Chinese yam skin aqueous extract, preparation thereof and application of Chinese yam skin aqueous extract in preparation of product for improving growth performance of immunosuppressed chicken

Abstract

The invention discloses a Chinese yam skin aqueous extract, preparation thereof and application of the Chinese yam skin aqueous extract in preparation of a product for improving the growth performance of immunosuppressed chickens. The optimal condition of the polysaccharide content in the Chinese yam skin aqueous extract is optimized through a single factor and response surface method, an immunosuppressive chicken model is established through cyclophosphamide (CTX), and the influence of the Chinese yam skin aqueous extract on growth and development of immunosuppressive chickens is researched. The result shows that the optimal extraction conditions of the Chinese yam peel water extract are that the material-liquid ratio is 1: 24, the extraction temperature is 75 DEG C, the extraction time is 4.4 hours, the polysaccharide extraction content of the Chinese yam peel water extract under the conditions is 101.43 mg / g, and the yield of the Chinese yam peel water extract is 19.46%. Meanwhile, the Chinese yam skin aqueous extract increases the weight of immunosuppressed chickens caused by cyclophosphamide, the immune organ index, the ratio of the intestinal villus height to the crypt depth, the level of serum immune globulin and the titer of Newcastle disease antibodies, and a theoretical basis is provided for utilization of the Chinese yam skin / Chinese yam skin aqueous extract.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a yam peel water extract, a preparation method thereof, and an application thereof in preparing a product for improving the growth performance of immunosuppressed chickens. Background Art

[0002] Chinese yam is a tonic Chinese medicine for tonifying the spleen and stomach. A large amount of yam peel will be produced during processing, but its research in the field of livestock and poultry is relatively small, and it is a resource with development potential. In livestock and poultry farming, many immunosuppressive diseases have a huge impact on animal health, damaging the immune organs and mucosal tissues of animals. Although there is a document (ZHAO ZW, WANG L, RUAN Y, et al. Physicochemical properties and biological activities of polysaccharides from the peel of Dioscorea opposita Thunb. extracted by four different methods [J]. Food Science and Human Wellness, 2023, 12 (1): 130-139. DOI: 10.1016 / j.fshw.2022.07.031.) reporting the use of hot water extraction to prepare yam peel extract, but the extraction conditions have not been optimized, nor has the function of yam peel extract been studied. In view of this, the present invention is proposed. Summary of the invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing yam peel water extract.

[0004] Another object of the present invention is to provide a yam peel water extract prepared by the above preparation method.

[0005] Another object of the present invention is to provide an application of the yam peel water extract.

[0006] The present invention aims to optimize the extraction process of yam peel water extract by single factor and response surface methodology, establish an immunosuppressive chicken model with cyclophosphamide (CTX), and explore the effects of yam peel water extract on the growth and development, immune regulation and intestinal barrier function of immunosuppressive chickens.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing a yam peel water extract comprises the following steps:

[0009] (1) drying, crushing and sieving the yam peel to obtain yam peel powder;

[0010] (2) adding the yam peel powder of step (1) to hot water at a material-liquid ratio of 1 g: 15-25 mL, soaking, and centrifuging to collect the supernatant, i.e., the yam peel water extract;

[0011] (3) The yam peel water extract of step (2) is subjected to rotary evaporation to obtain a yam peel water extract.

[0012] Preferably, in step (1), the drying condition is 50-60° C. for 5-8 h; further, 55° C. for 6 h.

[0013] Preferably, in step (1), the sieving is through a 50-100 mesh sieve; further, through a 60 mesh sieve.

[0014] Preferably, in step (2), the material-liquid ratio is 1:20-25; further 1:24.

[0015] Preferably, in step (2), the temperature of the hot water is 60-80°C; further 70-80°C; further 75°C.

[0016] Preferably, in step (2), the soaking time is 3 to 5 hours; further 4 to 5 hours; further 4.4 hours.

[0017] Preferably, in step (2), the centrifugation condition is 3000-5000 r / min for 5-15 min; further, 4000 r / min for 10 min.

[0018] A yam peel water extract is prepared by the above preparation method.

[0019] The application of the yam peel water extract is at least one of the following applications:

[0020] (a) Use in the preparation of products for improving the growth performance of immunosuppressed chickens;

[0021] (b) Use in the preparation of products for improving the immunity of immunosuppressed chickens;

[0022] (c) Use in the preparation of products that promote digestion and absorption in immunosuppressed chickens.

[0023] Preferably, the growth performance in (a) includes body weight.

[0024] Preferably, the improving the immunity of immunosuppressed chickens in (b) comprises improving the cellular immune function of immunosuppressed chickens and / or improving the humoral immune function of immunosuppressed chickens and / or increasing the titer of Newcastle disease antibodies.

[0025] Preferably, the promoting digestion and absorption of immunosuppressed chickens in (c) comprises increasing the depth of intestinal crypts of immunosuppressed chickens and / or increasing the ratio of intestinal villus height to crypt depth of immunosuppressed chickens.

[0026] Preferably, the immunosuppression is immunosuppression caused by cyclophosphamide (CTX).

[0027] Preferably, the product comprises feed, feed additive or drinking water additive.

[0028] Preferably, the amount of the yam peel water extract added to the feed is 0.5-2 g / kg; further 2 g / kg.

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] (1) The present invention optimizes the optimal conditions for the polysaccharide content in the yam peel water extract by single factor and response surface method. The results show that the optimal extraction conditions for the yam peel water extract are a solid-liquid ratio of 1:24, an extraction temperature of 75°C, and an extraction time of 4.4h. Under these conditions, the polysaccharide extraction content of the yam peel water extract is 101.43 mg / g, and the yam peel water extract yield is 19.46%.

[0031] (2) In order to explore the effect of yam peel extract on the growth and immune function of immunosuppressed chickens caused by CTX, the present invention randomly divided 200 one-day-old Ephedra chickens into five groups when they were isolated and raised until they were 7 days old: blank control group (NC), immunosuppressive model group (CTX), low-dose group of yam peel water extract (L), medium-dose group (M), and high-dose group (H). The experiment was divided into four stages: 1-7d, 8-14d, 15-21d, and 22-28d. The body weight and immune organ index of broilers at each stage were recorded, and the serum IgG, IgM content and Newcastle disease antibody titer were detected. The results are as follows: Compared with the CTX group, the body weight of each drug-treated group at each stage was significantly increased (P<0.05). At 28 days, the spleen index of each drug-treated group increased significantly (P<0.05), and the thymus and bursa index of the M group were significantly higher than those of the CTX group. For humoral immunity, at 28 days, compared with the CTX group, the IgG content of each drug-treated group could be significantly increased (P<0.05). The IgM content of the L and M groups was significantly higher than that of the CTX group at 14 and 21 days (P<0.05). The results of Newcastle disease antibody titer showed that the antibody titer of each drug-treated group was higher than that of the CTX group. At 14 days, the antibody titer of the H group was significantly higher than that of the CTX group (P<0.05). At 21 and 28 days, there was no significant difference between the groups.

[0032] In summary, the optimized conditions for the extraction process of yam peel are a solid-liquid ratio of 1:24, an extraction temperature of 75℃, and an extraction time of 4.4h. At the same time, the yam peel water extract increases the body weight, immune organ index, serum immunoglobulin levels, and Newcastle disease antibody titer of immunosuppressed chickens caused by cyclophosphamide, providing a theoretical basis for the utilization of yam peel / yam peel water extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the standard curve of glucose solution concentration-absorbance.

[0034] Figure 2 This is a graph of the extraction test results at different material-liquid ratios; Note: Data in the same column are marked with different letters, which means the difference is significant (P<0.05); data are not marked or marked with the same letter, which means the difference is not significant (P>0.05).

[0035] Figure 3 The figure is the result of the extraction test at different temperatures; Note: Data in the same column are marked with different letters, which means the difference is significant (P<0.05); data without letters or with the same letters are not significantly different (P>0.05).

[0036] Figure 4 The graphs are the test results extracted at different times; Note: Data in the same column are marked with different letters, which means the difference is significant (P<0.05); data are not marked or marked with the same letters, which means the difference is not significant (P>0.05).

[0037] Figure 5 The morphological structure of the jejunum of chickens in each group on the 14th day of the experiment (100×); Note: (a), (b), (c), (d), and (e) represent the blank control group (NC), cyclophosphamide group (CTX), low-dose yam peel water extract group (L), medium-dose yam peel water extract group (M), and high-dose yam peel water extract group (H), respectively. The scale bar is 50μm.

[0038] Figure 6 The morphological structure of the jejunum of chickens in each group on the 28th day of the experiment (40×); Note: (a), (b), (c), (d), and (e) represent the blank control group (NC), cyclophosphamide group (CTX), low-dose yam peel water extract group (L), medium-dose yam peel water extract group (M), and high-dose yam peel water extract group (H), respectively. The scale bar is 200μm.

[0039] Figure 7 The changes of serum Newcastle disease antibody titer, immunoglobulin IgG and IgM in chickens in each group at each stage; A: changes of serum Newcastle disease antibody titer; B: changes of immunoglobulin IgG; C: changes of immunoglobulin IgM; Note: groups at the same stage are marked with different letters, which means the difference is significant (P<0.05); groups without letters or with the same letters are not significantly different (P>0.05). DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples where specific experimental conditions are not specified are usually carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used are reagents and materials obtained from commercial sources unless otherwise specified.

[0041] Example 1 Optimization of the extraction process of yam peel water extract by response surface methodology

[0042] 1 Test methods

[0043] 1.1 Raw material pretreatment method

[0044] Take the iron stick yam peel and put it in a 55℃ oven to dry for 6 hours, then use a high-speed Chinese medicine grinder to make the dried yam peel into powder, pass it through a 60-mesh sieve, and mix it evenly. To avoid moisture, store the yam peel powder in a sealed bag.

[0045] 1.2 Single Factor Experiment

[0046] Three factors, extraction time, extraction temperature and solid-liquid ratio, were selected for single factor experiment to determine the influence of each factor on the total sugar content and water extract yield of yam peel water extract.

[0047] 1.2.1 Effect of solid-liquid ratio on polysaccharide content and water extract yield

[0048] 2 g of dried yam peel powder was weighed into different conical flasks, the extraction temperature was fixed at 70°C, the time was 2 h, the solid-liquid ratios were selected as 1:10, 1:15, 1:20, 1:25 and 1:30 (g / mL) for extraction, and the supernatant was collected by centrifugation at 4000 r / min for 10 min. The extraction was repeated 3 times in parallel to investigate the effect of the solid-liquid ratio on the polysaccharide content in the supernatant and the yield of water extract.

[0049] 1.2.2 Effect of extraction temperature on polysaccharide content and water extract yield

[0050] 2 g of dried yam peel powder was weighed into different conical flasks, and the solid-liquid ratio was fixed at 1:20 (g / mL). The extraction time was 2 h, and the temperatures were selected to be 50, 60, 70, 80, and 85 °C, respectively. The supernatant was collected by centrifugation at 4000 r / min for 10 min. The extraction was repeated 3 times in parallel to investigate the effect of extraction temperature on the polysaccharide content in the supernatant and the yield of water extract.

[0051] 1.2.3 Effect of extraction time on polysaccharide content and water extract yield

[0052] 2 g of dried yam peel powder was weighed into different conical flasks, with a fixed solid-liquid ratio of 1:20 (g / mL), a temperature of 70°C, and extraction times of 1, 2, 3, 4 and 5 h. The supernatant was collected by centrifugation at 4000 r / min for 10 min. The extraction was repeated 3 times in parallel to investigate the effect of extraction time on the polysaccharide content in the supernatant and the yield of water extract.

[0053] 1.3 Determination of polysaccharide content and water extract yield of yam peel water extract

[0054] The polysaccharide content of yam peel water extract was determined by phenol-sulfuric acid method. Accurately prepare 10 mg / mL glucose standard solution, dilute to 1 mg / mL, and dilute to 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL glucose standard solution, take 1 mL of each into a stoppered glass test tube, and take 1 mL of distilled water as a blank control. Then add 1.0 mL of 6% phenol solution to each tube, mix well and quickly add 5.0 mL of concentrated sulfuric acid, shake well and let stand at room temperature for 30 minutes, draw 200 μL into a 96-well plate, and measure the absorbance at a wavelength of 490 nm. With glucose concentration (mg / mL) as the horizontal coordinate and absorbance value as the vertical coordinate, the curve regression equation is drawn. The extraction effect is based on "polysaccharide content in water extract / total mass of yam peel powder" as an indicator, and the calculation formula is Y=(C×V×N) / M. In the formula: Y is the polysaccharide content of yam peel water extract, in mg / g; C is the polysaccharide concentration obtained by substituting into the standard curve, in mg / mL; V is the volume of the water extract, in mL; N is the dilution multiple; M is the mass of dry yam peel powder, in g.

[0055] The yam peel water extract is concentrated into an extract by pressure using a rotary evaporator, frozen at -80°C overnight, and then freeze-dried with a freeze dryer to obtain a solid powder, namely the yam peel water extract.

[0056] The calculation formula for the water extract yield is Y1(%)=m / M×100%, where Y1(%) is the yam peel water extract yield; m is the mass of the solid powder after freeze-drying, in mg; and M is the mass of the dry yam peel powder, in mg.

[0057] 1.4 Response surface experimental design

[0058] Combined with the effect of single factors on the polysaccharide content in the water extract of yam peel, the optimal test range was selected, and the Box-Behnken method in Design-Expert8.0 was used to design the experiment. The polysaccharide content of the water extract of yam peel was used as the response value, and the solid-liquid ratio, extraction temperature, and extraction time were used as the three factor levels of the response surface. The experiment was designed according to the three-factor three-level response surface analysis method, and the factors and levels were coded respectively to establish a model. A, B, and C were used to represent the independent variables of solid-liquid ratio, extraction temperature, and extraction time, respectively, and -1, 0, and 1 were used to represent the low, medium, and high levels of the independent variables. After the experiment, the extraction yield and fitting equation were calculated, and the optimal conditions were obtained to optimize the yam peel extraction process conditions, and they were verified. The coding and levels of the experimental design factors are shown in Table 1.

[0059] Table 1. Response surface analysis factor level table

[0060]

[0061] 2 Results and Analysis

[0062] 2.1 Drawing of glucose standard curve

[0063] Draw a glucose standard curve with glucose concentration (mg / mL) as the horizontal axis and absorbance measured at 490nm as the vertical axis. Figure 1 As shown, the calculated glucose standard curve equation is y=7.6786x-0.0128, where x is the concentration of the glucose solution, y is the absorbance value, and the correlation coefficient R 2 =0.9972, with good linear relationship, and can be used for the determination of total sugar content.

[0064] 2.2 Single factor test results

[0065] 2.2.1 Effect of solid-liquid ratio on polysaccharide content and water extract yield

[0066] The effects of different solid-liquid ratios on polysaccharide content and water extract yield are shown in Figure 2 .Depend on Figure 2It can be seen that in the range of the solid-liquid ratio of 1:10 to 1:30 (g / mL), the polysaccharide content in the yam peel water extract showed a trend of first increasing and then gradually flattening. When the solid-liquid ratio was between 1:10 and 1:20, the polysaccharide content gradually increased with the increase of the solid-liquid ratio and the difference was significant (P<0.05). When the solid-liquid ratio was increased to 1:25 and 1:30, the polysaccharide content of the water extract tended to be stable, reaching the measured extreme value and the difference was not significant (P>0.05), and when the solid-liquid ratio was 1:30, the yield of the yam peel water extract was significantly reduced (P>0.05). This may be because when the solid-liquid ratio was small, the aqueous solution quickly reached saturation and therefore could not precipitate more effective components, resulting in a low polysaccharide content and water extract yield; as the solid-liquid ratio increased, the effective components of the yam peel were fully dissolved in water, thereby increasing the polysaccharide content and water extract yield. However, when the volume of solvent is too large, the proton driving force affects the separation of effective components, the diffusion of polysaccharides tends to be balanced, and too much solvent affects the workload of subsequent rotary evaporation. Therefore, 1:15, 1:20, and 1:25 were finally selected as the optimal material-liquid ratios.

[0067] 2.2.2 Effect of extraction temperature on polysaccharide content and water extract yield

[0068] The effects of different extraction temperatures on polysaccharide content and water extract yield are shown in Figure 3 . Figure 3 The results showed that when the solid-liquid ratio was fixed at 1:20 (g / mL) and the extraction time was 2h, within the temperature range set in the experiment, the polysaccharide content in the water extract of yam peel increased with the increase of temperature. When the extraction temperature was 50-70℃, the polysaccharide content increased significantly, and the polysaccharide content in the water extract increased significantly (P<0.05). When the extraction temperature was 80℃ to 85℃, the polysaccharide content increased but there was no significant difference, indicating that the increase in temperature can increase the movement of solvent and solute molecules, promote diffusion, and thus increase the polysaccharide content. The water extract yield showed a trend of first increasing and then flattening at 50-80℃. After exceeding 80℃, the water extract yield decreased significantly (P<0.05). From the perspective of saving energy and preventing high temperature from reducing the activity of polysaccharides, the extraction temperatures of 60, 70, and 80℃ were selected as the factor levels of the response surface.

[0069] 2.2.3 Effect of extraction time on polysaccharide content and water extract yield

[0070] The effect of extraction time on polysaccharide content and water extract yield is shown in Figure 4 .Depend on Figure 4It can be seen that when the water bath time is 4h, the polysaccharide content in the yam peel water extract is significantly higher than that of 2h and 3h (P<0.05), and there is no significant difference in the water extract yield (P>0.05). The polysaccharide content of the yam peel water extract between the extraction time of 4 and 5h is not significantly different, but the water extract yield is significantly reduced (P<0.05). At 6h, the polysaccharide content and water extract yield are not significantly different from those at 5h (P>0.05), and the production time cost is increased. Therefore, 3, 4, and 5h are finally selected as the factor levels of the response surface.

[0071] 2.3 Response surface test results

[0072] 2.3.1 Box-Behnken optimization test design and results

[0073] Based on the single-factor experimental results, different extraction parameters of solid-liquid ratio (A), temperature (B), and time (C) were selected as independent variables, and the polysaccharide (Y) content of yam peel water extract was used as the response value. The Box-Behnken design principle and 3-factor 3-level response surface analysis method were used to design the experiment.

[0074] 2.3.2 Optimization test variance analysis and significance test

[0075] From the results of variance analysis in Table 2, it can be seen that the P value of the model is 0.0002, indicating that the regression model is extremely significant, the equation obtained by the model fits the actual data very well, and the model is meaningful. The model can be used to design experiments to optimize the extraction process of yam peel polysaccharide content. The first-order terms A solid-liquid ratio, B extraction temperature, and C extraction time have significant effects on the extracted polysaccharide content (P<0.05), and the interactions AB, AC, and NC among the factors are not significant (P>0.05); the second-order terms A 2 , B 2 , C 2 All of them reached a significant level (P<0.05), indicating that the three factors themselves had a significant effect on the polysaccharide content of yam peel water extract, but the interaction between the three factors was not significant. According to the F distribution and P value significance analysis, the order of influence of the three factors on the polysaccharide content of yam peel extract was: solid-liquid ratio (A)> time (C)> temperature (B).

[0076] Table 2 Results of variance analysis

[0077]

[0078]

[0079] Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0080] 2.3.3 Determination of optimal process conditions and verification test

[0081] Combined with the software calculation regression model, the predicted value of the polysaccharide content of the yam peel water extract can be predicted to be 102.02 mg / g. The optimal extraction process is a solid-liquid ratio of 1:23.87, an extraction temperature of 75.15℃, and an extraction time of 4.37h. According to the actual experimental operation, the optimal process parameters were adjusted to a solid-liquid ratio of 1:24, an extraction temperature of 75℃, and an extraction time of 4.4h. This condition was selected for the verification test. The experiment was repeated 3 times, and the average polysaccharide content of the yam peel water extract was measured to be 101.43 mg / g, with a relative deviation of about 0.58% from the theoretical prediction value, and the yield of the yam peel water extract was 19.46%. The results show that the extraction process optimized by the response surface method has a high degree of credibility, and the model established in this experiment can be used for experimental prediction.

[0082] 3 Discussions

[0083] Among the methods for extracting effective ingredients from traditional Chinese medicine, hot water extraction is the most commonly used and economical extraction method. This method is simple, easy to operate, and has strong feasibility. In this paper, hot water extraction was used. On the basis of a single factor experiment, the response surface optimization was carried out with the solid-liquid ratio, extraction temperature, and extraction time as independent variables and the polysaccharide content of the yam peel water extract as the response value. The response surface method is a method that can optimize the extraction process parameters. It can consider the interaction between influencing factors and display the functional relationship between independent variables and dependent variables in a graphical manner, which not only makes the data more intuitive and clear, but also improves the extraction rate. Finally, the optimal extraction conditions for the yam peel water extract were determined to be a solid-liquid ratio of 1:24, an extraction temperature of 75℃, and an extraction time of 4.4h. Under these conditions, the polysaccharide extraction content of the yam peel water extract was 101.43mg / g. This process provides a reference for the future utilization and development of yam peel resources.

[0084] 4 Summary

[0085] This paper preliminarily determined the optimal solid-liquid ratio, extraction temperature and extraction time of yam peel water extract through single factor experiment. Then, the response surface test analysis showed that the influence of each factor on the polysaccharide content of yam peel water extract was solid-liquid ratio, time and temperature. Among them, these three factors themselves had a significant effect on the polysaccharide content of yam peel water extract, but the interaction between the three factors was not significant. Finally, the optimal process of yam peel hot water extraction was obtained: solid-liquid ratio 1:24, extraction temperature 75℃, extraction time 4.4h. Under these conditions, the polysaccharide extraction content of yam peel water extract was 101.43mg / g, and the yield of yam peel water extract was 19.46%.

[0086] Example 2 Effect of yam peel aqueous extract on growth performance and immune function of immunosuppressed chickens

[0087] 1 Test methods

[0088] 1.1 Experimental treatment and grouping

[0089] Ephedra chicks with similar body weight at 1 day old were selected and isolated and raised until 7 days old. They were randomly divided into 5 groups, 40 in each group, and entered the formal experiment. The grouping is shown in Table 3. Group 1 is the blank control group (NC), fed with a basic diet; Group 2 is the cyclophosphamide group (CTX, i.e., the immunosuppressive model group), fed with a basic diet; Group 3 is the low-dose group of yam peel water extract (L), with 0.5g / kg yam peel water extract added to the basic diet; Group 4 is the medium-dose group of yam peel water extract (M), with 1g / kg yam peel water extract added to the basic diet; Group 5 is the high-dose group of yam peel water extract (H), with 2g / kg yam peel water extract added to the basic diet. The experiment is divided into four stages: Days 1 to 7 are the first stage, Days 8 to 14 are the second stage, Days 15 to 21 are the third stage, and Days 22 to 28 are the fourth stage. The low, medium and high dose groups of yam peel water extract were given yam peel water extract from the first day of the experiment. On the first day of the formal experiment, the chicks in each group were immunized with "Newcastle disease and infectious bronchitis two-in-one live vaccine (LaSota strain + H120 strain)" by eye drop. In addition, the chickens in the model group and each medication group were injected with cyclophosphamide (CTX) prepared with normal saline at 80 mg / kg body weight in the leg muscle to induce an immunosuppressive model, and the NC group was injected with an equal amount of normal saline for 3 consecutive days.

[0090] The experiment involved sampling on the 7th, 14th, 21st and 28th days for subsequent testing of various related indicators.

[0091] Table 3 Grouping

[0092]

[0093] 1.2 Detection methods of various related indicators

[0094] Calculate the average body weight of each group of experimental chickens;

[0095] After blood was collected from the jugular vein, the animals were killed by dislocation of the neck. The middle section of the jejunum (2-3 mm) was quickly taken and fixed for 48 h (maintaining pH = 7.4) with 20 mL of 4% paraformaldehyde phosphate buffer. The sections were then embedded in conventional paraffin and prepared into 10 continuous cross-sectional sections (5 μm thick). After conventional hematoxylin-eosin (HE) staining, the height of the small intestinal villi and the depth of the crypts were measured, and their ratios were calculated.

[0096] The spleen, thymus and bursa of Fabricius were collected, the surrounding fat was stripped and the water was absorbed with filter paper, then weighed and recorded, and the immune organ index [immune organ index (g / kg) = immune organ fresh weight (g) / live weight (kg)] was calculated.

[0097] The levels of serum immunoglobulins IgG and IgM were detected by enzyme-linked immunosorbent assay (ELASA) kits purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0098] The hemagglutination test (HA) and hemagglutination inhibition test (HI) were used to determine the Newcastle disease antibody titer.

[0099] 2 Results and Analysis

[0100] 2.1 Body weight measurement results

[0101] The changes in the weight of the chickens in each group are shown in Table 4. As can be seen from Table 4, the initial weights of the groups were close before the start of the experiment, and there was no difference between them. After the formal experiment began, the weight of the chicks in the CTX group was significantly lower than that in the NC group (P<0.01) throughout the experiment, and the inhibition of weight growth caused by cyclophosphamide was alleviated in different dosing groups, but the relief of different dosing groups was different at different stages. On the 7th day of the experiment, the weight of the chicks in each dosing group increased compared with the CTX group, and there was no significant difference between the three dosing groups (P>0.05). On the 14th and 21st days of the experiment, the weight of the chicks in each dosing group increased significantly compared with the CTX group. On the 14th day, the weight of the chicks in the L group was higher than that in the M and H groups, and there was a significant difference between the L group and the H group. On the 21st day, the weight of the chicks in the M group was higher than that in the L and H groups. In the last stage of the experiment, the weight of the chicks in each dosing group was significantly higher than that in the CTX group (P<0.05), and there was no significant difference between the dosing groups.

[0102] Table 4 Changes in body weight of chickens in each group at each stage

[0103]

[0104] Note: Data in the same column marked with different letters indicate significant differences (P<0.05); data without letters or marked with the same letters indicate insignificant differences (P>0.05), the same below.

[0105] 2.2 Test results of immune organ index

[0106] The spleen, thymus and bursa of Fabricius index can be used to preliminarily measure the immune status of the body and the development of the immune system. The changes in the immune organ index of each group of chickens are shown in Tables 5 to 7. As shown in Table 5, during the entire experimental period, the spleen index of the CTX group was significantly lower than that of the NC group (P<0.05), indicating that CTX can significantly inhibit the development of the spleen. On the 7th day of administration, compared with the CTX group, the spleen index of each administration group increased slightly, but the difference was not significant (P>0.05); on the 14th day of administration, the spleen index of the H group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the L and M groups and the CTX group (P>0.05); on the 21st day of administration, compared with the CTX group, the spleen index of the L group increased significantly (P<0.05), and there was no significant difference between the L group and the NC group. On the 28th day of administration, the spleen index of each administration group was significantly increased compared with the CTX group (P<0.05), but had no significant difference with the NC group (P>0.05).

[0107] As shown in Table 6, during the entire experimental period, the thymus index of the CTX group was lower than that of the NC group, indicating that CTX can significantly inhibit the development of the thymus. On the 7th day of administration, the thymus index of the L group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the M and H groups and the CTX group (P>0.05); on the 14th day of administration, the thymus index of the CTX group was significantly lower than that of the NC group (P<0.05), and the thymus index of each administration group increased, but there was no significant difference between the CTX group and the NC group (P>0.05); on the 21st day of administration, the thymus index of the CTX group was lower than that of the NC group, and the antibody titer of each administration group increased, but there was no significant difference between the groups; on the 28th day of administration, the thymus index of the M group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the L and H groups and the CTX group and the NC group (P>0.05).

[0108] As shown in Table 7, during the entire experimental period, the bursal index of the CTX group was significantly lower than that of the NC group (P<0.05), indicating that CTX can significantly inhibit the development of the bursa. On the 7th day of administration, compared with the CTX group, the bursal index of each dose group was slightly increased, but the difference was not significant (P>0.05); on the 14th day of administration, the bursal index of the L group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the M and H groups and the CTX group (P>0.05); on the 21st day of administration, the bursal index of the H group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the L and M groups and the CTX group (P>0.05); on the 28th day of administration, the bursal index of the M group was significantly higher than that of the CTX group (P<0.05), and there was no significant difference between the L and H groups and the CTX group (P>0.05).

[0109] Table 5 Changes of spleen index of chickens in each group at each stage

[0110]

[0111] Table 6 Changes of thymus index of chickens in each group at each stage

[0112]

[0113] Table 7 Changes of bursal index of chickens in each group at each stage

[0114]

[0115] 2.3 Histopathological changes of chicken jejunum

[0116] The quality of the intestinal mucosal structure will directly affect the animal's absorption and utilization of nutrients. The ratio of intestinal villus height to crypt depth is an important indicator for measuring intestinal digestion and absorption function. The changes in the morphological structure of the jejunum of each group of chickens on the 14th and 28th days of the pathological histological test were as follows: Figure 5 and Figure 6 The statistical results of the villus height, crypt depth and the ratio of villus height to crypt depth (V / C) of the jejunum of each group of chickens on the 14th and 28th days of the experiment are shown in Tables 8 and 9, respectively.

[0117] Depend on Figure 5 As shown in Table 8, at 14 days of the experiment, the jejunum of the NC group was intact in morphology, the intestinal villi were arranged in an orderly manner, the structure was complete and fine, and the crypts were shallow. The intestinal structure of the chickens in the CTX group was damaged, the intestinal villi were atrophied, there were different degrees of breakage, the crypts were deepened, and the villus height, crypt depth, and the ratio of villus height / crypt depth were significantly different from those in the NC group (P<0.01). After administration, the intestinal villus height of each group increased, and the symptoms of intestinal injury recovered. The jejunal villus height of immunosuppressed chickens in the L and H groups was significantly higher than that in the CTX group, but it had not yet fully recovered, and there was a significant difference from the NC group (P<0.05). After administration, the crypt depth of each group decreased, and there was no significant difference between the L and M groups and the NC group (P>0.05). For the villus height / crypt depth, the ratio of each medication group increased compared with the CTX group, among which the villus height / crypt depth of the L group was significantly higher than that of the M and H groups (P<0.05).

[0118] Combination Figure 6As shown in Table 9, with the increase of age, at 28 days, the height of the jejunal villi in the NC group increased compared with that at 14 days, and its structure was complete, tightly arranged and orderly. In contrast, the villi in the CTX group were loosely arranged, and its villus height and villus height / crypt depth were significantly lower than those in the NC group (P<0.05), but there was no significant difference in the crypt depth between the two groups at this time. After administration, the villus height of each administration group was higher than that of the CTX group, but there was no significant difference between them (P>0.05), while the crypt depth of each administration group was significantly lower than that of the NC group and the CTX group (P<0.05), and its villus height / crypt depth was significantly higher than that of the CTX group (P<0.05), among which the villus height / crypt depth of the M and H groups was not significantly different from that of the NC group (P>0.05).

[0119] Table 8 Morphological structure of the villi of the jejunum of chickens in each group on day 14 of the experiment

[0120]

[0121] Table 9 Morphological structure of the villi of the jejunum of chickens in each group at 28 days of the experiment

[0122]

[0123] 2.4 Changes in serum Newcastle disease antibody titers

[0124] Depend on Figure 7 A in the figure shows that on the 7th day of the experiment, compared with the NC group, the serum Newcastle disease antibody titer of the chicks in the CTX group was significantly lower (P<0.05), and the serum antibody titer of each drug-treated group increased, but the difference was not significant with the CTX group (P>0.05). On the 14th day of the experiment, the serum antibody titer of the CTX group was still significantly lower than that of the NC group (P<0.05), and the serum antibody titer of each drug-treated group increased. Among them, the L and M groups had no significant difference with the NC group and the CTX group (P>0.05), while the antibody titer of the H group was significantly higher than that of the CTX group (P<0.05), and was close to that of the NC group, with no significant difference (P>0.05). On the 21st and 28th days of the experiment, the antibody titer of the CTX group was lower than that of the NC group, and the antibody titer of each drug-treated group increased, but there was no significant difference between the groups.

[0125] 2.5 Changes in serum immunoglobulins (IgG, IgM)

[0126] Immunoglobulin and immune organ index are both important indicators for measuring animal immune function. Figure 7As shown in B, on the 7th day of the experiment, compared with the NC group, the serum immunoglobulin IgG content in the CTX group was significantly lower (P<0.05), and although the serum IgG content in each medication group increased, it was not significantly different from the CTX group (P>0.05). On the 14th day of the experiment, the serum IgG content in the CTX group was lower than that in the NC group, and the serum IgG content in each medication group was equivalent or slightly increased, but there was no significant difference between the groups. On the 21st and 28th days of the experiment, the serum IgG content in the CTX group was significantly lower than that in the NC group (P<0.05), and the serum IgG content in each medication group was significantly higher than that in the CTX group (P<0.05), which was equivalent to the serum IgG content in the NC group.

[0127] Depend on Figure 7 C in the figure shows that on the 7th day of the experiment, compared with the NC group, the serum immunoglobulin IgM content of the CTX group was significantly reduced (P<0.05), while the serum IgM content of each medication group was slightly increased, but the difference was not significant with the CTX group (P>0.05). On the 14th and 21st days of the experiment, the serum IgM content of the CTX group was still significantly lower than that of the NC group (P<0.05), and the serum IgM content of each medication group increased. Among them, the serum IgM content of the H group was not significantly different from that of the NC group and the CTX group (P>0.05), while the serum IgM content of the L and M groups was significantly higher than that of the CTX group (P<0.05), close to the IgM content of the NC group, and the difference was not significant (P>0.05).

[0128] 3 Summary

[0129] 1) Adding 0.5-2 g / kg of yam peel water extract to the basal diet can significantly increase the body weight and immune organ index, immunoglobulin (IgG, IgM), and Newcastle disease antibody titer of immunosuppressed chickens caused by cyclophosphamide, and regulate the body's humoral immune function, cellular immune function and Newcastle disease antibody titer.

[0130] 2) Adding 0.5-2g / kg of yam peel water extract to the basal diet can increase the villus height and the ratio of villus height to crypt depth in the jejunum, and reduce the crypt depth. Among them, the recovery effect of the 2g / kg yam peel water extract group was the most significant.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing yam peel water extract, characterized in that: The steps include: (1) drying, crushing and sieving the yam peel to obtain yam peel powder; (2) adding the yam peel powder of step (1) to hot water at a material-liquid ratio of 1 g: 15-25 mL, soaking, and centrifuging to collect the supernatant, i.e., the yam peel water extract; (3) The yam peel water extract of step (2) is subjected to rotary evaporation to obtain a yam peel water extract.

2. The preparation method according to claim 1, characterized in that: In step (1), the drying condition is 50-60° C. for 5-8 hours; In step (1), the sieving is through a 50-100 mesh sieve.

3. The preparation method according to claim 1, characterized in that: In step (2), the material-liquid ratio is 1:20-25; further 1:

24.

4. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the hot water is 60-80°C; further 70-80°C; further 75°C.

5. The preparation method according to claim 1, characterized in that: In step (2), the soaking time is 3 to 5 hours; further 4 to 5 hours; further 4.4 hours.

6. A yam peel water extract, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the yam peel water extract according to claim 6, characterized in that: At least one of the following applications: (a) Use in the preparation of products for improving the growth performance of immunosuppressed chickens; (b) Use in the preparation of products for improving the immunity of immunosuppressed chickens; (c) Use in the preparation of products that promote digestion and absorption in immunosuppressed chickens.

8. The use according to claim 7, characterized in that: (b) Improving the immunity of immunosuppressed chickens includes improving the cellular immune function of immunosuppressed chickens and / or improving the humoral immune function of immunosuppressed chickens; The promoting digestion and absorption of immunosuppressed chickens described in (c) includes increasing the depth of intestinal crypts of immunosuppressed chickens and / or increasing the ratio of intestinal villus height to crypt depth of immunosuppressed chickens.

9. The use according to any one of claims 7 to 8, characterized in that: The immunosuppression is immunosuppression caused by cyclophosphamide; The product comprises feed, feed additive or drinking water additive.

10. The use according to claim 9, characterized in that: The amount of the yam peel water extract added to the feed is 0.5-2 g / kg.

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