CN117903972A

CN117903972A - Bacterial enzyme synergistic preparation and application thereof in fruit residue-containing micro-storage feed for poultry

Info

Publication number: CN117903972A
Application number: CN202311782921.3A
Authority: CN
Inventors: 谈亚丽; 李啸; 秦涛; 刘玲彦
Original assignee: Angel Biotechnology Co ltd; Tibet Anqi Biotechnology Co ltd
Current assignee: Angel Biotechnology Co ltd; Tibet Anqi Biotechnology Co ltd
Priority date: 2023-12-18
Filing date: 2023-12-18
Publication date: 2024-04-19

Abstract

The invention provides a bacterial enzyme synergistic preparation and application thereof in fruit residue-containing micro-storage feed for poultry, wherein the bacterial enzyme synergistic preparation comprises, by weight, 30-60 parts of lactobacillus delbrueckii subspecies, 10-30 parts of candida utilis, 10-30 parts of enterococcus faecium, 300-500 parts of cellulase and 100-200 parts of pectase, and the bacterial enzyme synergistic preparation improves the palatability and the nutritional value of the micro-storage feed and reduces the problems of dyspepsia of young animals.

Description

Bacterial enzyme synergistic preparation and application thereof in fruit residue-containing micro-storage feed for poultry

Technical Field

The invention belongs to the technical field of fermented feeds, and particularly relates to a bacterial enzyme synergistic preparation and application thereof in fruit residue-containing micro-storage feeds for poultry.

Background

China has rich agricultural solid waste resources such as fruit residues, the annual output of citrus residues is up to 500 ten thousand tons, the annual output of pineapple residues is about 80 ten thousand tons, the output of tea residues is up to millions of tons, the fruit residues and the agricultural solid waste contain rich nutrients and metal elements such as magnesium elements, only a small part of the fruit residues and the agricultural solid waste are reused, the fruit residues and the agricultural solid waste are mainly used in the fields of functional substance extraction, biological composting or feed processing, and most of the fruit residues and solid waste resources are easy to deteriorate due to the fact that the moisture content is too high, and the fruit residues and the solid waste are inconvenient to store or transport for a long time and are directly abandoned.

For poultry, the slaughtering time of broiler chickens or meat ducks is only about 40-60 days, the requirements of the poultry on energy intake and nutrition absorptivity in the growth or egg laying period are higher, the coarse protein in the feed needs to reach more than 18%, the magnesium element in each kilogram of the feed needs to reach 400-600mg to reach the skeletal and nerve development requirements of the poultry, the calcium element can reach 3-5% to maintain the eggshell quality, and the pigment content has great influence on the flesh color and the yolk color. Meanwhile, the poultry is sensitive to temperature change and water source change, and often presents symptoms such as inappetence, intestinal dyspepsia, diarrhea and the like, and at the moment, medicines or antibiotics are needed to be used for auxiliary treatment to reduce the morbidity and mortality of the poultry, but the problems of animal residues, antibiotic resistance and the like of the poultry are caused, so that the food safety is seriously affected.

The agricultural solid waste contains astringent taste substances such as naringin, limonin, tea polyphenol and the like, and the raw materials which are represented by pomace also contain anti-nutritional ingredients such as pectin, phytic acid and the like, so that the problems of dyspepsia and poor nutrition absorption of poultry animals are easily caused, and the agricultural solid waste has a plurality of problems when applied to poultry animal cultivation.

Disclosure of Invention

Aiming at the problems that agricultural solid wastes are easy to spoil and inconvenient to transport and store for a long time, contain astringency flavor-developing substances, pectin, phytic acid and other anti-nutritional ingredients, have high nutrition requirements for poultry animals, are easy to cause diarrhea, dyspepsia and the like, the invention provides a bacterial enzyme synergistic preparation and application thereof in preparing fruit residue-containing micro-storage feed for poultry.

The technical scheme of the invention is as follows:

In a first aspect, the invention provides a synergistic formulation of a bacterial enzyme, which is characterized by comprising, by weight, 30-60 parts of lactobacillus delbrueckii subspecies lactis, 10-30 parts of candida utilis, 10-30 parts of enterococcus faecium, 300-500 parts of cellulase and 100-200 parts of pectase.

Preferably, the bacterial enzyme synergistic preparation is characterized by comprising 40-50 parts of lactobacillus delbrueckii subspecies, 15-25 parts of candida utilis, 15-25 parts of enterococcus faecium, 350-450 parts of cellulase and 125-175 parts of pectase in parts by weight.

Preferably, the bacterial enzyme synergistic preparation is characterized in that Lactobacillus delbrueckii subspecies are Lactobacillus delbrueckii subspecies (Lactobacillus delbrueckii subsp. Lactis) Dangxiong LB VIII strain, and the strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2023396.

Preferably, the Candida utilis is Candida utilis (Candida utilis) XZ-CU01 strain which is preserved in China center for type culture collection (cctccc) with the preservation number of cctccc No. M2023299.

Preferably, the enterococcus faecium is enterococcus faecium (Enterococcus faecium) XZ-EF01 strain which is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2023300.

Preferably, the bacterial enzyme synergistic preparation is characterized in that the viable count of lactobacillus delbrueckii subspecies, candida utilis and enterococcus faecium is more than or equal to 2 multiplied by 10 ¹¹ CFU/g, preferably, the enzyme activity of pectase is more than or equal to 60000U/g, further preferably, the enzyme activity of cellulase is more than or equal to 10000U/g.

In a second aspect, the invention provides a preparation method of the bacterial enzyme synergistic preparation, which comprises the following steps: mixing the lactobacillus delbrueckii subspecies lactis, candida utilis, enterococcus faecium, pectase and cellulase.

In a third aspect, the invention provides an application of the bacterial enzyme synergistic preparation or the bacterial enzyme synergistic preparation obtained by the preparation method in micro-storage feed;

preferably, the use of the synergistic formulation of the bacterial enzymes in the fermented micro-feed is characterized by comprising the use in the fermented micro-feed for poultry.

In a fourth aspect, the invention provides a micro-storage feed, which is characterized in that the raw materials of the micro-storage feed comprise fruit residues, tea residues, corn flour, bean pulp, feed-grade stone powder and amino acid mixture, and the microbial enzyme synergistic preparation or the microbial enzyme synergistic preparation obtained by the preparation method.

Preferably, the micro-feed is characterized in that the fruit residue comprises citrus residue and/or pineapple residue.

Preferably, the micro-feed is characterized in that the amino acid mixture comprises L-lysine hydrochloride, DL-methionine and L-threonine.

Preferably, the amino acid mixture comprises 4-6 parts by weight of L-lysine hydrochloride, 0.5-1.5 parts by weight of DL-methionine and 1-3 parts by weight of L-threonine.

Preferably, the micro-storage feed is characterized in that, according to parts by weight, the citrus slag, the pineapple slag, the tea slag, the corn meal, the soybean meal, the feed-grade stone powder, the amino acid mixture are 15-25 parts, 10-15 parts, 5-10 parts, 3-5 parts and 1-1.5 parts;

Preferably, the citrus pulp, pineapple pulp, tea pulp, corn meal, feed-grade stone powder and amino acid mixture are 18-22 parts, 12-14 parts, 6-8 parts, 3-5 parts and 1-1.5 parts by weight.

Preferably, the mass ratio of the microbial enzyme synergistic preparation to the total mass of the mixed substances containing fruit residues, tea residues, corn meal, bean pulp, feed-grade stone powder and amino acid mixture is 0.05-0.15 per mill, and preferably, the mass ratio of the microbial enzyme synergistic preparation to the total mass of the mixed substances containing fruit residues, tea residues, corn meal, bean pulp, feed-grade stone powder and amino acid mixture is 0.1-0.12 per mill.

In a fifth aspect, the present invention provides a method for preparing the micro-storage feed, the method comprising: culturing the Lactobacillus delbrueckii subspecies lactis, the candida utilis and the enterococcus faecium respectively, and mixing the obtained bacterial powder with pectase powder and cellulase powder to obtain the bacterial enzyme synergistic preparation.

Preferably, the preparation method of the micro-storage feed adopts anaerobic fermentation, and the fermentation temperature is preferably 25-30 ℃.

The invention has the beneficial effects that:

The lactobacillus probiotics can generate metabolites such as lactic acid and bacteriocin, can inhibit pathogenic bacteria from growing to prolong the shelf life of the feed, improve the immunity of animals and reduce the use of antibiotics; the yeast probiotics can improve the nutrition of the raw materials by improving the protein content and other nutrient substances in the raw materials; part of bitter and astringent substances in the raw materials can be used for improving the palatability of the fermentation raw materials in the fermentation process of probiotics; the related enzyme preparation further degrades substances such as cellulose and the like which can not be digested and utilized by poultry animals into oligosaccharides and glucose, and simultaneously provides more nutrient substances for the proliferation of probiotics in the feed and improves the nutritional value of the feed; the pectin rich in the pectin can be further degraded to relieve the problems of dyspepsia of young animals and the like.

The microbial enzyme synergistic preparation is used for preparing the micro-storage feed, so that a certain probiotic property of the feed is provided, the nutritional preference of poultry is aimed at, the nutritional value of the feed is improved, the content of anti-nutritional substances is reduced, the problems of the feed such as absorptivity, palatability, shelf life and quality stability are improved, the high-value utilization of low-value agricultural and sideline resources is finally realized, and an efficient preparation method is provided for the feed for poultry cultivation.

Information on preservation of strains

The Lactobacillus delbrueckii subspecies (Lactobacillus delbrueckii subsp. Lactis) Dangxiong LB VIII strain is preserved in China Center for Type Culture Collection (CCTCC) for type number of preservation (CCTCC NO: M2023396) in year 2023 and 23, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072, telephone: 027-68754052; and is disclosed in patent application publication number CN 116948884A.

The Candida utilis (Candida utilis) XZ-CU01 strain is preserved in China Center for Type Culture Collection (CCTCC) in the 13 th year of 2023, and the preservation number is CCTCC NO: M2023299, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072, telephone: 027-68754052.

The enterococcus faecium (Enterococcus faecium) XZ-EF01 strain provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) in the 13 th year of 2023, and the preservation number is CCTCC NO: M2023300, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072, telephone: 027-68754052.

Drawings

FIG. 1 is a graph showing the kinetics of lactic acid production during fermentation of micro-feed.

Detailed Description

Biological material source

The Lactobacillus delbrueckii subspecies (Lactobacillus delbrueckii subsp. Lactis) Dangxiong LB VIII strain provided by the invention is obtained by separation in milk pulled of herdsman at an altitude of about 4350m in Tibetan medicine xiongxian county, belongs to a natural wild type strain, has good stress resistance, can resist stronger adverse environment, has good acid production and acid resistance, has a certain repairing effect on damaged intestinal tracts of animals, can effectively regulate intestinal microorganisms of the animals so as to promote digestion and absorption, and has the separation and identification process disclosed in patent application with publication number of CN 116948884A.

The candida utilis (candida utilis) XZ-CU01 strain provided by the invention is collected from fermented feed of the palpus sheep town sedentary in zhongba county of the Tibetan day karst, and has higher protein production and good feed fermentation capacity.

The specific separation and identification method comprises the following steps:

The collected feed samples are diluted to a proper gradient and then coated in YPD solid culture medium, and are cultured at a constant temperature of 30 ℃, single colony is picked up for continuous streak purification, the finally obtained single colony samples are subjected to molecular biological identification and NCBI database comparison analysis by adopting 16S rRNA genes, and the strain with the final identification number XZ-CU01 is Candida utilis (Candida utilis) XZ-CU01 strain, and the 16S rRNA gene of the strain is 16S rDNA as shown in SEQ ID NO. 1:

GATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTTATACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATAGTACCTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCGACTGCTTGGGAGGGGTGTATTTATTAGATAAAAAATCAATGCCCTCGGGCTCTTTGATGATTCATAATAACTTGTCGAATCGCATGGCTTTACGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATGGTGGCAACGGGTAACGGGGAATAAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTAATTCAGGGAGGTAGTGACAATAAATAACGATACAGGGCCCTTCTGGGTCTTGTAATTGGAATGAGTACAATGTAAATACCTAGTGGCCTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACTTTGGGCCTGGCAGGCCGGTCCGCTTTTTGGCGAGTACTGACCCTGCCGGGCCTTTCCT TCTGGCTACCCTCCCCTCTGGAGAGGCGAACCAGGACTTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATGGAATAATAGAATAGGACGTGGCCTGGCAGTTTGTTGGTTTCTAGGACCATCGTAATGATTAATAGGGACGGTCGGGGGCATCAGTATTCAGTTGTCAGAGGTGAAATTCTTGGATTTACTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGACGTTTTCATTAATCAAGAACGAAAGTTAGGGGATCGAAGATGATCAGATACCCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGTGTTGTTTTTATAATGACTCACTCGGCACCTTACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCAGGGAAACTCACCAGGTCGGGGAAACTCACCAGGTCCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTACTAAATAGCATGGGTAATTTTTGCTGGTGCTGACGCTTCTTAGAGGGACTATCGATTTCAAGTCGATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGACGTTCTGGGCCGCACGCGCGCTACACTGACGGAGCCAGCGAGTCTAGCCTTGGCCGANAGGTCATGGGTAATCTTGTGAAACTCCGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGA

the enterococcus faecium (Enterococcus faecium) XZ-EF01 strain provided by the invention is collected from fresh feces of Tibetan chickens in the Tibetan xiongxian county plateau region, has better acid production, diacetyl and bacteriocin production capacity, can effectively inhibit growth and reproduction of intestinal pathogenic bacteria, and has a regulating effect on animal growth.

Diluting a collected chicken manure sample to a proper gradient, then coating the chicken manure sample in an MRS solid culture medium, culturing the chicken manure sample at a constant temperature of 37 ℃, picking single colonies, continuously streaking and purifying, carrying out molecular biological identification and NCBI database comparison analysis on the finally obtained single colony sample by adopting a 16S rRNA gene, wherein the strain with the final identification number XZ-EF01 is an enterococcus faecium (Enterococcus faecium) XZ-EF01 strain, and the gene of the 16S rRNA of the strain is 16S rDNA as shown in SEQ ID NO. 2:

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCT

in a specific embodiment of the invention, the preparation method of the bacterial enzyme synergistic preparation comprises the following steps:

Activating and expanding culture of Lactobacillus delbrueckii subspecies Dangxiong LB VIII strain with MRS liquid culture medium at 35-38deg.C, preferably 37deg.C; more preferably, the activation and propagation time is 10-12 hours; further preferably, the centrifugation speed of the bacterial liquid is 6000-10000r/min, and can be preferably 8000r/min; further preferably, the lyoprotectant is a combination of 30-50% skim milk powder and 50-70% maltodextrin, and may preferably be a combination of 40% skim milk powder and 60% maltodextrin; further preferably, the weight ratio of lyoprotectant to heavy phase is 2:1-4:1, and may preferably be 3:1; after 48h of freeze-drying, lactobacillus delbrueckii subspecies lactis powder is obtained.

Activating and expanding the candida utilis XZ-DU01 strain by adopting YPD liquid culture medium, wherein the activation and expanding temperature is 28-32 ℃, preferably 30 ℃; more preferably, the activation and propagation time is 24-36 hours; further preferably, the rotational speed of the shaking culture is 110 to 150r/min, and may preferably be 120r/min; further preferably, the centrifugation speed of the bacterial liquid is 6000-10000r/min, and can be preferably 8000r/min; further preferably, the lyoprotectant is a combination of 30-50% skim milk powder and 50-70% trehalose, and may preferably be a combination of 40% skim milk powder and 60% trehalose; further preferably, the weight ratio of lyoprotectant to heavy phase is 2:1-4:1, and may preferably be 3:1; after 48h of freeze-drying, candida utilis powder is obtained.

Activating and expanding enterococcus faecium XZ-EF01 strain with MRS liquid culture medium at 35-38deg.C, preferably 37deg.C; more preferably, the activation and propagation time is 10-12 hours; further preferably, the centrifugation speed of the bacterial liquid is 6000-10000r/min, and can be preferably 8000r/min; further preferably, the lyoprotectant is a combination of 30-50% skim milk powder and 50-70% maltodextrin, and may preferably be a combination of 40% skim milk powder and 60% maltodextrin; further preferably, the weight ratio of lyoprotectant to heavy phase is 2:1-4:1, and may preferably be 3:1; after 48h of freeze-drying, enterococcus faecium bacterial powder is obtained.

The preparation method of the bacterial enzyme synergistic preparation comprises the steps of weighing 30-60 parts of lactobacillus delbrueckii subspecies, 10-30 parts of candida utilis, 10-30 parts of enterococcus faecium, 300-500 parts of cellulase and 100-200 parts of pectase according to parts by weight, and fully mixing under the aseptic condition to obtain the bacterial enzyme synergistic preparation meeting the requirements.

In a specific embodiment of the invention, the method for preparing a fermented feed comprises the steps of:

The water content in the citrus slag, pineapple slag, tea slag, corn meal, bean pulp and feed-grade stone powder is measured according to the method in GB/T6435-2014, the mixture of 15-25 parts of 15-15 parts of 10-5 parts of 3-5 parts of 1-1.5 parts of amino acid is prepared according to the conditions that the citrus slag, the pineapple slag, the tea slag, the corn meal, the feed-grade stone powder and 15-25 parts of 15-15 parts of amino acid, and all materials are weighed and fully and uniformly mixed to prepare the mixture with the corresponding water content for standby.

(1) And (3) uniformly mixing the bacterial enzyme synergistic preparation and the mixed material: and (3) sprinkling (or spraying after water dissolution) the bacterial enzyme synergistic agent with a corresponding proportion into the mixed material, and stirring the mixed material and the bacterial enzyme synergistic agent until the mixed material and the bacterial enzyme synergistic agent are uniformly mixed for fermentation.

(2) Anaerobic fermentation: transferring the uniformly mixed materials to be fermented into an anaerobic fermentation bag (more than or equal to 20 threads) with a one-way exhaust valve, sealing and exhausting air in the fermentation bag as much as possible, placing the fermentation bag in a fermentation room at 25-30 ℃ for standing fermentation, and periodically checking the gas expansion condition of the fermentation bag in the fermentation process, and performing manual intervention for exhausting when necessary.

The reagents and raw material source information used in the examples of the present invention are shown in Table 1 below.

TABLE 1

Example 1

Preparing a mixed material: 2500g of orange residue particles, 2500g of pineapple residue, 1500g of tea residue, 1500g of corn flour, 1000g of soybean meal, 500g of feed stone powder and 150g of mixed amino acid (94 g of L-lysine hydrochloride, 19g of DL-methionine and 37g of L-threonine) are weighed and uniformly mixed to obtain a mixed material for standby, and the final moisture of the mixed material is about 60.7%, the calcium element content is 3.21% and the magnesium element content is 473mg/kg.

Preparing a bacterial enzyme synergistic preparation: 60g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII, 30g of candida utilis XZ-CU01, 30g of enterococcus faecium XZ-EF01, 500g of cellulase powder and 200g of pectase powder are accurately weighed under the aseptic condition, and are fully mixed, vacuum packaging is carried out, and the mixture is refrigerated and stored at 4 ℃ for standby. Wherein the viable count of the lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, the candida utilis XZ-CU01 bacterial powder and the enterococcus faecium XZ-EF01 bacterial powder is 2 multiplied by 10 ¹¹ CFU/g, the enzyme activity of the cellulase is 20000U/g, and the enzyme activity of the pectase is 60000U/g.

And (III) fermenting the mixed material: inoculating the bacterial enzyme synergistic preparation into the mixed material according to the inoculation amount of 0.15 per mill, uniformly mixing, and transferring into an anaerobic fermentation bag with an exhaust valve for anaerobic fermentation. And in the fermentation process, the fermentation condition of the material is monitored in real time, and the exhaust is manually interfered when necessary.

And (IV) fermentation quality monitoring: and detecting the lactic acid generation condition every day in the fermentation process, and when the continuous 2d lactic acid content is not obviously improved, treating as the fermentation is finished, detecting the moisture content, the crude ash content, the crude protein, the true protein, the neutral washing fiber, the bitter taste and taste substance content and the pH value of the micro-storage feed after the fermentation is finished, and carrying out sensory index analysis. The results are shown in fig. 1 and table 2.

The method comprises the steps of determining the content of crude protein in micro-storage by adopting a Kjeldahl nitrogen determination method GB/T6432-2018, determining the content of crude ash in the micro-storage by adopting a method in GB/T6435-2014, determining neutral washing fiber (NDF) in the micro-storage by adopting a method in GB/T20806-2022, determining the pH value in the micro-storage by adopting a method in DB 15/T1458-2018, determining naringin (naringin extract) in the micro-storage by adopting a method in GB 1886.262-2016, and detecting limonin in the micro-storage by referring to a method in spectrophotometry for determining limonin analog in orange pits in Tian Qingguo, ding Xiaolin, 1999'. . The method in GB/T8313-2008 is adopted for measuring tea polyphenol in the micro-storage feed, and the method in GB/T13885-2017 is adopted for measuring calcium and magnesium contents in the micro-storage feed.

Example 2

Preparing a mixed material: 2000g of orange residue particles, 2000g of pineapple residue, 1250g of tea residue, 1250g of corn meal, 1000g of soybean meal, 400g of feed stone powder and 125g of mixed amino acid (71.4 g of L-lysine hydrochloride, 17.9g of DL-methionine and 35.7g of L-threonine) are weighed and uniformly mixed to prepare a mixed material for standby, and the final moisture content of the mixed material is about 60.3%, the calcium element content is 2.82% and the magnesium element content is 393mg/kg.

Preparing a bacterial enzyme synergistic preparation: 45g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII, 20g of candida utilis XZ-CU01, 20g of enterococcus faecium XZ-EF01, 400g of cellulase powder and 150g of pectase powder are accurately weighed under the aseptic condition, and are fully mixed, vacuum packaging is carried out, and the mixture is refrigerated and stored at 4 ℃ for standby. Wherein the viable count of the lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, the candida utilis XZ-CU01 bacterial powder and the enterococcus faecium XZ-EF01 bacterial powder is 2 multiplied by 10 ¹¹ CFU/g, the enzyme activity of the cellulase is 20000U/g, and the enzyme activity of the pectase is 60000U/g.

And (III) fermenting the mixed material: inoculating the bacterial enzyme synergistic preparation into the mixed material according to the inoculation amount of 0.1 per mill, uniformly mixing, and transferring into an anaerobic fermentation bag with an exhaust valve for anaerobic fermentation. And in the fermentation process, the fermentation condition of the material is monitored in real time, and the exhaust is manually interfered when necessary.

And (IV) fermentation quality monitoring: the results are shown in FIG. 1 and Table 2, as in example 1.

Example 3

Preparing a mixed material: 1500g of orange residue particles, 1500g of pineapple residue, 1000g of tea residue, 1000g of corn flour, 500g of soybean meal, 300g of feed stone powder and 100g of mixed amino acid (73 g of L-lysine hydrochloride, 9g of DL-methionine and 18g of L-threonine) are weighed and uniformly mixed to prepare a mixed material for standby, and the final moisture content of the mixed material is measured to be about 61.8%, the calcium element content is 2.45% and the magnesium element content is 301mg/kg.

Preparing a bacterial enzyme synergistic preparation: 30g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII, 10g of candida utilis XZ-CU01, 10g of enterococcus faecium XZ-EF01, 300g of cellulase powder and 100g of pectase powder are accurately weighed under the aseptic condition, and are fully mixed, vacuum packaging is carried out, and the mixture is refrigerated and stored at 4 ℃ for standby. Wherein the viable count of the lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, the candida utilis XZ-CU01 bacterial powder and the enterococcus faecium XZ-EF01 bacterial powder is 2 multiplied by 10 ¹¹ CFU/g, the enzyme activity of the cellulase is 20000U/g, and the enzyme activity of the pectase is 60000U/g.

And (III) fermenting the mixed material: inoculating the bacterial enzyme synergistic preparation into the mixed material according to the inoculum size of 0.05 per mill, uniformly mixing, and transferring into an anaerobic fermentation bag with an exhaust valve for anaerobic fermentation. And in the fermentation process, the fermentation condition of the material is monitored in real time, and the exhaust is manually interfered when necessary.

Example 4

Preparing a mixed material: 1800g of orange slag particles, 1800g of pineapple slag, 1200g of tea slag, 1200g of corn flour, 600g of bean pulp, 300g of feed stone powder and 100g of mixed amino acid (73 g of L-lysine hydrochloride, 9g of DL-methionine and 18g of L-threonine) are weighed and uniformly mixed to prepare a mixed material for standby, and the final moisture content of the mixed material is about 60.2%, the calcium element content is 2.44% and the magnesium element content is 300mg/kg.

Preparing a bacterial enzyme synergistic preparation: 40g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII, 15g of candida utilis XZ-CU01, 15g of enterococcus faecium XZ-EF01, 350g of cellulase powder and 125g of pectase powder are accurately weighed under the aseptic condition, and are fully mixed, and then vacuum packaging is carried out, and the mixture is refrigerated and stored at 4 ℃ for standby. Wherein the viable count of the lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, the candida utilis XZ-CU01 bacterial powder and the enterococcus faecium XZ-EF01 bacterial powder is 2 multiplied by 10 ¹¹ CFU/g, the enzyme activity of the cellulase is 20000U/g, and the enzyme activity of the pectase is 60000U/g.

And (III) fermenting the mixed material: as in example 1.

Example 5

Preparing a mixed material: 2200g of orange residue particles, 2200g of pineapple residue, 1400g of tea residue, 1400g of corn flour, 800g of bean pulp, 500g of feed stone powder and 150g of mixed amino acid (94 g of L-lysine hydrochloride, 19g of DL-methionine and 37g of L-threonine) are weighed and uniformly mixed to prepare a mixed material for standby, and the final moisture content of the mixed material is about 60.3%, the calcium element content is 3.04% and the magnesium element content is 403mg/kg according to actual measurement.

Preparing a bacterial enzyme synergistic preparation: under aseptic condition, accurately weighing 50g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII, 25g of candida utilis XZ-CU01, 25g of enterococcus faecium XZ-EF01, 450g of cellulase powder and 175g of pectase powder, fully mixing, vacuum packaging, and refrigerating at 4 ℃ for storage for later use. The viable count of Lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, candida utilis XZ-CU01 bacterial powder and enterococcus faecium XZ-EF01 bacterial powder is 2X 10 ¹¹ CFU/g, the cellulase enzyme activity is 20000U/g, and the pectase enzyme activity is 60000U/g.

And (III) fermenting the mixed material: as in example 1.

Comparative example 1

The synergistic preparation of the bacterial enzymes in the example 1 is changed into 60g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, 30g of candida utilis XZ-CU01 bacterial powder and 30g of enterococcus faecium XZ-EF01 bacterial powder, and the rest steps are unchanged. The results are shown in fig. 1 and table 2.

Comparative example 2

The synergistic preparation of the bacterial enzymes in the example 1 is changed into the synergistic preparation according to 60g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, 30g of candida utilis XZ-CU01 bacterial powder, 500g of cellulase powder and 200g of pectase powder, and the rest steps are unchanged. The results are shown in fig. 1 and table 2.

Comparative example 3

The synergistic preparation of the bacterial enzymes in the example 1 is changed to be mixed according to 30g of candida utilis XZ-CU01 bacterial powder, 30g of enterococcus faecium XZ-EF01 bacterial powder, 500g of cellulase powder and 200g of pectase powder, and the rest steps are unchanged. The results are shown in fig. 1 and table 2.

Comparative example 4

The bacterial enzyme synergistic preparation in the example 1 is changed into Dangxiong LB VIII bacterial powder of Lactobacillus delbrueckii subspecies lactis 60, 30g of enterococcus faecium XZ-EF01 bacterial powder, 500g of cellulase powder and 200g of pectase powder, and the rest steps are unchanged. The results are shown in fig. 1 and table 2.

Comparative example 5

The bacterial enzyme synergistic formulation in example 1 was replaced with commercial micro-storage starter EM bacteria (the composition includes lactic acid bacteria, bacillus, bifidobacteria, etc.), and the rest steps were unchanged. The results are shown in FIG. 1 and Table 3. The results are shown in fig. 1 and table 2.

Comparative example 6

The natural fermentation was performed without inoculating a starter on the basis of the mixture in example 1 (one), i.e., the steps (III) (IV) were performed directly. The results are shown in fig. 1 and table 2.

Comparative example 7

The mixture of example 1 (one) was placed in a-20 ℃ refrigerator without inoculating the starter and sampled at any time for use. The results are shown in fig. 1 and table 2.

As can be seen from FIG. 1, lactic acid was rapidly accumulated in examples 1 to 4 and comparative examples 1 to 5, which were inoculated with the enzyme synergistic preparation and the commercial micro-storage starter EM bacteria, as compared with the natural fermentation comparative examples 6 to 7, in which the starter culture was not added. Based on non-lactic acid bacteria components such as yeast and enzyme preparation in the bacteria-enzyme synergistic preparation, the types of lactic acid bacteria are not as good as those of EM bacteria, but the yield of lactic acid can be quite even higher, and the visible bacteria-enzyme synergistic preparation has better lactic acid production capacity, so that the purposes of forming sour and fragrant flavor and accumulating organic acid in raw materials are achieved. In addition, lactic acid in the material after fermentation of the inoculated bacteria enzyme synergistic preparation and the commercial micro-storage starter EM bacteria is far more than 8000ug/g (the minimum sterilization concentration of common pathogenic microorganisms such as staphylococcus aureus, bacillus cereus or escherichia coli and the like), and the addition of the starter is helpful for the generation of lactic acid in the mixed material so as to inhibit the growth of other harmful bacteria, thereby achieving the purpose of prolonging the shelf life of the material.

TABLE 2 micro-storage feed index

As can be seen from table 2, the higher the magnesium element content in the micro-storage feed with higher addition ratio of the fruit residue mixture, the calcium element with a certain content provided by the feed stone powder is beneficial to the bone development of the poultry and the formation of eggshells. The real protein content of the fermented feed prepared in the example 1 is higher than that of the comparative examples 1-7, and in all the examples and comparative examples, the real protein content and the crude fiber content of the micro-storage feed prepared by inoculating the microbial enzyme synergistic agent in the example 1 are the highest, which means that in the microbial enzyme synergistic agent, according to parts by weight, 60g of Lactobacillus delbrueckii subspecies Dangxiong LB VIII bacterial powder, 30g of candida utilis XZ-CU01 bacterial powder, 30g of enterococcus faecium XZ-EF01 bacterial powder, 500g of cellulase powder and 200g of pectase powder, the number of active bacteria of candida utilis XZ-CU01 bacterial powder and enterococcus faecium XZ-EF01 bacterial powder is 2X 10 ¹¹ CFU/g, the cellulase activity is 20000U/g, and the pectinase activity is 60000U/g of the microbial enzyme synergistic agent is used for fermenting the micro-storage feed containing fruit residues, so that the nutritional value of the micro-storage feed can be remarkably improved. Compared with the natural fermentation comparative examples 6 and 7 without the addition of the fermentation inoculant, the true protein content in the inoculated inoculant enzyme synergistic preparation in examples 1 and comparative examples 1-5 is obviously improved, and the inoculant can effectively improve the true protein content in the mixed material so as to improve the nutrition. After fermentation, the moisture content, the crude ash content, the neutral washing fiber, the crude protein and the pH value index of the micro-storage feed all meet the standard limit (DB 15/T2410-2021), the contents of bitter taste flavor substances naringin, limonin and tea polyphenol are reduced along with the fermentation, and the sensory index shows that the micro-storage feed has the tea aroma and the fruit dreg aroma of the raw materials, and simultaneously has the fermented sour aroma and a certain fermented ester aroma, so that the food calling effect of the micro-storage feed can be effectively improved.

In conclusion, lactic acid in the materials can be rapidly accumulated by inoculating the microbial enzyme synergistic preparation in the mixed materials, so that the storage and transportation properties of the materials are ensured, the true protein content in the fermented feed is improved, so that the nutrition of the feed is ensured, the content of bitter taste and taste substances is reduced, and the sensory indexes meet the requirements, so that the flavor and palatability of the micro-storage feed are ensured. In the whole, the bacterial enzyme synergistic preparation has high feasibility in the use of citrus residues, pineapple residues, tea residues and other mixed materials for fermenting poultry feeds.

Application example 1

Test field and test animal selection: 90 hundred feather broilers (weighing 930-970g or so) of 21 days old are randomly selected and divided into 3 groups (noted as test ① group, test ② group and test ③ group) of 30 heads each, and the male and female groups are random. The 21-day-old cherry valley ducks (weight 1300-1400 g) were randomly selected 90 and divided into 3 groups (noted as test ④, test ⑤ and test ⑥), 30 heads per group, and the male and female were random.

(II) feeding scheme of meat poultry: after the physicochemical and sensory indexes of the examples and the comparative examples are comprehensively analyzed, the micro-storage feeds fermented in the first example and the fifth example are selected for the experiment of the breeding effect of the meat poultry, and the specific scheme is as follows:

The test ① group and the test ④ group are respectively fed with conventional feeds for the hundred-feather chickens and cherry valley ducks; both the test ② group and the test ⑤ group are fed with the fruit residue-containing micro-storage feed for poultry, which is fermented according to the first embodiment, namely the bacterial enzyme synergistic preparation; both the test ③ group and the test ⑥ group were fed with the fruit residue-containing micro-feed for poultry fermented according to comparative example five, commercial EM bacteria starter. The in-loop feeding mode is adopted, the conventional daily ration or the micro-storage feed containing fruit residues for poultry is fed for 3 times in the morning, in the middle and in the evening every day, the feed is freely ingested, and the residual material amount and free drinking water are ensured after the feeding is finished every day. The feeding experimental period was 21 days.

And (III) monitoring feeding effect: and counting the total head average feed intake, daily gain, feed weight ratio, meat yield and morbidity of each group of meat poultry of the test meat poultry within 21 days, and determining the feeding effect of the micro-storage feed on the meat poultry after studying and judging the feeding data. The experimental results are shown in Table 3.

TABLE 3 results of feeding experiments for meat poultry

As can be seen from table 3, the feeding results of white feather broilers and cherry valley ducks are consistent, namely, compared with the conventional feed only, the daily feed intake of broilers and cherry valley ducks is obviously improved after the poultry fruit residue-containing micro-storage feed is fed by the microbial enzyme synergistic agent or the commercial EM bacteria starter, and the feed-meat ratio is reduced. Meanwhile, the fruit dreg-containing micro-storage feed for feeding poultry also effectively reduces the morbidity (mortality) of broiler chickens and meat ducks, and can promote the immunity of animals to a certain extent. Finally, after the fruit dreg-containing micro-storage feed for poultry is fed, the total evisceration and the meat yield of broiler chickens and meat ducks are improved, and the feeding conversion rate of the micro-storage feed is correspondingly improved. In general, the fruit residue-containing micro-storage feed for poultry, which is fermented by adopting the bacterial-enzyme synergistic preparation, can meet the feeding requirements of broiler chickens and meat ducks, and has lower cost compared with the conventional feed, and is worthy of popularization and application.

Application example 2

Test field and test animal selection: the roman pink shell layer chickens 90 which are about 240 days old and have stable eggs are randomly selected and divided into 3 groups (marked as a test ① group, a test ② group and a test ③ group), and 30 heads are respectively arranged in each group. The golden ducks 90 which are about 240 days old and have stable eggs are randomly selected and divided into 3 groups (marked as a test ④ group, a test ⑤ group and a test ⑥ group), and 30 heads are respectively arranged in each group.

(II) feeding scheme of egg-laying poultry: after physical, chemical and sensory indexes of the examples and the comparative examples are comprehensively analyzed, the micro-storage feeds fermented in the first example and the fifth example are selected for carrying out an egg-laying fowl cultivation effect experiment, and the specific scheme is as follows:

The test ① group and the test ④ group are respectively fed with conventional feeds of roman pink shell layer chicken and golden ducks; both the test ② group and the test ⑤ group are fed with the fruit residue-containing micro-storage feed for poultry, which is fermented according to the first embodiment, namely the bacterial enzyme synergistic preparation; both the test ③ group and the test ⑥ group were fed with the fruit residue-containing micro-feed for poultry fermented according to the comparative example one, commercial EM bacteria starter. The mode of raising chicken cages or duck cages is adopted, 3 cages are fed, 3 conventional daily ration or fruit residue-containing micro-storage feed for poultry are fed in the morning, in the middle and in the evening each day, free feeding is realized, and the residual material amount and free drinking water are ensured after the daily feeding is finished. The feeding experimental period was 90 days.

And (III) monitoring feeding effect: and (3) counting indexes such as total feed intake, egg laying quantity, egg laying rate, egg average weight, egg quality, egg poultry morbidity and the like of the test egg poultry within 90 days, and determining the feeding effect of the micro-storage feed on the egg poultry after studying and judging the feeding data. The experimental results are shown in Table 4.

TABLE 4 results of feeding experiments for egg-laying birds

Note that: * The more representative egg yolk color was darker, the same number represents no difference in color to the naked eye.

As can be seen from Table 4, the feeding results of the Roman pink shell layer chicken and the Jinding duck are consistent, namely, compared with the conventional feed only, the daily feed intake of the layer chicken and the egg duck is improved after the fruit residue-containing micro-storage feed for the poultry is fed with the bacterial enzyme synergistic preparation or the commercial EM bacterial starter is fermented, the morbidity is reduced, and the immunity of the egg poultry is improved, and particularly, the bacterial enzyme synergistic preparation micro-storage feed is more obvious. In addition, after the fruit dreg-containing micro-storage feed for poultry is fed with the bacteria and enzyme synergistic preparation for fermentation, the laying rate of the egg poultry, the average weight of eggs and the quality of eggs are improved to different degrees, and the micro-storage feed has obvious gain effect on feeding the egg poultry. In conclusion, the conclusion can be drawn that the poultry fruit residue-containing micro-storage feed fermented by the bacterial enzyme synergistic preparation can meet the feeding requirements of laying hens and laying ducks, has lower cost compared with the conventional feed, and is worthy of popularization and application.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims

1. The synergistic bacterial enzyme preparation is characterized by comprising 30-60 parts of lactobacillus delbrueckii subspecies, 10-30 parts of candida utilis, 10-30 parts of enterococcus faecium, 300-500 parts of cellulase and 100-200 parts of pectase in parts by weight.

2. The synergistic formulation of claim 1, comprising, by weight, 40-50 parts of lactobacillus delbrueckii subspecies lactis, 15-25 parts of candida utilis, 15-25 parts of enterococcus faecium, 350-450 parts of cellulase and 125-175 parts of pectase.

3. The synergistic formulation of claim 1 or 2, wherein the lactobacillus delbrueckii subspecies lactis is lactobacillus delbrueckii subspecies lactis (Lactobacillus delbrueckii subsp. Lactis) Dangxiong LB VIII strain, which is preserved in China Center for Type Culture Collection (CCTCC), with a preservation number of cctccc No. M2023396;

preferably, the Candida utilis is Candida utilis (Candida utilis) XZ-CU01 strain which is preserved in China center for type culture collection (cctccc), and the preservation number is cctccc No. M2023299;

4. A synergistic formulation of claim 1 to 3, wherein the viable count of lactobacillus delbrueckii subspecies lactis, candida utilis and enterococcus faecium is equal to or greater than 2 x 10 ¹¹ CFU/g, preferably the enzyme activity of the pectase is equal to or greater than 60000U/g, more preferably the enzyme activity of the cellulase is equal to or greater than 10000U/g.

5. A method for preparing the synergistic formulation of bacterial enzymes according to any one of claims 1 to 4, comprising the steps of: culturing the Lactobacillus delbrueckii subspecies lactis, the candida utilis and the enterococcus faecium respectively, and mixing the obtained bacterial powder with pectase powder and cellulase powder to obtain the bacterial enzyme synergistic preparation.

6. Use of the synergistic formulation of a bacterial enzyme according to any one of claims 1 to 4 or the synergistic formulation of a bacterial enzyme according to the method of preparation of claim 5 in fermented micro-feed.

7. The use of a synergistic formulation of claim 6 in fermented micro-feed comprising the use in fermented poultry micro-feed.

8. A micro-storage feed, characterized in that the raw materials of the micro-storage feed comprise fruit residues, tea residues, corn flour, soybean meal, feed-grade stone powder and amino acid mixture, and the microbial enzyme synergistic agent obtained by the preparation method of any one of claims 1-4 or the microbial enzyme synergistic agent obtained by the preparation method of claim 5.

9. The micro-feed of claim 8, wherein the fruit residue comprises citrus residue, and/or pineapple residue.

10. The micro-feed of claim 8 or 9, wherein the amino acid mixture comprises L-lysine hydrochloride, DL-methionine and L-threonine;

11. The micro-storage feed according to any one of claims 8 to 10, wherein the weight parts of the citrus pulp, the pineapple pulp, the tea leaves and the corn meal, the feed-grade stone powder and the amino acid mixture are 15 to 25 parts, 10 to 15 parts, 5 to 10 parts, 3 to 5 parts and 1 to 1.5 parts;

12. The micro-feed according to any one of claims 8 to 11, wherein the mass ratio of the microbial enzyme synergistic formulation to the total mass of the mixture comprising fruit residues, tea residues, corn meal, bean pulp, feed grade stone dust and amino acid mixture is 0.05 to 0.15%o, preferably the mass ratio of the microbial enzyme synergistic formulation to the total mass of the mixture comprising fruit residues, tea residues, corn meal, bean pulp, feed grade stone dust and amino acid mixture is 0.1 to 0.12%o.

13. A method for producing a micro-feed according to any one of claims 8 to 12, which comprises adding the microbial enzyme synergistic formulation according to any one of claims 1 to 4 or the microbial enzyme synergistic formulation produced according to claim 5 to a mixed substance comprising fruit residues, tea residues, corn flour, soybean meal, feed-grade stone dust and an amino acid mixture for post-fermentation.

14. The method for preparing micro-feed according to claim 13, wherein the fermentation mode of the preparation method is anaerobic fermentation, preferably the fermentation temperature is 25-30 ℃.