WO2023108644A1 - 一种调控pH值的全价混合发酵饲料及其制备方法 - Google Patents

一种调控pH值的全价混合发酵饲料及其制备方法 Download PDF

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WO2023108644A1
WO2023108644A1 PCT/CN2021/139292 CN2021139292W WO2023108644A1 WO 2023108644 A1 WO2023108644 A1 WO 2023108644A1 CN 2021139292 W CN2021139292 W CN 2021139292W WO 2023108644 A1 WO2023108644 A1 WO 2023108644A1
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feed
roughage
mixed fermented
value
fermentation
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PCT/CN2021/139292
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English (en)
French (fr)
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李光春
何娇杨
严昌国
蔡洪范
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延边大学
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Priority to PCT/CN2021/139292 priority Critical patent/WO2023108644A1/zh
Priority to ZA2023/01212A priority patent/ZA202301212B/en
Publication of WO2023108644A1 publication Critical patent/WO2023108644A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/22—Compounds of alkali metals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A23K50/15—Feeding-stuffs specially adapted for particular animals for ruminants containing substances which are metabolically converted to proteins, e.g. ammonium salts or urea

Definitions

  • the invention relates to the technical field of feed fermentation, in particular to a full-price mixed fermented feed for regulating the pH value and a preparation method thereof.
  • Total Mixed Feed is to cut silage, hay and other roughages into a certain length according to the nutritional regulation technology according to the nutritional requirements and feeding purposes of ruminants in different physiological stages, and then add a certain proportion of concentrated feed, various minerals, and vitamins. It is a kind of ration with relatively balanced nutrition processed by stirring and mixing thoroughly. TMR feeding technology can effectively avoid the selective feeding of livestock and improve the palatability of some raw materials; it can also increase the dry matter intake, promote balanced feeding of livestock, and effectively reduce the occurrence of rumen diseases.
  • Total mixed fermented (TMF) feed refers to the feed produced by a special in vitro fermentation process that uses local feed resources to prepare a nutritionally balanced full mixed feed based on the nutritional needs of ruminants. TMF technology is to further improve the TMR feeding technology, so that the feeding method is more suitable for the physiological requirements of ruminants.
  • lactic acid bacteria are the most common bacterial agents for fermentation. Using lactic acid bacteria to ferment can produce a large amount of organic acids, which can reduce the pH value of TMF feed to 4.2-4.7, thereby inhibiting the growth of other spoilage bacteria and avoiding the mold and deterioration of feed.
  • the pH The difference between the pH value and the pH value of the rumen (6.2-6.8) is relatively large, resulting in low feed utilization and nutritional value.
  • the object of the present invention is to provide a kind of full-price mixed fermented feed and its preparation method that regulates the pH value. Utilization and nutritional value of fermented feed.
  • the invention provides a full-price mixed fermented feed for adjusting the pH value, including concentrated feed and roughage.
  • the concentrated feed is prepared from the raw material of the concentrated feed by lactic acid bacteria fermentation.
  • the bacillus MYB3 is prepared by fermentation; the pH value of the complete mixed fermented feed is 6.00-7.00.
  • the pH value of the concentrated feed is 4.20-4.80, and the pH value of the roughage is 8.00-9.00.
  • the nutritional indicators of the complete mixed fermented feed include: the mass content of crude fat is 3-15%, the mass content of ammoniacal nitrogen is 5-15%, the mass content of crude protein is 5-20%, and the mass content of medium
  • the mass content of neutral detergent fiber is 20-50%, the mass content of acid detergent fiber is 15-30%, and the content of lactic acid is 1000-5000mg/kg.
  • the preparation method of described roughage comprises the following steps:
  • Bacillus megaterium MYB3 is inoculated in the roughage raw material, and the first fermentation is carried out under the alkaline condition provided by the alkaline feed additive to obtain the roughage.
  • the roughage raw material includes straw.
  • the straw includes corn straw, rice straw or wheat straw.
  • the alkaline feed additive includes urea, baking soda, ammonium bicarbonate, ammonium sulfate or ammonium chloride.
  • the alkaline feed additive is used in the form of an alkaline feed additive aqueous solution, and the concentration of the alkaline feed additive aqueous solution is 5-15 wt%.
  • the first fermentation is carried out at a pH value of 8.00-9.00.
  • the temperature of the first fermentation is 30-40° C.
  • the time is 7-14 days.
  • the preparation method of described concentrated feed comprises the following steps:
  • the lactic acid bacteria are inoculated in the raw material of the concentrated feed, and the second fermentation is carried out to obtain the concentrated feed.
  • the concentrated feed raw materials include soybean meal, wheat bran, corn and premix, and the mass ratio of soybean meal, wheat bran, corn and premix is (30-45):(6-15):(240 ⁇ 255): (9 ⁇ 24).
  • the lactic acid bacteria are lactic acid bacteria BG-1.
  • the temperature of the second fermentation is 30-40° C.
  • the time is 7-14 days.
  • the present invention provides the preparation method of full-price mixed fermented feed described in the above technical scheme, comprising the following steps:
  • Concentrate feed raw materials are fermented by lactic acid bacteria to obtain concentrate feed;
  • Roughage raw materials are fermented by Bacillus megaterium MYB3 under alkaline conditions to prepare roughage;
  • the concentrated feed is mixed with the roughage to obtain a complete mixed fermented feed.
  • the invention provides a full-price mixed fermented feed for adjusting the pH value, including concentrated feed and roughage.
  • the concentrated feed is prepared from the raw material of the concentrated feed by lactic acid bacteria fermentation.
  • the bacillus MYB3 is prepared by fermentation; the pH value of the complete mixed fermented feed is 6.00-7.00.
  • the invention obtains the concentrated feed through the fermentation of lactic acid bacteria, which can provide a large amount of nutrients for the animal body, and a large amount of organic acids are produced during the fermentation process, thereby reducing the pH value of the concentrated feed, which is beneficial to inhibit the reproduction of pathogenic microorganisms, and can also reduce the degree of protein decomposition. , improve the quality of feed.
  • the present invention obtains roughage by fermenting Bacillus megaterium MYB3, said Bacillus megaterium MYB3 is used as a cellulolytic bacterium, can effectively decompose cellulose and hemifiber in roughage raw materials, and said Bacillus megaterium MYB3 is an alkaline microorganism, can be used in It grows and reproduces under alkaline conditions without affecting its physiological characteristics, and can decompose cellulose and hemicellulose under alkaline conditions to obtain alkaline roughage.
  • the present invention obtains TMF feed based on the concentrate feed and roughage, and the concentrate feed and roughage can regulate the pH value of the TMF feed, so that the pH value of the TMF feed is close to the optimum pH value (6.2-6.8) of the rumen, shortening the time of TMF feed.
  • the time spent in the rumen is conducive to improving the utilization rate and nutritional value of TMF feed.
  • Bacillus megaterium Bacillus megaterium (Bacillus megaterium) MYB3, the preservation date is December 07, 2016, and the preservation place is the General Microbiology Center of the China Committee for the Collection of Microbial Cultures.
  • the specific address is the Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, the deposit number is CGMCCNo.13492;
  • Lactic acid bacteria (Lactiplantibacillus plantarum) BG-1
  • the preservation date is September 27, 2021
  • the preservation place is the General Microbiology Center of the China Committee for the Collection of Microbial Cultures.
  • the specific address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, the deposit number is CGMCCNO.23493.
  • the invention provides a full-price mixed fermented feed for adjusting the pH value, including concentrated feed and roughage.
  • the concentrated feed is prepared from the raw material of the concentrated feed by lactic acid bacteria fermentation.
  • the bacillus MYB3 is prepared by fermentation; the pH value of the complete mixed fermented feed is 6.00-7.00.
  • the pH value of the complete mixed fermented feed is preferably 6.2-6.8.
  • the nutritional indicators of the complete mixed fermented feed include: the mass content of crude fat is preferably 3-15%, more preferably 10-13%; the mass content of ammoniacal nitrogen is preferably 5-15%, More preferably 10-13.51%; the mass content of crude protein is preferably 5-20%, more preferably 11.48-15.50%; the mass content of neutral detergent fiber is preferably 20-50%, more preferably 30-40%; The mass content of acid detergent fiber is preferably 15-30%, more preferably 16.67-20.00%; the content of lactic acid is preferably 1000-5000mg/kg, more preferably 3000-5000mg/kg.
  • the acetic acid content in the full-price mixed fermented feed is relatively low, 100-1500 mg/kg; in the embodiment of the present invention, it is specifically 849.59 mg/kg; the full-priced mixed fermented feed does not contain propionic acid and butyric acid.
  • the pH value of the concentrated feed is preferably 4.20-4.80, more preferably 4.37-4.50; the pH value of the roughage is preferably 8.00-9.00, more preferably 8.20-8.62.
  • the mass ratio of the concentrated feed to the roughage is based on ensuring that the pH value and nutritional indicators of the full-price mixed fermented feed meet the above requirements. Specifically, the mass ratio of the concentrated feed to the roughage can be 5:5 , can also be 4:6.
  • the preparation method of described roughage preferably comprises the following steps:
  • Bacillus megaterium MYB3 is inoculated in the roughage raw material, and the first fermentation is carried out under the alkaline condition provided by the alkaline feed additive to obtain the roughage.
  • the preservation number of the Bacillus megaterium MYB3 is CGMCC No.13492.
  • the roughage raw material preferably includes straw, and the straw is preferably corn straw, rice straw or wheat straw.
  • the alkaline feed additive preferably includes urea, baking soda, ammonium bicarbonate, ammonium sulfate or ammonium chloride; the alkaline feed additive is preferably used in the form of an alkaline feed additive aqueous solution, and the alkaline feed additive The concentration of the additive aqueous solution is preferably 5-15 wt%, more preferably 5-10 wt%.
  • the moisture content of roughage raw materials is preferably adjusted to 40-60%, inoculated with Bacillus megaterium MYB3 bacterial liquid, and then the pH value of the fermentation system is adjusted to be alkaline by using alkaline feed additive aqueous solution, and the first fermentation is carried out to obtain roughage.
  • the moisture content is preferably adjusted to meet the above requirements by adding distilled water to the roughage raw material; in the present invention, the moisture content of the roughage raw material is further preferably adjusted to 60%.
  • the moisture content of the roughage raw material is preferably adjusted to the above range, which is beneficial to achieve a better fermentation effect.
  • the number of live bacteria of the Bacillus megaterium MYB3 bacterial liquid is preferably 1 ⁇ 10 8 cfu/mL; in the present invention, 1 mL of the original bacterial liquid of Bacillus megaterium MYB3 is preferably inoculated into 100 mL of LB medium, at 37°C, Cultivate for 24 hours under the condition of 120r/min to obtain Bacillus megaterium MYB3 bacterial liquid.
  • the ratio of the roughage raw material to the bacterial solution of Bacillus megaterium MYB3 is preferably (100-1000) g: 100 mL, more preferably 200 g: 100 mL.
  • the alkaline feed additive aqueous solution is preferably sprayed into the roughage raw material inoculated with the Bacillus megaterium MYB3 bacterial liquid, so that the pH value of the fermentation system is alkaline.
  • the dosage of the alkaline feed additive aqueous solution there is no special limitation on the dosage of the alkaline feed additive aqueous solution, as long as the pH value of the fermentation system meets the requirements.
  • the first fermentation is preferably carried out at a pH value of 8.00-9.00, more preferably 8.20-8.80.
  • the temperature of the first fermentation is preferably 30-40°C, more preferably 30°C; the time is preferably 7-20 days, more preferably 7 days.
  • the temperature and time of the first fermentation are preferably controlled within the above-mentioned range, which is beneficial to achieve a better fermentation effect, better decompose the cellulose and hemicellulose in the roughage raw material, and control the pH value in the fermentation process at the same time, so that the final The pH value of the obtained roughage meets the above requirements.
  • the preparation method of the concentrated feed preferably comprises the following steps:
  • the lactic acid bacteria are inoculated in the raw material of the concentrated feed, and the second fermentation is carried out to obtain the concentrated feed.
  • the concentrated feed raw materials preferably include soybean meal, wheat bran, corn and a premix
  • the premix is preferably a compound premix of beef, cattle and sheep; in an embodiment of the present invention, the premix Specifically, the feed is 4% beef cattle and sheep fattening compound premixed feed EP476 of Huashou.
  • the mass ratio of the soybean meal, wheat bran, corn and the premix is preferably (30-45): (6-15): (240-255): (9-24), more preferably ( 35-40): (8-10): (244-250): (11-18), more preferably 35.42:8.11:244.53:11.94.
  • soybean meal, wheat bran and corn are preferably mixed with the premixed material to obtain the concentrated feed raw material.
  • the lactic acid bacteria are preferably lactic acid bacteria BG-1, and the preservation number of the lactic acid bacteria BG-1 is CGMCC NO.23493.
  • the moisture content of the concentrated feed raw material is preferably adjusted to 40-60%, the lactic acid bacteria BG-1 bacterial liquid is inoculated, and the second fermentation is carried out to obtain the concentrated feed.
  • the water content is preferably adjusted to meet the above requirements by adding distilled water to the raw material for concentrated feed; in the present invention, the water content of the raw material for concentrated feed is further preferably adjusted to 60%.
  • the moisture content of the concentrated feed raw material is preferably adjusted to the above range, which is beneficial to achieve a better fermentation effect.
  • the number of live bacteria of the lactic acid bacteria BG-1 liquid is preferably 1.09 ⁇ 10 8 cfu/mL; in the present invention, 1 mL of the original lactic acid bacteria BG-1 liquid is preferably inoculated into 100 mL of the MRS medium, and the Cultivate under the condition of 120r/min for 24h to obtain lactic acid bacteria BG-1 bacterial liquid.
  • the ratio of the concentrated feed raw material to the lactic acid bacteria BG-1 bacterial solution is preferably (100-5000) g: 50 mL, more preferably 300 g: 50 mL.
  • the temperature of the second fermentation is preferably 30-40°C, more preferably 30°C; the time is preferably 7-20 days, more preferably 10 days.
  • the temperature and time of the second fermentation are preferably controlled within the above-mentioned range, which is beneficial to achieve a better fermentation effect.
  • the present invention prepares concentrated feed with a lower pH value through fermentation of lactic acid bacteria, and prepares roughage with a higher pH value through fermentation of Bacillus megaterium MYB3 under alkaline conditions, wherein Bacillus megaterium MYB3 can still be produced under alkaline conditions.
  • the pH value of the TMF feed can be regulated by the concentrated feed and roughage, make the pH value of the TMF feed close to the optimum pH value (6.2 ⁇ 6.8) of the rumen, shorten the TMF feed
  • the time spent in the rumen is conducive to improving the utilization rate and nutritional value of TMF feed; and because Bacillus megaterium MYB3 can decompose cellulose and hemicellulose in roughage raw materials, it is conducive to improving the palatability of feed and reducing feed waste. It is of great significance to the livestock industry.
  • the present invention provides the preparation method of full-price mixed fermented feed described in the above technical scheme, comprising the following steps:
  • Concentrate feed raw materials are fermented by lactic acid bacteria to obtain concentrate feed;
  • Roughage raw materials are fermented by Bacillus megaterium MYB3 under alkaline conditions to prepare roughage;
  • the concentrated feed is mixed with the roughage to obtain a complete mixed fermented feed.
  • the concentrated feed and roughage are preferably prepared by referring to the aforementioned methods, which will not be repeated here.
  • Inoculate 1mL of lactic acid bacteria BG-1 (preservation number is CGMCC NO.23493) original bacterial solution into 100mL of MRS medium, and culture it at 37°C and 120r/min for 24h to obtain lactic acid bacteria BG-1 bacterial solution with a viable count of 1.09 ⁇ 10 8 cfu/mL; 35.42g of soybean meal, 8.11g of wheat bran, 244.53g of corn and 11.94g of beef cattle and sheep compound premix (specifically Huahu 4% beef cattle and sheep fattening compound premix feed EP476) were mixed to obtain For the mixed material, distilled water is used to adjust the water content of the mixed material to 60%, inoculate 50mL of lactic acid bacteria BG-1 bacterial liquid into the mixed material, and ferment for 10 days in a constant temperature incubator at 30°C to obtain a refined product with a pH value of 4.37. feed;
  • the concentrated feed and roughage are mixed according to the mass ratio of 5:5 to obtain a total mixed fermented (TMF) feed with a pH value of 6.243.
  • TMF mixed fermented
  • the TMF feed was prepared according to the method of Example 1, except that the mass ratio of the concentrated feed to the roughage was 4:6, and the pH value of the final TMF feed was 6.968.
  • the TMF feed was prepared according to the method of Example 1, except that the mass ratio of the concentrated feed to the roughage was 8:2, and the pH value of the final TMF feed was 4.815.
  • the TMF feed was prepared according to the method of Example 1, except that the mass ratio of the concentrated feed to the roughage was 7:3, and the pH value of the final TMF feed was 5.280.
  • the TMF feed was prepared according to the method of Example 1, except that the mass ratio of the concentrated feed to the roughage was 6:4, and the pH value of the final TMF feed was 5.863.
  • Crude protein Weigh 0.2g of the dried sample, add 2g of catalyst (specifically obtained by mixing copper sulfate and potassium sulfate at a mass ratio of 0.4:6) and 12mL of concentrated sulfuric acid (concentration: 98wt%), and carry out at 400°C Digest and stop until the solution is transparent; transfer the obtained digestion solution to a 100mL volumetric flask after cooling, and use distilled water to make up the volume; take 20mL of the digestion solution for semi-micro distillation, and titrate with 0.1mol/L hydrochloric acid after distillation (GB/ T 6432-2018).
  • catalyst specifically obtained by mixing copper sulfate and potassium sulfate at a mass ratio of 0.4:6
  • concentrated sulfuric acid concentration: 98wt%
  • Ammonia nitrogen Weigh 5g of sample, add 20mL of deionized water, put it in a refrigerator at 4°C for 24 hours, filter it with 4 layers of gauze to obtain the fermentation broth; take 50 ⁇ L of the fermentation broth, add phenol reagent and sodium hypochlorite reagent The mixture was heated in a water bath at 95°C for 5 minutes to develop color, and after cooling, the color was measured at a wavelength of 630 nm.
  • Neutral (acidic) detergent fiber Weigh 0.5g of the dried sample, add 100mL of neutral (acidic) detergent, a few drops of decahydronaphthalene and 0.5g of anhydrous sodium sulfate into a beaker, then put the beaker on The condensing device is placed on an electric furnace, boiled within 5-10 minutes, and kept at a slight boil for 60 minutes; after boiling, use a crucible of known weight to carry out suction filtration while it is hot, and rinse the crucible and filter residue with boiling water and acetone until the filtrate is neutral put the crucible containing the filter residue in an oven at 105°C for 2 hours, then take it out and put it in a desiccator to cool for 30 minutes and weigh it.
  • Fermentation broth preparation Weigh 5g sample, add 20mL deionized water, put it in a 4°C refrigerator for extraction for 24 hours, filter it with 4 layers of gauze to obtain the fermentation broth, pass through a 0.22 ⁇ m filter membrane, and use HPLC to measure the content of organic acids;
  • HPLC high performance liquid chromatography conditions of content determination are as follows:
  • Liquid chromatography column ZORBAX SB-C18, 5 ⁇ m ⁇ 4.6mm ⁇ 150mm;
  • Injection volume 10 ⁇ L.
  • Crude fat is the main substance for improving energy, and the content of crude fat in the TMF feed prepared in Example 1 is 13%.
  • the content of ammoniacal nitrogen may be affected by urea, which is slightly higher, but the effect is not significant.
  • Crude protein is an important basis for the feeding value of TMF feed and is the main bearer of life activities.
  • the content of crude protein in the TMF feed prepared in Example 1 is sufficient to meet daily nutritional needs.
  • Neutral detergent fiber is currently the most effective indicator to reflect the quality of fiber.
  • Acid detergent fiber is the key to determine feed energy, the lower the content, the higher the feeding value of silage.
  • acid detergent fiber is composed of lignin, and lignin is not easily degraded by microorganisms, so it is difficult to be absorbed and utilized by ruminants, and it will also reduce the digestibility of other nutrients.
  • the content of neutral detergent fiber and acid detergent fiber in the TMF feed prepared by Example 1 of the present invention is lower than that before fermentation (the content of neutral detergent fiber before fermentation is 45-55%, and the content of acid detergent fiber is 30-40%) ), thus proving that the addition of cellulolytic bacteria MYB3 effectively degrades cellulose and hemicellulose in corn stalks, and improves the feeding value and fermentation quality of feed.
  • the content and types of organic acids can reflect the quality of fermentation, the most important of which is lactic acid.
  • the organic acid produced by fermented feed can significantly increase the acidity of the feed, reduce the pH value of the feed, and can prevent the feed from mildew and inhibit bacteria, and improve the nutritional value of the feed.
  • the addition of lactic acid bacteria will promote the fermentation of feed.
  • lactic acid bacteria are the main fermentation bacteria, producing lactic acid, acetic acid or butyric acid, etc. However, if the content of acetic acid is too much, the fermentation quality may be reduced, and the content of butyric acid is too high. It will also have a negative impact on the fermented feed.
  • the proportion of butyric acid in the feed decreases, indicating that the reproductive activity of spoilage bacteria, mold and other harmful bacteria is inhibited.
  • the content of acetic acid in the TMF feed prepared in Example 1 of the present invention is relatively low, and does not contain butyric acid, indicating that the quality of the TMF feed is better.

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Abstract

一种调控pH值的全价混合发酵饲料，包括精饲料和粗饲料，所述精饲料为精饲料原料经乳酸菌发酵制备得到，所述粗饲料为粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到；所述全价混合发酵饲料的pH值为6.00～7.00。

Description

一种调控pH值的全价混合发酵饲料及其制备方法 技术领域
本发明涉及饲料发酵技术领域，具体涉及一种调控pH值的全价混合发酵饲料及其制备方法。
背景技术
全混合饲料(TMR)是根据反刍动物不同生理阶段的营养需要量以及饲养目的，按照营养调控技术把青贮、干草等粗饲料切成一定长度，再添加一定比例的精饲料、各种矿物质、维生素等充分搅拌、混合而加工成的一种营养相对平衡的日粮。TMR饲养技术可以有效避免家畜的选择性采食，改善一些原料的适口性；还可以提高干物质采食量，促使家畜均衡采食，有效减少瘤胃疾病的发生。
全价混合发酵(TMF)饲料是指根据反刍动物的营养需要，利用当地饲料资源配制营养均衡的全混合饲料，采用体外特殊发酵过程生产的饲料。TMF技术是进一步完善TMR饲养技术，使饲养方式更适合反刍动物的生理要求。目前乳酸菌是最为普遍的发酵用菌剂，利用乳酸菌发酵可以产生大量有机酸，使TMF饲料的pH值降低至4.2～4.7，从而抑制其他腐败菌的生长，避免了饲料的发霉变质，但是该pH值与瘤胃的pH值(6.2～6.8)相差较大，导致饲料利用率以及营养价值较低。
发明内容
本发明的目的在于提供一种调控pH值的全价混合发酵饲料及其制备方法，本发明提供的全价混合发酵饲料的pH值与瘤胃的最适pH值范围接近，有利于提高全价混合发酵饲料的利用率以及营养价值。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种调控pH值的全价混合发酵饲料，包括精饲料和粗饲料，所述精饲料为精饲料原料经乳酸菌发酵制备得到，所述粗饲料为粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到；所述全价混合发酵饲料的pH值为6.00～7.00。
优选地，所述精饲料的pH值为4.20～4.80，所述粗饲料的pH值为 8.00～9.00。
优选地，所述全价混合发酵饲料的营养指标包括：粗脂肪的质量含量为3～15％，氨态氮的质量含量为5～15％，粗蛋白的质量含量为5～20％，中性洗涤纤维的质量含量为20～50％，酸性洗涤纤维的质量含量为15～30％，乳酸的含量为1000～5000mg/kg。
优选地，所述粗饲料的制备方法包括以下步骤：
将巨大芽孢杆菌MYB3接种于粗饲料原料中，在碱性饲料添加剂提供的碱性条件下进行第一发酵，得到粗饲料。
优选地，所述粗饲料原料包括秸秆。
优选地，所述秸秆包括玉米秸秆、水稻秸秆或小麦秸秆。
优选地，所述碱性饲料添加剂包括尿素、小苏打、碳酸氢铵、硫酸铵或氯化铵。
优选地，所述碱性饲料添加剂以碱性饲料添加剂水溶液形式使用，所述碱性饲料添加剂水溶液的浓度为5～15wt％。
优选地，所述第一发酵在pH值为8.00～9.00的条件下进行。
优选地，所述第一发酵的温度为30～40℃，时间为7～14天。
优选地，所述精饲料的制备方法包括以下步骤：
将乳酸菌接种于精饲料原料中，进行第二发酵，得到精饲料。
优选地，所述精饲料原料包括豆粕、麦麸、玉米与预混料，所述豆粕、麦麸、玉米与预混料的质量比为(30～45)：(6～15)：(240～255)：(9～24)。
优选地，所述乳酸菌为乳酸菌BG-1。
优选地，所述第二发酵的温度为30～40℃，时间为7～14天。
本发明提供了上述技术方案所述全价混合发酵饲料的制备方法，包括以下步骤：
精饲料原料经乳酸菌发酵制备得到精饲料；
粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到粗饲料；
将所述精饲料与所述粗饲料混合，得到全价混合发酵饲料。
本发明提供了一种调控pH值的全价混合发酵饲料，包括精饲料和粗 饲料，所述精饲料为精饲料原料经乳酸菌发酵制备得到，所述粗饲料为粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到；所述全价混合发酵饲料的pH值为6.00～7.00。本发明通过乳酸菌发酵得到精饲料，可为动物机体提供大量营养物质，发酵过程中产生大量有机酸，从而降低了精饲料pH值，有利于抑制病原微生物的繁殖，同时也能够减少蛋白质的分解程度，提高饲料的品质。本发明通过巨大芽孢杆菌MYB3发酵得到粗饲料，所述巨大芽孢杆菌MYB3作为纤维素分解菌，可以有效分解粗饲料原料中的纤维素和半纤维，且所述巨大芽孢杆菌MYB3为碱性微生物，可以在碱性条件下生长繁殖，同时不影响其生理特性，能够在碱性条件下分解纤维素和半纤维素，得到呈碱性的粗饲料。本发明基于所述精饲料和粗饲料得到TMF饲料，所述精饲料和粗饲料能够调控TMF饲料的pH值，使TMF饲料的pH值接近瘤胃的最适pH值(6.2～6.8)，缩短了TMF饲料过瘤胃的时间，有利于提高TMF饲料的利用率以及营养价值。
生物保藏信息
巨大芽孢杆菌(Bacillus megaterium)MYB3，保藏日期为2016年12月07日，保藏地点为中国微生物菌种保藏管理委员会普通微生物中心，具体地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCCNo.13492；
乳酸菌(Lactiplantibacillus plantarum)BG-1，保藏日期为2021年09月27日，保藏地点为中国微生物菌种保藏管理委员会普通微生物中心，具体地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCCNO.23493。
具体实施方式
本发明提供了一种调控pH值的全价混合发酵饲料，包括精饲料和粗饲料，所述精饲料为精饲料原料经乳酸菌发酵制备得到，所述粗饲料为粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到；所述全价混合发酵饲料的pH值为6.00～7.00。
在本发明中，所述全价混合发酵饲料的pH值优选为6.2～6.8。在本发明中，所述全价混合发酵饲料的营养指标包括：粗脂肪的质量含量优选 为3～15％，更优选为10～13％；氨态氮的质量含量优选为5～15％，更优选为10～13.51％；粗蛋白的质量含量优选为5～20％，更优选为11.48～15.50％；中性洗涤纤维的质量含量优选为20～50％，更优选为30～40％；酸性洗涤纤维的质量含量优选为15～30％，更优选为16.67～20.00％；乳酸的含量优选为1000～5000mg/kg，更优选为3000～5000mg/kg。在本发明中，所述全价混合发酵饲料中乙酸含量较低，为100～1500mg/kg；在本发明的实施例中，具体为849.59mg/kg；所述全价混合发酵饲料中不含有丙酸以及丁酸。
在本发明中，所述精饲料的pH值优选为4.20～4.80，更优选为4.37～4.50；所述粗饲料的pH值优选为8.00～9.00，更优选为8.20～8.62。在本发明中，所述精饲料与粗饲料的质量比以保证全价混合发酵饲料的pH值以及营养指标满足上述要求为基准，具体的，所述精饲料与粗饲料的质量比可以为5：5，也可以为4：6。
在本发明中，所述粗饲料的制备方法优选包括以下步骤：
将巨大芽孢杆菌MYB3接种于粗饲料原料中，在碱性饲料添加剂提供的碱性条件下进行第一发酵，得到粗饲料。
在本发明中，所述巨大芽孢杆菌MYB3保藏编号为CGMCC No.13492。在本发明中，所述粗饲料原料优选包括秸秆，所述秸秆优选为玉米秸秆、水稻秸秆或小麦秸秆。在本发明中，所述碱性饲料添加剂优选包括尿素、小苏打、碳酸氢铵、硫酸铵或氯化铵；所述碱性饲料添加剂优选以碱性饲料添加剂水溶液形式使用，所述碱性饲料添加剂水溶液的浓度优选为5～15wt％，更优选为5～10wt％。
本发明优选将粗饲料原料的含水率调整到40～60％，接种巨大芽孢杆菌MYB3菌液，之后采用碱性饲料添加剂水溶液调节发酵体系的pH值为碱性，进行第一发酵，得到粗饲料。本发明优选通过向粗饲料原料中添加蒸馏水来调整含水率满足上述要求；本发明进一步优选将粗饲料原料的含水率调整到60％。本发明优选将粗饲料原料的含水率调整到上述范围，有利于达到更好的发酵效果。在本发明中，所述巨大芽孢杆菌MYB3菌液的活菌数优选为1×10 8cfu/mL；本发明优选将1mL巨大芽孢杆菌MYB3原菌液接种至100mLLB培养基中，在37℃、120r/min条件下培养24h，得到巨大芽孢杆菌MYB3菌液。在本发明中，所述粗饲料原料与巨大芽 孢杆菌MYB3菌液的用量比优选为(100～1000)g：100mL，更优选为200g：100mL。本发明优选将碱性饲料添加剂水溶液喷洒到接种有巨大芽孢杆菌MYB3菌液的粗饲料原料中，使发酵体系的pH值为碱性。本发明对所述碱性饲料添加剂水溶液的用量没有特殊限定，能够保证发酵体系的pH值满足要求即可。
在本发明中，所述第一发酵优选在pH值为8.00～9.00的条件下进行，更优选为8.20～8.80。在本发明中，所述第一发酵的温度优选为30～40℃，更优选为30℃；时间优选为7～20天，更优选为7天。本发明优选将第一发酵的温度以及时间控制在上述范围，有利于达到更好的发酵效果，更好的分解粗饲料原料中纤维素和半纤维素，并同时调控发酵过程中pH值，使最终所得粗饲料的pH值满足上述要求。
在本发明中，所述精饲料的制备方法优选包括以下步骤：
将乳酸菌接种于精饲料原料中，进行第二发酵，得到精饲料。
在本发明中，所述精饲料原料优选包括豆粕、麦麸、玉米与预混料，所述预混料优选为肉牛羊复合预混料；在本发明的实施例中，所述预混料具体为华畜4％肉牛羊育肥复合预混合饲料EP476。在本发明中，所述豆粕、麦麸、玉米与预混料的质量比优选为(30～45)：(6～15)：(240～255)：(9～24)，更优选为(35～40)：(8～10)：(244～250)：(11～18)，进一步优选为35.42：8.11：244.53：11.94。本发明优选将豆粕、麦麸、玉米与预混料混合，得到精饲料原料。在本发明中，所述乳酸菌优选为乳酸菌BG-1，所述乳酸菌BG-1的保藏编号为CGMCC NO.23493。
本发明优选将精饲料原料的含水率调整到40～60％，接种乳酸菌BG-1菌液，进行第二发酵，得到精饲料。本发明优选通过向精饲料原料中添加蒸馏水来调整含水率满足上述要求；本发明进一步优选将精饲料原料的含水率调整到60％。本发明优选将精饲料原料的含水率调整到上述范围，有利于达到更好的发酵效果。在本发明中，所述乳酸菌BG-1菌液的活菌数优选为1.09×10 8cfu/mL；本发明优选将1mL乳酸菌BG-1原菌液接种至100mLMRS培养基中，在37℃、120r/min条件下培养24h，得到乳酸菌BG-1菌液。在本发明中，所述精饲料原料与乳酸菌BG-1菌液的用量比优选为(100～5000)g：50mL，更优选为300g：50mL。
在本发明中，所述第二发酵的温度优选为30～40℃，更优选为30℃；时间优选为7～20天，更优选为10天。本发明优选将第二发酵的温度以及时间控制在上述范围，有利于达到更好的发酵效果。
本发明通过乳酸菌发酵制备得到具有较低pH值的精饲料，通过巨大芽孢杆菌MYB3在碱性条件下发酵制备得到具有较高pH值的粗饲料，其中，巨大芽孢杆菌MYB3在碱性条件下依然可以分解粗饲料原料中的纤维素和半纤维素，通过所述精饲料和粗饲料能够调控TMF饲料的pH值，使TMF饲料的pH值接近瘤胃的最适pH值(6.2～6.8)，缩短了TMF饲料过瘤胃的时间，有利于提高TMF饲料的利用率以及营养价值；而且由于巨大芽孢杆菌MYB3能够分解粗饲料原料中的纤维素和半纤维素，有利于改善饲料的适口性，减少了饲料的浪费，对畜牧行业有着重要的意义。
本发明提供了上述技术方案所述全价混合发酵饲料的制备方法，包括以下步骤：
精饲料原料经乳酸菌发酵制备得到精饲料；
粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到粗饲料；
将所述精饲料与所述粗饲料混合，得到全价混合发酵饲料。
在本发明中，所述精饲料和粗饲料优选参照前述方法制备得到，在此不再赘述。本发明对所述精饲料与粗饲料混合的方式没有特殊限定，能够将二者充分混合即可。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
将1mL乳酸菌BG-1(保藏编号为CGMCC NO.23493)原菌液接种至100mLMRS培养基中，在37℃、120r/min条件下培养24h，得到乳酸菌BG-1菌液，活菌数为1.09×10 8cfu/mL；将35.42g豆粕、8.11g麦麸、244.53g玉米与11.94g肉牛羊复合预混料(具体为华畜4％肉牛羊育肥复合预混合饲料EP476)混合，得到混合物料，采用蒸馏水将所述混合物料 的含水率调整在60％，向所述混合物料中接种50mL乳酸菌BG-1菌液，在30℃恒温培养箱发酵10天，得到pH值为4.37的精饲料；
将1mL巨大芽孢杆菌MYB3(保藏编号为CGMCC No.13492)原菌液接种至100mL LB培养基中，在37℃、120r/min条件下培养24h，得到巨大芽孢杆菌MYB3菌液，活菌数为1×10 8cfu/mL；称取200g玉米秸秆，采用蒸馏水将所述玉米秸秆的含水率调整到60％，之后接种100mL巨大芽孢杆菌MYB3菌液，同时喷洒浓度为5wt％的尿素水溶液，将发酵体系的pH值调节至8.7，在30℃恒温培养箱发酵7天，得到pH值为8.62的粗饲料；
将所述精饲料与粗饲料按照质量比为5：5的比例混合，得到pH值为6.243的全价混合发酵(TMF)饲料。
实施例2
按照实施例1的方法制备TMF饲料，不同之处仅在于所述精饲料与粗饲料的质量比为4：6，最终所得TMF饲料的pH值为6.968。
对比例1
按照实施例1的方法制备TMF饲料，不同之处仅在于所述精饲料与粗饲料的质量比为8：2，最终所得TMF饲料的pH值为4.815。
对比例2
按照实施例1的方法制备TMF饲料，不同之处仅在于所述精饲料与粗饲料的质量比为7：3，最终所得TMF饲料的pH值为5.280。
对比例3
按照实施例1的方法制备TMF饲料，不同之处仅在于所述精饲料与粗饲料的质量比为6：4，最终所得TMF饲料的pH值为5.863。
营养指标检测
以实施例1制备的TMF饲料为样品，对其营养指标进行检测，具体如下：
一、粗脂肪
称取65℃烘干的样品，装入105℃烘干至恒重的滤纸包内，放入抽提瓶内采用石油醚抽提50～70次。取出滤纸包，吹干石油醚，放入105℃烘箱内烘干2h，之后取出放入干燥器内冷却30min称重，滤纸包的减量为 粗脂肪的含量。
二、粗蛋白和氨态氮
粗蛋白：称取0.2g烘干后的样品，加入2g催化剂(具体为硫酸铜与硫酸钾按质量比0.4：6混合得到)和12mL浓硫酸(浓度为98wt％)，在400℃条件下进行消煮，直至溶液透明后停止；将所得消煮液冷却后转入100mL容量瓶，用蒸馏水定容；取20mL消煮液进行半微量蒸馏，蒸馏后用0.1mol/L盐酸进行滴定(GB/T 6432-2018)。
氨态氮：称取5g样品，加入20mL去离子水，放入4℃冰箱中浸提24h，用4层纱布过滤后得到发酵液；取50μL发酵液加入苯酚试剂和次氯酸钠试剂混匀，将所得混合液在95℃水浴中加热显色5min，冷却后在630nm波长下比色。
三、中性洗涤纤维和酸性洗涤纤维
中性(酸性)洗涤纤维：称取0.5g烘干后的样品，加入100mL中性(酸性)洗涤剂和数滴十氢化萘及0.5g无水硫酸钠置于烧杯中，之后将烧杯套上冷凝装置于电炉上，在5～10min内煮沸，并持续保持微沸60min；煮沸完毕后，趁热用已知重量的坩埚进行抽滤，并用沸水和丙酮冲洗坩埚和滤渣，直至滤液呈中性为止；将盛放有滤渣的坩埚置于105℃烘箱中烘干2h，之后取出放入干燥器内冷却30min称重。
四、有机酸
发酵液制备：称取5g样品，加入20mL去离子水，放入4℃冰箱中浸提24h，用4层纱布过滤后得到发酵液，过0.22μm滤膜后利用HPLC测定有机酸含量；有机酸含量测定的高效液相色谱条件如下：
液相色谱柱：ZORBAX SB-C18，5μm×4.6mm×150mm；
柱温：35℃；
流动相：乙腈25mL+0.1％(w/v)的磷酸水溶液975mL；
流速：0.8mL/min；
检测波长：UV 210nm；
进样量：10μL。
营养指标检测结果如表1所示。
表1实施例1制备的TMF饲料的营养指标
营养指标 数据
粗脂肪 13％
氨态氮 13.51％
粗蛋白 11.48％
中性洗涤纤维 40.00％
酸性洗涤纤维 16.67％
乳酸 3711.54mg/kg
乙酸 849.59mg/kg
丙酸 0mg/kg
丁酸 0mg/kg
粗脂肪是提高能量的主要物质，本实施例1制备的TMF饲料中粗脂肪含量为13％。氨态氮的含量可能受到尿素的影响，略微较高，但影响不大。粗蛋白为TMF饲料饲用价值的重要基础，是生命活动的主要承担者，本实施例1制备的TMF饲料中粗蛋白含量足够满足日常营养需求。
中性洗涤纤维是目前反映纤维质量好坏的最有效指标。酸性洗涤纤维是决定饲料能量的关键，含量越低，青贮饲料饲用价值越高。但是酸性洗涤纤维是由木质素构成，而木质素不易被微生物降解，从而很难被反刍动物吸收利用，同时还会降低其他养分的消化率。本发明实施例1制备的TMF饲料中的中性洗涤纤维和酸性洗涤纤维含量较发酵前都有所降低(发酵前中性洗涤纤维含量为45～55％，酸性洗涤纤维含量为30～40％)，由此证明添加了纤维素分解菌MYB3有效降解了玉米秸秆中的纤维素和半纤维素，提高了饲料的饲用价值以及发酵品质。
有机酸含量及其种类可以反映发酵品质的好坏，其中最主要的是乳酸。发酵饲料产生的有机酸可以显著提高饲料的酸度，使饲料的pH值下降，并且可以起到防止饲料霉变、抑菌等作用，提高饲料的营养价值。乳酸菌的添加会促进饲料发酵，发酵过程中乳酸菌为主要发酵菌，生成乳酸、乙酸或丁酸等，但乙酸含量过多时可能降低发酵品质，丁酸含量过高也会对发酵饲料产生负面影响，饲料中丁酸的比例降低，说明腐败菌、霉菌等 有害菌的繁殖活性受到抑制。本发明实施例1制备的TMF饲料中乙酸含量较低，且不含有丁酸，说明所述TMF饲料品质较好。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (15)

  1. 一种调控pH值的全价混合发酵饲料，包括精饲料和粗饲料，所述精饲料为精饲料原料经乳酸菌发酵制备得到，所述粗饲料为粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到；所述全价混合发酵饲料的pH值为6.00～7.00。
  2. 根据权利要求1所述的全价混合发酵饲料，其特征在于，所述精饲料的pH值为4.20～4.80，所述粗饲料的pH值为8.00～9.00。
  3. 根据权利要求1或2所述的全价混合发酵饲料，其特征在于，所述全价混合发酵饲料的营养指标包括：粗脂肪的质量含量为3～15％，氨态氮的质量含量为5～15％，粗蛋白的质量含量为5～20％，中性洗涤纤维的质量含量为20～50％，酸性洗涤纤维的质量含量为15～30％，乳酸的含量为1000～5000mg/kg。
  4. 根据权利要求1所述的全价混合发酵饲料，其特征在于，所述粗饲料的制备方法包括以下步骤：
    将巨大芽孢杆菌MYB3接种于粗饲料原料中，在碱性饲料添加剂提供的碱性条件下进行第一发酵，得到粗饲料。
  5. 根据权利要求4所述的全价混合发酵饲料，其特征在于，所述粗饲料原料包括秸秆。
  6. 根据权利要求5所述的全价混合发酵饲料，其特征在于，所述秸秆包括玉米秸秆、水稻秸秆或小麦秸秆。
  7. 根据权利要求4所述的全价混合发酵饲料，其特征在于，所述碱性饲料添加剂包括尿素、小苏打、碳酸氢铵、硫酸铵或氯化铵。
  8. 根据权利要求7所述的全价混合发酵饲料，其特征在于，所述碱性饲料添加剂以碱性饲料添加剂水溶液形式使用，所述碱性饲料添加剂水溶液的浓度为5～15wt％。
  9. 根据权利要求4所述的全价混合发酵饲料，其特征在于，所述第一发酵在pH值为8.00～9.00的条件下进行。
  10. 根据权利要求4所述的全价混合发酵饲料，其特征在于，所述第一发酵的温度为30～40℃，时间为7～14天。
  11. 根据权利要求1所述的全价混合发酵饲料，其特征在于，所述精饲料的制备方法包括以下步骤：
    将乳酸菌接种于精饲料原料中，进行第二发酵，得到精饲料。
  12. 根据权利要求11所述的全价混合发酵饲料，其特征在于，所述精饲料原料包括豆粕、麦麸、玉米与预混料，所述豆粕、麦麸、玉米与预混料的质量比为(30～45)：(6～15)：(240～255)：(9～24)。
  13. 根据权利要求1或11所述的全价混合发酵饲料，其特征在于，所述乳酸菌为乳酸菌BG-1。
  14. 根据权利要求11所述的全价混合发酵饲料，其特征在于，所述第二发酵的温度为30～40℃，时间为7～14天。
  15. 权利要求1～14任一项所述全价混合发酵饲料的制备方法，包括以下步骤：
    精饲料原料经乳酸菌发酵制备得到精饲料；
    粗饲料原料在碱性条件下经巨大芽孢杆菌MYB3发酵制备得到粗饲料；
    将所述精饲料与所述粗饲料混合，得到全价混合发酵饲料。
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CN105104857A (zh) * 2015-08-28 2015-12-02 方标嵩 一种喂养肉用野猪的发酵饲料及其生产方法
CN107365718A (zh) * 2017-04-27 2017-11-21 延边大学 巨大芽孢杆菌myb3及其在秸秆发酵饲料中的应用
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CN105104857A (zh) * 2015-08-28 2015-12-02 方标嵩 一种喂养肉用野猪的发酵饲料及其生产方法
CN107365718A (zh) * 2017-04-27 2017-11-21 延边大学 巨大芽孢杆菌myb3及其在秸秆发酵饲料中的应用
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