A Pichia membranifaciens and its application in ruminant farming

Abstract

The present invention discloses a strain of Pichia membranifaciens ( Pichia membranifaciens ), and its application in the breeding of ruminants. The Pichia membranifaciens ( Pichia membranifaciens ) M12 of the present invention was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on August 5, 2024, with the deposit number of CGMCC No. 31606. The present invention also discloses a breeding method for ruminants. The present invention discovers for the first time that the rumen-derived Pichia membranifaciens M12 can reduce the methane emissions of ruminants, and has no negative impact on feed intake, milk yield and milk quality. Therefore, the Pichia membranifaciens M12 of the present invention can be used for the breeding of ruminants.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of yeasts in ruminants, and particularly relates to a Pichia membranifaciens and its application in ruminant breeding. Background Art

[0002] At present, methane emitted from the gastrointestinal tract of ruminants accounts for about 6% of the global greenhouse gas emissions (40% of all livestock emissions), and the feed energy lost in the form of methane accounts for 2% to 12% of the total feed energy of ruminants. Methane emissions from ruminants are not only an important source of greenhouse gas emissions, but also reduce the utilization efficiency of feed energy. At present, various measures have been taken to reduce rumen methane emissions. In terms of nutritional regulation, adjusting the diet structure, such as increasing the proportion of concentrate 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. Using additives such as antibiotics like monensin can inhibit the activity of methanogens, but long-term use is likely to lead to the problem of rumen microbial drug resistance, endangering animal health and food safety. Plant extract additives, such as tannins and essential oils, although relatively green and safe, have disadvantages such as unstable effects and high costs, and are difficult to be popularized and applied on a large scale. In the field of microbial regulation, although some microorganisms or their metabolites that can regulate rumen methane production have been discovered, there are still problems such as difficult screening, unclear action mechanisms, and unstable application effects in actual application. To sum up, the existing rumen methane regulation methods have many limitations and are difficult to meet the needs of the sustainable development of the current livestock industry. 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 in ruminants and promote the green and sustainable development of the livestock industry.

[0003] At present, the research and application of Pichia membranifaciens ( Pichia membranifaciens ) mainly focus on its application in the prevention and control of postharvest fungal diseases of fruits, as well as its potential and mechanism as a biological control agent. There is no discovery of the application of this strain in animals, let alone its application in ruminant breeding. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a Pichia membranifaciens ( Pichia membranifaciens ).

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

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

[0007] Technical solution: To solve the above technical problems, the present invention provides Pichia membranifaciens ( Pichia membranifaciens ), and the Pichia membranifaciens ( Pichia membranifaciens ) was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 5, 2024, with the deposit number of CGMCC No. 31606 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0008] The present invention also includes a microbial preparation or a feed additive, and the microbial preparation or the feed additive contains the above-mentioned Pichia membranifaciens ( Pichia membranifaciens ).

[0009] The present invention also includes the application of the above-mentioned Pichia membranifaciens ( Pichia membranifaciens ) or the microbial preparation or the feed additive in the breeding of ruminants.

[0010] Among them, the ruminants include but are not limited to Holstein cows.

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

[0012] Among them, the dosage of the Pichia membranifaciens ( Pichia membranifaciens ) is 2.5×10 11 CFU / head / day to 5×10 11 CFU / head / day.

[0013] The present invention also includes a breeding method for ruminants, and the breeding method includes feeding the above-mentioned Pichia membranifaciens ( Pichia membranifaciens ) or the microbial preparation or the feed additive to ruminants.

[0014] Among them, the breeding method includes adding Pichia membranifaciens ( Pichia membranifaciens ) to the basal diet of ruminants for feeding.

[0015] Among them, the dosage of the Pichia membranifaciens ( Pichia membranifaciens ) is 2.5×10 11 CFU / head / day to 5×10 11 CFU / head / day.

[0016] In this invention, the detection method for the content of microbial protein in the fermentation broth 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 detection method for 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 detection of crude protein 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, this invention has the following advantages: This invention first discovers that Pichia membranifaciens ( Pichia membranifaciens can improve the crude fat digestibility of ruminant feed, significantly reduce methane emissions, and has no negative impact on feed intake, milk yield, and milk quality. Therefore, Pichia membranifaciens of this invention ( Pichiamembranifaciens ) It can be used for the breeding of ruminants. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the growth curve of yeast;

[0019] Figure 2 It is the determination chart of the lactic acid utilization ability of yeast;

[0020] Figure 3 It is the determination chart of the ability of yeast to produce microbial protein. DETAILED IMPLEMENTATION MANNER

[0021] The implementation plan of the present invention will be described in detail below in combination with the embodiments. The following embodiments are only used to illustrate the present invention. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0022] Example 1 Obtaining of Pichia membranifaciens ( Pichia membranifaciens ) M12

[0023] 1. Composition and preparation of the medium

[0024] YM liquid medium (Qingdao Haibo Biotechnology Co., Ltd.), each liter of the medium contains the following components: yeast extract powder 3.0 g, malt extract powder 3.0 g, peptone 5.0 g, glucose 10.0 g, distilled water 1000.0 mL. After autoclaving at 115 °C, it is reserved for use. YM solid medium (Qingdao Haibo Biotechnology Co., Ltd.), each liter of the solid medium contains the following components: yeast extract 3.0 g, malt extract powder 3.0 g, peptone 5.0 g, glucose 10.0 g, agar 20.0 g, distilled water 1000.0 mL. Sterilize at 121 °C for 15 min. After sterilization and cooling to about 60 °C, add streptomycin sulfate (2000 U / mL) and potassium penicillin (1600 U / mL) under aseptic operation.

[0025] 2. Sample collection and yeast isolation

[0026] Use an oral collector to collect fresh rumen fluid from Holstein cows (Teaching and Research Base of the College of Animal Science and Technology, Nanjing Agricultural University, Huai'an, Jiangsu). Gradient dilute the fresh rumen fluid with physiological saline, and then use the plate coating method to evenly coat the diluted rumen fluid on the YM solid medium. After culturing at 30 °C for 48 h, morphological observation is carried out. Use an inoculation loop to pick a single smooth round colony and purify it 3 times. Inoculate the single colony into the YM liquid medium and culture it at a shaker temperature of 30 °C and a rotation speed of 120 rmp for 48 h. Record the OD value at 48 h and conduct a preliminary screening of yeast, and select 11 strains of bacteria with the highest OD value. AsFigure 1 As shown in the growth curve of yeast, strain M12 began to enter the logarithmic phase at 16 h and the stationary growth phase at 36 h, with an OD600 greater than 1.0.

[0027] 3. Determination of lactic acid utilization ability

[0028] By adding lactic acid, the lactic acid concentration in the YM liquid medium was adjusted to 15 mmol / L. The yeast solution cultured for 24 h was inoculated into 10 mL of YM liquid medium containing 15 mmol / L lactic acid and in an aerobic environment at an inoculation amount of 0.5% (v / v), and cultured at 39 °C and 120 rmp for 48 h. The lactic acid concentration was measured using a lactic acid kit (product number A019-2-1, Jiancheng Bioengineering Institute, Nanjing, Jiangsu). The lactic acid utilization rate = (initial lactic acid concentration in the medium - lactic acid concentration in the fermentation broth) / initial concentration of the medium. As Figure 2 shown (Note: Different lowercase letters in the subscript indicate significant differences ( P < 0.05), Figure 3 the same), strain M12 had a strong ability to utilize lactic acid in the medium, with an average utilization rate of 41.08%.

[0029] 4. Determination of microbial protein production ability

[0030] The yeast solution cultured for 24 h was inoculated into the YM medium at an inoculation amount of 0.5% (v / v), and cultured at 39 °C and 120 rmp for 48 h. The microbial protein content in the fermentation broth was measured by the Coomassie brilliant blue colorimetric method. As Figure 3 shown, strain M12 had a strong ability to produce microbial protein, with an average microbial protein content of 108.36 mg / mL.

[0031] 5. Molecular biology identification

[0032] The genomic DNA of yeast M12 was extracted using a fungal genomic DNA extraction kit (product number D2300, Beijing Solarbio Science & Technology Co., Ltd., Beijing). The ITS region was amplified by PCR using primers ITS1F / ITS4R. The amplification system was as follows: 10 x Ex 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 (5 U / 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) was 5’-TCCGTAGGTGAACCTGCGG-3’, and ITS4R (shown in SEQ ID NO.2) was 5’-TCCTCCGCTTATTGATATGC-3’. The PCR program was 95 °C for 5 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s, with 30 cycles; and finally 72 °C for repair extension for 10 min.

[0033] Sequencing was performed using a 3730 sequencer. The sequencing results were subjected to BLAST alignment in the GenBank database on the NCBI platform. The alignment results showed that strain M12 had the highest similarity (100.00%) with Pichia membranifaciens and was identified as Pichia membranifaciens Pichia membranifaciens, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) on August 5, 2024, with the deposit number CGMCC No. 31606 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0034] Example 2 Application of rumen-derived Pichia membranifaciens Pichia membranifaciens M12 in Holstein cows

[0035] The test site for this example was the teaching and research base of the College of Animal Science and Technology, Nanjing Agricultural University (Huai'an, Jiangsu). The test time was from May 11 to June 14, 2024.

[0036] The raw data of this example was preliminarily processed using Excel (2021). SPSS 22.0 was used for data analysis. Among them, the data of feed intake, milk yield, milk composition, etc. measured weekly repeatedly were analyzed using a linear mixed model, with the corresponding production data obtained during the pre-feeding period as covariates. The model included treatment (CON, LPM, HPM), weeks 1 - 4, and treatment × week. The apparent digestibility of nutrients, OD value, lactic acid content, and microbial protein concentration were subjected to a significance test using one-way ANOVA, and Duncan's test was used for post hoc multiple comparisons to evaluate the differences between any two groups. When P <0.05 indicates a significant difference, P ≥0.05 indicates no significant difference.

[0037] 1. Experimental design and animal feeding

[0038] Thirty-six healthy Holstein cows were selected for this experiment. A completely randomized block design was adopted, with three treatment groups, namely the control group (CON), low-dose group (LPM), and high-dose group (HPM). The 36 cows were divided into three blocks according to the principle of similar parity (2.25 ± 0.13, 2.83 ± 0.34, 2.92 ± 0.26, P = 0.159), milk yield (38.25 ± 2.11 kg / d, 38.87 ± 1.80 kg / d, 38.43 ± 1.37 kg / d, P = 0.969). Twelve cows in each block were randomly assigned to the three treatment groups, with 12 cows in each treatment group. The live yeast solution used in the experiment was Pichia membranifaciens ( Pichia membranifaciens ), the strain M12 isolated from the rumen in Example 1. The cows in the CON group were fed a basal diet, the cows in the LPM group were fed a basal diet + 2.5×10 11 CFU / cow / day of M12 bacterial solution, and the cows in the HPM group were fed a basal diet + 5×10 11 CFU / cow / day of M12 bacterial solution. The formula and nutrient level of the basal diet are shown in Table 1. The pre-feeding period was 1 week, and the experimental period was 4 weeks. The experimental cows were housed in separate pens, fed ad libitum, and provided with water. The feeding management was carried out according to the daily management of the cattle farm. During the experimental period, the cows were fed twice a day (06:10, 13:10) and milked three times a day (06:10, 13:20, and 20:30).

[0039] Table 1 Diet composition and nutrient level (dry matter basis, DM)

[0040]

[0041] Premix 1Provided per kilogram of diet: VA 210 KIU, VD 70 KIU, VE 1165 mg; biotin 2 mg; β-carotene 3.3 mg; Cu 0.6 g; Fe 4.0 g; Mn 1.58 g; I 55 mg; Zn 3.25 g; Co 11 mg; Se 18 mg; copper methionine 120 mg; manganese methionine 300 mg; zinc methionine 660 mg; yeast selenium 3.3 mg; rumen-protected methionine 2.7%; total phosphorus 1.65%; calcium 12.3%; salt 18.3%; magnesium 5.5%; potassium carbonate 4.7%. Nutritional levels 2 Are measured values, and the net energy for lactation is calculated according to the NRC model (NRC, 2001).

[0042] 2. Effects of feeding M12 bacterial liquid on the production performance of dairy cows

[0043] 2.1 Dry matter intake and collection of diet samples:

[0044] During the experiment, on the last two days of each week, the dry matter intake of dairy cows was measured, and the feeding amount and remaining amount were recorded. Dry matter intake = (feeding amount - remaining amount) × dry matter content (%).

[0045] On the last two days of each week during the experimental period, 500 g of feed samples were collected at 06:10 and 13:20 respectively, and the collected feeds were mixed. The 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. The feed samples that could not pass through the sieve were ground by themselves and then passed through the sieve again, and were stored for subsequent determination of nutritional components.

[0046] 2.2 Milk yield and milk composition analysis

[0047] During the experiment, the daily milk yield of each dairy cow was recorded on the fourth and fifth days of each week during the experimental period; on the fourth and fifth days of each week during the experimental period, 50 ml of milk samples were collected from each dairy cow at 06:10, 13:20 and 20:30 respectively, and were mixed according to a volume ratio of 4:3:3. Using a FOSS full-automatic milk composition analyzer, the contents of various conventional milk components in milk were determined according to the standard procedure.

[0048] For the determination of feed nutritional components, crude protein was pretreated by digestion at 420 °C using a digestion furnace, and then determined using a full-automatic Kjeldahl apparatus. For crude ash, the feed samples first needed to be pretreated by carbonization on an electric furnace, and then burned to constant weight at 550 °C using a muffle furnace for determination. Neutral detergent fiber and acid detergent fiber were determined using a semi-automatic fiber analyzer.

[0049] 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 no significant effect on the feed intake and milk yield of dairy cows ( P > 0.05), and had no significant effect on milk solids ( P = 0.773), milk fat percentage ( P = 0.914), milk protein percentage ( P > 0.05), lactose percentage ( P = 0.236), milk fat yield ( P = 0.758), milk protein yield ( P = 0.385) and lactose yield ( P = 0.892).

[0050] Table 2 Effects of adding M12 bacterial solution to the diet on the production performance of dairy cows

[0051]

[0052] 3. Effects of feeding M12 bacterial solution on the apparent digestibility of feed nutrients in dairy cows

[0053] Collection and detection of fecal samples: In the last week of the experimental period, feces of 36 dairy cows were collected by rectal fecal collection method to detect the digestibility of nutrients. 200 g of fecal samples were collected from each dairy cow, and 10% sulfuric acid for nitrogen fixation was added to the self-sealing bag at a ratio of 10:1, and after mixing, they were stored at -20 °C for subsequent detection.

[0054] After sampling, the contents of conventional nutrients in feces were measured, including the contents of dry matter, neutral detergent fiber, acid detergent fiber, crude protein and crude fat. The apparent digestibility was measured by the endogenous indicator method. According to the acid-insoluble ash (AIA) contained in the feed and fecal samples themselves as the endogenous indicator, the digestibility of the diet was calculated and measured according to the following formula:

[0055] Apparent digestibility of feed nutrients % = 100 - 100 × [(b × c) / (a × d)]

[0056] Where: a is the content of a certain nutrient in the feed (%); b is the content of the nutrient in the fecal sample (%); c is the content of acid-insoluble ash in the feed (%); d is the content of acid-insoluble ash in the fecal sample (%).

[0057] The effects of adding M12 bacterial solution to the diet on the apparent digestibility of feed nutrients in dairy cows are 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), and significantly decreased 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), there was no significant effect on digestibility.

[0058] Table 3 Effects of adding M12 bacterial liquid to the diet on the digestibility of feed nutrients in heat-stressed dairy cows

[0059]

[0060] 4. Effects of feeding M12 bacterial liquid on methane emissions in dairy cows

[0061] The methane was measured using an AHC rumen methane analyzer (Subtropical Agricultural Ecology Institute, Chinese Academy of Sciences, Changsha, Hunan). Before the measurement began, the dairy cows were adapted to the AHC system for 7 days and continuously measured for 6 days. The methane measurement was carried out in two stages to ensure that the methane measurement of all dairy cows was completed within 1.5 h at each sampling time. A total of two groups were divided, with 18 dairy cows in each group divided into two stages for measurement. Methane emission data was collected continuously for 2 days in each group. d1: 7:00, 13:00, 19:00 and 1:00 (Group 1); 10:00, 16:00, 22:00 and 4:00 (Group 2); The second stage started immediately after the first stage, and the two groups exchanged time points for measurement, and the measurement was repeated once after the end. Ensure that the dairy cows have proper head positioning in the system during the measurement. After measuring the methane concentration for 5 minutes for each dairy cow, the average value was taken, and then the background gas was measured for 2 minutes.

[0062] The effects of adding M12 bacterial liquid to the diet on methane emissions in dairy cows are shown in Table 4. Adding a high dose of M12 bacterial liquid significantly reduced the daily methane emissions (g / d, P = 0.048), as well as methane / dry matter intake (g / kg of DMI, P = 0.009) and methane / energy-corrected milk (g / kg of ECM, P = 0.044).

[0063] Table 4 Effects of adding M12 bacterial liquid to the diet on methane emissions in dairy cows

[0064]

[0065] Energy-corrected 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];

[0066] Under the conditions of this experiment, adding M12 bacterial liquid (5×10 11 CFU / head / day) to the diet had no negative impact on the feed intake of dairy cows, increased the digestibility of crude fat in the feed, had no significant effect on milk yield and milk quality, and reduced methane emissions.

Claims

1. Pichia pastoris Pichia membranifaciens ) in ruminant breeding, wherein the ruminant is a Holstein cow, characterized in that: The Pichia pastoris ( Pichia membranifaciens ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (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. Contains Pichia pastoris ( Pichia membranifaciens ) in ruminant breeding, wherein the ruminant is a Holstein cow, 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.

3. The use according to claim 1 or 2, characterized in that: The applications include improving the digestibility and utilization of feed for ruminants and / or reducing methane emissions.

4. The use according to claim 1 or 2, 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.

5. A method for breeding ruminants, characterized in that: The culture method comprises the steps of: Pichia membranifaciens ) or containing the membrane-forming Pichia pastoris ( Pichia membranifaciens ) is fed to ruminants, wherein the ruminants are Holstein cows, and 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.

6. The method for breeding ruminants according to claim 5, characterized in that: The culture method comprises the steps of: Pichia membranifaciens ) added to the basic diet of ruminants for feeding.

7. The method for breeding ruminants according to claim 5, 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.

Images