Reduction of greenhouse gas emissions of ruminants by means of feed additives

Abstract

Feed composition for ruminants suitable for reducing methane gas and carbon dioxide emission in the rumen, and uses thereof as food additive for ruminants to reduce their emission of greenhouse gases.

Description

[0001] REDUCTION OF GREENHOUSE GAS EMISSIONS OF RUMINANTS BY MEANS OF

[0002] FEED ADDITIVES

[0003] Field of the Invention

[0004] The present invention relates to a feed composition for ruminants suitable for reducing methane gas and carbon dioxide emission in the rumen of ruminants, said composition comprising heat- treated lysozyme or a combination of heat-treated lysozyme and lysozyme as active ingredients. Background of the invention

[0005] Emissions of greenhouse gases, such as carbon dioxide and methane, into the atmosphere cause global warming and severe damage to the planet's ecosystem. Due to methane's relevance as a greenhouse gas (particularly considering its global warming potential that is 27.9 times greater that CO2 over a 100-year time horizon and its relatively short lifetime (~1 decade)) there is considerable interest in targeting methane emissions to mitigate climate change and improve air quality (https: / / doi.org / 10.1038 / s41561-023-01144-z). The ruminal livestock sector is estimated to be responsible for 14.5% of anthropogenic greenhouse gas emissions and methane emission from ruminant livestock is about the 30% of global anthropogenic methane emissions (Tackling Climate Change Through Livestock; FAO 2013). Beef and cattle milk production account for most of the emissions, respectively contributing 41 and 20% of the sector’s emissions. Supportive policies and more proactive governance fulfilled the mitigation potential and particularly the EU has recently agreed to reduce methane emissions by 36% by 2030 compared to 2005 levels (European Community Commission, 2020). New feeding strategies to mitigate enteric methane emissions in livestock systems are therefore a growing research topic. Recently new synthetic feed additives for reducing methane emission in ruminants were discovered and developed. These synthetic substances include nitro organics compounds, like 3-nitrooxypropanol (WO2018153702, DSM) and halogenated organic compounds, like bromoform (W02024250074, Number 8 Bio Holding PTY LTD).

[0006] 3-Nitrooxypropanol (3-NOP, Bovaer®) was approved by EFSA as feed additive for reducing methane emission and to increase the productive performance of mid-lactating dairy cows fed. This substance, useful to mitigate enteric methane production, generated an open discussion on the safety of the milk derived by these treated dairy cows. Even if the safety of this additive was certified by EFSA and FDA, which consider the use of this additive, at the employed dosage of 60-80 mg per kg of dry matter of the ration for lactating cows, a very low risk for the human health, some dairy companies confirmed that they do not use this additive, stating that the main components that make up Bovaer® are not included in the list of approved products / compounds for use in organic farming (https: / / www.yeovalley.co.uk / blog / does-yeo-valley-use-feed- additives). Also, the use of bromoform as antimethanogenic feed ingredient for ruminant livestock is studied for possible toxicological risks associated with its use and the possible impact on atmospheric chemistry (https: / / doi.Org / 10.1016 / j.algal.2022.102673).

[0007] Lysozyme (or muramidase or N-acetylmuramic acid hydrolase E.C. 3.2.1.17) is a natural protein that exerts its enzymatic activity through the hydrolysis of the P 1,4-glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamide (NAG) in the polysaccharide backbone of the peptidoglycans of the Gram-positive bacterial cell wall.

[0008] Preliminary evaluations of feed additives suitable to reduce tests methane emission in ruminants using lysozyme were recently published by Ashraf et al. (http: / / dx.doi.org / 10.5713 / ajas.16.0575 and KR2018044051) and Rodriguez et al. (https: / / doi.org / 10.18779 / cyt.vl5il.543) with several limitations, given the fact that they have been carried out using in vitro batch systems. These systems are carried out using rumen fluids used as an inoculum, collected at the slaughterhouse, while the fermentation processes are carried out in closed vessels in which the gas accumulation in the fermenters generate a pressure increase that causes changes in gas (CO2) dissolution. This kind of model is not suitable for testing gas emission of the microorganisms present in the rumen since the increase of the partial pressure of CO2 directly affects the equilibrium of methane emission (DOI: 10.1111 / jpn.13780). Ruminal methane production (PCH4) can be calculated using the equation:

[0009] PCH4 (mol / d) = Hy / 4.0 where 4.0 is the moles of H2 required to produce 1 mol of methane resulting from the reduction of CO2 and Hy is the excess hydrogen available for methanogenesis (Mills et al., 2001 Anim. Sci. 79, 1584-1597). These experimental conditions may interfere with the fermentation process with a consequent complication in the gas production assessment (Alvarez H. et al. Journal of the Science of Food and Agriculture, 99(1), 109-116, 2018; Cattani M. et al. Journal of Dairy Science, 97(3), 1736-1741, 2014). Moreover, these papers do not demonstrate their initial assumptions that the observed decrease in methane emission was due to the “antibiotic activity” of lysozyme against the methanogenic microorganisms (mainly domain archaea) present in the rumen. In fact, Ashraf et al. themselves did not find any sensible change in the methanogenic micro-organisms concentration in the tests carried out with and without lysozyme addition up to the dosage of 8.000 Unit of lysozyme / g dry matter feed substrate. Rodriguez et al. did not evaluate this aspect. Therefore, said papers neither teach that the observed methane reduction could be translated to in vivo trials on ruminants nor that a heat-treated lysozyme HC1 (LYSOHT®) could be used for carbon dioxide and methane reduction in ruminants, increasing also the feed conversion rate (FCR) (a conventional measure of livestock production efficiency expressed as weight of feed intake divided by weight gained by the animal) of the animals fed with said feed additives.

[0010] For these reasons, there is a need for a more effective, natural derived and safer feed additive useful to reduce methane emission in ruminants.

[0011] Description of the invention

[0012] The inventors of the present application have surprisingly found that heat-treated lysozyme (LYSOHT®), obtained through the production process disclosed in WO2021229430, optionally in combination with lysozyme (CAS registry number 9001-63-2), can be used as food additives in ruminants in order to reduce greenhouse gases, such as carbon dioxide and methane. These findings were confirmed by in in vivo and in vitro tests.

[0013] Description of the figures

[0014] Figure 1 shows the average live body weight (LBS) of each group over time (weeks).

[0015] Figure 2 shows the body condition score (BCS) of each group over time (weeks).

[0016] Figure 3 shows the methane emissions in ppm-methane for each group at day 0 and at day 42 of the trial.

[0017] Detailed description of the invention

[0018] Heat-treated lysozyme (LYSOHT®), obtained through the production process disclosed in WO2021229430, alone or in combination with lysozyme, were tested with success in in vitro and in in vivo tests in Italian dairy Holstein heifers as food additives in order to reduce greenhouse gases, such as carbon dioxide and methane. The use of these ovoproteins (i.e. LYSOHT® and lysozyme) as food additives in ruminants reduce sensibly the emission of these greenhouse gases.

[0019] A first object of the present invention is a feed composition for ruminants, suitable for reducing methane gas and carbon dioxide emission in the rumen, said composition comprising heat- treated lysozyme (LYSOHT®) alone or in combination with lysozyme as active ingredient.

[0020] In one embodiment, the composition contains a combination of heat-treated lysozyme to lysozyme in a weight ratio ranging from 1 :2 to 2: 1, preferably 1 : 1.

[0021] In one embodiment, the daily dosage of lysozyme (LYSOHT®) or a combination thereof with lysozyme is comprised between 2,000 and 10,000 FIP Unit / g of dry matter, preferably 10,000 FIP Unit / g of dry matter.

[0022] In a preferred embodiment, the feed composition comprises heat treated lysozyme (LYSOHT®) as the sole lysozyme. The heat-treated lysozyme (LYSOHT®) and lysozyme utilized for the preparation of the feed additives can be used as bases or as addition salts with hydrochloric acid or citric acid, preferably as hydrochloride salts.

[0023] In a preferred embodiment, the substrates for the preparation of the feed compositions are selected from barley meal, alfalfa hay, corn silage and soya bean hulls.

[0024] In another embodiment, heat-treated lysozyme or its combination with lysozyme are used as the sole feed additives.

[0025] The obtained in vivo and in vitro data confirmed the decrease of methane emission with respect to the controls for both heat-treated lysozyme (LYSOHT®) and combinations thereof with lysozyme used as single feed additives, with best performances for LYSOHT® which reached a decrease in methane emission of about of 18-21%.

[0026] In a further aspect, the invention provides a method of reducing the production of methane gas and carbon dioxide in ruminants, which comprises feeding ruminants with a composition comprising heat-treated lysozyme or a combination of heat-treated lysozyme and lysozyme, preferably in a 1 : 1 weight ratio, as active ingredients.

[0027] The invention embodiments disclosed above in connection with the feed composition apply to the method of invention as well.

[0028] Experimental section

[0029] Preparation of LYSOHT®

[0030] LYSOHT® utilized to perform the in vivo and in vitro trials for reducing greenhouse emission is produced according to the patent WO2021229430. According to this procedure LYSOHT® is isolated from HEW (hen egg white) by a purification process involving the following sequence of purification procedures: isolation of crude lysozyme base from other ovoproteins present in HEW, preparation of crude lysozyme HC1 solution, removal of inorganic salts, viral inactivation and isolation of solid lysozyme HC1 by spray-drying technique, followed by heat treatment. The crude HEW lysozyme was purified by the following purification process: undiluted HEW was loaded onto a polyacrylic cationic resin with a particle size ranging between 300 and 1600 pm and a capacity of > 2.7 eq / 1, preconditioned in a pH interval of 9.0-7.0. The relative ratio between HEW and resin ranges between 8 and 12 1 / 1, and the resin is washed twice with a 1.0- 1.5 bed volume of distilled water. The resin was then washed with a 1.5-2.1 bed volume of an aqueous solution of NaCl at a concentration ranging between 2 and 7% w / v nd a temperature ranging between 25 and 40°C. When said steps are performed, the resin can optionally be maintained under stirring at a stirring speed of up to 60 rpm. The fractions eluted with aqueous solution of NaCl were basified at a final pH value ranging between 10 and 11 with an aqueous solution of 4-8% w / v sodium hydroxide, and the resulting mixture was maintained under stirring at 0-8°C for 4-24 hours. The resulting precipitate was recovered by suction. The wet solid was dispersed under stirring in demineralised water (relative ratio between demineralised water and initial HEW 1 / 60-1 / 100 1 / 1), and the resulting mixture was maintained under stirring at a temperature ranging between 20 and 50°C for 30 minutes to 2 hours and corrected to a final pH interval of 2.5-3.5 with a 4-8% w / v aqueous solution of hydrochloric acid. The resulting solution was then heated under stirring at a temperature ranging between 20 and 60°C for 30 minutes to 2 hours, then cooled, and the final pH value corrected to an interval ranging between 8.0 and 10 by adding a 1-4% w / v aqueous solution of sodium hydroxide. Said solution was treated under stirring for 1-4 hours with activated charcoal (powder) and then filtered (the filter can optionally be washed with demineralised water). The filtrate (and optionally the washing water) was then corrected to a final pH interval of 3.0-4.0 with a 2-8% aqueous solution of hydrochloric acid, and ultrafiltered and / or diafiltered to remove the inorganic salts. The resulting aqueous solution was then optionally heated to a temperature ranging between 40°C and 100°C for a period of up to 7 days, then cooled to 40°C, or heated directly to 40°C, and treated with a spray-dryer (desolvation chamber temperature 160-220°C) to provide pure amorphous lysozyme hydrochloride as an ivory powder (> 99% recovery). The above- mentioned step involving heat treatment of lysozyme hydrochloride in solution at a temperature ranging between 40°C and 100°C for a period of up to 7 days (preferably at 74°C for 1 hour) can also alternatively / simultaneously be conducted on powdered lysozyme hydrochloride obtained after spray-drying treatment. Said controlled heat treatments give to LYSOHT®, prepared by the manufacturing process described herein, increase enzymatic and virucidal properties respect to the native lysozyme. In particular, said heat treatments that do not denature the lysozyme are preferably carried out on

[0031] 1. solid lysozyme hydrochloride: a treatment at 99°C for 40 minutes or at 74°C for 1 hour

[0032] 2. lysozyme hydrochloride in aqueous solution: a heat treatment at 90°C for between 2 and 6 minutes.

[0033] In vivo trials

[0034] Thirty-six 12 months Italian Holstein Friesian heifers were involved in the study and divided in four homogeneous groups based on live body weight (LBW) and age. The experimental groups have been housed in the same stall and fed the proper dietary treatment by Insentec RIC system, using the automated identification of animal’s ID and the attribution of the bin. Each bin is used to feed animals of the same group. Before the beginning of the trial, an adaptation period of two days to RIC bins is adopted for each experimental heifer. Groups have been characterized as follows: a control group fed with a control diet without LYSOHT® a group fed with a control diet supplemented with 2000 FIP U / g of LYSOHT® (T2) a group fed with a control diet supplemented with 10000 FIP U / g of LYSOHT® (T10). The trial has been carried out in a timeframe of 6 weeks plus one week of adaptation.

[0035] All the experimental animals have been fed using the same basal diet in the form of total mixed ration (TMR) throughout the trial period. TMR has been formulated to cover or exceed the nutritional requirements for dairy Holstein Friesian heifers, as reported by National Research Council (NRC 2021).

[0036] Live body weight (LBW) and body condition score (BCS)

[0037] Live body weight and body condition score have been individually recorded at the beginning of the trial and every 7 days during the whole trial period. Live body weight has been recorded after the morning milking by electronic scale, while body condition score has been evaluated based on a 5-points scale ( hin; 5=obese), as the mean of the evaluation sites using 0.25 points evaluation units.

[0038] In-vivo methane measurements

[0039] At the beginning, after three weeks, and at the end of the trial, methane emissions have been individually measured with a Laser Methane Detector Mini (LMD; Crowcon, Abingdon, UK) following the procedure described by Bore et al. (Animals (Basel), 2022. 12(10)). For this parameter an additional group fed with control diet was supplemented with 4000 FIP U / g of LYSOHT® (T4).

[0040] Results

[0041] After seven week the average final live body weight (LBW) of the three groups were of: C=403 Kg, T2=411 Kg and T10=410Kg.

[0042] After seven week the average final body condition score (BCS) of the three groups were of: C=3,50, T2=3,58 and T10=3,56.

[0043] In-vivo methane emission measurements: LYSOHT®

[0044] After 42 days of treatment the methane emission respect to the control group decreases respectively of: T2=8,62%, T4=18,23% and T10=21,0%. Table 1

[0045] In-vitro tests

[0046] Lysozyme (10.20 mg; 40,000 FIP Unit / mg) and heat-treated lysozyme (LYSOHT®; 10.20 mg; 40,000 FIP unit / mg) were also tested individually using a new rumen batch fermentation system that allows continuous measures of total gas (GP) and methane production (MP) (ref. DOI: 10.1111 / jpn.13780).

[0047] The fermentation system is composed of glass bottles connected to gas counters (Ritter Apparatebau GmbH & Co. KG) and an infrared gas analyser that measures the methane concentration. Com silage (CS) was used as substrate in four consecutive fermentation runs. Cumulative volumes of GP and MP and the percentage of methane on total GP were recorded continuously until 48 h and average values at 1 h intervals were fitted with an exponential model with a lag phase reaching a good fit (R2 > 0.992).

[0048] The rumen fluid for all of the fermentation runs was collected in the same slaughterhouse in controlled conditions: mixed fluid was collected within 20 min of slaughter from four culled dairy cows fed with total mixed rations based on CS. The fluid was delivered, within half an hour of it being collected, to the laboratory in airtight glass bottles refluxed with CO2 and maintained at 39°C. In these experiments the bottles of the fermentation system were filled with filtered rumen fluid and mixed with the Menke and Steingass (1988) buffer (ratio 1 :2, 500 ml in total). Substrate (CS 3300 mg of dry matter [DM]) was weighed, added with each single ovoprotein dissolved in water and introduced into each bottle. The bottles were closed and immersed in a water bath at 39°C for 48 h.

[0049] Results

[0050] Data of gas produced during the fermentation process were acquired using Rigamo Software (v4.26, Ritter Apparatebau GmbH & Co. KG) while data of methane concentration (as %) registered by the infrared gas analyzer sensor were acquired using Mars tool software (v0.76, Wi.Tec company). The total methane volume was calculated through the methane concentration registered at different time intervals using the following equation as suggested by Braidot et al. (2023):

[0051] In this equation, Ci and Ci+1 are the CH4 concentrations measured at time i +1 and i, respectively, AV is the difference between the volume of gas (ml) produced at i +1 and that at i, and n is the total number of CH4 detections. In each run, gas and methane production were adjusted subtracting the blank value to obtain gas and methane adjusted, respectively. Subsequently, the methane percentage (methane percentage: MP) was calculated using total gas adjusted blank and methane adjusted blank.

[0052] The two experimental additives (Lysozyme and LYSOHT®) were examined in four fermentation runs. In each fermentation, a fermenter with the feed substrate alone (control) or without the feed substrate (blank) was used. The data of total gas and methane gas (in ml) and the percentage of methane in total gas are shown in Table 2 (average data on 4 runs).

[0053] Table 2. Averages gas, methane, and methane percentage values achieved for each additive

[0054] The findings show that for these additives, and particularly for LYSOHT® a reduction of roughly 5-6% in methane percentage compared to control (22.36 vs 23.71%). Moreover, these data confirm a higher reduction of gas emission for LYSOHT® and a combination thereof with lysozyme compared to lysozyme alone.

[0055] Conclusions

[0056] The obtained in vivo and in vitro data confirmed the decrease of methane emission with respect to the controls in the set experimental conditions. Surprisingly, it was found that said decrease on the basis of the in vivo trials was about of 18-21% for LYSOHT® used as single feed additive. This trend was confirmed by the in vitro trials carried out on LYSOHT® used as single feed additive or in 1 : 1 w / w combination with lysozyme respect to the use of lysozyme alone.

Claims

CLAIMS1 . A feed composition for ruminants suitable for reducing methane gas and carbon dioxide emission in the rumen, said composition comprising heat-treated lysozyme or a combination of heat-treated lysozyme and lysozyme as active ingredients.

2. The feed composition according to claim 1 , wherein in said combination the heat-treated lysozyme to lysozyme weight ratio ranges from 1 :2 to 2: 1, preferably it is 1 : 1.

3. The feed composition according to claim 1 wherein heat-treated lysozyme or a combination of heat-treated lysozyme with lysozyme are administered at a dosage comprised between 2,000 and 10,000 FIP Unit / g of dry matter per day.

4. The feed composition according to claim 1 wherein the heat-treated lysozyme and lysozyme are addition salts with hydrochloric acid or citric acid.

5. The feed composition according to claim 1 further comprising, as the food substrates, barley meal, alfalfa hay, corn silage and / or soya bean hulls.

6. A method of reducing the production of methane gas and carbon dioxide in ruminants, which comprises feeding ruminants with a composition comprising heat-treated lysozyme or a combination of heat-treated lysozyme and lysozyme as active ingredients.

7. The method of claim 6, wherein said composition is as defined in claims 1-5.

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