Kluyveromyces marxianus SNJ1 for producing hypha for protein production and chicken manure treatment and application of Kluyveromyces marxianus SNJ1

Abstract

The invention relates to the technical field of environmental microorganisms and resource fermentation, and discloses Kluyveromyces marxianus SNJ1 for producing hyphae for protein production and chicken manure treatment and application of the Kluyveromyces marxianus SNJ1. The preservation number of the kluyveromyces marxianus SNJ1 is CCTCC NO: M 20251143, the kluyveromyces marxianus SNJ1 is obtained through adaptive evolution of a chicken manure culture medium, and single-cell protein can be efficiently synthesized under the condition that chicken manure is used as a main culture medium. The optimal growth conditions of the strain are as follows: the temperature is 28 DEG C, the pH value is 6.5, and the strain shows excellent proliferation performance and genetic stability in a chicken manure matrix with the water content of 71.4% and the 100-mesh grinding fineness. The invention provides a novel microbial tool for resource utilization of livestock and poultry manure, can synchronously realize organic waste treatment and high-value protein production, and has remarkable application potential in the fields of feed additives and biological fertilizers.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms and resource-based fermentation, and particularly relates to mycelium-producing Kluyveromyces marxianus SNJ1 for protein production and chicken manure treatment and applications thereof. Background Art

[0002] The continuous growth of the global population and the increase in demand for dietary protein have jointly driven the need for transformation of the livestock protein supply system. Traditional livestock protein production methods have obvious resource and environmental constraints. Its high resource consumption, large amounts of greenhouse gas emissions and environmental pollution not only affect food safety, but also put continuous pressure on the ecosystem. In this context, the development of sustainable alternative protein sources has become a current research focus. Single-cell protein (SCP) technology has shown significant advantages due to its unique biological characteristics. Microbial-derived SCP has the characteristics of rapid proliferation, high protein content and broad substrate adaptability. It can not only realize the resource utilization of organic waste, but also significantly reduce the ecological and environmental impact of traditional protein production. It is a sustainable protein solution with great development prospects.

[0003] When the "Kluyveromyces marxianus_CICC 32865" of the China Industrial Microorganism Culture Collection Center is cultured on a conventional glucose yeast extract peptone agar slant, it forms pseudohyphae without producing ascospores, which indicates that under conventional culture conditions, it has the ability to differentiate into hyphae-like structures. However, Kluyveromyces marxianus usually presents a round, milky white, moist colony morphology under standard culture conditions and does not produce hyphae. Studies have reported that this yeast can form pseudohyphae under specific induction conditions and exhibit functional characteristics such as strong lactose fermentation ability and high temperature tolerance. Therefore, the phenomenon of the CICC 32865 strain forming pseudohyphae under conventional culture conditions may be related to its unique genetic background or environmental adaptability.

[0004] Chicken manure, a major by-product of livestock and poultry farming, is rich in organic matter and nitrogen nutrients and has important resource value. However, its effective utilization faces many technical challenges, including complex matrix composition, the presence of inhibitory substances, and nutritional imbalance. Chicken manure contains a high proportion of difficult-to-degrade organic matter such as cellulose, hemicellulose, and lignin, as well as inhibitory components such as urate. These factors lead to a complex matrix and unbalanced nutrients, which restricts the growth and reproduction of most microorganisms and reduces the efficiency of its resource utilization.

[0005] Yeast strains are usually limited in their growth and protein synthesis abilities in complex substrates such as chicken manure. The present invention screened out a mycelium-producing Kluyveromyces marxianus SNJ1 for protein production and chicken manure treatment, which can be used to promote the resource utilization of chicken manure. Summary of the Invention

[0006] The present invention aims to provide a strain of Kluyveromyces marxianus SNJ1 for protein production and chicken manure treatment, wherein the Kluyveromyces marxianus strain has a deposit number of CCTCCNO: M 20251143.

[0007] Another object of the present invention is to provide the use of Kluyveromyces marxii SNJ1 in chicken manure treatment. The strain of the present invention has the characteristic of growing in the form of hyphae, and can effectively degrade organic matter in chicken manure, reduce N, K, and P content, and efficiently synthesize bacterial protein, providing theoretical support for the recycling and harmless treatment of agricultural waste.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] Obtaining Kluyveromyces marxianus SNJ1 strain:

[0010] The applicant isolated a fast-growing yeast strain from four samples of yogurt using chicken manure culture medium. This strain was identified as Kluyveromyces marxianus by ITsrDNA analysis. Subsequently, after adaptive evolution using chicken manure culture medium and comprehensive comparison of bacterial biomass and protein content, a Kluyveromyces marxianus strain was identified that produces high levels of protein in chicken manure. This strain was deposited with the China Center for Type Culture Collection on May 21, 2025, under the classification name Kluyveromyces marxianus SNJ1 and the deposit number CCTCC NO: M 20251143. The address is Wuhan University, Wuhan, China.

[0011] Under the growth conditions of temperature of 28-32° C., the morphology of Kluyveromyces marxii SNJ1 on a chicken manure culture medium with a moisture content of 60.0-71.4% and a pH of 6-7 is white creeping hyphae.

[0012] The protection content of the present invention also includes:

[0013] The invention relates to a Kluyveromyces marxianus SNJ1 fermentation broth, wherein the fermentation broth contains live Kluyveromyces marxianus SNJ1 bacteria.

[0014] A composition containing Kluyveromyces marxianus SNJ1 or the fermentation broth thereof.

[0015] Application of Kluyveromyces marxianus SNJ1, Kluyveromyces marxianus SNJ1 fermentation liquid or the above composition in chicken manure treatment.

[0016] In the above application, preferably, the ambient temperature of the application is 28-32°C, more preferably 28°C;

[0017] In the above application, preferably, the pH of the chicken manure culture medium is 6-7, more preferably 6.5;

[0018] In the above application, preferably, the moisture content of the chicken manure culture medium is 60.0-71.4%, more preferably 71.4%;

[0019] In the above application, specifically, the chicken manure treatment includes processing organic matter, N, P and / or K in the chicken manure.

[0020] Application of Kluyveromyces marxianus SNJ1, Kluyveromyces marxianus SNJ1 fermentation liquid or the above composition in preparing a microbial preparation for treating chicken manure.

[0021] Application of Kluyveromyces marxianus SNJ1, Kluyveromyces marxianus SNJ1 fermentation broth or the above composition in bacterial protein production in a high-nitrogen substrate.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides, for the first time, a yeast strain, Kluyveromyces marxii SNJ1, that produces high levels of bacterial protein in chicken manure and exhibits excellent organic matter removal efficiency and N, P, and K removal. This strain provides a high-quality seed strain for single-cell protein production and chicken manure treatment. Furthermore, it forms white, creeping hyphae in chicken manure, making it easy to collect. Testing has shown that this strain performs optimally under optimal temperature and pH conditions, exhibits environmental tolerance, and is suitable for industrial applications.

[0024] The present invention provides a new microbial tool for resource utilization of livestock and poultry manure, which can simultaneously achieve organic waste treatment and high-value protein production, and has significant application potential in the fields of feed additives and biofertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of white mycelium growth screened on chicken manure culture medium during the initial screening process.

[0026] Figure 2 This is the morphology of the mycelium of four fungal strains screened from chicken manure culture medium.

[0027] Figure 3 The dry weight and protein yield of Kluyveromyces marxianus SNJ1 at different temperatures.

[0028] Figure 4 The dry weight and protein yield of Kluyveromyces marxianus SNJ1 at different pH values.

[0029] Figure 5 The dry weight and protein yield of Kluyveromyces marxianus SNJ1 at different moisture contents.

[0030] Figure 6 The dry weight and protein yield of Kluyveromyces marxianus SNJ1 under different substrate grinding degrees.

[0031] Figure 7 This is an orthogonal test of the dry weight and protein yield of Kluyveromyces marxianus SNJ1.

[0032] Figure 8 The removal efficiency of organic matter, N, P and K in chicken manure after treatment with Kluyveromyces marxii SNJ1 under optimal fermentation conditions. DETAILED DESCRIPTION

[0033] In the technical solutions of the present invention, the reagents, unless otherwise specified, were purchased from biochemical stores, and the technical solutions, unless otherwise specified, are conventional techniques in the art.

[0034] YPD medium: Weigh 20 g of peptone, 10 g of yeast extract powder, and 20 g of glucose, and add 1000 mL of pure water.

[0035] YPD solid medium: weigh 20 g of peptone, 10 g of yeast extract powder, 20 g of glucose, and 15 g of agar, and add 1000 mL of pure water.

[0036] Chicken manure culture medium: Dry and grind chicken manure and pass it through a 100-mesh sieve. Weigh 5 g of chicken manure and put it into a culture dish. Add appropriate amount of pure water to adjust the moisture content to 60.0%, 66.6%, and 71.4%, respectively. Adjust the pH by adding NaOH.

[0037] Example 1:

[0038] Screening, Isolation and Purification of Kluyveromyces marxianus SNJ1

[0039] 1. Sample Collection and Pretreatment

[0040] Weigh 10g of yogurt sample into a conical flask, add 90mL of sterile water, mix thoroughly, and incubate overnight at 37°C. The next day, filter the sample with filter paper, and perform a gradient dilution of the filtrate with sterile water in a clean bench.

[0041] 2. Enrichment of bacterial strains

[0042] Use a pipette to draw up 100 μL of the diluted solution and inoculate it onto YPD solid medium. Spread the solution evenly using a glass spreading rod. Incubate the medium in an inverted position at 37°C for 24 hours. After 24 hours, pick a single colony from the medium and inoculate it onto a fresh plate of YPD solid medium for streak isolation and purification. Incubate the plate in an inverted position at 37°C for 24 hours. Repeat this process three times to obtain pure colonies.

[0043] 3. Initial screening of chicken manure-resistant bacteria

[0044] After purification and culture, single colonies were selected and expanded in liquid YPD medium, and then inoculated into chicken manure medium for coating. Chicken manure medium: Chicken manure was dried and ground, then passed through a 100-mesh sieve. 5g of chicken manure powder was weighed and placed in a culture dish. 10ml of pure water was added to adjust the moisture content to 66.6%, and the mixture was sterilized at 115°C for 30 minutes. 500μL of the bacterial solution was taken with a pipette and added to the chicken manure medium. The culture was then cultured in a 30°C incubator for 5 days. Finally, only four strains of bacteria had mycelium formed. Figure 1 Show some strain results.

[0045] 4. Rescreening of chicken manure-tolerant yeast

[0046] Weigh 5g of chicken manure powder and put it into a culture dish. Add 10ml of pure water to adjust its moisture content to 66.6%. Stir well and sterilize at 115℃ for 30min. Use a pipette to pick up the hyphae of the four fungi in step 3 and inoculate the hyphae of the strains on the chicken manure culture medium respectively. Culture in a constant temperature incubator at 30℃ for 5 days. Observe the growth of hyphae and the size of the hyphae. The results are as follows: Figure 2 shown.

[0047] 5. Growth Rate Determination

[0048] The screened strains were inoculated into chicken manure culture medium in the form of mycelium, cultured in a 30°C incubator for 7 days, and measured every 24 hours. The mycelium diameter of the strain was used as the standard for the strain growth rate, and the mycelium diameter (unit: cm) at different culture times was measured. The strain with the largest mycelium diameter was selected for subsequent tests. As can be seen from the table below, the strain diameter of SNJ1 was the largest after seven days of culture, so it was selected as the candidate strain.

[0049]

[0050] Purification and identification of a bacterial strain with high protein production from chicken manure

[0051] After the isolated SNJ1 strain was isolated through multiple streaking to obtain a pure single colony, colony morphology was observed for biochemical identification. Genomic DNA was extracted and its ITSrDNA sequenced and analyzed. Comprehensive identification and analysis confirmed that the strain was Kluyveromyces marxianus. The strain was deposited with the China Center for Type Culture Collection on May 21, 2025, under the classification name Kluyveromyces marxianus SNJ1 and the deposit number CCTCC NO: M 20251143. The address is Wuhan University, Wuhan, China.

[0052] Kluyveromyces marxianus SNJ1 was inoculated on YPD solid medium and cultured in a 30°C incubator for 12 h. The colonies were milky white and opaque.

[0053] Kluyveromyces marxianus SNJ1 was inoculated into YPD liquid medium and cultured at 30°C and 180 rpm for 12 h. The effective bacterial concentration of the final fermentation liquid was 1.6 x 10 7 cfu / ml.

[0054] Example 2:

[0055] Optimal Treatment Conditions for Kluyveromyces marxianus SNJ1

[0056] 1. Optimal Temperature Selection Test for Kluyveromyces marxianus SNJ1

[0057] The optimal temperature for the growth of the fungus in chicken manure was selected through a selection test, and the mycelia were transferred to a chicken manure medium (air-dried chicken manure was passed through a 100-mesh sieve, 5.00 g of chicken manure powder was weighed, 10 ml of pure water was added to adjust the moisture content to 66.6%, and a 10 mol / L NaOH solution was added to adjust the pH to 6.0, and sterilized. The mycelia were inoculated with the above chicken manure medium and placed in a constant temperature incubator at 28°C, 30°C, and 22°C for 7 days. The mycelia were scraped and separated with a blade, and the mycelia were collected, washed repeatedly by centrifugation with pure water, and then dried and ground in a 60°C oven. The dry weight and protein content of the mycelia were measured.

[0058] Weigh the dried mycelium sample and scale it up 200 times to get the mycelium yield in g / kg. For example, at 28°C, the mycelium yield is: mycelium yield (1.28 g / kg) = 0.064 mg (mycelium) / 5 g (chicken manure) × 200.

[0059] The total nitrogen content of the dried sample was determined by Kjeldahl nitrogen determination and converted into crude protein: crude protein (%) = total nitrogen content of mycelium (%) × 6.25.

[0060]

[0061] 2. Optimal pH test for Kluyveromyces marxianus SNJ1

[0062] The optimal pH for the growth of this fungus in chicken manure was determined through a selective experiment. Using 5g of chicken manure powder (passed through a 100-mesh sieve), 10ml of pure water was added to adjust the moisture content to 66.6%. Subsequently, 10mol / L NaOH solution was added to adjust the pH of the culture medium to 6, 6.5, and 7, respectively. The culture medium was sterilized at 115°C for 30 minutes. Mycelium was inoculated into the sterilized chicken manure culture medium at different pH values ​​and incubated in a 30°C incubator for 7 days. The mycelia were separated with a razor blade, collected, washed repeatedly with pure water by centrifugation, dried in a 60°C oven, and ground. The dry weight and protein content of the mycelium were then measured.

[0063]

[0064] 3. Selection test of optimal moisture content of Kluyveromyces marxianus SNJ1

[0065] The optimal moisture content for the growth of this fungus in chicken manure was determined through a selection experiment. Using 5g of chicken manure powder (passed through a 100-mesh sieve), 7.5ml, 10ml, and 12.5ml of pure water were added to adjust the moisture content to 60.0%, 66.6%, and 71.4%, respectively. The pH was adjusted to 6.0 with a 10mol / L NaOH solution, and the culture was sterilized at 115°C for 30 minutes. Mycelium was inoculated into the sterilized chicken manure culture medium with different moisture contents and incubated in a 30°C incubator for 7 days. The mycelia were separated with a razor blade, collected, washed repeatedly with pure water, dried in a 60°C oven, ground, and then measured for dry weight and protein content.

[0066]

[0067] 4. Experimental study on the optimal grinding degree of Kluyveromyces marxianus SNJ1

[0068] A selection experiment was conducted to determine the growth rate of the fungus in chicken manure of varying degrees of grinding. After sun-drying, the manure was passed through 18-, 60-, and 100-mesh sieves. 5g of chicken manure powder was weighed, and 10ml of pure water was added to adjust the moisture content to 66.6%. The pH was adjusted to 6.0 with a 10mol / L NaOH solution, and the mixture was sterilized at 115°C for 30 minutes. Mycelium was inoculated into the sterilized chicken manure culture medium with varying degrees of grinding and incubated in a 30°C incubator for 7 days. The mycelia were separated with a razor blade, collected, washed repeatedly with pure water by centrifugation, dried in a 60°C oven, and ground. The dry weight and protein content of the mycelium were then measured.

[0069]

[0070] 5. Orthogonal test

[0071] We selected optimal experimental conditions and designed orthogonal experimental groups. We then combined these conditions to create a four-factor, three-level orthogonal experiment. During the experiment, we controlled the range of variation for each factor to identify the optimal combination of conditions. By comparing the changes in bacterial biomass in the biogas slurry culture medium under different combinations, we ultimately determined the optimal fermentation conditions to be: a temperature of 28°C, a pH of 6.5, a moisture content of 71.4%, and a chicken manure grind size of 100 mesh.

[0072] factor A B C Temperature (℃) 28 30 32 pH 6 6.5 7 Moisture content (%) 60 66.6 71.4 Grinding degree (mesh) 18 60 100

[0073] The test results are shown in the figure below. In the table below, factor B is the moisture content. 66% is actually 66.6% and 71% is actually 71.4%.

[0074]

[0075]

[0076]

[0077] A: Temperature; B: Moisture content; C: pH; D: Grinding degree

[0078] Example 3:

[0079] Application of Kluyveromyces marxii SNJ1 in chicken manure treatment

[0080] After determining the optimal fermentation conditions through orthogonal experiments, mycelial culture experiments were conducted in 200mm Petri dishes. The optimal fermentation conditions, determined based on the orthogonal results, were group Z3 (pH 6.5, 71.4% moisture content, and 100-mesh grind). After autoclaving, the selected SNJ1 fungal strain was inoculated into the aforementioned chicken manure substrate to ensure uniform mycelial growth. Following inoculation, the dishes were placed in a 28°C incubator for 7 days.

[0081] During this period, chicken manure samples were collected every 24 hours, dug with a key under the mycelium, and mixed at different points. The changes in N, P, K, organic matter and crude ash in the chicken manure matrix were then measured.

[0082] Under the optimal conditions of Z3, the mycelium yield was 14.8 g / kg and the mycelial protein content was 55.89%.

[0083]

[0084] During the growth of the fungal mycelium, the nitrogen (2.88% → 2.51%), phosphorus (1.19% → 1.01%), and organic matter (60.36% → 45.67%) contents in the chicken manure decreased significantly, indicating that the mycelial growth preferentially utilized these nutrients to synthesize proteins, nucleic acids, and energy substances, while phosphorus (1.51% → 1.32%) was consumed less, primarily to maintain cellular metabolism. Simultaneously, the crude ash content increased (15.2% → 19.3%), indicating that the fermented chicken manure still has mineral fertilizer value. This process not only reduces the environmental pollution risk of chicken manure but also achieves the dual benefits of bacterial protein production and resource utilization of organic waste.

Claims

1. A strain of Kluyveromyces marxii obtained by adaptive evolution in chicken manure substrate ( Kluyveromyces marxianus )SNJ1, the deposit number of the Kluyveromyces marxianus strain is: CCTCC NO: M 20251143.

2. The fermentation broth of Kluyveromyces marxianus SNJ1 according to claim 1, wherein the fermentation broth contains live bacteria of Kluyveromyces marxianus SNJ1.

3. A composition comprising the Kluyveromyces marxianus SNJ1 according to claim 1 or the fermentation broth according to claim 2.

4. Use of the Kluyveromyces marxianus SNJ1 according to claim 1, the fermentation liquid according to claim 2 or the composition according to claim 3 in chicken manure treatment.

5. The use according to claim 4, wherein the ambient temperature of the application is 28-32°C.

6. The use according to claim 4, wherein the pH of the chicken manure is 6-7.

7. The use according to claim 4, wherein the moisture content of the chicken manure is 60.0-71.4%.

8. The use according to claim 4, wherein the chicken manure treatment comprises reducing organic matter, N, P and K in the chicken manure.

9. Use of the Kluyveromyces marxianus SNJ1 according to claim 1, the fermentation broth according to claim 2, or the composition according to claim 3 in preparing a microbial preparation for treating chicken manure.

10. Use of the Kluyveromyces marxianus SNJ1 according to claim 1, the fermentation broth according to claim 2, or the composition according to claim 3 in the production of bacterial protein in a high-nitrogen substrate.

Images