Candida krusei and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a Candida krusei strain and application thereof. The invention provides a Candida krusei strain which can effectively degrade antinutritional factors in corn steep liquor through a microbial fermentation process, improve the acid environment of the corn steep liquor and improve the application value of the corn steep liquor, and has a wide application prospect in the field of feeds.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Candida krusei and application thereof. Background Art

[0002] Corn syrup is one of the by-products of corn starch production by wet process. It is made by soaking corn in water, dehydrating it, and then soaking it in 0.2%-0.25% sulfurous acid solution for 60-80 hours before evaporation and concentration. Every ton of corn processed will produce 0.8m 3 The annual output of corn pulp reaches 45 million tons. Corn pulp is rich in nutrients, including protein, peptides, amino acids, polysaccharides, vitamins, trace elements and growth factors. Adding a proper amount can increase the energy value and protein content of feed, enhance the nutritional content of feed, and is beneficial to the healthy growth of animals.

[0003] However, the pH of corn syrup is extremely low (about pH 3.5), and it contains anti-nutritional factors such as sulfites and phytic acid, which seriously restricts its application in feed. Microbial fermentation can degrade the anti-nutritional factors in corn syrup, improve the acidic environment of corn syrup, and enhance its application value.

[0004] In the prior art, CN114027435A discloses a method for eliminating anti-nutritional factors in sunflower meal by mixed fermentation of probiotics. In this method, Bacillus subtilis is used in combination with Saccharomyces cerevisiae and Trichoderma longibrachiatum to eliminate anti-nutritional factors in sunflower meal through liquid fermentation. This method uses many types of microorganisms and has a high cost. In addition, it targets anti-nutritional factors in sunflower meal, not corn pulp.

[0005] Therefore, how to provide a microorganism that can effectively degrade anti-nutritional factors in corn steep liquor has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] The present invention provides a strain of Candida krusei, which can effectively degrade anti-nutritional factors in corn steep liquor through a microbial fermentation process. Based on this, the following technical scheme is proposed.

[0007] First, the present invention provides a strain of Candida krusei ( Candida krusei ), which is Candida krusei 109, and its preservation number is CGMCC No.32031.

[0008] The Candida krusei 109 of the present invention is isolated and screened from fermented food. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 20, 2024. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Candida krusei Candida krusei, the deposit number is CGMCC No.32031.

[0009] Furthermore, the present invention provides a microbial agent, which comprises the Candida krusei 109.

[0010] Preferably, the microbial agent is freeze-dried powder of fermentation broth of Candida krusei 109.

[0011] Preferably, the fermentation broth freeze-dried powder also includes a freeze-drying protectant.

[0012] Furthermore, the present invention provides a product, which comprises the Candida krusei 109 or the microbial agent.

[0013] Furthermore, the present invention provides the use of the Candida krusei 109 or the microbial agent in preparing products.

[0014] Preferably, the product is feed or feed additive.

[0015] Furthermore, the present invention provides the use of the Candida krusei 109, the microbial agent or the product in improving the pH of a YPD culture medium.

[0016] At the same time, the present invention also provides the use of the Candida krusei 109, the microbial agent or the product in improving the pH of corn steep liquor.

[0017] At the same time, the present invention also provides the use of the Candida krusei 109, the microbial agent or the product in degrading anti-nutritional factors in corn steep liquor.

[0018] Preferably, the anti-nutritional factors are phytic acid and / or sulfite ions.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a strain of Candida krusei, which can effectively degrade anti-nutritional factors in corn steep liquor through a microbial fermentation process, improve the acidic environment of corn steep liquor, enhance its application value, and has broad application prospects in the field of feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a growth curve of Candida krusei 109.

[0021] Figure 2 This is a growth curve of Candida krusei 109 in YPD medium with different pH values.

[0022] Figure 3 This is a graph showing the test results of the ability of Candida krusei 109 to increase the pH of YPD medium.

[0023] Figure 4 This is a graph showing the test results of the tolerance of Candida krusei 109 to sulfite.

[0024] Figure 5 This is a test result of the ability of Candida krusei 109 to increase the pH of corn syrup at a 1% inoculation rate.

[0025] Figure 6 This is a graph showing the time required for Candida krusei 109 to raise the pH of corn slurry to neutral at a 5% inoculation rate.

[0026] Figure 7 This is a statistical chart showing the changes in anti-nutritional factor content of Candida krusei 109 before and after fermentation in corn steep liquor medium. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0029] Example 1 Growth curve of Candida krusei 109 A single clone of Candida krusei 109 was selected and placed in YPD medium. The culture was carried out at 180 rpm and 28 °C for 24 h to obtain the seed solution of Candida krusei. The Candida krusei was inoculated into YPD medium at a 0.1% inoculation rate and cultured at 180 rpm and 28 °C. Samples were taken at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. The absorbance of Candida krusei was detected at 600 nm, and the growth curve was finally drawn.

[0030] Growth curve Figure 1 As shown, Candida krusei 109 was in the logarithmic growth phase from 12 to 48 h and reached the stationary phase after 48 h.

[0031] Example 2 Growth of Candida krusei 109 in YPD medium at different pH values A single clone of Candida krusei 109 was selected and put into YPD medium. The culture was carried out at 180 rpm and 28 °C for 24 h to obtain the seed solution of Candida krusei. The Candida krusei was inoculated into YPD medium with different pH values ​​at a 0.1% inoculation rate and cultured at 180 rpm and 28 °C. Samples were taken at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. The absorbance of Candida krusei was detected at 600 nm, and the growth curve of Candida krusei in YPD medium with different pH values ​​was finally drawn.

[0032] like Figure 2 As shown in the figure, pH 12 and pH 13 can significantly inhibit the growth of Candida krusei in YPD medium. After continuous monitoring for 96 hours, the OD value has no significant change; while pH 2, pH 10 and pH 11 can slow down the growth of Candida krusei. When the pH of YPD medium is between 3 and 9, Candida krusei can grow normally.

[0033] Example 3 Ability of Candida krusei 109 to increase pH of YPD medium A single clone of Candida krusei 109 was selected and placed in YPD medium. The culture was carried out at 180 rpm and 28°C for 24 h to obtain Candida krusei seed solution. The Candida krusei was inoculated into YPD medium with different pH values ​​at a 0.1% inoculation rate. The culture was carried out at 180 rpm and 28°C. Samples were taken at 96 h to measure the pH change.

[0034] like Figure 3 As shown, Candida krusei can significantly increase the pH of acidic culture medium. For example, the pH of YPD medium with a pH of 3 reaches 3.98 after 96 hours of culture, and the pH of culture medium with pH of 4, 5, 6 and 7 reaches 7.75-8.35 after 96 hours of culture.

[0035] Example 4 Growth of Candida krusei 109 at different sulfite concentrations A single clone of Candida krusei 109 was selected and placed in YPD medium. The culture was carried out at 180 rpm and 28°C for 24 h to obtain a Candida krusei seed solution. The Candida krusei was inoculated at a 0.1% inoculation rate into YPD medium containing different concentrations of sulfite (CK is a control group without sulfite). The culture was carried out at 180 rpm and 28°C. Samples were taken at 0 h, 12 h and 24 h. The absorbance of Candida krusei was detected at 600 nm and the curve was drawn.

[0036] The results are as follows Figure 4As shown in the figure, Candida krusei has a certain tolerance to sulfite. It can grow at a sulfite concentration of 0.05%-2%, but as the sulfite concentration increases, the growth rate is inhibited. When the sulfite concentration rises to 10%, the growth of Candida krusei is completely inhibited.

[0037] Example 5 Ability of Candida krusei 109 to increase the pH of corn syrup A single clone of Candida krusei 109 was selected and placed in YPD medium. The culture was carried out at 180 rpm and 28°C for 24 h to obtain Candida krusei seed solution. The Candida krusei was inoculated into corn slurry medium with different concentrations (4%-40%, corn slurry medium was corn slurry of different concentrations, sterilized at 121°C for 15 min) at an inoculation rate of 1% and 5%. The pH of the culture medium was measured every day. When the pH reached above 6.5, the culture and measurement were terminated.

[0038] The results are as follows Figure 5 As shown in the figure, Candida krusei can significantly increase the pH of corn steep liquor. After 3 days of culture, the pH of 4% corn steep liquor can reach neutral. After 10 days of culture, the pH of 20% corn steep liquor can reach neutral. After 10 days of culture, the pH of 40% corn steep liquor has no significant change. After inoculating Candida krusei into corn steep liquor at a 5% inoculation rate, its ability to increase the pH of corn steep liquor is significantly improved. Figure 6 As shown, the time for raising the pH to neutrality with 4% corn steep liquor culture medium is shortened to 2 days, and the time for raising the pH to neutrality with 20% corn steep liquor culture medium is shortened to 8 days. Meanwhile, after 28 days of cultivation, the pH of 40% corn steep liquor culture medium can reach neutrality.

[0039] Example 6 Effect of Anti-Nutrient Factors on Fermentation of Candida krusei 109 in Corn Stew Medium A single clone of Candida krusei 109 was selected and placed in YPD medium. The culture was carried out at 180 rpm and 28°C for 24 h to obtain Candida krusei seed solution. The Candida krusei was inoculated into 16% corn steep liquor medium at a 5% inoculation rate. When the pH of the corn steep liquor medium reached neutral, the concentrations of phytic acid and sulfite in the corn steep liquor were determined.

[0040] The determination of sulfite in corn steep liquor refers to GB 5009.34-2022 Determination of sulfur dioxide in food. Determination of phytic acid: weigh 10g of sample and place it in a conical flask, add 40mL of sodium sulfate-hydrochloric acid extraction solution (sodium sulfate concentration is 10%, hydrochloric acid concentration is 1.2%), shake and extract for 2h, centrifuge the extract at 5000rpm for 5min, collect the supernatant, and make up to 50mL with sodium sulfate-hydrochloric acid extraction solution, filter through rapid filter paper for later use. Take 0.5g of anion exchange resin (AG1-X4106µm-250µm, ion exchange capacity 3.5mmol / g) and wet-load it into an empty column tube, wash the ion exchange column with 15mL of 0.7mol / L sodium chloride solution and 20mL of water respectively. Take 5mL of filtrate, add 1mL of 30g / L sodium hydroxide solution, dilute to 30mL with water, mix well and transfer to the activated ion exchange column, then elute the exchange column with 15mL of water and 15mL of 0.05mol / L sodium chloride solution at a flow rate of 1mL / min, and discard the effluent. Finally, elute with 25mL of 0.7mol / L sodium chloride solution, collect the eluate, and dilute to 25mL. Accurately draw 5mL of the dilute eluate obtained in the previous section into a 10mL colorimetric tube, add 4mL of ferric chloride-sulfosalicylic acid reaction solution, mix well, let stand for 20min, draw part of the mixed solution into a 1mL colorimetric dish, measure the absorbance at 500nm, and determine the phytic acid content according to the standard curve.

[0041] The results are as follows Figure 7 As shown, the contents of phytic acid and sulfite in the corn steep liquor culture medium were significantly reduced before and after fermentation of Candida krusei, with the phytic acid content decreasing from 1.81 mg / mL to 0.89 mg / mL and the sulfite content decreasing from 0.37 mg / mL to 0.27 mg / mL, indicating that Candida krusei has the ability to degrade anti-nutritional factors in corn steep liquor culture medium.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Candida krusei ( Candida krusei ), characterized in that, It is Candida krusei 109, and its deposit number is CGMCC No.32031.

2. A microbial agent, characterized in that: The method comprises the Candida krusei 109 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent is freeze-dried powder of fermentation broth of Candida krusei 109.

4. The microbial agent according to claim 3, characterized in that: The fermentation broth freeze-dried powder also includes a freeze-drying protectant.

5. A product, characterized in that: The product comprises the Candida krusei 109 according to claim 1 or the microbial agent according to any one of claims 2 to 4.

6. Use of the Candida krusei 109 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in preparing a product.

7. Use of the Candida krusei 109 according to claim 1, the microbial agent according to any one of claims 2 to 4, or the product according to claim 5 in improving the pH of a YPD medium.

8. Use of the Candida krusei 109 according to claim 1, the microbial agent according to any one of claims 2 to 4, or the product according to claim 5 in improving the pH of corn steep liquor.

9. Use of the Candida krusei 109 according to claim 1, the microbial agent according to any one of claims 2 to 4, or the product according to claim 5 in degrading anti-nutritional factors in corn steep liquor.

10. The use according to claim 9, characterized in that: The anti-nutritional factors are phytic acid and / or sulfite ions.

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