Pichia membranaefaciens and application thereof in ruminant breeding

Abstract

The invention discloses pichia membranaefaciens and application of the pichia membranaefaciens in the aspect of breeding ruminants. The pichia membranaefaciens M12 is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on August 5, 2024, and the preservation number of the pichia membranaefaciens M12 is CGMCC (China General Microbiological Culture Collection Center) No.31606. The pichia membranaefaciens M12 has the advantages that the pichia membranaefaciens M12 is preserved in the pichia membranaefaciens M12, and the pichia membranaefaciens M12 is preserved in the pichia membranaefaciens M12; the invention further discloses a ruminant breeding method. The rumen-source pichia membranaefaciens M12 is found to be capable of reducing methane emission of ruminants for the first time, and has no negative influence on food consumption, milk yield and milk quality. Therefore, the pichia membranaefaciens M12 disclosed by the invention can be used for breeding ruminants.

Description

Technical Field

[0001] The invention belongs to the technical field of application of yeast in ruminants, and specifically relates to a strain of Pichia membranaceus and application thereof in ruminant breeding. Background Art

[0002] At present, methane emitted from the gastrointestinal tract of ruminants accounts for about 6% of global greenhouse gas emissions (accounting for 40% of all livestock emissions), and feed energy lost in the form of methane accounts for 2% to 12% of the total energy of ruminant feed. Ruminant methane emissions are not only an important source of greenhouse gas emissions, but also reduce the efficiency of feed energy utilization. At present, a variety of measures have been taken to reduce rumen methane emissions. In terms of nutritional regulation, adjusting the diet structure, such as increasing the proportion of concentrated feed and optimizing the quality of roughage, can reduce methane production to a certain extent, but the effect is limited, and it may cause other problems such as rumen acidosis, affecting the health and production performance of ruminants. The use of additives such as antibiotics such as monensin can inhibit the activity of methanogens, but long-term use can easily lead to rumen microbial resistance problems, endangering animal health and food safety. Plant extract additives, such as tannins and essential oils, are relatively green and safe, but they have disadvantages such as unstable effects and high costs, making them difficult to promote and apply on a large scale. In the field of microbial regulation, although some microorganisms or their metabolites that can regulate rumen methane production have been found, there are still problems such as difficulty in screening, unclear mechanism of action, and unstable application effect before practical application. In summary, the existing rumen methane regulation methods have many limitations and are difficult to meet the needs of the current sustainable development of animal husbandry. Therefore, there is an urgent need to develop an efficient, safe, environmentally friendly and low-cost rumen methane regulator to solve the energy waste and environmental problems caused by rumen methane emissions from ruminants and promote the green and sustainable development of animal husbandry.

[0003] Currently, there are Pichia membranificans ) is mainly focused on its application in the prevention and control of postharvest fungal diseases in fruits, as well as its potential and mechanism as a biological control agent. The strain has not yet been found to be used in animals, let alone in ruminant farming. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Pichia pastoris ( Peach membrane-forming ).

[0005] Another technical problem to be solved by the present invention is to provide a microbial preparation or a feed additive.

[0006] The final technical problem to be solved by the present invention is to provide a method for breeding ruminants.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides Pichia membranacea ( Peach membrane-forming ), the membrane-forming Pichia pastoris ( Pichia membranificans ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on August 5, 2024, with the deposit number CGMCC No. 31606, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0008] The present invention also includes a microbial preparation or feed additive, wherein the microbial preparation or feed additive contains the Pichia membranaceus ( Pichia membranificans ).

[0009] The present invention also includes the Pichia pastoris ( Pichia membranificans ) or application of microbial preparations or feed additives in ruminant farming.

[0010] The ruminants include but are not limited to Holstein cows.

[0011] Among them, the application includes improving the digestibility of nutrients in ruminant feed and / or reducing methane emissions.

[0012] Wherein, the Pichia pastoris ( Pichia membranificans ) is used in an amount of 2.5×10 11 CFU / head / day~5×10 11 CFU / head / day.

[0013] The present invention also includes a method for breeding ruminants, which comprises the steps of: Pichia membranificans ) or microbial preparations or feed additives for feeding ruminants.

[0014] Wherein, the breeding method comprises the following steps: Pichia membranificans ) added to the basic diet of ruminants for feeding.

[0015] Wherein, the Pichia pastoris ( Pichia membranificans ) is used in an amount of 2.5×10 11 CFU / head / day~5×10 11 CFU / head / day.

[0016] The method for detecting the bacterial protein content in the fermentation broth in the present invention refers to the literature (Makkar HPS, Sharma OP, Dawra RK, Negi SS. Simple determination of microbial protein in rumen liquor. Journal of Dairy Science, 1982.). The method for detecting methane production refers to the literature (Hristov AN, Oh J, Giallongo F, Frederick T, Weeks H, Zimmerman PR, Harper MT, Hristova RA, Zimmerman RS, Branco AF. The use of an automated system (GreenFeed) to monitor enteric methane and carbon dioxide emissions from ruminant animals. Journal of Visualized Experiments, 2015). The calculation of methane emissions refers to (Wang R, Cao YR, Zhang XM, Zhang F, Tian X, Zhong RZ, Tan ZL, Wang M. Relationship between daily variations of methane emissions and eructation peaks in dairy cows measured with an automated head-chamber system. Animal Feed Science and Technology, 2023). The determination of neutral detergent fiber in feed and feces refers to "Determination of neutral detergent fiber in feed, GBT20806-2022", the determination of acid detergent fiber refers to "Determination of acid detergent fiber in feed, NYT1459-2022", the crude protein detection refers to "Determination of crude protein in feed-Kjeldahl method, GBT6432-2018", and the determination of moisture refers to "Determination of moisture in feed, GBT6435-2014".

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention first discovered that Pichia membranacea ( Pichia membranificans ) can improve the crude fat digestibility of ruminant feed, significantly reduce methane emissions, and have no negative impact on feed intake, milk production and milk quality. Peach membrane-forming ) can be used in the breeding of ruminants. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the growth curve of yeast; Figure 2 This is a diagram for determining the lactic acid utilization ability of yeast; Figure 3 This is a diagram for determining the ability of yeast to produce bacterial protein. DETAILED DESCRIPTION

[0019] The embodiments of the present invention will be described in detail below in conjunction with the examples, and the following examples are only used to illustrate the present invention. If the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0020] Example 1 Pichia pastoris ( Pichia membranificans ) Acquisition of M12 1. Culture medium composition and preparation YM liquid medium (Qingdao Haibo Biotechnology Co., Ltd.), each liter of medium contains the following ingredients: yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, glucose 10.0 g, distilled water 1000.0 mL. Sterilize at 115℃ and set aside. YM solid medium (Qingdao Haibo Biotechnology Co., Ltd.), each liter of solid medium package contains the following ingredients: yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, glucose 10.0 g, agar 20.0 g, distilled water 1000.0 mL. Sterilize at 121℃ for 15 min. Sterilize and cool to about 60℃, and add streptomycin sulfate (2000 U / mL) and penicillin potassium (1600 U / mL) aseptically.

[0021] 2. Sample collection and yeast isolation Fresh rumen fluid from Holstein cows (Teaching and Research Base of College of Animal Science and Technology, Nanjing Agricultural University, Huai'an, Jiangsu) was collected using an oral collector. The fresh rumen fluid was gradiently diluted with physiological saline, and then the diluted rumen fluid was evenly spread on YM solid culture medium using the plate spreading method. After culturing at 30°C for 48 h, morphological observation was performed. A single smooth round colony was picked using an inoculation loop and purified three times. A single colony was inoculated into YM liquid culture medium and cultured for 48 h at a shaker temperature of 30°C and a speed of 120 rpm. The OD value at 48 h was recorded for initial screening of yeast, and the 11 strains with the highest OD values ​​were selected. Figure 1The growth curve of yeast showed that strain M12 entered the logarithmic phase at 16 h and the stable growth phase at 36 h, with OD600 greater than 1.0.

[0022] 3. Using lactic acid capacity determination By adding lactic acid, the lactic acid concentration of the YM liquid medium was adjusted to 15 mmol / L. The yeast culture solution that had been cultured for 24 hours was inoculated at a 0.5% inoculation amount (v / v) into 10 mL YM liquid medium containing 15 mmol / L lactic acid and in an aerobic environment, and cultured at 39°C and 120 rpm for 48 hours. The lactic acid concentration was determined using a lactic acid kit (Cat. No. A019-2-1, Jiancheng Biological Research Institute, Nanjing, Jiangsu). Lactic acid utilization rate = (initial lactic acid concentration of the culture medium - lactic acid concentration of the fermentation liquid) / initial concentration of the culture medium. Figure 2 As shown (Note: Different lowercase letters on the shoulders indicate significant differences ( P <0.05), Figure 3 The strain M12 had a strong ability to utilize the lactic acid in the culture medium, with an average utilization rate of 41.08%.

[0023] 4. Determination of bacterial protein production ability The yeast culture liquid after 24 h of culture was inoculated into YM medium at a 0.5% inoculum (v / v) and cultured at 39°C and 120 rpm for 48 h. The protein content of the fermentation liquid was determined by the Coomassie brilliant blue colorimetric method. Figure 3 As shown, strain M12 has a strong ability to produce microbial protein, and the average bacterial protein content is 108.36 mg / mL.

[0024] 5. Molecular Biology Identification The fungal genomic DNA extraction kit (Cat. No. D2300, Beijing Solebow Technology Co., Ltd., Beijing) was used to extract the DNA of yeast M12. The primers ITS1F / ITS4R were used to amplify the ITS region by PCR. The amplification system was as follows: 10 xEx Taq buffer, 5.0 µL; 2.5 mmol / L dNTP Mix, 4.0 µL; primer ITS1F, 2.0 µL; primer ITS4R, 2.0 µL; DNA polymerase (5U / µL), 0.5 µL; bacterial solution, 2.0 µL; double distilled water, 34.5 µL. The sequence of primer ITS1F (shown in SEQ ID NO.1) is 5'-TCCGTAGGTGAACCTGCGG-3', and that of ITS4R (shown in SEQ ID NO.2) is 5'-TCCTCCGCTTATTGATATGC-3'. The PCR program was 95 °C, 5 min; 95 °C, 30 s, 55 °C, 30 s, 72 °C, 45 s, 30 cycles; and finally repair and extension at 72 °C for 10 min.

[0025] The sequencing was performed using a 3730 sequencer. The sequencing results were compared with the GenBank database on the NCBI platform using BLAST. The comparison results showed that strain M12 was Pichia membranificans The highest similarity (100.00%) was identified as Pichia pastoris ( Pichia membranificans ), and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on August 5, 2024, with the deposit number CGMCC No. 31606, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0026] Example 2 Rumen-derived Pichia membranaceus ( Pichia membranificans )Application of M12 on Holstein Cows The test location of this embodiment is: Teaching and Research Base of College of Animal Science and Technology of Nanjing Agricultural University (Huai'an, Jiangsu). Test time: May 11-June 14, 2024.

[0027] The raw data of this example were preliminarily processed using Excel (2021). SPSS 22.0 was used for data analysis. The weekly repeated measurement data of feed intake, milk production, milk composition, etc. were analyzed using a linear mixed model, and the corresponding production data obtained during the pre-feeding period were used as covariates. The model included treatments (CON, LPM, HPM), 1 to 4 weeks, and treatment × week. The apparent digestibility of nutrients, OD value, lactic acid content, and bacterial protein concentration were tested for significance using one-way analysis of variance, and the Duncan test was used for post hoc multiple comparisons to evaluate the differences between any two groups. PWhen <0.05, it means the difference is significant. P When ≥0.05, it means that the difference is not significant.

[0028] 1. Experimental design and animal husbandry This study selected 36 healthy Holstein cows and adopted a completely randomized block design. Three treatment groups were set up, namely the control group (CON), the low-dose group (LPM) and the high-dose group (HPM). The 36 cows were divided into the control group (CON), the low-dose group (LPM) and the high-dose group (HPM) according to their parity (2.25 ± 0.13, 2.83 ± 0.34, 2.92 ± 0.26, P = 0.159), milk production (38.25 ± 2.11 kg / d, 38.87 ± 1.80 kg / d, 38.43 ±1.37 kg / d, P = 0.969) were divided into three blocks, and the 12 cows in each block were randomly divided into three treatment groups, with 12 cows in each treatment group. The live yeast liquid used in the experiment was Pichia pastoris ( Peach membrane-forming ), strain M12 isolated from the rumen in Example 1. Cows in the CON group were fed a basal diet, and cows in the LPM group were fed a basal diet + 2.5×10 11 CFU / head / day of M12 bacterial solution, cows in the HPM group were fed the basic diet + 5×10 11 CFU / head / day of M12 bacterial liquid. The basic diet formula and nutritional level are shown in Table 1. The pre-feeding period was 1 week and the experimental period was 4 weeks. The experimental dairy cows were raised in stalls, fed and drank water freely, and the feeding management was carried out according to the daily management of the cattle farm. During the experimental period, they were fed twice a day (06:10, 13:10) and milked three times a day (06:10, 13:20 and 20:30).

[0029] Table 1 Diet composition and nutrient levels (dry matter basis, DM)

[0030] Premix 1 Each kg of feed provides: VA 210KIU, VD 70KIU, VE 1165 mg; biotin 2 mg; beta-carotene 3.3 mg; Cu 0.6 g; Fe 4.0 g; Mn 1.58 g; I 55 mg; Zn 3.25g; Co 11 mg; Se 18 mg; methionine copper 120 mg; methionine manganese 300 mg; methionine zinc 660 mg; yeast selenium 3.3 mg; rumen bypass methionine 2.7%; total phosphorus 1.65%; calcium 12.3%; salt 18.3%; magnesium 5.5%; potassium carbonate 4.7%. Nutritional level 2The net energy for lactation is calculated based on the NRC model (NRC, 2001).

[0031] 2. Effect of feeding M12 bacterial solution on the production performance of dairy cows 2.1 Dry matter intake and collection of feed samples: During the experiment, the dry matter intake of dairy cows was measured on the last two days of each week, and the feed amount and the remaining feed amount were recorded. Dry matter intake = (feed amount - remaining feed amount) × dry matter content (%).

[0032] During the last two days of each week of the trial period, 500 g of feed samples were collected at 06:10 and 13:20, respectively, and the collected feeds were mixed. Freshly collected feed samples were dried in an oven at 65 °C for 48 h to obtain air-dried samples, which were crushed and passed through a 40-mesh sieve. Feed samples that could not pass the sieve were ground by themselves and sieved again, and stored for subsequent nutrient composition determination.

[0033] 2.2 Milk production and milk composition analysis During the experiment, the daily milk production of each cow was recorded on the fourth and fifth days of each week during the experiment; 50 ml of milk samples were collected from each cow at 06:10, 13:20 and 20:30 on the fourth and fifth days of each week during the experiment, and mixed according to the volume ratio of 4:3:3. The content of each conventional milk component in milk was determined using a FOSS fully automatic milk component analyzer according to standard procedures.

[0034] For the determination of feed nutritional components, crude protein was digested and pretreated at 420°C in a digestion furnace, and then determined using a fully automatic nitrogen analyzer. Crude ash content first requires the feed sample to be carbonized on an electric furnace, and then burned to constant weight in a muffle furnace at 550°C for determination. Neutral detergent fiber and acid detergent fiber were determined using a semi-automatic fiber analyzer.

[0035] The effects of adding M12 bacterial solution to the diet on the production performance of dairy cows are shown in Table 2. Compared with the CON group, feeding high-dose M12 had a significant effect on the feed intake and milk production of dairy cows ( P >0.05) had no significant effect on milk solids ( P = 0.773), milk fat rate ( P = 0.914), milk protein rate ( P > 0.05), lactose rate ( P = 0.236), milk fat yield ( P = 0.758), milk protein yield ( P = 0.385) and lactose production ( P = 0.892) had no significant effect.

[0036] Table 2 Effects of adding M12 bacterial solution to the diet on the production performance of dairy cows

[0037] 3. Effect of feeding M12 bacterial solution on the apparent digestibility of nutrients in dairy cow feed Fecal sample collection and testing: In the last week of the trial, feces of 36 dairy cows were collected by rectal fecal collection to test the digestibility of nutrients. 200 g of fecal sample was collected from each dairy cow, and 10% sulfuric acid was added to the nitrogen fixation in a 10:1 ratio in a ziplock bag, mixed and stored at -20°C for subsequent testing.

[0038] After sampling, the content of conventional nutrients in feces was determined, and the content of dry matter, neutral detergent fiber, acid detergent fiber, crude protein and crude fat was determined. The apparent digestibility was determined using the endogenous indicator method. The acid insoluble ash (AIA) contained in the feed and feces samples was used as an endogenous indicator, and the feed digestibility was calculated and determined according to the following formula: Apparent digestibility of feed nutrients % = 100-100 × [(b×c) / (a ​​× d)] In the formula: a is the content of a certain nutrient in feed (%); b is the content of the nutrient in feces (%); c is the content of acid-insoluble ash in feed (%); d is the content of acid-insoluble ash in feces (%).

[0039] The effect of adding M12 bacterial solution to the diet on the apparent digestibility of nutrients in dairy cow feed is shown in Table 3. Compared with the control group, feeding high-dose M12 bacterial solution significantly increased the apparent digestibility of crude fat in dairy cow feed ( P = 0.016), significantly reduced the apparent digestibility of neutral detergent fiber ( P = 0.020), but for dry matter ( P = 0.126), crude protein ( P > 0.05) and acid detergent fiber ( P = 0.698) had no significant effect on digestibility.

[0040] Table 3 Effect of adding M12 bacterial solution to the diet on the digestibility of nutrients in heat-stressed dairy cows

[0041] 4. Effect of feeding M12 bacterial solution on methane emission of dairy cows Methane was measured using an AHC rumen methane meter (Institute of Subtropical Agriculture and Ecology, Chinese Academy of Sciences, Changsha, Hunan). Cows were acclimated to the AHC system for 7 days before the measurement began, and the measurement was performed continuously for 6 days. Methane measurement was performed in two phases to ensure that methane measurements for all cows were completed within 1.5 h of each sampling time. The cows were divided into two groups, and 18 cows in each group were measured in two phases. Methane emission data were collected for 2 consecutive days for each group, day 1: 7:00, 13:00, 19:00 and 1:00 (group 1); 10:00, 16:00, 22:00 and 4:00 (group 2); the second phase started immediately after the first phase, and the two groups exchanged time points for measurement, and the measurement was repeated once after the end. It was ensured that the cows had proper head positioning in the system during the measurement. The methane concentration was averaged after 5 minutes of measurement for each cow, and then a 2-minute background gas measurement was performed.

[0042] The effect of adding M12 bacterial solution to the diet on the methane emission of dairy cows is shown in Table 4. Adding high-dose M12 bacterial solution significantly reduced the daily methane emission (g / d, P = 0.048), and CH4 / DMI (g / kg of DMI, P =0.009) and methane / energy corrected milk (g / kg of ECM, P = 0.044).

[0043] Table 4 Effect of adding M12 bacterial solution to the diet on methane emissions of dairy cows

[0044] Energy correction milk (kg / d) 1 = milk yield × [(38.3 × % milk fat rate × 10 + 24.2 × % milk protein rate × 10 + 16.54 × % lactose rate × 10 + 20.7) / 3,140]; Under the conditions of this experiment, M12 bacterial solution (5×10 11 CFU / head / day) had no negative impact on the feed intake of dairy cows, increased the digestibility of crude fat in feed, had no significant effect on milk production and milk quality, and reduced methane emissions.

Claims

1. A strain of Pichia pastoris ( Pichia membranifaciens ), characterized in that, The Pichia pastoris ( Pichia membranifaciens ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on August 5, 2024, with the deposit number CGMCC No. 31606, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

2. A microbial preparation or feed additive, characterized in that: The microbial preparation or feed additive contains the Pichia membranaceus according to claim 1 ( Pichia membranifaciens ).

3. The Pichia pastoris according to claim 1 ( Pichia membranifaciens ) or the use of the microbial preparation or feed additive according to claim 2 in ruminant breeding.

4. The use according to claim 3, characterized in that: The ruminant is a Holstein cow.

5. The use according to claim 3, characterized in that: The applications include improving the digestibility and utilization of feed for ruminants and / or reducing methane emissions.

6. The use according to claim 3, characterized in that: The Pichia pastoris ( Pichia membranifaciens ) is used in an amount of 2.5×10 11 CFU / head / day~5×10 11 CFU / head / day.

7. A method for breeding ruminants, characterized in that: The cultivation method comprises the steps of: Pichia membranifaciens ) or the microbial preparation or feed additive according to claim 2 is fed to ruminants.

8. The method for breeding ruminants according to claim 7, characterized in that: The culture method comprises the steps of: Pichia membranifaciens ) added to the basic diet of ruminants for feeding.

9. The method for breeding ruminants according to claim 7, characterized in that: The Pichia pastoris ( Pichia membranifaciens ) is used in an amount of 2.5×10 11 CFU / head / day~5×10 11 CFU / head / day.

10. The method for breeding ruminants according to claim 7, characterized in that: The ruminant is a Holstein cow.

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