 2024, the address of the preservation unit is No. 3, Yuanmingyuan Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences; the postcode is 100101, and the classification name is Devosia Devosia sp. , and the preservation number is CGMCC No. 32030.

[0021] Compared with the prior art, the beneficial effects of the application are that:

[0022] The application provides a Devosia capable of efficiently degrading vomitoxin in corn steep liquor, and the corn steep liquor is fermented by the Devosia and the Candida krusei in cooperation, so that the pH of the corn steep liquor is increased, the content of vomitoxin and anti-nutritional factors in the corn steep liquor is reduced, and the content of choline in the corn steep liquor is significantly increased, the quality of the corn steep liquor is obviously improved, the corn steep liquor after the cooperative fermentation can be better applied in the field of animal feed, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a growth curve diagram of the Devosia C2-8.

[0024] Figure 2 is a result diagram of the Devosia C2-8 degrading DON standard products.

[0025] Figure 3 is a result diagram of the Devosia C2-8 degrading DON in neutral corn steep liquor medium.

[0026] Figure 4 is a result diagram of the Candida krusei degrading DON in corn steep liquor medium in cooperation with the Devosia.

[0027] Figure 5 is a result diagram of the influence of the Candida krusei fermentation in cooperation with the Devosia on anti-nutritional factors in corn steep liquor medium.

[0028] Figure 6 is a result diagram of the influence of the Candida krusei fermentation in cooperation with the Devosia on nutritional factors in corn steep liquor medium. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the application more clear, the technical scheme in the application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0030] In the examples provided in the present specification, if a specific technique or condition is not specified, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0031] Example 1 Growth curve of Devosia C2-8

[0032] Devosia C2-8 monoclonal was picked into LB medium and cultured at 180 rpm, 37°C for 24 h to obtain Devosia C2-8 seed liquid. Devosia C2-8 was inoculated into LB medium at an inoculation amount of 0.1%, and cultured at 180 rpm, 37°C. Samples were taken at 0, 6, 12, 24, 30, 36, and 42 h, and the absorbance of Devosia was detected at 600 nm. Finally, the growth curve was drawn.

[0033] As shown in Figure 1 , the logarithmic phase of Devosia was 6-24 h, and the stationary phase was reached after 24 h.

[0034] Example 2 Degradation of DON standard by Devosia C2-8

[0035] Devosia C2-8 monoclonal was picked into LB medium and cultured at 180 rpm, 37°C for 24 h to obtain Devosia C2-8 seed liquid. Devosia C2-8 was inoculated into LB medium at an inoculation amount of 1%, and cultured at 180 rpm, 37°C. Samples were taken at 18, 20, 22, 24, 26, 28, 42, and 48 h, and DON standard was added at a final concentration of 100 µg / mL. Incubation was performed at 180 rpm, 37°C. Samples were taken at 1, 3, 5, 10, and 60 min, and the DON content was detected.

[0036] As shown in Figure 2 , the 24 h cultured Devosia could completely degrade 100 µg / mL of DON standard within 10 min.

[0037] Example 3 Degradation of DON in neutral pH corn steep liquor by Devosia C2-8

[0038] The Dvorstia C2-8 monoclonal was inoculated into LB medium and cultured at 180 rpm and 37°C for 24 h to obtain a Dvorstia C2-8 seed solution, which was inoculated into corn steep liquor medium (corn steep liquor pH was adjusted to 7.0, and corn steep liquor medium was configured at 4%, 8%, 12%, 16%, 20% and 24% concentrations, and sterilized at 121°C for 15 min) at inoculation amounts of 1% and 5%, respectively, and cultured at 180 rpm and 37°C. Samples were taken at 24 h, and the DON content in the corn steep liquor was detected.

[0039] As shown in Figure 3 , the Dvorstia C2-8 can completely degrade DON in 8% corn steep liquor at an inoculation amount of 1% in 24 h; and the Dvorstia C2-8 can completely degrade DON in 12% corn steep liquor at an inoculation amount of 5% in 24 h.

[0040] Example 4 Dvorstia C2-8 degrading DON in corn steep liquor after the pH of the corn steep liquor is raised by Candida krusei 109

[0041] The Candida krusei monoclonal was inoculated into YPD medium and cultured at 180 rpm and 28°C for 24 h to obtain a Candida krusei seed solution, which was inoculated into corn steep liquor medium at a concentration of 4%-32% at an inoculation amount of 5%. When the pH of the medium reached 6.5 or above, it was sterilized, and then Dvorstia C2-8 was inoculated at an inoculation amount of 1%. The medium was cultured at 180 rpm and 37°C until the DON in the corn steep liquor was completely degraded.

[0042] As shown in Figure 4 , when the pH of 4%-24% corn steep liquor medium is raised to neutral by Candida krusei, inoculation of Dvorstia can completely degrade DON in the corn steep liquor, but as the concentration of the corn steep liquor increases, the time required for Dvorstia to degrade DON in the corn steep liquor increases. When the concentration of the corn steep liquor medium is raised to 28%, the ability of Dvorstia to degrade DON in the medium is significantly reduced, and the degradation rate of DON in the 28% corn steep liquor medium is only 22% after 3 d of incubation.

[0043] Example 5 Synergistic fermentation of Candida krusei 109 and Dvorstia C2-8 on anti-nutritional factors in corn steep liquor

[0044] The Candida krusei monoclonal was inoculated into YPD medium and cultured at 180 rpm and 28°C for 24 h to obtain a Candida krusei seed solution. The Candida krusei was inoculated into a 16% corn syrup medium at a 5% inoculation amount, the medium was sterilized after the pH reached 6.5 or above, and then the Dvorst's bacteria C2-8 was inoculated at a 1% inoculation amount. The medium was cultured at 180 rpm and 37°C for 24 h, and the phytic acid and sulfite concentrations in the corn syrup medium were detected. The sulfite in the corn syrup was determined according to GB 5009.34-2022 Determination of sulfur dioxide in food. The sample was weighed at 10 g and placed in a triangular flask, 40 mL of sodium sulfate-hydrochloric acid extraction solution (sodium sulfate concentration of 10%, hydrochloric acid concentration of 1.2%) was added, and oscillation extraction was performed for 2 h. The extraction liquid was centrifuged at 5000 rpm for 5 min, the supernatant was collected, and the sodium sulfate-hydrochloric acid extraction solution was used to dilute to 50 mL, and then filtered with a rapid filter paper for standby. 0.5 g of anion exchange resin (AG1-X4106 µm-250 µm, ion exchange capacity 3.5 mmol / g) was wet-packed into an empty column tube, and the ion exchange column was washed with 15 mL of 0.7 mol / L sodium chloride solution and 20 mL of water, respectively. 5 mL of filtrate was taken, 1 mL of 30 g / L sodium hydroxide solution was added, and water was used to dilute to 30 mL, and then mixed and transferred into the activated ion exchange column. Then, 15 mL of water and 15 mL of 0.05 mol / L sodium chloride solution were used to elute the exchange column at a flow rate of 1 mL / min, and the effluent was discarded. Finally, 25 mL of 0.7 mol / L sodium chloride solution was used for elution, and the eluate was collected and diluted to 25 mL. 5 mL of the above eluate was accurately taken into a 10 mL colorimetric tube, 4 mL of ferric chloride-sulfosalicylic acid reaction solution was added, mixed well, and stood for 20 min. Part of the mixed solution was poured into a 1 mL cuvette, and the absorbance was measured at 500 nm. The phytic acid content was determined according to the standard curve.

[0045] As shown in Figure 5 , after fermentation of Candida krusei and Dvorst's bacteria, the contents of phytic acid and sulfite were significantly reduced. The concentration of phytic acid decreased from 1.81 mg / mL to 0.98 mg / mL, and the concentration of sulfite decreased from 0.37 mg / mL to 0.28 mg / mL.

[0046] Example 6 Effect of synergistic fermentation of Candida krusei 109 and Dvorst's bacteria C2-8 on nutritional factors in corn syrup

[0047] Single clones of *Candida cruzi* were selected and cultured in YPD medium at 180 rpm and 28°C for 24 h to obtain *Candida cruzi* seed culture. *Candida cruzi* was then inoculated into 16% corn steep liquor medium at a 5% inoculum rate. The medium was sterilized after reaching a pH above 6.5, and then *Devrosella dysporum* C2-8 was inoculated at a 1% inoculum rate and cultured at 180 rpm and 37°C for 24 h. Changes in crude protein, inositol, and choline content in the corn steep liquor medium were then measured. Crude protein content was determined according to GB / T 6432-2018, inositol content according to NY / T 1345-2007, and choline content according to NY / T 1819-2009.

[0048] The results are as follows Figure 6 As shown, after fermentation by Candida cruzi and Dvora, the content of crude protein and inositol in corn steep liquor both decreased, but the difference in content was not significant; while the content of choline increased to a certain extent, from 2.35 g / L to 3.33 g / L.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fermenting corn steep liquor, characterized in that, Inclusive: Candida crus-galli ( Candida krusei )109 was inoculated into corn steep liquor for fermentation, and sterilized after the pH reached above 6.

5. Then, it was inoculated with Dvora bacteria C2-8 for secondary fermentation; the Candida crus-galli ( Candida krusei The accession number of 109 is CGMCC No. 32031; the accession number of Dvora bacteria C2-8 is CGMCC No. 32030; the processing method can increase the pH of corn steep liquor, reduce the content of vomitoxin and anti-nutritional factors in corn steep liquor, and increase the content of choline in corn steep liquor; the anti-nutritional factors are phytic acid and sulfite ions.

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