A composition for improving the activity of digestive enzymes in the intestine and use thereof

Abstract

The application provides a composition for improving intestinal digestive enzyme activity and application thereof, and belongs to the technical field of animal medicine. The composition is composed of double-kidney polysaccharide, ginsenoside Rb1 and ginsenoside Rg1. In the composition, the mass ratio of the double-kidney polysaccharide, the ginsenoside Rb1 and the ginsenoside Rg1 is 90:5:5 or 95:2.5:2.5, preferably 90:5:5, and the intestinal digestive enzyme is specifically duodenal trypsin and amylase. Experimental results show that the composition can significantly synergistically improve the activities of the duodenal trypsin and amylase of laying hens, improve the feed utilization rate, thereby improve the egg production rate, improve the egg weight and the eggshell strength, and effectively improve the breeding benefit of the laying hens.

Description

Technical Field

[0001] This invention belongs to the field of animal medicine technology, and particularly relates to a composition for improving the activity of intestinal digestive enzymes and its application. Background Technology

[0002] Egg production performance is a crucial indicator for evaluating the economic efficiency and production level of poultry farming, directly reflecting the overall level of breeding management, feed utilization, and the health status of laying hens. Among the many factors affecting egg production performance, digestive enzyme activity, as a key physiological indicator determining nutrient absorption and metabolic efficiency, plays a vital role. Highly efficient digestive enzyme activity not only ensures the full decomposition and absorption of nutrients in feed, providing laying hens with sufficient energy and essential nutrients, but also maintains the overall health of laying hens, enhancing their immunity and disease resistance.

[0003] By enhancing the digestive enzyme activity of laying hens, feed utilization can be significantly optimized, reducing feed waste and costs. This optimization process not only lowers feeding costs and improves farming efficiency but also promotes rapid growth and high egg production by increasing nutrient absorption efficiency. Furthermore, enhanced digestive enzyme activity directly impacts egg quality, thereby meeting market demand for high-quality eggs and further increasing farmers' economic returns. Therefore, enhancing the digestive enzyme activity of laying hens is a key approach to improving overall farming efficiency. Summary of the Invention

[0004] This invention aims to provide a composition, feed, and its application for improving the activity of intestinal digestive enzymes, egg production performance, and egg quality in laying hens. Addressing the shortcomings of existing technologies, this invention synergistically enhances the activity of intestinal digestive enzymes in laying hens by combining *Dioscorea bulbifera* polysaccharide with ginsenosides Rb1 and Rg1 in a specific ratio, thereby improving the egg production performance and egg quality.

[0005] Terminology: In this invention, the term "Dicranopteris polysaccharide" refers to a polysaccharide extracted from the plant Dicranopteris; the terms "ginsenoside Rb1" and "ginsenoside Rg1" refer to the active ingredients extracted from ginseng. Ginsenoside Rb1 has the CAS number 41753-43-9 and the molecular formula C 54 H 92 O 23 The CAS number for ginsenoside Rg1 is 22427-39-0, and its molecular formula is C. 42 H 72 O 14 The term "intestinal digestive enzymes" refers to the enzymes in the intestines of laying hens that participate in the digestion of proteins, fats, and starches, mainly including trypsin, lipase, and amylase.

[0006] The technical solution of the present invention includes: In a first aspect, the present invention provides a composition for improving the activity of digestive enzymes in the intestinal tract of laying hens, characterized in that the composition is composed of ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1.

[0007] Preferably, the kidney polysaccharide is prepared according to the following preparation method:

[0008] (1) After washing and crushing the dried kidney seeds, mix them with water at a solid-liquid ratio of 1:15;

[0009] (2) After adding cellulase for enzymatic hydrolysis, heat to inactivate, and then perform ultrasonic extraction under the conditions of ultrasonic power 250w, temperature 60℃, and time 45 minutes.

[0010] (3) After centrifuging and collecting the supernatant, decolorize it with activated carbon.

[0011] (4) Filter and collect the filtrate, concentrate it, and use Sevage reagent to remove the protein to obtain a crude polysaccharide solution of *Dictyophora indica*.

[0012] (5) After adding 3 times the amount of ethanol for alcohol precipitation, centrifuge to collect the precipitate and wash it to obtain crude polysaccharide of Dioscorea cirrhosa.

[0013] (6) After dissolving the crude polysaccharide of *Lysimachia christinae*, collect the 10kDa-50kDa *Lysimachia christinae* polysaccharide solution using an ultrafiltration membrane;

[0014] (7) After concentration, freeze-dry to obtain ginseng polysaccharide.

[0015] Preferably, in the composition, the mass ratio of ginsenoside Rb1 and ginsenoside Rg1 is 90:5:5 or 95:2.5:2.5.

[0016] Preferably, in the composition, the mass ratio of ginsenoside Rb1 and ginsenoside Rg1 is 90:5:5.

[0017] Preferably, the intestinal digestive enzymes are duodenal trypsin and amylase.

[0018] Preferably, the laying hen is a Hy-Line Brown laying hen.

[0019] Secondly, the present invention provides a feed for improving the activity of intestinal digestive enzymes in laying hens. The feed comprises a basal diet for laying hens and the aforementioned composition, wherein the composition is added to the feed at an amount greater than or equal to 0.05%. The feed of the present invention can enhance the digestive capacity of laying hens by increasing the activity of intestinal digestive enzymes, thereby improving feed conversion ratio and overall production efficiency.

[0020] Preferably, the composition is added to the feed at an amount of 0.05%.

[0021] Thirdly, the present invention provides the application of the above composition in the preparation of feed that improves the activity of intestinal digestive enzymes in laying hens, wherein the intestinal digestive enzymes are duodenal trypsin and amylase.

[0022] Fourthly, the present invention provides a composition for improving the egg production performance and quality of laying hens, characterized in that the composition comprises ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1, wherein the mass ratio of ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1 in the composition is 90:5:5 or 95:2.5:2.5.

[0023] Preferably, in the composition, the mass ratio of ginsenoside Rb1 and ginsenoside Rg1 is 90:5:5.

[0024] Preferably, the egg production performance refers to the egg production rate, and the egg production quality refers to egg weight and eggshell strength;

[0025] The kidney polysaccharide was prepared according to the above preparation method.

[0026] Fifthly, the present invention provides a feed for improving the egg production performance and quality of laying hens, characterized in that the feed comprises a basic diet for laying hens and the above-mentioned composition, wherein the composition is added to the feed at an amount greater than or equal to 0.05%.

[0027] Preferably, the egg production performance refers to the egg production rate, and the egg production quality refers to egg weight and eggshell strength.

[0028] In a sixth aspect, the present invention provides the application of a composition in the preparation of feed that improves the egg production performance and quality of laying hens, the composition being composed of ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1;

[0029] The nephrolepis polysaccharide was prepared by the above-described preparation method.

[0030] Preferably, in the composition, the mass ratio of ginsenoside Rb1 and ginsenoside Rg1 is 90:5:5 or 95:2.5:2.5.

[0031] Preferably, in the composition, the mass ratio of ginsenoside Rb1 and ginsenoside Rg1 is 90:5:5.

[0032] Preferably, the egg production performance refers to the egg production rate, and the egg production quality refers to egg weight and eggshell strength.

[0033] The beneficial effects of this invention are as follows: through the rational ratio of kidney polysaccharide to ginsenosides Rb1 and Rg1, the composition of this invention significantly enhances the activity of digestive enzymes in the intestinal tract of laying hens, especially trypsin and amylase activities. Enhanced digestive enzyme activity helps improve the efficiency of feed digestion and absorption in laying hens, promoting more efficient absorption of nutrients from the feed.

[0034] Meanwhile, experimental data show that feeding hens with the feed of this invention can significantly improve the egg production rate of laying hens and reduce feed waste. Due to the increased activity of digestive enzymes, laying hens can utilize the nutrients in the feed more effectively, thereby increasing egg production.

[0035] Secondly, the use of the composition can not only increase the egg production rate, but also significantly improve egg weight and eggshell strength, helping laying hens produce more and better quality eggs to meet the market demand for high-quality eggs. Attached Figure Description

[0036] Figure 1 This reflects the differences in digestive enzyme activity among different experimental groups of laying hens;

[0037] in, Figure 1 (a) shows the differential results for trypsin. Figure 1 (b) shows the differential results for lipase. Figure 1 (c) shows the differential results for amylase;

[0038] Figure 2 This is the result of the difference in egg production rate among different experimental groups of laying hens;

[0039] Figure 3 This reflects the differences in egg quality among different experimental groups of laying hens;

[0040] in, Figure 3 (a) shows the results of the difference in egg weight. Figure 3 (b) shows the results of the difference in eggshell strength. Detailed Implementation

[0041] The optional embodiments of this invention will now be described in more detail. Although optional embodiments of this invention have been described, it should be understood that this invention can be implemented in many forms and should not be limited to the embodiments listed herein. In fact, these embodiments are provided to make the content of this invention clearer and more comprehensive, thereby ensuring that those skilled in the art can accurately and completely understand the scope of this invention.

[0042] In this application, the terminology used is merely for describing specific embodiments and is not intended to limit the scope of the application. Furthermore, the singular forms "a," "the," and "the" used in this application and the appended claims, unless the context clearly indicates otherwise, encompass the majority forms. Additionally, it should be understood that the term "and / or" as used herein refers to any or all possible combinations including one or more of the associated listed items.

[0043] Example 1

[0044] (1) After washing the dried kidney tuber with clean water three times, grind the kidney tuber into fine powder using a grinder and pass it through an 80-mesh sieve to obtain kidney tuber powder.

[0045] (2) Add 15 times the amount of deionized water to the double kidney powder, and add 2.5% cellulase at the same time. React in a water bath at 50°C for 60 minutes.

[0046] (3) After inactivation at 90℃ for 10 minutes, ultrasonic extraction was performed at 250W power and 60℃ for 45 minutes.

[0047] (4) Centrifuge at 4000 rpm for 15 min, collect the supernatant, add activated carbon at a solid-liquid ratio of 5:100, and stir to decolorize for 30 min;

[0048] (5) Filter and collect the filtrate, concentrate the filtrate to 1 / 3 of the original volume, add an equal volume of Sevage reagent to remove protein 5 times, and obtain a crude polysaccharide solution of *Dictyophora indica*.

[0049] (6) Add three times the amount of 95% ethanol cooled to 4°C to the double kidney solution, and let it stand at 4°C for 16 hours for alcohol precipitation.

[0050] (7) Centrifuge at 4000 rpm for 20 minutes, collect the precipitate, wash twice with pre-cooled 95% ethanol, then wash twice with pre-cooled acetone, and air dry at room temperature to obtain crude polysaccharide of *Dictyophora indicum*.

[0051] (8) Dissolve the crude polysaccharide extract of *Dictyophora indicum* in 10 times the amount of water, filter it using a 50 kDa ultrafiltration membrane, and collect the permeate;

[0052] (9) The permeate was filtered using a 10 kDa ultrafiltration membrane, and the retentate was collected to obtain the nephrolepis polysaccharide extract;

[0053] (10) After being concentrated under reduced pressure to 1 / 3 of the original volume, it was freeze-dried in a freeze dryer to obtain bis-nephrine polysaccharide.

[0054] Example 2

[0055] The formula for layer hen feed 1 consists of 99.95% basal diet and 0.05% composition 1;

[0056] The basal diet consists of 67.00%, 3.00% wheat bran, 14.00% soybean meal, 5% cottonseed meal, 4% rapeseed meal, 2.00% meat floss powder, 4.00% limestone powder, and 1.00% premix.

[0057] The premix provides the following per kilogram of diet: Vitamin A 10000 IU, Vitamin D 32500 IU, Vitamin E 18 IU, Vitamin K 31 mg, Vitamin B1 2 mg, Vitamin B2 6 mg, Vitamin B6 3.5 mg, Vitamin B1 4 mg, Vitamin B2 6 mg, Vitamin B6 3.5 mg, Vitamin B2 6 mg, Vitamin B2 7 mg, Vitamin B2 8 mg, Vitamin B1 8 mg, Vitamin B2 ... 12 15μg, pantothenic acid 18mg, niacin 30mg, biotin 0.15mg, folic acid 0.4mg, Fe 80mg, Cu 9mg, Zn 70mg, Mn 80mg, I 0.6mg, Se 0.3mg.

[0058] The composition consists of ginsenoside Rb1 and Rg1, with a mass ratio of ginsenoside Rb1 and Rg1 of 90:5:5.

[0059] Example 3

[0060] The formula for layer hen feed 2 consists of 99.95% basal diet and 0.05% composition 2;

[0061] The basic diet composition is the same as in Example 2;

[0062] Composition 2 consists of ginseng polysaccharide, ginsenoside Rb1, and Rg1, with a mass ratio of ginseng polysaccharide, ginsenoside Rb1, and Rg1 of 95:2.5:2.5.

[0063] Comparative Example 1

[0064] The formula for layer hen feed 3 consists of 99.95% basal diet and 0.05% nephrolepis polysaccharide.

[0065] Comparative Example 2

[0066] The formula for layer hen feed 4 consists of 99.95% basal diet and 0.05% composition 3;

[0067] The basic diet composition is the same as in Example 2;

[0068] Composition 3 is composed of Ganoderma lucidum polysaccharide, ginsenoside Rb1 and Rg1, with a mass ratio of Ganoderma lucidum polysaccharide, ginsenoside Rb1 and Rg1 of 90:5:5.

[0069] Comparative Example 3

[0070] The formula for layer hen feed 5 consists of 99.95% basal diet and 0.05% composition 4;

[0071] The basic diet composition is the same as in Example 2;

[0072] Composition 4 consists of ginseng polysaccharide and ginsenoside Rb1, with a mass ratio of ginseng polysaccharide to ginsenoside Rb1 of 90:10.

[0073] Comparative Example 4

[0074] The formula for layer hen feed 6 consists of 99.95% basal diet and 0.05% composition 5;

[0075] The basic diet composition is the same as in Example 2;

[0076] Composition 5 consists of *Pseudolarix amabilis* polysaccharide and ginsenoside Rg1, with a mass ratio of 90:10.

[0077] Example 4

[0078] (1) Select healthy Hy-Line Brown laying hens aged 56 weeks with consistent egg production performance and randomly divide them into 7 groups (control group and experimental group af), with 3 replicates in each group and 20 birds in each replicate;

[0079] (2) The control group was fed a basic diet, experimental group a was fed layer hen feed 1, experimental group b was fed layer hen feed 2, experimental group c was fed layer hen feed 3, experimental group d was fed layer hen feed 4, and experimental group e was fed layer hen feed 5.

[0080] (3) The laying hens were raised in individual cages, with one waterer in each cage. The temperature was set at 25℃ and the relative humidity at 65% during the experiment, with 6 hours of light and 8 hours of darkness.

[0081] (4) One week of preliminary experiment to allow laying hens to adapt to the environment and feed, and six weeks of formal experiment.

[0082] Example 5

[0083] (1) On the day the experiment ended, five laying hens were randomly selected from each replicate, euthanized and dissected, and their duodenums were removed.

[0084] (2) Gently squeeze the intestinal segment and collect the contents into a pre-cooled sterile centrifuge tube;

[0085] (3) The duodenal contents were diluted 1:10 with 0.1% physiological saline on ice and homogenized using a sterile homogenizer to obtain a homogenate.

[0086] (4) Centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and use an ELISA kit (Nanjing Jiancheng Biotechnology Co., Ltd.) to detect the differences in the activities of trypsin, lipase, and amylase. The results are shown in Table 1 and... Figure 1 As shown.

[0087] Table 1. Differences in the activity of digestive enzymes in the duodenum of laying hens in different experimental groups.

[0088]

[0089] As shown in Table 1, the trypsin and amylase activities of experimental groups a and b were significantly higher than those of other experimental groups and the control group, indicating that the digestive enzyme activities of laying hens fed with the laying hen feed 1 and laying hen feed 2 provided in Examples 2 and 3 of this invention were significantly improved. Specifically, the trypsin and amylase activities of laying hens in experimental group a were significantly higher than those in experimental group b, indicating that the combination of *Dioscorea bulbifera* polysaccharide, ginsenoside Rb1, and Rg1 according to the formula of laying hen feed 1 can fully exert the synergistic effect of the three in promoting the activity of protease and amylase in laying hens.

[0090] Further observation revealed that the trypsin and amylase activities in experimental group c were somewhat increased compared to the control group, but the effect was not significant. This indicates that while using only laying hen feed containing ginseng polysaccharide 3 can promote trypsin and amylase activities to some extent, the effect is limited and fails to achieve the expected significant increase. This suggests that the strategy of simply adding functional polysaccharides to feed formulations is insufficient to fully realize their potential.

[0091] The trypsin and amylase activities in experimental group d were the lowest among all experimental groups, indicating that even when Ganoderma lucidum polysaccharide was replaced with Ganoderma lucidum polysaccharide, the combination of Ganoderma lucidum polysaccharide with ginsenosides Rb1 and Rg1 could not exert a synergistic effect in promoting the digestive enzyme activity of laying hens. This may be because under the experimental conditions, Ganoderma lucidum polysaccharide failed to form an effective synergistic effect with ginsenosides Rb1 and Rg1, and could not replace the function of Ganoderma lucidum polysaccharide in enhancing digestive enzyme activity.

[0092] Furthermore, the results of experimental groups e and f further illustrate that while the combined use of ginsenoside Rb1 or Rg1 with *Lysimachia christinae* polysaccharide was more effective than that of laying hen feed supplemented with only *Lysimachia christinae* polysaccharide, it still showed a highly significant difference compared to the combined effect of ginsenoside Rb1 and Rg1. This indicates that relying solely on the combined use of a single ginsenoside (Rb1 or Rg1) with *Lysimachia christinae* polysaccharide cannot fully realize its synergistic effect. Therefore, the combined use of ginsenoside Rb1 and Rg1 is a key condition for achieving the synergistic enhancement of digestive enzyme activity in laying hens with *Lysimachia christinae* polysaccharide.

[0093] Example 6

[0094] (1) Throughout the experiment, the number of eggs laid by the hens was collected daily and the egg production rate was calculated. Detailed egg production rate results are shown in Table 2 and... Figure 2 As shown.

[0095] (2) Egg weight and shell strength were tested, and the results are shown in Table 3 and... Figure 3 As shown.

[0096] Table 2. Differences in egg production rate among different experimental groups of laying hens.

[0097] detection indicators control group Experimental group a Experimental group b Experimental group c Experimental group d Experimental group e Experimental group f Egg production rate / % 85.33±1.28 93.99±1.53 91.11±1.36 87.18±1.21 86.13±1.32 88.72±1.31 88.87±1.54

[0098] Table 3. Differences in egg quality among different experimental groups of laying hens.

[0099] detection indicators control group Experimental group a Experimental group b Experimental group c Experimental group d Experimental group e Experimental group f Egg weight / g 61.47±0.46 67.02±0.60 65.12±0.71 62.20±0.51 61.71±0.69 62.71±0.39 63.17±0.56 Eggshell strength / kgf 3.129±0.045 3.674±0.062 3.505±0.043 3.203±0.035 3.199±0.029 3.284±0.044 3.328±0.028

[0100] Table 2 shows the differences in egg production rates among the different experimental groups. The egg production rate of the control group was 85.33%, while that of experimental groups a and b were 93.99% and 91.11%, respectively, significantly higher than the control group and other experimental groups. The egg production rate of experimental group c was 87.18%, slightly higher than the control group, but the difference was not significant. The egg production rate of experimental group d was only 86.13%, similar to the control group, indicating that the Ganoderma lucidum polysaccharide replacing the kidney polysaccharide had a limited effect on the egg production rate. The egg production rates of experimental groups e and f were 88.72% and 88.87%, respectively, both higher than the control group, but lower than experimental groups a and b.

[0101] These results further validate the findings in Table 1, namely that the combination of *Lysimachia foenum-graecum* polysaccharide with ginsenosides Rb1 and Rg1 significantly improved the egg production rate of laying hens. Experimental group a had the highest egg production rate, indicating that the combination of *Lysimachia foenum-graecum* polysaccharide with ginsenosides Rb1 and Rg1, according to the formula of laying hen feed 1, can effectively promote the physiological functions of laying hens and improve egg production efficiency.

[0102] Table 3 shows the differences in egg weight and eggshell strength among the different experimental groups. The control group had an egg weight of 61.47 g and an eggshell strength of 3.129 kgf. Experimental group a had an egg weight of 67.02 g and an eggshell strength of 3.674 kgf, significantly higher than the control group. Experimental group b had an egg weight of 65.12 g and an eggshell strength of 3.505 kgf, also significantly better than the control group. Experimental group c had an egg weight of 62.20 g and an eggshell strength of 3.203 kgf, slightly higher than the control group but not significantly. Experimental group d had an egg weight of 61.71 g and an eggshell strength of 3.199 kgf, similar to the control group. Experimental groups e and f had egg weights of 62.71 g and 63.17 g, and eggshell strengths of 3.284 kgf and 3.328 kgf, respectively, both better than the control group but lower than experimental groups a and b.

[0103] These data indicate that the combination of *Dioscorea bulbifera* polysaccharide with ginsenosides Rb1 and Rg1 not only enhances the digestive enzyme activity and egg production rate in laying hens, but also significantly improves egg quality, specifically through increased egg weight and shell strength. This further demonstrates the effectiveness of the synergistic effect of the three components, enhancing the overall production performance of laying hens.

Claims

1. Use of a composition in the manufacture of a feed for increasing the activity of digestive enzymes in the intestine of a laying hen, characterized in that, The composition is made from ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1; The kidney polysaccharide was prepared according to the following method: (1) After washing and crushing the dried kidney seeds, mix them with water at a solid-liquid ratio of 1:15; (2) After adding cellulase for enzymatic hydrolysis, heat to inactivate, and then perform ultrasonic extraction under the conditions of ultrasonic power 250w, temperature 60℃, and time 45 minutes. (3) After centrifuging and collecting the supernatant, decolorize it using activated carbon; (4) Filter and collect the filtrate, concentrate it, and use Sevage reagent to remove the protein to obtain a crude polysaccharide solution of *Dictyophora indica*. (5) After adding 3 times the amount of ethanol for alcohol precipitation, centrifuge to collect the precipitate and wash it to obtain crude polysaccharide of Dioscorea nipponica; (6) After dissolving the crude polysaccharide of *Lysimachia christinae*, collect the 10kDa-50kDa *Lysimachia christinae* polysaccharide solution using an ultrafiltration membrane; (7) After concentration, freeze-dry to obtain ginseng polysaccharide; In the composition, the mass ratio of *Dictyophora indica* polysaccharide, ginsenoside Rb1, and ginsenoside Rg1 is 90:5:5 or 95:2.5:2.

5. The intestinal digestive enzymes are duodenal trypsin and amylase; The composition is added to the feed at a rate of 0.05% or greater.

2. A composition for improving egg laying performance and egg quality in laying hens, characterized by, The composition comprises ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1, wherein the mass ratio of ginseng polysaccharide, ginsenoside Rb1 and ginsenoside Rg1 in the composition is 90:5:5 or 95:2.5:2.

5. The egg production performance refers to the egg production rate, and the egg production quality refers to egg weight and eggshell strength. The kidney polysaccharide was prepared according to the following method: (1) After washing and crushing the dried kidney seeds, mix them with water at a solid-liquid ratio of 1:15; (2) After adding cellulase for enzymatic hydrolysis, heat to inactivate, and then perform ultrasonic extraction under the conditions of ultrasonic power 250w, temperature 60℃, and time 45 minutes. (3) After centrifuging and collecting the supernatant, decolorize it using activated carbon; (4) Filter and collect the filtrate, concentrate it, and use Sevage reagent to remove the protein to obtain a crude polysaccharide solution of *Dictyophora indica*. (5) After adding 3 times the amount of ethanol for alcohol precipitation, centrifuge to collect the precipitate and wash it to obtain crude polysaccharide of Dioscorea nipponica; (6) After dissolving the crude polysaccharide of *Lysimachia christinae*, collect the 10kDa-50kDa *Lysimachia christinae* polysaccharide solution using an ultrafiltration membrane; (7) After concentration, freeze-dry to obtain ginseng polysaccharide.

Images