A bacterial enzyme synergistic fermented feed containing stevia residue and application thereof

Abstract

The invention provides a bacterial enzyme synergistic fermented feed containing stevia residue and application thereof. The feed comprises 38-43 parts of stevia residue pre-fermented by Aspergillus niger, 6-12 parts of corn, 4.5-12 parts of soybean meal, 12-20 parts of DDGS, 11-20 parts of rice bran meal, 0-15 parts of bran, 0-8 parts of stone powder, 0-0.3 parts of 30% betaine, 0-0.7 parts of calcium hydrogen phosphate, 0-0.5 parts of sodium chloride and 1-2 parts of glucose, and a composite bacterial agent is added in an amount of 0.05-0.1% of the stevia residue, mixed evenly, put into a breathing bag, sealed, and fermented for 5-7 days at 25-30 DEG C. The feed can improve the production performance of laying hens and the air quality in laying hen houses; it can increase the slaughter rate and lean meat rate of pigs and reduce the backfat thickness and incidence rate; it can effectively increase the dry matter intake and milk production of dairy cows, reduce the somatic cell count in milk, and reduce the incidence of mastitis in dairy cows.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed, and in particular relates to a bacterial enzyme-cooperative fermented feed containing stevia residue and application thereof. Background Art

[0002] Stevia sugar products produce a large amount of stevia residue in the sugar production process. my country produces more than 200,000 tons of stevia residue each year. Stevia residue is industrial waste and the cost of treatment is very high. At present, the main treatment methods for stevia residue are incineration and composting. Incineration pollutes the environment, and composting prevents the nutrients and active substances in it from being reasonably and efficiently utilized, resulting in a waste of resources. How to effectively treat stevia residue is not only related to environmental issues, but also affects the extension of the stevia deep processing industry chain and the benefits of enterprises.

[0003] Research data show that stevia residue contains a variety of nutrients such as crude protein, crude fiber, crude fat, trace elements and vitamins, and the total flavonoid content is as high as 4%. It is very valuable as feed or additive. Studies have shown that the appropriate addition of stevia can improve the index of animal immune organs and enhance the immune effect. Using 5% as poultry feed can prevent poultry diarrhea, regulate poultry digestion function, and increase egg production rate; adding it to feed for dairy cows and sheep can increase the sweetness of milk, improve milk quality and trace elements, amino acids and other substances in milk, and have a certain promoting effect on milk production. For beef cattle, it can improve the quality and taste of meat. However, stevia residue is a high-humidity material. The added value of selling it directly or after drying as a feed product is low, and the enthusiasm of enterprises is not high. It is urgent to develop high-value-added products. In addition, stevia residue has a rough texture, poor palatability, low acid-soluble protein content, and high fiber content, which affects its feeding value. The synergistic fermentation of bacteria and enzymes can effectively increase the acid-soluble protein content in stevia residue, reduce its crude fiber content, and produce some digestive enzymes and vitamins, which greatly improves its nutritional value and palatability, and explores a new way to expand feed resources and reduce feed costs. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a bacterial enzyme synergistic fermentation feed containing stevia residue.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A bacterial enzyme cooperative fermented feed containing stevia residue comprises raw materials, measured by mass fraction, of 38-43 parts of stevia residue pre-fermented by Aspergillus niger, 6-12 parts of corn, 4.5-12 parts of soybean meal, 12-20 parts of DDGS, 11-20 parts of rice bran meal, 0-15 parts of bran, 0-8 parts of stone powder, 0-0.3 parts of 30% betaine, 0-0.7 parts of calcium hydrogen phosphate, 0-0.5 parts of sodium chloride and 1-2 parts of glucose, a composite bacterial agent is added in an amount of 0.05-0.1% of the total mass of the above raw materials, the mixture is mixed, the mixture is sealed in a breathing bag, and the mixture is fermented at 25-30°C for 5-7 days to obtain the bacterial enzyme cooperative fermented feed.

[0007] In the bacterial-enzyme synergistic fermented feed as described above, preferably, the composite bacterial agent comprises Lactobacillus casei, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae, wherein the strain preservation numbers of Lactobacillus casei, Lactobacillus plantarum P-8 and Candida utilis are CGMCC No.18913, CGMCC No.5468 and CGMCC No.18917 respectively.

[0008] In the above-mentioned bacterial enzyme synergistic fermentation feed, preferably, the bacteria Lactobacillus casei, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae are freeze-dried powders, which are mixed in a mass ratio of 1-3:1-3:5-10:5-10, and the number of viable bacteria of Lactobacillus casei, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae in the composite bacterial agent is ≥1×10 9 CFU / g, ≥1×10 9 CFU / g, ≥1×10 8 CFU / g and ≥1×10 8 CFU / g.

[0009] As described above, in the bacterial enzyme synergistic fermented feed, preferably, the stevia residue after Aspergillus niger pre-fermentation is the material obtained by fully mixing stevia residue, molasses and urea in a mass ratio of 93:6:1 and inoculating Aspergillus niger for fermentation 36 to 48 hours.

[0010] In the above-mentioned bacterial enzyme synergistic fermented feed, preferably, the spore content of Aspergillus niger is ≥ 15 billion / g, and the feed is inoculated at 0.01% to 0.05% of the mass of stevia residue, and the fermentation temperature is lower than 40°C.

[0011] The above-mentioned application of bacterial enzyme-co-fermented feed containing stevia residue in animal breeding.

[0012] In the application as described above, preferably, the bacterial enzyme synergistically fermented feed is added to the diet of laying hens at a mass fraction of 3% to 5%.

[0013] In the above application, preferably, the bacterial enzyme synergistically fermented feed is added to the diet of fattening pigs at a mass fraction of 10-15%.

[0014] In the application as described above, preferably, the bacterial enzyme synergistically fermented feed is added to the daily diet of dairy cows at a mass fraction of 10% to 20%.

[0015] The beneficial effects of the present invention are:

[0016] The present invention provides a bacterial enzyme cooperative fermented feed containing stevia residue, which uses stevia residue as one of the raw materials to produce fermented feed, which can improve the feeding value of stevia residue and increase the crude protein and acid-soluble protein content of the fermented feed, reduce fiber, NDF and ADF, improve the palatability of the feed, greatly improve the nutritional value of stevia residue, and turn stevia residue from waste into treasure.

[0017] The bacterial enzyme synergistically fermented feed containing stevia residue provided by the present invention can be used for raising poultry and laying hens, thereby improving the production performance of laying hens and improving the air quality in laying hen houses; it can be used for raising fattening pigs, thereby increasing the slaughter rate and lean meat rate of pigs and reducing the backfat thickness and incidence rate; it can be used for raising dairy cows, thereby effectively increasing the dry matter intake and milk production of dairy cows, reducing the somatic cell count in milk, and reducing the incidence rate of mastitis in dairy cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the pH value of acid production by Lactobacillus casei BFC190201 over time;

[0019] Figure 2 The fermentation supernatant of Lactobacillus casei BFC190201 has the function of inhibiting mold activity. DETAILED DESCRIPTION

[0020] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] Example 1 Screening of lactic acid bacteria attached to stevia residue

[0023] Stevia residue (DM=60%) naturally piled in the factory area of ​​Shandong Zhucheng Haotian Pharmaceutical Co., Ltd. was used as a strain source for lactic acid bacteria enrichment culture. 1.0g of stevia residue was added to 9mL MRS culture solution (sucrose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract powder 5g / L, sodium acetate 5g / L, ammonium citrate 2g / L, K2HPO4 2g / L, MgSO4·7H2O0.58g / L, MnSO4·4H2O 0.25g / L, Tween 80 1.0mL / L, pH adjusted to 6.3, sterilized at 115℃ for 20min) for enrichment culture: after static culture at 30℃ for 24h, the culture was transferred at an inoculum amount of 1% (volume fraction ratio) and continuously subcultured for 20 generations to obtain a lactic acid bacteria group with stable properties and strain composition.

[0024] The enriched culture fluid was diluted and spread on MRS agar medium at 30℃ for 36h, and a single colony was picked to isolate the pure strain. The isolated pure strain was inoculated into MRS liquid medium and cultured at 30℃ for 24h, and the strain BFC190201 with the highest L-lactic acid production was selected to complete the screening. After measurement, the production of L-lactic acid and D-lactic acid in the culture broth of the strain was 36.3 g / L and 2.56 g / L, respectively. The DNA was extracted and PCR amplified with universal primers 27F / 1492R. The sequencing result was shown in SEQ ID NO.1. It was identified as Lactobacillus casei after comparison and preserved in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Administration, with the preservation address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Lactobacillus casei, the preservation number is CGMCC No.18913, and the preservation date is November 7, 2019.

[0025] Acid production test: Pick a single colony of strain BFC190201 and inoculate it into MRS medium (sucrose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, sodium acetate 5g / L, ammonium citrate 2g / L, K2HPO4 2g / L, MgSO4·7H2O0.58g / L, MnSO4·4H2O 0.25g / L, Tween 80 1.0mL / L, pH adjusted to 6.3, sterilized at 115℃ for 20min), culture at 30℃ for 48h, and measure the pH value of the bacterial solution every 2h. The results of measuring the pH value within 48h are shown in Figure 1 The results showed that the strain could quickly produce acid and lower the pH value.

[0026] Antifungal test: Inoculate mold spores onto the slope of PDA solid culture medium and culture at 37°C for 4 days (based on the growth of mold spores). Rinse the slope with sterile water containing 0.05% Tween-80 by volume to prepare a suspension. Inoculate Lactobacillus casei BFC190201 into liquid MRS culture medium, culture at 37°C for 24 hours, centrifuge at 4,000r / min for 15 minutes, and aspirate 0, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL, 1.2mL, 1.4mL, 1.6mL, 2.0mL and 20mL of PDA agar culture medium, mix well, pour into a plate, and after solidification, aspirate 30μL of the spore suspension into the solid culture medium, spread evenly, and culture at 28°C to observe the antibacterial ability of Lactobacillus casei BFC190201. Results Figure 2 shown. Figure 2 The first culture dish is a solid culture medium inoculated with only spore suspension but not inoculated with supernatant, i.e., 0 mL of supernatant was absorbed. The following are marked: 1% (0.2 mL of supernatant was absorbed), 2% (0.4 mL of supernatant was absorbed), 3% (0.6 mL of supernatant was absorbed), 4% (0.8 mL of supernatant was absorbed), 5% (1.0 mL of supernatant was absorbed), 6% (1.2 mL of supernatant was absorbed), 7% (1.4 mL of supernatant was absorbed), 8% (1.6 mL of supernatant was absorbed), 9% (1.8 mL of supernatant was absorbed), and 10% (2.0 mL of supernatant was absorbed), indicating that the metabolites of Lactobacillus casei BFC190201 have the characteristics of inhibiting the growth of mold spores, and the higher the concentration, the stronger the inhibition ability.

[0027] Pathogenic bacteria inhibition test: Inoculate 3 pathogenic bacteria, Escherichia coli, Salmonella and Staphylococcus aureus, into 50mL LB medium and culture at 37℃ and 200r / min for 18h. Take a culture dish with a diameter of about 90mm, add 20mL of melted LB medium, let it be evenly distributed in the plate, and let it solidify for a while as the bottom layer. Then take a semi-solid nutrient agar medium (agar content is 1%) and heat it to melt, wait for it to cool to 48-50℃, add 0.1mL of indicator bacteria suspension to every 50mL of semi-solid, and add 5mL of semi-solid medium to the bottom layer so that it can be evenly distributed on the first layer as the second layer. Then, four Oxford cups were placed in the plate with the same spacing. After cooling, the fermentation supernatant of Lactobacillus casei BFC190201 after centrifugation at 8,000 r / min for 5 minutes was taken, and 200 μL of the sample was added to the Oxford cup in each double-layer plate. After culturing at 37°C for 24 hours, the diameter of each inhibition zone was measured for evaluation. The results are shown in Table 1.

[0028] Table 1 The inhibitory effect of the supernatant of BFC190201 fermentation broth on pathogens

[0029] Bacteria Antibacterial diameter mm Staphylococcus aureus 16.28±0.26 salmonella 15.88±0.24 Escherichia coli 15.36±0.28

[0030] The results showed that the metabolites of Lactobacillus casei BFC190201 could broadly inhibit the growth of Staphylococcus aureus, Salmonella, Escherichia coli and mold.

[0031] Example 2

[0032] The preserved Lactobacillus casei BFC190201 is inoculated into an MRS liquid culture medium, and statically cultured at 25-37°C for 18-24 hours to obtain a primary seed liquid; the primary seed liquid is transferred into a new MRS culture medium again at an inoculation amount of 5%-10% (v / v) for secondary activation, and statically cultured for 18-24 hours to obtain a secondary seed liquid; the secondary seed liquid is separately inoculated into a fermentation tank culture medium at an inoculation amount of 5%-10% (v / v), and statically cultured at a temperature of 25-37°C for 18-24 hours to obtain a Lactobacillus casei BFC190201 fermentation liquid. The fermentation liquid of Lactobacillus casei BFC190201 was centrifuged at 8000r / min for 10min at 4℃ to obtain bacterial sludge. Skim milk was added to the bacterial sludge at a ratio of 1:2-4 (m / v). The mixture was shaken and mixed on a vortex oscillator and vacuum freeze-dried to obtain freeze-dried powder of Lactobacillus casei BFC190201. The effective viable cell count was ≥1×10 12 CFU / g.

[0033] Lactobacillus plantarum P-8 was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on November 18, 2011, with the deposit number of CGMCC No. 5468. (The strain has been disclosed in the Chinese invention patent "A probiotic that can promote SIgA secretion in the intestine and its detection method" with the publication number of CN102618461B). The strain in the deposited state was activated step by step as described above and then freeze-dried to obtain Lactobacillus plantarum P-8 freeze-dried powder, with an effective viable count of ≥1×10 12 CFU / g.

[0034] The isolated and identified Candida utilis BFC190301 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on November 7, 2019. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Candida utilis, and the deposit number is CGMCC No.18917. The preserved Candida utilis BFC190301 was diluted with sterile water and mixed to prepare the original bacterial liquid, and the original bacterial liquid was spread on YPD solid medium (glucose 2%, peptone 2%, yeast extract powder 1%, agar 1.5%), and cultured at 30-35°C for 3-5 days, and a single colony was picked and inoculated on a YPD solid medium slant test tube. After the strains grew all over the slant of the test tube, it was transferred once for use; strains were picked from the slant culture medium, inoculated into YPD liquid culture medium, shaken evenly, and placed in a shaking incubator for shaker fermentation culture for 30-3 The method comprises the following steps: culturing the mixture in a shaking table at 5° C. for 20 to 24 hours at a rotation speed of 130 to 150 r / min to obtain a primary seed solution; transferring the primary seed solution into a new YPD medium at an inoculation amount of 5% to 10% (v / v) for secondary activation, and obtaining a secondary seed solution after standing for 18 to 24 hours; and separately inoculating the secondary seed solution into a fermentation tank medium at an inoculation amount of 5% to 10% (v / v), culturing the mixture at 30 to 35° C. for 20 to 24 hours at a rotation speed of 130 to 150 r / min to obtain a fermentation solution of Candida utilis BFC190301. The fermentation broth of Candida utilis BFC190301 was centrifuged at 8000r / min for 10min at 4℃ to obtain bacterial sludge. Skim milk was added to the bacterial sludge at a ratio of 1:2-4 (m / v), and the mixture was shaken and mixed on a vortex oscillator. The mixture was then vacuum-freezed to obtain freeze-dried powder of Candida utilis BFC190301. The effective viable count was ≥1×10 10 CFU / g.

[0035] Saccharomyces cerevisiae was provided by Angel Yeast Co., Ltd., with a viable count of ≥ 2 × 10 10 CFU / g.

[0036] The freeze-dried powder of Lactobacillus casei BFC190201, the freeze-dried powder of Lactobacillus plantarum P-8, the freeze-dried powder of Candida utilis and the freeze-dried powder of Saccharomyces cerevisiae are mixed in a mass ratio of 2:1:8:6, and the freeze-dried powder mixture is mixed with an organic carrier in a mass ratio of 1:58 to obtain a composite bacterial agent, wherein the organic carrier is any one or more of glucose, sucrose, fructose or dextrin. The number of viable cells of Lactobacillus casei BFC190201, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae in the final composite bacterial agent is ≥1×10 9 CFU / g, ≥1×10 9 CFU / g, ≥1×10 8CFU / g, ≥1×10 8 CFU / g.

[0037] Example 3 Processing of fermented feed containing stevia residue

[0038] (1) Aerobic fermentation pretreatment of stevia residue

[0039] Stevia residue (DM = 30%), molasses and urea were fully mixed in a mass ratio of 93:6:1, inoculated with Aspergillus niger, placed in a cement pool or fermentation tank, and fermented aerobically for 42 hours, and the temperature was raised to 40° C. and tossed to ensure that the fermentation temperature was below 40° C. As the fermentation time increased, the true protein content increased and the crude fiber content decreased. The results are shown in Table 2. The true protein content increased by 13.77%, 36.96% and 51.12% after fermentation for 24 hours, 36 hours and 48 hours, respectively.

[0040] Table 2

[0041] True Protein % Crude fiber % Before fermentation 15.61 18.32 Fermentation 24h 17.76 17.54 Fermentation 36h 21.38 15.55 Fermentation 48h 23.59 12.84

[0042] Among them, Aspergillus niger was provided by Shandong Kangyuan Biotechnology Co., Ltd., with a spore content of ≥15 billion / g, and the inoculation amount during the culture preparation process was 0.01% to 0.05%. This strain has a strong enzyme production ability, including cellulase, β-glucanase, xylanase, glucanase, β-mannanase, cellulase, protease, phytase and pectinase. After 48 hours of fermentation, the composition and activity of the enzymes in the stevia residue were measured as shown in Table 3.

[0043] Table 3 Composition and activity of enzymes in stevia residue after 48h fermentation

[0044] type Enzyme activity U / g Cellulase 7.5±0.37 Xylanase 62.5±1.22 β-Glucanase 5.0±0.73 β-Mannanase 10.0±0.65 Pectinase 7.5±0.29 Phytase 7.5±0.18 Protease 25.0±0.15

[0045] (2) Stevia residue fermented feed raw materials

[0046] The stevia residue pretreated by Aspergillus niger is added with 0.1% of the composite bacterial agent prepared by the method in Example 2, wherein the freeze-dried powder of Lactobacillus casei BFC190201, the freeze-dried powder of Lactobacillus plantarum P-8, the freeze-dried powder of Candida utilis and the freeze-dried powder of Saccharomyces cerevisiae are prepared in a mass ratio of 2:1:8:6, and a carrier: glucose is added, and the composite bacterial agent is prepared by mixing the total amount of the freeze-dried powder with glucose in a mass ratio of 1:58, and fermented in a breathing bag at 25-30° C. for 5-7 days to obtain the fermented stevia residue stevia residue fermented feed raw material.

[0047] The components of stevia residue (i.e., without any treatment) before and after fermentation were measured. The results are shown in Table 4. After aerobic and anaerobic fermentation, the moisture content of stevia residue decreased to about 60% suitable for fermentation by beneficial microorganisms. On a fresh basis, the crude protein and acid-soluble protein contents were significantly increased. As shown in Table 5, on a dry matter basis, the crude ash, calcium, and phosphorus contents in stevia residue before and after fermentation did not change significantly, but the crude protein and acid-soluble protein contents increased by 46.90% and 345.79%, respectively, and the crude fiber, neutral detergent fiber (NDF), and acid detergent fiber (ADF) decreased by 19.11%, 13.36%, and 5.57%, respectively. This shows that fermentation greatly improves the utilization rate of nutrients in stevia residue.

[0048] Table 4 Changes in components of stevia residue before and after fermentation (fresh basis)

[0049] index Stevia residue before fermentation Fermented stevia residue Moisture (%) 71.7 60.51 Crude ash (%) 2.11 3.01 Crude protein (%) 5.54 11.76 Acid soluble protein (g / 100g) 0.09 0.56 Crude fiber (%) 6.51 7.35 Neutral detergent fiber (%) 15.34 18.55 Acid detergent fiber (%) 7.42 9.78 calcium(%) 0.59 0.84 Total phosphorus (%) 0.04 0.06 Crude fat (%) 0.62 0.89

[0050] Table 5 Changes in components of stevia residue before and after fermentation (dry matter basis)

[0051] index Stevia residue before fermentation Fermented stevia residue DM (dry matter) (%) 28.30 39.49 Crude ash (%) 7.46 7.63 Crude protein (%) 19.58 20.04 Acid soluble protein (g / 100g) 0.32 1.42 Crude fiber (%) 23.00 18.61 Neutral detergent fiber (%) 54.20 46.96 Acid detergent fiber (%) 26.22 24.76 calcium(%) 2.08 2.13 Total phosphorus (%) 0.14 0.14 Crude fat (%) 2.19 2.24

[0052] Example 4-8: Feed containing stevia residue and bacterial enzyme synergistic fermentation

[0053] According to the mass fractions shown in Table 6, stevia residue aerobically fermented for 48 hours, i.e. stevia residue pre-fermented by Aspergillus niger, corn, soybean meal, DDGS, rice bran meal, bran, stone powder, 30% betaine, calcium hydrogen phosphate, sodium chloride and glucose were weighed, and the composite bacterial agent used in Example 3 was added.

[0054] Table 6 Formula of fermented feed containing stevia residue

[0055]

[0056] After being fully mixed, the mixture was placed in a breathing bag and sealed, and fermented at 25-30°C for 5-7 days to obtain fermented feed. The fermented feed was tested and the physical and chemical indicators were shown in Table 7.

[0057] Table 7 Physical and chemical indicators of feed containing stevia residue after fermentation

[0058]

[0059]

[0060] Example 9 Effect of using stevia residue and bacterial enzyme to ferment feed on laying hens

[0061] 367-day-old Hailan brown laying hens were selected, and 12,000 laying hens were randomly divided into three treatment groups, each with 4,000 hens, and each group was set up with 5 replicates. Four-layer ladder cages were used for free feeding and drinking water. They were vaccinated and immunized according to the immunization program and disinfected regularly. The experimental group I was the control group, which was fed with a basic diet, and the groups II and III added 3% and 5% of the bacterial enzyme synergistic fermented feed prepared according to Example 8 to the basic diet, respectively. The formula was adjusted according to the nutritional needs of laying hens so that the nutritional composition of the daily diet formula of each group was basically the same.

[0062] 1 Effect of stevia residue fermented feed on laying hens' production performance

[0063] The results are shown in Table 8. It can be seen that on the 45th day of the experiment, the addition of 3% and 5% stevia residue fermented feed in the diet showed an upward trend in egg production and daily feed intake, a significant decrease in feed-egg ratio (P<0.05), a significant increase in egg weight (P<0.05), a decrease in egg breakage rate of 36.67% and 61.05%, and a decrease in mortality rate of 6.06% and 9.68%, respectively.

[0064] Table 8 Effects on laying hen production performance

[0065]

[0066]

[0067] Note: Compared with the same industry indicators, * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01), the same as in the following table.

[0068] 2 Effect of stevia residue fermentation feed on air quality in laying hen houses

[0069] As shown in Table 9, compared with the control period, the carbon dioxide concentration increased significantly on the 35th day of the test period (P<0.05); the carbon monoxide, nitrous oxide, ammonia, and methane concentrations decreased significantly on the 15th and 35th days of the test period (P<0.01); compared with the 15th day of the test, the carbon monoxide, nitrous oxide, ammonia, and methane concentrations decreased significantly on the 35th day of the test (P<0.01). It can be seen that stevia residue fermented feed has the effect of improving the air quality in the laying hen house.

[0070] Table 9 Gas concentration in laying hen house

[0071]

[0072] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and the same lowercase letters indicate insignificant differences (P>0.05).

[0073] Example 10 Application effect of stevia residue bacterial enzyme synergistic fermentation feed on fattening pigs

[0074] 320 fattening pigs (Du × Chang × Da) weighing about 80 kg and in good health were selected and randomly divided into 4 groups, each with 4 pens and 20 pigs in each pen. The experimental group I was the control group, fed with a basic diet, and groups II, III and IV added 5%, 10% and 15% of the bacterial enzyme synergistic fermented feed (30% moisture) prepared in Example 4 to the basic diet, respectively. The formula was adjusted according to the nutritional needs of growing and fattening pigs so that the nutritional composition of the daily diet formula of each group was basically the same. The pre-trial period of the experiment was 3 days and the formal period was 4 weeks. Except for the different feeds fed to the four groups of pigs, other feeding conditions and management methods were consistent. During the trial period, artificial feeding was adopted, and the pigs were free to eat and drink water. The weekly feed intake of fattening pigs was recorded in pens. On the first day of the test and the morning of the fourth week of the test, the pigs were weighed on an empty stomach, and the average weight was calculated in pens. At the end of the experiment, one pig was randomly selected from each pen in each group for slaughter, and the slaughter rate and meat quality were measured.

[0075] 1. Effect on the production performance of fattening pigs

[0076] The measurement results are shown in Table 10. Adding 5%, 10% and 15% stevia residue fermented feed to the diet will increase the dry matter intake of finishing pigs, increase the average daily weight gain, and reduce the feed-to-weight ratio. Among them, the experimental group with 10% addition had the highest daily weight gain and the lowest feed-to-weight ratio.

[0077] Table 10 Effects on production performance of fattening pigs

[0078]

[0079] Note: Different letters in the same row indicate significant differences at the level of P=0.05.

[0080] 2. Impact on slaughter performance

[0081] The measurement results are shown in Table 11. Adding 5%, 10% and 15% of stevia residue fermented feed to the fattening pig diet will increase the slaughter rate and lean meat rate and reduce the back fat thickness. When the addition amount is 10% and 15%, the slaughter rate, lean meat rate and back fat thickness are significantly different from the control group, but the difference between the experimental groups with 10% and 15% addition is not significant. Therefore, the 10% addition amount is preferred.

[0082] Table 11 Effects on slaughter performance

[0083]

[0084] Note: Different letters in the same row indicate significant differences at the level of P=0.05.

[0085] 3 Impact on health status

[0086] The incidence rate is (the number of pigs with disease in each group / the total number of pigs in each group) × 100%. The results are shown in Table 12, indicating that adding 5%, 10% and 15% of stevia residue fermented feed to the diet of finishing pigs can improve immunity and reduce the incidence rate. The more stevia residue fermented feed is added to the diet, the lower the incidence rate of pigs.

[0087] Table 12 Impact on health status

[0088]

[0089] Example 11 Effect of using stevia residue fermented feed on dairy cows

[0090] 100 high-yielding Holstein lactating cows with similar age, parity, milk yield and lactation days in good health were selected and randomly divided into 4 groups, 25 in each group. Group I was the control group, fed with a basic diet, and groups II, III and IV were fed with 10%, 15% and 20% of the fermented stevia residue feed (30% moisture) prepared in Example 8 added to the basic diet, respectively. The pre-test period was 1 week and the full test period was 7 weeks.

[0091] The test results of dry matter, milk production, somatic cell count and mastitis are shown in Table 13, indicating that the fermented feed of the present invention significantly increases the dry matter intake and milk production of dairy cows, reduces the somatic cell count in milk, and reduces the incidence of mastitis in dairy cows. The best effect is achieved when the usage accounts for 10% of the daily diet.

[0092] Table 13 Effects on dairy cow production performance

[0093]

[0094]

[0095] Note: Different letters in the same row indicate significant differences at the level of P=0.05. Sequence Listing <110> Boyide (Beijing) Biotechnology Co., Ltd. <120> A bacterial enzyme synergistic fermented feed containing stevia residue and application thereof <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1393 <212> DNA <213> Artificial Sequence <400> 1 tacgccaccg gcttcgggtg ttacaaactc tcatggtgtg acgggcggtg tgtacaaggc 60 ccgggaacgt attcaccgcg gcgtgctgat ccgcgattac tagcgattcc gacttcgtgt 120 aggcgagttg cagcctacag tccgaactga gaatggcttt aagagattag cttgacctcg 180 cggtctcgca actcgttgta ccatccattg tagcacgtgt gtagcccagg tcataagggg 240 catgatgatt tgacgtcatc cccaccttcc tccggtttgt caccggcagt cttactagag 300 tgcccaactg aatgctggca actagtcata agggttgcgc tcgttgcggg acttaaccca 360 acatctcacg acacgagctg acgacaacca tgcaccacct gtcattttgc ccccgaaggg 420 gaaacctgat ctctcaggtg atcaaaagat gtcaagacct ggtaaggttc ttcgcgttgc 480 ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc 540 aaccttgcgg tcgtactccc caggcggaat gcttaatgcg ttagctgcgg cactgaaggg 600 cggaaaccct ccaacaccta gcattcatcg tttacggcat ggactaccag ggtatctaat 660 cctgttcgct acccatgctt tcgagcctca gcgtcagtta cagaccagac agccgccttc 720 gccactggtg ttcttccata tatctacgca tttcaccgct acacatggag ttccactgtc 780 ctcttctgca ctcaagtttc ccagtttccg atgcgcttcc tcggttaagc cgagggcttt 840 cacatcagac ttaaaaaacc gcctgcgctc gctttacgcc caataaatcc ggataacgct 900 tgccacctac gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttggat 960 accgtcacgc cgacaacagt tactctgccg accattcttc tccaacaaca gagttttacg 1020 acccgaaagc cttcttcact cacgcggcgt tgctccatca gacttgcgtc cattgtggaa 1080 gattccctac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca atgtggccga 1140 tcaacctctc agttcggcta cgtatcatcg ccttggtgag ccgttacctc accaactagc 1200 taatacgccg cgggtccatc caaaagcgat agcttgcgcc atctttcagc caagaaccat 1260 gcggttcttg gatttatgcg gtattagcat ctgtttccaa atgttatccc ccacttaagg 1320 gcaggttacc cacgtgttac tcacccgtcc gccactcgtt ttaagttgaa tcacagtgca 1380 agcaccgttc atc 1393

Claims

1. A bacterial enzyme-co-fermented feed containing stevia residue, characterized in that: The raw materials include 38-43 parts of stevia residue pre-fermented by Aspergillus niger, 6-12 parts of corn, 4.5-12 parts of soybean meal, 12-20 parts of DDGS, 11-20 parts of rice bran meal, 0-15 parts of bran, 0-8 parts of stone powder, 0-0.3 parts of 30% betaine, 0-0.7 parts of calcium hydrogen phosphate, 0-0.5 parts of sodium chloride and 1-2 parts of glucose, and a composite bacterial agent is added in an amount of 0.05-0.1% of the total mass of the above raw materials. After mixing, the mixture is sealed in a breathing bag and fermented at 25-30°C for 5-7 days to obtain bacterial enzyme synergistic fermented feed. The stevia residue after Aspergillus niger pre-fermentation is a material obtained by fully mixing stevia residue, molasses and urea in a mass ratio of 93:6:1 and inoculating Aspergillus niger for fermentation 36 to 48 hours; The composite bacterial agent comprises lactobacillus casei, lactobacillus plantarum P-8, Candida utilis and saccharomyces cerevisiae, wherein the strain preservation numbers of lactobacillus casei, lactobacillus plantarum P-8 and Candida utilis are CGMCC No.18913, CGMCC No.5468 and CGMCC No.18917 respectively.

2. The bacterial enzyme synergistic fermented feed according to claim 1, characterized in that: The Lactobacillus casei, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae are freeze-dried powders, which are mixed in a mass ratio of 1-3:1-3:5-10:5-10. The number of viable bacteria of the freeze-dried powders of Lactobacillus casei, Lactobacillus plantarum P-8, Candida utilis and Saccharomyces cerevisiae in the composite bacterial agent after activation is ≥1×10 9 CFU / g, ≥1×10 9 CFU / g, ≥1×10 8 CFU / g and ≥1×10 8 CFU / g.

3. The bacterial enzyme cooperative fermentation feed according to claim 1, characterized in that: The spore content of Aspergillus niger is ≥15 billion / g, and the spores are inoculated at 0.01%-0.05% of the mass of stevia residue, and the fermentation temperature is 25-40°C.

4. Use of the bacterial enzyme-co-fermented feed containing stevia residue according to any one of claims 1 to 3 in raising animals.

5. The use according to claim 4, characterized in that: The bacterial enzyme synergistic fermented feed is added to the daily diet of laying hens at a mass fraction of 3% to 5%.

6. The use according to claim 4, characterized in that: The bacterial enzyme synergistic fermented feed is added to the daily diet of fattening pigs at a mass fraction of 10-15%.

7. The use according to claim 4, characterized in that: The mass fraction of the bacterial enzyme synergistic fermented feed added to the daily diet of dairy cows is 10% to 20%.

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