Application of black ink fruit extract in preparation of product for reducing methane emission and / or nitrogen emission of animals

Abstract

The invention belongs to the technical field of agricultural waste resource utilization, and particularly relates to an application of a black ink fruit extract in preparation of a product for reducing methane emission and / or nitrogen excretion of animals, a preparation method of the black ink fruit extract comprises the following steps: drying black ink fruits, and crushing the dried black ink fruits to obtain black ink fruit powder; taking ethanol with a volume fraction of 60% as an extraction solvent, adding the black ink fruit powder for extraction, and collecting an extract liquid; and centrifuging, decolorizing, filtering, concentrating and freeze-drying the leaching solution to obtain the black ink fruit extract. According to the invention, it is proved for the first time that the black ink fruits can inhibit a methane generation key approach and reduce ammonia nitrogen concentration by regulating rumen fermentation while not damaging feed basic digestibility and animal health. The method not only directly brings emission reduction and environmental protection benefits, but also can improve the utilization efficiency of feed energy and protein, improve the milk protein rate and the feed conversion rate, and realize double improvement of environment and production benefits.

Description

Technical Field

[0001] This invention belongs to the field of agricultural waste resource utilization technology, specifically relating to the application of black moss fruit extract in the preparation of products that reduce animal methane emissions and / or nitrogen excretion. Background Technology

[0002] During digestion, rumen microorganisms in rumen produce large amounts of methane through fermentation. Methane is a potent greenhouse gas, and its emissions exert significant pressure on climate change. Simultaneously, rumen microbial fermentation results in the loss of approximately 6%–12% of the energy in feed as methane, leading to significant energy waste. Furthermore, ruminants generally have low nitrogen utilization efficiency in their feed, averaging only about 25%. Large amounts of unabsorbed nitrogen are excreted in feces and urine as urea and ammonia, not only wasting valuable protein resources but also contributing to environmental problems such as soil eutrophication, water pollution, and ammonia emissions. Therefore, developing technologies that can simultaneously reduce methane and nitrogen excretion in ruminants is of urgent importance for addressing climate change, improving livestock efficiency, and reducing environmental pollution.

[0003] Currently, the industry has explored various strategies to address these challenges, mainly including chemical inhibitors such as 3-nitrooxypropanol, plant-derived natural additives such as saponins, tannins, and essential oils, and dietary nutrient regulation. However, existing technologies often face many limitations in practical applications: while chemical inhibitors are highly effective, they have potential residue issues, animal tolerance problems, and consumer acceptance issues; many plant extracts have unstable effects, and their high cost limits their large-scale application; some additives, while reducing methane emissions, may inhibit overall rumen fermentation, leading to decreased feed digestibility and reduced volatile fatty acid production, thus affecting animal production performance. More importantly, few products can synergistically and stably achieve the dual goals of reducing methane emissions and nitrogen excretion without compromising rumen health and feed utilization efficiency.

[0004] Therefore, there is an urgent need to find a new solution that is safe in origin, cost-controllable, has stable effects, and can balance environmental benefits and production performance. Summary of the Invention

[0005] This invention provides an application of black moss fruit extract in the preparation of products that reduce methane emissions and / or nitrogen excretion in animals, in order to solve the problem of the lack of safe and economical additives in the prior art that can synergistically and stably achieve dual emission reduction without affecting animal production performance.

[0006] It solves the problems existing in the current technology.

[0007] The technical solution adopted in this invention is: This invention provides an application of *Curcuma longa* fruit extract in the preparation of products that reduce methane emissions and / or nitrogen excretion in animals. The preparation method of the *Curcuma longa* fruit extract is as follows: The black ink fruit is dried and then pulverized to obtain black ink fruit powder; Using 60% ethanol as the extraction solvent, black ink fruit powder was added for extraction, and the extract was collected. The extract was centrifuged, decolorized, filtered, concentrated, and freeze-dried to obtain the extract of black ginseng fruit.

[0008] Preferably, the product also includes a total mixed ration; the total mixed ration is composed of the following ingredients by dry matter weight percentage: The composition of the meal is as follows: corn silage 34.59%, alfalfa hay 11.85%, oat hay 4.33%, corn kernels 17.73%, flaked corn 3.98%, soybean meal 10.46%, rapeseed meal 2.90%, beet kernel meal 0.56%, whole cottonseed 7.12%, fat meal 1.33%, vitamin and mineral premix 1.28%, and molasses 1.28%.

[0009] Preferably, the amount of black moss fruit extract added is 0.5% to 2% of the total mixed diet weight.

[0010] Preferably, the amount of black berry extract added is 1% of the total mixed ration by weight.

[0011] Preferably, the ratio of black ink fruit powder to extraction solvent is 1g: 20mL~30mL.

[0012] Preferably, the conditions for extracting the blackcurrant powder are as follows: Extract at 70℃ and 150r / min for 60-90 minutes.

[0013] Preferably, the conditions for centrifuging the extract are as follows: Centrifuge at 4℃, 8000g for 15 minutes.

[0014] Preferably, the animal is a ruminant.

[0015] Preferably, the ruminant includes any one of cattle, sheep, and camels.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an application of *Curcuma longa* fruit extract in the preparation of products that reduce methane and / or nitrogen excretion in animals. The preparation method of the *Curcuma longa* fruit extract is as follows: *Curcuma longa* fruit is dried and pulverized to obtain *Curcuma longa* fruit powder; 60% ethanol (volume fraction) is used as the extraction solvent, and the *Curcuma longa* fruit powder is added for extraction, and the extract is collected; the extract is centrifuged, decolorized, filtered, concentrated, and freeze-dried to obtain the *Curcuma longa* fruit extract. *Curcuma longa* fruit, with its natural plant-derived characteristics, overcomes the safety and residue concerns of chemical inhibitors; through standardized extraction processes, the stability of the active ingredients and emission reduction and nitrogen-lowering effects is ensured, achieving synergistic and efficient inhibition of methane and ammonia nitrogen, overcoming the bottleneck of difficult dual regulation in existing technologies. Most importantly, while providing environmental benefits, this extract does not inhibit normal rumen fermentation and feed digestibility; on the contrary, it can improve milk protein synthesis and feed conversion efficiency, ensuring production performance. Furthermore, this invention uses *Curcuma longa* fruit, a major agricultural byproduct, as raw material, making the cost controllable and opening up a new avenue for the high-value utilization of agricultural waste resources, combining economic efficiency, safety, and sustainability. Meanwhile, the extract of *Curcuma longa* fruit of the present invention is safe and applicable to various ruminant animal breeding systems such as cattle, sheep, and camels, and has both resource recycling value and good application prospects. Attached Figure Description

[0017] Figure 1 The extract of *Curcuma longa* prepared for this invention. A: *Curcuma longa*; B: *Curcuma longa* extract.

[0018] Figure 2 This describes the extraction process of black ginseng extract. Detailed Implementation

[0019] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0020] The inventive concept of this invention is as follows: Black ink ( Syzygium cumini(L.) Skeels is an underutilized plant resource. This species produces a large yield of fruit with a concentrated ripening period, often growing in dense clusters when ripe. However, currently, Skeels is primarily cultivated as an ornamental plant, and its agricultural and medicinal value has not been fully developed, nor has the potential application of its fruit been widely explored. Although Skeels fruit has certain medicinal value—for example, the *Yunnan Traditional Chinese Medicine* records its use for relieving coughs and asthma, and for promoting diuresis and reducing swelling—traditional applications are limited and actual consumption is small. Every year, a large amount of ripe Skeels fruit is discarded without timely utilization, resulting in significant resource waste. Based on these issues, there is an urgent need to develop new application methods and uses for Skeels fruit to achieve the recycling of this resource and increase its added value. Expanding innovative uses of Skeels fruit in feed, additives, and other fields holds promise for transforming a large amount of idle Skeels fruit resources into useful products.

[0021] Based on this, the present invention provides an application of *Curcuma longa* fruit extract in the preparation of products that reduce methane emissions and / or nitrogen excretion in animals. *Curcuma longa* fruit contains active substances such as polyphenols, which, upon entering the rumen of ruminants, may inhibit the activity of methanogenic bacteria, thus helping to reduce methane production. Furthermore, polyphenols may bind to feed proteins in the rumen, thereby reducing the degradation rate of proteins in the rumen, decreasing ammonia production, and reducing nitrogen excretion.

[0022] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0023] The list of abbreviations for this invention is shown in Table 1.

[0024] Table 1. List of abbreviations for this invention Example 1 The application of *Curcuma longa* fruit extract in the preparation of products that reduce methane and / or nitrogen excretion in animals is as follows: 1. Preparation of extract from black ink fruit.

[0025] Select mature black ink fruits, remove stems and impurities, rinse clean, and drain at room temperature for 15 minutes. Spread the raw material evenly on a stainless steel tray and dry in a 65℃ forced-air drying oven until constant weight. After drying, pulverize using a high-speed pulverizer, pass through a 60-mesh sieve, and store the resulting black ink fruit powder in a brown polyester resealable bag, sealed and protected from light for later use.

[0026] Prepare 60% ethanol by volume as the extraction solvent: Measure 600 mL of anhydrous ethanol in a clean graduated cylinder and mix it with 400 mL of purified water to obtain 60% ethanol by volume. Prepare and use it on the same day.

[0027] Preparation of *Curcuma longa* fruit extract: 1) Weigh 5g of black moss fruit powder into a 250mL stoppered conical flask and add 100mL of 60% ethanol solvent. Place it in a 40kHz ultrasonic cleaner and sonicate at 45℃ for 30min to break the plant cell walls; then transfer it to a 70℃ constant temperature water bath shaker at 150r / min for 90min; after cooling to room temperature, pour off the extract for later use.

[0028] 2) Add 100 mL of 60% ethanol solvent to the residue and repeat the process of sonication at 45°C for 30 min and immersion at 70°C for 60 min; after cooling to room temperature, pour out the extract for later use.

[0029] 3) Combine the two extracts. Centrifuge the combined extracts at 8000g, 15min, and 4℃, and collect the supernatant. Add 0.5g of activated carbon to every 100mL of supernatant and stir magnetically for 10min for decolorization. Then, vacuum filter through a 0.45μm polyethersulfone membrane to obtain a clear filtrate. Concentrate the filtrate under reduced pressure in a rotary evaporator at -0.08MPa (water bath 45℃) to one-tenth of its original volume, approximately 20mL. Then, slowly dry it with nitrogen at 40℃ until it becomes a viscous paste. Spread the concentrated paste evenly on a freeze-drying tray, first quickly freeze at -80℃ for 4h, then freeze-dry under vacuum in a freeze dryer at -55℃ for 36h to obtain a light brown, loose, porous dry powder. Quickly transfer the dry powder to a pre-drying mortar, lightly crush it, and pass it through a 60-mesh sieve to obtain a homogeneous freeze-dried powder, which is the *Curcuma longa* extract. Figure 1 As shown. The preparation method flow provided by this invention is shown in the figure. Figure 2 .

[0030] After sampling and testing, the samples were repackaged into aluminum-plastic composite self-sealing bags, each with a net content of 10g and containing 2g of silica gel desiccant. The bags were then sealed and stored at 4℃ in a dark and dry environment.

[0031] Calculate the extraction rate using the following formula: .

[0032] In the formula, N For extraction rate, A The mass of the freeze-dried powder is in grams. B The quantity refers to the mass of the dry powder being fed, expressed in grams.

[0033] This invention prepared three batches of *Ipomoea aquatica* extract, with freeze-dried powder masses of 0.94g, 1.02g, and 0.97g, respectively, corresponding to extraction rates of 18.8%, 20.4%, and 19.4%, with an average of 19.5% ± 0.8%, and n=3.

[0034] 2. In vitro test of the inhibition of methane and ammonia formation by extract of *Curcuma longa* fruit.

[0035] 2.1. In vitro fermentation test of rumen.

[0036] 2.1.1 Preparation of fermentation inoculum.

[0037] The rumen fluid used in the experiment was obtained from four lactating Holstein cows with permanent rumen fistulas. These cows had an average daily milk yield of approximately 32 kg. The cows were fed a total mixed ration (TMR), the composition of which is shown in Table 2. The ration was fed once daily at 09:00 AM, with free access to feed. Before the morning feed, rumen contents were collected from different parts of the rumen, with a total sample size of approximately 2 L. After thorough mixing, the rumen fluid was filtered through four layers of sterile gauze and immediately placed in a preheated 39°C thermos flask and brought back to the laboratory. The rumen fluid was mixed with artificial saliva at a 1:2 volume ratio to prepare the fermentation inoculum.

[0038] Each liter of artificial saliva contains: 0.013g CaCl2·2H2O, 0.001g CoCl2·6H2O, 0.010g MnCl2·4H2O, 0.008g FeCl3·6H2O, 0.832g NH4HCO3, 7.280g NaHCO3, 1.966g Na2HPO4·12H2O, 0.125g MgSO4·7H2O, 1.290g KH2PO4, 0.390g L-cysteine ​​hydrochloride, 0.390g Na2S·9H2O, and 0.1mg resazurin indicator. CO2 is continuously bubbled into the artificial saliva during preparation and use to maintain an anaerobic environment.

[0039] 2.1.2. Rumen in vitro fermentation test.

[0040] The test substrate was a representative sample of TMR fed to the donor dairy cows. The sample was freeze-dried, pulverized, and passed through a 1 mm sieve. 500 mg of the test substrate was weighed and placed in a 135 mL fermentation flask, and 75 mL of preheated fermentation inoculum was added. After bottling, the gas phase inside the flask was flushed with CO2, and then sealed with a rubber stopper and aluminum cap.

[0041] Different groups used different test substrates. A total of five groups were set up, including one control group and four treatment groups. The treatment groups received *Curcuma longa* extract at 0.5%, 1.0%, 1.5%, and 2.0% of the total mixed diet dry matter as the test substrate. Specifically: Control group: 500mg total mixed diet.

[0042] Treatment group 1: 500 mg total mixed diet + 2.5 mg blackberry extract; Treatment group 2: 500 mg total mixed diet + 5 mg blackberry extract; Treatment group 3: 500 mg total mixed diet + 7.5 mg blackberry extract; Treatment group 4: 500mg total mixed diet + 10mg blackberry extract.

[0043] Each treatment was replicated eight times. Fermentation was carried out at 39°C for 24 h, and the cumulative gas production was continuously recorded using an automated trace gas production recording system. The system automatically triggered a 3.0 mL standard venting when the pressure inside the bottle reached 100 Pa, with a differential pressure range of 20 Pa to 300 Pa. The experiment was conducted in three batches, with three blank bottles in each batch used to calibrate the net gas production and dry matter degradation rate.

[0044] 2.1.3. Indicator Measurement.

[0045] After fermentation, the fermentation flask was immersed in cold water to stop the reaction, and the pH value was measured immediately. Then, the fermentation broth in the flask was transferred to a centrifuge tube and centrifuged at 2000g for 20 min. The supernatant was collected and stored at -20℃ for the determination of volatile fatty acids (VFA) and ammonia nitrogen (NH3-N). The precipitate was retained for the determination of in vitro dry matter digestibility (IVDMD), neutral detergent fiber digestibility (IVNDFD), and acid detergent fiber digestibility (IVADFD).

[0046] After fermentation, a portion of the supernatant was collected for the determination of volatile fatty acids (VFAs). VFA analysis was performed using a gas chromatograph equipped with a flame ionization detector (FID) and a DB-FFAP capillary column. The analytical procedure was as follows: The frozen sample was thawed on ice, centrifuged at 10000g for 10 min, and the supernatant was collected. 100 μL of formic acid was added as an internal standard, mixed well, and centrifuged again to remove the precipitate. The clear supernatant was injected to determine the content of major VFAs such as acetic acid, propionic acid, butyric acid, and valeric acid. Another portion of the supernatant was used to determine the ammonia nitrogen content.

[0047] Gas composition analysis of fermentation gas was performed using gas chromatography, equipped with an FID detector and a GS-CarbonPLOT capillary column. At the end of fermentation, gas samples were collected from the flask head for analysis to determine the methane volume fraction.

[0048] The above measurements were used to obtain indicators such as VFA composition, NH3-N concentration, CH4 emission and nutrient degradation rate for each group, which were used to evaluate the effect of black ginseng extract on rumen fermentation characteristics.

[0049] Table 2. Ingredient composition and nutrient levels of total mixed rations for dairy cows Note: In Table 2, " / " indicates that this item is not present.

[0050] Table 3. Effects of *Curcuma longa* extract on in vitro rumen fermentation parameters. Note: In Table 3, TVFA: total volatile fatty acids; VFA: volatile fatty acids; NH3-N: ammonia nitrogen. ab Different letters on the shoulder labels of numerical values ​​within the same category indicate significant differences. P <0.05.

[0051] 2.1.4 Results.

[0052] Table 3 shows the effects of different levels of *Curcuma longa* extract on the parameters of rumen in vitro fermentation. The results indicated that the pH of the fermentation broth was maintained between 6.53 and 6.59, with no significant differences among treatments. The total volatile fatty acid concentrations ranged from 88.7 mmol / L to 94.6 mmol / L, also with no significant differences among treatments.

[0053] Regarding the composition of VFAs, the proportion of acetic acid decreased with increasing levels of *Curcuma longa* extract, decreasing from 53.8% in the control group to 49.3%–50.4% in the 1.0%–2.0% addition group, showing a significant and linear effect. The proportion of propionic acid, however, increased significantly, from 29.1% to 35.1%, exhibiting a significant linear trend. The proportions of butyric acid, isobutyric acid, valeric acid, and isovaleric acid did not differ significantly among the groups. The acetic acid-propionic acid ratio decreased significantly from 1.85 to 1.43, showing a significant linear decreasing trend, indicating that *Curcuma longa* extract promoted a relative enhancement of the propionic acid production pathway.

[0054] The ammonia nitrogen concentration decreased significantly with increasing dosage of *Curcuma longa* extract, from 25.6 mg / dL in the control group to 18.5 mg / dL in the 2.0% addition group, showing a significant linear effect. These results indicate that *Curcuma longa* extract can effectively reduce nitrogen release and improve nitrogen utilization efficiency.

[0055] Table 4 shows that the extract of *Ipomoea aquatica* had no significant effect on total gas production. However, with increasing addition levels, the total gas production decreased slightly from 37.6 mL to 33.3 mL–33.9 mL, exhibiting a certain linear trend. Methane production decreased significantly, from 1.64 mg to 1.28 mg–1.37 mg, showing a significant linear relationship. Methane production calculated per gram of degraded organic matter (DOM) also decreased significantly, from 8.36 mg / g DOM to 7.02 mg / g DOM, showing a significant linear effect, although the quadratic term also had some influence.

[0056] Regarding the nutrient degradation rate, there were no significant differences in DMD, OMD, NDFD, and ADFD among the treatments, indicating that the extract of *Curcuma longa* fruit had no negative impact on feed degradation efficiency.

[0057] Comprehensive analysis showed that *Curcuma longa* extract significantly reduced rumen methane production and ammonia nitrogen concentration in vitro within the range of 0.5%–2.0% DM addition, while improving the acetic acid to propionic acid ratio, without affecting fermentation stability or substrate degradation rate, demonstrating its potential application in rumen emission reduction in ruminants. The 1.0% DM addition showed the most significant effect; therefore, this level was used for further validation in animal experiments.

[0058] Table 4. Effects of *Curcuma longa* extract on in vitro rumen gas production parameters and nutrient digestibility. Note: In Table 4, DOM: Degradable organic matter; DMD: Dry matter degradation rate; OMD: Organic matter degradation rate; NDFD: Neutral detergent fiber degradation rate; ADFD: Acid detergent fiber degradation rate. ab Different letters on the shoulder labels of the same numerical values ​​indicate significant differences. P <0.05).

[0059] 3. Animal studies on the reduction of rumen methane emissions in dairy cows by extract of *Citrus aurantium*.

[0060] Thirty-two multiparous Holstein dairy cows in mid-lactation were selected, with an average lactation duration of 162±15 days, an average body condition score of 3.0±0.5, a parity of 3.3±1.2, and a body weight of 677±27 kg. A randomized complete block design was used, with cows grouped by parity, lactation duration, and milk yield, and then randomly assigned to two treatments:

[0061] (1) Control group: basal diet; (2) Black Moss Fruit Extract Group: Black Moss Fruit Extract was added to the basal diet, 250g / head / day, accounting for about 1% of the daily dry matter intake.

[0062] The trial lasted for 9 weeks, including a 7-day covariate period, a 14-day adaptation period, and a 42-day formal sampling period. All dairy cows were fed the same total mixed ration (TMR). The ration composition is shown in Table 2. Feeding was conducted twice daily at 08:00 and 17:30, with free access to feed and water. Feed intake was maintained at 5%–10% of the cow's feed remaining. Individual feed intake was recorded daily, and dry matter intake (DMI) was calculated based on the difference between the amount fed and the amount of feed remaining.

[0063] The extract of *Curcuma longa* fruit was mixed evenly into the TMR twice daily, morning and evening, at a dose of 250g / head. Each time, it was thoroughly mixed with approximately 3kg of TMR and fed separately for 2 hours, with the feed intake recorded. If there was any leftover, it was added directly to the remaining TMR for the day. The control group was fed the same time and method, but without the extract.

[0064] Dairy cows were milked three times daily at 06:30, 13:00, and 22:00, and milk yield was recorded. Two mixed milk samples were collected at the end of the covariate period and on days 28, 35, 42, 49, 56, and 63 of the experiment. These samples were preserved with preservatives and their composition (fat percentage, protein percentage, lactose, total solids, blood urea nitrogen, and somatic cell count) were determined using a MilkoscanFT 6000 milk composition analyzer.

[0065] During the covariate period and on days 28, 35, 42, 49, 56, and 63 of the experiment, rumen fluid was collected 4 hours after feeding using a sterilized gastric tube sampler. To avoid saliva contamination, the first 200 mL of fluid was discarded, and approximately 50 mL of rumen fluid was collected. The pH value was measured immediately after sampling. Subsequently, 10 mL of the sample was transferred to a centrifuge tube and frozen at -20°C for analysis of ammonia nitrogen, volatile fatty acids, and dissolved hydrogen.

[0066] Methane emissions were measured using a handheld laser methane detector. Measurements were taken once before and once after feeding, and continuously at the end of the covariate period and from weeks 4 to 9 of the formal trial. During each measurement, the laser beam was aimed at the cow's nostril at a distance of approximately 1 meter, and methane concentration changes were recorded continuously for 8 minutes, ensuring that each cow included 3-4 belching events. Two trained personnel held the instrument in place to minimize head movement interference. Data were recorded in ppm·m.

[0067] The AMPD algorithm in R language was used to extract the belching peak signal, and a binormal distribution mixture model was used to distinguish between the respiration peak and the belching peak. The mean of the belching peak was used as the representative methane concentration. Measurements were taken four times daily, and the average value was used as the individual's daily average CH4 concentration. Finally, it was converted into daily methane emissions.

[0068] Each indicator was analyzed using a mixed linear model. Treatment (control / addition) was a fixed effect, while individual cow and time effects were random effects. The significance test threshold was set to... P <0.05.

[0069] Table 5. Effects of *Curcuma longa* extract on milk production performance in lactating cows. In Table 5, DMI stands for Dry Matter Intake; FCM stands for Fat Corrected Milk; and ECM stands for Energy Corrected Milk.

[0070] The formula for calculating ECM is: ECM = (0.327 × milk yield) + (12.95 × milk fat yield) + (7.2 × milk protein yield); 3.5% FCM is: (0.432 × milk yield) + (16.23 × milk fat yield).

[0071] Table 5 shows the effects of *Curcuma longa* extract on the milk production performance of lactating dairy cows. The addition of *Curcuma longa* extract had no significant effect on the dry matter intake and milk yield of dairy cows; the DMI was 26.6 kg / d and 26.9 kg / d, respectively, and the milk yield was 42.6 kg / d and 44.5 kg / d, respectively. Energy-corrected milk and 3.5% fat-corrected milk were slightly higher in the *Curcuma longa* extract group than in the control group, but the differences were not significant (P>0.05).

[0072] Regarding milk composition, the milk fat percentage in the *Curcuma longa* extract group was not significantly different from the control group, but the time effect was significant, indicating that milk fat content fluctuates with the lactation cycle. The milk protein percentage showed significant differences between treatments, with the *Curcuma longa* extract group being slightly higher than the control group, and the time effect was also significant. Lactose content remained stable at 5.13%–5.15%, with no significant difference. Urea nitrogen levels decreased in the added groups, with a significant time effect, suggesting that *Curcuma longa* extract may improve nitrogen metabolism efficiency.

[0073] In terms of feed utilization efficiency, the milk yield / DMI, 3.5%FCM / DMI, and ECM / DMI of the black berry extract group were all higher than those of the control group. Among them, the differences in 3.5%FCM / DMI and ECM / DMI were significant, suggesting that black berry extract has a positive trend in improving energy utilization efficiency.

[0074] Table 6. Effects of *Curcuma longa* extract on rumen fermentation and methane emission in lactating dairy cows. In Table 6, TVFA: Total Volatile Fatty Acids; VFA: Volatile Fatty Acids; NH3-N: Ammonia Nitrogen; DMI: Dry Matter Intake; ECM: Energy Corrected Milk.

[0075] Table 6 reflects the effects of *Curcuma longa* extract on rumen fermentation parameters and methane emissions in lactating dairy cows. The pH of the fermentation broth was not significantly affected between 6.59 and 6.65, and the total acetic acid (TVFA) concentration was between 94.8 mmol / L and 96.0 mmol / L. In the TVFA composition, compared with the control group, the proportion of acetic acid was significantly decreased, the proportion of propionic acid was significantly increased, and the acetic acid-to-propionic acid ratio was significantly increased in the *Curcuma longa* extract group. The rumen ammonia nitrogen concentration was significantly decreased in the *Curcuma longa* extract group, while the dissolved hydrogen (dH2) level was significantly increased, indicating that *Curcuma longa* extract may improve hydrogen utilization efficiency and promote the conversion of hydrogen to propionic acid.

[0076] Regarding methane emissions, the daily methane emissions of the group with black moss extract were significantly reduced, and the proportion of methane production to DMI decreased from 21.8 g / kg DMI to 19.6 g / kg DMI. The methane emissions corrected by ECM were also significantly reduced, and the time effect was also significant, indicating that the emission reduction effect remained stable during continuous feeding.

[0077] The results showed that continuous feeding of *Curcuma longa* extract had no adverse effects on feed intake and milk production performance in dairy cows, but could increase milk protein content and energy-corrected milk production efficiency, significantly reduce methane emissions and ammonia nitrogen concentration, and promote rumen hydrogen metabolism efficiency, demonstrating a good dual regulatory effect of emission reduction and nitrogen utilization.

[0078] 4. Animal studies on the effect of extract of black moss fruit on reducing nitrogen excretion in dairy cows.

[0079] Thirty-two multiparous Holstein dairy cows in mid-lactation were selected, with an average parity of 2.7±0.9, lactation days of 103±12 days, average daily milk yield of 45.2±4.8 kg, and average body weight of 693±42 kg. A randomized complete block design was used. The cows were paired according to milk yield and lactation days and then randomly assigned to two treatment groups, with 16 cows in each group.

[0080] (1) Control group: fed with basal diet; (2) Black Moss Fruit Extract Group: Black Moss Fruit Extract was added to the basal diet, 250g / head / day, accounting for about 1% of the dry matter intake.

[0081] Both groups of dairy cows were fed the same total mixed ration (TMR) with the same formulation and nutrient levels as in the previous experiment. The ration was fed twice daily at 08:00 and 17:30, with free access to feed and water. 5%–10% of the feed was leftover, and individual daily feed intake was recorded to calculate dry matter intake. *Gynura divaricata* extract was pre-fed at a dose of 250g / head / day, mixed into 3kg of TMR twice daily, ensuring it was consumed within 2 hours.

[0082] The entire experiment lasted 16 weeks, including a 3-week pretreatment period, a 3-week acclimatization period, and a 10-week formal sampling period. During the last week of the pretreatment period and the 13th week of the formal experiment, complete collection of feces and urine was conducted for 6 consecutive days. Cows were fitted with specialized feces and urine separation and collection devices. Urine was introduced into a stainless steel container via a catheter, with a sufficient volume of 0.5% sulfuric acid solution pre-added to maintain the urine pH below 2.0 to prevent ammonia volatilization. The total urine volume was recorded daily, thoroughly stirred, and then frozen at -20°C for the determination of total nitrogen.

[0083] Fecal samples were collected daily in pre-weighed wooden collection boxes lined with plastic. After weighing and mixing, a representative sample of 2% was taken and frozen at -20°C. All samples were thawed, combined daily, then freeze-dried, ground, and sieved through a 1 mm sieve for analysis.

[0084] Fecal samples were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), and total energy (GE) according to the AOAC (2005) standard method. Urine samples were analyzed for total nitrogen using the Kjeldahl method. Nitrogen intake and nitrogen excretion were calculated based on feed intake, fecal nitrogen, and urinary nitrogen data.

[0085] Data were analyzed using a mixed linear model, with treatments (control and black moss extract) as fixed effects and individual cows as random effects. The significance level was set at P ≤ 0.05.

[0086] Table 7. Effects of *Curcuma longa* extract on nutrient intake and digestibility in lactating dairy cows. As shown in Table 7, the addition of *Gynostemma pentaphyllum* extract had no significant effect on the intake and digestibility of dry matter, organic matter, crude protein, and neutral detergent fiber in dairy cows. This indicates that at an addition level of 250 g / head / day, *Gynostemma pentaphyllum* extract does not alter the feeding behavior or nutrient digestibility of dairy cows, demonstrating that the extract has good feeding safety and dietary compatibility.

[0087] The addition of *Ipomoea aquatica* extract significantly affected nitrogen excretion in dairy cows. Compared with the control group, the urinary nitrogen excretion in the *Ipomoea aquatica* extract group was significantly lower, and its proportion of nitrogen intake was also significantly reduced, indicating a significant decrease in the volatile components of nitrogen loss. Total nitrogen excretion was significantly lower than that in the control group, with the proportion of nitrogen excretion to nitrogen intake decreasing from 66.6% to 62.4%. Furthermore, milk nitrogen output was significantly increased, and its proportion of nitrogen intake was also significantly higher, indicating that more ingested nitrogen was converted into milk protein for export.

[0088] Overall, black moss fruit extract can effectively reduce urinary nitrogen and total nitrogen emissions in dairy cows, increase the proportion of nitrogen in milk, thereby improving nitrogen use efficiency and reducing the potential environmental pollution caused by nitrogen excretion.

[0089] Table 8. Effects of *Curcuma longa* extract on nitrogen excretion in lactating dairy cows The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0090] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of *Curcuma longa* fruit extract in the preparation of products that reduce methane emissions and / or nitrogen excretion in animals, characterized in that, The preparation method of the black ink fruit extract is as follows: The black ink fruit is dried and then pulverized to obtain black ink fruit powder; Using 60% ethanol as the extraction solvent, black ink fruit powder was added for extraction, and the extract was collected. The extract was centrifuged, decolorized, filtered, concentrated, and freeze-dried to obtain the extract of black ginseng fruit.

2. The application as described in claim 1, characterized in that, The product also includes a total mixed ration; the total mixed ration is composed of the following ingredients by dry matter weight percentage: The composition of the meal is as follows: corn silage 34.59%, alfalfa hay 11.85%, oat hay 4.33%, corn kernels 17.73%, flaked corn 3.98%, soybean meal 10.46%, rapeseed meal 2.90%, beet kernel meal 0.56%, whole cottonseed 7.12%, fat meal 1.33%, vitamin and mineral premix 1.28%, and molasses 1.28%.

3. The application as described in claim 2, characterized in that, The amount of black berry extract added is 0.5% to 2% of the total mixed diet weight.

4. The application as described in claim 3, characterized in that, The amount of black berry extract added was 1% of the total mixed ration by weight.

5. The application as described in claim 1, characterized in that, The ratio of black ink fruit powder to extraction solvent is 1g: 20mL~30mL.

6. The application as described in claim 1, characterized in that, The conditions for extracting black bean powder are as follows: Extract at 70℃ and 150r / min for 60-90 minutes.

7. The application as described in claim 1, characterized in that, The conditions for centrifuging the extract are as follows: Centrifuge at 4℃, 8000g for 15 minutes.

8. The application as described in claim 1, characterized in that, The animal in question is a ruminant.

9. The application as described in claim 8, characterized in that, The ruminants include any one of cattle, sheep, and camels.

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