Application of hemicellulose and mannan in regulation of growth performance of sheep

Abstract

The invention relates to the technical field of animal husbandry, and discloses application of hemicellulose and mannan in regulation of growth performance of sheep. The content of hemicellulose is 10.3% DM or 17% DM, and the addition amount of mannan is 80-160 mg / kg DM. According to the application of the hemicellulose and the mannan in regulating the growth performance of the sheep, the mannan is added into daily ration, so that the digestibility of animals to dry matters, crude proteins, neutral detergent fibers and acid detergent fibers can be increased, and the content of total acid, acetic acid, propionic acid, butyric acid and other volatile fatty acids in rumen can be increased; according to the present invention, with the feed, the richness and the diversity of the flora are increased, the richness of the firmicophyta, the spirochete and the like is improved, the intestinal flora of the sheep is adjusted, the blood sugar is reduced, and the daily ration with different hemicellulose levels has different influences on the growth performance, the rumen microorganisms, the metabolite and the lipid metabolism of the sheep; and a theoretical basis is provided for research and development of the yeast-source functional feed additive.

Description

Technical Field

[0001] The present invention relates to the field of animal husbandry technology, and particularly to the application of hemicellulose in combination with mannan in regulating the growth performance of sheep. Background Art

[0002] Cellulose and hemicellulose are the main components of plant cell walls and also the main components of roughage. Their main function is to control animal feed intake and maintain normal rumen function. Ruminants mainly use roughage as their food source and can utilize anaerobic microorganisms and fungi in the rumen to degrade and ferment it to produce volatile fatty acids (VFA). Moreover, the degradation of cellulose and hemicellulose is the largest source of changes in the digestibility and nutritional value of ruminant diets. Mannan is one of the main effective components of yeast cultures, mainly derived from yeast cell walls. Adding yeast cultures to feed is beneficial to the digestion, absorption, and utilization of cellulose and hemicellulose.

[0003] Therefore, to explore whether adding different doses of mannan to diets with different hemicellulose levels can play a positive regulatory role in the rumen function of sheep, the present invention conducts in vitro rumen fermentation simulation experiments to deeply explore the effects of hemicellulose levels and mannan in the diet on the rumen function, microbial community, and metabolites of sheep, aiming to further reveal the regulatory mechanism of mannan on sheep nutritional metabolism and provide a scientific basis for the research and development of yeast-derived functional feed additives. Summary of the Invention

[0004] The object of the present invention is to provide the application of hemicellulose in combination with mannan in regulating the growth performance of sheep. Adding mannan to the diet can increase the digestibility of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber in animals, increase the content of volatile fatty acids such as total acid, acetic acid, propionic acid, and butyric acid in the rumen, increase the richness and diversity of the flora, increase the richness of Firmicutes, Spirochaetes, etc., regulate the intestinal flora of sheep, reduce blood sugar, and different hemicellulose levels in the diet have different effects on the growth performance, rumen microorganisms and metabolites, and lipid metabolism of sheep; it provides a theoretical basis for the research and development of yeast-derived functional feed additives.

[0005] To achieve the above object, the present invention provides the application of hemicellulose in combination with mannan in regulating the growth performance of sheep. The content of hemicellulose is 10.3% DM or 17% DM, and the addition amount of mannan is 80 - 160 mg / kg DM.

[0006] Further, the addition amount of mannan is 160 mg / kg DM.

[0007] Further, it is applied to:

[0008] ①Improve the disappearance rates of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF), reduce the content of NH₃-N, and increase gas production;

[0009] ②When adding mannan to hemicellulose at 10.3% DM, increase the concentration of volatile fatty acids (VFA) in the rumen fluid;

[0010] ③Increase the abundances of hemicellulose-degrading bacteria and cellulose-degrading bacteria;

[0011] ④Regulate the rumen flora structure;

[0012] ⑤Increase the abundance of Prevotella and the content of epigallocatechin gallate metabolites to prevent diseases. The present invention also provides a yeast-derived functional feed additive comprising 160 mg / kg DM of mannan and 10.3% DM or 17% DM of hemicellulose.

[0013] The present invention also provides a functional feed, the active ingredients of which comprise 160 mg / kg DM of mannan and 10.3% DM or 17% DM of hemicellulose.

[0014] The advantages and positive effects of the application of the hemicellulose in synergy with mannan in regulating the growth performance of sheep in the present invention are as follows:

[0015] Adding mannan to the diet can increase the digestibility of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber in animals, increase the contents of total acids, acetic acid, propionic acid, butyric acid and other volatile fatty acids in the rumen, increase the richness and diversity of the flora, increase the richness of Firmicutes, Spirochaetes, etc., regulate the intestinal flora of sheep, reduce blood sugar, and different levels of hemicellulose in the diet have different effects on the growth performance, rumen microorganisms and metabolites, and lipid metabolism of sheep. The above results provide a theoretical basis for the research and development of yeast-derived functional feed additives.

[0016] The technical solution of the present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0017] Figure 1 This is the result of the abundance of bacteria in vitro fermentation in the embodiment of the present invention, where LHM0 is diet one (low hemicellulose) with 0 mg / kg DM of mannan added; LHM160 is diet one (low hemicellulose) with 160 mg / kg DM of mannan added; HHM0 is diet two (high hemicellulose) with 0 mg / kg DM of mannan added; HHM160 is diet two (high hemicellulose) with 160 mg / kg DM of mannan added;

[0018] Figure 2Results of the abundance of in vitro fermented bacteria in the embodiments of the present invention. Here, LHM0 is diet one (low hemicellulose) with 0 mg / kg DM mannan added; LHM160 is diet one (low hemicellulose) with 160 mg / kg DM mannan added; HHM0 is diet two (high hemicellulose) with 0 mg / kg DM mannan added; HHM160 is diet two (high hemicellulose) with 160 mg / kg DM mannan added.

[0019] Figure 3 In vitro fermentation Alpha diversity index in the embodiments of the present invention. Here, LHM0 is diet one (low hemicellulose) with 0 mg / kg DM mannan added; LHM160 is diet one (low hemicellulose) with 160 mg / kg DM mannan added; HHM0 is diet two (high hemicellulose) with 0 mg / kg DM mannan added; HHM160 is diet two (high hemicellulose) with 160 mg / kg DM mannan added.

[0020] Figure 4 Analysis of the correlation between rumen microbial phyla and fermentation parameters during 6 h of in vitro fermentation in the embodiments of the present invention. Here, A is diet one + 0 mg / kg DM, B is diet one + 160 mg / kg DM, C is diet two + 0 mg / kg DM, and D is diet two + 160 mg / kg DM.

[0021] Figure 5 Analysis of the correlation between rumen microbial genera and fermentation parameters during 6 h of in vitro fermentation in the embodiments of the present invention. Here, A is diet one + 0 mg / kg DM, B is diet one + 160 mg / kg DM, C is diet two + 0 mg / kg DM, and D is diet two + 160 mg / kg DM.

[0022] Figure 6 Statistical classification of metabolites in the embodiments of the present invention.

[0023] Figure 7 OPLS-DA analysis of metabolites during 6 h of in vitro fermentation in the embodiments of the present invention.

[0024] Figure 8 Volcano plot of differential metabolites in the embodiments of the present invention. Here, A is LHM0 vs HHM0, B is LHM0 vs LHM160, C is HHM0 vs HHM160, and D is LHM160 vs HHM160.

[0025] Figure 9This is the bubble chart of the differential metabolite enrichment pathway in the embodiments of the present invention, where A is LHM0 vs HHM0, B is LHM0 vs LHM160, C is HHM0 vs HHM160, and D is LHM160 vs HHM160. Detailed implementation manners

[0026] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0028] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. The experimental methods without specific conditions indicated in the following embodiments are usually determined according to national standards. The experimental instruments, equipment and reagents not indicated the sources in the following embodiments are all commercially available raw materials.

[0029] Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention.

[0030] Example: In vitro simulated rumen fermentation test

[0031] I. Effects of adding mannan to diets with different hemicellulose levels on in vitro rumen fermentation parameters of sheep:

[0032] 1. Test method:

[0033] 1.1 Test animals and location:

[0034] In this test, 4 healthy and quarantined male Small Tail Han sheep rams (36 ± 1.5 kg) were selected as test animals, and permanent rumen fistula installation surgeries were performed on them. After the surgeries, the health status of the sheep was observed regularly, the fistulas were nursed and cleaned. After the surgical wounds healed and the sheep's status recovered, the test was carried out. All test animals were kept in single cages, the fistulas were cleaned regularly and the health status of the test animals was observed, and the sheep pens were cleaned regularly to keep the pens hygienic. The test animals were fed at 8:00 am and 5:00 pm every day and were allowed to drink fresh and clean water freely. The test was carried out at the scientific research and teaching base west of Jilin Agricultural University, and all test steps were strictly implemented in accordance with the "Guidelines for the Care and Use of Laboratory Animals in Jilin Agricultural University".

[0035] 1.2 Test diets:

[0036] Table 1 Formulation and nutritional composition of test pellet feeds

[0037]

[0038] Note: a Premix ingredients (per kilogram): FeSO4 179 mg, CuSO4·5H2O 23 mg, ZnSO4·5H2O 92 mg, MnSO4 70 mg, Vitamin A 16 KIU, Vitamin D 111 KIU, Vitamin E 915 IU.

[0039] a Premix composition (per kilogram): FeSO4 179mg, CuSO4·5H2O 23mg, ZnSO4·5H2O92mg, MnSO4 70mg, Vitamin A 16KIU, Vitamin D 111KIU, Vitamin E 915IU.

[0040] 1.3 Experimental design:

[0041] The in vitro experiment adopted a 2×4 full factorial experimental design, which was divided into two factors. Factor one was two diets with different hemicellulose levels, with hemicellulose levels of 10.3% and 17% respectively; factor two was four different doses of mannan (0, 80, 160, 320 mg / kg) with four replicates in each group, three batches, and a total of 12 replicates. Sampling was carried out after 6 hours of in vitro culture time, and digestion parameters, fermentation parameters and rumen contents were analyzed to screen out the appropriate mannan dose that has a positive regulatory effect on rumen fermentation.

[0042] 1.4 Rumen fluid collection:

[0043] On the day of the experiment, CO2 was introduced into a thermos bottle preheated to 39°C for two minutes to ensure a suitable living environment for anaerobic microorganisms in the rumen fluid. Before morning feeding of fistula sheep, mixed rumen fluid was collected from different parts of the rumen using a hard PVC tube. The collected rumen fluid was quickly put into the thermos bottle, the bottle cap was closed, and the laboratory was immediately returned to filter the rumen fluid with multiple layers of gauze. The temperature was kept appropriate and CO2 was continuously introduced during the entire operation to ensure an anaerobic environment.

[0044] 1.5 In vitro culture:

[0045] The gas production was monitored and recorded using the ANKOM RFS device. Before the test, 2.000 g of substrate (formula see Table 1) was placed in 800-mesh nylon bags, which were sealed with a sealing machine and placed in ANKOM bottles, one in each bottle. Mannan was added to the bottle, and rumen fluid and buffer were added to the bottle in a ratio of 1:2. CO2 was continuously introduced into the bottle for one minute, and the bottle cap was quickly tightened and placed in a 39°C constant temperature air bath incubator for shaking culture (80 rpm).

[0046] Buffer preparation: 1 hour before the start of the experiment, mix Solution A, Solution B, and Solution C evenly at a ratio of 494:5:1 according to the required amount of the experiment. After mixing, continuously introduce CO2 into the bottom of the buffer solution until the buffer solution turns colorless. Then place the buffer solution in a water bath at 39 °C for preheating and standby. The buffer solution formula is shown in Table 2. Solution A is prepared one day before the experiment. After Solution B is prepared, CO2 needs to be continuously introduced for 18 hours. After sealing, it can be stored in the refrigerator for long-term use. After Solution C is prepared, CO2 needs to be continuously introduced for 20 minutes. After sealing, it can be stored in the refrigerator for long-term use.

[0047] Table 2 Buffer solution formula

[0048]

[0049]

[0050] 1.6 Determination of rumen fluid pH value:

[0051] The pH value is measured using a SANXIN MP523-04 portable pH meter (Shanghai Sanxin Instrumentation Co., Ltd., Shanghai, China). The pH value of the in vitro culture solution is measured at three time points. The pH meter is placed in the rumen fluid, and the value is recorded after the displayed value is stable.

[0052] 1.7 Determination of NH3-N concentration:

[0053] The ammonia nitrogen in the rumen fluid is determined by colorimetry, and colorimetric determination is carried out using an ultraviolet spectrophotometer.

[0054] 1.8 VFA determination:

[0055] The determination of volatile fatty acids is carried out using a gas chromatograph (Agilent Technologies 7890B).

[0056] 1.9 Determination of in vitro fermentation gas production:

[0057] The gas production is measured using an ANKOM RFS device, and the data of the gas production in the fermentation bottle is saved and recorded by using software GPM.

[0058] The calculation formula for gas production is: Vy = Vx * Ppsi * 0.068004084, where Vy is the gas production volume (ml), Vx is the remaining volume in the fermentation bottle (ml), and Ppsi is the cumulative pressure of fermentation (psi).

[0059] 1.10 Determination of in vitro nutrient disappearance rate:

[0060] Take out the nylon bags filled with substrates after fermentation in in vitro culture from the bottles, wash them repeatedly, and wash the rumen fluid on the bags until the water becomes clear. After air-drying the bags, put them into an oven and dry them at 105 °C until constant weight. Determine the dry matter (DM) in the feed raw materials, calculate the in vitro dry matter disappearance rate (IVDMD). Mix the fermentation substrates at the same time point and determine the contents of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE) and starch. Further calculate the in vitro crude protein digestibility (IVCPD), neutral detergent fiber digestibility (IVNDFD), acid detergent fiber digestibility (IVADFD), ether extract digestibility (IVEED) and starch digestibility (IVStarchD) of each substrate.

[0061] 2. Result analysis:

[0062] 2.1 In vitro fermentation nutrient disappearance rate:

[0063] As can be seen from Table 3, after 6 h of in vitro fermentation, adding different doses of mannan had a highly significant effect on IVDMD (P<0.01). Among them, the mannan added at 160 mg / kg DM in Diet 1 group was significantly higher than that in the control group and other experimental groups (P<0.05), while the mannan added at 320 mg / kg DM was significantly lower than that in the 160 mg / kg DM dose group (P<0.05). However, for Diet 2 group, adding different doses of mannan had no significant effect on IVDMD.

[0064] As shown in Table 3, adding different doses of mannan to diets with different hemicellulose levels all had a highly significant effect on IVCPD (P<0.01). After 6 h of in vitro fermentation, the mannan added at 40 mg / kg DM in Diet 1 was significantly higher than that in the control group and the groups adding 80 and 320 mg / kg DM mannan (P<0.05).

[0065] As shown in Table 3, adding different doses of mannan to diets with different hemicellulose levels all had a highly significant effect on IVADFD (P<0.01). After 6 h of in vitro fermentation, the control group and the group adding 160 mg / kg DM mannan in Diet 2 group were significantly higher than the group adding 320 mg / kg DM mannan (P<0.05).

[0066] As shown in Table 3, after 6 h of in vitro fermentation, diets with different hemicellulose levels and adding different doses of mannan did not have a significant effect on IVNDFD.

[0067] As shown in Table 3, diets with different hemicellulose levels and diets supplemented with different doses of mannan had highly significant effects on IVEED (P<0.01), and the interaction effect was also highly significant (P<0.01). After 6 h of in vitro fermentation, the IVEED of the diet group 1 supplemented with 80 mg / kg DM mannan was significantly higher than that of the control group and other experimental groups (P<0.01). The IVEED of the control group in diet group 2 was significantly higher than that of the other experimental groups supplemented with mannan.

[0068] As shown in Table 3, after 6 h of in vitro fermentation, adding different doses of mannan had highly significant effects on IVStarchD (P<0.01). Diets with different hemicellulose levels and their interaction effects both had significant effects on IVStarchD (P<0.05).

[0069] Table 3 (unit / %)

[0070]

[0071] 2.2 In vitro fermentation VFA:

[0072] As shown in Table 4, after 6 h of in vitro fermentation, the total acid (TVFA) content of the diet group 1 supplemented with 160 mg / kg DM mannan was significantly higher than that of the control group and the group supplemented with 320 mg / kg DM mannan (P<0.01). In diet group 2, the group supplemented with 160 mg / kg DM mannan was significantly higher than the control group (P<0.05). Different hemicellulose diets had significant effects on the total acid (P<0.05), adding different doses of mannan had highly significant effects on it (P<0.01), and their interaction effects had significant effects on the total acid (P<0.05).

[0073] Table 4

[0074]

[0075] 2.3 In vitro fermentation pH value, ammonia nitrogen concentration and gas production:

[0076] Table 5 shows the effects of adding different doses of mannan to two diets with different hemicellulose levels on the pH value, ammonia nitrogen concentration and gas production after 6 h of in vitro fermentation. As can be seen from Table 5, after 6 h of in vitro fermentation, the pH value of the control group in diet group 1 was significantly higher than that of the group supplemented with 160 mg / kg DM (P<0.01), and there was no significant difference in diet group 2. After 6 h of in vitro fermentation, different hemicellulose diets had highly significant effects on the pH value (P<0.01).

[0077] For the ammonia nitrogen (NH3-N) concentration, as shown in Table 5, after 6 h of in vitro fermentation, diets with different hemicellulose levels had highly significant effects (P<0.01).

[0078] As shown in Table 5, after 6 h of in vitro fermentation, the gas production of the diet group 1 with 320 mg / kg DM addition was extremely significantly higher than that of the control group (P<0.01), while the difference in gas production of the diet group 2 was not significant. After 6 h of in vitro fermentation, different levels of dietary hemicellulose and different doses of mannan added had extremely significant effects on it (P<0.01), and the interaction effect between the two also had an extremely significant effect (P<0.01).

[0079] Table 5

[0080]

[0081] Therefore, the results of the present invention show that different levels of hemicellulose and different doses of mannan added have extremely significant effects on CPD, NDFD, ADFD and EED, and the two have extremely significant interaction effects on NDFD and ADFD. Adding 160 mg / kg DM mannan to the diets with two different levels of hemicellulose improved the digestibility of DM, CP, NDF and ADF to varying degrees, and the NDFD and ADFD in the high hemicellulose level diet group were higher than those in the low hemicellulose level diet group. This indicates that adding mannan to the diet with a high hemicellulose level can better improve the utilization of cellulose and hemicellulose by ruminants.

[0082] The results show that adding mannan to the diet with a low hemicellulose level reduced the concentration of NH3-N to varying degrees, and the concentration was the lowest when adding 160 mg / kg DM mannan. While in the high hemicellulose level diet group, adding 160 mg / kg DM mannan reduced the concentration of NH3-N, but the concentration increased at 6 h of fermentation in other dose addition groups. This indicates that adding an appropriate amount of mannan can positively regulate the utilization of nitrogen. The concentration of NH3-N in the low hemicellulose level diet group was higher than that in the high hemicellulose level diet group, which may be due to the higher content of protein-degrading bacteria in it, thus decomposing excessive protein to generate ammonia.

[0083] The results show that adding mannan to the diet with a low hemicellulose level affected the gas production to varying degrees. Adding 160 and 320 mg / kg DM mannan were both significantly higher than the non-added group. This indicates that an appropriate dose of mannan can play a positive regulatory role in gas production, while adding mannan to the diet with a high hemicellulose level also reduced the gas production, which indicates that adding mannan to the diet with a high hemicellulose content did not have a positive effect on it.

[0084] The results show that after adding mannan, the pH value of the rumen fluid was effectively reduced. Especially after adding 160 mg / kg DM mannan, the reduction of the pH value was more significant. This indicates that adding an appropriate amount of mannan plays a positive regulatory role in balancing the rumen environment.

[0085] The results showed that adding 160 mg / kg DM mannan to diets with two different hemicellulose levels significantly increased the content of VFA in rumen fluid.

[0086] II. Effects of adding mannan to diets with different hemicellulose levels on rumen microorganisms in vitro fermentation of sheep:

[0087] 1.1 The collection of rumen fluid was the same as above.

[0088] 1.2 Extraction and detection of total microbial DNA:

[0089] The nucleic acid in rumen microorganisms was extracted using the OMEGA Soil DNA Kit (D5625-01) kit, and the DNA was quantified using an ultraviolet spectrophotometer.

[0090] 1.3 Determination of rumen microorganisms:

[0091] 16S rDNA, as a characteristic nucleic acid sequence revealing biological species, is considered the most suitable indicator for bacterial phylogeny and taxonomic identification. Therefore, 16S rDNA amplicon sequencing was selected. Specific primers F: GTGCCAGCMGCCGCGG and R: CCGTCAATTCMTTTRAGTTT were designed for PCR amplification of the 16S V4-V5 region to obtain an amplification fragment of about 450 bp. Using the Illumina Novaseq6000 platform, 2X250 bp paired-end data were sequenced. By splicing, longer sequences could be obtained for 16S analysis.

[0092] 1.4 Experimental design:

[0093] The optimal mannan addition dose of 160 mg / kg DM was selected, and 0 mg / kg DM was used as the control group for comparison, and rumen microorganisms were analyzed after 6 h of fermentation.

[0094] 2. Result analysis:

[0095] 2.1 Relative abundances of rumen fermentation microorganisms at the phylum level in vitro:

[0096] As Figure 1 shown, Figure 1For the in vitro fermentation of diets with different hemicellulose levels for 6 h, the abundances of the phyla of the in vitro fermentation bacteria of 160 mg / kg DM mannan are as follows. The top ten phyla in terms of abundance are Bacteroidota, Firmicutes, Spirochaetota, Fibrobacterota, Proteobacteria, Actinobacteriota, Cyanobacteria, Synergistota, Verrucomicrobiota, and Planctomycetota. The phyla with a relative abundance ratio greater than 1% are the dominant phyla, and there are a total of 5 dominant phyla, namely Bacteroidota, Firmicutes, Spirochaetota, Fibrobacterota, and Proteobacteria.

[0097] Table 6 In vitro phylum abundances of diets with different hemicellulose levels supplemented with mannan

[0098]

[0099] As can be seen from Table 6, after 6 h of in vitro fermentation, the abundances of Bacteroidota and Actinobacteriota in diet group 1 are extremely significantly higher than those in diet group 2 (P < 0.01), while the abundances of the main dominant phyla such as Firmicutes, Spirochaetota, Fibrobacterota, and Proteobacteria in diet group 2 are significantly higher than those in diet group 1 (P < 0.05). Adding 160 mg / kg DM mannan to diet group 1 significantly increased the abundances of Spirochaetota and Synergistota compared with the control group (P < 0.05), and significantly decreased the abundances of Firmicutes and Cyanobacteria (P < 0.05), while having no significant effect on Fibrobacterota, Proteobacteria, Actinobacteriota, and Verrucomicrobiota. Adding 160 mg / kg DM mannan to diet group 2 significantly increased the abundances of Firmicutes and Spirochaetota (P < 0.05), and significantly decreased the abundances of Cyanobacteria, Synergistota, and Verrucomicrobiota (P < 0.05), while having no significant effect on Bacteroidota, Fibrobacterota, Actinobacteriota, etc.

[0100] 2.2 Relative abundances of in vitro rumen fermentation microorganisms at the genus level:

[0101] As Figure 2 shown, Figure 2For the in vitro fermentation of diets with different hemicellulose levels supplemented with 160 mg / kg DM mannan for 6 h, the top ten genera with the highest in vitro fermentation abundances were Prevotella, F082, Muribaculaceae, Rikenellaceae, Treponema, Shuttleworthia, Fibrobacter, Succiniclasticum, Sphaerochaeta, and Lachnospiraceae.

[0102] Table 7 In vitro genus abundances of diets with different hemicellulose levels supplemented with mannan

[0103]

[0104] As can be seen from Table 7, the abundances of Prevotella, Muribaculaceae, Succiniclasticum, and Lachnospiraceae in diet group 1 were all extremely significantly higher than those in diet group 2 (P < 0.01), while the abundances of Treponema, Shuttleworthia, and Fibrobacter in diet group 2 were all significantly higher than those in diet group 1 (P < 0.05). Adding 160 mg / kg DM mannan to both different diets increased the abundances of Rikenellaceae, Treponema, and Lachnospiraceae, but decreased the abundance of Fibrobacter. Adding 160 mg / kg DM mannan to diet group 1 increased the abundances of Prevotella, Shuttleworthia, and Sphaerochaeta, and decreased the abundances of F082, Muribaculaceae, and Succiniclasticum. However, adding 160 mg / kg DM mannan to diet group 2 had the opposite effects on the genus abundances compared to adding mannan to diet group 1. The genera with increased abundances in diet group 1 decreased in diet group 2, while the genera with decreased abundances in diet group 1 increased in diet group 2.

[0105] 2.3 Analysis of Alpha diversity of in vitro rumen fermentation microorganisms:

[0106] Figure 3 This was the Alpha diversity analysis of in vitro rumen fermentation microorganisms with different doses of mannan added to two different diets. Table 8 shows the analysis of various indices of in vitro rumen fermentation microorganisms with different mannan additions to different diets.

[0107] Table 8 In vitro fermentation Alpha diversity indices

[0108]

[0109] As can be seen from Table 8, there were extremely significant differences in the Alpha diversity index between the diets with two different hemicelluloses (P<0.01), and the indexes of the diet group 2 were extremely significantly higher than those of the diet group 1 (P<0.01). However, adding mannan to the diet had no significant effect on it, and there was no interaction effect between different hemicelluloses and different doses of mannan added.

[0110] 2.4 In vitro fermentation parameters and correlation analysis with rumen microorganisms:

[0111] As Figure 4 shown, under the condition of in vitro fermentation for 6 h, by performing a correlation analysis between the in vitro fermentation parameters and the bacterial phyla with the top abundance rankings, it was judged whether adding mannan to the two diets promoted or inhibited them. In the diet group 1, when no mannan was added, Bacteroidetes was significantly negatively correlated with isovaleric acid (P<0.05), Spirochaetes was significantly negatively correlated with propionic acid, total acid and pH value (P<0.05), Firmicutes was significantly positively correlated with acetic acid and isovaleric acid (P<0.05), Fibrobacteres and Verrucomicrobia were significantly negatively correlated with isobutyric acid, butyric acid and valeric acid (P<0.05), Fibrobacteres was significantly positively correlated with the acetic acid to propionic acid ratio (P<0.05), Proteobacteria was significantly negatively correlated with isobutyric acid, butyric acid, valeric acid and total acid (P<0.05), while Actinobacteria was significantly positively correlated with isobutyric acid, butyric acid, valeric acid and total acid (P<0.05), and Cyanobacteria was significantly negatively correlated with propionic acid and pH value (P<0.05); after adding 160 mg / kg DM mannan, Cyanobacteria was significantly negatively correlated with pH value (P<0.05), and Verrucomicrobia was significantly negatively correlated with valeric acid (P<0.05). In the diet group 2, when no mannan was added, Firmicutes was significantly positively correlated with acetic acid, butyric acid, isovaleric acid and total acid (P<0.05), and was significantly negatively correlated with gas production (P<0.05), Actinobacteria was significantly positively correlated with acetic acid, isovaleric acid, total acid and the acetic acid to propionic acid ratio (P<0.05), and was significantly negatively correlated with gas production (P<0.05), Cyanobacteria was significantly positively correlated with acetic acid, isobutyric acid, butyric acid, isovaleric acid and total acid (P<0.05), and was significantly negatively correlated with gas production; after adding 160 mg / kg DM mannan, Firmicutes, Actinobacteria and Cyanobacteria were all significantly negatively correlated with gas production (P<0.05), and Cyanobacteria was significantly negatively correlated with pH value (P<0.05).

[0112] Figure 5Heatmap of the correlation analysis between in vitro fermentation parameters and the top-ranked genera after 6 hours of in vitro fermentation. In Diet Group 1, when mannan was not added, there was a significant negative correlation between genus F082 and isobutyric acid (P<0.05), a significant positive correlation between genus Fibrobacter and acetic acid (P<0.05), a significant positive correlation between genus Sphaerochaeta and valeric acid (P<0.05), and a significant negative correlation between genus Treponema and pH value (P<0.05). In Diet Group 2, when mannan was not added, there was a significant negative correlation between genus Lachnospira and isobutyric acid (P<0.05). After adding 160 mg / kg DM mannan, there were significant positive correlations between genus Lachnospira and propionic acid and valeric acid (P<0.05).

[0113] Therefore, the results of the present invention show that under in vitro fermentation conditions, the microbial diversity and richness of diets with high hemicellulose levels are significantly higher than those of diets with low hemicellulose levels, which may be due to the higher contents of corn and soybean meal in diets with low hemicellulose levels.

[0114] In the present invention, the contents of Bacteroidetes, Actinobacteria, Prevotella, Succiniclasticum, and Lachnospira in diets with low hemicellulose levels are all significantly higher than those in diets with high hemicellulose levels, while the contents of Firmicutes, Spirochaetes, Fibrobacteres, F082, Sutterella, and other microbial communities are significantly lower than those in diets with high hemicellulose levels.

[0115] After adding 160 mg / kg DM mannan to diets with low hemicellulose levels, Bacteroidetes, Spirochaetes, Synergistetes, Prevotella, Sutterella, etc. increased, while Firmicutes, Cyanobacteria, F082, and Succiniclasticum decreased. The opposite was true for the microbial abundance after adding mannan to diets with high hemicellulose levels.

[0116] Analysis of the correlation results shows that when mannan was not added, most phyla were negatively correlated with rumen fermentation parameters in diets with low hemicellulose levels, and genera were positively correlated with acids. After adding mannan, the correlation of phyla turned positive, and the genera did not change. In diets with high hemicellulose levels, the situation was opposite to that in diets with low hemicellulose levels. When mannan was not added, phyla were positively correlated with VFAs, and there was no significant correlation for genera. After adding mannan, a small number of genera were positively correlated with fermentation parameters. This indicates that adding mannan to diets with low hemicellulose levels has a positive regulatory effect on the phyla in rumen fluid and the production of VFAs, while adding mannan to diets with high hemicellulose levels has a positive regulatory effect on the abundance of genera in rumen fluid and the correlation of fermentation parameters.

[0117] III. Effects of adding different mannans to diets with different hemicellulose levels on rumen metabolism of in vitro fermentation in sheep rumen:

[0118] 1. Test method:

[0119] 1.1 Metabolite extraction:

[0120] Take 100 μL of the sample and place it in an EP tube. Add 400 μL of 80% methanol aqueous solution, vortex, let it stand in an ice bath for 5 min, and centrifuge at 15000 g and 4 °C for 20 min; Take a certain amount of the supernatant and dilute it with mass spectrometry-grade water until the methanol content is 53%; Centrifuge at 15000 g and 4 °C for 20 min, take the supernatant, and perform analysis.

[0121] 1.2 Data processing:

[0122] Use the software SIMCA (V14.1) to perform principal component analysis (PCA) on QC samples and experimental samples. Parameter settings: Use the R (V3.6.2) corrplot package and plotrix package in auto-fit to perform Pearson correlation coefficient analysis and relative standard deviation (RSD) analysis on QC samples and draw the correlation heat map and RSD distribution map.

[0123] 2. Test results:

[0124] 2.1 Metabolite classification statistics:

[0125] In this experiment, the rumen fluid fermented in vitro for 6 h was detected. A total of 1915 metabolites were detected, and the top nine in terms of proportion were analyzed. As Figure 6 shown, among them, organic acids and their derivatives accounted for the highest proportion, which was 27%. Lipids and lipid-like molecules accounted for 22.9% of the total. Organic heterocyclic compounds accounted for 17.3%. Benzenoid compounds accounted for 11.3%. Organic oxygen compounds accounted for 9.2%. Phenylpropanoids and polyketides accounted for 4.8%. Nucleosides, nucleotides and analogs accounted for 4.2%. The remaining two with relatively small proportions were organic nitrogen compounds accounting for 2.1% and alkaloids and their derivatives accounting for 1.16%.

[0126] 2.2 Metabolite OPLS-DA analysis:

[0127] The results are as Figure 7 shown. In the figure, the abscissa t[1]p represents the predicted principal component score of the first principal component, and the ordinate t[1]o represents the orthogonal principal component score. The scatter plot shapes and colors represent different experimental groups. It can be seen that the two groups of samples are very significantly distinguished, and all the samples are within the 95% confidence interval (Hotelling’s T-squared ellipse).

[0128] 2.3 Screening of differential metabolites:

[0129] As Figure 8As shown, the screening of differential metabolites is described by a volcano plot, and the screening criteria are p-value < 0.05 and VIP > 1 or p-value < 0.05 and FC > 1.2 | FC < 0.83.

[0130] Table 9

[0131] Total Increase Decrease LHM0 vs HHM0 546 81 465 LHM0 vs LHM160 125 121 4 HHM0 vs HHM160 20 2 18 LHM160vs HHM160 733 41 692

[0132] Table 9 shows the number of up-regulated and down-regulated differential metabolites. A total of 546 differential metabolites were detected in the LHM0 vs HHM0 group, of which 81 were up-regulated and 465 were down-regulated; 125 differential metabolites were detected in the LHM0 vs LHM160 group, of which 121 were up-regulated and 4 were down-regulated; 20 differential metabolites were detected in the HHM0 vs HHM160 group, of which 2 were up-regulated and 18 were down-regulated; 733 differential metabolites were detected in the LHM160 vs HHM160 group, of which 41 were up-regulated and 692 were down-regulated.

[0133] 2.4 Enrichment analysis of KEGG metabolic pathways of differential metabolites:

[0134] Figure 9 It is a bubble chart of the enriched metabolic pathways of the differential metabolites in four groups. The KEGG enrichment degree is measured by Enrichment factor, pvalue and the number of metabolites enriched in this pathway. The larger the Enrichment factor, the greater the enrichment degree; the closer the p-value is to zero, the more significant the enrichment.

[0135] The differential metabolites in the LHM0 vs HHM0 group were mainly enriched in sulfur metabolism, carbon metabolism, microbial metabolism in diverse environments, nucleotide metabolism, biosynthesis of secondary metabolites, biosynthesis of plant hormones, chlorocyclohexane and chlorobenzene degradation, pentose phosphate pathway, histidine metabolism, pyruvate metabolism, and pyrimidine metabolism; in the LHM0 vs LHM160 group, the differential metabolites were mainly enriched in pyrimidine metabolism, vitamin B6 metabolism, pentose phosphate pathway, isoflavonoid biosynthesis, and nucleotide metabolism;The differential metabolites in the HHM0 vs HHM160 group were mainly concentrated in purine metabolism, carbon metabolism, arginine and proline metabolism, glycine, serine and threonine metabolism, methane metabolism, glyoxylate and dicarboxylate metabolism, C5-branched dibasic acid metabolism, carbon fixation pathways in prokaryotes, caffeine metabolism, microbial metabolism in diverse environments, and histidine metabolism; the differential metabolites in the LHM160 vs HHM160 group were mainly concentrated in amino sugar and nucleotide sugar metabolism, antifolate resistance, biosynthesis of alkaloids derived from histidine and purine, biosynthesis of amino acids, biosynthesis of nucleotide sugars, biosynthesis of plant hormones, biosynthesis of plant secondary metabolites, carbon metabolism, histidine metabolism, nucleotide metabolism, and purine metabolism.;

[0136] 2.5 In vitro combined analysis of rumen microorganisms and metabolites:

[0137] The interactions between gut microbiota and their metabolites were explored through metabolomics analysis and 16S sequencing analysis.

[0138] The Spearman correlation coefficients were calculated using the psych package in R (v3.6.2) with the relative abundance table of ASV (top 30) and the eigenvector values of differential metabolites, and visualization was performed using the pheatmap package. In the LHM0 group, metabolites such as (2,5-dioxo-1-pyrrolidinyl)acetic acid and (2E)-4-hydroxybut-2-enoic acid were extremely significantly positively correlated with Prevotella (P < 0.01), while significantly negatively correlated with Xylobacter and NK4A214 genus (P < 0.05); in the LHM160 group, metabolites such as (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and (6E,8E)-3-hydroxy-10-methoxy-4,9-dimethyl-10-oxododeca-6,8-dienoic acid were significantly positively correlated with Prevotella (P < 0.05), while significantly negatively correlated with Succinivibrio and Xylobacter (P < 0.05); in the HHM0 group, metabolites such as epigallocatechin gallate and (1S)-2-amino-1-(4-chlorophenyl)-1-[4-(1H-pyrazol-4-yl)phenyl]ethanol were extremely significantly positively correlated with Prevotella and Treponema (P < 0.01), significantly negatively correlated with Wenkenella (P < 0.05), and metabolites such as (1-hydroxycyclohexyl)acetic acid and (3R)-3-hydroxy-L-proline were extremely significantly positively correlated with Sutterella and Prevotella (P < 0.01), significantly negatively correlated with Christensenella (P < 0.05); in the HHM160 group, metabolites such as (2R)-2-(2,5-difluorophenyl)pyrrolidine and (2R)-6-oxo-2-piperidinecarboxylic acid were extremely significantly positively correlated with Sphaerochaeta, Lachnospiraceae, Succinivibrio, etc. (P < 0.01), significantly negatively correlated with Prevotella, Xylobacter, etc. (P < 0.05).

[0139] The results showed that the differential metabolites among the dietary groups with different hemicellulose levels were mainly enriched in pathways such as the pentose phosphate pathway, nucleotide metabolism, and pyrimidine metabolism. After adding mannan to the dietary groups with different hemicellulose levels, the differential metabolites were mainly enriched in pathways such as amino sugar and nucleotide sugar metabolism, biosynthesis of nucleotide sugars, and carbon metabolism. The differential metabolites between the low hemicellulose level group and the group with 160 mg / kg DM mannan added were mainly enriched in pathways such as nucleotide metabolism, vitamin B6 metabolism, and pentose phosphate pathway. The differential metabolites between the high hemicellulose level group and the group with 160 mg / kg DM mannan added were mainly enriched in pathways such as purine metabolism, arginine and proline metabolism, methane metabolism, and histidine metabolism. The content of aspartic acid was upregulated in the high hemicellulose level dietary group compared with the low hemicellulose group, and it plays a crucial role in the neuroendocrine and endocrine systems as well as the central nervous system. After adding mannan to the diets with two different hemicellulose levels, the content of lysine was upregulated in the high hemicellulose level dietary group, and lysine can effectively inhibit the tubular reabsorption of albumin and protect the kidneys from further damage. The content of aspartic acid was upregulated in the low hemicellulose level dietary group, indicating that adding mannan improved the number of metabolites in the low hemicellulose level group compared with the non-added group.

[0140] For the joint analysis of the flora and metabolites, the results showed that 3-hydroxy-L-proline was extremely significantly positively correlated with Prevotella and Sutterella, and epigallocatechin gallate was extremely significantly positively correlated with Prevotella and Treponema. Among them, 3-hydroxy-L-proline can be degraded into ornithine and glutamate, thus preserving dietary and endogenously synthesized proline and arginine. Epigallocatechin gallate has protective antioxidant and anti-neuroinflammatory anti-inflammatory effects, and also has anti-tumor effects.

[0141] Therefore, the metabolites after adding mannan to the diets with different hemicellulose levels were mainly enriched in amino sugar and nucleotide sugar metabolism. Adding mannan to the high hemicellulose level dietary group can effectively prevent the occurrence of diseases. Prevotella was extremely significantly positively correlated with metabolites such as epigallocatechin gallate and can resist the occurrence of diseases.

[0142] Therefore, the present invention adopts the above application of hemicellulose in combination with mannan in regulating the growth performance of sheep. Adding mannan to the diet can increase the digestibility of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber in animals, increase the contents of total acid, acetic acid, propionic acid, butyric acid and other volatile fatty acids in the rumen, increase the richness and diversity of the flora, increase the richness of Firmicutes, Spirochaetes, etc., regulate the intestinal flora of sheep, reduce blood sugar, and different levels of hemicellulose in the diet have different effects on the growth performance, rumen microorganisms and metabolites, and lipid metabolism of sheep. The above results provide a theoretical basis for the research and development of yeast-derived functional feed additives.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of hemicellulose and mannan in regulating the growth performance of sheep is characterized by: The hemicellulose content is 10.3% DM or 17% DM, and the added amount of mannan is 80-160 mg / kg DM.

2. The use of hemicellulose and mannan in regulating sheep growth performance according to claim 1, characterized in that: The added amount of mannan was 160 mg / kg DM.

3. The use of hemicellulose and mannan in regulating sheep growth performance according to claim 1, characterized in that: Applies to: ① Increase the disappearance rate of dry matter DM, crude protein CP, neutral detergent fiber NDF and acid detergent fiber ADF, reduce the content of NH3-N and increase gas production; ② When mannan is added to hemicellulose with 10.3% DM, the concentration of VFA in rumen fluid is increased; ③Increase the abundance of hemicellulose-degrading bacteria and cellulose-degrading bacteria; ④Regulate the structure of rumen flora; ⑤Increase the abundance of Prevotella and the content of epigallocatechin gallate metabolites to prevent diseases.

4. A yeast-derived functional feed additive, characterized in that: Includes 160 mg / kg DM and hemicellulose at 10.3% DM or 17% DM.

5. A functional feed, characterized in that: The effective ingredient comprises the yeast-derived functional feed additive according to claim 4.

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