Microencapsulation efficiency of fruit peel phytonutrient-based antimicrobial to mitigate rumen emission using in vitro fermentation technique

Abstract

This research investigated the protecting properties of polyphenols and flavonoids in phytonutrient pellets formulated from lemongrass powder and mangosteen peel (LEMANGOS pellets) through the microencapsulation and named microencapsulated LEMANGOS (mLEMANGOS). For this purpose, the effects of mLEMANGOS supplementation at various R:C ratios of 60:40 and 20:80 were evaluated and compared with monensin (antibiotic) supplementation under an in vitro study technique. Treatments were randomly assigned in a 2 × 4 × 2 factorial arrangement in a completely randomised design consisting of factors A: R:C ratios (60:40 and 80:20), factor B: mLEMANGOS supplementation (0, 2, 4, and 6% DM), and factor C: monensin supplementation (0 and 20% DM). There was an interaction between the R:C ratio and both mLEMANGOS and monensin supplements on the in vitro gas production kinetics, ruminal by-product fermentation, methane production, and rumen microbial population (p < 0.001, 0.01, 0.05). Results indicated that each supplementation influenced the gas production kinetics, while there was decreased cumulative gas production in the mLEMANGOS supplemented. Consequently, the supplemented group buffered ruminal pH and increased the in vitro dry matter degradability (IVDMD) and ammonia nitrogen (NH₃-N) concentrations. Moreover, the additional treatment of mLEMANGOS supplementation (6% DM at R:C ratios of 60:40 and 20:80) significantly reduced the number of Methanobacteriales to 53.5% and 50.4% after 24 h, respectively. Results from those supplements can reduce methane production to 99.2% and 97.9% (p < 0.001), respectively. This research suggests that phytonutrient-based antimicrobial in the mLEMANGOS supplement could potentially be used as ruminant feed additives and as antimicrobial substances.

HIGHLIGHT

• Microencapsulated LEMANGOS was formulated by biopolymer using green technique to retain the phytonutrients and their long-term release.

• The mLEMANGOS supplementation (at 6% of total DM) can be used as a synthetic bio-antibiotic for inhibiting methanogens-archaea population.

• The mLEMANGOS supplementation (at 6% of total DM) can enhance rumen nutrients degradability, ruminal end-products, and mitigate methane production.

Keywords:

• Microencapsulation
• lemongrass oil
• mangosteen peel
• feed innovation
• rumen fermentation
• methane mitigation

Introduction

Currently, research is being conducted on mitigation techniques to lower methane (CH₄) emissions and enhance the productivity of ruminants. The previous research has demonstrated that altering the rumen microbiota with dietary supplements is one of the most effective mitigation techniques and that it has positive effects on both the environment and animal production (Wanapat et al. 2013; Honan et al. 2021). The type of feed nutrition, such as forage-roughage- and/or concentrate-based diets, is one of the main causes of the uneven effectiveness (Vázquez-Carrillo et al. 2020). It is frequently believed that diet composition in terms of chemical components affects the production of CH₄ through the activity of rumen microbiota in particular the consortium species of protozoa and methanogenic archaea (Ku-Vera et al. 2020). Numerous feed additives and supplements have been utilised to reduce CH₄ production and ammonia formation in the rumen by influencing microbial fermentation (Honan et al. 2021). Consequently, numerous research teams worldwide are looking into various feed supplements (such as plant -based natural compounds or plant secondary metabolites) and additives with antimicrobial activity that case of the above mention (Wanapat et al. 2013; Ku-Vera et al. 2020; Vázquez-Carrillo et al. 2020). The effects of different secondary metabolites, such as condensed tannin, saponin, resveratrol, emodin, berberine, curcumin, and quercetin, have been investigated in a variety of alternative plant-based bioactive compounds as supplements (Yücel and Karatoprak 2022). Especially, for example, plant- or fruit peel-based phytonutrients obtained from tropical, herb oils, e.g. lemongrass (Cymbopogon citratus Stapf.) and fruit peels, e.g. mangosteen (Garcinia mangostana L.), which have been popularly studied in terms of their use as an antimicrobial agent (Wanapat et al. 2008; Poungchompu et al. 2009). Development of a feed additive in this research which evaluated fruit peel-based phytonutrients extract in terms of phytochemical characteristics, sum of essential oils (EOs) 67.98% derived from lemongrass which contain citral component combined with a key natural antimicrobial activity obtained from mangosteen peel extract consisting of polyphenolic compounds (e.g. condensed tannins) 39.87% and flavonoids (e.g. saponins) and some antioxidant activity, which inhibit the growth and metabolism of protozoa and methanogens archaea population (Wanapat et al. 2008; Poungchompu et al. 2009; Yücel and Karatoprak 2022). Thus, there is an urgent need for a feed supplement that can effectively reduce emissions with various ruminant diets without impairing rumen fermentation (Poungchompu et al. 2009; Ahmed et al. 2021). Additionally, supplements should be natural because there is a global interest in using plants and their secondary metabolites as alternatives to phytochemical compounds to bring about antimicrobial activity when used as a supplement in animal feed, and because natural supplements are well-liked by consumers (Ku-Vera et al. 2020; Honan et al. 2021). Using phytonutrients offers the promise of reducing the damaging effects of animal agriculture on the environment, but more strong supplements are still needed through using encapsulation technology for applying in feed supplement formulation and to contain active substances within nano- or microparticles (Amin et al. 2021; Phupaboon et al. 2022a). Due to the different wall or core materials, the principles of microencapsulation can be divided into chemical and physical processes. Through applying mechanical and physical principles, the physical process produces microcapsules are widely used the methods of extrusion, emulsification, and spray-drying techniques (Bah et al. 2020). Another advantage of microencapsulation technique is their stability, ensuring a site-specific release of the intended phytonutrients and EOs in the animals’ gastrointestinal tracts as well as in the rumen, abomasum, and small intestine simulated buffers at different pH (6.5, 2.9 and 7.4) (Amin et al. 2021; Ibrahim and Hassen 2021). According to the research of Yoshimaru et al. (1999) reported that mechanism of releasing encapsulated substances in the ruminant’s rumen via rumen bypass microcapsules, which prevent microbial hydrogenation processes (in neutral pH (6.8–7.0) condition), was formulated from carbohydrate as an encapsulant by spray-drying method. Furthermore, an excellent approach to encapsulating the value of exposed phytonutrients into tiny particles is the microencapsulation of film-forming and acid-resistant wall materials. It can enhance the intestinal barrier’s performance, preserve the activity of healthy flora, and increase the bacteria’s ability to colonise the mucosa of the intestine and as a protective rumen (Wei et al. 2022).

The manufacture of stable products consisting of probiotics (lactic acid bacteria and some yeast strains), vitamins with functional properties, fatty acids (polyunsaturated), and bioactive components (antimicrobial or antioxidant activities) via microencapsulation is a rapidly developing technology that is currently widely employed in human and animal nutrition (Kim et al. 2020). In order to achieve the desired stability and site-specific delayed release of the by-products, research have been carried out on various encapsulated plant-based extracts and EOs utilising various encapsulant matrices (for example, chitosan, plant-based protein, and insect-based protein) via spray-drying technique (Ahmed et al. 2021; Amin et al. 2021). For instance, accordance with Amin et al. (2021), who reported the capability of changing rumen fermentation, potentially reducing CH₄ emissions and the most frequently employed technique for successfully delivering their microparticles to the in vitro rumen fermentation. In a another study, microencapsulation using hydrogenated fatty acids, calcium carbonate, and starch effectively protects EOs from breakdown in the rumen, minimising microbial degradation. The EOs extracted from herbal plant leave (e.g. Mitragyna speisosa) in the microspheres can be used as a rumen enhancer. In accordance with Matra et al. (2024), who reported the effect of microencapsulated Mitragyna leave-oil extract (mMLOE) supplements was to investigate on nutrient degradability, rumen ecology, microbial dynamics, and CH₄ production in an in vitro studyat 12, 24, and 48 h. When supplemented with 6% of the total DM substrate, the mMLOE produced the most propionate and total volatile fatty acids, as well as boosted the concentration of ammonia-nitrogen (NH₃-N), while less CH₄ production (12, 24, and 48 h). Additionally, the microbial population of cellulolytic bacteria and Butyrivibrio fibrisolvens were increased, whilst Methanobacteriales was decreased with mMLOE feeding.

Consequently, it was hypothesised that microencapsulated fruit peel-based phytonutrients extracted from lemongrass mixture with mangosteen peel in diet pellets, which is called microencapsulated LEMANGOS (mLEMANGOS) supplementation could increase nutrient digestibility, favourably alter rumen fermentation characterisation and the dynamics of the microbial population, and might have the potential to reduce ruminal in vitro CH₄ production with the same action as a commercial antimicrobial reagent such as monensin (MNS). Therefore, the objective of this in vitro experiment was to evaluate the effect of mLEMANGOS supplementation in comparison with commercial MNS supplemented into feed for comparing and improving efficacy of phytonutrients, particularly total polyphenolic, total flavonoids components on ruminal fermentation in Thai native cattle rumen fluid and real-time-PCR technique to analyse microbial rumen population.

Materials and methods

Animal ethics

All procedures involving animals providing rumen fluid collection were approved by the ethics committee of Khon Kaen University, with authorisation from the Khon Kaen University’s guidelines of the Institutional Animals Care and Use Committee of Khon Kaen University and the Institute of Animals for Scientific Purpose Development (IAD), Thailand (record no. IACUC-KKU 110/66 and U1-10937-2566).

LEMANGOS pellet preparation

The LEMANGOS pellet was prepared by adapting the procedures used by Wanapat et al. (2008) and Poungchompu et al. (2009) with slightly formulated ingredients containing lemongrass powder (500 g/kg), mangosteen peel powder (450 g/kg), and cassava starch (20 g/kg) on a dry matter basis. All ingredients were well mixed with molasses (30 g/kg) and added water make up the moisture content (600 g/kg). Pellet products were processed through a pellet maker and then dried to contain at least 850 g/kg dry matter following which, dried pellets were stored in a closed drum for use on the phytonutrient’s extraction step.

Phytonutrients extraction and microencapsulation process

The methodology of chitosan-mLEMANGOS extraction for this study followed the modified method of Phupaboon et al. (2022a). Briefly, dried LEMANGOS pellets were crushed and extracted at the concentration of 10% (v/w) with DI water using microwave extraction under the optimal conditions; 100 W, at the temp. ≤ 60 °C for 20 min, to enhance the phytonutrients compounds and antioxidant capacity. Then, the supernatant was separated and collected by filtration using a suction pump. For the microencapsulation process, LEMANGOS microcapsules were formulated at 1:1 ratio consisting of extracted supernatant mixed with the encapsulants prepared by 2% (w/v) chitosan media containing 1% (v/v) acetic acid solution. Subsequently, homogeneously media processed through the spray-drying technique at the processing conditions; operation speed (10 mL/min), drying airflow (600 L/h), pressure drop (0.75 bar), inlet temp. (160 °C), and outlet temp. (90 °C). The microcapsules were kept in vacuum bag and stored at −20 °C until use in the in vitro gas fermentation.

Experimental design, dietary treatments, and chemical analysis

The experimental treatment of this study was comprised 2 × 4 × 2 factorial arrangement in a Completely randomised design (CRD). Factor A was 2 levels of roughage to concentrate (R:C) ratio (60:40, 20:80), Factor B was 4 levels of additives by mLEMANGOS supplementation at 0, 2, 4, 6% of total DM substrate on basal diet, and Factor C was 2 levels of MNS premix (Bio Agri Mix, Canada) supplementation at 0 and 20% of total DM substrate on basal diet. The details of this experimental work were designed as gas production parameter (total 48 bottles from 3 bottles/treatment), nutrient degradability and CH₄production parameters (total 64 bottles from 2 bottles/treatment, for 2 assay times: 12 and 24 h), and the last experiment to analyse NH₃-N concentration, along with microbial population parameters (total 64 bottles from 2 bottles/treatment, for 2 assay times: 12 and 24 h).

The dietary treatments concentrate was formulated with the combination of ingredients and mineral mix as shown in Table 1. Each of the dried materials were chemically analysed for DM, ash, CP, and fibre fractions following the methodology of Thiex et al. (2012). The fibre contents (neutral detergent fibre; NDF and acid detergent fibre; ADF) were analysed using the method of Van Soest (1994). In addition, the mLEMANGOS supplement was analysed for the bioactive compounds and antioxidative activities following the protocol described by Phupaboon et al. (2022b). The bioactive values consisting of total polyphenolic content (TPC) following by Al-Duais et al. (2009), total flavonoid content (TFC) (Topçu et al. 2007) and antioxidative values in term of 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) (Gali and Bedjou 2019), (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS assay) (Re et al. 1999) radical scavenging inhibition and Ferric Reducing Antioxidant Power (FRAP assay) (Benzie and Strain 1996) were measured in the extract juice obtained from mLEMANGOS as shown in Table 1. In addition, the percentage of encapsulation efficacy (EE) was calculated as following the equation of EE (%) = (Amount of TPC in extract/Amount of TPC in entrapped) × 100 (Phupaboon et al. 2022a).

Table 1. Characterisation of feed ingredients, chemical composition, and phytochemical components of feeds used in this study.

Items	Concentrate	Rice straw	mLEMANGOS
Ingredients (% as fed)
Cassava chip	54.0
Rice bran meal	17.0
Palm kernel meal	13.0
Soybean meal	10.5
Urea	2.5
Sulphur	1.0
Salt	1.0
Mineral mixed^1	1.0
Chemical composition
DM, %	90.5	89.4	82.8
	% of DM
OM, %	92.2	85.4	88.6
CP, %	14.6	2.4	18.6
NDF, %	20.5	78.9	43.1
ADF, %	8.2	52.6	23.2
Ether extract, %			2.6
Phytonutrition content
TPC (g GAE/kg DM)	–	–	2.6
TFC (g QUE/kg DM)	–	–	0.2
Antioxidative values
DPPH inhibition (%)	–	–	84.2
ABTS inhibition (%)	–	–	70.6
FRAP capacity (g TROE/kg DM)	–	–	20.6

Notes: mLEMONGOS microencapsulated of lemongrass combined with mangosteen peel obtained from basal diet pellet extracts; DM dry matter; OM organic matter; CP crude protein; NDF neutral detergent fibre; ADF acid detergent fibre; TPC total phenolic content; TFC total flavonoid content; DPPH (2, 2-diphenyl-1-picrylhydrazyl) DPPH radical scavenging activity; ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] ABTS radical scavenging activity; FRAP ferric reducing antioxidant power; GAE gallic acid equivalent; QUE quercetin equivalent; TROE Trolox equivalent.¹ Mineral premix (contains per kg) vitamin A 10,000,000 IU, vitamin D 1,600,000 IU, vitamin E 70,000 IU, Iron (Fe) 50 g, Manganese 40 g, Zinc 40 g, Copper 10 g, I 0.5 g, Selenium 0.1 g, Cobalt 0.1 g.

Rumen fluid and in vitro fermentation

Three Thai native cattle with an average live weight of 300 ± 10 kg were used as rumen fluid donors. Approximately 1000 mL of rumen fluid from each animal was collected using a suction pump and blended prior to the morning feeding. The sample was combined with the prepared artificial saliva solution (2:1), incubated at 39 °C with continuous CO₂ flushing. A 500 mg of total DM substrate mixture including substrates and additives was weighed and combined with 40 mL of rumen fluid medium into 50 mL in vitro bottles. The procedure for in vitro fermentation was modified and described by the methodology of Menke and Steingass (1988). Gas production during incubation was measured at 1, 2, 4, 6, 8, 12, 24, 48, 72 and 96 h and data was fitted to the cumulative gas production model of Ørskov and McDonald (1979) as following the equation of Y = a + b (1 – e ^(-ct)), where Y = gas produced at time ‘t’ (mL), a = the gas production from the immediately soluble fraction (mL), b = the gas production from the insoluble fraction (mL), and c = the gas production rate constant for insoluble fraction (mL/h).

For the kinetic analysis during the fermentation of the ruminal fluid, the inoculum was directly measured following the inoculation at 12 and 24 h for the gas production and the pH changes. For the other parameters the rumen fluid samples were divided into two portions; the first supernatant portion mixed with a pre-filtered sample and used for DNA extraction to identify the rumen microbiota using real time-PCR (RT-PCR) technique. The second supernatant portion provided insoluble fibre by filtration followed by centrifugation and kept at −20 °C containing 2 mL of 10% H₂SO₄ solution for analysis of the ammonia nitrogen (NH₃-N) content using an NH₃-N kit (FUJIFILM Wako Pure Chemical Corp, Japan) (Ahmed et al. 2021). The in vitro DM degradability (g/kg) after the incubation times of 12 and 24 h was calculated according to the method of Van Soest (1994). Additionally, CH₄ production was measured by taking 3 mL of gas sample from the nutrient’s degradability experiment for CH₄ analysis using GC instrument (model: GC-17A System, Shimadzu, Kyoto, Japan; thermal conductivity detector; column ShinCarbon; column size 3 m × 3 mm, activated charcoal 60/80 mesh) and calculated as following the equation of CH₄ production (% v/v) = (Peak area/slope)/volume of gas sample in bottle.

DNA extraction

Genomic DNA was extracted from 1.0 mL of rumen fluid obtained from the pellet portion (12 and 24 h) using the GF-1 DNA extraction kit (Vivantis, Malaysia) in accordance with some modifications of Phupaboon et al. (2023). The extracted DNA template was eluted by elution buffer and normalised using free-Ranse DI water to measure the DNA concentration using DS-11 Spectrophotometers (DeNovix Inc., USA).

Specific primers and quantitative real-time PCR analysis

The species-specific PCR primers consisting of Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens (Koike and Kobayashi 2001), Megasphaera elsdenii (Ouwerkerk et al. 2002), Butyribrio fibrisolvens (Fernando et al. 2010), and Methanobacteriales (Yu et al. 2005), were used to amplify 16S rDNA regions through qPCR technique using real time-PCR equipment.

The PCR amplification was performed using CFX Connect TM Real-Time System (Bio-Rad, Singapore). The optimal conditions used for annealing temperature at 55 °C and reaction mixture was conducted in a final volume of 10 µL containing the following: Luna® Universal qPCR Master Mix (New England Biolabs GmbH, Germany) (5.0 µL), Fw and Rv primers (0.2 µL), purified DNA template (final DNA quantity = 0.01 µg/µL, 3 µL) and water (1.6 µL). The thermal cycling protocol was modified according to the procedure of Lee et al. (2006). Quantification of the number of each species was counted in duplicate from each sample and the mean value was calculated. The 10-fold serial dilutions of each standard DNA containing the target gene sequences of the relevant microbial group were used to create standard curves. The log10 gene copies number/mL of rumen fluid was used to express the absolute abundance of each microbial group or specific species (Whelan et al. 2003).

Statistical analysis

Data management and analysis were performed using the GLM procedure of SAS (2013) for 2 × 4 × 2 factorial arrangement via a CRD platform. Comparison between R:C ratio, mLEMANGOS, monensin addition and interaction were tested by orthogonal polynomials at differences amongst means with p < 0.05, < 0.01, and < 0.001.

Results

Chemical and phytochemical components of experimental feeds

Table 1 presents the details of the nutritive, phytonutrient, and antioxidant values of feeds and mLEMANGOS supplementation used in the experiment. In addition, the information data on the percentage of encapsulation efficiency (%EE) obtained from mLEMANGOS microcapsules was calculated from the majority content in terms of total phenolic (TPC) content at 23.8% in microcapsules.

In vitro gas production characteristics

Table 2 shows the results of gas kinetics production, including gas production from the insoluble fraction (b) and the potential extent of gas production (a + b) was an interaction effect between the R:C ratio and additives of either mLEMANGOS or MNS supplementation (p < 0.05). Particularly, gas kinetics production consisting of gas production from the insoluble fraction (b), gas production rate constant for the insoluble fraction (c), potential extent of gas production (a + b), and cumulative gas were significantly increased (p < 0.001) by mLEMANGOS supplementation, while the immediately soluble fraction (a) was not affected (p < 0.05). Additionally, the treatment of MNS supplementation was affected (p < 0.01) in gas production from the insoluble fraction (b) and potential extent of gas production (a + b), nonetheless, it was not affected (p < 0.05) in the immediately soluble fraction (a), gas production rate constant for the insoluble fraction (c), or cumulative gas at 96 h of post-fermentation (Table 2).

Table 2. Effect of roughage to concentrate (R:C) ratio with the different levels of microencapsulated LEMANGOS (mLEMANGOS) and monensin (MNS) premix supplementation on in vitro gas production kinetics.

T^a	R:C^b	mLEMANGOS^c (% of total DM substrate)	MNS^d (% of total DM substrate)	Gas Kinetics^e				Cumulative Gas at 96 h^f
				a	B	c	a + b
1	60:40	0	0	−2.6	114.7	0.013	112.1	105.4
2		2	0	−6.0	83.2	0.043	77.1	76.0
3		4	0	−5.6	78.5	0.035	92.9	73.2
4		6	0	−7.3	90.2	0.041	82.8	76.7
5		0	20	−5.3	82.2	0.034	76.9	51.5
6		2	20	−7.9	80.7	0.034	72.8	66.2
7		4	20	−3.9	57.8	0.037	53.9	43.9
8		6	20	−3.2	22.9	0.044	19.7	35.4
9	20:80	0	0	−4.6	115.1	0.016	110.5	105.6
10		2	0	−4.8	79.7	0.041	74.8	81.0
11		4	0	−8.2	61.0	0.052	52.8	68.5
12		6	0	−7.6	64.5	0.057	56.9	69.3
13		0	20	−3.6	53.7	0.023	50.1	49.0
14		2	20	−6.4	54.3	0.047	47.9	44.8
15		4	20	−4.6	82.0	0.057	77.5	43.8
16		6	20	−4.6	61.8	0.039	57.2	38.7
SEM				0.65	3.14	0.002	2.94	4.42
Comparisons
R:C ratio				ns	ns	ns	ns	ns
mLEMANGOS				ns	***	**	***	***
MNS				ns	*	ns	**	ns
Interactions
R:C ratio × mLEMANGOS				ns	**	ns	***	ns
R:C ratio × MNS				ns	ns	ns	ns	ns
mLEMANGOS × MNS				ns	*	ns	*	ns
R:C ratio × mLEMANGOS × MNS				ns	ns	ns	ns	ns

^a T each treatment in this experiment.

^b R:C roughage to concentrate ratio.

^c mLEMANGOS microencapsulated lemongrass combined with mangosteen peel pellet extract (% of total DM substrate on basal diet).

^d MNS 20% monensin premix for animal feeding (% of total DM substrate on basal diet).

^e Gas production kinetics: a the gas production from the immediately soluble fraction (mL); b the gas production from the insoluble fraction (mL); c the gas production rate constant for the insoluble fraction (mL/h); a + b the potential extent of gas production (mL).

^f Cumulative gas at 96 h (mL/0.5 g DM substrate).

* Means indicating significant differences (p < 0.05).

** Means indicating significant differences (p < 0.01).

*** Means indicating significant differences (p < 0.001); ns means non-significant among treatments; SEM, standard error of mean.

Ruminal pH, nutrient degradability, and ammonia nitrogen (NH₃-N) concentration

Figures 1 and 2, indicate the ruminal pH, nutrient degradability, and NH₃-N concentration, respectively. There was an interaction between the R:C ratio and mLEMANGOS supplementation on ruminal pH at 12 h post-fermentation (p < 0.05). Interestingly, the ruminal pH was influenced (p < 0.001) by the R:C ratio (12 and 24 h) and was increased (p < 0.01) by mLEMANGOS supplementation (12 h) after fermentation, while it was not affected (p < 0.05) by 20% MNS supplementation (Figure 1). As Figure 1 shows nutrient degradability (in vitro dry matter degradability; IVDMD) at different incubation times. There was no interaction between the R:C ratio and the addition of both mLEMANGOS and MNS supplementation. The R:C ratio and mLEMANGOS supplementation have affected (p < 0.001) IVDMD (12 and 24 h), and additionally, MNS supplementation has influenced (p < 0.001) IVDMD (12 h). In addition, the ruminal NH₃-N concentration interacts with the R:C ratio × mLEMANGOS (p < 0.01), mLEMANGOS × MNS (p < 0.001), and the combination of the R:C ratio × mLEMANGOS × MNS (p < 0.001) after incubation at 12 and 24 h as shown in Figure 2. This finding result of the concentrations at 24 h of the R:C ratio (20:80) were significantly higher (p < 0.001) than at 12 h of the R:C ratio (60:40) by correlation with the level of mLEMANGOS and MNS supplementation (Figure 2).

Figure 1. Effect of roughage to concentrate (R:C) ratio with the different levels of on microencapsulated LEMANGOS (mLEMANGOS) and monensin (MNS) premix supplementation on nutrients degradability (left side in a bar chart) ruminal pH (right side in the line chart) at different times of post fermentation.

Figure 2. Effect of roughage to concentrate (R:C) ratio with the different levels of microencapsulated LEMANGOS (mLEMANGOS) and monensin (MNS) premix supplementation on nutrients ruminal ammonia nitrogen (NH₃-N) concentration at different times of fermentation.

Methane (CH₄) production

The effect of mLEMANGOS and MNS supplementation on CH₄ production is presented in Table 3. There was an interaction (p < 0.001) between mLEMANGOS and MNS supplementation in the substrate diet after 24 h of fermentation. The average amount of CH₄ production (at 12 and 24 h) steadily decreased (p < 0.01, 0.001) due to increasing the level of feed additives of mLEMANGOS (6% of total DM substrate) and MNS (20% of total DM substrate) supplementations (at 24 h; p < 0.001), while the R:C ratio did not influence (p < 0.05) to reduce CH₄ production under this experiment of in vitro gas fermentation. The comparative analysis of the efficacy of supplementing with mLEMANGOS without MNS versus supplementing with mLEMANGOS substances plus MNS supplementation resulted in the reduction of CH₄ concentration from 20% (at R:C of 20:80) to 33.3% (at R:C of 60:40) (Table 3).

Table 3. Effect of roughage to concentrate (R:C) ratio with the different levels of microencapsulated LEMANGOS (mLEMANGOS) and monensin (MNS) premix supplementation on methane (CH₄) production.

T^a	R:C^b	mLEMANGOS^c (% of total DM substrate)	MNS^d (% of total DM substrate)	CH4 production (%)
				12 h	24 h
1	60:40	0	0	17.0	23.9
2		2	0	15.9	4.6
3		4	0	14.7	0.6
4		6	0	16.8	0.3
5		0	20	13.1	1.5
6		2	20	11.4	0.5
7		4	20	13.1	0.5
8		6	20	10.9	0.2
9	20:80	0	0	15.3	23.3
10		2	0	13.9	1.0
11		4	0	14.2	1.5
12		6	0	12.8	0.5
13		0	20	12.8	0.3
14		2	20	14.0	0.7
15		4	20	12.9	0.5
16		6	20	12.3	0.4
SEM				0.37	0.22
Comparisons
R:C ratio				ns	ns
mLEMANGOS				**	***
MNS				ns	***
Interactions
R:C ratio × mLEMANGOS				ns	ns
R:C ratio × MNS				ns	ns
mLEMANGOS × MNS				ns	***
R:C ratio × mLEMANGOS × MNS				ns	ns

^aT each treatment in this experiment.

^b R:C roughage to concentrate ratio.

^c mLEMANGOS microencapsulated lemongrass combined with mangosteen peel pellet extract (% of total DM substrate on basal diet).

^d MNS 20% monensin premix for animal feeding (% of total DM substrate on basal diet).

* Means indicating significant differences (p < 0.05).

** Means indicating significant differences (p < 0.01).

*** Means indicating significant differences (p < 0.001); ns means non-significant among treatments.

Rumen microbial population

There was an interaction with different factors by the R:C ratio × mLEMANGOS, mLEMANGOS × MNS, and R:C ratio × mLEMANGOS × MNS supplementations (p < 0.01, 0.05), as presented in Table 4. The dynamic of the rumen microbiota population consisting of cellulolytic, proteolytic, and hydrogenation bacteria was influenced and increased (p < 0.001, 0.01, 0.05) the amount of gene copy numbers by feed addition of the R:C ratio, mLEMANGOS, and MNS supplementation. Interestingly, for those subjected to increasing the level of mLEMANGOS supplementation without MNS supplementation. It was significantly increased (p < 0.01, 0.001) the gene copy number including R. albus, R. flavefaciens, M. elsdenii, and B. fibrisolvens at 24 h of post-fermentation, followed by the gene number of F. succinogens, which was influenced by the R:C ratio and mLEMANGOS supplantation after 12 h of incubation time. In addition, the gene copy number of Methanobacteriales was decreased (p < 0.001, 0.05) when increasing the amount of supplemented by mLEMANGOS (6%) mixed with MNS premix (20%) on total DM substrate after fermentation (at 24 h). From this result, when comparing the efficiency of action between supplementing with mLEMANGOS without MNS substances compared to supplementing mLEMANGOS mixed with MNS substances obtained from the R:C ratios of 20:80 and 60:40 at 24 h of post-fermentation time, it was found that their Methanobacteriales or methanogen populations could be reduced to 29.4% and 43.5%, respectively (Table 4).

Table 4. Effect of roughage to concentrate (R:C) ratio with the different levels of microencapsulated LEMANGOS (mLEMANGOS) and monensin (MNS) premix supplementation on dynamic of the rumen microbiota population.

T^a	R:C^b	mLEMANGOS^c (% of total DM substrate)	MNS^d (% of total DM substrate)	Rumen microbiota population (Log copies/mL)
				F. succinogenes		R. albus		R. flavefaciens		M. elsdenii		B. fibrisolvens		Methanobacteriales
				12 h	24 h	12 h	24 h	12 h	24 h	12 h	24 h	12 h	24 h	12 h	24 h
1	6	0	0	9.9	9.5	10.4	10.1	10.2	10.2	9.7	9.1	9.9	9.5	8.1	9.9
2	0	2	0	8.9	10.5	9.2	11.0	9.9	11.1	9.1	9.8	9.2	9.8	7.5	5.1
3	:	4	0	9.3	9.7	9.2	10.5	9.9	9.6	9.4	9.2	9.6	9.6	7.1	5.1
4	4	6	0	9.1	9.0	9.2	10.2	9.6	10.6	9.2	9.0	9.6	9.8	7.0	4.6
5	0	0	20	7.5	8.3	8.4	8.0	9.3	8.9	8.2	8.1	8.6	8.1	8.0	9.8
6		2	20	7.9	7.8	8.3	7.4	9.1	8.4	8.3	7.8	8.6	8.0	7.8	4.8
7		4	20	7.7	7.8	8.4	7.7	9.1	9.0	8.1	8.5	8.5	7.0	7.8	3.8
8		6	20	7.9	10.1	7.8	7.4	9.1	9.0	8.3	8.2	8.5	8.2	7.1	2.6
9	2	0	0	9.5	9.6	9.9	9.5	9.4	9.2	8.9	9.2	8.7	9.5	7.9	10.2
1	0	2	0	8.2	8.6	8.5	7.2	8.7	7.6	9.5	8.0	9.6	8.7	7.8	5.8
0	:
1	8	4	0	7.9	8.2	8.0	8.1	8.4	8.6	9.4	8.8	9.4	9.1	7.9	5.9
1	0
1		6	0	8.3	9.0	8.2	9.4	9.9	9.4	9.0	9.0	8.6	9.6	7.6	5.1
2
1		0	20	7.5	8.9	8.1	7.2	9.4	8.4	7.9	7.6	8.1	8.0	7.9	8.9
3
1		2	20	7. 4	8.3	7.6	6.8	8.5	7.4	7.9	7.6	8.0	7.7	7.7	4.8
4
1		4	20	7.4	8.7	7.6	6.6	8.3	6.4	8.1	7.5	7.6	7.7	7.5	3.6
5
1		6	20	7.7	9.3	7.9	6.9	9.0	6.3	8.2	7.7	8.2	8.1	7.6	3.6
6
SEM				0.08	0.14	0.08	0.10	0.19	0.14	0.06	0.06	0.12	0.06	0.04	4.08
Comparisons
R:C ratio				***	ns	***	***	ns	***	ns	**	*	*	*	ns
mLEMANGOS				***	ns	***	***	ns	**	***	***	**	***	ns	***
MNS				ns	ns	*	ns	ns	ns	ns	ns	ns	ns	ns	*
Interactions
R:C ratio × mLEMANGOS				*	ns	ns	*	ns	*	ns	ns	ns	ns	ns	*
R:C ratio × MNS				ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
mLEMANGOS × MNS				*	ns	*	ns	ns	ns	ns	ns	ns	ns	ns	ns
R:C ratio × mLEMANGOS × MNS				ns	ns	ns	**	ns	ns	ns	*	ns	*	*	ns

^a T each treatment in this experiment.

^b R:C roughage to concentrate ratio.

^c mLEMANGOS microencapsulated lemongrass combined mangosteen peel pellet extract (% of total DM substrate on basal diet).

^d MNS 20% monensin premix for animal feeding (% of total DM substrate on basal diet).

* Means indicating significant differences (p < 0.05).

** Means indicating significant differences (p < 0.01).

*** Means indicating significant differences (p < 0.001); ns means non-significant among treatment.

Discussion

In vitro gas production characteristics

In this study, we hypothesised that the efficacy of phytonutrient components like TPC (such as condensed tannins; CT), TFC (like saponins; SP) contents, and antioxidant capacity to be retained in capsules using the microencapsulation technique from the combination of LEMANGOS pellets consisting of lemongrass and mangosteen peel can reduce gas production caused by ruminal microbial activity to the same extent as antimicrobials or antibiotics such as monensin, which was selected in this experiment. The results obtained in this study indicated that increasing the mLEMANGOS supplementation from 2 to 6% of total DM substrate could increase the gas production rate constant for the insoluble fraction (c) and decreased cumulative gas at 96 h. The impact of mLEMANGOS on gas generation can be attributed to their phytonutrients, which possess the ability to interact with fibre and protein components. This interaction subsequently influences microbial activity (Pal et al. 2015). Another potential factor that may contribute to starch degradation is its crucial function in the regulation of energy utilisation for the growth of rumen microorganisms, the augmentation of rumen population, and the digestion of feed (Phesatcha et al. 2020). Similarly, Wanapat et al. (2014), discovered that unencapsulated Garcinnia mangostana peel supplementation including the content of CT ≥ 60 g/kg DM, reduced total gas production in an in vitro experiment. This finding of the interaction between the R:C ratio × mLEMANGOS supplement is consistent with the study conducted by Júnior et al. (2020), which showed that the inclusion of unencapsulated lemongrass essential oil at intermediate levels (ranging from 1.53 to 2.22 mL/kg) in silage resulted in the reduction of total gas production. The interaction of these chemical compositions and the nutritional values of feeds has been described previously, according to the evidence of Pal et al. (2015). Additionally, the present findings of the interaction between additives of mLEMANGOS × MNS seem to be consistent with other research which found that the phytochemicals: CT and SP components from tropical fruit peel powder including red dragon (Matra et al. 2021), rambutan (Gunun et al. 2018), and banana flower (Kang et al. 2016), increased gas kinetics production on the gas production rate constant for the insoluble fraction (c) and decreased total gas or cumulative gas production of in vitro study. According to Foiklang et al. (2016) and Cherdthong et al. (2019), who reported the rich phytonutrients of CT and SP from grape pomace powder and pellets containing Delonix regia supplementation at 2, 4, and 6% of total DM substrate increased gas kinetics and cumulative gas production tended to be lower than non-supplementation group.

In vitro fermentation characteristics

Under this investigation, the addition of mLEMANGOS supplementation (2 to 6% of total DM substrate) supplemented into the R:C ratio (20:80) affected reducing ruminal pH lower than the R:C ratio (60:40) ranged from 6.72 to 6.94 across all treatments. In addition, this result can be explained by the phytochemical substances in mLEMANGOS microcapsules obtained from mangosteen peel and lemongrass extracts including CT, SP, and EOs. They influence the control of rumen ecology and the growth of microorganisms in acid-producing bacteria to prevent the development of rumen acidosis in an environment with a high concentration of the R:C ratio (20:80) at 12 h post-fermentation. The current results are consistent with those from earlier research, which showed that the primary factors contributing to the elevated soluble fraction (carbohydrate) in the concentrate and the fermentation of fibre carbohydrates by ruminal microorganisms are the main drivers of the swift generation of volatile fatty acids (VFAs) and lactic acids. Consequently, the accumulation of these acids in the rumen fluid results in a decline in ruminal pH (Aschenbach et al. 2009). The pH under this experiment agrees with other studies of Van Soest (1994) and Phupaboon et al. (2022b, 2023), reported the optimal ruminal pH of 6.2–7.2 for microbial activity especially cellulolytic and/or proteolytic bacteria to degrade fibre and protein for increasing carbon and nitrogen sources for their microbiota growth under in vitro fermentation study. According to Wanapat (2000), who reported that the supplementation of feedstuffs containing strategic CT and SP has the potential to improve rumen efficiency by sustaining a higher pH, enhancing NH₃-N concentration, and augmenting microbial protein synthesis: the mode of action remains unidentified. In addition, IVDMD at 12 and 24 h post-fermentation was influenced by increasing dose of mLEMANGOS supplementation at 6% of total DM substrate, which was significantly higher than MNS supplementation that was slowly affected after 24 h of fermentation. This phenomenon could be attributed to a concomitant rise in microbial populations (mLEMANGOS supplied crucial nutrients to enhance ruminal microbial activity), potentially leading to enhanced degradability of nutrients. The results are similar to those studies of Matra et al. (2021, 2024), which showed that adding dragon fruit feel extract or Mitragyna leaf-based oil extract increased IVDMD at 12 and 24 h after fermentation. This was because the supplements and roughage sources had more phytonutrients (e.g. CT and SP components), which could help the microbes break down more feeds.

Under this study, NH₃-N concentration at 12 and 24 h was increased when the R:C ratio × mLEMANGOS, mLEMANGOS × MNS, and R:C ratio × mLEMANGOS × MNS supplementations were increased. The current finding accords with our earlier observations, which conclude that CP content degradable from the concentrate and microencapsulated, with respective percentages of 14.6% and 18.6%, together with the utilisation of urea as a non-protein nitrogen source, it has been observed to have a favourable impact for increasing the NH₃-N concentration during in vitro fermentation (Wanapat 2000; Ampapon et al. 2019). Moreover, the study conducted by Wanapat and Pimpa (1999) focused on the NH₃-N concentration in rumen fluid. This concentration is influenced by various factors, including NH₃-N synthesis from the feed, protein breakdown, absorption through the rumen wall, passage out of the rumen, and utilisation by microorganisms. Interestingly, this data obtained from NH₃-N concentration correlation is related to increasing dynamics of rumen microbial population, particularly R. albus, R. flavefaciens, F. succinogens, M. elsdenii, and B. fibrisolvens based on phytonutrient values retained in mLEMANGOS. These findings further support the idea of typical concentration of rumen NH₃-N for the dietary treatments was 19.6 mg%, which is within the recommended range of 15 to 30 mg% NH₃-N for promoting microbial growth in tropical ecology environments (Wanapat and Pimpa 1999; Gunun et al. 2016).

Consortia of rumen microbial population on methane (CH₄) production

The ruminants depend on a symbiotic relationship with a diverse community of microbes in the anaerobic environment of the rumen to facilitate the breakdown of complex nutrients such as carbohydrates, proteins, and some organic polymers into their constituent monomers. The monomers undergo fermentation, resulting in the production of end-products including volatile fatty acids (VFAs), free ammonia (NH₃), carbon dioxide (CO₂), and hydrogen (H₂). Additionally, the CO₂ and H₂ gases are transformed into CH₄ by the activity of methanogens, including Methanopyrales, Methanocellales, and Methanomicrobiales (Paul et al. 2012). Therefore, it is imperative to develop cost-effective strategies for moderating CH₄ emissions while simultaneously promoting efficient energy utilisation. The phytonutrient of TPC content like CT serve as rumen modifiers, and there have been four proposed mechanisms for how CT effectively suppress CH₄ production; (i): Methanogens are directly affected by these chemicals (Díaz Carrasco et al. 2017); (ii): the protozoal group is influenced by CT (Bhatta et al. 2009); (iii): CT serve as a hydrogen sink (Becker et al. 2014); and (iv): they have an impact on reducing fibre degradation (Carulla et al. 2005). In the current investigation, the amount of Methanobacteriales was reduced based on the phytonutrients, TPC and TFC contents followed by antioxidative capacities, DPPH, ABTS, and FRAP activity (as shown in Table 1) from the supplementation of mLEAMANGOS level to the diet clearly depressed production of CH₄ when compared with 20% MNS supplement. Several studies have indicated that the inclusion of tropical fruit peels, such as mangosteen peel (Wanapat et al. 2013; 2014; Polyorach et al. 2016) and rambutan peel and/or Mao (Antidesma thwaitesianum Muell. Arg.) (Gunun et al. 2016; Ampapon et al. 2019), in the diets of ruminants can lead to the reduction of CH₄ emissions. These peels are known to include phytonutrients, including CT and SP, which have been found to have an inhibitory effect on CH₄ production. Additionally, the findings of the current study are consistent with those of Matra et al. (2024), who found that the effect of feed supplementation of microencapsulated Mitragyna leaves-based oil extracts (mMLOE) was added, in vitro dry matter degradability rose considerably at 12, 24, and 48 h, while their supplementation boosted the NH₃-N concentration. When supplemented with 6% of the total DM substrate, the mMLOE produced the most propionate and total volatile fatty acids while producing less CH₄ (12, 24, and 48 h). Furthermore, with mMLOE feeding, the consortia of microbial rumen population of cellulolytic bacteria and B. fibrisolvens grew while that of Methanobacteriales declined with potential alternative plant/EOs-based bioactive compound supplements to be used as ruminant feed additives.

Conclusions

Microencapsulated LEMANGOS supplementation, which are made by combining lemongrass oil extract with mangosteen peel extract, exhibit a high concentration of phytochemical and phytonutrient components including the TPC (e.g. CTs), TFC (e.g. SPs), and demonstrate the significant antioxidant potential. In conclusion, the mLEMANGOS at a concentration of 6% of the total DM substrate, together with an R:C ratio of 60:40, led to enhancements in the in vitro rumen fermentation, higher fermentation end-products, enhancement of microbial protein synthesis, as well as, reducing methanogen population and CH₄ production. Hence, the mLEMANGOS has a notable impact on the mechanism of antibiotic action, particularly in comparison to MNS. This finding suggests the potential use of the mLEMANGOS as a supplementation of antibiotics to increase fermentation efficiency and to mitigate rumen CH₄ emission.

Ethics approval

The experiment relevant to rumen fluid collection was officially agreed upon and approved by the Khon Kaen University Committee of Animal Care and Use for Research (recode no. IACUC-KKU-86/66).

Author statement

Srisan Phupaboon: Conceptualisation, Methodology, Investigation, Data curation, analysis, Supervision, Writing—original draft & editing; Maharach Matra: Conceptualisation, Investigation, Data curation & analysis; Sukruthai Sommai: Methodology, Investigation; Gamonmas Dagaew: Methodology, Investigation; Chaichana Suriyapha: Methodology, Investigation; Rittikeard Prachumchai: Methodology, Investigation; Metha Wanapat: Conceptualisation, Resources, Supervision, Writing—Review & editing. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no completing interests.

Acknowledgements

The authors would like to express sincere thanks to Professor Dr. Peter Rowlinson Independent Animal Science Consultant his in valuable suggestion to improve the manuscript and to the Tropical Feed Resources Research and Development Center (TROFEC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand for providing the research facilities and facilitation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Additional information

Funding

This research was supported by the Program Management Unit Human & Resources Institutional Development Research and Innovation (PMU-B) (PMU project no. 660000050309).