CN114532452B - Method for preparing ruminal fermented feed by using defective fruits and application - Google Patents
Method for preparing ruminal fermented feed by using defective fruits and application Download PDFInfo
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Abstract
The invention relates to a method for preparing a ruminant fermented feed by using defective fruits and application thereof, wherein the method comprises the following steps: collecting defective fruits, crushing and extruding, and performing solid-liquid separation; preparing a strain for fermentation; and (3) filling the mixed materials into a one-way valve breathing bag through a packaging machine, transferring the bag into a culture room, and fermenting for 3-5 days at 25-28 ℃ to obtain the ruminant fermented feed prepared by using defective fruits. The method provided by the invention can be used for squeezing the defective fruits and separating the pomace and the fruit juice, so that the water in the pomace can be effectively reduced, the sugar in the pomace can be indirectly reduced, and the utilization rate of microorganisms on the sugar can be increased; the fermentation strain adopts the compounding of two kinds of mould and two kinds of microzyme, can effectively utilize residual sugar in the pomace, decomposes macromolecular substances of the raw materials, and improves the digestion utilization rate of the raw materials. The fermented feed prepared by the invention can reduce the content of lipopolysaccharide and improve the milk yield of acid poisoning sub-healthy dairy cows, has higher pH value, and is easy to popularize in commercial application.
Description
Technical Field
The invention relates to the technical field of feeds, in particular to a method for preparing a ruminal fermented feed by using defective fruits and application of the ruminal fermented feed.
Technical Field
Defective fruit refers to fruit that has been removed before marketing or in the market, and generally refers to fruit that has fallen to the ground, is not particularly good in quality or condition. Although the fruits cannot enter the dining table of people, the fruits have high nutritional value, and if the fruits can be developed and used as feed, the fruit waste can be greatly reduced. For example, the total output of apples in 2021 years in China is 4500 ten thousand tons, but the output of apples entering a human dining table is about equal to or slightly larger than the output of apples entering the human dining table, and if the defective fruits are well utilized, the feed pressure of the animal husbandry can be relieved. Fruits contain a large amount of carbohydrates, vitamins, minerals, bioactive ingredients and dietary fibers, and can be used as feed for ruminants, but the fruits are rotten and deteriorated in the collection, storage and transportation of the inferior fruits, and the inferior fruits are fallen fruits caused by diseases, and the factors have certain toxic effects on the ruminants, so that the safe treatment process of the inferior fruits needs to be developed when the inferior fruits are successfully applied to the feed for the ruminants. For example, the defective fruits contain a large amount of acidic substances (malic acid), phenols, pectin, saccharides and other substances, and if the defective fruits are directly fed in a large amount, rumen acidity is easily too large, and rumen acidosis is easily caused.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preparing a ruminant fermented feed by using defective fruits and application thereof.
The invention provides a method for preparing a ruminant fermented feed by using defective fruits, which comprises the following steps:
step one, collecting defective fruits, crushing and extruding, and then carrying out solid-liquid separation: soaking the defective fruits with clear water, drying in the air under natural conditions, crushing by a crusher, and extruding by a screw extrusion dehydrator to obtain fruit residues serving as raw materials of the ruminant fermented feed; collecting juice during the squeezing process;
step two, preparation of fermentation strains: selecting 4 strains, namely saccharomyces cerevisiae, candida utilis, geotrichum candidum and aspergillus niger; wherein the saccharomyces cerevisiae and the candida utilis are prepared into a saccharomyces cerevisiae-candida utilis mixed culture solution by adopting a liquid fermentation method; respectively preparing an aspergillus niger solid culture and a geotrichum candidum solid culture by adopting a solid fermentation process;
step three, fully mixing 50-60% of pomace, 15-25% of wheat bran, 5-15% of soybean germ powder, 0-10% of palm meal, 0.1-0.5% of light calcium carbonate, 0.1-1.0% of ammonium sulfate, 2-8% of glutamic acid residue, 0.5-3% of aspergillus niger solid culture, 0.5-3% of geotrichum candidum solid culture and 0.5-3% of saccharomyces cerevisiae-candida utilis mixed culture solution in a stirrer, filling the mixed materials into a breather bag of a one-way valve, transferring the bag into a culture room, and fermenting for 3-5 days at 25-28 ℃ to obtain the ruminant fermented feed prepared from inferior fruits.
Further, the preparation method of the saccharomyces cerevisiae-candida utilis mixed culture solution comprises the following steps of:
(1) preparing a saccharomyces cerevisiae or candida utilis strain inclined plane: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH (potential of Hydrogen) adjusted to 6.4 +/-0.2, 20 g/L of agar, water with constant volume of 1L, sterilizing at 121 ℃ for 30min, preparing a strain inclined plane, dipping saccharomyces cerevisiae or candida utilis strain from a glycerol tube by using an inoculating loop, respectively inoculating the strain on the inclined plane at 28-30 ℃, and culturing for 3-5 days for later use;
(2) preparing a saccharomyces cerevisiae or candida utilis seed culture solution: 5g/L of peptone, 2 g/L of yeast extract powder, 40 g/L of apple juice, 0.5g/L of magnesium sulfate and 1 g/L of dipotassium phosphate, adjusting the pH to 6.4 +/-0.2, fixing the volume to 1L of distilled water, sterilizing for 30min at 121 ℃, inoculating the saccharomyces cerevisiae or candida utilis strain, placing the strain in a constant-temperature oscillation incubator, and culturing for 20-24h at 30 ℃ and 200rpm for later use;
(3) preparing a saccharomyces cerevisiae-candida utilis mixed culture solution: weighing 50g/L of high-temperature bean cake powder, 40 g/L of fruit juice, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate and 0.5g/L of ammonium sulfate, adjusting the pH value to 6.4 +/-0.2, fixing the volume of distilled water to 1L, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 10mL of saccharomyces cerevisiae seed culture solution and 10mL of candida utilis seed culture solution into each liter of liquid culture medium under an aseptic condition, placing the culture medium in a constant-temperature shaking flask for shaking culture, wherein the culture temperature is 28-30 ℃, the shaking rotation speed is 200rpm, and the culture time is 24 h.
Further, the preparation method of the aspergillus niger solid culture comprises the following steps:
(1) preparing an aspergillus niger strain inclined plane: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH (potential of Hydrogen) adjusted to 6.4 +/-0.2, 20 g/L of agar, water constant volume of 1L, 121 ℃, sterilization for 30min, preparation of a strain inclined plane, dipping an Aspergillus niger strain from a glycerol tube by using an inoculating loop, inoculating the Aspergillus niger strain on the inclined plane, and culturing for 3-5 days for later use;
(2) preparing an aspergillus niger culture solution: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium hydrogen phosphate, pH (potential of Hydrogen) adjusted to 6.4 +/-0.2, constant volume of distilled water to 1L, 121 ℃, sterilization for 30min, cooling to room temperature, inoculating Aspergillus niger colonies on the inclined plane of the Aspergillus niger strain under aseptic condition, inoculating 5-10 Aspergillus niger colonies per liter of liquid culture medium, and performing shaking culture for 3-5 days at the temperature of 28-30 ℃ and the rpm of 200 in a constant-temperature shaking table for later use;
(3) preparation of aspergillus niger solid culture: mixing the wheat bran, the corn flour and the soybean meal according to the proportion of 80%, 15% and 5% to prepare a mixed material, mixing the mixed material with the fruit juice obtained in the step one according to the proportion of 3-4:1, uniformly mixing, sterilizing at the temperature of 115 ℃ and 121 ℃ for 30min, and inoculating the Aspergillus niger culture solution, wherein the inoculation amount of the Aspergillus niger culture solution is 5%, and the calculation is carried out based on the weight of the wet material; placing the material inoculated with Aspergillus niger into a culture tank, culturing for 5-7 days, drying at 50-60 deg.C, and using the dried Aspergillus niger solid culture within 4-6 months.
Further, the preparation method of the geotrichum candidum solid culture comprises the following steps:
(1) preparing a geotrichum candidum strain inclined plane: 20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH value to 6.4 +/-0.2, 20 g/L of agar, fixing the volume to 1L of water, sterilizing at 121 ℃ for 30min, preparing a strain inclined plane, dipping a geotrichum candidum strain from a glycerol tube by using an inoculating loop, inoculating the strain on the inclined plane, and culturing at 28-30 ℃ for 2-3 days for later use;
(2) preparing a geotrichum candidum culture solution: 20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH to 6.4 +/-0.2, 20 g/L of agar, fixing the volume to 1L of water, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating Geotrichum candidum colonies on the inclined plane of the Geotrichum candidum strain under the aseptic condition, inoculating 3-5 Geotrichum candidum colonies per liter of liquid culture medium, and performing shaking culture at 28-30 ℃ and 200rpm in a constant-temperature shaking table for 2-3 days for later use;
(3) preparation of Geotrichum candidum solid culture: mixing wheat bran, corn flour and soybean meal according to the proportion of 80%, 15% and 5%, adding ammonium sulfate of 0.5-1.0% based on the weight of the materials to prepare a mixed material, mixing the mixed material with the fruit juice obtained in the step one according to the proportion of 3-4:1, uniformly mixing, sterilizing at the temperature of 115 ℃ and 121 ℃ for 30min, and inoculating the geotrichum candidum culture solution, wherein the inoculation amount of the geotrichum candidum culture solution is 5%, and the calculation is carried out based on the weight of the wet materials; placing the material inoculated with the Geotrichum candidum culture solution in a culture pond, culturing for 3-5 days, drying at 50-60 deg.C, and using the dried Geotrichum candidum solid culture within 2-3 months.
Further, the material proportion in the third step is as follows: 55% of pomace, 20% of wheat bran, 10% of soybean germ powder, 5% of palm meal, 0.2% of light calcium carbonate, 0.8% of ammonium sulfate, 5% of glutamic acid residue, 1% of aspergillus niger solid culture, 1% of geotrichum candidum solid culture and 2% of a saccharomyces cerevisiae-candida utilis mixed culture solution.
Further, the fermentation time of the third step is 4 days.
The invention provides application of a ruminant fermented feed prepared by using defective fruits in ruminant feeding.
The invention has the following beneficial effects:
(1) the fermentation of the defective fruits has the advantages that after the fermentation of the pomace, the anti-nutritional factors and pesticide residues in the pomace can be obviously reduced, the digestibility of acidic and neutral detergent fibers is improved, and active substances such as proteins, amino acids, vitamins, nucleotides, unknown growth factors and the like are improved. Meanwhile, raw materials can be well controlled, a cleaning mode can be adopted, impurities and mould on the surface of the defective fruit can be effectively removed, and then the influence of diseases, germs and mould inside the defective fruit can be removed through fermentation. This patent adopts solid-liquid separation, squeezes the junior fruit, separates pomace and fruit juice, can effectively reduce the moisture in the pomace, prevents that the pomace from depositing the in-process, because the too high production of moisture mildenes and rot, perhaps the assay goes out to pollute the storehouse place in the pomace. The water in the pomace is extruded out through the screw, so that the water in the pomace can be effectively reduced, and the sugar in the pomace is also indirectly reduced, because the sugar can be partially lost along with the water, and the sugar which can be effectively utilized by microorganisms is about 20% -40% of the sugar content of the pomace, so that the pomace which is not subjected to water extrusion is directly fermented, and the sugar cannot be thoroughly utilized, so that loss and waste of nutrient substances can be caused.
(2) The fermentation strain disclosed by the invention adopts the compounding of two kinds of mould and two kinds of saccharomycetes, so that residual sugar in fruit residues can be effectively utilized, macromolecular substances of raw materials are decomposed, the digestion utilization rate of the raw materials is improved, oxygen in the check valve breathing bag can be consumed during the propagation process of the mould and the saccharomycetes, other microorganisms cannot grow, and mixed bacteria cannot be bred even when the air leakage phenomenon exists in the instant breathing valve, so that the oxygen stability of a product can be improved, and the shelf life of the product is prolonged.
(3) At present, the tank type fermentation or the tank type fermentation is mostly adopted for fermenting the apple pomace, the temperature is not easy to control in the fermentation process, and the risk of fermentation and bacterial contamination is large. This patent adopts the check valve to breathe the bag fermentation, ferments at the constant temperature culture medium, can the temperature of the whole fermentation in-process of effective control, adopts this mode also can effectively reduce the mould risk of fermentation substrate dyeing.
(4) The invention extrudes and crushes the defective fruits to separate the fruit juice from the pulp, the fruit juice is efficiently utilized as a carbon source for culturing fermentation strains, the pulp is used as a solid fermentation substrate raw material, and the mixed bacterial strains are adopted for fermentation to prepare a biological fermentation feed product with high added value. The inferior fruit is not limited to apple, can also be pear, kiwi fruit and the like, and the method of the invention efficiently utilizes the pulp and the juice in the inferior fruit, thereby reducing the waste to the maximum extent.
(5) The fermented feed prepared by the invention has obviously better effect of improving the yield of milk than the fermented feed in the prior art, has certain improvement effect of relieving the acidosis of the dairy cows, can reduce the content of lipopolysaccharide and improve the milk yield of the sub-healthy dairy cows suffering from the acidosis, has higher pH value, and is easier to popularize in commercial application.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1A method for preparing a fermented ruminant feed using cull fruit
1. Collecting defective fruits (falling apples), extruding, and performing solid-liquid separation.
Soaking the fallen apples in clear water, drying the apples in the air under natural conditions, crushing the apples by a crusher, extruding the apples by a screw extrusion dehydrator, and collecting juice in the extrusion process. The fruit residue is used as raw material matrix for solid state fermentation, and the fruit juice is used for culturing fermentation strain.
2. Preparation of Aspergillus niger strains (solid state fermentation seed production)
(1) Preparation of Aspergillus niger slant strain
Preparation of aspergillus niger slant strain: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH adjusted to 6.4 +/-0.2, 20 g/L of agar, water constant volume to 1L, 121 ℃, sterilizing for 30min, preparing a strain inclined plane, dipping an Aspergillus niger strain from a glycerol tube by using an inoculating loop, inoculating the Aspergillus niger strain on the inclined plane, and culturing for 3-5 days for later use.
(2) Preparation of Aspergillus niger culture solution
The preparation method of the Aspergillus niger culture solution comprises the following steps: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate and 1 g/L of dipotassium phosphate, adjusting the pH to 6.4 +/-0.2, fixing the volume of distilled water to 1L, sterilizing at 121 ℃ for 30 min. Cooling to room temperature, inoculating 5-10 Aspergillus niger colonies per liter of liquid culture medium under aseptic condition, and shake-culturing at constant temperature and 200rpm in a shaking table at 28-30 deg.C for 3-5 days.
(3) Preparation of Aspergillus niger solid culture
Mixing wheat bran, corn flour and soybean meal according to the proportion of 80%, 15% and 5% to prepare a mixed material, mixing the mixed material and the extruded apple juice according to the proportion of 3-4:1, uniformly mixing, sterilizing at the temperature of 115 ℃ and 121 ℃ for 30min, and inoculating an Aspergillus niger culture solution. Wherein the inoculum size of the Aspergillus niger culture solution is 5%, calculated based on the weight of the wet material. Placing the Aspergillus niger inoculated material in a culture tank, culturing for 5-7 days, drying at low temperature (air inlet temperature < 60 deg.C), and allowing the dried culture to be used for 4-6 months.
3. Geotrichum candidum strain preparation (solid fermentation seed production)
(1) Preparation of Geotrichum candidum culture solution
Preparation of geotrichum candidum slant strains: 20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH to 6.4 +/-0.2, 20 g/L of agar, fixing the volume to 1L of water, sterilizing at 121 ℃ for 30min, preparing a strain inclined plane, dipping a geotrichum candidum strain from a glycerol tube by using an inoculating loop, inoculating the strain on the inclined plane, and culturing at 28-30 ℃ for 2-3 days for later use.
(2) Preparation of Geotrichum candidum culture solution
20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH to 6.4 +/-0.2, adjusting the content of agar to 20 g/L, fixing the volume of water to 1L, and sterilizing at 121 ℃ for 30 min. Cooling to room temperature, inoculating under aseptic condition, inoculating 3-5 Geotrichum candidum colonies per liter of liquid culture medium, and shake culturing at 28-30 deg.C and 200rpm in a constant temperature shaking table for 2-3 days.
(3) Preparation of Geotrichum candidum solid cultures
Mixing wheat bran, corn meal and bean pulp according to the proportion of 80%, 15% and 5%, adding ammonium sulfate of 0.5-1.0% based on the weight of the materials to prepare a mixed material, mixing the mixed material and the extruded apple juice according to the proportion of 3-4:1, uniformly mixing, sterilizing at the temperature of 115 ℃ plus 121 ℃ for 30min, and inoculating geotrichum candidum culture solution. Wherein the inoculation amount of the geotrichum candidum culture solution is 5 percent, and the calculation is carried out based on the weight of the wet material. Placing the geotrichum candidum inoculated material in a culture pond, culturing for 3-5 days, drying in a low-temperature drying mode (the air inlet temperature is less than 60 ℃), and using the dried culture for 2-3 months.
4. Preparation of Saccharomyces cerevisiae and Candida utilis strains (liquid fermentation seed production)
Because the saccharomyces cerevisiae and the candida utilis belong to the saccharomyces, a production strain is prepared by adopting a mixed fermentation mode.
(1) Preparation of slant strain of Saccharomyces cerevisiae or Candida utilis
Preparing saccharomyces cerevisiae or candida utilis slant strains: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH adjusted to 6.4 +/-0.2, 20 g/L of agar, water constant volume to 1L, 121 ℃, sterilizing for 30min, preparing a strain inclined plane, dipping an Aspergillus niger strain from a glycerol tube by using an inoculating loop, inoculating the Aspergillus niger strain on the inclined plane, and culturing for 3-5 days for later use.
(2) Preparation of seed culture solution of Saccharomyces cerevisiae or Candida utilis
The preparation method of the seed culture solution of the saccharomyces cerevisiae or the candida utilis comprises the following steps: 5g/L of peptone, 2 g/L of yeast extract powder, 40 g/L of apple juice, 0.5g/L of magnesium sulfate and 1 g/L of dipotassium phosphate, adjusting the pH to 6.4 +/-0.2, fixing the volume to 1L of distilled water, sterilizing for 30min at 121 ℃, inoculating the saccharomyces cerevisiae or candida utilis strain, putting the strain into a constant-temperature oscillation incubator, and culturing for 20-24h at 30 ℃ and 200rpm for later use;
(3) preparation of mixed culture solution of saccharomyces cerevisiae and candida utilis
Weighing 50g/L of high-temperature bean cake powder, 40 g/L of apple juice, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium hydrogen phosphate and 0.5g/L of ammonium sulfate, adjusting the pH value to 6.4 +/-0.2, fixing the volume of distilled water to 1L, sterilizing at 121 ℃ for 30 min. Cooling to room temperature, inoculating 10mL of Saccharomyces cerevisiae seed culture solution and 10mL of Candida utilis culture solution per liter of liquid culture medium under aseptic condition, placing in a constant-temperature shaking flask for shaking culture at 28-30 ℃, at a shaking speed of 200rpm for 24h, and culturing for later use.
5. Preparation of solid-state fermented feed containing pomace
The method comprises the following steps of fully mixing 55% of pomace, 20% of wheat bran, 10% of soybean germ powder, 5% of palm meal, 0.2% of light calcium carbonate, 0.8% of ammonium sulfate, 5% of glutamic acid residue, 1% of aspergillus niger culture, 1% of geotrichum candidum culture and 2% of mixed culture solution of saccharomyces cerevisiae/candida utilis in a stirrer, filling the mixed materials into a one-way valve breathing bag through a packing machine, transferring the materials into a culture medium, and fermenting for 3-5 days at 25-28 ℃ to obtain a fermented feed product.
The prepared fermented feed product has mellow smell, good loose, soft and free-running property, no bad smell and no caking; the water content of the material is 50-51 percent, and the crude protein is 11-14 percent.
Example 2 preparation of common strains for fermentation preparation of ruminally fermented feed
Apple pomace obtained by extrusion in example 1 is used as a raw material of a solid fermentation medium, lactobacillus plantarum, saccharomycetes and bacillus licheniformis are used as fermentation strains to prepare the ruminant fermentation feed of the apple pomace, and the strains used in the method are also common strains for pomace fermentation. The specific method comprises the following steps:
apple pomace, palm meal, wheat bran, corn germ powder, light calcium carbonate and glutamic acid pomace are mixed according to a certain proportion, wherein the specific proportion is 55% of apple pomace, 20% of wheat bran, 10% of soybean germ powder, 5% of palm meal, 0.2% of light calcium carbonate, 0.8% of ammonium sulfate and 5% of glutamic acid pomace. Mixing the mixed bacterial liquid and the solid fermentation substrate in a mixer at a ratio of 20:1000, wherein each ml of the mixed bacterial liquid contains Lactobacillus plantarum 1 × 107CFU/mL, yeast 1X 108CFU/mL, B.subtilisBacillus 1X 106CFU/mL. Mixing for 1-2min, packaging the mixed materials into a one-way valve breathing bag by a packaging machine, transferring into a culture medium, and fermenting at 25-28 deg.C for 3-5 days to obtain the ruminant fermented feed product obtained by fermenting apple pomace in the prior art.
Example 3 preparation of a fermented ruminant feed not extruded but prepared using the strains of the invention
The defective fruits (ground apples) are soaked in clear water and dried in the air under natural conditions, and after being crushed by a crusher, the fermentation strains are inoculated according to the process of the embodiment 1, and the rumination fermentation feed is prepared according to the fermentation process.
Example 4 detection of ruminally fermented feed
The fermented feeds prepared in examples 1 to 3 were examined and analyzed to compare the effects of different preparation methods on the quality of the fermented feeds. The detection method of each nutrient component comprises the following steps:
the detection of the water content is carried out according to GB/T6435-2014;
the pH was measured according to GB 5009.237-2016;
the detection of the content of the crude protein is carried out according to GB/T6432-2018;
the detection of the content of the acid soluble protein is carried out according to GB/T22492-2008;
the detection of the total acid content is carried out according to GB/T12456-2008;
the detection of the content of the lactic acid bacteria is carried out according to GB/T4789.35-2016;
the detection of the content of the yeast is carried out according to GB/T4789.15-2016;
the detection of the content of the bacillus is carried out according to GB/T26428-2010;
the detection of the mold content is carried out according to GB/T13092-2006.
As can be seen from the test analysis in Table 1, the fermented feed production process has a significant effect on the acid production of the same material. In examples 1 and 2, the acidity of the fermented feed in example 2 is high, while the acidity of the fermented feed in example 1 is low, and the analysis is mainly that the fermented strains are greatly different, and the compound strain used in example 1 does not contain lactobacillus, so that the pH value is not rapidly reduced in the fermentation process; the fungi and the yeast can utilize the sugar in the pomace to produce more mycoprotein and improve the proportion of crude protein in the fermentation material; the strain used in the fermentation of the embodiment 2 contains lactic acid bacteria, and the lactic acid bacteria can fully utilize the sugar metabolism in the pomace to generate a large amount of lactic acid, so that the pH of the fermented pomace product is reduced, and the total acid content is high. For example 1 and example 3, the same strains are used for fermentation in both examples, but the fermentation substrate in example 3 is not subjected to the step of squeezing to remove fruit juice, the acidity of the fermented feed in example 3 is high, while the acidity of the fermented feed in example 1 is low, mainly because the fermentation process has great difference, the water in the pomace is squeezed in the preparation process of the fermented pomace product in example 1, and the sugar in the inferior fruit is partially lost along with the water in the squeezing process, so the process of squeezing to remove fruit juice reduces the water and the sugar in the pomace, slows down the metabolism of the fermentation strains, and avoids the generation of a large amount of organic acid in the fermentation process to influence the quality of the fermented feed. Example 3, by adopting a non-extrusion process, the moisture of the fermentation material is obviously increased by 6.12%, the pH is also obviously reduced to 3.92, and the total acid is increased to 7.21, which indicates that the higher moisture can affect the reproductive metabolism of yeast, and in addition, the wild lactic acid bacteria in the pomace can also be metabolized to generate a large amount of organic acid under the conditions of high sugar and high moisture, so that the pH in the fermentation material is obviously reduced; in addition, the high moisture condition is not favorable for the proliferation of the microorganism, which results in low mycoprotein content in the fermented feed, and the crude protein content is obviously lower than that in the example 1. Therefore, by comparing example 1 and example 3, it can be seen that whether the pomace is dehydrated or not has a significant effect on the quality of the fermented feed under the same fermentation strain and fermentation process conditions. Therefore, the pH value of the conventional pomace fermented feed is between 3.5 and 4.0, the acidity is too high, a small amount of cattle with good body conditions can be added, and the conventional pomace fermented feed is not suitable for adding cattle with acidosis. The pomace fermented feed has the pH value of over 5.3 and low acidity, and is more suitable for being used in ruminant feed.
Example 5 mixing uniformity study of the ruminally fermented feeds of examples 1-3 in TMR diets
The ruminally fermented feeds of examples 1-3 were mixed with dairy farm ingredients in the following table proportions in a TMR ration mixer and apple pomace was analyzed for uniformity of mixing in the TMR ration by sieving.
The proportion of the apple pomace in each layer of oversize products is analyzed by adopting a screening method, and the content of the apple pomace in the oversize products of the B group daily ration and the C group daily ration is found to be not obviously different from that of the apple pomace in the A group every time, so that the ruminant fermented feed prepared by fermenting the apple pomace by adopting the technical scheme of the invention can be mixed by adopting a TMR daily ration stirrer, the operation is simple, and the manpower is saved. The apple pomace in the oversize material of the D group TMR daily ration is more, the surface of the fermented feed is stained with more powder, such as soybean meal or premix in the concentrate supplement, and the phenomenon can cause the feeding preference of the dairy cows, further influence the nutritional balance of the dairy cows, easily cause the poor body condition of the dairy cows or reduce the milk yield and influence the production benefit.
The screened substances of the TMR daily ration in the group E contain the most caking materials, and the phenomenon can cause the similar result with the TMR daily ration in the group D, finally causes the poor body condition of the dairy cow or the reduction of the milk yield and influences the production benefit.
The above table shows that the fermented feed obtained by the fermentation processes of examples 2 and 3 has high acidity and high moisture content, and affects the stirring effect of TMR, and for large-scale dairy farms, it is not recommended to feed cows in TMR ration directly with the non-dehydrated pomace fermented feed or the dehydrated fermented feed having high acidity, because the nutrient intake is already accurate to the mouth, and thus, adverse effects are avoided.
Example 6 evaluation of application of fermented feed of defective fruits to cow breeding
(1) 300 test cows of an ecological cow farm in Henan are selected, single-factor test design is adopted, and cows with good health degree are randomly divided into 4 groups according to the principle that the milk yield, the fetal number and the lactation days are similar, wherein each group comprises 75 cows. A, B, C, D four TMR diets of example 5 were fed separately for a period of 40 days. A loose feeding mode is adopted, TMR daily ration is fed for 3 times in the morning, in the middle and at night every day, the feed is freely taken, the feeding residual amount is guaranteed to be 3% -5% every day, the milk is milked for 3 times every day, and water is freely drunk.
(2) Milk yield and milk composition: and (3) measuring the milk yield by using a Laval flowmeter from the beginning to the end of the test, performing statistical analysis on the milk yield data of the test cattle after the test is finished, and measuring the nutritional components of the milk by using a UL40BC milk component rapid detector. The original data are sorted by using excel2016 software, and the milk yield and milk component indexes are subjected to variance analysis by using a sps 16.0 repeated measurement module.
From the analysis of the test results in table 3, it can be seen that the milk yield of the group C and the group D is significantly increased (P <0.05), the milk yield of the group C and the group B is significantly decreased (P <0.05), the milk yield of the group C is increased by 3.98%, and the milk yield of the group D is increased by 1.59%, compared to the control group, the milk yield of the group C and the group D is significantly increased (P <0.05), the milk yield of the group C is increased by 8.90%, and the milk yield of the group D is increased by 7.89% at the end of the test, and from the milk production data, the milk yield can be increased and the milk peak period can be prolonged by adding 6% of the feed of the present invention to the TMR ration.
Note that the difference between the lower case letters on the same row indicates significant difference (P <0.05), the difference between the lower case letters on the same row and the same letter indicates insignificant difference (P >0.05), the difference between the upper case letters on the same column and the same letter indicates significant difference (P <0.05), and the difference between the upper case letters on the same column and the same letter indicates insignificant difference (P > 0.05).
As shown in table 4, before and after the test, there is no significant difference (P >0.05) between milk fat, lactose, total solids, somatic cells, and milk protein, and at the end of the test, there is no significant difference (P >0.05) between milk fat, lactose, total solids, somatic cells, and milk protein compared to the control group, so that the fermented feed prepared by the fermentation method of the present invention and the fermentation method of the prior art has no significant influence on the milk quality when the cow is fed compared to the control group.
Note that the difference of the lower case letters of the shoulder marks of the same row indicates that the difference is obvious (P <0.05), the difference is not obvious when the letters or the letters are the same (P >0.05)
Therefore, the fermented feed prepared from the inferior fruits can be used in the TMR daily ration of the dairy cows, the promotion effect of 6% of the addition amount on the milk yield is better than 3%, and meanwhile, the fermented feed (group C) prepared by squeezing the fruit residues after removing the fruit juice and performing combined fermentation by the strain disclosed by the invention has a significantly better effect on improving the milk yield than the fermented feed (group D) prepared by conventional zymophyte on the same fruit residues.
Example 7 evaluation of the use of a ruminally fermented feed prepared from cullet in the cultivation of sub-healthy cows
At present, in order to improve the milk yield of dairy cows, dairy farmers add more concentrate supplement to daily ration, so that the cows only have acidosis. The fermented feed has effects of regulating rumen flora balance, and improving intestinal health. Few people verify the effect of the pomace fermented feed under the condition because the pH value of the pomace fermented feed is lower. Because the pH value of the fermented feed is higher than that of the conventional pomace fermented feed, the effect is verified by considering that the selected sub-healthy cattle with acidosis are only fed for test.
(1) Selecting 30 test cows in an ecological cow farm in Henan, wherein the cows only have rumen acidosis, adopting single-factor test design, and randomly dividing the test cows into 3 groups of 10 cows each according to the principle that the milk yield, the number of fetuses and the number of lactation days are similar. A, C, D three TMR diets in example 4 were fed separately for a period of 30 days. A loose feeding mode is adopted, TMR daily ration is fed for 3 times in the morning, in the middle and at night every day, the feed is freely taken, the feeding residual amount is guaranteed to be 3% -5% every day, the milk is milked for 3 times every day, and water is freely drunk.
(2) Milk yield and milk composition: and (3) measuring the milk yield by using a Laval flowmeter from the beginning to the end of the test, performing statistical analysis on the milk yield data of the test cattle after the test is finished, and measuring the nutritional components of the milk by using a UL40BC milk component rapid detector. The original data are sorted by using excel2016 software, and the milk yield and milk component indexes are subjected to variance analysis by using a sps 16.0 repeated measurement module.
(3) Biochemical indexes of serum: on the day of the beginning of prefeed and the end of the pilot period, 20 mL/head of blood was collected by feeding the anterior jugular vein in the morning, centrifuged at 3500r/min, and the upper serum was collected and stored by freezing at-20 ℃. The ELISA kit and the scientific research kit are used for detecting the content of lactic acid and lipopolysaccharide in serum, and the kit is purchased from Nanjing to certain biological Limited company.
As can be seen from Table 5, although the milk production performance of the acidotic cows is low, the ruminal fermented feed prepared by different processes has no significant influence on the milk production and milk components of the cows (P >0.05), but at the end of the test, compared with the control group, the milk production of the group C is improved by 3.38%, the difference is not significant (P >0.05), before and after the test, the milk production of the control group is reduced by 1.28%, the difference is not significant (P >0.05), while the milk production of the test group C is improved by 1.7%, the difference is not significant (P >0.05), which indicates that the group C has a certain improvement effect on the milk production performance of the cows. Compared with the control group, the milk yield of the group C is improved by 3.38 percent, and the milk yield of the group D is improved by 0.28 percent, which shows that the group C has a good effect of improving the milk production performance of the acidotic cows.
Lipopolysaccharide is a measurement index of acidosis, and when the test is carried out on the weak acidosis cows selected in the test, the lipopolysaccharide content of normal cows is 200-300 ng/L, the acidosis condition content can be increased, and the lipopolysaccharide content of the acute acidosis cows can reach about 2000 ng/L. From table 6, it can be seen that, for the cows with acidosis, the ruminal fermented feed prepared by different processes has no significant influence on lactic acid in serum (P >0.05), but the ruminal fermented feed prepared by different processes has significant influence on the lipopolysaccharide content in serum (P <0.05), and at the end of the test, compared with the control group, the lipopolysaccharide content in the test group C is reduced by 39.96%, and the difference is significant (P <0.05), which indicates that the group C has a certain improvement effect on alleviating the cow acidosis. The pH value of the fermented feed in the group D is 3.89, the pH value of rumen juice is less than 5.5 under the condition of rumen acidosis, in this case, a common farm cannot adopt the biological fermented feed with low pH value, the pH value of the biological fermented feed in the group C (the biological fermented feed prepared by the process) is higher (pH value is 5.37), the possibility of using the fermented feed in the farm is increased, the field use effect also shows that the fermented feed can obviously improve the body condition of acidosis cows, reduce the content of lipopolysaccharide and improve the milk yield, and the preparation method of the fermented feed prepared from the fruits of the patent is better than the preparation method of the conventional fruit residue fermented feed.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (5)
1. A method for preparing a ruminant fermented feed by using defective fruits is characterized by comprising the following steps:
step one, collecting defective fruits, crushing and extruding, and then carrying out solid-liquid separation: soaking the defective fruits with clear water, airing under natural conditions, crushing by a crusher, and extruding by a screw extrusion dehydrator to obtain fruit residues serving as raw materials of the ruminant fermentation feed; collecting juice during the squeezing process;
step two, preparation of fermentation strains: selecting 4 strains, namely saccharomyces cerevisiae, candida utilis, geotrichum candidum and aspergillus niger; wherein the saccharomyces cerevisiae and the candida utilis are prepared into a saccharomyces cerevisiae-candida utilis mixed culture solution by adopting a liquid fermentation method; respectively preparing an aspergillus niger solid culture and a geotrichum candidum solid culture by adopting a solid fermentation process;
step three, fully mixing 50-60% of pomace, 15-25% of wheat bran, 5-15% of soybean germ powder, 0-10% of palm meal, 0.1-0.5% of light calcium carbonate, 0.1-1.0% of ammonium sulfate, 2-8% of glutamic acid residue, 0.5-3% of aspergillus niger solid culture, 0.5-3% of geotrichum candidum solid culture and 0.5-3% of saccharomyces cerevisiae-candida utilis mixed culture solution in a stirrer, filling the mixed materials into a breather bag of a one-way valve, transferring the bag into a culture room, and fermenting for 3-5 days at 25-28 ℃ to obtain the ruminant fermented feed prepared from inferior fruits;
the preparation method of the saccharomyces cerevisiae-candida utilis mixed culture solution comprises the following steps:
(1) preparing a saccharomyces cerevisiae or candida utilis strain inclined plane: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH adjusted to 6.4 +/-0.2, 20 g/L of agar, water constant volume to 1L, sterilization at 121 ℃ for 30min, preparing a strain inclined plane, dipping saccharomyces cerevisiae or candida utilis strain from a glycerol tube by using an inoculating loop, respectively inoculating the strain on the inclined plane, and culturing for 3-5 days for later use at 28-30 ℃;
(2) preparing a saccharomyces cerevisiae or candida utilis seed culture solution: 5g/L of peptone, 2 g/L of yeast extract powder, 40 g/L of apple juice, 0.5g/L of magnesium sulfate and 1 g/L of dipotassium phosphate, adjusting the pH to 6.4 +/-0.2, fixing the volume to 1L of distilled water, sterilizing for 30min at 121 ℃, inoculating the saccharomyces cerevisiae or candida utilis strain, placing the strain in a constant-temperature oscillation incubator, and culturing for 20-24h at 30 ℃ and 200rpm for later use;
(3) preparing a saccharomyces cerevisiae-candida utilis mixed culture solution: weighing 50g/L of high-temperature bean cake powder, 40 g/L of fruit juice, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate and 0.5g/L of ammonium sulfate, adjusting the pH value to 6.4 +/-0.2, fixing the volume of distilled water to 1L, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 10mL of saccharomyces cerevisiae seed culture solution and 10mL of candida utilis seed culture solution into each liter of liquid culture medium under the aseptic condition, placing the culture medium in a constant-temperature shaking flask for shaking culture, wherein the culture temperature is 28-30 ℃, the shaking rotation speed is 200rpm, and the culture time is 24 h;
the preparation method of the Aspergillus niger solid culture comprises the following steps:
(1) preparing an aspergillus niger strain inclined plane: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium phosphate, pH adjusted to 6.4 +/-0.2, 20 g/L of agar, water constant volume to 1L, 121 ℃, sterilizing for 30min, preparing a strain inclined plane, dipping an Aspergillus niger strain from a glycerol tube by using an inoculating loop, inoculating the Aspergillus niger strain on the inclined plane, and culturing for 3-5 days for later use;
(2) preparing an aspergillus niger culture solution: 5g/L of peptone, 2 g/L of yeast extract powder, 20 g/L of glucose, 0.5g/L of magnesium sulfate, 1 g/L of dipotassium hydrogen phosphate, pH (potential of Hydrogen) adjusted to 6.4 +/-0.2, constant volume of distilled water to 1L, 121 ℃, sterilization for 30min, cooling to room temperature, inoculating Aspergillus niger colonies on the inclined plane of the Aspergillus niger strain under aseptic condition, inoculating 5-10 Aspergillus niger colonies per liter of liquid culture medium, and performing shaking culture for 3-5 days at the temperature of 28-30 ℃ and the rpm of 200 in a constant-temperature shaking table for later use;
(3) preparation of aspergillus niger solid culture: mixing wheat bran, corn meal and soybean meal according to the proportion of 80%, 15% and 5% to prepare a mixed material, mixing the mixed material with the fruit juice according to the proportion of 3-4:1, uniformly mixing, sterilizing for 30min at the temperature of 115-; placing the material inoculated with the Aspergillus niger in a culture tank, culturing for 5-7 days, drying at low temperature of 50-60 ℃, and using the dried Aspergillus niger solid culture within 4-6 months;
the preparation method of the geotrichum candidum solid culture comprises the following steps:
(1) preparing a geotrichum candidum strain inclined plane: 20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH value to 6.4 +/-0.2, 20 g/L of agar, fixing the volume to 1L of water, sterilizing at 121 ℃ for 30min, preparing a strain inclined plane, dipping a geotrichum candidum strain from a glycerol tube by using an inoculating loop, inoculating the strain on the inclined plane, and culturing at 28-30 ℃ for 2-3 days for later use;
(2) preparing a geotrichum candidum culture solution: 20 g/L of peptone, 10 g/L of yeast extract powder and 20 g/L of glucose, adjusting the pH to 6.4 +/-0.2, 20 g/L of agar, fixing the volume to 1L of water, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating Geotrichum candidum colonies on the inclined plane of the Geotrichum candidum strain under the aseptic condition, inoculating 3-5 Geotrichum candidum colonies per liter of liquid culture medium, and performing shaking culture at 28-30 ℃ and 200rpm in a constant-temperature shaking table for 2-3 days for later use;
(3) preparation of Geotrichum candidum solid culture: mixing wheat bran, corn flour and soybean meal according to the proportion of 80%, 15% and 5%, adding ammonium sulfate of 0.5-1.0% based on the weight of the materials to prepare a mixed material, mixing the mixed material and the fruit juice according to the proportion of 3-4:1, sterilizing at the temperature of 115 ℃ and 121 ℃ for 30min after uniformly mixing, and inoculating the geotrichum candidum culture solution, wherein the inoculation amount of the geotrichum candidum culture solution is 5%, and the calculation is carried out based on the weight of the wet materials; placing the material inoculated with the Geotrichum candidum culture solution in a culture pond, culturing for 3-5 days, drying at 50-60 deg.C, and using the dried Geotrichum candidum solid culture within 2-3 months.
2. The method for preparing the ruminant fermented feed by using the defective fruits as claimed in claim 1, wherein the material ratio in the third step is as follows: 55% of pomace, 20% of wheat bran, 10% of soybean germ powder, 5% of palm meal, 0.2% of light calcium carbonate, 0.8% of ammonium sulfate, 5% of glutamic acid residue, 1% of aspergillus niger solid culture, 1% of geotrichum candidum solid culture and 2% of a saccharomyces cerevisiae-candida utilis mixed culture solution.
3. The method for preparing the ruminant fermented feed from the defective fruit as claimed in claim 1, wherein the fermentation time of the third step is 4 days.
4. A fermented ruminant feed prepared in accordance with the method of claim 1.
5. Use of the ruminally fermented feed of claim 4 for ruminant animal farming.
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