Application of red alga and brown alga composition in inhibition of ruminant rumen methane generation

Abstract

The invention discloses a composite seaweed additive with red algae and brown algae as core components and application of the composite seaweed additive in inhibition of ruminant rumen methane generation. In the composition, the ratio of red algae is 30-60%, the ratio of brown algae is 20-50%, and auxiliary algae and the like can also be added (the total ratio is less than or equal to 20%). In-vitro and in-vivo experiments prove that by adding the composition, the methane emission can be reduced by 55-65%, meanwhile, the dry matter degradation rate (DMD) is kept stable, and the rumen microbial community structure is remarkably optimized. Compared with the prior art, through the synergistic effect of the halogenated compound of the red algae and the brown algae polyphenol, the dosage limitation of single algae is broken through, efficient emission reduction is achieved at low cost, and no side effect exists. The method is suitable for breeding ruminants such as cattle and sheep, and assists low-carbon transformation in animal husbandry.

Description

Technical Field

[0001] The present invention belongs to the technical field of ruminant nutrition regulation and emission reduction, and specifically relates to an application of a red algae and brown algae composition in inhibiting rumen methane production in ruminants, and a feed additive based on the synergistic effect of red algae and brown algae, which is used to directionally inhibit the activity of rumen methanogens and reduce greenhouse gas emissions. Background Art

[0002] According to existing technical analysis, ruminant methane emissions account for 14.5% of the world's total anthropogenic methane. Its emission reduction technologies mainly include:

[0003] 1. Chemical inhibitors: such as 3-nitrooxypropanol (3-NOP), which can reduce methane by 30%, but there is a risk of drug residues, and the EU has restricted its use (Roque et al., 2021);

[0004] 2. Single seaweed additives: such as the red algae patent (WO2015109362A2), which can achieve a 14.7% reduction, but requires a high dose (≥5% dry weight), and long-term use may inhibit fiber-degrading bacteria, leading to a decrease in DMD (Lorenna Machado et al., 2015);

[0005] 3. Plant extracts: Tannins, saponins, etc. have limited methane inhibition effects (<15%) and poor palatability (Kinley et al., 2020).

[0006] At present, the technical bottleneck in this field is the limitation of single seaweed: although red algae (such as Asparagopsistaxiformis) contain powerful methane inhibitors such as bromoform, their resources are scarce and expensive (Roque et al., 2019); brown algae (such as Sargassum) are cheap, but the effect is unstable when used alone (Kuenzel et al., 2022).

[0007] ● Unexplored synergistic effects: Existing patents have not explored the combined synergistic mechanism of different seaweed groups, resulting in difficulty in achieving breakthroughs in emission reduction efficiency. Summary of the invention

[0008] The present invention solves the defects of single seaweed through the synergistic mechanism of red algae and brown algae:

[0009] 1. Red algae directional inhibition of methanogens: bromoform (CHBr3) in red algae directly inhibits methyl coenzyme M reductase (Mcr), blocking the final step of methane production (Cheong et al., 2023);

[0010] 2. Brown algae regulate microbial balance: Brown algae polyphenols (such as alginic acid) promote the proliferation of propionic acid-producing bacteria (such as Succinivibrio) and reduce the supply of hydrogen substrates (Li et al., 2023);

[0011] 3. Balance between cost and effect: the proportion of red algae is reduced to 30%-60%, combined with cheap brown algae, the total cost is reduced by 40%,

[0012] And the emission reduction efficiency is increased to 55%-65%.

[0013] Specifically, it is an application of a composition of red algae and brown algae in inhibiting rumen methane production in ruminants, or an application of a composition of red algae and brown algae as a feed additive in inhibiting rumen methane production in ruminants, characterized in that: the red algae in the composition accounts for 30%-60%, the brown algae accounts for 20%-50%, and the remainder is auxiliary seaweed (accounting for ≤20%).

[0014] The red algae are mainly Asparagopsis taxiformis or Rhodomelaconfervoides, etc.; the brown algae are mainly Sargassum serratum, Laminaria japonica, Undaria pinnatifida Suringar, etc.

[0015] The auxiliary seaweed is one or more of Enteromorpha prolifera, Gracilaria or Ulva lactuca, and the total proportion does not exceed 20%.

[0016] The composition is added to the basic daily diet feed of ruminants as a feed additive in the form of dry powder, freeze-dried particles or extracts, and the added amount is 0.5%-10% of the dry weight of the daily diet.

[0017] The composition preparation method comprises the following steps:

[0018] (1) Rinse the red algae, brown algae and auxiliary seaweed with fresh water, dry at 60°C and then crush to 40 mesh;

[0019] (2) Mix the components in proportion and grind them by ultrafine grinding technology to obtain a composition, the particle size of the composition is ≤100

[0020] μm.

[0021] The invention comprises a basic diet and any one of the above-mentioned compositions, wherein the added amount of the composition is 0.5%-10% of the dry weight of the feed.

[0022] Technical advantages of the present invention

[0023] ● High efficiency: methane emissions were reduced by 52% at a 5% addition, which is better than single red algae (18%) or brown algae (12%);

[0024] Safety: No significant change in DMD (P>0.05), avoiding the risk of rumen acidosis;

[0025] ●Universality: Applicable to a variety of ruminants such as dairy cows, beef cattle, goats, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Heat map of the abundance analysis of key bacterial genera in diets supplemented with different doses of complex algae.

[0027] Figure 2 PCoA analysis of rumen microbial communities in diets supplemented with different doses of complex algae. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0029] Example 1: Effect of red algae-brown algae composition on in vitro rumen fermentation

[0030] 1.1 Materials and Design

[0031] The composition of the basic diet in this study (by weight) was 50% corn silage, 26.5% corn kernels, 13% soybean meal, 9.5% wheat bran, 0.5% sodium bicarbonate, and 0.5% salt.

[0032] This study used red algae - Asparagopsis taxiformis collected from the South China Sea, which was freeze-dried, crushed, and sieved through a 40-mesh sieve before being added to the basic diet. This study used brown algae - Sargassum serratum collected from the Yellow Sea, dried at 60°C, ultra-finely crushed, and sieved through a 40-mesh sieve before being added to the basic diet.

[0033] Experimental animals: Three healthy Holstein cows with similar body weight and lactation age were selected from China Agricultural University as rumen fluid donors. The donor cows were fed twice a day (7:00 and 19:00) and had free access to water. The donor cow diet met the nutritional requirements of the National Research Council (NRC) for 30 kg lactating cows. All animal experimental procedures were approved by the Animal Welfare and Ethics Committee of the Institute of Oceanology, Chinese Academy of Sciences (No: IOAS2022-93).

[0034] 1.2 In vitro fermentation test

[0035] In this study, rumen fluid was collected strictly according to the collection of about 1L of rumen fluid from the rumen fistula of 3 healthy Holstein cows with an average weight of 500 kg 1 hour after feeding. The rumen fluid was filtered through 4 layers of gauze and evenly mixed for use. A total of 4 dietary grouping schemes were designed in this study, and the specific schemes are as follows: the control group was a blank control group fed only with a basic diet; the low-dose combination supplement group (L group) was a combination of a basic diet + 3% of the basic diet (red algae: brown algae = 1:1); the high-dose combination supplement group (H group) was a combination of a basic diet + 5% of the basic diet (red algae: brown algae = 3:2).

[0036] After rumen fluid collection and processing, this study strictly followed the Menke standard process, accurately weighed approximately 200 mg of each diet regimen, and placed it in a 100 mL glass syringe equipped with a plunger. The syringe contained a mixed solution containing 10 mL of rumen fluid and 20 mL of buffer solution. There were 6 replicates for each treatment, and the other 3 replicates served as blanks without diet. All glass syringes were incubated at 39°C in a biochemical incubator (SHP 205; Peiyin, Shanghai, China). The gas production (GP) was monitored and recorded at 2, 4, 6, 8, 12, 24, and 48 h of incubation. After 48 h of incubation, the culture fluid samples were collected for volatile fatty acid (VFA) analysis, and gas samples were collected to evaluate the concentration of CH4.

[0037] Analysis of Volatile Fatty Acids and Gas Components Gas chromatography-assisted analysis was used in this study. After 48 h of incubation, the glass syringes were placed on ice to terminate the microbial fermentation. About 5 mL of total gas in each glass syringe was collected, and the concentration of CH4 was determined using gas chromatography (GC-8600; FTIR, Beijing, China).

[0038] In this study, 10 mL of culture fluid was collected and stored at -20 °C as a preparatory sample for DNA extraction and VFA analysis. The concentration of VFA was determined using gas chromatography (GC-8600; FTIR, Beijing, China), including major components such as acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate. To prepare the sample for VFA analysis, 1 mL of culture fluid was mixed with 250 μL of 25% HPO3, and then the mixture was centrifuged at 15,000 × g and 4 °C for 15 min. Subsequently, 5 μL of the supernatant was injected into the gas chromatograph to evaluate the VFA profile. The specific analysis conditions of the gas chromatography were as follows: the injector temperature was set at 200 °C, the column temperature was increased from 45 °C to 130 °C at a rate of 10 °C / min, and then kept at 130 °C for 20 min. The sample VFA analysis was carried out immediately after the column state was stable.

[0039] In addition, this study investigated the effect of combined diets on improving dry matter degradability. 500 mg of dietary sample was added to a 50 mL centrifuge tube, and 10 mL of rumen fluid and 20 mL of buffer solution were added for incubation. The residuals after 48 h of fermentation were collected to measure dry matter degradability (DMD), and each treatment was repeated 5 times.

[0040] The data in this study were analyzed using SAS 9.4 for ANOVA analysis, and when the overall treatment effect was found to be significant, multiple pairwise comparisons were performed using Tukey's test.

[0041] 1.3 Experimental Results

[0042] As shown in Appendix 1, the composite seaweed additives composed of the above-mentioned red algae and brown algae were supplemented in the basal diet. The low-dose combination supplement group (L group) was a combination of basal diet + 3% basal diet (red algae: brown algae = 1:1); the high-dose combination supplement group (H group) was a combination of basal diet + 5% basal diet (red algae: brown algae = 3:2). Compared with the control group, the total gas production after 24 hours of culture was reduced, and the high-dose combination supplement group was better than the low-dose combination supplement group. Compared with the control group, the addition of high and low doses of composite seaweed can reduce the gas production rate of methane by about 65% and 55%, respectively. Through the analysis of the total volatile fatty acid concentration, the supplementation of composite seaweed additives can significantly reduce the volatile fatty acids produced by experimental dairy cows. Compared with the control group (110.26mmol / L), the high-dose composite seaweed additive can reduce the concentration of volatile fatty acids by 23% (85.08mmol / L). After 48 hours of incubation, there was no significant difference in dry matter degradation rate between the basal diet supplemented with high and low doses of composite seaweed additives and the control group.

[0043] Example 2: Effect of the composition on rumen microbiota

[0044] 2.1 Target amplification fragment sequencing and bioinformatics analysis

[0045] In this study, the experimental culture medium based on the basic diet and supplemented with a composite seaweed additive composed of red algae and brown algae was subjected to target amplification fragment sequencing and bioinformatics analysis. After the culture medium was incubated for 48 hours, based on the CH4 emission reduction effect of each experimental group, this study selected the culture medium of each experimental group (n=6) to extract DNA and further perform directional amplification fragment sequencing analysis. DNA was extracted using a magnetic soil and feces DNA kit. In this study, 357F (5'-ACTCCTACGGRAGGCAGCAG-3') and 806R (5'-GGACTACHVGGTWTCTAAT-3') were used to amplify the V3-V4 region of 16S rRNA. All PCR reactions used 15 μL High-Fidelity in PCR Master Mix was used. PCR products were mixed at equal density and purified using a DNA purification kit (Tianjian Biotechnology Co., Ltd., China). Ultra TM II FS DNA PCR-free library preparation kit (New England Biolabs, USA) was used to generate sequencing libraries according to the manufacturer's instructions and add indexes. The library was quantified using Qubit and real-time PCR, and the size distribution of the library was detected using a bioanalyzer. The quantitative libraries were pooled and sequenced on the Illumina Miseq sequencing platform using 250 bp paired-end reads.

[0046] In this study, paired-end reads after sequencing were assigned to samples according to their unique barcodes and truncated by removing their barcodes and primer sequences using Python. At the same time, Cutadapt (V3.3) was used to remove adapters, and FLASH was used to merge paired-end reads. In this study, FASTP was used to perform quality filtering on the original tags to obtain high-quality clean reads. The clean reads were then aligned with the Silva database using the UCHIME algorithm to detect chimeric sequences. Subsequently, VSEARCH software was used to remove chimeric sequences and obtain valid reads. Uparse software was used for sequence analysis, and sequences with a similarity of ≥97% were classified into the same operational taxonomic unit (OTU). The BLAST online software in the Silva 128 database was used to classify and annotate the OTU representative sequences into phylum, class, order, family, genus, and species.

[0047] 2.2 Experimental Results

[0048] As attached Figure 1As shown in the figure, a total of 239 genera of bacteria were identified from all samples. The rumen fluid was dominated by Bacteroidetes, Firmicutes and Proteobacteria, accounting for 50.24%, 30.90% and 9.19%, respectively. At the genus level, the most dominant was Rikenellaceae_RC9_gut_group, followed by Prevotella, ruminobacter, Succiniclasticum, Methanobrevibacter, Succinvibrio and UCG-005. Compared with the control group, the supplementation of high and low doses of composite seaweed based on the basic daily feed had a significant effect on the bacterial genus level. Compared with the control group, the high-dose supplementation of composite algae significantly inhibited the level of methanogenic bacteria, and the abundance of Methanobrevibacter decreased by 62%. At the same time, it significantly promoted the proliferation of functional bacteria, with the propionic acid-producing bacteria Succinivibrio increasing by 1.5 times and the hydrogen-consuming bacteria Anaerovibrio increasing by 47%.

[0049] As attached Figure 2 As shown, CoA analysis showed that the group supplemented with high-dose compound seaweed in the basal diet was significantly separated from the control group at the phylum level (Bray-Curtis distance = 0.32, P = 0.002) and genus level (distance = 0.28, P = 0.005). The addition of compound seaweed can significantly improve the rumen community structure.

[0050] Example 3: Comparative test with existing patents

[0051] Compared with the existing single addition of red algae additives (patent WO2015109362A2), this study compared the CH4 gas production index and dry matter degradability after 48 hours of incubation in the culture medium by adding the same basic diet weight addition amount (5% dry weight). As shown in Appendix 2, by adding a high dose of composite algae additives [basic diet + 5% composition (red algae: brown algae = 3:2)] to the basic diet, the composite algae additive showed significant advantages, which can significantly increase the CH4 emission reduction rate from 14.7% to 55%; DMD decreased significantly.

[0052] Table 1: Effect of red algae-brown algae composition on gas production dynamics

[0053]

[0054] Table 2: Comparison of gas production indexes of red algae-brown algae composite and single red algae

[0055]

[0056] Industrial application prospects

[0057] The composition of the present invention can be produced on a large scale and is suitable for the following scenarios:

[0058] 1. Add directly to the pasture: Mix into the total mixed ration (TMR) at a ratio of 1%-5% for feeding ruminants such as dairy cows, beef cattle, and sheep;

[0059] 2. Premixes for feed enterprises: made into granules or powders and combined with concentrate supplements;

[0060] 3. Emission reduction certified products: Combined with the carbon trading market, enhance the green added value of livestock products.

[0061] Economic benefits: Taking a dairy farm with 10,000 cows as an example, annual methane emissions are reduced by about 480 tons (CO2 equivalent), saving more than one million yuan in carbon tax expenditure.

[0062] in conclusion

[0063] The present invention breaks through the technical bottleneck of single seaweed through the synergistic effect of red algae and brown algae, and achieves efficient methane emission reduction at low cost without affecting feed utilization. Combined with clear experimental data and industrialization path, it has significant environmental and economic benefits and provides core technical support for the low-carbon transformation of animal husbandry.

Claims

1. Use of a composition of red algae and brown algae in inhibiting rumen methane production in ruminants, characterized in that: In the composition, red algae account for 30%-60%, brown algae account for 20%-50%, and the remainder is auxiliary seaweed (accounting for ≤20%).

2. The use according to claim 1, characterized in that: The red algae are mainly Asparagopsis taxiformis or Rhodomela confervoides, etc.; the brown algae are mainly Sargassum serratum, Laminaria japonica, Undaria pinnatifida Suringar, etc.

3. The use according to claim 1, characterized in that: The auxiliary seaweed is one or more of Enteromorpha prolifera, Gracilaria or Ulva lactuca, and the total proportion does not exceed 20%.

4. The use according to claim 1, characterized in that: The composition is added to the basic daily diet feed of ruminants as a feed additive in the form of dry powder, freeze-dried particles or extracts, and the added amount is 0.5%-10% of the dry weight of the daily diet.

5. A use according to claims 1 to 4, characterized in that: The composition preparation method comprises the following steps: (1) Rinse the red algae, brown algae and auxiliary seaweed with fresh water, dry at 60°C and then crush to 40 mesh; (2) The components are mixed in proportion and pulverized by ultrafine pulverization technology to obtain a composition, wherein the particle size of the composition is ≤100 μm.

6. A feed for reducing emissions, characterized in that: The invention comprises a basic diet and the composition according to any one of claims 1 to 3, wherein the addition amount of the composition is 0.5% to 10% of the dry weight of the feed.

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