Stevia rebaudiana residue fermented feed additive for improving livestock and poultry productivity, and preparation method therefor

Abstract

A Stevia rebaudiana residue fermented feed additive for improving livestock and poultry productivity, a preparation method therefor, and a use thereof. Raw materials of the Stevia rebaudiana residue fermented feed additive comprise: Stevia rebaudiana residue, fermented brown sugar, a microbial agent, and water. The microbial agent is composed of EM bacteria, white rot fungus and Phanerochaete chrysosporium, in a ratio of 1:1:1.

Description

A stevia residue fermented feed additive for improving livestock and poultry production performance and a preparation method thereof Technical Field

[0001] The present invention relates to the field of Chinese medicinal fermented feed additives, and more particularly to a Chinese medicinal fermented feed additive for improving the production performance of meat ducks and laying hens and a preparation method thereof. Background Art

[0002] Stevia rebaudiana is a perennial herbaceous plant in the Asteraceae family native to South America. Its extracts possess numerous medicinal benefits, including antibacterial, antioxidant, lipid-lowering, blood sugar-lowering, and anti-tumor properties. Consequently, my country has begun to introduce, cultivate, and utilize stevia on a large scale. Stevia residue is a waste product generated during this utilization process. In southern Jiangxi alone, over 80,000 tons of stevia residue are produced annually. The vast majority of this waste is directly incinerated or landfilled, failing to be effectively utilized. This not only pollutes the environment but also represents a waste of resources. Bio-fermentation cell wall-breaking technology can degrade lignin, cellulose, and other substances in the cell walls of stevia residue, promoting the release of active ingredients within the cells. Therefore, developing a fermented stevia residue feed additive for use in the aquaculture industry would not only improve the comprehensive utilization of traditional Chinese medicine waste but also modulate the intestinal flora of animals, promote nutrient absorption, and increase feed conversion rates.

[0003] The characteristics and advantages of the present invention are that stevia residue is rich in crude fiber, crude protein, and bioactive ingredients, which can promote the proliferation of beneficial intestinal flora, inhibit the growth of harmful bacteria, and thus improve the balance of intestinal flora. It can also improve the body's antioxidant activity, immunity, and anti-stress ability by promoting the expression of related genes such as antioxidant enzymes. It is a feed raw material with great development and utilization value. Bio-fermentation wall-breaking technology can utilize microorganisms to degrade crude fiber, crude protein, and other macromolecular substances that are difficult to digest and absorb in fermented products into monosaccharides, small peptides, amino acids, and other small molecular substances that are easily absorbed by the body, thereby improving the absorption and utilization rate of nutrients. Adding certain traditional Chinese medicines and their preparations to feed can not only counteract the stress response of livestock and poultry, but also increase the immunity of livestock and poultry, increase feed intake, and improve meat quality. Overall, stevia residue is a feed raw material with extremely high development and utilization value, with broad application prospects and important economic and ecological benefits.

[0004] In summary, how to provide a stevia residue fermented feed additive is a problem that needs to be solved urgently by those skilled in the art.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides a stevia residue fermented feed additive and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention provides a stevia residue fermented feed additive. The stevia residue fermented feed additive is obtained by mixing and fermenting 100 parts stevia residue, 2.7 parts fermented brown sugar, 3 parts bacterial agent and 75 parts water as raw materials.

[0009] A method for preparing a stevia residue fermented feed additive comprises the following steps:

[0010] (1) Screening of fermentation agents: Agent 1 (EM bacteria), Agent 2 (EM bacteria: yeast powder = 5:1), and Agent 3 (EM bacteria: white rot fungi: Phanerochaete chrysosporium = 1:1:1) were selected as the agents. 60% water and 5% of each agent were added to stevia residue, mixed evenly, and allowed to ferment at 28°C for 24 days. Samples were taken every 3 days to detect the chlorogenic acid content in each sample, and a biological agent suitable for stevia residue was screened out;

[0011] (2) Screening of factors affecting the fermentation process: Using the bacterial agent screened in step (1), the inoculation amount, fermentation temperature, liquid-to-material ratio, brown sugar addition amount, soybean meal addition amount, and initial pH value of fermentation were used as screening targets, and the chlorogenic acid in stevia residue was quantitatively analyzed to screen out factors affecting the fermentation of stevia residue;

[0012] (3) Response surface optimization: Based on the factors affecting the fermentation quality of stevia residue screened in step (2), a response surface experimental design was conducted. Static fermentation was performed under conditions of a temperature of 4-37°C, a liquid-to-solid ratio of 60%-100%, and an addition of brown sugar of 1%-5%. Fermentation was performed under the optimized conditions to obtain a fermented stevia residue feed additive.

[0013] Furthermore, in the above step (1), the bacterial agent obtained by screening is: bacterial agent 3; in steps (2) and (3), the optimal fermentation process is: the inoculation amount is 3%, the amount of fermentation brown sugar added is 2.7%, the liquid-to-material ratio is 75%, the fermentation temperature is 25°C, and the fermentation time is 9 days.

[0014] The present invention also seeks to protect the use of the stevia residue fermented feed additive as described above or the stevia residue fermented feed additive prepared by the preparation method as described above in livestock and poultry breeding.

[0015] It can be seen from the above technical solution that compared with the existing technology, the beneficial effects achieved by the present invention are: after feeding stevia residue fermented feed additive to meat ducks, the feed-to-meat ratio of the ducks is reduced, and the average daily weight gain is significantly improved; after feeding laying hens, the laying performance and egg quality of the laying hens are improved, and the antibody concentration and antioxidant activity in the serum are also improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Corrected 19.04.2024 according to Rule 91] Figure 1 shows the microscopic structure of the cell wall of stevia residue fermentation. DETAILED DESCRIPTION

[0017] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0018] Example 1

[0019] Screening of fermentation agents

[0020] 5% of bacterial agent 1, bacterial agent 2, and bacterial agent 3 were added to stevia residue respectively, and then 60% of water was added, mixed evenly, and fermented at 28°C for 24 days. Samples were taken every 3 days to detect the chlorogenic acid content in the stevia residue after fermentation with different bacterial agents.

[0021] Table 1 Determination of chlorogenic acid content in stevia residue fermentation

[0022] The chlorogenic acid content of stevia residue increased after fermentation with all three inoculants. Compared to pre-fermentation, the chlorogenic acid content of stevia residue fermented with inoculant 3 increased by 79.53% on day 9. Fermenting stevia residue with inoculant 3 not only significantly increased the chlorogenic acid content but also significantly shortened the fermentation time. Therefore, in all subsequent fermentations, inoculant 3 was used for stevia residue, and the fermentation time was set at 9 days.

[0023] Example 2

[0024] Screening of influencing parameters of stevia residue wall-breaking fermentation process

[0025] The bacterial agent 3 was selected to carry out cell wall fermentation on stevia residue. The inoculation amount (1%, 3%, 5%, 7%, 9%), fermentation temperature (4°C, 28°C, 37°C), liquid-to-solid ratio (20%, 40%, 60%, 80%, 100%), brown sugar addition amount (1%, 2%, 3%, 4%, 5%), soybean meal addition amount (1%, 2%, 3%, 4%, 5%) and initial fermentation pH value (2, 3, 4, 5, 6, 7) were selected as screening targets. The chlorogenic acid in stevia residue was quantitatively analyzed to screen out the optimal fermentation conditions for stevia residue.

[0026] The chlorogenic acid content in the fermented stevia residue was higher than that in the unfermented stevia residue. The chlorogenic acid content in the fermented stevia residue was significantly increased when the inoculum level was 3%, the temperature was 28°C, and the liquid-to-solid ratio was 80%. The chlorogenic acid content in the fermented stevia residue was highest when the brown sugar addition level was 4%, but the difference in chlorogenic acid content was not significant when the brown sugar addition level was 3% and 4%. There was no significant difference in chlorogenic acid content at different soybean meal addition levels or initial fermentation pH values. To reduce costs, the conditions in subsequent fermentation experiments were: 3% inoculum level, 28°C temperature, 80% liquid-to-solid ratio, 3% brown sugar addition, no soybean meal addition, and no adjustment of the initial fermentation pH.

[0027] Example 3

[0028] Response surface optimization experimental design

[0029] Based on the screening of parameters influencing the fermentation process, a significant difference analysis was conducted on the chlorogenic acid content of stevia residue fermented under different conditions. Among all conditions, the liquid-to-material ratio, brown sugar addition, and temperature had the greatest impact on the stevia residue wall-breaking fermentation process. Therefore, appropriate levels of liquid-to-material ratio, brown sugar addition, and temperature were selected to optimize the fermentation conditions. Regression analysis of the experimental results was performed using Design-Expert software, resulting in a quadratic polynomial regression equation for chlorogenic acid content on coded variables A, B, and C, which was then validated against the fermentation results.

[0030] Table 2 Response surface experimental design and results

[0031] Table 3 Response surface variance analysis

[0032] The pairwise interactions between liquid-to-solid ratio, brown sugar addition, and temperature were all significant. Combined with the results of the response surface design experiment, the optimal conditions for stevia residue wall-breaking fermentation were: a liquid-to-solid ratio of 75.6%, a brown sugar addition of 2.7%, and a fermentation temperature of 25°C, with an estimated chlorogenic acid content of 4.888 mg / g. Repeated experiments confirmed that the actual chlorogenic acid content was not significantly different from the expected chlorogenic acid content. During large-scale fermentation, the peak chlorogenic acid content in stevia residue was advanced from 9 days to 6 days, but the chlorogenic acid content did not differ significantly.

[0033] Example 4

[0034] Changes in cell wall structure of stevia residue fermentation products

[0035] [Corrected 19.04.2024 according to Rule 91] Stevia residue was added with 3% clean water (CK) and 3% stevia residue cell-breaking fermentation inoculum. Under the optimal conditions, the fermentation was allowed to proceed for 15 days, with samples collected every three days. After sampling, the cell wall microstructure of each sample was observed using a scanning electron microscope. The results are shown in Figure 1.

[0036] [Corrected 19.04.2024 according to Rule 91] As shown in Figure 1, the cell walls of the stevia residue in both the CK and inoculum fermentation groups tended to degrade with increasing fermentation time, with the degree of cell wall degradation increasing with fermentation time. However, compared with the CK, the optimized fermentation group showed a higher degree of cell wall degradation, demonstrating a more significant effect.

[0037] Analysis of the activities of enzymes related to cell wall degradation in fermentation products of stevia rebaudiana

[0038] Stevia residue fermentation products at different stages (0, 3, 6, 9, 12 and 15 days) were taken as samples, and their laccase activity was determined using a laccase kit; their cellulase activity was determined using an endo-β-1,4-glucanase / cellulase kit.

[0039] Table 4 Results of determination of enzyme activities related to cell wall degradation of stevia residue

[0040] Weak laccase activity was detected on the sixth day of fermentation and continued to increase with fermentation time, reaching its peak on the 15th day. Cellulase activity showed an initial upward and then downward trend, reaching its peak on the 12th day, significantly higher than at other fermentation stages. Laccase and cellulase activity measurements in stevia residue fermentation revealed that cellulase primarily degraded the stevia residue cell walls during the first six days of fermentation, while after the ninth day, both enzymes synergistically degraded the cell walls.

[0041] Analysis of nutritional components of stevia residue fermentation products

[0042] The stevia residues before and after fermentation were taken to determine the contents of soluble sugar, crude fat, crude fiber, ash, acid-soluble protein, alkali-soluble protein, etc.

[0043] Table 5 Nutritional content of stevia residue before and after fermentation

[0044] Compared with pre-fermentation, the soluble sugar, crude fat, crude fiber, and other nutrients in the fermented stevia residue were significantly increased. Soluble sugar content increased by 52.79%, crude fat content increased by 56.96%, crude fiber content decreased by 43.09%, and other nutrients increased by 95.97%. Acid-soluble protein, alkali-soluble protein, and ash content increased by 153.79%, 8.71%, and 11.76%, respectively, but the increases were not significant.

[0045] Example 5

[0046] Effects of stevia residue fermented feed additive on production performance of broiler ducks

[0047] Three hundred and sixty healthy, 30-day-old Cherry Valley ducks were randomly divided into five groups, each containing three replicates and 24 ducks per replicate. The CK group served as a control and was fed a basal diet. Treatment 1 supplemented the diet with 1% stevia slag, Treatment 2 supplemented the diet with 2% stevia slag, Treatment 3 supplemented the diet with 1% fermented stevia slag, and Treatment 4 supplemented the diet with 2% fermented stevia slag for 38 days. Ducks were weighed before and after feeding, and average daily gain (ADR) (ADR = ADR / number of days fed) and feed-to-meat ratio (FCR = total feed intake / total ADR × 100%) were calculated during the experimental period.

[0048] Table 6 Effects of stevia residue on the production performance of broiler ducks

[0049] According to the test results of broiler duck production performance, compared with CK, the average daily weight gain of broiler ducks in treatment 1, treatment 2 and treatment 3 was significantly improved, which increased by 21.76%, 16.99% and 22.48% respectively; the feed-to-meat ratio of treatment 1, treatment 2 and treatment 3 was significantly lower than that of CK, which decreased by 17.99%, 14.25% and 18.22% respectively.

[0050] Example 6

[0051] Feeding of stevia residue fermentation products to laying hens

[0052] A total of 560 healthy, 50-week-old laying hens were randomly divided into seven groups: control (CK, fed a basal diet), treatment 1 (0.5% stevia residue added to the basal diet), treatment 2 (1.0% stevia residue added to the basal diet), treatment 3 (1.5% stevia residue added to the basal diet), treatment 4 (0.5% stevia residue fermentation product added to the basal diet), treatment 5 (1.0% stevia residue fermentation product added to the basal diet), and treatment 6 (1.5% stevia residue fermentation product added to the basal diet). Each group had eight replicates, with 10 hens per replicate. The experimental period was 28 days.

[0053] Effects of stevia residue on the production performance of laying hens

[0054] The number of eggs laid by each replicate was recorded daily, and the egg production rate was calculated. Eggs were weighed using an electronic balance and the average egg weight was calculated to the nearest 0.01 g. Feed intake was calculated for each replicate during the experiment, and the feed-to-egg ratio (feed-to-egg ratio = feed material / egg mass) was calculated. The number of laying hens that died in each replicate was counted, and the survival rate (%) was calculated (survival rate = number of surviving laying hens / total number of laying hens × 100). The number of soft and broken eggs was counted, and the defective rate (%) was calculated (defective rate = (number of soft eggs + number of broken eggs) / total number of eggs laid × 100).

[0055] Table 7 Effects of stevia residue on laying hen production performance

[0056] Results showed that stevia residue significantly affected feed intake in laying hens, with hens in Treatment 2 consuming the highest feed intake and hens in Treatment 1 consuming the lowest. Compared with the CK group, the egg production rate of hens in Treatment 1 increased by 0.18%, significantly higher than that in Treatment 3, but not significantly different from the other groups. The average egg weight of hens in Treatments 1, 2, 5, and 6 increased by 2.10%, 0.56%, 0.75%, and 0.73%, respectively. The feed-to-egg ratio of hens in Treatment 1 decreased by 4.69%, significantly lower than that in Treatments 3, 5, and 6, but not significantly different from the other groups. There were no significant differences in total egg weight, defective egg rate, or survival rate among the groups.

[0057] Effects of stevia residue fermentation on egg quality

[0058] After the feeding period, three eggs were randomly selected from each group. The vertical and horizontal diameters of the eggs were measured with a vernier caliper, and the egg shape index (egg shape index = horizontal diameter / vertical diameter) was calculated. The ends and middle of the eggs were measured with a spiral micrometer, and the average value was used to calculate the eggshell thickness to the nearest 0.01 mm. Eggshell strength was measured using an eggshell strength meter. Yolk color, albumen height, and Haugh units were measured using a multifunctional egg quality tester.

[0059] Table 8 Effect of stevia residue on egg quality

[0060] Compared with CK, the eggshell strength of laying hens in treatment 5 increased by 1.05%, which was significantly higher than that in treatment 4, but there was no significant difference with the other groups; the eggshell thickness of laying hens in treatment 1 increased by 2.94%, which was significantly higher than that in other groups; the yolk color of laying hens in treatment 4 and treatment 5 increased by 1.92% and 1.33% respectively, and the Haugh unit of laying hens in treatment 1 and treatment 4 increased by 3.76% and 1.63% respectively; the egg shape index of laying hens in treatment 1, treatment 2 and treatment 3 increased by 1.50%, 0.75% and 0.75% respectively, but the differences were not significant.

[0061] Effects of stevia residue fermentation products on immunoglobulin levels in laying hens' serum

[0062] After feeding, one chicken from each group was taken, blood was drawn from the wing vein, and the blood was centrifuged at 4000 rpm. The serum was aliquoted. The concentrations of infectious rhinitis antibodies, IgA, IgM, and IgY in the serum of laying hens were measured using chicken infectious rhinitis antibody, chicken immunoglobulin IgA, immunoglobulin IgM, and immunoglobulin IgY detection kits.

[0063] Table 9 Effects of stevia residue fermentation on immunoglobulin content in laying hen serum

[0064] Adding stevia residue and stevia residue fermentation product to the laying hen diet had no significant effect on the concentration of rhinitis antibodies and IgM in the laying hen serum; the concentration of IgA in the laying hen serum of treatment 6 was the highest, which was significantly improved compared with the control group; the concentration of IgY in treatment 3 was the highest, which was improved compared with the control group, but the effect was not significant.

[0065] Effects of fermented stevia residue on antioxidant activity in laying hens

[0066] The total antioxidant capacity (T-AOC) kit and glutathione peroxidase (GPx) activity kit were used to determine the T-AOC and GPx in laying hen serum.

[0067] Table 10 Effects of stevia residue fermentation on antioxidant enzyme activities in laying hens

[0068] When stevia residue and fermented stevia residue were added to laying hens' diets, the total antioxidant enzyme content was significantly increased compared to the control. This suggests that stevia residue supplementation can significantly enhance the total antioxidant capacity of laying hens. However, there was no significant difference in glutathione peroxidase activity compared to the control.

[0069] In summary, the fermented stevia residue in this application has the efficacy of a microbial feed additive. Adding an appropriate amount of fermented stevia residue to the diet of meat ducks and laying hens can improve the immunity, antioxidant activity and production performance of livestock and poultry.

Claims

1. A stevia residue fermented feed additive, characterized in that: The invention comprises the following raw materials in parts by weight: 100 grams of stevia residue, 2.7 grams of fermented brown sugar, 3 grams of bacterial agent and 75 grams of water.

2. The stevia residue fermented feed additive according to claim 1, characterized in that: The specific composition of the bacterial agent 3 is: EM fungus, white rot fungus and Phanerochaete chrysosporium, and the ratio of each component is 1:1:

1.

3. A method for preparing a stevia residue fermented feed additive, characterized in that: The specific steps include: (1) Screening of fermentation agents: Agents 1, 2, and 3 were selected. 60% water and 5% of each agent were added to stevia residue, mixed evenly, and allowed to ferment at 28°C for 24 days. Samples were taken every 3 days to detect the chlorogenic acid content in each sample, and a biological agent suitable for stevia residue was screened out. (2) Screening of factors affecting the fermentation process: Using the bacterial agent screened in step (1), the inoculation amount, fermentation temperature, liquid-to-material ratio, brown sugar addition amount, soybean meal addition amount and initial pH value of fermentation were used as screening targets, and the chlorogenic acid in stevia residue was quantitatively analyzed to screen out the best influencing factors on stevia residue; (3) Response surface optimization: Based on the factors affecting the fermentation quality of stevia residue screened in step (2), a response surface experimental design was conducted. Static fermentation was performed under conditions of a temperature of 4-37°C, a liquid-to-solid ratio of 60%-100%, and an addition of brown sugar of 1%-5%. Fermentation was performed under the optimized conditions to obtain a fermented stevia residue feed additive.

4. The method for preparing a stevia residue fermented feed additive according to claim 3, characterized in that: In step (1), the bacterial agent obtained by screening is: bacterial agent 3; in steps (2) and (3), the optimal fermentation process is: the inoculation amount is 3%, the amount of fermentation brown sugar added is 2.7%, the liquid-to-material ratio is 75%, the fermentation temperature is 25°C, and the fermentation time is 9 days.

5. Use of the stevia residue fermented feed additive according to claim 1 or the stevia residue fermented feed additive prepared by the preparation method according to claims 3-4 in livestock and poultry breeding.

Images