WO2024120068A1 - 基于全麦糟的发酵饲料及其制备方法 - Google Patents

基于全麦糟的发酵饲料及其制备方法 Download PDF

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WO2024120068A1
WO2024120068A1 PCT/CN2023/128347 CN2023128347W WO2024120068A1 WO 2024120068 A1 WO2024120068 A1 WO 2024120068A1 CN 2023128347 W CN2023128347 W CN 2023128347W WO 2024120068 A1 WO2024120068 A1 WO 2024120068A1
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grains
fermented feed
spent grains
lactic acid
fermentation
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PCT/CN2023/128347
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English (en)
French (fr)
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徐楠
尹花
余俊红
黄淑霞
闫鹏
刘佳
秦青青
王剑锋
胡淑敏
王书谦
刘明丽
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青岛啤酒股份有限公司
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Priority to EP23899649.0A priority Critical patent/EP4467003A1/en
Publication of WO2024120068A1 publication Critical patent/WO2024120068A1/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
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • 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
    • A23K10/38—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 from distillers' or brewers' waste
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present application belongs to the technical field of fermented feed, and in particular, relates to a fermented feed based on whole wheat grains and a preparation method thereof.
  • Brewer's grains are the main by-product in the beer brewing industry, accounting for about 8-10% of beer production (wet weight, water content of about 80%). China's beer production has ranked first in the world for 20 consecutive years. Taking the 2021 beer production of 35.62 million kiloliters as an example, the output of brewer's grains is about 3 million tons, which is a very large volume. Brewer's grains are the product of the saccharification process in beer brewing. Barley malt and auxiliary materials such as rice are saccharified in the saccharification pot. The starch is decomposed into maltose to form liquid wort. The saccharified mash is separated into solid and liquid in the filter tank.
  • the wort enters the fermentation tank and yeast is added to ferment, and finally beer is produced.
  • the insoluble residue mainly composed of husks is brewer's grains. Unlike white wine lees, there is no yeast and alcohol in brewer's grains, so it is also called wheat grains in the industry. Wheat spent grains have high water content and are easy to spoil, making them difficult to store for a long time and inconvenient to transport.
  • most manufacturers in my country sell them directly to farmers as feed at low prices. A few manufacturers dry them and use them as feed raw materials, and some even discharge them directly as waste, wasting resources and seriously damaging the ecological environment, especially in the hot summer season.
  • Spent grains contain a large amount of dietary fiber and protein, accounting for about 50% and 30% respectively. In addition, they also contain phenols, vitamins, mineral elements and other substances. They are very nutritious and are high-quality feed resources. Spent grains are sold directly for animal feeding without processing, which has low added value, is difficult for animals to digest, and has low utilization rate. In recent years, people's demand for meat, eggs and milk has increased dramatically, which has promoted the vigorous development of the breeding industry and the feed industry. The market demand for the feed industry is large, and there is a serious shortage of protein feed. The commonly used daily ration soybean meal is overly dependent on soybean imports, and the price of feed has risen rapidly. It is urgent to find new feed resources. Spent grains are rich in nutrients and large in volume, so they are a good choice. How to turn waste into treasure, make rational use of spent grains, increase added value, and effectively solve environmental pollution and resource waste problems has become a major issue facing the beer industry.
  • reports on the use of spent grains to make fermented feed generally adopt two methods: one is to directly add wet spent grains to a dozen or dozens of feed raw materials such as corn flour, bran, etc., and then add some microbial agents or enzyme preparations for fermentation; the other is to dry the wet spent grains first, then add them to a dozen or dozens of feed raw materials, add water to adjust, and then add some microbial agents or enzyme preparations for fermentation.
  • the above processing methods have some problems in production technology, and are also difficult problems that need to be solved in the feed industry.
  • wet spent grains have a high water content and are rich in nutrients, they will be contaminated by harmful microorganisms in the air if left for a few hours in the hot summer. They are very easy to spoil, and the nutrients are destroyed and toxins are produced. If the distance between the brewery and the feed processing plant is far, or the amount of wet spent grains is large, and the feed production capacity is limited, there will be great risks, large losses, and adverse effects on animals.
  • the first way is to quickly mix the wet spent grains, and the second way is to quickly dry the wet spent grains. If the processing is not timely, it will lead to corruption. After drying, the shelf life of the spent grains can be extended, and it is not limited by the time of later mixing and processing, but the cost of drying the whole is high, which will increase by 600-1000 yuan per ton or even higher.
  • malt grains are used to make feed or fermented feed, they are mostly used as one of a dozen or dozens of raw materials, and the amount added is very small. It is not considered a feed with malt grains as the main body in the true sense. Due to the low starch content in malt grains (about 3-6%), in the process of preparing fermented feed from malt grains, starch-containing nutrients such as corn flour are often added to supplement the carbon source for animals, but the price of corn flour is relatively high, which leads to increased production costs. If some low-cost substances are added at will, although the cost and moisture content are reduced, the nutritional value of the malt grains themselves is also lowered, and the quality is unstable and difficult to control. The malt grains themselves contain 10-15% cellulose and 10-15% hemicellulose, and this part of the nutrients is not well utilized.
  • spent grains are mostly used to feed animals directly, which is relatively extensive and lacks systematic experimental data.
  • spent grains, especially whole spent grains are rarely used in animal experiments as fermented feed, which limits the application scope of spent grains as animal feed.
  • the present application proposes a fermented feed based on whole wheat grains and a preparation method thereof.
  • the first aspect of the present application provides a method for preparing a fermented feed, comprising a fresh-keeping treatment step, a baking step, an aerobic fermentation step, and an anaerobic fermentation step;
  • the fresh-keeping treatment step includes: adding lactic acid bacteria to the wet grains immediately after taking the wet grains;
  • the aerobic fermentation step includes: adding mixed bacteria to the spent grains obtained in the baking step; species and enzyme preparation, aerobic fermentation, the mixed bacteria species include yeast and bacillus, the enzyme preparation includes cellulose complex enzyme, protease and xylanase;
  • the anaerobic fermentation step comprises: adding lactic acid bacteria to the spent grains obtained by the aerobic fermentation step, and performing anaerobic fermentation to obtain fermented feed.
  • the baking step includes: dehydrating the spent grains obtained through the preservation step until the moisture content of the spent grains is 60-70%, and then baking at 55-60° C. until the moisture content of the spent grains is 40-50%.
  • lactic acid bacteria culture solution is added to the wet grains, the amount of the lactic acid bacteria culture solution added is 0.5-1% of the weight of the wet grains, and the concentration of the lactic acid bacteria culture solution is not less than 10 8 cells/mL.
  • a mixed strain culture solution is added to the spent grains obtained through the baking step, and the added amount of the mixed strain culture solution accounts for 10-20% of the dry weight of the spent grains, and the mixed strains are composed of the yeast and the bacillus in a ratio of 1:1, and the concentration of the yeast and the bacillus culture solution is not less than 10 8 cells/mL.
  • the enzyme preparation in the aerobic fermentation step, is added in an amount of 1-3% of the dry weight of the spent grains, and the enzyme preparation is composed of the cellulose complex enzyme, the protease and the xylanase in a ratio of 5:3:2, and the enzyme activities of the cellulose complex enzyme, the protease and the xylanase are greater than 100,000 U/g.
  • the aerobic fermentation in the aerobic fermentation step includes: adding the spent grains to which the mixed bacteria and the enzyme preparation are added into a one-way valve anaerobic fermentation bag, and aerobic fermenting at 28-32° C. for 1-3 days.
  • a lactic acid bacteria culture solution is added to the spent grains obtained by the aerobic fermentation step, the amount of the lactic acid bacteria culture solution added is 10-20% of the dry weight of the spent grains, and the concentration of the lactic acid bacteria culture solution is not less than 10 8 cells/mL.
  • the anaerobic fermentation in the anaerobic fermentation step comprises anaerobic fermentation at 35-38° C. for 4-6 days.
  • the second aspect of the present application provides a fermented feed based on whole wheat grains, which is prepared by the fermented feed preparation method described in any of the above technical solutions.
  • the number of viable bacteria is ⁇ 10 8 /mL
  • the crude protein is ⁇ 30%
  • the neutral detergent fiber is 28% ⁇ 40%
  • the acetic acid is 0.5% ⁇ 2.0%
  • the lactic acid is 3% ⁇ 5%
  • the pH is 3.5 ⁇ 4.5.
  • the method for preparing fermented feed avoids the risk of contamination by harmful bacteria by limiting the preservation treatment step, thereby achieving the purpose of preserving wet grains and laying the foundation for the subsequent fermentation treatment process; by limiting the aerobic fermentation step and the anaerobic fermentation step, it not only provides a carbon source for beneficial bacteria, but also makes the fermented feed have the function of prebiotics.
  • the fermented feed is used for animal feeding, it can improve the immune ability of the animal and greatly improve the quality of the fermented feed.
  • the fermented feed provided in at least one embodiment of the present application is obtained by fermenting whole wheat grains, can be used for animal feeding without adding any additional substances, and is functional and beneficial to improving animal immunity; in addition, the feed has good feed palatability and digestibility of nutrients. Based on the nutrition of wheat grains and the digestion or milk production mechanism of animals, the nutritional value and effectiveness of whole wheat grains fermented feed are confirmed.
  • Figure 1 is a diagram of dairy cows eating fermented feed
  • Fig. 2 is a diagram of the culture medium for preparing samples and ruminants
  • FIG3 is a graph showing the bionic digestibility of 0# malt grains and 2# whole malt grains fermented feed in a monogastric animal (duck);
  • FIG4 is a graph showing the bionic digestibility of 0# malt grains and 2# whole malt grains fermented feed in monogastric animals (chickens);
  • Figure 5 is a bionic digestibility diagram of monogastric animals (pigs) of malt grains 0# and whole malt grains fermented feed 2#.
  • the present application provides a method for preparing fermented feed, comprising a fresh-keeping treatment step, a baking step, an aerobic fermentation step and an anaerobic fermentation step;
  • the fresh-keeping treatment step includes: adding lactic acid bacteria to the wet grains immediately after taking the wet grains;
  • the aerobic fermentation step comprises: adding mixed bacteria and enzyme preparation to the spent grains obtained in the baking step, and aerobic fermentation, wherein the mixed bacteria include yeast and bacillus, and the enzyme preparation includes cellulose complex enzyme, protease and xylanase;
  • the anaerobic fermentation step comprises: adding lactic acid bacteria to the spent grains obtained by the aerobic fermentation step, and performing anaerobic fermentation to obtain fermented feed.
  • the above preparation method processes the beer brewing by-product malt grains into fermented feed, which can not only greatly increase the added value of the by-products and alleviate the shortage of protein resources in my country, but also increase the shelf life of wet malt grains, prevent the malt grains from becoming corrupt and smelly, reduce environmental pollution, and increase animal palatability and improve nutritional value.
  • the digestibility and utilization rate of food quality are of great significance.
  • the present application quickly adds lactic acid bacteria during or after taking the wet spent grains from the distiller's grains station, that is, before harmful bacteria are produced in the wet spent grains.
  • the presence of a small amount of lactic acid bacteria can form a dominant bacterial community, thereby avoiding the risk of contamination by harmful bacteria.
  • the lactic acid bacteria is Pediococcus acidilactici, which produces acid and bacteriocin, has a wide antibacterial spectrum, inhibits the growth of harmful bacteria, and regulates the gastrointestinal flora.
  • the wet spent grains are dehydrated and dried by the baking step.
  • the baking step includes: dehydrating the spent grains obtained by the fresh-keeping treatment step until the water content of the spent grains is 60-70%, and then baking at 55-60°C until the water content of the spent grains is 40-50%. Compared with drying all the wet spent grains, the cost is significantly reduced.
  • the technical solution specifically defines that the wet spent grains are first dehydrated to a water content of 60-70% by using a dehydrating device, and then baked at 55-60°C until the water content of the spent grains is 40-50%. The reason is that this technical means can effectively reduce costs compared with pure drying means.
  • the present application uses malt grains as the single raw material, makes full use of 10-15% cellulose and 10-15% hemicellulose in malt grains, adds cellulose complex enzyme and xylanase, degrades them into glucose, xylose, arabinose, etc., which not only provides a carbon source, but also has the function of prebiotics, activates beneficial intestinal bacteria and promotes their growth, and improves the body's immune ability.
  • the enzyme preparation has a small amount of addition, is easy to operate, and significantly reduces costs.
  • this application screened the beneficial bacteria and enzyme preparations suitable for wheat spent grains fermentation, determined the optimal formula of each based on the mixture design, studied the influence of key fermentation processes on the fermented wheat spent grains indicators, determined the optimal process parameters for wheat spent grains fermentation based on the five-factor experiment, and based on the wheat spent grains raw materials and animal digestion mechanism, formed a set of wheat spent grains fermentation technology system covering bacterial agent formula, enzyme preparation formula and key process parameters, which greatly improved the quality of fermented wheat spent grains.
  • the technical solution of the present application also defines the fermentation process as aerobic fermentation followed by anaerobic fermentation.
  • yeast and Bacillus are aerobic bacteria or facultative anaerobes. Under aerobic conditions, yeast and Bacillus grow rapidly, produce proteases and flavor substances, and quickly consume free oxygen in the fermentation environment to form a hypoxic or anaerobic environment; then anaerobic lactic acid bacteria are added, and the anaerobic environment formed before is suitable for their growth, producing organic acids such as lactic acid and acetic acid and antibacterial substances, lowering the pH value, and inhibiting the growth of pathogenic bacteria.
  • a lactic acid bacteria culture solution is added to the wet grains, the amount of the lactic acid bacteria culture solution added is 0.5-1% of the weight of the wet grains, and the concentration of the lactic acid bacteria culture solution is not less than 10 8 cells/mL.
  • This technical solution specifically limits the amount of lactic acid bacteria added in the fresh-keeping treatment step, because it not only ensures that the content of lactic acid bacteria in the treated grains is not less than 10 5 cells/mL, but also controls the lactic acid bacteria.
  • the cost of adding is that as long as the lactic acid bacteria form the dominant bacteria and play a role in temporarily inhibiting harmful bacteria.
  • a mixed bacterial culture solution is added to the spent grains obtained in the baking step, and the added amount of the mixed bacterial culture solution accounts for 10-20% of the dry grain weight (i.e., the dry weight of spent grains), and the mixed bacterial species are composed of the yeast and the bacillus in a ratio of 1:1, and the concentration of the yeast and the bacillus culture solution is not less than 10 8 /mL.
  • This technical solution specifically limits the amount of mixed bacterial species added in the aerobic fermentation step, because it ensures that the number of live bacteria of the mixed bacteria after the spent grains fermentation is not less than 10 8 /mL, produces sufficient fermentation products, and controls costs.
  • the addition amount of the mixed strains can also be 12%, 14%, 16%, 18% and any point value within the range; the technical solution also specifically defines that the mixed strains are composed of yeast and bacillus, the yeast is optionally brewer's yeast, and the bacillus is optionally bacillus subtilis, wherein the yeast produces fragrance, increases palatability, consumes free oxygen, promotes the growth of beneficial bacteria, and synthesizes a variety of enzymes; Bacillus subtilis produces spores, has strong stress resistance, consumes free oxygen, promotes the growth of beneficial bacteria, and synthesizes a variety of enzymes. The synthesized enzymes can decompose the macromolecular substances in the malt grains and provide nutrition and prebiotics for the beneficial bacteria.
  • the amount of the enzyme preparation added is 1-3% of the dry weight of the spent grains, and the enzyme preparation is composed of the cellulose complex enzyme, the protease and the xylanase in a ratio of 5:3:2, and the enzyme activities of the cellulose complex enzyme, the protease and the xylanase are greater than 100,000 U/g.
  • This technical solution specifically limits the amount of enzyme preparation added, because the amount of enzyme added can degrade the cellulose, hemicellulose and protein in the spent grains to generate enough glucose, protein peptides, xylooligosaccharides, etc., provide carbon source and nitrogen source nutrition and prebiotics for microorganisms, and control costs.
  • the amount of the enzyme preparation added can also be 2%.
  • the enzyme preparation is composed of cellulose complex enzyme, protease and xylanase, and the protease can be optionally acidic protease, wherein the enzyme preparation is added to decompose arabinoxylan to obtain extractable arabinoxylan, which has a certain prebiotic effect.
  • cellulose complex enzyme is used to degrade macromolecular cellulose to generate glucose, providing an available carbon source for beneficial bacteria
  • acidic protease is used to degrade macromolecular protein to generate polypeptides and amino acids, providing an available nitrogen source for beneficial bacteria.
  • the acidic protease is selected because a small amount of lactic acid bacteria is added to preserve the sorghum grains in order to prevent the contamination of harmful microorganisms and cause rancidity during the pretreatment of the sorghum grains. Lactic acid bacteria produce acid, and the sorghum grains are acidic; xylanase is used to degrade arabinoxylan to generate a certain amount of xylo-oligosaccharides and xylose with prebiotic effects; after the above enzymes are added in the aerobic fermentation step, The enzyme reaction is carried out in the aerobic fermentation stage (28-32°C aerobic fermentation for 1-3 days), and also in the subsequent anaerobic fermentation stage (35-38°C anaerobic fermentation for 4-6 days).
  • the aerobic fermentation of the aerobic fermentation step includes: adding the spent grains to which the mixed strains and the enzyme preparation are added to a one-way valve anaerobic fermentation bag, and aerobic fermentation at 28-32°C for 1-3 days.
  • the aerobic fermentation of the present technical solution is to first dehydrate the spent grains and then bake them to obtain spent grains with a water content of 40-50% and then perform solid-state fermentation.
  • the present technical solution adopts solid-state fermentation. Compared with liquid fermentation, solid-state fermentation has lower cost, lower mechanization requirements, higher fermentation product concentration, lower pressure, and lower energy consumption.
  • a lactic acid bacteria culture solution is added to the spent grains fermented in the aerobic fermentation step, and the amount of the lactic acid bacteria culture solution added accounts for 10-20% of the dry weight of the spent grains, and the concentration of the lactic acid bacteria culture solution is not less than 10 8 /mL.
  • This technical solution further limits the amount of lactic acid bacteria added in the anaerobic fermentation step, because it ensures that the number of live lactic acid bacteria after the spent grains fermentation is not less than 10 8 /mL, produces sufficient fermentation products, and controls costs. It is understandable that the amount of lactic acid bacteria added can also be 12%, 14%, 16%, 18% and any point value within the range.
  • the anaerobic fermentation step includes anaerobic fermentation at 35-38° C. for 4-6 days.
  • the technical solution further limits the fermentation conditions of anaerobic fermentation, because the lactic acid bacteria can grow and reproduce at a suitable temperature, produce enough fermentation products within a suitable time, minimize the fermentation cycle, and reduce costs.
  • the present application provides a fermented feed based on whole wheat grains, which is prepared by the preparation method of the fermented feed described in any of the above technical solutions.
  • the fermented feed is obtained by fermenting whole wheat grains, can be used for animal feeding without adding any additional substances, and is functional and beneficial to improving animal immunity.
  • the results show that the fermented feed based on whole wheat grains of the present application significantly increases the feed intake of animals, improves the palatability of feed and the digestibility of nutrients, and based on the nutrition of wheat grains and the digestion or milk production mechanism of animals, the nutritional value and effectiveness of the fermented feed based on whole wheat grains are confirmed.
  • the fermented feed prepared by the above preparation method has an obvious and pleasant fermentation sour aroma, without moldy smell and other peculiar smell, is softer and looser than the spent grains before fermentation, the husk becomes softer, and the palatability is significantly improved.
  • the number of viable bacteria is ⁇ 108 /mL
  • crude protein is ⁇ 30%, 28% ⁇ neutral detergent fiber ⁇ 40%, 0.5% ⁇ acetic acid ⁇ 2.0%, 3% ⁇ lactic acid ⁇ 5%, 3.5 ⁇ pH ⁇ 4.5.
  • the bacterial liquid was expanded according to the weight of the wet grains. 5 kg of wet grains were taken from the brewery's grains station. According to the total amount of bacterial liquid added being 10% of the weight of the wet grains and the three bacterial liquids each accounting for about 1/3, it was roughly calculated that the three bacterial liquids needed to be expanded to about 200 mL.
  • Turbidimetric method Take 200 ⁇ L of the mixed bacterial solution and place it in an ELISA plate. Use the corresponding blank liquid culture medium as a control and measure the optical density at 600 nm. The requirement is met only when the optical density is greater than 2.0.
  • the results of the turbidity method and the dilution plate method are highly correlated.
  • the turbidity method is simple and quick, but relatively rough.
  • the dilution plate method is accurate, but the method is cumbersome and lagging. The two methods complement each other.
  • the turbidity method is used to determine the number of viable bacteria in the culture suspension
  • the dilution plate method is used to determine the number of viable bacteria in the whole grain fermented feed.
  • OD Optical Density
  • Table 1 The number of viable bacteria in three bacterial suspensions (turbidimetric method)
  • the dry weight of the baked spent grains was calculated to be 825 g. 10% (82 mL) of saccharomyces cerevisiae and Bacillus subtilis culture solution were added to the baked spent grains at a ratio of 1:1. The actual addition amount was calculated based on the OD value of the culture solution, 40 mL of saccharomyces cerevisiae and 42 mL of Bacillus subtilis, and the mixture was stirred evenly.
  • the prepared whole grain spent grain fermented feed was compared with the initial unfermented grain spent grain and commercially available fermented feed, as shown in Table 2. All indicators are based on dry weight.
  • Lactic acid bacteria produce fatty acids, and the pH value decreases, which can effectively inhibit harmful bacteria.
  • Short-chain volatile fatty acids are involved in the regulation of feeding, among which acetic acid is a precursor for milk fat synthesis.
  • Protein is the main nutrient required by animals.
  • Neutral detergent fiber and acid detergent fiber are used to evaluate dietary fiber nutrition. Too high a content is not conducive to animal digestion and affects palatability.
  • the whole grain spent grain fermented feed had higher acetic acid, lactic acid and crude protein contents, and lower neutral and acid detergent fiber contents.
  • the whole grains fermented feed of the present application is superior to unfermented grains and two commercially available fermented feeds in all indicators.
  • the whole grain fermented feed prepared in this application, unfermented grain, and 3 commercially available fermented feeds were used for feeding experiments on dairy cows, as shown in Figure 1, to evaluate the application effect of the whole grain fermented feed on animals and investigate the palatability.
  • the 5 samples were unfermented grain 1#, whole grain fermented feed 2#, and commercially available fermented feeds 3-5#, each weighing about 1 kg.
  • the whole grain grain fermented feed of the present application has a pleasant sour aroma and is eaten the fastest by cows, indicating that cows like it the most and it has the best palatability.
  • 3# is fermented soybean meal, which is one of the daily rations for dairy cows. After fermentation, the aroma is increased and the palatability is improved. Unfermented grain grains are also feed for dairy cows, but they have a sour smell and a high water content, so cows don’t like them too much.
  • 4# is a fermented feed containing grain grains, but the proportion of grain grains is very small, and it is mainly silage.
  • 5# fermented feed does not contain grain grains, but contains bran, corn stalks, etc., and has a slight ammonia smell.
  • the whole grains fermented feed prepared in this application was subjected to in vitro animal bionic evaluation tests together with unfermented grains and two commercially available fermented feeds.
  • the pH of the fermentation medium was adjusted to 7.0-7.3, and the fermentation medium and rumen fluid were mixed in a ratio of 2:1 and poured into a 2 L beaker, placed in a 39°C water bath, and stirred at a constant speed to prepare an artificial culture medium.
  • Nutrient digestibility (nutrient content before reaction - nutrient content after reaction) ⁇ nutrient content before reaction ⁇ 100%
  • the digestibility of commercially available fermented feed 2 is relatively low, and its fiber content is relatively high, which reduces its digestibility. It is speculated that the addition of high-fiber raw materials during the preparation process is not conducive to the digestion and utilization of animals.
  • the SDS-III monogastric animal bionic digestion system was used to automatically simulate the gastrointestinal digestion process of pigs and poultry, and the differences in digestion indicators between unfermented grains 0# and whole grains fermented feed 2# were analyzed. The results are shown in Figures 3-5.
  • the SDS-III monogastric animal bionic digestion system consists of a simulated digester and a control system.
  • the inside of the dialysis bag in the simulated digester is regarded as the internal environment of the stomach, small intestine, and large intestine (digestive environment), and the outside of the dialysis bag is regarded as the capillary body fluid environment (absorption environment).
  • Configuration software technology is used to enter the buffer automatic input-emptying-switching system, digestive fluid automatic secretion system, hydrolyzate automatic cleaning system, constant temperature control system, mixing intensity automatic control system, electronic component signal detection system and other modules in the simulated digester through computer program control to achieve full automatic simulation of the gastrointestinal digestion process of pigs and poultry.
  • the rumen degradation rate of dry matter can reflect the ease of digestion of feed in the rumen, and is also an important factor affecting the DM intake of dairy cows.
  • the results showed that the rumen degradation rate and effective degradation rate (ED) of whole grains fermented feed increased significantly over time and were significantly higher than those of unfermented grains. Its DM was mainly degraded within 36 hours, and the degradation rate of 48 hours was about 70%.
  • the rumen degradation rate and degradation parameters of crude protein are shown in Table 5.
  • the degradation of crude protein (CP) in the rumen is mainly determined by its retention time in the rumen and the difficulty of degradation.
  • the degradation of CP in the rumen is also directly related to its internal structure composition, non-protein nitrogen (NPN) and true protein content, the physical and chemical properties of true protein, the presence of cell wall inert barriers and anti-nutritional factors.
  • NPN non-protein nitrogen
  • true protein content the physical and chemical properties of true protein
  • the presence of cell wall inert barriers and anti-nutritional factors The results of this experiment showed that the rumen degradation rate, a value and ED value of CP in whole wheat grain fermented feed increased significantly. It may be that the reduction in DM content relatively increased the concentration of proteolytic enzymes in the rumen, resulting in an increase in CP degradation rate. It may also be because the fermentation process can increase the NPN and soluble protein content, increase the a value of CP in the feed,
  • the rumen degradation rate and degradation parameters of neutral detergent fiber of unfermented spent grains and whole spent grains fermented feed are shown in Table 6.
  • the rumen degradation rate of NDF is an important indicator of the nutritional value of feed, and the composition of NDF will affect the rumen degradation rate of NDF.
  • the rumen degradation rate of NDF of whole spent grains fermented feed at each time point increased significantly. It may be that the NDF and DM content of spent grains decreased after microbial fermentation, resulting in an increase in the concentration of cellulase and hemicellulase that decompose NDF, thereby increasing the rumen degradation rate and ED value of NDF.

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Abstract

一种基于全麦糟的发酵饲料，其制备方法包括保鲜处理步骤、烘制步骤、好氧发酵步骤以及厌氧发酵步骤；所述保鲜处理步骤包括：取湿麦糟后立即向所述湿麦糟中添加乳酸菌；所述好氧发酵步骤包括：向经过所述烘制步骤得到的麦糟中加入混合菌种和酶制剂，好氧发酵，所述混合菌种包括酵母菌和芽孢杆菌，所述酶制剂包括纤维素复合酶、蛋白酶以及木聚糖酶；所述厌氧发酵步骤包括：向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌，厌氧发酵，得到发酵饲料。该方法可避免麦糟容易变质、麦糟养分无法充分利用、发酵技术不完善的问题。

Description

基于全麦糟的发酵饲料及其制备方法
本申请要求在2022年12月28日提交中国专利局、申请号为202211693065.X、发明名称为“一种基于全麦糟的发酵饲料及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请属于发酵饲料技术领域，尤其涉及一种基于全麦糟的发酵饲料及其制备方法。
背景技术
啤酒糟是啤酒酿造工业中的主要副产物，约占啤酒产量的8-10％(湿重，含水量80％左右)。中国的啤酒产量己连续20年位居世界第一，以2021年啤酒产量3562万千升为例计算，啤酒糟的产量约300万吨，体量非常大。啤酒糟是啤酒酿造中糖化工序的产物，大麦芽与辅料大米等在糖化锅中进行糖化，淀粉分解成麦芽糖形成液体麦汁，糖化醪液在过滤槽中进行固液分离，麦汁进入发酵罐添加酵母后发酵，最终生成啤酒，而以皮壳为主的不溶性残渣则是啤酒糟。和白酒糟不同，啤酒糟中没有酵母和酒精，因此行业内也称其为麦糟。麦糟因其含水率高，容易腐败，存在不宜长期贮藏、不便于运输的问题，目前我国大多数厂家以低价直接将麦糟出售给农户作饲料，少数厂家将其烘干作饲料原料，有的甚至直接当废物排放，浪费资源的同时严重破坏了生态环境，尤其在夏季高温季节。
麦糟含有大量的膳食纤维和蛋白质，分别约占50％和30％，另外还含有酚类、维生素、矿物元素等物质，营养非常丰富，是优质的饲料资源。麦糟不经过加工直接出售用于饲喂动物，附加值低，且动物难以消化，利用率低。近年来，人们对肉蛋奶的需求量急剧增加，促进了养殖业与饲料工业的蓬勃发展，饲料行业市场需求量大，蛋白饲料严重短缺，而常用日粮豆粕过度依赖大豆进口，饲料价格涨幅迅猛，亟需寻找新的饲料资源。麦糟营养丰富且体量大，是很好的选择。如何变废为宝，合理利用麦糟，提高附加值，有效地解决环境污染问题和资源浪费问题，已经成为啤酒工业面临的重大课题。
目前使用麦糟制作发酵饲料的报道，一般采取两条途径：一是将湿麦糟直接加入到十几种或者几十种饲料原料如玉米粉、麸皮等中，再添加一些微生物菌剂或酶制剂进行发酵；二是将湿麦糟先烘干，再加入到十几种或者几十种饲料原料中，加水调制，再添加一些微生物菌剂或酶制剂进行发酵。但 以上处理方式在制作技术上存在一些问题，同时也是饲料行业需要解决的难题。
首先，关于加工方式。由于湿麦糟含水量大且营养丰富，在高温夏季放置几个小时就会被空气中的有害微生物污染，非常容易腐败变质，营养物质被破坏且产生毒素。如果啤酒厂到饲料加工厂的距离较远，或者湿麦糟的量比较大，而饲料生产能力有限，就存在很大风险，损失较大，且对动物产生不良影响。第一条途径必须迅速对湿麦糟进行混料加工，第二条途径必须迅速对湿麦糟进行烘干，如果加工不及时都会导致腐败。烘干后可以延长麦糟的保存期，不受后期混料加工时间的限制，但全部烘干的成本较高，每吨会增加600-1000元甚至更高。
其次，关于营养物质。目前，麦糟用于制作饲料或者发酵饲料时，大多作为十几种或者几十种原料里的一种，添加量非常少，不算真正意义上的以麦糟为主体的饲料。由于麦糟淀粉含量低(约3-6％)，在将麦糟制备发酵饲料的过程中，常添加玉米粉等含淀粉的营养物质，给动物补充碳源，但玉米粉价格较高，导致生产成本升高。如果随意添加一些低成本的物质，虽然成本和水分有所降低，却也拉低了麦糟自身的营养价值，而且质量不稳定不容易控制。麦糟自身含有10-15％的纤维素和10-15％的半纤维素，这部分营养物质没有被很好地利用。
再次，关于发酵技术。在添加微生物菌剂、酶制剂进行固态发酵的过程中，菌种的选择及其添加量，酶制剂的选择及其添加量，固态发酵工艺条件比如发酵温度、时间、好氧或厌氧等都非常重要，需要系统全面地考虑，但目前这些参数还比较粗放。
最后，关于动物实验。目前，麦糟多用于直接饲喂动物，比较粗放，缺少系统实验数据，而以麦糟尤其是全麦糟作为发酵饲料，用于动物实验的少之又少，限制了麦糟作为动物饲料的应用范围。
发明内容
针对现有技术存在的至少一个不足之处，本申请提出一种基于全麦糟的发酵饲料及其制备方法。
本申请第一方面提供了一种发酵饲料的制备方法，包括保鲜处理步骤、烘制步骤、好氧发酵步骤以及厌氧发酵步骤；
所述保鲜处理步骤包括：取湿麦糟后立即向所述湿麦糟中添加乳酸菌；
所述好氧发酵步骤包括：向经过所述烘制步骤得到的麦糟中加入混合菌 种和酶制剂，好氧发酵，所述混合菌种包括酵母菌和芽孢杆菌，所述酶制剂包括纤维素复合酶、蛋白酶以及木聚糖酶；
所述厌氧发酵步骤包括：向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌，厌氧发酵，得到发酵饲料。
在第一方面的一些实施例中，所述烘制步骤包括：对经过所述保鲜处理步骤得到的麦糟进行脱水处理至麦糟含水量为60-70％，然后55-60℃下烘制至麦糟含水量为40-50％。
在第一方面的一些实施例中，所述保鲜处理步骤中，向所述湿麦糟中添加乳酸菌培养液，所述乳酸菌培养液的添加量为湿麦糟重量的0.5-1％，乳酸菌培养液的浓度不小于108个/mL。
在第一方面的一些实施例中，所述好氧发酵步骤中，向经过所述烘制步骤得到的麦糟中加入混合菌种培养液，所述混合菌种培养液的添加量占麦糟干重的10-20％，所述混合菌种由1:1的所述酵母菌和所述芽孢杆菌组成，所述酵母菌和所述芽孢杆菌培养液的浓度不小于108个/mL。
在第一方面的一些实施例中，所述好氧发酵步骤中，所述酶制剂的添加量占麦糟干重的1-3％，所述酶制剂由5:3:2的所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶组成，所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶的酶活力大于10万U/g。
在第一方面的一些实施例中，所述好氧发酵步骤的好氧发酵包括：将添加了所述混合菌种和所述酶制剂的麦糟加入单向阀厌氧发酵袋，28-32℃好氧发酵1-3天。
在第一方面的一些实施例中，所述厌氧发酵步骤中，向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌培养液，所述乳酸菌培养液的添加量占麦糟干重的10-20％，所述乳酸菌培养液的浓度不小于108个/mL。
在第一方面的一些实施例中，所述厌氧发酵步骤中厌氧发酵包括35-38℃厌氧发酵4-6天。
本申请第二方面提供了一种基于全麦糟的发酵饲料，由上述任一技术方案所述的发酵饲料的制备方法制备得到。
在第二方面的一些实施例中，所述发酵饲料中：活菌数≥108个/mL，粗蛋白质≥30％，28％≤中性洗涤纤维≤40％，0.5％≤乙酸≤2.0％，3％≤乳酸≤5％，3.5≤pH≤4.5。
与现有技术相比，本申请的有益效果在于：
(1)本申请至少一个实施例所提供的发酵饲料的制备方法，通过限定保鲜处理步骤，避免了有害菌的污染风险，达到保鲜湿麦糟的目的，为后续的发酵处理过程奠定基础；通过限定好氧发酵步骤和厌氧发酵步骤，既为有益菌提供了碳源，又使得发酵饲料具有益生元的功能，将该发酵饲料用于动物喂养时能够提高动物的免疫能力，大大提升了发酵饲料的质量。
(2)本申请至少一个实施例所提供的发酵饲料，基于全麦糟发酵得到，不需要额外添加任何物质即可用于动物喂养，且具有功能性，有利于提高动物免疫力；此外，该饲料具有较好的饲料适口性和营养物质的消化率，基于麦糟营养和动物消化或产奶机理，证实了全麦糟发酵饲料的营养价值及有效性。
附图说明
图1为奶牛采食发酵饲料图；
图2为制备样品与反刍动物的培养液图；
图3为麦糟0#与全麦糟发酵饲料2#的单胃动物(鸭)仿生消化率图；
图4为麦糟0#与全麦糟发酵饲料2#的单胃动物(鸡)仿生消化率图；
图5为麦糟0#与全麦糟发酵饲料2#的单胃动物(猪)仿生消化率图。
具体实施方式
下面将对本申请具体实施例中的技术方案进行详细、完整的描述。显然，所描述的实施例仅仅是本申请总的技术方案的部分具体实施方式，而非全部的实施方式。基于本申请的总的构思，本领域普通技术人员所获得的所有其他实施例，都落于本申请保护的范围。
本申请一方面提供了一种发酵饲料的制备方法，包括保鲜处理步骤、烘制步骤、好氧发酵步骤以及厌氧发酵步骤；
所述保鲜处理步骤包括：取湿麦糟后立即向所述湿麦糟中添加乳酸菌；
所述好氧发酵步骤包括：向经过所述烘制步骤得到的麦糟中加入混合菌种和酶制剂，好氧发酵，所述混合菌种包括酵母菌和芽孢杆菌，所述酶制剂包括纤维素复合酶、蛋白酶以及木聚糖酶；
所述厌氧发酵步骤包括：向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌，厌氧发酵，得到发酵饲料。
上述制备方法将啤酒酿造副产物麦糟加工成发酵饲料，既可以大大提高副产物的附加值，缓解我国蛋白质资源的紧缺问题，又能提高湿麦糟的保存期，防止麦糟腐败变臭，减少环境污染，还能增加动物适口性，提高营养物 质的消化率和利用率，具有重要意义。
具体的，针对湿麦糟的预处理和加工方式存在较大风险或成本高的问题，本申请在从酒糟站取湿麦糟的过程中或取后，即，在湿麦糟中产生有害菌之前，迅速添加乳酸菌，有少量乳酸菌存在，就能形成优势菌群，避免了有害菌的污染风险；可选的，乳酸菌为乳酸片球菌，乳酸片球菌产酸，产细菌素，具有较宽抑菌谱，抑制有害菌生长，调节胃肠道菌群。
另外，通过烘制步骤对湿麦糟进行脱水烘干，可选的，所述烘制步骤包括：对经过所述保鲜处理步骤得到的麦糟进行脱水处理至麦糟含水量为60-70％，然后55-60℃下烘制至麦糟含水量为40-50％，与烘干全部湿麦糟相比，显著降低了成本。该技术方案具体限定了先采用脱水设备将湿麦糟脱水至含水量为60-70％，然后55-60℃烘制至麦糟含水量为40-50％，原因在于，该种技术手段相比于纯烘干的手段能够有效降低成本。
针对添加其他营养物质导致成本高的问题，本申请使用麦糟作为单一原料，充分利用麦糟中10-15％的纤维素和10-15％的半纤维素，添加纤维素复合酶、木聚糖酶，降解成葡萄糖、木糖、阿拉伯糖等，既提供了碳源，又具有益生元的功能，活化肠道有益菌并促其生长，提高机体的免疫能力。与玉米粉等相比，酶制剂添加量少，操作简便，且显著降低成本。
另外，本申请分别筛选了适合麦糟发酵的有益菌种和酶制剂种类，基于混料设计确定了各自的最佳配方，研究了关键发酵工艺对发酵麦糟指标的影响，基于五因子实验确定了麦糟发酵的最佳工艺参数，基于麦糟原料和动物消化机理，形成了一套涵盖菌剂配方、酶制剂配方和关键工艺参数的麦糟发酵技术体系，大大提升了发酵麦糟的质量。
另外，本申请技术方案还限定了发酵过程为先好氧发酵后厌氧发酵，原因在于，酵母菌和芽孢杆菌是好氧菌或兼性厌氧菌，在好氧条件下，酵母菌和芽孢杆菌快速生长，产生蛋白酶及风味物质，且迅速消耗发酵环境中的游离氧，形成低氧或无氧环境；然后加入厌氧菌乳酸菌，正好之前形成的厌氧环境适合其生长，产生乳酸、乙酸等有机酸类以及抑菌物质，降低pH值，抑制致病菌的生长。
在一些实施例中，所述保鲜处理步骤中，向湿麦糟中添加乳酸菌培养液，乳酸菌培养液的添加量为湿麦糟重量的0.5-1％，乳酸菌培养液的浓度不小于108个/mL。该技术方案具体限定了保鲜处理步骤中乳酸菌的添加量，原因在于，既保证处理后麦糟中乳酸菌的含量不小于105个/mL，又控制乳酸菌的 添加成本，只要乳酸菌形成优势菌，起到暂时抑制有害菌的作用即可。可以理解的是，该乳酸菌的添加量还可以是0.6％、0.7％、0.8％、0.9％及其范围内的任意点值。可以理解的是，在本申请的描述中，有关菌种培养液浓度的单位“个/mL”中的个是指菌落个数，即，个/mL＝CFU/mL。
在一些实施例中，所述好氧发酵步骤中，向经过烘制步骤得到的麦糟中加入混合菌种培养液，所述混合菌种培养液的添加量占干糟重量(即，麦糟的干重)的10-20％，所述混合菌种由1:1的所述酵母菌和所述芽孢杆菌组成，所述酵母菌和所述芽孢杆菌培养液的浓度不小于108个/mL。该技术方案具体限定了好氧发酵步骤中混合菌种的添加量，原因在于，保证麦糟发酵后混合菌的活菌数不小于108个/mL，产生足够的发酵产物，又控制成本。可以理解的是，该混合菌种的添加量还可以是12％、14％、16％、18％及其范围内的任意点值；该技术方案还具体限定了混合菌种由酵母菌和芽孢杆菌组成，所述酵母菌可选地为酿酒酵母，所述芽孢杆菌可选地为枯草芽孢杆菌，其中，酵母菌产香，增加适口性，消耗游离氧，促进有益菌生长，合成多种酶类；枯草芽孢杆菌产芽孢，抗逆性强，消耗游离氧，促进有益菌生长，合成多种酶类。合成的多种酶类又可以分解麦糟中的大分子物质，为有益菌提供营养和益生元。
在一些实施例中，所述好氧发酵步骤中，所述酶制剂的添加量占麦糟干重的1-3％，所述酶制剂由5:3:2的所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶组成，所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶的酶活力大于10万U/g。该技术方案具体限定了酶制剂的添加量，原因在于，所加酶量既能降解麦糟中的纤维素、半纤维素和蛋白质生成足够的葡萄糖、蛋白肽、木寡糖等，为微生物提供碳源氮源营养和益生元，又控制成本。可以理解的是该酶制剂的添加量还可以是2％。该技术方案还进一步限定了酶制剂由纤维素复合酶、蛋白酶以及木聚糖酶组成，蛋白酶可选地为酸性蛋白酶，其中，添加酶制剂是为了将阿拉伯木聚糖分解，获得可提取的阿拉伯木聚糖，有一定益生作用，具体的，利用纤维素复合酶降解大分子纤维素，生成葡萄糖，为有益菌提供可利用的碳源；利用酸性蛋白酶降解大分子蛋白质，生成多肽和氨基酸，为有益菌提供可利用的氮源，之所以选择酸性蛋白酶是因为在对麦糟前处理时为了防止有害微生物污染导致酸败，添加了少量乳酸菌进行保鲜，乳酸菌产酸，麦糟为酸性；利用木聚糖酶降解阿拉伯木聚糖，生成一定量的具有益生作用的木寡糖和木糖；上述酶在好氧发酵步骤中加入后， 在好氧发酵阶段(28-32℃好氧发酵1-3天)进行酶反应，同时也在接下来的厌氧发酵阶段(35-38℃厌氧发酵4-6天)进行酶反应。
在一些实施例中，所述好氧发酵步骤的好氧发酵包括：将添加了所述混合菌种和所述酶制剂的麦糟加入单向阀厌氧发酵袋，28-32℃好氧发酵1-3天。需要说明的是，本技术方案的好氧发酵是将麦糟先脱水再烘制，得到含水量40-50％的麦糟后再进行的固态发酵，本技术方案采用固态发酵，与液态发酵相比，固态发酵成本较低，机械化程度要求低，发酵产物浓度高，压力低，能耗小。
在一些实施例中，所述厌氧发酵步骤中，向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌培养液，所述乳酸菌培养液的添加量占麦糟干重的10-20％，所述乳酸菌培养液的浓度不小于108个/mL。该技术方案进一步限定了厌氧发酵步骤中的乳酸菌的添加量，原因在于，保证麦糟发酵后乳酸菌的活菌数不小于108个/mL，产生足够的发酵产物，又控制成本。可以理解的是，该乳酸菌的添加量还可以是12％、14％、16％、18％及其范围内的任意点值。
在一些实施例中，所述厌氧发酵步骤中厌氧发酵包括35-38℃厌氧发酵4-6天。该技术方案进一步限定了厌氧发酵的发酵条件，原因在于，使乳酸菌在适宜的温度下生长繁殖，在合适的时间内产生足够的发酵产物，尽量减少发酵周期，降低成本。
本申请另一方面提供了一种基于全麦糟的发酵饲料，由上述任一技术方案所述的发酵饲料的制备方法制备得到。该发酵饲料基于全麦糟发酵得到，不需要额外添加任何物质即可用于动物喂养，且具有功能性，有利于提高动物免疫力。通过对该基于全麦糟的发酵饲料进行系统多维度动物实验，包括奶牛的采食饲喂试验、反刍和单胃动物体外仿生试验、瘘管牛瘤胃消化试验等，结果显示本申请的基于全麦糟的发酵饲料显著增加了动物的采食量，提高了饲料适口性和营养物质的消化率，基于麦糟营养和动物消化或产奶机理，证实了全麦糟发酵饲料的营养价值及有效性。
进一步的，通过上述制备方法制备得到的发酵饲料具有明显的、愉悦的发酵酸香味，无霉味和其它异味，比发酵前的麦糟松软松散，皮壳变软，适口性明显增加，具体的：活菌数≥108个/mL，粗蛋白质≥30％，28％≤中性洗涤纤维≤40％，0.5％≤乙酸≤2.0％，3％≤乳酸≤5％，3.5≤pH≤4.5。
为了更清楚详细地介绍本申请实施例所提供的基于全麦糟的发酵饲料及其制备方法，下面将结合具体实施例进行描述。
实施例1
1、菌种
对实验室筛选出的抑菌能力和产蛋白酶能力俱佳的乳酸片球菌、酿酒酵母和枯草芽孢杆菌分别进行逐级活化和扩培。根据湿麦糟的重量扩培菌液。从啤酒厂酒糟站取5kg湿麦糟，按照菌液总添加量为湿糟重量的10％以及三种菌液各占约1/3粗算，三种菌液大约需要扩培出200mL。
(1)第一次活化。从-80℃冰箱取出菌种的甘油冷冻保存管，冷冻管融化后分别取100μL到5mL液体试管培养基中，进行活化(乳酸片球菌用MRS液体培养基，于37℃恒温箱中静置培养24h；酿酒酵母用YPD液体培养基，于30℃恒温箱中静置培养24h；枯草芽孢杆菌用LB液体培养基，于37℃摇床中振荡培养24h)
(2)第二次活化。从一级活化液体试管中各取200μL到20mL三角瓶液体培养基中，进行二次活化(乳酸片球菌用MRS液体培养基，于37℃恒温箱中静置培养24h；酿酒酵母用YPD液体培养基，于30℃恒温箱中静置培养24h；枯草芽孢杆菌用LB液体培养基，于37℃摇床中振荡培养24h)
(3)扩培。从二级活化液体试管中各取10mL到200mL三角瓶液体培养基中，进行扩培(乳酸片球菌用MRS液体培养基，于37℃恒温箱中静置培养24-48h；酿酒酵母用YPD液体培养基，于30℃恒温箱中静置培养24-48h；枯草芽孢杆菌用LB液体培养基，于37℃摇床中振荡培养24-48h)
(4)扩培菌悬液的活菌数测定。
有三种常用方法测定活菌数：
1)浊度法。取混匀的菌液200μL于酶标板中，以相应的空白液体培养基分别作为对照，在600nm下测定光密度，当光密度大于2.0时才能满足要求。
2)稀释平板计数法。从菌悬液中取100μL，加900μL无菌水，振荡混匀，为1#管，稀释倍数为10；从1#管中取100μL加900μL无菌水，振荡混匀，为2#管，稀释倍数为100；依次稀释到106，共稀释6个梯度。准备MRS、YPD、LB固体平板培养基，挑选104、105、106三个稀释度涂布平板。将涂布后的MRS和LB平板置于37℃培养48h，YPD平板置于30℃培养48h。记录稀释倍数和平板上相应的菌落数量。计算两个平行平板菌落数的 平均值，再将平均值乘以相应稀释倍数，作为每mL中菌落总数结果。浊度法与稀释平板法的结果具有高度相关性。浊度法简便快捷，但相对粗一些，稀释平板法精确，但方法繁琐、滞后。两个方法相辅相成，一般测定扩培菌悬液的活菌数用浊度法，测定全麦糟发酵饲料中活菌数用稀释平板法。
3)自动计数仪法。目前有酵母自动计数仪，测定酵母菌数的方法比较成熟，已经广泛应用。但细菌尺寸较小，还没有成熟的仪器。
用浊度法测定三种菌悬液在600nm的OD(Optical Density)值，如表1所示。
表1 三种扩培菌悬液的活菌数(浊度法)
2、麦糟的前处理
(1)从啤酒厂的酒糟站取5kg湿麦糟，立即加入25mL扩培的乳酸片球菌培养液，搅拌均匀。
(2)将湿麦糟运到实验室后，测量湿麦糟的水分：准确称取5g于称量瓶中，称量空瓶和带麦糟的总重，于105℃烘箱中3.5h，测量烘干后的总重，计算得出麦糟的水分为81％，干重为19％。
(3)将湿麦糟使用脱水机进行脱水，按照上述方法测定脱水麦糟的实际水分，显示水分从81％降低到了65％；将脱水麦糟置于55℃烘箱烘制4h，按照上述方法测定烘制麦糟的实际水分，计算出水分从65％降低到了45％，除去损失和中间取样，获得1.5kg的烘制麦糟。
3、全麦糟发酵饲料的制备
(1)计算烘制麦糟的干重为825g，向烘制麦糟中加入10％(82mL)的酿酒酵母和枯草芽孢杆菌扩培菌液，比例为1:1，根据菌液OD值计算出实际添加量，酿酒酵母40mL，枯草芽孢杆菌42mL，搅拌均匀。
(2)向加菌后的麦糟中加入2％的酶制剂，同样以干重计，共16.5g，包括纤维素复合酶、酸性蛋白酶、木聚糖酶，比例为5:3:2，三种酶的实际添加量依次为8.25g、4.95g和3.3g。
(3)将加菌加酶后的麦糟盛入带有单向阀的厌氧发酵袋，搅拌均匀，不封口，30℃好氧发酵2天。
(4)向麦糟中加入10％的乳酸菌，同样以干重计，共82mL，搅拌均匀，将发酵袋封口，37℃厌氧发酵5天，即得到以麦糟为单一原料的全麦糟发酵饲料。
实施例2
将制备的全麦糟发酵饲料，与起始的未经发酵的麦糟以及市售发酵饲料进行比较。如表2所示。所有指标都是以干重计。
表2 全麦糟发酵饲料与未发酵麦糟、市售发酵饲料的对比
乳酸菌产生脂肪酸，pH值下降，可有效抑制有害菌。短链挥发性脂肪酸参与采食调控，其中乙酸是乳脂合成的前体物质。蛋白是动物需要的主要营养物质。中性洗涤纤维、酸性洗涤纤维用于评估日粮纤维营养，太高了不利于动物消化，影响适口性。
首先对比麦糟发酵前后的差异。发酵后，各项指标都有明显改善。短链脂肪酸(乙酸、乳酸)升高，粗蛋白含量升高，中性与酸性洗涤纤维明显下降。发酵后有愉悦的酸香味。
与两款市售发酵饲料相比，全麦糟发酵饲料的乙酸、乳酸和粗蛋白含量都较高，中性与酸性洗涤纤维较低。
本申请的全麦糟发酵饲料各项指标都优于未发酵麦糟和两款市售发酵饲料。
实施例3
将本申请制备的全麦糟发酵饲料，与未发酵麦糟，以及3款市售发酵饲料，开展奶牛采食饲喂试验，如图1所示，评价全麦糟发酵饲料在动物上的应用效果，考察适口性。5个样品依次是未发酵麦糟1#、全麦糟发酵饲料2#、及市售发酵饲料3-5#，分别约1公斤。
在奶牛养殖场，选择多头奶牛分别饲喂5款样品，观察奶牛对5款样品的喜好度，以及对单个奶牛进行多次饲喂，观察重复性。最终也可以通过样品的剩余量进行评判。
结果显示：5款饲料的采食优先级依次是2#>3#>1#>4#>5#。本申请的全麦糟发酵饲料有愉悦的酸香味，是奶牛吃得速度最快的，表明奶牛最喜欢，适口性最好。3#是发酵豆粕，豆粕本身就是奶牛的日粮之一，发酵后增加了香味，适口性增加。未发酵的麦糟本身也是奶牛的饲料，但有些酸馊味，且含水量高，奶牛不是太喜欢。4#是含有麦糟的发酵饲料，但麦糟占比非常少，主要是青贮类物质。5#发酵饲料不含麦糟，有麸皮、玉米秸秆等，有轻微的氨臭味。
表明饲料原料、合适的菌酶协同、合适的发酵工艺条件都是制备发酵饲料的关键因素。
实施例4
将本申请制备的全麦糟发酵饲料，与未发酵麦糟，以及2款市售发酵饲料，开展动物体外仿生评价试验。
1、反刍动物体外消化率的评价方法
(1)将各物料分别于65℃烘箱干燥12h，至恒重为止，置于封口袋中备用。
(2)将各物料分别用植物粉碎机过40目筛。
(3)测定原料的干物质(DM)含量和粗蛋白质(CP)含量，参考国标。
(4)配制发酵培养液，参考Menke氏的方法。
(5)晨饲前屠宰奶牛，收集瘤胃液。使用4层医用纱布过滤瘤胃液到通有CO2的家用暖瓶中。
(6)调节发酵培养液的pH为7.0-7.3，将发酵培养液与瘤胃液按照2：1的比例混合均匀倒入2L的烧杯内，置于39℃水浴锅内，匀速搅拌。制备出人工培养液。
(7)称取1.0000g±0.0010g样品于一次性样品瓶中，加入60mL人工培养液，密封，置于39℃水浴摇床，转速为180rpm，反应24h。如图2所示。
(8)反应结束后测定反应液的pH，收集沉淀，测定其DM和CP含量。
(9)计算
养分的消化率＝(反应前养分的含量-反应后养分的含量)÷反应前养分的含量×100％
4款发酵饲料的反刍动物瘤胃体外消化率，如表3所示。
表3 不同发酵饲料的反刍动物瘤胃体外消化率
动物消化率高，采食量增加，吸收好，就会合成更多的营养，生产性能得以提升。与未发酵麦糟相比，全麦糟发酵饲料的干物质(DM)和中性洗涤纤维(NDF)消化率都明显升高，表明发酵后提高了反刍动物的瘤胃消化率。麦糟发酵后，大分子物质被降解，更容易被奶牛消化。同时也优于两款市售发酵饲料。
市售发酵饲料1中没有添加枯草芽孢杆菌，可能分泌的纤维素酶较少，降低了纤维素类物质的降解，影响了消化率。
市售发酵饲料2的消化率较低，其纤维较高，降低了其消化率，推测制备过程中添加了高纤维的原料，不利于动物的消化利用。
2、单胃动物(鸭、鸡、猪)的仿生体外消化率评价
采用SDS-III单胃动物仿生消化系统，全自动模拟猪禽胃肠道消化过程，分析未发酵麦糟0#和全麦糟发酵饲料2#的消化指标差异。结果如图3-5所示。
SDS-III单胃动物仿生消化系统由模拟消化器和控制系统组成。模拟消化器中透析袋内视为胃、小肠、大肠的内环境(消化环境)，透析袋外视为毛细血管体液环境(吸收环境)。采用组态软件技术通过电脑程控进入模拟消化器内的缓冲液自动输入-排空-切换系统、消化液自动分泌系统、水解产物自动清洗系统、恒温控制系统、混合强度自动控制系统、电子元件信号检测系统等模块，实现对猪禽胃肠道消化过程的全自动模拟。
结果显示：与未发酵麦糟相比，全麦糟发酵饲料的干物质(DM)消化率、能量消化率和酶水解物能值都明显升高。表明全麦糟发酵饲料同样也适用于单胃动物鸭、鸡、猪。三种单胃动物对全麦糟发酵饲料的仿生消化率从高到低依次为：鸭＞鸡＞猪。
实施例5
(1)全麦糟发酵饲料和未发酵麦糟的瘘管牛瘤胃降解效果评价
选用3头健康、安装永久性瘘管的荷斯坦奶牛，称取样品(全麦糟发酵饲料和未发酵麦糟)于特制尼龙袋内，每头牛每个时间点设3个平行，早晨饲喂前投入瘤胃中，分别在2h、4h、8h、12h、24h、36h、48h培养后取出。置于55℃烘箱烘48h至恒量，测定样品在不同时间点DM、NDF和 CP的瘤胃降解率。计算快速降解部分a、慢速降解部分b、瘤胃有效降解率ED。
未发酵麦糟和全麦糟发酵饲料的干物质瘤胃降解率及降解参数如表4所示。
表4 未发酵麦糟和全麦糟发酵饲料的干物质瘤胃降解率及降解参数
干物质(DM)瘤胃降解率可以反映出饲料在瘤胃中消化的难易程度，同时也是影响奶牛DM采食量的重要因素。结果表明，全麦糟发酵饲料的DM瘤胃降解率和有效降解率(ED)值随时间变化明显增加，且明显高于未发酵麦糟。其DM主要在36h之内降解，且48h的降解率均在70％左右。
粗蛋白瘤胃降解率及降解参数如表5所示。粗蛋白(CP)在瘤胃中的降解主要由其在瘤胃内的滞留时间和降解的难易程度决定。此外，CP在瘤胃中的降解与其内部结构组成、非蛋白氮(NPN)和真蛋白质含量以及真蛋白的理化特性、细胞壁惰性屏障的存在和抗营养因子等也有直接关系。本试验结果表明，全麦糟发酵饲料的CP瘤胃降解率、a值和ED值明显增加，可能是DM含量的降低相对提高了瘤胃中蛋白质分解酶的浓度，导致CP降解率提高。也可能是因为通过发酵处理能够提高NPN和可溶性蛋白含量，提高了饲料中CP的a值，从而提高了CP的降解率。
表5 未发酵麦糟和全麦糟发酵饲料的粗蛋白瘤胃降解率及降解参数

未发酵麦糟和全麦糟发酵饲料的中性洗涤纤维瘤胃降解率及降解参数如表6所示。NDF瘤胃降解率是反映饲料营养价值的一个重要指标，NDF的组成会影响到NDF瘤胃降解率。经发酵后，全麦糟发酵饲料其各个时间点的NDF瘤胃降解率显著提高，可能是麦糟的NDF和DM含量经微生物发酵后降低，造成分解NDF的纤维素酶和半纤维素酶浓度升高，从而提高了NDF的瘤胃降解率和ED值。
表6 未发酵麦糟和全麦糟发酵饲料的中性洗涤纤维瘤胃降解率及降解参数
(2)常规日粮和全麦糟发酵饲料的瘘管牛瘤胃降解效果评价
在日粮中添加全麦糟发酵饲料4.35公斤，占日粮干物质的14.41％，替代0.52公斤日粮(包括豆粕和其他发酵饲料等)。替代方案是根据CPM奶牛配方软件计算出来的，代替前后蛋白含量、干物质含量、纤维含量基本不变。将常规日粮和添加全麦糟发酵饲料的日粮做瘘管牛实验，分析蛋白、纤维的瘤胃降解率和有效降解率，如表7和表8所示。结果显示：与常规日粮 相比，全麦糟发酵饲料提高了粗蛋白的瘤胃降解率，中性洗涤纤维的瘤胃降解率基本不变。
表7 粗蛋白瘤胃降解率及降解参数
表8 中性洗涤纤维瘤胃降解率及降解参数

Claims (10)

  1. 一种发酵饲料的制备方法，其特征在于，包括保鲜处理步骤、烘制步骤、好氧发酵步骤以及厌氧发酵步骤；
    所述保鲜处理步骤包括：取湿麦糟后立即向所述湿麦糟中添加乳酸菌；
    所述好氧发酵步骤包括：向经过所述烘制步骤得到的麦糟中加入混合菌种和酶制剂，好氧发酵，所述混合菌种包括酵母菌和芽孢杆菌，所述酶制剂包括纤维素复合酶、蛋白酶以及木聚糖酶；
    所述厌氧发酵步骤包括：向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌，厌氧发酵，得到发酵饲料。
  2. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述烘制步骤包括：对经过所述保鲜处理步骤得到的麦糟进行脱水处理至麦糟含水量为60-70％，然后55-60℃下烘制至麦糟含水量为40-50％。
  3. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述保鲜处理步骤中，向所述湿麦糟中添加乳酸菌培养液，所述乳酸菌培养液的添加量为湿麦糟重量的0.5-1％，乳酸菌培养液的浓度不小于108个/mL。
  4. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述好氧发酵步骤中，向经过所述烘制步骤得到的麦糟中加入混合菌种培养液，所述混合菌种培养液的添加量占麦糟干重的10-20％，所述混合菌种由1:1的所述酵母菌和所述芽孢杆菌组成，所述酵母菌和所述芽孢杆菌培养液的浓度不小于108个/mL。
  5. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述好氧发酵步骤中，所述酶制剂的添加量占麦糟干重的1-3％，所述酶制剂由5:3:2的所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶组成，所述纤维素复合酶、所述蛋白酶以及所述木聚糖酶的酶活力大于10万U/g。
  6. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述好氧发酵步骤的好氧发酵包括：将添加了所述混合菌种和所述酶制剂的麦糟加入单向阀厌氧发酵袋，28-32℃好氧发酵1-3天。
  7. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述厌氧发酵步骤中，向经过所述好氧发酵步骤发酵得到的麦糟中加入乳酸菌培养液，所述乳酸菌培养液的添加量占麦糟干重的10-20％，所述乳酸菌培养液的浓度不小于108个/mL。
  8. 根据权利要求1所述的发酵饲料的制备方法，其特征在于，所述厌氧 发酵步骤中厌氧发酵包括35-38℃厌氧发酵4-6天。
  9. 一种基于全麦糟的发酵饲料，其特征在于，由权利要求1-8任一项所述的发酵饲料的制备方法制备得到。
  10. 根据权利要求9所述的基于全麦糟的发酵饲料，其特征在于，所述发酵饲料中：活菌数≥108个/mL，粗蛋白质≥30％，28％≤中性洗涤纤维≤40％，0.5％≤乙酸≤2.0％，3％≤乳酸≤5％，3.5≤pH≤4.5。
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