Research Article

Innovative Strategies for Methane Mitigation: The Role of Durian Seed Nanoemulsions (DSN) in In vitro Studies

Efi Rokana1*, Zein Ahmad Baihaqi1,2, Ahsin Daroini3, Fadlilatul Taufany4

1Program of Animal Husbandry, Faculty of Agriculture, Universitas Islam Kadiri, Kediri, East Java, Indonesia; 2Research Center for Animal Husbandry, Research Organization for Agriculture and Food, National Research and Innovation Agency (BRIN), Bogor, West Java, Indonesia; 3Program of Agribusiness, Post-graduates program, Universitas Islam Kadiri, Kediri, East Java, Indonesia; 4Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS Sukolilo, , Surabaya, East Java, Indonesia.

Received | September 28, 2024; Accepted | January 25, 2025; Published | May 15, 2025

*Correspondence | Efi Rokana, Program of Animal Husbandry, Faculty of Agriculture, Universitas Islam Kadiri, Kediri, East Java, Indonesia; Email: [email protected]

Citation | Rokana E, Baihaqi ZA, Daroini A, Taufany F (2025). Innovative strategies for methane mitigation: The role of durian seed nanoemulsions (DSN) in In vitro studies. J. Anim. Health Prod. 13(2): 340-345.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.2.340.345

ISSN (Online) | 2308-2801

Copyright © 2025 Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The ruminant farming sector is a major source of greenhouse gas emissions, particularly methane (CH4), which significantly contributes to global warming. Methane has a global warming potential approximately 23 times greater than carbon dioxide (Terefe et al., 2024). Rising global temperatures pose a critical environmental challenge (Richardson et al., 2023). Omeme and Mona (2025) stated that methane possesses a significantly higher warming potential, being 21 times more effective at trapping heat in the atmosphere than carbon dioxide, thereby accelerating global warming. Additionally, Debbarma et al. (2023) identify various sources of methane, with emissions from methanogenic bacteria during the digestion process in ruminants being a significant contributor.

Baihaqi et al. (2023) demonstrated that Carica fruit seeds can effectively reduce methane emissions, control Haemonchus contortus, and are safe for rumen fermentation in ruminants. Additionally, research by Baihaqi et al. (2020a), (2020b), (2020c), along with studies by Sakti et al. (2024); Rokana et al. (2024); Prasetyo et al. (2024); Lokapirnasari et al. (2024); Prayudi et al. (2023); Lisnanti et al. (2023) and Baihaqi et al. (2024a), (2024b), highlighted that various active compounds derived from plant-based agro-industrial waste have been successful as alternative treatments, including bio-anthelmintics, bio-antibacterials, methane-reduction agents, and enhancers of livestock productivity.

The selection of durian seeds for this study is based on the fact that the waste from these seeds causes environmental pollution and is abundant in quantity (Gamay et al., 2024). Strategies to reduce excessive methane emissions can involve the use of active plant compounds (Baihaqi et al., 2023). Gamay et al. (2024) stated that durian seed waste contains active plant compounds that are largely underutilized. Zhao et al. (2024) reported that flavonoid compounds inhibit populations of methanogenic bacteria and protozoa in the rumen, improving organic matter digestibility and increasing populations of Fibrobacter succinogenes. Nascimento et al. (2021) noted that tannins promote microbial protein synthesis by forming protein-tannin complexes that inhibit methanogenic bacteria. Rira et al. (2019) suggested that plant bioactives could function as feed additives to reduce CH4 production and greenhouse gas emissions. Furthermore, the formulation of nanoemulsion preparations enhances the solubility of active substances, facilitating their absorption and bioavailability in the gastrointestinal tract (Singh et al., 2009). Dhakal et al. (2024) advocate for using plant bioactives as feed additives to reduce CH4 emissions and overall greenhouse gas output. Therefore, this research aims to investigate the potential of compounds from Durio zibethinus seed nanoemulsions in mitigating methane emissions.

MATERIALS AND METHODS

Location and Material

Research on the analysis of Durio zibethinus seed nanoemulsions for methane reduction was conducted at the National Research and Innovation Agency. The Durio zibethinus seeds were collected from community gardens in Kediri Regency, Indonesia, and were used for the extraction process..

Preparation of Durio zibethinus Seeds Nanoemulsions

The Durio zibethinus seeds were collected during the durian fruit season in June 2024 through direct collection, then oven-dried at 55°C and ground into powder. The powder was extracted using ethanol as a solvent. The nanoemulsion formulation consisted of the ethanol extract of Durio zibethinus seeds, Tween 80, and phosphate buffer. The preparation of this formulation followed the method outlined in previous research by Harmi (2014). The extract of Durio zibethinus seeds at concentrations of 5% and 10%, along with 3 ml of Tween 80, was dissolved in the buffer solution and homogenized to a final volume of 100 ml. The extract mixture, along with the other ingredients, was blended until homogeneous using a centrifuge for 30 minutes at a speed of 10,000 rpm, with temperatures ranging from 10°C to 30°C.

Identification of Bioactive Compounds In Nanoemulsions

The bioactive components of the Durio zibethinus seeds nanoemulsions were qualitatively identified using the method outlined by Adeyemi et al. (2017). Quantitative analysis of the total phenolic content in fruit peel waste was performed using the Folin-Ciocalteu method and expressed in mg of gallic acid. Concurrently, the total flavonoid content in fruit peel waste was analyzed using a colorimetric method, with results expressed in mg of rutin, following the procedure by Sari et al. (2023).

Prepare for In vitro Gas Production

Bali cattle rumen fluid samples were collected from Faculty of Animal Science, Universitas Gadjah Mada (Yogyakarta, Indonesia). This research used a feed ingredient design consisting of complete feed and the addition of Durio zibethinus seeds nanoemulsions with percentages of 3%, 5% and 7%. The results of the proximate analysis of the feed ingredients used are presented in Table 1.

 

Table 1: Chemical composition of complete feed.

Feed name	DM %	Percentage Based on Dry Matter (DM) (%)
		Ash	CP	CF	CFiber	NFE	TDN
Complete feed	89.91	4.21	14.32	4.83	26.71	59.60	65.11

 

Note: CP= Crude Protein; CF = Crude Fat; Cfiber = Crude Fiber; NFE= Nitrogen-Free Extract; TDN= Total Digestible Nutrient .

 

The fermentation substrate used in each treatment was 300 mg. All samples were put into a syringe filled with rumen buffer fluid in triplicate. Then, according to Theodorou methods (Theodorou et al., 1994), incubation using a water bath. Gas production was measured at 0, 2, 4, 6, 8, 12, 24, 36, and 48 hours and the gas collection for methane analysis was conducted at hour 24.

Statistical Analysis

The research design in this study is divided into 4 treatments, consisting of a control treatment and 3 different concentrations of Durian Seed Nanoemulsion (DSN). One treatment in this study consists of 7 replications. The research results related to rumen fermentation characteristics were analyzed using a two-way analysis of variance and Duncan’s multiple range test. Significant research results were stated with p <0.05. The analyses were performed using IBM Statistical Package for the Social Sciences version 20 (IBM Corp., Chicago, USA). Data were presented as mean ± standard deviation (SD).

RESULTS

The results of qualitative observations (Table 2) of plant bioactivity showed that Durio zibethinus seeds tested positive for the presence of tannins, flavonoids, alkaloids, saponins, and steroids.

 

Table 2: Analyses of qualitative phytochemicals of Durio zibethinus seeds (DSN).

Secondary metabolite	DSN
Tannin	+
Flavonoid	+
Alkaloid	+
Saponin	+
Steroid	+

 

The results of the quantitative analysis of bioactive compounds in this study, including flavonoids, total phenols, and tannins, are presented in Table 3. The flavonoid content of the Durio zibethinus seed nanoemulsion was 4.2 mg RE/g dry weight (dw), the total phenolic content was 9.1 mg GAE/g dw, and the total tannin content was 7.6%, with condensed tannins accounting for 4.4% and hydrolysable tannins at 3.2%.

As shown in Table 4, statistical analyses revealed that incorporating DSN did not significantly affect gas production kinetics from soluble feed fractions (a), potentially degradable feed fractions (b), the fractional rate of gas production (c), or total gas production (a+b) (p > 0.05). However, DSN significantly reduced methane production, with a notable decrease in methane emissions observed when DSN was administered at higher levels (p < 0.05).

DISCUSSION

The results of this quantitative plant bioactive study are consistent with the research found by Aisyah et al. (2024), who studied the same plant parts, specifically the durian seeds. Baihaqi et al. (2023) stated that Carica pubescens seeds contain bioactive compounds both qualitatively and quantitatively, with details of flavonoid content at 3.1 mg RE/g dw, total phenol at 9.5 mg GAE/g dw, and total tannin at 5.4% have successfully provided a significant methane reduction effect, positively causing death to worms in the abomasum of ruminant livestock, and also did not disrupt rumen fermentation characteristics. Króliczewska et al. (2023) stated that plant parts containing active plant compounds have the potential to be utilized as herbal medicine, aiding in methane emission reduction and serving various sectors.

 

Table 3: The quantification of Durio zibethinus seeds.

Material	Flavonoids content (mg RE/g dw)	Total phenolic (mg GAE/g dw)	Tannin total (%)	Condensed tannin, CT (%)	Hydrolyzed tannin, HT(%)
DSN	4.2	9.1	7.6	4.4	3.2

 

Table 4: In vitro assessment of rumen gas production following the addition of DSN.

Parameter	Treatment group
	DSN 0%	DSN 3%	DSN 5%	DSN 7%
Gas production from soluble feed fraction (a; ml/300 mg/dry matter)ns	11.27 ±0.31	11.26 ±0.30	11.24 ±0.29	11.23 ±0.28
Gas production from potentially degraded feed fractions (b; ml/300 mg/dry matter)ns	114.01 ±0.30	113.99 ±0.28	113.97 ±0.27	113.95 ±0.26
Fractional rate of gas production (c; ml/hour)	0.05 ±0.00	0.05 ±0.00	0.05 ±0.00	0.05 ±0.00
Potential gas production (a+b; ml/300 mg dray matter)ns	125.28 ±0.38	125.27 ±0.36	125.25 ±0.34	125.24 ±0.32
CH4 (ppm)	15.31 ±0.32a	11.15 ±0.49ᵇ	8.37 ±0.18ᶜ	6.21 ±0.14ᵈ

 

a-d: Different superscripts in a row indicate significant difference (p<0.05) between means.

 

Table 4 may demonstrate these effects of DSN supplementation on rumen gas production kinetics. The findings of the recent study align with prior research conducted by Acosta-Lozano et al. (2023), which stated that the addition of Acacia mearnsii, which contains tannins, did not significantly affect gas production parameters but resulted in a significant decrease in methane production along with the tannin content in the plant. This effect is likely due to a reduction in methanogenic archaea and ruminal protozoa and the formation of tannin complexes. Additionally, Baihaqi et al. (2023) added that the addition of Carica pubescens seeds also did not significantly change gas production but could significantly reduce methane production. In contrast to the findings of Liu et al. (2023), who found that the isolated silibinin from the fruit of Silybum marianum (chrysanthemum plant) resulted in a decrease in total gas production and methane in line with the high silibinin content.

The active compounds in DSN aqueous extract successfully provided an effect for methane reduction. The findings were in line with those reported by previous researchers, Martins et al. (2024), who conducted a meta-analysis of various references, stated that several methane mitigation interventions using β-cyclodextrin, Leptospermum pattersoni, Salix caprea, Heracleum spp., nitroglycerin, Fructulus Ligustri, and Sesbania grandiflora. According to Lambo et al. (2024), they highlighted the regulatory mechanisms of medicinal plants (MP) and emphasized the critical gaps that need addressing to enhance their efficacy. MP has the potential to decrease enteric methane production by 8-50% through regulating rumen fermentation pathways, shifting hydrogen towards propionogenesis, and modifying the diversity, structure, and population of methanogens and protozoa in the rumen.

Lins et al. (2018) noted that the inclusion of Moringa seeds containing tannins did not significantly affect (p>0.05) total gas production. In vitro gas testing serves as a parameter for evaluating rumen microbial conditions and digestibility. This study demonstrated that administering DSN effectively reduced methane emissions, particularly as the percentage of DSN increased. These outcomes align with previous research suggesting that certain active plant compounds can lower methane production. For instance, tannic acid, salicylic acid, and quercetin have been shown to decrease rumen methane formation after 48 hours of incubation, exhibiting a significant reduction compared to the control using corn silage (Nørskov et al., 2023). Furthermore, the addition of tannin extract from Acacia mearnsii has been proposed as a strategy for methane reduction, although it did not affect the production of short-chain fatty acids, despite the decrease in rumen CH4 levels (Junior et al., 2022).

CONCLUSIONS AND RECOMMENDATIONS

The results of the exploration using nanoemulsion from Durio zibethinus seeds (DSN) significantly reduced methane levels, likely due to the active compounds in the durian seeds. Further research is needed to determine the optimal concentration for direct application to ruminant livestock (in vivo).

ACKNOWLEDGEMENTS

This study was supported by Kementerian Pendidikan dan Kebudayaan, Direktorat Jenderal Pendidikan Tinggi, Riset dan Teknologi Republik Indonesia, Regular Fundamental Research Scheme, with Grant number 029/SP2H/PT/LL7/2024

NOVELTY STATEMENTS

This study pioneers the use of Durian Seed Nanoemulsions (DSN) as a cutting-edge nanomaterial-based approach for methane mitigation in in vitro conditions. Unlike conventional methods, DSN harnesses nanotechnology to enhance bioavailability, reactivity, and targeted efficacy, offering a highly efficient, plant-derived, and sustainable alternative. By optimizing nanoparticle properties, this research provides breakthrough insights into methane reduction mechanisms, paving the way for next-generation, nano-based strategies in sustainable livestock production and environmental conservation.

AUTHOR’S CONTRIBUTIONS

ER, ZAB, AD, FT: Designed the study and

collected samples and performed examinations. All authors

have drafted and revised the manuscript. All authors have

read, reviewed, and approved the final manuscript.

Conflict of Interest

The authors declare that they have no competing interests.

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