Polygonatum sibiricum stem and leaf fermented feed additive and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a rhizoma polygonati stem and leaf fermented feed additive and application thereof. The compound probiotics of bacillus, saccharomycetes and lactic acid bacteria are used as a feed additive, the additive for the rhizoma polygonati stem and leaf feed is provided and applied to preparation of the rhizoma polygonati stem and leaf fermented feed, the fermented feed prepared by the additive remarkably improves the dissolution of the content of total phenols and polysaccharides in the rhizoma polygonati stem and leaf feed, and the content of the total phenols and polysaccharides in the rhizoma polygonati stem and leaf feed is increased. The palatability of the feed is improved, the immunity of animals is favorably enhanced, the health and production performance of the animals are improved, the development and industrial application of medicinal and edible medicinal materials in the animal feed are promoted, and the feed has a wide prospect.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to a polygonatum stem and leaf fermented feed additive and application thereof. Background Art

[0002] In view of the serious threat posed by excessive use of antibiotics in animal feed to animal and human health, the current research focus of animal husbandry has shifted to finding and developing feed additives that can effectively replace antibiotics.

[0003] Fermented strains play an important role in the field of feed additives due to their unique biological characteristics and have been included in the "Feed Additive Variety Catalog". These strains can significantly improve the conversion efficiency of feed, enhance the nutritional content of feed, including protein, amino acids, vitamins, polysaccharides and total phenols, and reduce the content of potential harmful substances in feed. In addition, fermented strains can also increase the number of probiotics in feed and reduce the dependence on antibiotics in feed production, thus becoming one of the key trends in the sustainable development of the feed industry.

[0004] At present, the production process of biological feed additives mainly relies on the fermentation of probiotics, among which compound probiotics show more significant fermentation advantages compared with single strains. Therefore, the use of mixed strain fermentation technology occupies an important position in the research and development of feed additives and is a key research direction to improve the effectiveness of feed additives.

[0005] For feed raw materials from different sources, such as stem and leaf feed and soybean meal feed, due to the significant differences in their composition and structure, animals have different requirements for feed nutrition at different stages of growth and development. For example, young animals have higher requirements for feed palatability and small molecule nutrients that are easy to digest and absorb, while sick animals need feed rich in antibacterial and anti-inflammatory active ingredients. Therefore, for specific feed raw materials or feed formulas for specific nutritional needs, it is particularly important to carry out optimization research on the ratio of mixed microbial strains and their fermentation conditions. This not only helps to improve the nutritional value and bioavailability of the feed, but also enhances the functionality of the feed to meet the nutritional needs of different animal groups under different physiological conditions.

[0006] As a traditional medicinal plant, Polygonatum cyrtonema has been proven to have multiple biological activities such as hypoglycemic, anti-fatigue, immunity enhancement, anti-tumor, antibacterial, antiviral, anticoagulant and anti-depressant. At present, its roots are widely used as health products as the main medicinal parts, while the stems and leaves containing active ingredients such as polysaccharides and flavonoids are often discarded as waste. Therefore, the development of a feed additive that can promote the dissolution of these active ingredients in the stems and leaves of Polygonatum cyrtonema is of great significance for improving animal health and production performance.

[0007] In summary, although the stems and leaves of Polygonatum cyrtonema have similar bioactive ingredients as the roots, no optimal formula has been developed for its use as a feed additive to improve the immunity, antiviral and antibacterial capabilities of animals. Research and development in this field has important industrial value and application prospects. Summary of the invention

[0008] In view of the problems of the prior art, the present invention provides a polygonatum stem and leaf fermented feed additive and application thereof.

[0009] The present invention provides a feed additive, which is composed of lactic acid bacteria, bacillus and yeast;

[0010] The weight ratio of the lactic acid bacteria, bacillus and yeast is 2-3:1-6:1-6;

[0011] The lactic acid bacteria are selected from Lactobacillus casei and Lactobacillus plantarum;

[0012] The bacillus is selected from Bacillus subtilis and Bacillus amyloliquefaciens;

[0013] The yeast is selected from Saccharomyces cerevisiae and Candida utilis.

[0014] Preferably, the weight ratio of the lactic acid bacteria, Bacillus and yeast is 2:2:3.

[0015] Preferably, the Bacillus is selected from Bacillus amyloliquefaciens; and / or, the lactic acid bacteria is selected from Lactobacillus casei; and / or, the yeast is selected from Saccharomyces cerevisiae.

[0016] The invention provides use of the feed additive in fermenting feed.

[0017] The invention provides a fermentation liquid prepared from the feed additive, which is prepared according to the following steps: mixing raw materials and the feed additive and fermenting to obtain the fermentation liquid.

[0018] Preferably, the raw material is stems and leaves of Polygonatum sibiricum.

[0019] Preferably, the raw material is stems and leaves of Hubei Polygonatum cyrtonema.

[0020] Preferably, the initial inoculation amount of the bacteria in the feed additive is 3%-5% by volume mass ratio; and / or the water content of the mixture is 50-60% by volume mass ratio.

[0021] Preferably, the fermentation temperature is 25-35° C.; and / or the fermentation time is 3-5 days.

[0022] The invention provides a method for preparing the fermentation liquid, which comprises the following steps: mixing raw materials and feed additives, and fermenting to obtain the fermentation liquid.

[0023] The present invention provides a polygonatum stem and leaf fermented feed additive and its application. The present invention uses a composite probiotic of bacillus, yeast and lactic acid bacteria as a feed additive, provides an additive for polygonatum stem and leaf feed, and is applied to prepare polygonatum stem and leaf fermented feed. The fermented feed prepared by the additive significantly increases the dissolution of the total phenol and polysaccharide content of the polygonatum stem and leaf feed, improves the palatability of the feed, is beneficial to enhance the immunity of animals, improves the health and production performance of animals, promotes the development and industrial application of medicinal and edible materials in animal feed, and has broad prospects.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The total phenolic and polysaccharide contents of Polygonatum sibiricum stem and leaf fermented feed after fermentation by different strains.

[0027] Figure 2 This study is the effect of different inoculation rates of bacterial strains on the total phenolic and polysaccharide contents in the fermented feed of Polygonatum cyrtonema stems and leaves.

[0028] Figure 3 This study is the effect of different bacterial strain ratios on the total phenol and polysaccharide contents in the fermented feed of Polygonatum cyrtonema stems and leaves.

[0029] Figure 4 To investigate the effects of different water contents on the total phenolic and polysaccharide contents in fermented feed from Polygonatum cyrtonema stems and leaves.

[0030] Figure 5 To investigate the effects of different fermentation temperatures on the total phenolic and polysaccharide contents in fermented feed from Polygonatum cyrtonema stems and leaves.

[0031] Figure 6 To investigate the effects of different fermentation times on the total phenolic and polysaccharide contents in fermented feed from Polygonatum cyrtonema stems and leaves.

[0032] Figure 7 This is the effect of the interaction of various factors on the total phenol extraction rate in the fermented feed of Polygonatum cyrtonema stems and leaves.

[0033] Figure 8 This is the effect of the interaction of various factors on the polysaccharide extraction rate in the fermented feed of Polygonatum cyrtonema stems and leaves.

[0034] Fig. 9This is the form of Polygonatum sibiricum stem and leaf feed before fermentation.

[0035] Fig.10 It is the form of fermented Polygonatum sibiricum stem and leaf feed. DETAILED DESCRIPTION

[0036] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0037] Experimental Materials

[0038] 1. Strains

[0039] Saccharomyces cerevisiae ATCC 9763, Candida utilis ACCC 20060, Bacillus subtilis ATCC 6633, Bacillus amyloliquefaciens AS1.892, Lactobacillus casei ATCC 334, Lactobacillus plantarum ATCC 8014 (Beijing Baozang Biotechnology Co., Ltd.).

[0040] 2. Culture medium

[0041] LB liquid culture medium and CM187 MRS broth culture medium (Beijing Luqiao Technology Co., Ltd.) were sterilized at 121°C for 30 min before use.

[0042] 3. Feed raw materials

[0043] Polygonatum sibiricum stem and leaf samples: collected from Group 15, Hongqi Village, Daoming Town, Chongzhou City, Sichuan Province in mid-March 2022, and identified by Associate Professor Long Fei from the Pharmacy Teaching and Research Section of Chengdu University of Traditional Chinese Medicine as the above-ground part of Polygonatum zanlanscianense Pamp. of the Liliaceae family.

[0044] The stems and leaves of the samples were separated, washed, dried in an oven at 60°C to constant weight, crushed to pass through a No. 5 sieve, and stored in a refrigerator at -20°C for later use.

[0045] Embodiment 1 A kind of Polygonatum cyrtonema stem and leaf fermented feed additive

[0046] The present embodiment provides a fermented feed additive of polygonatum stems and leaves, which comprises the following strains in weight ratio:

[0047] Lactobacillus casei: Bacillus amyloliquefaciens: Saccharomyces cerevisiae in a weight ratio of 2:2:3

[0048] In other embodiments, the corresponding weight ratio of the strains can also be 1:1:1, 1:2:2, 1:3:3, 2:1:2, 3:2:1, 3:2:3. Lactobacillus casei can also be Lactobacillus plantarum; Bacillus amyloliquefaciens can also be Bacillus subtilis; Saccharomyces cerevisiae can also be Candida utilis.

[0049] Example 2 Preparation method of a fermented feed made from stems and leaves of Polygonatum cyrtonema

[0050] The present embodiment provides a method for preparing a fermented feed of polygonatum stems and leaves, which comprises the following steps:

[0051] 1. Strain expansion

[0052] The lactic acid bacteria in the feed additives were cultured in an MRS liquid culture medium at 37°C, and the yeast and Bacillus in the feed additives were cultured in an LB liquid culture medium at 30°C. Each strain was cultured until OD 600 =0.8.

[0053] 2. Preparation of fermented feed

[0054] Take 5 g of stem and leaf powder, inject the expanded feed additive into distilled water at an initial inoculation rate of 3% (V / W) of the strain to make its water content 55% (V / W), stir evenly, place it in a fermentation bag with a one-way exhaust port, and then transfer it to a 31°C constant temperature incubator for anaerobic fermentation for 4 days.

[0055] In other embodiments, the initial inoculation amount of the strain can also be 4%-5%, the water content can also be 50-60% (V / W), the fermentation temperature can also be 25-35° C., and the fermentation time can also be 3 to 5 days.

[0056] The technical solution of the present invention is further illustrated by experiments below.

[0057] Experimental Example 1: Fermented feed additives for fermenting Polygonatum cyrtonema stems and leaves

[0058] 1. Experimental Materials and Instruments

[0059] 1. Reagents

[0060] Anthrone, sulfuric acid (Chengdu Kelong Chemical Reagent Factory); anhydrous glucose (Batch No.: 21011805, Sichuan Weikeqi Biotechnology Co., Ltd.).

[0061] 2. Instruments

[0062] SW-CJ-1D clean bench (Suzhou Bolaier), SPX-150B-Z biochemical incubator (Shanghai Boxun Medical Biological Instrument Co., Ltd.), high pressure sterilizer (Shanghai Boxun Instrument Co., Ltd.).

[0063] 2. Experimental Methods

[0064] 1. Fermentation strain screening

[0065] The bacterial liquid of each single strain (Saccharomyces cerevisiae, Candida utilis, Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus casei, and Lactobacillus plantarum) was taken for fermentation test of feed additive. It was added to the aboveground powder at a dosage of 3% (V / W), and then distilled water was injected to make its water content 50% (V / W). After stirring evenly, it was placed in a fermentation bag with a one-way exhaust port, and finally transferred to a constant temperature incubator at 30°C for anaerobic fermentation. The changes in total phenol and polysaccharide contents were detected after 3 days of fermentation.

[0066] 2. Single Factor Test

[0067] Single factor experiments were conducted to optimize the five influencing factors of inoculation amount, bacterial species ratio, water content, fermentation temperature and fermentation time. The initial fermentation conditions were: inoculation amount of 3%, bacterial species ratio (lactic acid bacteria: spore bacteria: yeast) of 2:2:3, fermentation time of 3d and fermentation temperature of 30℃. During the single factor optimization, the inoculation amount increased by 1% each time, and they were 1%, 2%, 3%, 4% and 5% respectively; the bacterial species ratio was 1:1:1, 1:2:2, 1:3:3, 2:1:2, 2:2:3, 2:3:1, 3:1:3, 3:2:1 and 3:2:3; the water content interval was 10%, and they were 30%, 40%, 50%, 60% and 70% respectively; the fermentation time interval was 1d, and they were 1d, 2d, 3d, 4d, 5d and 6d respectively; the fermentation temperature interval was 5℃, and they were 25℃, 30℃, 35℃, 40℃ and 45℃ respectively. Each group of experiments was repeated three times.

[0068] 3. Optimization of fermentation conditions by response surface

[0069] Through the results of the single factor test, as shown in Table 1, the three main influencing factors, fermentation temperature, water content, and inoculum size, were selected as independent variables, and the total phenol and polysaccharide contents in the fermentation broth were used as dependent variables to conduct a Box-Behnken response surface experimental design and a response surface experiment.

[0070] Table 1 Factors and levels of response surface experiment

[0071]

[0072] 4. Determination of active ingredients

[0073] Take 5 g of the fermented feed into a 100 mL conical flask, add 50 mL of distilled water, perform ultrasonic extraction for 60 min, and filter to obtain the fermentation liquid.

[0074] Take 5 g of stem and leaf powder each and put it in a 100 mL conical flask, add 50 mL of distilled water, and perform ultrasonic extraction for 60 min. Filter to obtain the water extract.

[0075] The fermentation broth and water extract were respectively used to determine the total phenolic and polysaccharide contents.

[0076] Determination of total phenol content: The total phenol extraction rate was determined by the Folin-Ciocalteu method, and a gallic acid standard solution with a mass concentration of 0.25 mg / mL was prepared. Gradient concentrations (0, 0.08, 0.12, 0.16, 0.20, 0.24 and 0.28 mL) of standard solutions were respectively drawn and placed in a 10 mL volumetric flask. To each volumetric flask, 6.4 mL of distilled water and 0.4 mL of Folin-phenol reagent were added in sequence. After shaking evenly, 1 mL of 20% Na2CO3 solution was added respectively, and the mixture was thoroughly shaken and placed at room temperature in the dark for 2 h. The absorbance at 764 nm was measured and a standard curve was drawn.

[0077] The determination of polysaccharide content is carried out in accordance with the sulfuric acid-anthrone method of the "Chinese Pharmacopoeia 2020 Edition".

[0078] 5. Calculation of recovery rate

[0079] 30 g of Hubei Polygonatum cyrtonema stem and leaf powder was fermented and extracted according to the optimal fermentation process, the extract was concentrated under reduced pressure at 50°C, freeze-dried, weighed, and the recovery rate was calculated.

[0080] 6. Data statistics and analysis

[0081] All experiments were repeated 3 times. Excel 2010 software was used for data processing; Origin 2019 was used for drawing; SPSS 26 professional mathematical statistics software was used for data difference analysis. During the analysis, the data variables were selected in the form of mean ± standard deviation. Before the significant analysis T-test, ANOVA single-factor variance homogeneity test was required to ensure that multiple independent samples had the same variance. In the significant result analysis, when the P value was greater than 0.05, the difference between the groups was not significant; when the P value was less than 0.05, the difference between the groups was significant.

[0082] 3. Experimental Results

[0083] 1. Screening of fermentation strains

[0084] The results are as follows Figure 1As shown: Through the determination results of polysaccharide and total phenol content, it can be observed that different types of fermentation bacteria can improve the content of polysaccharides and total phenols. Among lactic acid bacteria, the total phenol content has been significantly improved, and the use of Lactobacillus casei fermentation to improve the polysaccharide content is more significant, which can increase the polysaccharide content to 270.83% before fermentation. Among yeasts, the increase in total phenol content by Saccharomyces cerevisiae and Candida utilis is close to 178.57% before fermentation. Saccharomyces cerevisiae has a stronger ability to improve polysaccharide content, which can be increased to 235.71% before fermentation. Among Bacillus, Bacillus amyloliquefaciens has a better effect on improving the content of polysaccharides and total phenols, which can be increased to 277.98% and 159.29% before fermentation, respectively. The above results show that Lactobacillus casei, Saccharomyces cerevisiae and Bacillus amyloliquefaciens are preferred strains that can be used to ferment Polygonatum cyrtonema stem and leaf feed.

[0085] 2. Effect of strain inoculation amount on fermentation

[0086] The results are as follows Figure 2 As shown: when the inoculation amount of the strain is 3% (V / W), the total phenol and polysaccharide contents of the fermented feed are the highest, which are 5.29% and 4.85% respectively. When the inoculation amount of the fermentation bacteria is small, the secondary metabolites produced by metabolism are small, and the function of promoting the release of active ingredients is weak, so the contents are low. When the inoculation amount exceeds the appropriate range, a large number of microorganisms will compete for limited nutrients to support their growth in the initial stage of fermentation. However, due to the insufficient supply of nutrients, this competition will inhibit the growth of microorganisms. In addition to the above effects, excessive inoculation will produce a large amount of organic acids during the fermentation process, which will rapidly reduce the pH value in the feed. This sharp drop in pH has a significant inhibitory effect on the growth of fermented strains and the production of secondary metabolites. The above results show that the preferred volume-to-mass ratio of the strain inoculation amount of the feed additive is 3%.

[0087] 3. Effect of strain ratio on fermentation

[0088] The results are as follows Figure 3 As shown in the figure: when the ratio of inoculated lactic acid bacteria: spore bacteria: yeast is 2:2:3, the total phenol and polysaccharide contents of the fermented feed are the highest, which are 5.96% and 5.06% respectively, indicating that the preferred bacterial species inoculation ratio of feed additives is Lactobacillus casei: Bacillus amyloliquefaciens: Saccharomyces cerevisiae = 2:2:3.

[0089] 4. Effect of water content on fermentation

[0090] The results are as follows Figure 4As shown in the figure: With the gradual increase of water content, the total phenol and polysaccharide content of fermented feed both show a trend of first increasing and then decreasing. When the water content reaches 50%, the content of each component reaches a peak, and then the content decreases with the increase of water content. When the water content increases, water-soluble total phenol and polysaccharide are more easily dissolved and released, and at the same time provide a suitable growth environment for fermentation bacteria. When the water content is too high, the excessive reproduction of miscellaneous bacteria will affect the growth of fermentation bacteria. Therefore, the preferred water content of feed additives is 50%.

[0091] 5. The influence of fermentation temperature on fermentation

[0092] Fermentation temperature is a key factor affecting the fermentation effect. Figure 5 As shown: With the increase of temperature, the polysaccharide and total phenol content of the fermented feed reached a peak at 30°C, reaching 4.48% and 5.30% respectively, and then showed a downward trend with the increase of temperature. It is speculated that this trend is related to the optimal growth temperature of each fermentation bacteria. The growth temperature range of brewer's yeast is best between 25°C and 30°C. Once the temperature exceeds 30°C, the activity of its strains will gradually weaken until it dies. On the other hand, the growth temperature of lactic acid bacteria is relatively moderate, and its optimal growth temperature range is 30°C to 40°C. The optimal growth temperature of Bacillus is between 30°C and 37°C. Therefore, at a temperature of 30°C, each strain can grow better, indicating that the preferred fermentation temperature of feed additives is 30°C.

[0093] 6. The influence of fermentation time on fermentation

[0094] During the fermentation process, there will be a series of material changes, such as the growth and proliferation of fermentation bacteria, as well as the metabolism and transformation of substances. These processes all require a certain amount of time to complete. However, the extension of fermentation time does not always mean an improvement in fermentation effect. In fact, too long fermentation time may cause a series of problems, such as the consumption of active ingredients, contamination by harmful bacteria, and increased fermentation costs. Therefore, reasonable control of fermentation time is crucial to ensure fermentation effect and product quality. Figure 6 As shown in the figure, as the fermentation time increases, the total phenol and polysaccharide content of the fermented feed increases. In the first 3 days, the increase rate is obvious. At the 4th day of fermentation, the total phenol content reaches 5.45%, and the polysaccharide content reaches 4.77%, and then tends to be stable. The results show that the optimal fermentation time for feed additives is 4 days.

[0095] 7. Response surface test results

[0096] According to the results of the single factor experiment, the total phenol content (Y1) and polysaccharide content (Y2) were taken as the response values, and the three investigation factors of fermentation temperature (A), inoculation amount (B) and water content (C) were set to design the response surface experiment. The experimental results are shown in Table 2.

[0097] Table 2 Response surface test results

[0098]

[0099] The Design-Expert 10.0 software was used for multivariate regression fitting, and the quadratic multinomial regression equations of total phenol content (Y1) and polysaccharide content (Y2) on fermentation temperature (A), inoculation amount (B), and water content (C) were obtained as follows:

[0100] Y1=5.87+0.58A+0.28B+0.30C-0.37AB+0.24AC+0.056BC-1.68A 2 -0.78B 2 -0.49C 2

[0101] Y2=5.34+0.59A+0.45B+0.37C-0.38AB+0.23AC+0.13BC-1.22A 2 -0.90

[0102] B 2 -0.62C 2

[0103] As shown in Tables 3 and 4, the P value of the significance test of the model is <0.05, indicating that the model has statistical significance and can accurately reflect the effects of the three factors of fermentation temperature, inoculation amount, and water content on the total phenolic and polysaccharide contents of the stems and leaves of Polygonatum cyrtonema. Regression analysis of the fitted model showed that the model reached a significant level, and the determination coefficient R 2 (total phenol content) = 0.9952, R 2 (polysaccharide content) = 0.9864, the adjusted values ​​are 0.9891 and 0.9690, the coefficients of variation are 2.71% and 4.76%, the lack of fit value P (total phenol content) is 0.2480, and the lack of fit value P (polysaccharide content) is 0.1260, which are not significant, indicating that the measured values ​​and predicted values ​​of the total phenol content and polysaccharide content of the fermented feed have a good fit, so this model has a good fitting effect and can be used to analyze and predict the process parameters of the fermentation of Hubei Polygonatum stems and leaves. From the P value, it can be observed that A, B, C, AB, AC, A 2 , B 2 , C 2 The total phenol content was significantly affected by A, B, AB, and A 2 , B 2 , C 2 The effect on polysaccharide content is extremely significant, and AC has a significant effect on polysaccharide content.

[0104] Table 3 Analysis of variance of regression model for total phenol content

[0105]

[0106] Table 4 Variance analysis of polysaccharide content regression model

[0107]

[0108]

[0109] The steepness of the response surface 3D graph can reflect the influence of a certain factor on the response value. The steeper the graph, the more significant the influence of the factor on the response value. In addition, the distribution of contour lines also provides us with important information about the interaction between two variables. The density of contour lines and whether they tend to be elliptical are important indicators for judging the strength of the interaction. Specifically, the denser the contour lines are and the more they tend to be elliptical, the stronger the interaction between the two variables. The response surface 3D graph results are as follows: Figure 7 As shown: the interaction between fermentation temperature and inoculation amount, and the interaction between fermentation temperature and water content have significant effects on the total phenol and polysaccharide contents.

[0110] 8. Verification experiment

[0111] According to the response surface analysis, the optimal fermentation conditions were obtained as follows: fermentation temperature 31.041℃, inoculation volume 3.195%, and water content 53.621%. Based on this result and combined with actual conditions, the fermentation conditions were changed to: fermentation temperature 31℃, inoculation volume 3%, and water content 55%. The theoretical predicted values ​​of total phenol content were 5.993% and polysaccharide content 5.528%. The actual test results were total phenol content 6.03±0.11%, and polysaccharide content 5.41±0.27%, which were close to the predicted values.

[0112] 9. Recovery rate

[0113] Comparison of extracts from stems and leaves of Polygonatum cyrtonema before and after fermentation Fig. 9 , 10 In three parallel tests, 30.00 g of Hubei Polygonatum cyrtonema stem and leaf powder was finally converted into 9.32 g, 9.75 g, and 9.44 g of brown freeze-dried products, with a recovery rate of 31.68 ± 0.74%.

[0114] The present invention uses Hubei Polygonatum stem and leaf as feed culture medium for single bacteria solid fermentation, and screens out the best fermentation strains by total phenol and polysaccharide content. The above results show that Lactobacillus casei, Saccharomyces cerevisiae, and Bacillus amyloliquefaciens have more significant improvements on the content of effective ingredients in each strain, and these three strains are selected as late fermentation strains. Through single factor ANOVA test and response surface optimization, the fermentation process conditions with the maximum extraction rate of effective ingredients in Polygonatum stem and leaf are established: Lactobacillus casei: Bacillus amyloliquefaciens: Saccharomyces cerevisiae ratio is 2:2:3, fermentation temperature is 31 ° C, inoculation amount is 3%, water content is 55%, and fermentation is 4 days. Compared with unfermented Polygonatum stem and leaf feed, the total phenol content of the fermented stem and leaf feed is increased by 1.15 times, the polysaccharide content is increased by 2.22 times, the content of effective ingredients is increased, and the palatability of the feed additive after fermentation is better, which is conducive to promoting animal growth and development.

[0115] Through the above embodiments and experimental examples, it can be seen that the present invention provides a fermented feed additive of stems and leaves of polygonatum and its application. The present invention uses a composite probiotic of bacillus, yeast and lactic acid bacteria as a feed additive, provides an additive for polygonatum stem and leaf feed, and is used to prepare a fermented feed of polygonatum stem and leaf. The fermented feed prepared by the additive significantly improves the dissolution of the total phenol and polysaccharide content of the polygonatum stem and leaf feed, improves the palatability of the feed, is conducive to enhancing the immunity of animals, improving the health and production performance of animals, and promoting the development and industrial application of medicinal and edible medicinal materials in animal feed, which has broad prospects.

Claims

1. A feed additive, characterized in that: It is composed of lactic acid bacteria, bacillus and yeast; The weight ratio of the lactic acid bacteria, bacillus and yeast is 2-3:1-6:1-6; The lactic acid bacteria are selected from Lactobacillus casei and Lactobacillus plantarum; The bacillus is selected from Bacillus subtilis and Bacillus amyloliquefaciens; The yeast is selected from Saccharomyces cerevisiae and Candida utilis.

2. The animal feed additive according to claim 1, characterized in that: The weight ratio of the lactic acid bacteria, bacillus and yeast is 2:2:

3.

3. The animal feed additive according to claim 1, characterized in that: The Bacillus is selected from Bacillus amyloliquefaciens; and / or the lactic acid bacteria is selected from Lactobacillus casei; and / or the yeast is selected from Saccharomyces cerevisiae.

4. Use of the feed additive according to any one of claims 1 to 3 in fermented feed.

5. The fermentation liquid obtained from the feed additive according to any one of claims 1 to 3, characterized in that: The preparation is carried out according to the following steps: the raw materials and feed additives are mixed and fermented to obtain the product.

6. The fermentation liquid according to claim 5, characterized in that: The raw materials are stems and leaves of Polygonatum cyrtonema.

7. The fermentation broth according to claim 6, characterized in that: The raw materials are stems and leaves of Hubei Polygonatum cyrtonema.

8. The fermentation broth according to claim 5, characterized in that: The initial inoculation amount of the bacteria in the feed additive is 3%-5% by volume by mass; and / or the water content of the mixture is 50%-60% by volume by mass.

9. The fermentation broth according to claim 5, characterized in that: The fermentation temperature is 25-35° C.; and / or the fermentation time is 3-5 days.

10. The method for preparing the fermentation broth according to any one of claims 5 to 9, characterized in that: The method comprises the following steps: mixing raw materials and feed additives and fermenting the mixture to obtain the product.

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