Candida utilis strain suitable for cassava broth fermentation for producing feed protein and application thereof

Abstract

The application relates to the technical field of microbial fermentation, and discloses a Candida utilis, a microbial agent, application of the Candida utilis or the microbial agent in a feed additive, a feed additive, and a preparation method of the feed additive. The Candida utilis has high protein yield, high small peptide content, and rich amino acid types, and the Candida utilis can be used to produce cell protein by using cassava supernatant, can be used as a high-value feed additive, and can realize high-value utilization of the cassava supernatant.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Candida utilis, an inoculum, the application of Candida utilis or its inoculum in feed additives, feed additives, and methods for preparing feed additives. Background Technology

[0002] With fluctuating raw material prices, fuel ethanol companies are dynamically adjusting their feedstocks, using diversified raw materials to maximize profits. Currently, some ethanol producers use 100% cassava as a raw material for fermentation. However, using 100% cassava for fermentation produces a large amount of cassava supernatant. Cassava supernatant is the supernatant obtained after centrifuging distiller's grains. Its substances cannot be utilized by brewer's yeast, and its complex composition makes it a high-concentration organic wastewater. While it does not contain heavy metals or toxic substances, it decomposes rapidly depending on storage conditions. Direct discharge as waste not only results in significant resource waste but also environmental pollution.

[0003] In the feed additive industry, *Candida utilis* is a crucial microorganism, commonly used in the production of glutathione, amino acids, and certain enzymes. *Candida utilis* can proliferate and ferment using non-food industrial resources and waste materials, exhibiting a strong ability to convert inorganic nitrogen into organic nitrogen and producing microbial proteins rich in protein, nucleotides, growth factors, various enzymes, and vitamins. Simultaneously, it stimulates the rumen of ruminants to produce a large number of beneficial microorganisms, regulating their gut microbiota and enhancing their immunity. *Candida utilis* has a high proliferation density and can be used alone as an excellent microbial protein feed, showing broad development prospects.

[0004] Currently, some reports have studied the cultivation of *Candida utilis* using waste liquids from different factories, but these are all laboratory studies with limited reference value for industrial applications. Some researchers have found that inoculating 5% *Candida utilis* into distillery waste can achieve good fermentation results, with the crude protein content of the waste reaching 18.28% after fermentation (Study on the Influence of Different Additives on the Nutritional Value of Fermented Feed from Distillery Waste, Sun Yanan et al., 2022). However, distillery waste is already rich in protein and other nutrients, so the crude protein production capacity of this *Candida utilis* strain is not very high. Furthermore, unlike distillery waste, the clear liquid after centrifugation has a lower content of crude protein and other nutrients, making it more difficult for the strain to utilize. Existing strains show better fermentation results using distillery waste than clear liquid, and no research has yet publicly disclosed *Candida utilis* strains capable of efficiently utilizing and degrading cassava clear liquid. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a strain of *Candida utilis*, an inoculum, the application of *Candida utilis* or its inoculum in feed additives, feed additives, and a method for preparing feed additives. This *Candida utilis* strain has high protein yield, high small peptide content, and a rich variety of amino acids. Furthermore, using this *Candida utilis* strain allows for the production of cell protein from cassava extract, which can be used as a high-value-added feed additive, thereby realizing the high-value utilization of cassava extract.

[0006] To achieve the above objectives, the present invention provides a Candida utilis yeast, which has the accession number CGMCC NO.22631.

[0007] A second aspect of the present invention provides a microbial agent containing Candida utilis as described above.

[0008] A third aspect of the present invention provides the use of the aforementioned Candida utilis or the aforementioned inoculum in feed additives.

[0009] The fourth aspect of the present invention provides a method for preparing a feed additive, the method comprising: inoculating the aforementioned Candida utilis or the aforementioned inoculum into a fermentation medium for fermentation to obtain a fermentation broth.

[0010] The fifth aspect of the present invention provides a feed additive prepared by the preparation method described above.

[0011] The sixth aspect of the present invention provides the application of the aforementioned Candida utilis or the aforementioned inoculum in the treatment of cassava broth.

[0012] The seventh aspect of the present invention provides a method for treating cassava extract, the method comprising: inoculating the cassava extract with the aforementioned Candida utilis yeast or the aforementioned inoculum.

[0013] The beneficial effects obtained by the present invention through the above technical solution include:

[0014] 1. Compared with other Candida species, the Candida utilis strain of this invention has a faster growth rate, higher protein yield, higher small peptide yield, and shorter fermentation time, making it more suitable for industrial production.

[0015] 2. Compared with other Candida species, the Candida utilis of this invention has a better ability to utilize cassava extract (especially oligosaccharides (maltodextrin, cellulose oligosaccharides), xylose, glycerol and organic acids in cassava extract), and can have good growth ability in cassava extract containing organic acids and glycerol. It can utilize cassava extract to produce up to 53.47 wt.% crude protein and up to 25.26 wt.% small peptides, thereby achieving high-value utilization of cassava extract and reducing environmental pollution.

[0016] 3. The protein produced by the Candida utilis of the present invention is rich in a variety of amino acids, which can meet the nutritional needs of livestock such as cattle and sheep, and is therefore very suitable for the preparation of feed additives.

[0017] Biological Preservation

[0018] The Candida utilis strain of this invention was deposited on May 28, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCCNo.22631, abbreviated as CUNX-01. Attached Figure Description

[0019] Figure 1 This is a 400x optical microscope image of the Candida utilis of the present invention;

[0020] Figure 2 This is a contour plot of the nitrogen source screening in shake-flask fermentation in Example 3, with yeast count as the response value;

[0021] Figure 3 This is a graph showing the change in OD value of the fermentation broth of the present invention at different rotation speeds as of fermentation time in Example 3.

[0022] Figure 4 This is a graph showing the change in yeast count in the fermentation broth of *Candida utilis* of the present invention with fermentation time under different dissolved oxygen (DO) parameters in Example 3.

[0023] Figure 5 This is a graph showing the change in yeast count in the fermentation broth of *Candida utilis* of the present invention with fermentation time at different feeding times in Example 3.

[0024] Figure 6 This is a graph showing the change in yeast count in the fermentation broth of *Candida utilis* of the present invention over fermentation time at different feeding rates in Example 3. Detailed Implementation

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] In this invention, "small peptide" refers to a small-molecule active protein that lies between amino acids and high-molecular-weight proteins (large proteins), specifically with a molecular weight below 1000 Daltons. Small peptides are superior not only to amino acids but also to high-molecular-weight proteins (large proteins). Compared to large-molecular-weight proteins with molecular weights generally ranging from tens of thousands to hundreds of thousands of Daltons, small peptides with a molecular weight below 1000 Daltons can be directly absorbed without digestion. The content of the small peptides is determined according to Appendix B (Determination of Peptide Content) of the national standard GB / T 22492-2008 "Soybean Peptide Powder".

[0027] The first aspect of this invention provides a Candida utilis yeast, which has the accession number CGMCC NO.22631.

[0028] A second aspect of the present invention provides a microbial agent containing Candida utilis as described above.

[0029] In this invention, the form of the microbial agent can be a conventional form of microbial agent in the art, such as a solid, liquid or semi-solid form.

[0030] In some embodiments of the present invention, the bacterial agent contains live cells of the said Candida utilis.

[0031] In this invention, the number of live bacteria in the bacterial agent can be selected within a wide range, as long as it meets the requirements of relevant standards.

[0032] The preparation method of the bacterial agent can refer to the conventional preparation methods in this field, and will not be described in detail here.

[0033] A third aspect of the present invention provides the use of the aforementioned Candida utilis or the aforementioned inoculum in feed additives.

[0034] In some embodiments of the present invention, the feed additive is a livestock feed additive, preferably a ruminant feed additive and / or a monogastric animal feed additive.

[0035] The fourth aspect of the present invention provides a method for preparing a feed additive, the method comprising: inoculating the aforementioned Candida utilis or the aforementioned inoculum into a fermentation medium for fermentation to obtain a fermentation broth.

[0036] In this invention, the inoculation amount of the Candida utilis or the inoculum can be selected within a wide range, for example, it can be inoculated at a final concentration of 0.05-100 million yeast cells / mL.

[0037] In this invention, the fermentation medium can be a commonly used culture medium for Candida utilis fermentation, such as YPD medium, molasses medium, etc. The Candida utilis of this invention can utilize cassava extract. When inoculated into cassava extract, the resulting fermentation broth has high protein and small peptide content, and the protein in the fermentation broth is rich in various amino acids, which can meet the nutritional needs of livestock. Therefore, in some embodiments of this invention, the fermentation medium can also be a cassava extract fermentation medium. Preferably, the fermentation medium is a cassava extract fermentation medium.

[0038] In this invention, the "molasses culture medium" refers to a culture medium containing molasses. Besides molasses, the molasses culture medium may also contain other components commonly used in culture media for Candida utilis fermentation, such as nitrogen sources and / or inorganic salts. Preferably, the nitrogen source content and inorganic salt content of the molasses culture medium are 0-10 g / L and 0-10 g / L, respectively. Preferably, the nitrogen source in the molasses culture medium is ammonium sulfate and / or urea; the inorganic salt in the molasses culture medium is at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

[0039] In this invention, the "cassava extract fermentation medium" refers to a culture medium containing cassava extract. Besides cassava extract, the cassava extract fermentation medium may also contain other components commonly used in Candida utilis fermentation media, such as at least one of carbon sources, nitrogen sources, and inorganic salts. Unless otherwise specified, the carbon sources, nitrogen sources, and inorganic salts refer to components added to the cassava extract fermentation medium in addition to those already present in the cassava extract itself. It should be understood that, preferably, these components can further increase the yeast count, protein content, and small peptide content of Candida utilis in the fermentation broth.

[0040] In some embodiments of the present invention, the pH value of the cassava extract fermentation medium is 5-7.

[0041] In some embodiments of the present invention, the cassava broth fermentation medium also contains an antibacterial agent. The type of antibacterial agent is not particularly limited, as long as it does not inhibit the growth of *Candida utilis*. The amount of antibacterial agent added is selected using conventional techniques in the art, as long as it can inhibit the growth of other microorganisms, for example, 5-20 mg / L.

[0042] In some embodiments of the present invention, the cassava extract fermentation medium contains cassava extract, an optional carbon source, an optional nitrogen source, an optional inorganic salt, and an antibacterial agent.

[0043] In some embodiments of the present invention, the method for preparing the cassava broth fermentation medium is as follows: an optional carbon source, an optional nitrogen source, an optional inorganic salt and an antibacterial agent are dissolved in cassava broth to obtain the cassava broth fermentation medium.

[0044] In this invention, the carbon source content in the cassava broth fermentation medium can be selected within a wide range, for example, it can be any one of 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g, 8 g / L, 9 g / L, and 10 g / L, or a value within a range of any two of the above. The inventors have found that when the cassava broth fermentation medium contains a carbon source, the yeast count of *Candida utilis* in the fermentation broth can be further increased. Therefore, preferably, the carbon source content in the cassava broth fermentation medium is 2-10 g / L.

[0045] In some embodiments of the present invention, the carbon source in the cassava broth fermentation medium is molasses. Preferably, the molasses is soybean molasses and / or sugarcane molasses. More preferably, the molasses is soybean molasses.

[0046] In some embodiments of the present invention, the nitrogen source content in the cassava broth fermentation medium can be any one of 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g, 8 g / L, 9 g / L, or 10 g / L, or a value within a range of any two of the above values. To further increase the number of Candida utilis yeasts in the fermentation broth, preferably, the nitrogen source content in the cassava broth fermentation medium is 2-6 g / L.

[0047] In some embodiments of the present invention, the nitrogen source in the cassava broth fermentation medium is ammonium sulfate and / or urea. Preferably, in order to further increase the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth, the nitrogen source is preferably ammonium sulfate.

[0048] In some embodiments of the present invention, the content of inorganic salts in the cassava broth fermentation medium can be selected within a wide range. For example, the content of inorganic salts can be any one of 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g, 8 g / L, 9 g / L, 10 g / L, or a value within a range of any two of the above values.

[0049] In some embodiments of the present invention, the inorganic salt in the cassava broth fermentation medium is at least one selected from potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate. Preferably, when the fermentation medium is a cassava broth fermentation medium, the inorganic salt is preferably potassium dihydrogen phosphate.

[0050] In this invention, the "cassava clear liquid" refers to the supernatant obtained after centrifugation of the lees obtained during cassava fermentation to produce alcohol.

[0051] In some embodiments of the present invention, the cassava extract contains at least one of soluble sugar, organic acid, and glycerol. Specifically, the soluble sugar content in the cassava extract is 15-20 g / L, the organic acid content is 10-15 g / L, and the glycerol content is 5-10 g / L. The inventors have found that when the soluble sugar content is within the above range, the growth rate of *Candida utilis* is faster.

[0052] In some embodiments of the present invention, the soluble sugar contains monosaccharides, and the monosaccharide content in the cassava extract is 1-3 g / L. When the monosaccharide content is within the above range, the number of *Candida utilis* at the end of fermentation is the highest. In some embodiments of the present invention, the monosaccharide is glucose and / or xylose, wherein the glucose content in the cassava extract is 0.5-1 g / L and the xylose content is 1-2 g / L.

[0053] In some embodiments of the present invention, the soluble sugar further contains soluble sugars (i.e., oligosaccharides) with a degree of polymerization of 2 or higher. The content of the oligosaccharides in the cassava extract is 12-18 g / L. Preferably, the oligosaccharides are maltodextrin and / or cellulose oligosaccharides. In the present invention, the oligosaccharides refer to soluble sugars with a degree of polymerization of 2 or higher.

[0054] In some preferred embodiments of the present invention, the soluble sugar content in the cassava extract is 15-20 g / L. Preferably, the soluble sugar is a monosaccharide and an oligosaccharide, with the monosaccharide content being 1-3 g / L and the oligosaccharide content being 12-18 g / L. Preferably, the monosaccharide is glucose and xylose, with the glucose content in the cassava extract being 0.5-1 g / L and the xylose content being 1-2 g / L. Preferably, the oligosaccharide is maltodextrin and cellulose oligosaccharide.

[0055] In some preferred embodiments of the present invention, the cassava broth fermentation medium contains cassava broth, soybean molasses, and ammonium sulfate, and does not contain any other inorganic salts besides ammonium sulfate; preferably, the soybean molasses content is 2-5 g / L, and the ammonium sulfate content is 5-10 g / L. When the cassava broth fermentation medium is within this preferred range, it can further increase the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth. Preferably, the pH value of the cassava broth fermentation medium is 5-7.

[0056] In some preferred embodiments of the present invention, the cassava broth fermentation medium contains cassava broth, sugarcane molasses, and optionally ammonium sulfate, and does not contain any other inorganic salts besides ammonium sulfate; preferably, the sugarcane molasses content is 5-10 g / L, and the ammonium sulfate content is 0-5 g / L. More preferably, the cassava broth fermentation medium contains cassava broth and sugarcane molasses, and does not contain ammonium sulfate or inorganic salts. Within this preferred range, the cassava broth fermentation medium can further increase the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth. Preferably, the pH value of the cassava broth fermentation medium is 5-7.

[0057] In some embodiments of the present invention, the method includes: the Candida utilis is activated before inoculation.

[0058] In this invention, the activation treatment can be a conventional preparation method in the art, such as primary seed culture or secondary seed culture. Those skilled in the art can choose the culture method according to the actual situation. Preferably, the culture medium used for seed culture is a conventionally selected one in the art, such as YPD medium.

[0059] In some embodiments of the present invention, the activation treatment is as follows: (1) inoculating a single colony of the Candida utilis strain onto a plate and culturing it in YPD liquid medium to obtain a primary seed culture; (2) inoculating the primary seed culture into a new YPD liquid medium and culturing it to obtain a secondary seed culture.

[0060] In some embodiments of the present invention, in step (1), the single colony of the Candida utilis strain can be directly inoculated into 30-50 mL of YPD liquid medium and cultured at 28-30℃ and 150-200 rpm for 16-20 h to form a primary seed culture. Alternatively, it can be first inoculated into 5-10 mL of YPD liquid medium and cultured at 28-30℃ and 150-200 rpm for 16-24 h, and then inoculated into new YPD liquid medium at an inoculation rate of 0.5-1 vol.% and cultured at 28-30℃ and 150-200 rpm for 16-20 h to form a primary seed culture.

[0061] In some embodiments of the present invention, step (2) includes: inoculating the primary seed solution into a new YPD liquid culture medium at an inoculation amount of 0.5-1 vol.%, and culturing at 28-30°C and 150-200 rpm for 16-20 h to form a secondary seed solution.

[0062] In some preferred embodiments of the present invention, the activation treatment is as follows: a single colony of the Candida utilis strain is first inoculated into 5-10 mL of YPD liquid medium and cultured at 28-30°C and 150-200 rpm for 16-24 h. Then, it is inoculated into new YPD liquid medium at an inoculation rate of 0.5-1 vol.% and cultured at 28-30°C and 150-200 rpm for 16-20 h to form a primary seed culture. The primary seed culture is then inoculated into new YPD liquid medium at an inoculation rate of 0.5-1 vol.% and cultured at 28-30°C and 150-200 rpm for 16-20 h to form a secondary seed culture.

[0063] In some embodiments of the present invention, the fermentation conditions include: a temperature of 28-30°C, dissolved oxygen of 10-40%, and fermentation time of 18-24 hours. Preferably, to further increase the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth, the fermentation conditions include: a temperature of 28-30°C, dissolved oxygen of 10-30%, and fermentation time of 18-24 hours. The dissolved oxygen level in the fermentation system can be controlled by aeration and stirring (or shaking). To achieve the above dissolved oxygen levels, the aeration rate and stirring (or shaking) rate can be selected within a wide range; for example, the aeration rate can be selected within the range of 0.5-10 L / min, and the stirring (or shaking) rate can be selected within the range of 300-900 rpm. The inventors have found that when the stirring (or shaking) rate is within the above range, the yeast count of *Candida utilis* in the fermentation broth can be further increased. More preferably, the stirring (or shaking) rate can be selected within the range of 500-900 rpm.

[0064] In some embodiments of the present invention, the fermentation conditions further include a pH of 5-7. The pH control is a conventional technique in the art, for example, pH can be controlled using ammonia or sulfuric acid solution, and will not be elaborated further here.

[0065] In some embodiments of the present invention, the method further includes feeding, the feeding comprising: adding a carbon source during fermentation.

[0066] In some embodiments of the present invention, the feeding rate is 2-20 g / h relative to 1 L of initial fermentation medium. The inventors have found that when the feeding rate is 10-20 g / h relative to 1 L of initial fermentation medium, the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth can be further increased. More preferably, the feeding rate is 15-20 g / h relative to 1 L of initial fermentation medium.

[0067] The inventors discovered that feeding the yeast during the 5th-12th hour of fermentation can further increase the yeast count, protein content, and small peptide content of *Candida utilis* in the fermentation broth. Therefore, in a preferred embodiment, the method further includes feeding the yeast during the 5th-12th hour of fermentation. More preferably, the method further includes feeding the yeast during the 8th-12th hour of fermentation.

[0068] In some embodiments of the present invention, the feeding time is 5-10 hours.

[0069] In this invention, the feed additive can be in the form of a solid and / or a liquid. When the feed additive is in the form of a solid, the method may further include: performing solid-liquid separation on the fermentation broth and low-temperature drying, with the resulting solid being the feed additive. The solid-liquid separation method can be a conventional technique in the art, such as centrifugation, filtration, or rotary evaporation. The low-temperature drying method can be a conventional technique in the art, as long as the moisture content of the material after low-temperature drying reaches below 12% (v / v), for example, vacuum low-temperature drying can be used.

[0070] The fifth aspect of the present invention provides a feed additive prepared by the preparation method described above.

[0071] In some embodiments of the present invention, the feed additive contains crude protein. Preferably, the crude protein content is 40-55 wt.% on a dry basis, based on the total weight of the feed additive. More preferably, the crude protein contains small peptides, and the small peptide content is 20-30 wt.% on a dry basis, based on the total weight of the feed additive. The crude protein content is determined according to the national standard GB / T 6432-1994 "Determination of Crude Protein in Feed", and the small peptide content is determined according to Appendix B of the national standard GB / T 22492-2008 "Soybean Peptide Powder".

[0072] The sixth aspect of the present invention provides the application of the aforementioned Candida utilis or the aforementioned inoculum in the treatment of cassava broth.

[0073] The seventh aspect of the present invention provides a method for treating cassava liquid, characterized in that the method includes: inoculating the cassava liquid with the aforementioned Candida utilis yeast or the aforementioned inoculum.

[0074] The present invention will be described in detail below through examples. Unless otherwise specified, the methods used in the following examples are conventional methods in the art, and the reagents and raw materials used are commercially available.

[0075] In the following embodiments:

[0076] The crude protein content was determined in accordance with the national standard GB / T 6432-1994 "Method for Determination of Crude Protein in Feed".

[0077] The content of small peptides was determined according to the method in Appendix B of the national standard GB / T 22492-2008 "Soybean Peptide Powder".

[0078] Cassava extract was sourced from Guangxi COFCO Biomass Energy Co., Ltd. The oligosaccharide (soluble sugars with a degree of polymerization greater than 2) content in the cassava extract was approximately 18 g / L. Based on component analysis, the oligosaccharides were identified as maltodextrin and fiber oligosaccharides. The total glucose and xylose content was approximately 2 g / L, with glucose at 0.5 g / L and xylose at 1.5 g / L. Glycerol content was approximately 9 g / L, and organic acid content was approximately 15 g / L. All these substances were detected using an Aminex HPX–87H liquid chromatography column (purchased from Agilent Technologies, model 300 x 7.8 mm).

[0079] Soybean molasses was purchased from Yihai Kerry; ammonium sulfate and potassium dihydrogen phosphate were purchased from Sinopharm Biotechnology; and the antibacterial agent was the fungicide product Bacteriocin Treasure purchased from Shandong Anmaokang Biotechnology Co., Ltd.

[0080] Both strains, *Candida utilis* (CGMCC 2.3047) and *Candida tropicalis* (CGMCC 2.3967), were purchased from the China General Microbiological Culture Collection Center.

[0081] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract.

[0082] YPD solid medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 20 g / L agar powder.

[0083] Malt extract agar medium: 1L malt extract, 2wt.% agar.

[0084] Example 1

[0085] This example illustrates how the Candida utilis strain CUNX-01 was obtained.

[0086] 1. Isolation and identification of the starting strain of Candida utilis CUNX-01

[0087] Take 2g of mud sample from the molasses storage area of ​​Guangxi Sugar Factory, dilute it with sterile water to 100mL to obtain a diluted solution, take 100uL of the diluted solution and spread it on malt extract agar plates (containing 1wt‰ penicillin and streptomycin), and incubate it in a constant temperature incubator at 28-30℃ for 48h. Select single clones and conduct multiple rounds of streak culture to finally isolate and obtain purified strains.

[0088] The strain was characterized by its microbial morphology. On YPD solid plates, the colonies were milky white, smooth, glossy, and had regular edges. After incubation in YPD liquid medium at 28°C for 24 hours, bacterial sediment was observed at the bottom of the tube after a period of static time.

[0089] Subsequently, the bacteria were sequenced using 18S rDNA and ITS. Through sequencing comparison and analysis, combined with morphological identification, the bacteria were finally identified as Candida utilis strain, which is also the starting strain.

[0090] 2. Mutagenesis of Candida utilis strain CUNX-01

[0091] Mutagenesis was performed on the isolated and identified Candida utilis strain (i.e., the starting strain) in order to improve the growth rate and protein content of the strain.

[0092] 1) Preparation of bacterial suspension

[0093] 50 μL of *Candida utilis* glycerol culture was transferred to 10 mL of YPD liquid medium and activated overnight at 200 rpm and 28-30 °C. 1 mL of the overnight activated culture was transferred to 100 mL of YPD medium and cultured until the logarithmic growth phase. The culture was then centrifuged and washed twice. The bacterial pellet was collected and dissolved in physiological saline, thoroughly mixed, and a bacterial suspension was prepared. Before mutagenesis, the cells were directly counted under a microscope using a hemocytometer. The suspension was then diluted to a cell concentration of 10-1. 7 The bacterial culture was obtained at a concentration of 100 cells / mL, resulting in an unmutated bacterial culture.

[0094] 2) Exploration of conditions for plasma (ARTP) mutagenesis treatment

[0095] In a clean bench, place a metal slide on a sterile agar plate and spread 10 μL of bacterial suspension evenly onto the slide. Use sterile forceps to place the slide in the corresponding well, positioning it 2 mm from the airflow port, and close the chamber door. The instrument power is 120 W, the gas flow rate is 10 SLM, and the mutagenesis times are set to 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s. After sample processing, use sterile forceps to place the slide into an EP tube containing 1 mL of physiological saline.

[0096] Shake the centrifuge tube thoroughly for 1 minute to ensure that the microorganisms on the carrier are fully washed off and a new bacterial solution (the mutagenized bacterial solution) is formed.

[0097] 3) Dilute coating plate

[0098] Unmutated bacterial culture diluted 10 -4 10 -5 10 -6 After dilution, the bacterial suspension was spread. The mutagenized bacterial suspension was diluted 10 times. -1 10 -2 10 -3 After coating, count the samples to calculate the lethality rate.

[0099] 4) Formal mutagenesis experiment

[0100] Three mutagenesis times (30s, 35s, 40s) were selected around the mutagenesis time with a lethality of 90% for mutagenesis, and the mutagenesis was diluted and plated.

[0101] 5) High-throughput screening

[0102] Larger single colonies from the plate were transferred to 96-well plates and incubated overnight at 30°C and 750 rpm. OD was then measured. 600nm The selected colonies with high OD values ​​were subjected to four more rounds of ARTP mutagenesis, ultimately yielding strains selected through five rounds of ARTP mutagenesis.

[0103] 6) Secondary screening of mutagenic strains

[0104] Using unmutated strains as controls and strains selected through 5 rounds of ARTP mutagenesis screening, 100 mL of YPD medium was inoculated and cultured at 30°C and 200 rpm for 24 h. The OD of the bacterial culture was then measured. 600nm The bacterial cells were collected, and the crude protein content (i.e., the protein content of the bacterial cells themselves) was determined.

[0105] After the above operations, a strain with a cell protein content of 55.97 wt.% was finally obtained, which is 20.97% higher than the starting strain (35 wt.%). The newly obtained strain underwent 18S rDNA and ITS sequencing. Sequencing comparison analysis, combined with morphological identification, confirmed that this strain still belongs to the Candida utilis strain and was named *Candida utilis* CUNX-01. Under a microscope at 400x magnification, the cells of this strain are oval in shape, measuring 3.5-4.5 μm × 7.0-13.0 μm. Figure 1 ).

[0106] This Candida utilis was deposited on May 28, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22631, abbreviated as CUNX-01.

[0107] Example 2

[0108] This example illustrates the ability of Candida utilis CUNX-01 to utilize and degrade cassava extract.

[0109] Two Candida strains purchased from the China General Microbiological Culture Collection Center—Candida utilis CGMCC 2.3047 and Candida tropicalis CGMCC 2.3967—along with the Candida utilis CUNX-01 of this invention, were used as test strains. The three strains were used together to ferment simple cassava liquid, and their utilization capacity of the cassava liquid was compared.

[0110] 1. Activation of the strain

[0111] Three strains of Candida albicans were streaked on YPD solid plates and cultured in a constant temperature incubator at 30℃ for 48 h. Single clones from the plates were picked and transferred to 30 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 18 h to form primary seed culture. 1 vol.% was inoculated into new YPD liquid medium and cultured under the same conditions for 18 h to form secondary seed culture.

[0112] 2. Cassava liquid shake-flask fermentation

[0113] Take 300 mL of cassava extract, add 5 ppm of antibacterial agent, and dispense into three 250 mL Erlenmeyer flasks (100 mL in each flask). Then, inoculate the three activated Candida strains at a final yeast count of 0.4 billion / mL and incubate at 30℃ and 200 rpm for 24 h. After incubation, perform yeast counting and determine the crude protein content of the precipitate after centrifugation. The results are shown in Table 1.

[0114] Table 1 shows that *Candida utilis* CUNX-01 exhibited the fastest growth after 24 hours of culture in cassava broth, reaching a yeast count of 230 million / mL. In contrast, *Candida tropicalis* CGMCC2.3967 and *Candida utilis* CGMCC2.3047 had lower yeast counts, at 146 million / mL and 98 million / mL, respectively. The yeast precipitate was collected after centrifugation of the fermentation broth, and its mass, crude protein content, and moisture content (measured using a rapid moisture analyzer) were determined. The oven-dry content of crude protein in the yeast precipitate was calculated. *Candida utilis* CUNX-01 also showed the highest oven-dry crude protein content, at 33.69 wt.%.

[0115] The dry weight of crude protein in yeast precipitate = crude protein content of yeast precipitate / [mass of yeast precipitate × (1 - moisture content of yeast precipitate)] × 100%

[0116] Therefore, Candida utilis CUNX-01 has advantages in utilizing cassava extract.

[0117] Table 1. Detection of indicators in centrifuged precipitates from fermentation of three different Candida species.

[0118]

[0119] Example 3

[0120] This example illustrates the optimization of the composition of the cassava broth fermentation medium for Candida utilis CUNX-01.

[0121] 1. Nitrogen source screening for shake-flask fermentation

[0122] Candida utilis generally exhibits good utilization of inorganic nitrogen sources, such as urea and ammonium sulfate. Therefore, sugarcane molasses was initially used as the carbon source, and urea and ammonium sulfate were screened. Using response surface methodology in Minitab software, the culture medium composition was designed within the following range: 100 mL cassava extract, sugarcane molasses content 0-10 g / L, ammonium sulfate 0-10 g / L, urea 0-10 g / L, and antibacterial agent addition of 5 ppm, with the overall pH adjusted to 5-7. The specific culture medium composition is shown in Table 1. Candida utilis CUNX-01 was activated according to the "1. Strain Activation" step in Example 2. Activated Candida utilis CUNX-01 was inoculated into the prepared culture medium at a final yeast count of 0.4 billion / mL, and fermented at 30℃ and 200 rpm for 24 h. A fermentation experiment of Candida utilis cultured in cassava extract was conducted, and the yeast count was calculated after fermentation.

[0123] The experimental results are shown in Table 2. Based on the data in Table 2, contour plots with yeast count as the response value were drawn using Mintab software, as shown below. Figure 2 As shown. From Figure 2 As can be seen, the amount of urea added has almost no effect on the number of yeasts after fermentation, while the amount of ammonium sulfate added is relatively high within a certain range.

[0124] Therefore, the addition of ammonium sulfate within a certain range promoted the growth of Candida utilis, indicating that ammonium sulfate is more suitable as a nitrogen source for the fermentation of Candida utilis CUNX-01 cassava liquid.

[0125] Table 2

[0126]

[0127]

[0128] 2. Screening of carbon sources for shake-flask fermentation

[0129] Candida utilis can generally utilize inexpensive molasses as a carbon source. Therefore, based on the molasses actually obtained, sugarcane molasses and soybean molasses were screened as carbon sources. Since sugarcane molasses and soybean molasses are byproducts of sugarcane and soybean processing, respectively, they contain not only large amounts of polysaccharides and monosaccharides but also other inhibitory factors that may affect the growth of Candida utilis. Therefore, we need to optimize the type and amount of molasses added.

[0130] Two sets of experiments were designed using response surface methodology in Minitab software. The first set used sugarcane molasses as the carbon source, and the culture medium composition was designed within the following range: 100 mL cassava extract, sugarcane molasses content 2-10 g / L, ammonium sulfate 0-10 g / L, potassium dihydrogen phosphate 0-5 g / L, and antibacterial agent added at 5 ppm. The overall pH was adjusted to 5-7. The specific culture medium composition is shown in Table 3. The second set used soybean molasses as the carbon source, and the culture medium composition was designed within the following range: 100 mL cassava extract, soybean molasses content 2-10 g / L, ammonium sulfate 0-10 g / L, potassium dihydrogen phosphate 0-5 g / L, and antibacterial agent added at 5 mg / L. The overall pH was adjusted to 5-7. The specific culture medium composition is shown in Table 4. *Candida utilis* CUNX-01 was activated according to the "1. Strain Activation" step in Example 2 to obtain a secondary seed culture. Activated Candida utilis CUNX-01 was inoculated into the prepared culture medium at a final concentration of 0.4 billion yeast cells / mL. Fermentation was carried out at 30℃ and 200 rpm for 24 h. Fermentation experiment of Candida utilis cultured in cassava broth was carried out. The yeast count was calculated after fermentation.

[0131] The experimental results are shown in Tables 3 and 4.

[0132] Table 3

[0133]

[0134] Table 4

[0135]

[0136]

[0137] Using the data in Table 3, model fitting analysis was performed using Mintab software. The maximum response was optimized with the yeast count as the target. It was found that when the carbon source was sugarcane molasses, the formula of the cassava broth fermentation medium was cassava broth with 8 g / L sugarcane molasses added, without adding ammonium sulfate and potassium dihydrogen phosphate. According to the software model fitting analysis results, the yeast count was relatively high, reaching 406 million / mL.

[0138] Using the data in Table 4, model fitting analysis was performed using the mintab software. Similarly, the maximum response was optimized with the yeast count as the target. It was found that when the carbon source was soybean molasses, the formula of the cassava broth fermentation medium was cassava broth with 2 g / L soybean molasses, 10 g / L ammonium sulfate, and no potassium dihydrogen phosphate added. According to the software model fitting analysis results, the yeast count was relatively high, at 436 million / mL.

[0139] 3. Optimization of the optimal fermentation formula and parameters for the fermentation tank

[0140] Based on the above optimization results of the cassava broth fermentation medium formula, a cassava broth fermentation medium was prepared, and four parameters were optimized: maximum rotation speed, dissolved oxygen, feeding time point, and feeding rate.

[0141] The formula for cassava broth fermentation medium is: 0.6L cassava broth, 2g / L soybean molasses, 10g / L ammonium sulfate, and 5mg / L antibacterial agent.

[0142] Method for strain activation: The Candida utilis CUNX-01 was activated according to the steps in "1. Strain activation" in Example 2.

[0143] 1) Speed ​​optimization

[0144] To optimize the rotation speed range in the culture of Candida utilis CUNX-01, the following experiments were conducted:

[0145] The activated Candida utilis CUNX-01 was inoculated into four parallel 1L fermenters (containing 0.6L of cassava extract fermentation medium) at a final yeast count of 0.4 billion / mL. The aeration rate was controlled at 1L / min, the fermentation temperature was 30℃, and the fermentation pH was controlled at around 6. The rotation speeds of the four fermenters were set to 300rpm, 500rpm, 700rpm, and 900rpm, respectively. OD values ​​and yeast counts were measured at 4h, 8h, 12h, 14h, 20h, and 22h. Fermentation was stopped after 22h of culture.

[0146] Statistical analysis of yeast counts at different time points revealed that yeast counts were higher when the rotation speed was controlled between 500-900 rpm, with the highest OD value observed at 700 rpm. Figure 3 Furthermore, the yeast count was the highest (468 million / mL). Therefore, the fermenter's rotation speed range was optimized to 500-900 rpm.

[0147] 2) Dissolved oxygen optimization

[0148] The activated Candida utilis CUNX-01 was inoculated into four parallel 1L fermenters (containing 0.6L of cassava extract fermentation medium) at a final yeast count of 0.4 billion / mL. The aeration rate was controlled at 1L / min, the fermentation temperature at 30℃, and the fermentation pH at around 6. The dissolved oxygen in the four fermenters was controlled at around 10%, 20%, 30%, and 40% by adjusting the rotation speed within the range of 500-900rpm. Yeast counts were measured at different time points, and fermentation was stopped after 24 hours of culture.

[0149] Analysis of the yeast growth curves from four fermenters revealed that when dissolved oxygen was controlled at around 10%, the yeast count at different time points remained consistently higher compared to the other three dissolved oxygen levels, with the yeast count reaching approximately 790 million / mL at the fermentation endpoint. Figure 4 When the dissolved oxygen parameter is 20% and 30%, the yeast count at the fermentation endpoint is also relatively high, reaching 550 million / mL and 690 million / mL, respectively. Therefore, the dissolved oxygen parameter of the fermenter is optimized to 10-30%, with 10% being the optimal value.

[0150] 3) Optimization of material replenishment timing

[0151] After activating the *Candida utilis* strain CUNX-01, it was inoculated into four parallel 1L fermenters (containing 0.6L of cassava extract fermentation medium) at a final concentration of 0.4 billion yeast cells / mL. Aeration was controlled at 1L / min, fermentation temperature at 30℃, fermentation pH at approximately 6, rotation speed at 500-900 rpm, and dissolved oxygen at approximately 10%. The four fermenters were subjected to three different inoculation processes: no feeding, feeding starting at 8 hours, feeding starting at 10 hours, and feeding starting at 12 hours, with a feeding rate of 10 g / L. 初始的木薯清液发酵培养基 The feeding experiment was conducted at a rate of / h, with soybean molasses as the feed and a feeding duration of 8 hours. Fermentation was stopped after 24 hours.

[0152] Analysis of the yeast growth curves from four fermenters revealed that the yeast count remained consistently high at different time points during the 8th-10th hour of fermentation when feeding began. Specifically, at the 10th hour of fermentation, the yeast count reached a peak of 1.18 billion / mL. Figure 5 ).

[0153] 4) Optimization of feeding rate

[0154] After activating the *Candida utilis* strain CUNX-01, it was inoculated into four parallel 1L fermenters (containing 0.6L of cassava extract fermentation medium) at a final concentration of 0.4 billion yeast cells / mL. Aeration was controlled at 1L / min, fermentation temperature at 30℃, fermentation pH at approximately 6, rotor speed at 500-900 rpm, and dissolved oxygen at approximately 10%. Feeding was performed in all four fermenters at 10 hours of fermentation, with a feeding period of 8 hours. The feed consisted of soybean molasses at a feeding rate of 2 g / L. 初始的木薯清液发酵培养基 / h, 10g / L 初始的木薯清液发酵培养基 / h, 15g / L 初始的木薯清液发酵培养基 / h and 20g / L 初始的木薯清液发酵培养基 / h.

[0155] Analysis of the yeast growth curves from four fermenters revealed that at a concentration of 15 g / L... 初始的木薯清液发酵培养基 / h and 20g / L 初始的木薯清液发酵培养基 At a feed rate of 15 g / L, the yeast count remained at a high level. 初始的木薯清液发酵培养基 At a feed rate of 20 g / L, the yeast count can reach 1.63 billion / mL. 初始的木薯清液发酵培养基 The yeast count reached 1.71 billion / mL at a feed rate of 15 g / L. 初始的木薯清液发酵培养基 At / h, costs can be further reduced ( Figure 6 ).

[0156] Through optimization of fermentation parameters, the final fermentation parameters were determined as follows: yeast was inoculated into the fermenter at a final concentration of 0.4 billion / mL; aeration rate was controlled at 1 L / min; fermentation temperature was 30℃; fermentation pH was controlled at approximately 6; rotation speed was controlled at 500-900 rpm; dissolved oxygen was controlled at approximately 10%; and to increase the yeast count, feeding was initiated after 10 hours at a feeding rate of 15 g / L. 初始的木薯清液发酵培养基 / h, replenishment time is 8h.

[0157] Example 4

[0158] This embodiment illustrates the fermentation of *Candida utilis* strain CUNX-01 in cassava broth fermentation medium, using a 7L feed-fed fermenter.

[0159] In this embodiment, the formula of the cassava broth fermentation medium is: 4L cassava broth, 2g / L soybean molasses, 10g / L ammonium sulfate, and 5mg / L antibacterial agent.

[0160] Seed activation: Single colonies of *Candida utilis* CUNX-01 on agar plates were first cultured on 5 mL of YPD medium at 30°C and 200 rpm for 18 h. Then, they were inoculated into 100 mL Erlenmeyer flasks (containing 20 mL of YPD medium) at a ratio of 1 vol.% and cultured at 30°C and 200 rpm for 18 h to form primary seed culture. The primary seed culture was then inoculated into 500 mL Erlenmeyer flasks (containing 100 mL of YPD medium) at a ratio of 1 vol.% and cultured at 30°C and 200 rpm for 18 h to form secondary seed culture.

[0161] Fermentation in a 7L fermenter: The secondary seed culture was inoculated into a 7L fermenter (containing 4L of cassava extract fermentation medium) at a final yeast concentration of 0.4 billion / mL. Fermentation lasted 24 hours under the following conditions: aeration rate of 0.6L / min, fermentation temperature of 30℃, fermentation pH of approximately 6, agitator speed of 500-900 rpm, and dissolved oxygen of approximately 10%. Feeding with soybean molasses began at the 10th hour of fermentation at a rate of 15g / L. 初始的木薯清液发酵培养基 / h, replenishment time is 8h.

[0162] After 24 hours of fermentation, the number of yeasts in the fermentation broth was measured, and the yeast count reached 2.2 billion / mL. The solids, i.e., the bacterial protein precipitate, were collected by centrifugation. The bacterial protein precipitate was then dried in a vacuum low-temperature oven to obtain bacterial protein powder. The mass, moisture content (measured by a rapid moisture analyzer), crude protein content, and small peptide content of the bacterial protein powder were measured. The oven-dry yield of the bacterial protein was calculated according to the following formula, and the oven-dry content of crude protein and small peptides in the bacterial protein powder were also calculated.

[0163] Oven-dried yield of bacterial protein = Mass of bacterial protein powder × (1 - Moisture content of bacterial protein powder) / Volume of fermentation broth

[0164] The oven-dry content of crude protein in bacterial protein powder = crude protein content of bacterial protein powder / [mass of bacterial protein powder × (1 - moisture content of bacterial protein powder)] × 100%

[0165] The absolute dry content of small peptides in bacterial protein powder = Small peptide content of bacterial protein powder / [Mass of bacterial protein powder × (1 - Moisture content of bacterial protein powder)] × 100%

[0166] The experimental results are shown in Table 5. As can be seen from Table 5, after fermentation, the oven-dry yield of the bacterial protein was 40.4 g / L of fermentation broth. The oven-dry crude protein content of the bacterial protein powder was 53.47 wt.%, and the oven-dry small peptide content was 25.39 wt.%. Its crude protein content was higher than the index in patent ZL 201310327937.5 (51 wt.%), and its small peptide content was higher than the acid-soluble protein (small peptides + amino acids) index in ZL 201310327937.5 (6 wt.%).

[0167] Table 5

[0168]

[0169] Amino acid analysis of the bacterial protein powder (the types and mass of amino acids were determined according to the method described in Appendix B of the national standard GB / T 22492-2008 "Soybean Peptide Powder" for "Determination of Free Amino Acid Content") revealed a rich variety of amino acids, including 17 kinds (Table 6). The relative proportion of amino acids was calculated with lysine content as 100% (Table 6), indicating that the bacterial protein powder is suitable for the nutritional needs of animals such as cattle, chickens, and sheep.

[0170] Table 6

[0171]

[0172]

[0173] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining the various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing a feed additive, characterized by, The method comprises inoculating Williopsis saturnus in a fermentation medium for fermentation to obtain a fermentation liquor; The preservation number of the Williopsis saturnus is CGMCC NO. 22631; The medium is a cassava clear liquid fermentation medium; The cassava clear liquid fermentation medium is cassava clear liquid, a carbon source, a nitrogen source and a bacteriostatic agent; The carbon source is soybean molasses, and the content of the soybean molasses is 2 g / L; The nitrogen source is ammonium sulfate, and the content of the ammonium sulfate is 10 g / L; The content of oligosaccharides in the cassava clear liquid is 18 g / L, and the oligosaccharides are malt oligosaccharides and fiber oligosaccharides with a polymerization degree of 2 or more; the total content of glucose and xylose in the cassava clear liquid is 2 g / L, the content of glucose is 0.5 g / L, and the content of xylose is 1.5 g / L; the content of glycerol is 9 g / L; and the content of organic acid is 15 g / L; The method comprises feeding during the 8th-12th hour of fermentation; the feeding comprises adding a carbon source during the fermentation process; The feeding rate is 10-20 g / h with respect to 1 L of the initial fermentation medium; and the feeding duration is 5-10 h.

2. The method of claim 1, wherein, The fermentation conditions comprise a temperature of 28-30 ℃, a dissolved oxygen of 10-40%, and fermentation for 18-24 h.

3. The method of claim 2, wherein, The fermentation conditions comprise a temperature of 28-30 ℃, a dissolved oxygen of 10-30%, and fermentation for 18-24 h.

4. Use of the method according to any one of claims 1-3 in cassava clear liquid treatment.

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