Application of alpha-ketoglutaric acid in improvement of rumen fermentation and improvement of microbial protein synthesis

Abstract

The invention discloses an application of alpha-ketoglutaric acid in improving ruminant rumen fermentation and promoting microbial protein synthesis. The invention discloses application of alpha-ketoglutaric acid in preparation of a feed additive for improving ruminant rumen fermentation and promoting microbial protein synthesis. The invention discloses an application of alpha-ketoglutaric acid in preparation of a feed for improving ruminant rumen fermentation and promoting microbial protein synthesis. The alpha-ketoglutaric acid can increase the concentration of total volatile fatty acids (including acetic acid, propionic acid and butyric acid), reduce the concentration of ammonia nitrogen in rumen, increase the concentration of microbial protein, improve the feed efficiency, promote the synthesis of the microbial protein and improve rumen fermentation.

Description

Technical Field

[0001] The invention belongs to the field of ruminant feed additives and relates to the application of alpha-ketoglutaric acid in improving rumen fermentation and enhancing microbial protein synthesis. Background Art

[0002] Currently, the use of non-protein nitrogen as a substitute for soybean meal is being actively promoted. This provides a nitrogen source for ruminant rumen microbes to synthesize microbial protein (MCP), converting non-protein nitrogen into high-quality protein in milk, beef, and lamb. This reduces ruminant farming costs and reduces dependence on high-quality protein feed ingredients. However, its potential advantages and value have not yet been fully explored and utilized.

[0003] The stomach and small intestine of monogastric animals mainly rely on their own enzymes to digest food (such as soybean meal and fish meal), but there is a complex microbial community in the hindgut (cecum, colon - the end of the digestive system). These microorganisms can ferment undigested fiber and other substrates (such as resistant starch and oligosaccharides) and synthesize microbial protein in the process. Monogastric animals have low absorption efficiency of hindgut microbial protein, and most microbial protein will be excreted with feces, with low utilization rate. Microbial protein cannot become the main source of protein for monogastric animals.

[0004] Ruminants (cattle and sheep) rely on microbial protein as their main source of protein. Microbial protein plays a vital role in ruminants and is an important source of digestible protein in the small intestine of ruminants. Rumen microorganisms can break down cellulose, starch, fat, and protein in feed into small molecules, which are used by microorganisms to synthesize microbial protein. After the microbial protein formed in the rumen enters the true stomach and intestine with the chyme, it is broken down into small peptides or free amino acids by the digestive enzymes secreted by the animal for use by the animal body. The amino acids that can be absorbed by the small intestine of ruminants mainly come from three parts: rumen microbial protein, non-degradable protein in feed (including small peptides), and endogenously secreted protein. Microbial protein is the primary source of amino acids absorbed by the small intestine. For most diets, microbial protein synthesized in the rumen accounts for approximately 60%-85% of the total amino acid nitrogen entering the small intestine. Furthermore, the amino acid composition of microbial protein is superior to that of plant protein, closely matching animal requirements. Microbial protein is particularly rich in limiting amino acids such as lysine and methionine, and its degradation rate in the small intestine is as high as 80%-85%. This can compensate for deficiencies in plant-based feeds, contributing over 60% of ruminants' protein needs and serving as a core nutrient source for maintaining growth, milk production, and reproduction. Microbial protein plays a central role in the nutrition, metabolism, and overall health of ruminants, serving not only as a primary source of amino acids but also in multiple aspects such as feed conversion efficiency and protein metabolism.

[0005] Compared to monogastric animals, ruminants utilize only about 25% less protein. This not only results in a significant waste of high-quality protein but also contributes to environmental pollution, leading to acid deposition, eutrophication, human respiratory diseases, and climate change. Furthermore, rumen microbes can reconvert urea from the host's blood (via saliva or diffusion through the rumen wall) into microbial protein, significantly improving nitrogen utilization efficiency and reducing nitrogen excretion (e.g., urea in urine).

[0006] During rumen fermentation, the degradation of feed protein and the efficiency of microbial protein synthesis are limited by multiple factors, resulting in low nitrogen utilization efficiency. This is not only a waste of protein resources, but also causes certain environmental pollution. During rumen fermentation, the synthesis of microbial protein is an important way for ruminants to obtain high-quality protein. However, the existing technology lacks effective means to significantly improve the efficiency of microbial protein synthesis, especially in the complex rumen environment. Therefore, improving the nitrogen utilization efficiency of ruminants is an urgent problem that needs to be solved. Summary of the Invention

[0007] The present invention aims to address the problems of low nitrogen utilization efficiency and insufficient microbial protein synthesis during ruminant rumen fermentation by providing an application of α-ketoglutarate (AKG) in improving ruminant rumen fermentation and promoting microbial protein synthesis. AKG can be used as a feed additive for efficient production in the ruminant breeding industry, thereby improving nitrogen utilization efficiency in ruminants and improving rumen fermentation.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The purpose of the present invention is to provide the application of α-ketoglutarate in improving rumen fermentation of ruminants and promoting microbial protein synthesis.

[0010] Preferably, the application is: α-ketoglutarate is mixed evenly with the basic diet as a feed additive and fed, and the added dosage of α-ketoglutarate is 1-5g α-ketoglutarate per 1kg of the basic diet.

[0011] Another object of the present invention is to provide the use of α-ketoglutarate in the preparation of a feed additive for improving rumen fermentation and promoting microbial protein synthesis in ruminants.

[0012] Another object of the present invention is to provide the use of α-ketoglutarate in preparing feed for improving rumen fermentation and promoting microbial protein synthesis in ruminants.

[0013] Preferably, the application is as follows: α-ketoglutarate is mixed evenly with a basal diet as a feed additive and fed to the feed, with the additive dosage being 1-5 g α-ketoglutarate per 1 kg of feed intake. For example, 1-2 g, 2-3 g, or 3-5 g α-ketoglutarate is added per 1 kg of basal diet.

[0014] The ruminants are cattle, sheep, camels, deer, alpacas and antelopes.

[0015] Another object of the present invention is to provide the use of α-ketoglutarate in improving rumen fermentation in dairy cows and promoting microbial protein synthesis.

[0016] The application has at least one of the following:

[0017] (1) α-ketoglutaric acid reduces the concentration of NH3-N in the rumen and increases the content of microbial protein;

[0018] (2) α-ketoglutarate promotes the synthesis of volatile fatty acids;

[0019] (3) α-ketoglutarate promotes the synthesis of milk protein production in ruminants;

[0020] (4) α-Ketoglutaric acid improves the digestibility of crude protein, dry matter and organic matter in ruminants.

[0021] The volatile fatty acids include acetic acid, propionic acid, butyric acid and branched-chain fatty acids.

[0022] α-Ketoglutaric acid can be purchased from the market or prepared by the user, and the purity requirement is relatively high, at least above 90%.

[0023] The beneficial effects of the present invention are:

[0024] 1. α-ketoglutaric acid is easily soluble in water and has high stability in aqueous solution. When α-ketoglutaric acid is mixed with the basal diet and fed, it can be utilized by the animal body and can stably exert its functions and effects due to its high stability.

[0025] 2. α-ketoglutaric acid can perfectly play the physiological function of α-ketoglutaric acid, which can increase the concentration of total volatile fatty acids (including acetic acid, propionic acid, and butyric acid), reduce the concentration of ammonia nitrogen in the rumen, increase the concentration of microbial protein, improve feed efficiency, promote microbial protein synthesis, and improve rumen fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the effect of α-ketoglutarate on the dynamic changes of gas production during 24h in vitro fermentation.

[0027] Figure 2 The effect of adding different doses of α-ketoglutaric acid on gas production.

[0028] Figure 3 The effect of adding different doses of α-ketoglutarate on pH.

[0029] Figure 4 This is the effect of adding different doses of α-ketoglutarate on ammonia nitrogen concentration.

[0030] Figure 5 The effect of adding different doses of α-ketoglutarate on the content of microbial protein. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0032] Table 1. Basic diet composition

[0033]

[0034]

[0035] Note: Premix: Jianbida 5% compound premix feed for dairy cows - M505A (Jiangsu Jianhe Animal Husbandry Technology Co., Ltd.); Dry yeast: Aofu feed additive brewer's yeast [for livestock and poultry], yeast viable cell count ≥ 20 billion / g; De-mold agent: mycotoxin scavenger for dairy cows (Xinkemei) (Manufacturer: American International Bio-Nutrition Co., Ltd.): Crude ash (hydrated sodium calcium aluminosilicate) ≤ 90%, brewer's yeast ≥ 1.0×10 10 cfu / kg.

[0036] Example 1

[0037] Using the in vitro static simulated rumen fermentation method, Holstein cows were selected as rumen fluid donors, and different doses of α-ketoglutaric acid were added to the rumen fluid of the cows. The gas production, total volatile fatty acids, acetic acid, propionic acid, butyric acid concentrations, rumen ammonia nitrogen concentration, and microbial protein concentration were investigated to evaluate whether α-ketoglutaric acid can regulate rumen fermentation and improve the utilization of nitrogen and energy in the feed.

[0038] 1 Materials and Methods

[0039] 1.1 Experimental Design

[0040] This experiment used a single-factor design with one control group and three experimental groups, with each treatment replicated five times. The control group (NC) included no additives to the rumen fluid. The three α-ketoglutarate (AKG) experimental groups, A-1, A-2, and A-3, were as follows: A-1 added 5 mg of AKG to a final concentration of 5 mg / dL; A-2 added 15 mg of AKG to a final concentration of 15 mg / dL; and A-3 added 45 mg of AKG to a final concentration of 45 mg / dL.

[0041] 1.2 Rumen fluid inoculation and in vitro culture

[0042] Three Holstein cows were used as rumen fluid donors. The cows' diets are shown in Table 1. Cows were fed three times daily at 06:00, 13:00, and 18:00, with free access to food and water. Before morning feeding, fresh rumen fluid was collected orally using a sample collector, mixed thoroughly, and then poured into a sterile bottle (1500 mL), placed in a 39°C incubator, and quickly brought back to the laboratory. The mixed rumen fluid sample was then filtered through four layers of gauze in a 39°C water bath under CO2. The filtered rumen fluid was used for inoculation. Strict anaerobic conditions were maintained throughout the collection and handling of rumen fluid.

[0043] In vitro fermentation was carried out in 180 mL serum bottles, each containing 1 g of substrate. The substrate composition is shown in Table 2. The buffer was prepared according to Zhou Yaqi et al. [1] Preparation: Under anaerobic conditions, filtered rumen fluid was thoroughly mixed with buffer at a ratio of 1:9 (v / v). Subsequently, 100 mL of the mixture was dispensed into serum bottles containing 1 g of substrate and the corresponding dose of α-ketoglutaric acid. The bottles were sealed with butyl rubber stoppers and secured with aluminum caps and incubated in a 39.0°C incubator for 24 h.

[0044] Table 2. Substrate composition

[0045]

[0046] Note: Premix: Jianbida 5% compound premix feed for dairy cows - M505A (Jiangsu Jianhe Animal Husbandry Technology Co., Ltd.).

[0047] 1.3 Index determination

[0048] Gas production is a key indicator to measure the degree of feed fermentation in the rumen and the activity of rumen microorganisms. An increase in gas production usually indicates that the growth rate of rumen microorganisms is accelerated, the activity is enhanced, and the metabolism is vigorous, thereby improving the fermentation level and increasing the utilization rate of nutrients (especially energy and nitrogen). During the fermentation process, a pressure sensor was used to measure the gas production at 3, 6, 9, 12, and 24 hours of fermentation. At the end of fermentation, a portable pH meter (EcoScan pH 5, Eutech Instruments, Singapore) was immediately used to measure the pH value of the fermentation broth in each serum bottle. The serum bottle was then placed in an ice bath to terminate the fermentation, and a fermentation broth sample was collected for the determination of ammonia nitrogen (NH3-N), volatile fatty acids (VFA), and microbial protein (MCP). 0.2 mL of 25% HPO3 was added to 1 mL of fermentation broth and frozen overnight. According to Mao et al. [2] VFA concentrations were determined using gas chromatography (7890A, Agilent, United Kingdom); ammonia nitrogen concentrations were determined using a colorimetric method; and microbial protein concentrations in the fermentation broth were determined using the Coomassie Brilliant Blue method. All samples were stored at −20°C until analysis.

[0049] 1.4 Data Statistics and Analysis

[0050] All experimental data were analyzed for variance and significance using SPSS, with Duncan's method used for multiple comparisons. Orthogonal polynomial contrasts were also used to analyze the linear and quadratic effects of different α-ketoglutarate doses. The significance level was P < 0.05.

[0051] 2 Results

[0052] As shown in Table 3 and Figure 1 、 Figure 2 、 Figure 3 As shown, gas production increased linearly with increasing AKG supplementation. Compared with the control group, α-ketoglutarate supplementation significantly increased rumen gas production in dairy cows. The pH of the A-2 group (AKG dose of 15 mg / dL AKG) and the A-3 group (AKG dose of 45 mg / dL) decreased significantly. The A-3 group also significantly increased TVFA, acetic acid, propionic acid, and butyric acid concentrations.

[0053] Table 3. Effects of different doses of α-ketoglutarate on rumen fermentation parameters in vitro

[0054]

[0055] Note: Different letters indicate significant differences.

[0056] like Figure 4 、 Figure 5 As shown in the figure, with the increase of AKG addition, the ammonia nitrogen concentration decreased linearly and the microbial protein content increased linearly. Compared with the control group, the ammonia nitrogen concentration of groups A-2 and A-3 decreased significantly, and the microbial protein content of group A-3 increased significantly.

[0057] This shows that AKG significantly improves the feed utilization efficiency of dairy cows, improves rumen fermentation, and promotes the synthesis of microbial protein.

[0058] Example 2

[0059] Holstein cows were selected and fed with α-ketoglutaric acid to investigate whether it could increase the microbial protein content and fatty acid concentration of the cows, the crude protein digestibility of the cows, the milk protein content in the milk, and whether it could improve the nitrogen utilization efficiency of the cows.

[0060] 1 Materials and Methods

[0061] 1.1 Experimental Materials and Animals

[0062] Twenty-four Holstein cows with similar lactation days, milk production, and parity were selected for the experiment.

[0063] α-Ketoglutaric acid (purchased from Shaanxi Xinpai Biotechnology Co., Ltd., purity ≥99%).

[0064] 1.2 Experimental design

[0065] Twenty Holstein cows were randomly divided into two groups: a control group (CON group), fed a basal diet; and an α-ketoglutarate group (AKG group), fed a basal diet supplemented with 25 g / d of AKG (approximately 0.125% of the AKG concentration, based on a DMI (Dry Matter Intake) of 20 kg / d). The α-ketoglutarate was mixed evenly with the basal diet and fed to the cows. Considering production practices and cost factors, and considering the results in Example 1 showing that both 15 mg / dL and 45 mg / dL of AKG exhibited good rumen regulation, the final AKG supplementation dose for the animal feeding trial was 25 g / d. The trial consisted of a two-week pilot phase and an eight-week final phase. Cows in both treatment groups were fed three times daily at 6:00 AM, 1:00 PM, and 7:00 PM, with a feed residue rate of 5-10% and free access to water.

[0066] 1.3 Sample collection and analysis

[0067] During the formal period, the feed intake and milk production of each cow were measured continuously for two days every two weeks; three hours after the morning feeding on the 56th day, rumen fluid samples were collected using an oral catheter for the determination of rumen fermentation parameters; in the eighth week of the formal experiment, cow fecal samples were collected continuously for three days using the spot sampling method for the subsequent determination of nutrient digestibility. In the 2nd, 4th, 6th, and 8th weeks of the experimental period, two whole mixed ration feed samples of the treatment groups were collected continuously for two days in the morning, middle, and evening respectively (using the quartering method, 500 g was collected each time after充分 mixing the feed), and stored sealed at -20 °C for the subsequent determination of the nutrient composition of the diet.

[0068] To determine the nutrient digestibility and the nutrient composition of the diet, the feed samples and fecal samples were dried in a ventilated oven at 65 °C for 48 h, then pulverized using a pulverizer, and passed through a 40-mesh sieve. Referring to Zhang Yingli et al. [3] The methods were used to determine the nutrient contents of dry matter (DM), organic matter (OM), crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), crude fat (EE), crude ash (Ash), and acid-insoluble ash (AIA). AIA in feces was used as an endogenous indicator to determine the digestibility of each nutrient, and the apparent digestibility of nutrients was calculated as follows:

[0069] Nutrient digestibility (%) = 100% - (acid-insoluble ash content in feed % / acid-insoluble ash content in feces %) * (% nutrient content in feces % / nutrient content in feed %) * 100%

[0070] After the rumen fluid samples were thawed from the rumen, NH4Cl was used as a standard product, and the colorimetric method was used to measure the NH3-N concentration with the help of an enzyme-labeled instrument, and the Coomassie Brilliant Blue method was used to measure the microbial protein concentration in the fermentation broth. 0.2 mL of 25% HPO3 was added to 1 mL of rumen fluid respectively, and a gas chromatograph (7890A, Agilent, UK) was used to measure the content of volatile fatty acids (VFA).

[0071] 1.4 Data statistics and analysis

[0072] The data obtained from the experiment were sorted out by Excel 2024 and analyzed using the independent samples T-test in SPSS. P < 0.05 was considered significantly different, and 0.05 < P < 0.1 indicated a trend of change.

[0073] 2 Results

[0074] 2.1 Production performance

[0075] As can be seen from Table 4, compared with the control group, the addition of α-ketoglutaric acid significantly increased the milk protein yield in cow milk, and there was a trend of increase in feed intake, milk production, ECM, and lactose yield.

[0076] Table 4. Effects of feeding α-ketoglutarate on dry matter intake, milk production and milk composition of dairy cows

[0077]

[0078] Note: SEM, standard error of the mean; Trt, treatment; Wk, week; Trt×Wk, interaction between treatment and week.

[0079] 2.2 Nutrient digestibility

[0080] As shown in Table 5, compared with the control group, α-ketoglutaric acid can significantly improve the digestibility of crude protein, dry matter and organic matter in dairy cows, thereby improving the utilization of feed protein in dairy cows.

[0081] Table 5. Effects of feeding α-ketoglutarate on nutrient intake and apparent digestibility of dairy cows

[0082]

[0083]

[0084] 2.2 Rumen fermentation parameters

[0085] As shown in Table 6, the addition of AKG increased the microbial protein content, butyric acid, valeric acid, isobutyric acid, isovaleric acid and total branched-chain fatty acid concentrations of dairy cows, and the ammonia nitrogen concentration and total volatile fatty acids showed an increasing trend.

[0086] Table 6. Effects of feeding α-ketoglutarate on rumen fermentation parameters of dairy cows

[0087]

[0088]

[0089] α-Ketoglutaric acid significantly promotes milk production and nutritional metabolism. It significantly increases the microbial protein content and volatile fatty acid (VFA) concentration in dairy cows, thereby improving the rumen fermentation process. At the same time, AKG significantly increases the digestibility of crude protein in feed, thereby improving the efficiency of feed protein utilization in dairy cows. Furthermore, AKG significantly increases the milk protein content in cow milk and improves the nitrogen utilization efficiency of dairy cows, providing strong support for efficient dairy production.

[0090] References:

[0091] [1] Zhou Yaqi, Cheng Yanfen, Zhu Weiyun. (2021). Rumen microbial in vitro fermentation process and precautions. Microbiome Experiment Manual. Bio-101:e2003663. DOI:10.21769 / BioProtoc.2003663.

[0092] [2]Mao SY, Zhang G, Zhu W Y. Effect of disodium fumarate on ruminalmetabolism and rumen bacterial communities as revealed by denaturing gradientgel electrophoresis analysis of 16Sribosomal DNA[J]. Animal feed science and technology, 2008, 140(3-4): 293-306.

[0093] [3] Zhang Liying. Feed Analysis and Feed Quality Testing Technology (2nd Edition)[M]. Beijing: China Agricultural University Press, 2007.

Claims

1. Application of α-ketoglutarate in improving rumen fermentation and promoting microbial protein synthesis in ruminants.

2. The use according to claim 1, characterized in that: The application has at least one of the following: (1) α-ketoglutaric acid reduces the concentration of NH3-N in the rumen and increases the content of microbial protein; (2) α-ketoglutarate promotes the synthesis of volatile fatty acids; (3) α-ketoglutarate promotes the synthesis of milk protein production in ruminants; (4) α-Ketoglutaric acid improves the digestibility of crude protein, dry matter and organic matter in ruminants.

3. The use according to claim 1 or 2, characterized in that: α-ketoglutaric acid is mixed evenly with the basic diet as a feed additive and fed. The added dosage of α-ketoglutaric acid is 1 to 5 g of α-ketoglutaric acid per 1 kg of basic diet.

4. Application of α-ketoglutaric acid in the preparation of feed additives for improving rumen fermentation and promoting microbial protein synthesis in ruminants.

5. The use according to claim 4, characterized in that: The application has at least one of the following: (1) α-ketoglutaric acid reduces the concentration of NH3-N in the rumen and increases the content of microbial protein; (2) α-ketoglutarate promotes the synthesis of volatile fatty acids; (3) α-ketoglutarate promotes the synthesis of milk protein production in ruminants; (4) α-Ketoglutaric acid improves the digestibility of crude protein, dry matter and organic matter in ruminants.

6. Application of α-ketoglutaric acid in the preparation of feed for improving rumen fermentation and promoting microbial protein synthesis in ruminants.

7. The use according to claim 6, characterized in that: The application has at least one of the following: (1) α-ketoglutaric acid reduces the concentration of NH3-N in the rumen and increases the content of microbial protein; (2) α-ketoglutarate promotes the synthesis of volatile fatty acids; (3) α-ketoglutarate promotes the synthesis of milk protein production in ruminants; (4) α-Ketoglutaric acid improves the digestibility of crude protein, dry matter and organic matter in ruminants.

8. The use according to claim 6, characterized in that: α-ketoglutaric acid was mixed evenly with the basic diet and fed. The additive dosage of α-ketoglutaric acid was 1 to 5 g per 1 kg of basic diet.

9. The use according to any one of claims 1 to 8, characterized in that: The ruminants are cattle, sheep, camels, deer, alpacas and antelopes.

10. Application of α-ketoglutaric acid in improving rumen fermentation and promoting microbial protein synthesis in dairy cows.

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