Acid-resistant cyberlindnera jadinii strain and use thereof

Abstract

An acid-resistant Cyberlindnera jadinii strain and the use thereof, belonging to the technical field of microorganisms. The Cyberlindnera jadinii strain is Cyberlindnera jadinii C-12-2 and is preserved in Guangdong Microbial Culture Collection Center on March 05, 2024 and assigned the accession number GDMCC NO: 64388. The Cyberlindnera jadinii strain can grow in mediums having a pH value of 3.5, and can achieve a crude protein content of 70.02% after fermentation for 24 h. The Cyberlindnera jadinii strain is obtained by first undergoing heavy ion irradiation mutagenesis and then acid resistance domestication. A test illustrates that the strain can grow in acidic mediums having a pH value of 3.5, overcoming the obstacle of an original strain failing to grow under low pH values. The Cyberlindnera jadinii strain has higher acid resistance and protein production capabilities, and thus can be provided as a high-quality strain resource for mixed bacterial fermentation for producing mycoproteins, mycoprotein feeds or yeast cultures, etc.

Description

Acid-tolerant cyberlindnera jadinii and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to an acid-tolerant Cyberlindnera jadinii and application thereof. BACKGROUND

[0002] With the rapid development of animal husbandry and the entry of the feed industry into the era of banning antibiotics, the demand for protein feed in China is increasing. At present, the annual import volume of protein feed in China exceeds 100 million tons, and the shortage of protein feed seriously affects the development of the breeding and animal husbandry industries in China, so more and more protein feed resources need to be put into the production of animal husbandry. Single-cell protein has high protein content, rich in nutrients, and is not affected by seasons and climate, which is an important way to solve the shortage of protein feed resources and realize the substitution of protein feed resources. Candida utilis is one of the excellent strains for producing microbial fermentation feed high-cell protein, which is rich in vitamin B and protein, and has the effects of regulating intestinal microecological balance, improving feed digestibility, and enhancing animal immune function. In addition, Candida utilis has the advantages of short cultivation cycle, good safety and fermentation performance, and can use molasses, starch plant waste liquid and vinasse as carbon sources, and use urea or nitrate as nitrogen source to grow and ferment feed. However, the acid tolerance of the existing strains is relatively low, so it is necessary to breed strains with higher acid tolerance. SUMMARY

[0003] The present application aims to provide an acid-tolerant Cyberlindnera jadinii and application thereof to solve the technical problem of relatively low acid tolerance of the existing Candida utilis strains.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0005] The acid-tolerant Cyberlindnera jadinii is Cyberlindnera jadinii C-12-2, which was preserved in the Guangdong Microbial Culture Collection Center on March 5, 2024, with the preservation number GDMCC NO:64388 and the preservation address being No. 59 Building, 5th Floor, 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0006] The Cyberlindnera jadinii C-12-2 provided in the application is obtained by heavy ion irradiation mutagenesis and acid-tolerant domestication, can grow in an acidic culture medium with pH 3.5, and the crude protein content can reach 70.02% after 24 h fermentation, overcoming the obstacle that the original strain cannot grow at low pH values. The performance stability test shows that the change range of the crude protein yield of the mutant strain is not significant after 12 times of continuous subculture, proving that the mutant strain has stable genetic performance and is a strain with higher acid-tolerant protein production capacity, which can provide high-quality strain resources for the production of bacterial protein by mixed fermentation, bacterial protein feed or yeast culture, etc.

[0007] Based on the above-mentioned strain, the application also provides the application of the acid-tolerant Cyberlindnera jadinii as a fermentation strain in the preparation of bacterial protein by single or mixed fermentation, or as a raw material for the preparation of yeast culture.

[0008] The application has the following beneficial effects:

[0009] Cyberlindnera jadinii is the sexual or asexual form of Candida utilis, and the application provides a high-yield acid-tolerant Cyberlindnera jadinii (Cyberlindnera jadinii C-12-2) with high bacterial protein production capacity, which can grow in a culture medium with pH 3.5, and the crude protein content can reach 70.02% after 24 h fermentation. The Cyberlindnera jadinii described in the application is obtained by heavy ion irradiation mutagenesis and acid-tolerant domestication, and can grow in an acidic culture medium with pH 3.5, overcoming the obstacle that the original strain cannot grow at low pH values. It is a strain with higher acid-tolerant protein production capacity, which can provide high-quality strain resources for the production of bacterial protein by mixed fermentation, bacterial protein feed or yeast culture, etc. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a strain domestication flowchart;

[0011] Figure 2 is a comparison chart of the growth of strains before and after domestication, in which the left is Candida utilis C-12 bacterial solution, and the right is the bacterial solution of Cyberlindnera jadinii C-12-2 after growth in YPD liquid medium with pH 3.5;

[0012] Figure 3 is a growth curve chart of Cyberlindnera jadinii C-12-2 strain;

[0013] Figure 4 is a comparison chart of the crude protein content of Candida utilis C-0, C-12 and Cyberlindnera jadinii C-12-2 respectively;

[0014] Figure 5 is a performance stability test result chart of the bacterial protein yield of Cyberlindnera jadinii C-12-2. DETAILED DESCRIPTION

[0015] The present application is further illustrated in conjunction with the embodiments and the accompanying drawings, which include but are not limited to the following embodiments.

[0016] Embodiment 1

[0017] This embodiment uses the Heavy Ion Research Facility in Lanzhou (HIRFL) of the Institute of Modern Physics, Chinese Academy of Sciences to carry out irradiation mutation screening of strains with high yield of cell protein, and the specific method is as follows:

[0018] 1. The Candida utilis (purchased from China General Microbiological Culture Collection Center, preservation number CGMCC 2.2878) was taken out from the -70°C refrigerator, slowly thawed at room temperature, inoculated into 100 mL YPD liquid medium at an inoculation amount of 1%, and cultured overnight in a constant temperature shaker; the culture conditions were: temperature 30°C, rotation speed 180 rpm, culture time 24 h, and the OD 600 was measured to obtain the OD 600 =0.8 of the bacterial liquid, which was recorded as the starting bacterial liquid;

[0019] 2. 2 mL of the starting bacterial liquid was taken and placed in a sterile irradiation dish and sealed with a sealing film, and then irradiated with an 80 MeV / u 12 C 6+ ion beam, and the irradiation doses were 0 Gy, 40 Gy, 80 Gy, 120 Gy, 160 Gy and 200 Gy, respectively;

[0020] 3. The bacterial liquid with different irradiation doses was diluted to 10 -5 , and bacterial suspensions with different irradiation doses were obtained; 50 μL of the bacterial suspensions with different irradiation doses were respectively spread on YPD solid medium, and incubated at 30°C for 48 h to obtain different irradiated strains;

[0021] 4. The different irradiated strains were inoculated into 100 mL YPD medium and cultured overnight in a constant temperature shaker; the culture conditions were: temperature 30°C, rotation speed 180 rpm, and the OD 600 was measured to obtain the OD 600 =0.8 of the activated bacterial liquid;

[0022] 5. Primary screening of the strains: according to the morphological characteristics and growth of the colonies, strains with acid tolerance and high yield of cell protein were screened, and the specific method was as follows: the activated bacterial liquid obtained in step 4 was diluted, and the dilution gradients were 10 -3 , 10 -4 , 10 -5 , 10 -6, 50 μL of the diluted bacterial solution was coated on the starch screening medium, and placed in a 30°C constant temperature incubator for culture; after the colonies grew, the colonies with large diameters were selected and transferred to YPD liquid medium, cultured at 30°C, 180 rpm for 48 h, and the growth rate of the bacterial body was determined by measuring the OD 600 value at a wavelength of 600 nm, and the strain with faster growth rate and higher bacterial body protein was selected for further screening.

[0023] In this experiment, 21 strains with larger diameters than the original strain were selected from 2080 single colonies, and were named according to the strain irradiation dose. The liquid culture results are shown in Table 1. 21 strains of mutagenic strains were selected for further screening.

[0024] Table 1. Results of initial screening colony diameter and bacterial concentration (OD 600 )

[0025]

[0026] 6. Screening of strains: 22 strains in Table 1 were activated respectively, and the specific method is as follows:

[0027] Take out from -70°C refrigerator, slowly thaw at room temperature, then inoculate into 100 mL YPD liquid medium with a volume ratio of 1%, and incubate in a constant temperature shaker overnight; the culture conditions are 30°C, 180 rpm, and the OD 600 is measured, and the activated bacterial solution with OD 600 =0.8 is obtained.

[0028] Prepare the culture solution of 22 strains respectively, and the specific method is as follows:

[0029] After the 22 strains of Candida utilis were activated, they were inoculated into YPD liquid medium with a volume ratio of 1%, and cultured at 30°C, 180 rpm for 48 h to obtain the culture solution, and then the bacterial biomass and bacterial protein content were measured respectively, and the specific method is as follows:

[0030] (1) Centrifuge the bacterial suspension at 8000 rpm for 5 min, discard the supernatant, then wash the bacterial body with sterile water, centrifuge at 8000 rpm for 5 min, discard the supernatant, and wash continuously for 3 times, collect the bacterial body, and dry at 90°C to constant weight, measure the sample mass (dry weight), which is the biomass of Candida utilis.

[0031] (2) According to the method of GB / T 6432-2018, the crude protein content (crude protein (%)) in the sample is detected, which is the single cell protein content. The calculation formula is as follows:

[0032]

[0033] In the formula: V2 - the volume of standard hydrochloric acid solution consumed by the sample mL; V1 - the volume of standard hydrochloric acid solution required by the blank control mL; C - the concentration of standard hydrochloric acid solution; m - the mass of the sample g; V - the total volume of the sample digestion solution mL; V' - the volume of digestion solution for distillation mL; 0.0140 - the number of grams per milliequivalent of nitrogen; 6.25 - the average coefficient for converting nitrogen into protein.

[0034] The results of the determination of the biomass and the crude protein content of the 22 strains are shown in Table 2.

[0035] Table 2 Biomass of the rescreened colonies and the crude protein content thereof in the fermentation broth

[0036]

[0037] As can be seen from Table 2, the crude protein content of the 5 strains of mutagenized bacteria that have positive mutations is increased to different degrees compared with the crude protein content of the original strains. When the irradiation dose reaches 120 Gy, a strain of positive mutant with the highest crude protein content is obtained, which is named Candida utilis C-12, and the crude protein content thereof reaches 64.88%, which is increased by 24.08% compared with the crude protein content of 52.29% of the original Candida utilis CGMCC 2.2878.

[0038] Example 2

[0039] In this example, the Candida utilis C-12 strain with high cell protein yield screened in the previous stage through heavy ion mutagenesis is subjected to acid-tolerant domestication by gradually reducing the pH value of the culture medium.

[0040] The specific process is as follows:

[0041] 1. Strain domestication

[0042] The strain is domesticated by gradually reducing the pH value of the culture medium (as shown in Figure 1). The Candida utilis C-12 strain with high cell protein yield is inoculated into YPD liquid medium with a pH of 4.5 at an inoculation amount of 1%, and then cultured at 30°C and 180 rpm for 24 h, after which the culture is transferred into fresh YPD liquid medium with a pH of 4.5, and the operation is continuously performed for one month, during which the strain is purified by plate streaking every other week. According to the above process, the yeast strain adapted to pH 4.5 is transferred into YPD liquid medium with a pH of 3.5 for further domestication, and finally a Candida utilis strain with high cell protein yield and low pH tolerance is obtained, which is named Candida utilis C-12-2.

[0043] Figure 2 shows the growth of Candida utilis before and after acid acclimation. The high-protein-producing Candida utilis C-12 was acclimated to the acid-tolerant, high-protein-producing Candida utilis C-12-2. The acid-acclimated Candida utilis C-12-2 was found to be well adapted to low-pH growth conditions, overcoming the inability of Candida utilis C-12 to grow in YPD medium at a pH of 3.5.

[0044] 2. Growth curve determination

[0045] The acclimated yeast C-12-2 seed solution was diluted to 1% (WV) (OD 600 =0.8) were inoculated into 100 mL / 250 mL YPD liquid culture medium, and a blank control was made at the same time. The culture was shaken at 30°C and 180 rpm. 1 mL of samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, and 28 h after inoculation, and the zero value was adjusted with uninoculated culture medium. The absorbance of the two yeast strains after acclimation at 600 nm was measured, and the growth curve was plotted with the yeast absorbance as the ordinate and the growth time as the abscissa.

[0046] The growth curve of the Lindnerella jardinis strain C-12-2 was determined by three replicate experiments. The data are shown in FIG3 .

[0047] Figure 3 shows the growth trend of the Lindnerella jardiniseiberi C-12-2 strain. The lag phase lasts from 0 to 4 hours; the cell number increases exponentially from 4 to 22 hours, marking the logarithmic growth phase. After 22 to 28 hours of culture, the bacterial yield reaches its peak, marking the stationary growth phase. Strains in the logarithmic growth phase experience adequate nutrition, rapid growth, and active metabolism. Cell morphology and physiological characteristics are relatively stable, but they are also more sensitive to changes in physical and chemical factors and prone to mutation. Therefore, cells in this growth phase are generally selected for experimental research. Determining growth curves can be used to assess the growth and metabolic patterns of different strains and determine the optimal inoculation time. Under the same conditions, a faster growth rate indicates better nutrient utilization and greater application advantages.

[0048] 3. Determination of single cell protein

[0049] The seed solution of Lindnerella jardinis C-12-2 was diluted to 1% (WV) (OD 600The inoculation amount of each strain (OD=0.8) was inoculated into 100 mL / 250 mL YPD liquid medium, and cultured at 30°C, 180 rpm for 24 h. The fermentation culture was centrifuged at 4500 rpm for 10 min, and the supernatant was discarded. After washing with deionized water, the sample was centrifuged at 4500 rpm for 10 min, and the supernatant was discarded. The above operation was repeated three times, and the sample was placed in a 105°C oven and dried to constant weight. After cooling, 0.1 g of the sample was accurately weighed, and the crude protein content was determined according to the method in the standard "GB / T 6432-2018 Determination of crude protein in feed - Kjeldahl method".

[0050] The crude protein content of each strain of Candida utilis C-0, C-12 and Starmerena C-12-2 was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. The crude protein content of each strain of Candida utilis C-0, C-12 and Starmerena C-12-2 is shown in Figure 4.

[0051] As shown in Figure 4, the single cell protein content of Starmerena C-12-2 after acid adaptation was 70.02%, which was increased by 33.91% compared with the original strain Candida utilis CGMCC 2.2878 (i.e. C-0). Moreover, the adapted yeast strain had the ability to grow at low pH, so it could be used as a subsequent fermentation strain.

[0052] 4. Genetic stability test of mutagenized and adapted strain

[0053] The genetic stability of mutagenized and adapted Starmerena C-12-2 was analyzed. The method was as follows: Starmerena C-12-2 was continuously subcultured for 12 generations. The mutagenized and adapted strain was recorded as generation 0. The generation 0 liquid was inoculated into YPD liquid medium with pH 3.5 at an inoculation amount of 1%, and cultured at 30°C, 180 rpm for 12 h (OD 600 =2) to obtain generation 1 seed liquid. The generation 1 seed liquid was inoculated into YPD liquid medium with pH 3.5 at an inoculation amount of 1%, and cultured at 30°C, 180 rpm for 12 h (OD 600 =2) to obtain generation 2 seed liquid. In this way, 12 generations were continuously cultured, and the crude protein content was detected every 2 generations according to the method of GB / T 6432-2018.

[0054] The crude protein content of Starmerena C-12-2 strain of 0, 2, 4, 6, 8, 10 and 12 generations was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. The crude protein content of Starmerena C-12-2 strain was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. The crude protein content of Starmerena C-12-2 strain was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. The crude protein content of Starmerena C-12-2 strain was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. The crude protein content of Starmerena C-12-2 strain was determined by three parallel repeated experiments, and the data results were expressed by mean ± standard deviation. 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[0055] As shown in Figure 5, the crude protein content of the C-12-2 strain of Jadinia cyberlindneri is about 70.02%, and the difference is not significant through difference significance analysis, which proves that the performance of the C-12-2 strain of Jadinia cyberlindneri in producing single-cell protein crude protein has good stability. Through the performance stability experiment, it can be known that the crude protein content of the C-12-2 strain of Jadinia cyberlindneri selected by heavy ion mutagenesis has good stability.

[0056] In summary, the present application provides an acid-tolerant Jadinia cyberlindneri C-12-2, which is obtained by 12 C 6+ The present application provides an acid-tolerant Jadinia cyberlindneri C-12-2, which is obtained by

[0057] The above examples are only preferred embodiments of the present application, and should not be used to limit the protection scope of the present application. Any modification or polishing made within the main design idea and spirit of the present application, which does not have substantial significance, and which still solves the technical problems consistent with the present application, should be included in the protection scope of the present application.

Claims

1. An acid-tolerant Cyberlindnera jadinii strain, characterized in that: The Cyberlindnera jadinii C-12-2 was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 5, 2024, with a deposit number of GDMCC NO: 64388. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

2. Use of the acid-tolerant Saccharomyces jardinisiiberlindnerii according to claim 1 as a fermentation strain in the preparation of bacterial protein by single-bacterial or mixed-bacterial fermentation.

3. Use of the acid-tolerant Lindnerella jardinischibryl yeast according to claim 1 as a raw material for preparing a yeast culture.

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