The additional effect of cinnamon leaf powder (Cinnamomum burmanni Ness ex. BI) as a source of cinnamaldehyde on in vitro gas and methane production

Fitriana Permatasari, Chusnul Hanim and Asih Kurniawati

Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
c.hanim@ugm.ac.id

Abstract

Ruminant farming produces massive amounts of methane which contributes to global warming, while methane production also indicates inefficient use of feed nutrients. Cinnamaldehyde can reduce methane production by binding to feed protein. One example of a source of cinnamaldehyde is cinnamon leaves. This study aimed to determine the effect of a natural bioactive compound in the form of cinnamaldehyde from cinnamon leaf using in vitro method for rumen fermentation on gas production and methane mitigation. The fermentation gas production method by Menke and Steingass was used in this research with an incubation period of 48 hours. The sample used was cinnamon leaf powder. The proportion of forage and concentrate is 60:40, where the concentrate consists of 90% wheat bran pollard and 10% soybean meal. The treatments applied in this research were different levels of addition of cinnamon leaf powder, namely levels of 0%, 1%, 2%, 3%, and 4% DM of feed. The observed parameters included gas production, digestible dry matter (DDM), digestible organic matter (DOM), as well as methane (CH₄) and carbon dioxide (CO₂) production. The data collected will be examined through one-way analysis of variance (ANOVA) followed by Duncan Multiple Range Test (DMRT). The results of the research showed the production of gas when adding cinnamon leaf powder with a percentage of 2% DM of feed showed the highest results (p<0.05) compared to adding other percentages. The addition of cinnamon leaf powder with a percentage of 2% DM of feed showed the lowest CH₄ and CO₂production results compared to the addition of cinnamon leaf powder at other percentages (p<0.05). DDM and DOM values in the rumen showed the lowest results (p<0.05) due to the addition of cinnamon leaf powder at a percentage of 4% DM of feed compared to all treatments. The research brought out the conclusion that the addition of 2% cinnamon leaf powder which is equivalent to cinnamaldehyde of 32 mg/kg DM can increase gas production in the rumen and reduce CH₄ production also CO₂production in the rumen in vitro.

Keywords: cinnamon leaves, cinnamaldehyde, global warming, methane mitigation

Introduction

Livestock farming is a significant source of greenhouse gas emissions globally. According to O’Mara (2011), emissions from industries associated with livestock production account for 8% to 10.8% of total global greenhouse gas emissions. Production of methane in the rumen requires energy obtained from the feed digestion process. Alterations in the efficiency of feed energy use can impact methane emissions produced by the enteric fermentation in ruminant (Shibata and Terada, 2010). Broucek (2014) stated that volatile fatty acids (VFA) can be produced as the result of feed digestion in rumen by microorganisms such as bacteria, protozoa and fungi. The primary energy source for livestock is provided by acetic, propionic, and butyric acids which are predominantly produced in rumen. Some of the structural carbohydrates, proteins, and organic materials in the feed will be converted into H₂ and CO₂ which will then be converted into methane by methanogenic bacteria (Morgavi et al 2010). The methane gas produced will be wasted with other gases in the rumen, resulting in inefficient use of feed energy.

Manipulation of feed fermentation through protein protection is necessary to reduce methane production while increasing the efficiency of ruminant feed protein use. Feed protein protection can reduce the level of protein degradation by rumen bacteria and provide protein and amino acids for host livestock (Suhartanto et al 2014; Kumari and Kumar, 2015). This is because protein protection reduces the degradation of protein which in turn enchances rumen microbes, serving as a vital nutrient source for protozoa. Tan et al (2011) stated that the reduction in the number of methanogenic bacteria is related to the protozoa population, because some methanogens have symbiosis with protozoa. Methanogenesis serves as the principal pathway for hydrogen (H₂) consumption in the rumen, with the partial pressure of H₂ thermodynamically regulating the redox balance and this regulation is essential for the fermentation process, facilitating the production of volatile fatty acids, heat, and microbial biomass (van Lingen et al 2016).

Formalin, as a form of aldehyde, is the most effective protein protecting ingredients, but the availability of formalin is currently limited so it is difficult for the public to obtain. Apart from that, formaldehyde can cause allergies and irritation to the skin of humans and livestock (Hadianto, 2020). The use of natural protein protection ingredients is very necessary in order to increase the use of safe and environmentally friendly feed protein. Cinnamaldehyde, a secondary metabolite from cinnamon plant, can be utilized to protect proteins by forming complex compounds that are resistant to proteases, which subsequently reduces protein degradation in the rumen. The use of essential oil from cinnamon plants with the active compound in the form of cinnamaldehyde as a feed additive can manipulate nitrogen (N) metabolism in the rumen by reducing the peptidolysis process without affecting the concentration of VFA, microbial protein and the activity of enzymes in rumen (Calsamiglia et al 2007). Wendakoon and Sakaguchi (1994) reported that the carbonyl group in cinnamaldehyde will inhibit the decarboxylation process of amino acids, thereby inhibiting the deamination of amino acids. The aldehyde-protein reaction can be reversed or reversible at low pH and does not change the amino acid composition and interfere the post-rumen tract digestibility of protein (Antoniewicz et al 1992).

Material and method

Preparation and evaluation of the nutrient composition in feed ingredients

Pennisetum purpureum and fresh cinnamon leaves, cut into pieces measuring ± 5 cm and dried in a 55°C oven for three days. Pennisetum purpureum and cinnamon leaves are ground to form powder. The results of cinnamon leaf powder were then analyzed for the total bioactive compound content of phenols, saponins, total tannins, and total flavonoids. The total phenols, flavonoids, and tannins were analyzed using UV-vis spectrophotometry, while the saponin content was determined using UV-vis spectrophotometry with saponin standards derived from Quillaja bark. Dry matter (DM), organic matter (OM), crude protein (CP), crude fiber (CF) and ether extract (EE), as the nutrient content were analyzed proximately according to AOAC (2005). Calculation formula from Hartadi et al (2005) was used to calculating the nitrogen free extract (NFE) and the total digestible nutrient (TDN).

Photo 1. Cinnamon leaf (Cinnamomum burmanni Ness ex. BI) used in the research was from Kulon Progo region,Yogyakarta, Indonesia

This research used the addition of cinnamon leaf powder at 0%, 1%, 2%, 3%, and 4% DM or the equivalent of 0; 16; 32; 48; and 64 mg/kg DM feed with cinnamaldehyde content contained in cinnamon leaf powder of 64.38% (essential oil content of 0.25%). The 60:40 ratio used for the basal ration which is consists of forage and concentrate. The forage used was Pennisetum purpureum, while the concentrate used consists of 90% pollard bran and 10% soybean meal as a protein source. Table 1 shows the composition of the feed ingredients used in each treatment.

The composition of chemicals in the feed ingredients and the bioactive compounds in cinnamon leaves used in this research are presented in Table 2 and Table 3, respectively, while Table 4 provides chemical composition of feed utilized.

Adaptation of donor livestock

Two Bali cows weighing around 300 kg with fistulas were adapted by being fed twice daily, in the morning and evening, with a dry matter amount of 3% of their body weight. Feeding according to the cow's needs follows Kearl (1982) with a feed composition of 60% forage and 40% concentrate. Meanwhile, drinking water for donor livestock was provided ad libitum.

Collection of rumen fluid

A thermos filled with water at a temperature of 39°C was used to collect rumen fluid in the morning before feeding. The water was completely removed from the thermos before adding the rumen fluid, which was then the rumen fluid poured into the thermos until it is full. The rumen fluid was brought to the laboratory. Layered gauze was used to filter the rumen fluid in the laboratory, with the feed particles left on the gauze and the filtrate collected in an Erlenmeyer flask. An anaerobic environment was sustained within the Erlenmeyer flask by introducing CO₂ gas.

Preparation of in vitro fermentation medium

Medium and rumen fluid were added in a ratio of 2:1 to conduct the in vitro fermentation. The medium was made with a composition in the form of a macro mineral solution (main elements). Na₂HPO₄ (5.7 g), KH₂PO (6.2 g) and MgSO₄·7H₂O (0.6 g) were used to prepare the macro minerals which were poured to measuring flask to reach the limit with 1 l of distilled water. Micro minerals (trace elements) were prepared with CaCl₂.2H₂O (13.2 g), MnCl₂.4H₂O (10 g), CoCl₂.6H₂O (1 g) and 0.8 g of FeCl₃. 6H₂O placed at measuring flask with a capacity of 100 ml using distilled water. Resazurin was prepared as a color indicator by dissolving 100 mg of resazurin in distilled water until it reached the 100 ml mark in the measuring flask. A carbonate buffer solution was prepared as a pH balancer with 35 g of NaHCO₃ and 4 g of (NH₄)HCO₃which were adjusted to 1 l in a measuring flask using distilled water. The reducing solution was prepared with 285 mg of Na₂S.7H₂O dissolved in 2 ml of 1 N NaOH then added with 47.5 ml of distilled water. The reductant solution should be prepared immediately before adding the rumen fluid into the fermentation medium.

Once prepared, fermentation solution was mixed in a 500 ml capacity Erlenmeyer flask, then heated and homogenized using amagnetic stirrer and hot plate at a temperature of 39°C. The solution entered began with 237 ml of distilled water, 0.06 ml of micro minerals, 118.5 ml of carbonate buffer solution, 118.5 ml of macro minerals, 0.61 ml of resazurin, and 24.75 ml of reducing solution. CO₂ gas was introduced before adding reductant solution. The rumen fluid is then added to the buffer in the ratio of 1:2, with CO₂ gas continuously introduced to maintain anaerobic environment.

Gas production test using the in vitro method by Menke and Steingass (1988)

Medium consisted of rumen fluid and buffer solution in a ratio of 1:2. A syringe with the volume of 100 ml was filled with 300 mg of basal ration (Pennisetum purpureum, wheat bran pollard, soybean meal) in a 60:36:4 DM ratio and cinnamon leaf powder at varying concentrations (0%, 1%, 2%, 3%, 4% DM) or equivalent to a cinnamaldehyde content of 0; 16; 32; 48; and 64 mg/kg dry matter feed. Syringes contained no feed used for blank treatment, and standard syringes were filled with 300 mg of pangola grass.

A mixture of rumen fluid and buffer solution was added as much as 30 ml to the syringe. Incubation was carried out at 39°C for 48 hours. Gas volume was observed at time intervals of 0, 2, 4, 6, 12, 24, 36 and 48 hours and used for analysis of gas production from feed fractions and total gas production with the Fit Curve Program (Chen, 1994). Gas production, digestible organic matter or DDM, digestible organic matter or DOM, also emissions of methane and carbon dioxide were observed as parameters.

Gas production, methane and carbon dioxide measurement

Gas production from observations starting from 0, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours were used for analysis using the Fit Curve Program, so that an equation was obtained Y = ax+b (Chen, 1994). Gas volume from the easily fermentable fraction (a), gas volume from the possibly degradable fraction (b), total gas production from both the easily degradable and possibly degradable fractions (a+b), and the gas production rate of fraction b (c) were measured. For the measurement of methane and carbon dioxide, samples were taken at 12^th hour. Samples were collected using 10 ml venoject plain tubes with a total amount of 10 ml of gas. After that,venoject plain covered with parafilm. Then the measurement of methane concentration was carried out using Portable Micro Gas Chromatography (GC) CP 4900 Varian, Inc.'s. Thermal Conductivity Detector (TCD) was used as the detector and gas carrier using Helium. The production of methane gas was calculated by multiplying the CH₄ concentration with total gas production.

Digestible dry matter (DDM)

The fermentation residue was filtered using a crucible containing glass wool, then it was placed in the oven for 12 hours in the temperature of 105ºC (AOAC, 2005), after that it moved into desiccator and left to set for one hour. Then, the filtered residue was weighed in order to measure weight of bypass or not digested DM (residual DM).

Digestible organic matter (DOM)

The feed residue after being oven-baked to determine the DM content was used to determine the OM content by ashing it two hours in a kiln at 600ºC until only ash remains (AOAC, 2005), then place it for one hour in desiccator and weigh it to determine the weight of undigested organic matter (residual OM).

Ethical approval

The inclusion of living animals in this study was under check and approval by the Research Ethics Committee, Faculty of Veterinary Medicine, Gadjah Mada University, with the number 053/EC-FKH/Eks./2023.

Statistic analysis

This research obtained results analyzed by completely randomized design (CRD) with one-way ANOVA. According to Rosner (1990) Duncan’s Multiple Range Test (DMRT) assessment was needed in order to determine average differences for each treatment. These analysis has carried out using software named Statistical Product and Service Solutions (SPSS) with the version of 26.

Results

Gas production

The result proved the significant effect of addition of cinnamon leaf powder as a source of cinnamaldehyde (P<0.05) on gas production resulting from in vitrofeed fermentation (Table 5).Fermentation gas production based on feed dry matter (DM) showed a significant increase in the addition of cinnamon leaf powder as a source of cinnamaldehyde at 2% feed DM.

Based on the analysis results presented above, it can be observed that feed fermentation treated with the addition of cinnamon leaves 2% DM to feed has the highest gas production. Cinnamaldehyde has different antibacterial properties against several species of rumen amylolytic bacteria so that it indirectly affects the amylase activity of rumen fluid resulting from feed fermentation in this study. Mehta and Satyanarayana (2016) stated that even though they belong to the same species, each bacterial strain can produce amylase enzymes with different activities. Cinnamaldehyde reduces protozoa that are predators of rumen microbes. A rise in the number of amylolytic bacteria, which play a role in starch digestibility and produce propionate, can be caused by the population suppression of protozoa in the rumen (Sairullah et al 2016).

Based on the presented analysis results at Table 5, it can be concluded that there is a significant impact from the addition of cinnamon leaf powder as source of cinnamaldehyde (p<0.05) on production of fermentation gas based on digestible dry matter (DDM) with the highest value being the percentage of addition of cinnamon leaf powder at 4% DM feed. This is possible due to the low digestible dry matter (DDM) in this percentage as a comparison of the gas production value. The low DDM at the percentage of adding cinnamon leaf powder of 4% DM to feed is possible because the lignin content at this percentage begins to inhibit the digestibility of feed organic matter. Cinnamaldehyde is the compound most commonly found in the bark of cinnamon plants because it functions as a compound that supports the growth of lignin or wood substances (Budiarti et al 2018). The statistical analysis results demonstrated that there is significant effect on the addition of cinnamon leaf powder (p<0.05) the reduction of DDM, particularly at an additional percentage of 4% DM feed, thereby confirming this effect.

According to the analysis results showed at Table 5, it can be concluded that there is significant impact on the addition of cinnamon leaf powder as a source of cinnamaldehyde (p<0.05) on gas production based on DOM with the highest effect observed at 4% DM feed. This is possible due to the low digestible organic matter (DOM) in this percentage as a comparison of the gas production value. The low DOM at the percentage of adding cinnamon leaf powder of 4% DM to feed is possible because the lignin content at this percentage begins to inhibit the digestibility of feed organic matter. Cinnamaldehyde is the compound most commonly found in the bark of cinnamon plants because it functions as a compound that supports the growth of lignin or wood substances (Budiarti et al 2018). Analysis results on cinnamon leaf powder addition effect to DOM was significantly different (p<0.05) with the largest decrease in the percentage of adding 4% DM to feed also proves this. Providing rations with high levels of crude fiber can cause low nutrient utilization in the ration and a decrease in body weight (Hsu et al 2000).

Gas production from easily degradable fraction (a), possibly degradable fraction (b) and gas production rate from fraction b (c)

According to the analysis showed in Table 5, it can be can concluded that cinnamon leaf powder addition to provides cinnamaldehyde significantly affected the gas production from the easily degraded fraction (a) with lowest value observed at 3% cinnamon leaf powder addition (p<0.05). This is possible because the a value has not been detected due to the lag phase which affects the performance of rumen microbes in degrading feed nutrients. According to Agus et al (2006) negative fraction a values are possible due to the lag time in microbes so it is possible that a values have not been detected. Negative a values occur if there is no dissolved material at all or only a small amount (Orskov and Ryle, 1990). Fraction (a) in the treatment with the addition of cinnamon leaf powder as a source of cinnamaldehyde at 0%, 1%, 2% and 4% DM of feed did not have a significant difference. The activity of the amylase enzyme may be higher than that of protease, so that the easily degraded fraction in this treatment has a high value. According to Hadianto (2020) lackness of the activity of protease in rumen fluid caused by the feed fermentation is possible due to the formation of a complex bond between the substrate protein and cinnamaldehyde so that the substrate protein cannot be hydrolyzed by the protease enzyme.

The analysis in Table 5 revealed that cinnamon leaf powder addition as a source of cinnamaldehyde had no significant effect (p>0.05) on gas production resulting from the fraction (b). It is possible that cinnamaldehyde does not bind to the CMC-ase enzyme protein so it does not affect the stability and structure of the CMCase enzyme, especially the active site (Hadianto, 2020). Enzymes have an active site whose shape matches the shape of the substrate (Silverman, 2002). The active side of the CMC-ase enzyme can still bind to feed substrates, especially cellulose, so it does not interfere with CMCase activity in degrading cellulose (Hadianto, 2020). This is in line with the analysis results which show that the addition of cinnamon leaf powder as a source of cinnamaldehyde has no significant effect on the gas production rate of the possibly degraded fraction (c) (p>0.05).

Total gas production of easily degradable and potentially degradable fractions (a+b)

The analysis results in Table 5 showed cinnamon leaf powder addition to provides cinnamaldehyde did not significantly influence easily degraded fraction and the potentially degraded fraction total gas production (a+b) during 48 hours of incubation (p>0.05). Based on Riyanto et al (2017) protection of soybean meal in feed rations with formaldehyde did not significantly affect CMC-ase enzyme activity. The emergence of CMC-ase enzyme activity can occur due to the presence of fiber originating from forage substances or substances that can be fermented for the growth of CMC-ase producing bacteria which play a role in degrading cellulose as a component of feed fiber (Weimer, 1996).

Production of methane

Based on the research result showed in Table 6, cinnamon leaf addition in order to provides cinnamaldehyde significantly affect the CH₄ production with detail in CH₄ concentration and emissions (p<0.05). The lowest was the addition of cinnamon leaf powder at 2% DM feed . The decrease in protozoa may be related to the low CH₄ values in this study. Lozano et al (2017) reported that the decline in protozoa populations is related to a decrease in methane gas production because protozoa are producers of H₂ which is a precursor for the formation of methane gas. According to McGuffey et al (2001) and Ferme et al (2004) cinnamaldehyde has the same antibacterial properties as monensin and ionophore which can inhibit rumen microbes growth which affect the of lactic, butyric, acetic, formic acids and hydrogen production as the primary final product but stimulates the growth of bacteria which produce propionate, namely Succinimonas amylolitica, Fibrobacter succinogenes, Selenomonas ruminantium and Ruminobacter amylophilus. The analysis of emissions of CH₄ (methane) and CO₂ carbon dioxide showed in Table 6 below.

Production of carbon dioxide

According to the results of the research presented in Table 6, cinnamon leaves addition as a source of cinnamaldehyde significantly influence (p<0.05) the CO₂ production and concentration from fermentation. McGuffey et al (2001) and Ferme et al (2004) explained that cinnamaldehyde has the same antibacterial properties as monensin and ionophore hich plays a role in restricting the rise of microbes in rumen which affect production of lactic, acetic, butyric, formic acid and hydrogen as the main final product but stimulates the growth of bacteria which produce propionate. Based on McDonald et al (2011) CH₄in the rumen is formed due to the reduction of CO₂ by H₂. Propionate is the result of fermentation of soluble carbohydrates, especially starch or starch, which is catalyzed by amylase produced by rumen microbes (Hadianto, 2020). Holmes et al (2014) stated that symbiosis will be formed in ciliated protozoa with organelles in the form of hydrogenosomes which will ferment pyruvate to produce acetic acid, H₂ and CO₂, where acetic acid is used by protozoa as an energy source and carbon source, while H₂ and CO₂ re utilized by symbionts, namely methanogens, for methanogenesis. Wallace et al (1994) stated that optimizing the number of rumen protozoa can increase efficiency of the metabolism of nitrogen, reduce methane gas production and cause microbial protein amount improvement of that enters post-rumen channel.

Conclusion

According to the research conducted, it can be concluded that cinnamon leaf powder addition as source of cinnamaldehyde at 2% DM of feed which is equivalent to cinnamaldehyde of 32 mg/kg DM of feed is able to increase gas production and reduce CH₄ and CO₂ emissions resulting from fermentation in the rumen in vitro. The addition of cinnamon leaf powder as a source of cinnamaldehyde at 4% DM of feed which is equivalent to cinnamaldehyde of 64 mg/kg DM of feed is able to reduce the digestibility of feed ingredients with the lowest DDM and DOM values among other treatments.

Acknowledgement

The researcher extends gratitude and thanks to Gadjah Mada University (Universitas Gadjah Mada) through the Batch I of the 2024 Rekognisi Tugas Akhir (RTA) Program who have helped, support, and facilitate this research activities, leading to its successful completion.

Conflict of interest

The authors declare no conflicts of interest.

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