AAVS_14_3_553-566

Introduction

Agricultural waste is now recognized as a global issue, affecting the environment, the economy, and food security (FAO, 2019). Following the concept of a circular bioeconomy, fruit and vegetable waste can be converted into valuable resources, such as animal feed, thereby reducing emissions and feed costs (Sahoo et al., 2021). Utilizing fruit and vegetable waste as ruminant feed offers a dual solution: Increasing nutrient supply while reducing the environmental burden of organic waste. Several studies have confirmed that fruit and vegetable waste fractions are rich in readily fermentable carbohydrates, fiber, minerals, vitamins, and phytochemicals, which can support rumen fermentation both after drying and during the ensiling process (Nath et al., 2023; Dou et al., 2022). In vitro, the use of fruit and vegetable waste exhibits a favorable fermentation profile and the potential to reduce emissions, indicating that horticultural waste has the potential to be developed as a sustainable alternative feed ingredient (Giller et al., 2022; Flanagan et al., 2024).

In addition to the solid fraction, fermentation broth (FB) is the liquid fraction produced during the fermentation of fruit and vegetable waste and contains dissolved sugars, minerals, and bioactive compounds such as flavonoids and polyphenols that may modulate the rumen microbiota (Vasta et al., 2019; Salminen et al., 2021; Ogbuewu et al., 2023). In practical on-farm fermentation systems, FB is often produced using mixed organic substrates to ensure adequate microbial activity, buffering capacity, and fermentable energy, which may include non-plant ingredients as fermentation starters or nutrient enhancers. Research by Nurnaningsih et al. (2025) demonstrated that the addition of FB from fruit and vegetable waste can impact rumen metabolites, thereby offering potential as a feed additive. The technological aspect of food waste ensilage feed is seen as promising for maintaining the availability of easily digestible substrates in liquid form, although variability in composition and water content needs to be managed (Hansen et al., 2025). Physiologically, the success of FB is largely determined by the synchronization of rapid energy supply and nitrogen degradation in the rumen, which enables optimal microbial growth and nutrient utilization efficiency (Zhang et al., 2020).

In the present study, the FB was produced using a complex formulation that included fruit and vegetable waste as the primary carbon source, while ingredients such as coconut water, cassava yeast, and fish gut waste were incorporated to initiate fermentation, supply soluble nitrogen, and enhance microbial activity. The rationale for this formulation was not to evaluate each ingredient individually, but to simulate a realistic small-scale waste fermentation system commonly practiced in tropical regions, where mixed organic wastes are co-fermented to stabilize the process and improve metabolite yield. The FB was formulated to reflect practical, mixed-waste systems, acknowledging that this introduces complexity. Therefore, we characterized the net metabolite profile and interpret effects at the functional level.

Rice straw has nutritional limitations, including a high cell wall fiber content, lignification, and low crude protein, which limit its digestibility and negatively impact livestock performance (Sarnklong et al., 2010). Various chemical and biological treatments have been developed to improve these conditions, including breaking the lignin–hemicellulose ester bond, increasing the formation of ammonia-bound nitrogen, and enhancing the availability of the fiber fraction, ultimately improving digestibility both in vitro and in vivo (Gunun et al., 2013). However, the release of nitrogen from urea must be balanced with a rapid energy supply for optimal nutrient utilization efficiency (Ma et al., 2020). Therefore, combining an energy source in the form of fermentation broth derived predominantly from fermented fruit and vegetable waste with ammoniated rice straw has the potential to create synergy by providing rapidly available energy to rumen microbes when ammonia nitrogen is released, while simultaneously improving the utilization of the straw fiber fraction.

The direct use of fermentation broth as a ruminant feed additive remains relatively underexplored compared to the use of solid fractions or ensilages (Lee et al., 2023). On the other hand, although ammoniated rice straw has been shown to improve digestibility, several studies emphasize that when ammoniated straw is the primary feed, supplementation strategies are still necessary to balance nutrient supply and stabilize the rumen ecosystem (Gunun et al., 2013; Ma et al., 2020). Therefore, this study aimed to determine the bioactive compound profile of fermentation broth and to evaluate its effects as an additive to ammoniated rice straw on in vitro using sheep rumen fluid.

Materials and Methods

Study location and experimental design

This research was conducted in the Animal Nutrition Laboratory of the Faculty of Animal Science at Universitas Jenderal Soedirman for the in vitro analysis. The rumen fluid was collected from a slaughtered lamb at the Sokaraja slaughterhouse (RPH), Department of Fisheries and Husbandry, Banyumas Regency.

The experimental design of this study was a 2 x 3 factorial (Table 1) randomized design with five replications. The first factor is the volume of fermentation broth added, and the second factor is the type of rice straw. The treatment details were as follows:

FB0= without adding FB
FB300= addition of 300 µL FB
FB600= addition of 600 µL FB
JP= non-ammoniated rice straw
JPa= ammoniated rice straw

Preparation of fermentation broth

The inoculum is composed of vegetable and fruit waste, coconut sugar, and clean water (a 1:10 ratio). The inoculum is prepared in a 150-liters plastic container. A total of 30 kg of vegetable and fruit waste, consisting of 0.67% water spinach, 0.67% papaya, 3.33% orange, 8.67% pineapple peel waste, 13.33% starfruit, 6.67% brown sugar, and 66.67 liters of clean water, is used. The fermentation broth is prepared in a 40-liter container. The required ingredients include: 12.47% fish gut waste (without gallbladder and feces); 37.41% coconut water; 0.25% fermented cassava yeast; 24.94% young sweet corn; 12.47% GMT (sweet cane sugar); and 12.47% inoculum. The ingredients are fermented anaerobically for three months at room temperature, protected from rain or direct sunlight. After three months, the barrel is opened, and the fermented liquid is filtered to separate the liquid from the sediment. The liquid (fermentation broth) is then bottled and stored in the refrigerator until use. The visual figure of the FB preparation workflow is shown in Figure 1.

Table 1: Experimental layout of the 2 × 3 factorial design consisting of two rice straw types.

Straw type

FB0 (0 µL)

FB300

(300 µL)

FB600

(600 µL)

JP (Non-ammoniated)

JP–FB0

JP–FB300

JP–FB600

JPa (Ammoniated)

JPa–FB0

JPa-FB300

JPa–FB600

Note: rice straw types (JP = non-ammoniated, JPa = ammoniated) and three levels of fermentation broth (FB0 = 0 µL, FB300 = 300 µL, FB600 = 600 µL) with five replications per treatment.

Rumen fluid preparation

Rumen fluid is collected directly from sheep using a stomach tube with the aid of a vacuum pump. The rumen fluid is then filtered through gauze, and the filter is collected in a beaker immersed in warm water (40 °C) and supplied with CO2 gas. The time between rumen fluid collection and use is a maximum of two hours under anaerobic conditions and warm temperatures (40 °C).

In vitro fermentation procedure

The in vitro fermentation was performed using the standard batch culture procedure described by Tilley and Terry (1963). FB was pipetted directly into each fermentation tube in addition to the 40 mL McDougall’s buffer and 10 mL rumen fluid, resulting in final FB inclusion rates of 0, 0.6, and 1.2% (v/v) for the FB0, FB300, and FB600 treatments, respectively, with a negligible effect on total incubation volume (<1.2%). The mixture was then subjected to CO2 gas for 30 seconds to create an anaerobic environment. The tube was sealed with a vented rubber stopper and incubated for 48 hours at 39°C in a water bath shaker. During incubation, the pH was checked after 4 hours. After 48 hours, the stopper was removed, and 1 mL of 5% HgCl2 was added. The fermenter tube and mixture were centrifuged for 15 minutes. Afterward, 50 mL of pepsin-HCl solution was added, and the tube was incubated for a second time at 39°C in a shaker water bath. The results obtained from the filtering process are in the form of sediment and supernatant. The visual figure of the FB preparation workflow is shown in Figure 2.

Observation parameters

The sediment is the residue from which nutrient digestibility will be measured. Four parameters were analyzed: Identification of bioactive compounds, digestibility, NH3 concentration, and volatile fatty acid (VFA) concentration. The measurements were as follows:

Identification of bioactive compounds in fermentation broth. The identification of bioactive compounds in vegetable and fruit waste was conducted using liquid chromatography-mass spectrometry (LC-MS) with a Shimadzu 8040, Japan. LC–MS analysis was untargeted and semi-quantitative, and the percentages reported represent relative ion intensities, not absolute concentrations.
Digestibility: In vitro digestibility tests were performed for dry matter (DM), organic matter (OM), crude protein (CP), crude fiber (CF), and crude fat (CFa) according to the procedure described by Tilley and Terry (1963).
NH3 Concentration: NH3 concentration was measured using the Conway method (General Laboratory Procedures, 1966), and N-NH3 concentration using the formula:

Volatile Fatty Acid (VFA) Concentration. VFA concentration was measured using the steam distillation method (General Laboratory Procedures, 1966).

Statistical analysis

The LCMS results of the study are presented in a descriptive analysis. Residuals were tested for normality (Shapiro-Wilk test) and Homogeneity of variances (Levene’s test) before ANOVA, and that no major violations were detected. Digestibility data, NH3 concentrations, and VFA were analyzed using analysis of variance (ANOVA). If differences between treatments were found, Duncan’s test was performed using Genstat version 12.0.

Results

Bioactive characterization of fermented broth from vegetable and fruit waste

The three major bioactive components of fermented broth from vegetable and fruit waste were identified using LC-MS analysis, and the resulting compounds are presented in Table 2 and Figure 3. LC-MS analysis was untargeted and semi-quantitative. The percentages reported represent relative ion intensities and should not be interpreted as absolute concentration percentages. The analysis identified 122 different compounds (Supplementary Table 1), dominated by flavonoids (26.7%), followed by sugars/oligosaccharides (16.8%), amino acids (10.4%), organic acids (9.4%), phenolic acids (8.6%), catechins (6.6%), anthocyanins (4.0%), triterpenoids (3.0%), sesquiterpene terpenoids (2.5%), sterols (1.5%), and others (9.5%).

The flavonoid group in fermented broth from vegetable and fruit waste consisted of kaempferol (2.603%), myricetin (2.485%), and quercetin (1.778%), along with various glycosides. Phenolic acids are composed of gallic acid (1.815%), caffeic acid (1.630%), ellagic acid (1.488%), and chlorogenic acid (1.232%). Catechins consist of epigallocatechin (1.567%) and epigallocatechin gallate (1.210%). Anthocyanins produced from the fermentation process, such as malvidin, cyanidin, and delphinidin, contribute to the natural red-purple color of the broth. The presence of flavonoids, phenolic acids, catechins, and anthocyanins indicates that fermented vegetable and fruit waste broth has antioxidant activity.

Table 2: Three major bioactive compounds found groups in fermentation broth.

Peak	Bioactive compound	%	RT min	Group	Peak	Bioactive compound	%	RT_min	Group
1	Glycine	0.599	1,049	Amino acid	33	Rhamnetin	1.208	12,001	Flavonoid
2	Alanine	0.756	1,158	Amino acid	34	Melibiose	0.539	12,291	Oligosaccharides
3	Serine	0.309	1,204	Amino acid	35	Trehalose	0.628	12,293	Oligosaccharides
4	Proline	0.480	1,232	Amino acid	36	Isovitexin	1.205	21,385	Flavonoid
5	Valine	0.658	1,241	Amino acid	37	Kaempferol -3-O-rhamnoside	1.395	21,429	Flavonoid
6	Threonine	0.605	1,248	Amino acid	38	Quercetin -3-arabinoside	0.696	21,436	Flavonoid
7	Cystein	0.147	1,286	Amino acid	39	Dihydrokaem-pferol -3-O-α-L-	1.198	21,453	Flavonoid
8	Arabinose	0.766	1,602	Oligosacc-harides	40	Quercetin -3-O-rhamnoside	1.706	22,616	Flavonoid
9	Isoleucine	0.725	1,752	Amino acid	41	Luteolin -7- glucoside	0.830	22,628	Flavonoid
10	Leucine	1.041	1,766	Amino acid	42	Myricitrin	1.019	24,119	Flavonoid
11	Asparagine	0.629	1,773	Amino acid	43	Maltotriose	0.630	26,302	Oligosaccharides
12	Glutamine	0.105	2,204	Amino acid	44	1-kestose	1.277	26,303	Oligosaccharides
13	Lysine	0.705	2,228	Amino acid	45	Raffinose	0.827	26,305	Oligosaccharides
14	Methionine	0.423	2,538	Amino acid	46	Inulotriose	1.496	26,309	Oligosaccharides
15	Xylose	0.829	2,539	Oligosacc-harides	47	Kaempferol -3-O-(6-	0.744	30,865	Flavonoid
16	Histidine	0.641	2,598	Amino acid	48	Quercetin -3- O-(6-	0.996	31,822	Flavonoid
17	Rhamnose	0.630	2,649	Oligosacc-harides	49	Kaempferol -7-rhamnoside -4'-	1.209	34,003	Flavonoid
18	Phenylalanine	0.512	2,687	Amino acid	50	Quercetin -3-glucoside -7-	0.744	35,511	Flavonoid
19	Arginine	1.310	3,094	Amino acid	51	Rutin	0.310	35,517	Flavonoid
20	Fructose	2.066	4,647	Oligosacc-harides	52	Isorhamnetin -3-O-rutinoside	0.535	36,852	Flavonoid
21	Glucose	1.488	4,709	Oligosacc-harides	53	Quercetin -3,7-diglucoside	0.859	36,872	Flavonoid
22	Tyrosine	0.557	4,745	Amino acid	54	Isorhamnetin -3,4' diglucoside	0.744	38,017	Flavonoid
23	Tryptophan	0.204	5,485	Amino acid	55	Inulotetraose	1.246	43,21	Oligosaccharides
24	Γ-glutamyl alanine	0.405	6,702	Peptide	56	Nystose	1.128	43,213	Oligosaccharides
25	Luteolin	1.448	10,265	Flavonoid	57	Stachyose	0.894	46,176	Oligosaccharides
26	Kaempferol	2.603	10,322	Flavonoid	58	1(F)-α-D-galactosylraffinose	1.491	46,18	Oligosaccharides
27	Glutathione	0.068	10,511	Peptide	59	Quercetin -3,7,4' -triglucoside	0.650	49,892	Flavonoid
28	Kaempferide	1.205	11,021	Flavonoid	60	1F-fructofurano-sylnystose	0.839	49,954	Oligosaccharides
29	Rhamnocitrin	1.138	11,024	Flavonoid
30	Quercetin	1.778	11,427	Flavonoid
31	Myricetin	2.485	11,514	Flavonoid
32	Glutamyl tyrosine	0.099	11,55	Peptide

Total sugars/oligosaccharides reached 16.8%, with the main simple sugars being fructose (2.066%) and glucose (1.488%). Fructooligosaccharides (FOS) such as 1-kestose, nystose, 1F-fructofuranosylnystose, inulotriose, and inulotetraose were also found. These findings suggest the potential of fermented vegetable and fruit waste broth as a prebiotic that supports the growth of beneficial microbiota. Twenty-three amino acids were produced, with the highest concentrations being arginine (1.310%), leucine (1.041%), isoleucine (0.725%), lysine (0.705%), and valine (0.658%). Fifty-three percent of the resulting amino acid fraction was essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). Peptides such as γ-glutamyl-alanine and glutathione, which have potential as cellular antioxidants, were also identified.

Minor lipid components produced from vegetable and fruit waste broth fermentation liquid include triterpenoids (lupeol, betulin), sesquiterpene terpenoids (β-caryophyllene/epoxide), sterols (β-sitosterol, stigmasterol), polyols (myo-inositol), phenolics (biflorin/isobiflorin), and aromatic esters (benzyl benzoate). These compounds, although small in number, contribute to the aroma, bioactivity, and stability of the resulting fermentation broth.

In vitro digestibility

The level of fermentation broth (FB) significantly affected (P < 0.001) all in vitro digestibility parameters of rice straw (Table 3). Increasing FB supplementation progressively improved dry matter (DM), organic matter (OM), crude protein (CP), crude fiber (CF), and crude fat (CFa) digestibility. Digestibility values increased markedly from 0 to 300 µL FB and reached their highest levels at 600 µL FB, with digestibility of DM, OM, CP, CF, and CFa attaining 63.47%, 71.97%, 64.97%, 63.66%, and 70.87%, respectively. These values were significantly higher (P < 0.001) than those observed in the control treatment (0 µL FB), indicating a strong positive response to the inclusion of fermentation broth in terms of fiber degradation and nutrient availability.

Table 3: Effect of the amount of fermentation broth and type of rice straw on in vitro digestibility (%).

Treatment		Digestibility
Treatment		DM	OM	CP	CF	CFa
FB (μL)
0		42.77a	51.76a	51.80a	39.22a	50.01a
300		54.19b	59.92b	59.90b	49.36b	56.06b
600		63.47c	71.97c	64.97c	63.66c	70.87c
Rice straw
Non-ammoniated		48.20a	57.17a	55.83a	45.19a	55.76a
Ammoniated		58.75b	65.26b	61.96b	56.30b	62.19b
Interaction
FB	Rice Straw
0	Non-ammoniated	36.27	44.38a	51.76a	36.61a	47.26
300	Non-ammoniated	47.68	57.13b	56.75b	41.58b	51.22
600	Non-ammoniated	60.66	70.01d	58.96b	57.38c	68.80
0	Ammoniated	49.26	59.15bc	51.84a	41.82b	52.76
300	Ammoniated	60.69	62.71c	63.05c	57.14c	60.87
600	Ammoniated	66.28	73.92e	70.98d	69.94d	72.97
SEM		2.23	1.83	1.26	2.18	1.79
P values
FB		<0.001	<0.001	<0.001	<0.001	<0.001
Rice Straw		<0.001	<0.001	<0.001	<0.001	<0.001
FB x Rice Straw		0.430	<0.001	<0.001	<0.001	0.051

Note: Different superscripts in the same column indicate differences between treatments; FB = Fermentation Broth, DM = Dry Matter, OM = Organic Matter, CP = Crude Protein, CF = Crude Fiber, CFa = Crude Fat

Rice straw ammoniation also significantly enhanced (P < 0.001) the digestibility of all nutrients compared with non-ammoniated straw. Ammoniated straw exhibited higher DM (58.75 vs 48.20%), OM (65.26 vs 57.17%), CP (61.96 vs 55.83%), CF (56.30 vs 45.19%), and CFa digestibility (62.19 vs 55.76%). Significant FB × rice straw interactions were observed for OM, CP, and CF digestibility (P < 0.001), indicating that the beneficial effect of FB was more pronounced in ammoniated straw. The highest overall digestibility was recorded in the combination of 600 µL FB with ammoniated straw, yielding OM, CP, CF, and CFa digestibility values of 73.92%, 70.98%, 69.94%, and 72.97%, respectively, demonstrating a synergistic effect between ammoniation and fermentation broth supplementation on nutrient utilization.

In vitro NH3 and VFA concentrations

NH₃ concentration showed a numerically lower value in FB-treated groups, but the effect of FB and straw type on NH₃ was not statistically significant (P>0.05). Therefore, these data should be interpreted as a non-significant decreasing trend rather than a confirmed reduction. However, adding 600 μL of fermentation broth to ammoniated straw reduced NH₃ concentration by 24.51. In contrast, VFA concentrations (including acetic, propionic, and butyric acids) increased markedly (P<0.001) with higher amounts of fermentation broth, and ammoniated straw consistently produced higher VFA levels than non-ammoniated straw. A significant interaction (P<0.001) between fermentation broth volume and straw type was observed for VFA production, with the highest concentrations (132.43 acetic, 110.82 propionic, and 3.06 butyrate) obtained from ammoniated straw supplemented with 600 μL of fermentation broth. Overall, increasing the amount of fermentation broth in both ammoniated and non-ammoniated rice straw reduced NH₃ concentration while enhancing VFA production, as shown in Table 4.

Table 4: Effect of adding fermentation broth to rice straw on NH3 and VFA in vitro.

Treatments		NH3	VFA
Treatments		NH3	Acetate	Propionate	Butyrate
FB (μL)
0		29.51	81.85a	49.22a	1.69a
300		27.93	96.08b	77.70b	1.91a
600		25.24	122.92c	90.34c	2.74b
Rice straw
Non amoniasi		27.75	78.85a	58.98a	1.93a
Amoniasi		27.37	121.71b	85.87b	2.29b
Interaction
FB	Rice straw
0	Non-ammoniated	29.34	49.40a	43.78a	1.54a
300	Non-ammoniated	27.94	73.75b	63.29c	1.84b
600	Non-ammoniated	25.96	113.41c	69.86d	2.41d
0	Ammoniated	29.68	114.30c	54.67b	1.83b
300	Ammoniated	27.92	118.41d	92.12e	1.99c
600	Ammoniated	24.51	132.43e	110.82f	3.06e
SEM		1.21	5.38	4.22	0.09
P values
FB		0.402	<0.001	<0.001	<0.001
Rice Straw		0.884	<0.001	<0.001	<0.001
FB x Rice Straw		0.956	<0.001	<0.001	<0.001

Note: Different superscripts in the same column indicate differences between treatments; FB = Fermentation Broth, VFA = Volatile Fatty Acid

Discussion

Fermentation broth (FB), produced from vegetable and fruit waste, contains diverse bioactive compounds including flavonoids, sugars/oligosaccharides, amino acids, organic acids, phenolic acids, catechins, anthocyanins, triterpenoids, terpenoids, and sterols. These metabolites contribute antioxidant activity and demonstrate potential to stabilize the rumen ecosystem, enhance nitrogen utilization (Hristov et al., 2010), improve fermentation and VFA production (Izuddin et al., 2018), mitigate methane (Doyle et al., 2019), and support antimicrobial and digestive functions (Guo et al., 2020). However, because the LC–MS analysis used in this study was untargeted and semi-quantitative, the detected metabolites represent a compositional profile rather than a precise quantification of compounds entering the fermentation system. Therefore, the present study demonstrates the net functional effect of a complex, multi-component fermentation broth rather than the causal effect of specific isolated metabolites. During fermentation, lactic acid bacteria enzymatically degrade plant cell walls and glycosidic bonds, increasing the release of phenolic compounds and flavonoids (Li et al., 2018; Putra et al., 2023), with flavonoids comprising 26.7% of FB components (Table 2). Fermentation also increases amino acids, peptides, and organic acids via protein breakdown and microbial metabolism (Hughes et al., 2014; Wang et al., 2022), producing 23 amino acids and elevated levels of organic acids such as glutamic and lactic acids (Islam et al., 2020). Anaerobic microbial action further produces VFAs such as acetate and propionate (Li et al., 2023; Somers et al., 2018), along with citric acid, oligosaccharides, and aromatic compounds like terpenoids and esters (Jia et al., 2023; Khandaker et al., 2021), enriching FB with functional metabolites beneficial for livestock.

It is important to emphasize that the present study demonstrates the net functional effect of a complex, multi-component fermentation broth rather than the effects of individual metabolites. Fermentation broths and postbiotic products typically consist of heterogeneous mixtures of organic acids, sugars, amino acids, phenolics, minerals, and microbial metabolites that may act synergistically within the rumen ecosystem. Recent reviews on postbiotics and fermentation-derived products in ruminant nutrition highlight the difficulty of attributing observed responses to single molecules within such complex matrices, and instead recommend interpreting outcomes at the functional or system level (Marlida et al., 2024).

Accordingly, improvements in digestibility and VFA production observed in this study are best interpreted as system-level responses to the provision of a readily fermentable liquid substrate enriched with organic acids, sugars, amino acids, and microbial metabolites, rather than as evidence for specific actions of flavonoids or oligosaccharides. It is plausible that FB acted primarily as an additional source of rapidly available carbon and metabolic intermediates, stimulating microbial growth and fermentative activity and thereby increasing total VFA production and fiber degradation efficiency.

The addition of 600 μL FB significantly increased feed digestibility and VFA production (P < 0.001), whereas NH₃ concentration showed only a non-significant decreasing trend (Tables 3 and 4). This pattern indicates that the dominant physiological response was enhanced carbon fermentation rather than demonstrable improvement in nitrogen capture into microbial biomass. FB likely provided readily fermentable carbon sources and organic acids that stimulated microbial growth and metabolic activity, resulting in higher total VFA production. Similar enhancements in VFA production without marked reductions in NH₃ concentration have been reported for postbiotic fermentation products and plant-derived additives evaluated in vitro (Guo et al., 2022).

Previous studies have reported that flavonoids and phenolic compounds may reduce the negative effects of tannins and modulate rumen microbial populations, potentially favoring VFA production and reducing NH₃ accumulation (Zhan et al., 2017; Abid et al., 2023). Organic acids stimulate fibrolytic activity by reducing rumen pH (Nayohan et al., 2024) and support microbial protein synthesis (Bernard et al., 2004). Meanwhile, amino acids provide readily available nitrogen for microbial growth (Harmon and Swanson, 2020), thereby jointly improving digestibility. However, in the present study, such compound-specific mechanisms were not directly evaluated. Therefore, these reported effects should be interpreted as contextual evidence from the literature rather than as confirmed causal mechanisms for the responses observed herein.

The non-significant change in NH₃ concentration despite increased digestibility and VFA production suggests that the primary physiological response to FB supplementation was enhancement of fermentable carbon metabolism rather than a demonstrable improvement in nitrogen capture into microbial biomass. In in vitro systems where baseline NH₃ availability is relatively high, stimulation of carbon fermentation can substantially increase VFA output while producing only modest changes in the NH₃ pool, due to the simultaneous occurrence of deamination and ammonium assimilation. Accordingly, improved nitrogen utilization in the present study should be regarded as a plausible but unconfirmed hypothesis, because microbial biomass or microbial protein synthesis was not directly measured. Oligosaccharides selectively stimulate fermentative bacteria that efficiently convert them into VFAs (Zhu et al., 2020), helping maintain a favorable rumen pH and improving microbial survival (Suningsih and Sadjadi, 2020). Organic acids regulate microbial balance, increase VFA production, and inhibit pathogenic microbes (Saro et al., 2020). Amino acids enhance microbial protein synthesis and nitrogen utilization efficiency (Tan et al., 2017), thereby contributing to reduced NH₃ emissions and improved digestibility.

Ammoniated straw also significantly increased digestibility and VFA production (P<0.001) (Table 4). Ammoniation alters the lignocellulose structure by breaking lignin–hemicellulose bonds, decreasing NDF, and enhancing microbial accessibility (Ilham et al., 2018; Datsomor et al., 2022). This structural modification enhances nutrient availability and improves fermentation end products, such as VFAs (Bata et al., 2021). Additionally, urea-based ammoniation reduces NH₃ accumulation by enhancing nitrogen capture for microbial protein synthesis, promoting a more balanced rumen metabolism, and supporting more sustainable feeding systems. Overall, the most robust and consistent finding of this study is the improvement in feed digestibility and VFA production following FB supplementation. Effects on nitrogen utilization should be interpreted cautiously and are limited to a non-significant trend in NH₃ concentration under the present experimental conditions.

A significant interaction (P<0.001) between FB and straw type demonstrated synergistic improvements in digestibility and VFA production. Ammoniation increases straw porosity and exposes cellulose and hemicellulose surfaces (Datsomor et al., 2022), while organic acids in FB (lactic, acetic, citric) promote ester hydrolysis that further breaks ferulic acid linkages within the fiber matrix (Tarasov et al., 2018). These combined effects enhance dry matter and organic matter digestibility, as also supported by studies showing improved degradability with fermentation extracts in high-fiber feeds (Li et al., 2022, 2023; Wang et al., 2024).

Improvements in crude protein digestibility (KcPK) arise from the increased nitrogen content following ammoniation (Ma et al., 2020) and the activity of lactobacilli proteinases that hydrolyze proteins into oligopeptides (Oskoueian et al., 2021). Increased microbial activity accelerates fiber breakdown and boosts VFA production, which enhances nutrient absorption (Rosmalia et al., 2022). The synergy between fermentable sugars and organic acids in rice straw, combined with nitrogen from ammoniation, results in more efficient NH₃ utilization for microbial protein synthesis, as reflected in increased KcPK and reduced NH₃. Ammoniation also increases crude fiber digestibility by reducing lignin content (Ma et al., 2020), and FB bacteria promote further degradation of complex carbohydrates (Zhang et al., 2020). Combined ammonia-microbial treatments similarly improve NDF and ADF digestibility (Ningrat et al., 2019), while increased lipid accessibility and microbial lipase activity enhance crude fat digestibility (Kholif, 2019).

The superior digestibility and VFA production observed when FB was combined with ammoniated straw are therefore most plausibly explained by a complementary interaction between enhanced substrate accessibility caused by ammoniation and increased availability of rapidly fermentable substrates supplied by FB. This synergistic response is best interpreted as a structural–energetic interaction rather than as selective stimulation by specific secondary metabolites (Ma et al., 2020; Zhang et al., 2025). FB bioactive compounds stimulate lactate-utilizing bacteria, enhancing propionate production and increasing overall VFA yield while suppressing NH₃ accumulation (Cabral and Weimer, 2024).

In addition to organic metabolites, the complex composition of FB suggests that minerals may have contributed to the observed fermentation responses. Ingredients such as coconut water and fish–plant fermentation materials are known to contain appreciable levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, and trace elements, which can influence rumen osmolarity and microbial efficiency. Although the mineral composition of FB was not analyzed in the present study, previous work on food-waste-derived feeds suggests that elevated mineral content can exert either beneficial or adverse effects on rumen fermentation, depending on the dose and balance (Ominski et al., 2021). In this study, the relatively low inclusion rate of FB (1.2% v/v) and the absence of adverse effects on rumen pH, VFA production, or digestibility suggest that osmolarity remained within a tolerable range for microbial activity. Nevertheless, mineral profiling represents an important additional dimension for future FB formulations to optimize dosing strategies and avoid potential negative osmotic or ionic effects.

Overall, the most robust outcome of this study is the consistent improvement in feed digestibility and VFA production. It should be acknowledged that rumen fluid used in this in vitro study was obtained from a single lamb at a municipal slaughterhouse, which may limit the generalizability of the findings to broader ruminant populations. A single-donor inoculum may not fully represent the diversity of rumen microbial ecosystems, particularly when compared with pooled rumen fluid from multiple fistulated animals maintained on standardized diets. However, recent studies and reviews (Ma et al., 2021; Aprilia et al., 2021) suggest that rumen fluid collected from slaughtered animals can serve as an acceptable alternative inoculum for in vitro fermentation and digestibility studies when fistulated donors are unavailable, provided that sampling and handling are conducted promptly and consistently. Moreover, the use of a single donor allows reliable comparison of treatment effects within a uniform microbial background, which was the primary objective of this study. Nevertheless, future studies should combine pooled rumen fluid from multiple donors with targeted quantification of major fermentation broth metabolites (e.g., organic acids and flavonoids), as the absence of compound-specific concentration data in the present study limits the interpretation of dose-response relationships and the broader applicability of the findings.

Conclusion

FB derived from fruit–vegetable waste and complementary substrates improved in vitro digestibility and total VFA production of rice straw–based substrates, particularly when combined with ammoniated straw, indicating a synergistic effect on rumen fermentation. NH₃ concentrations showed only a non-significant decreasing trend, so our findings support enhanced carbon fermentation and a possible better synchronization of energy and nitrogen supply, but do not demonstrate a definitive optimization of nitrogen utilization. Further in vivo and mechanistic studies, including targeted metabolomics and microbial analyses, are required before recommending FB as a nitrogen-efficiency strategy in practice.

Acknowledgments

The authors thank the Animal Nutrition Laboratory of the Faculty of Animal Science at Universitas Jenderal Soedirman for providing the laboratory equipment for analysis and the Sokaraja slaughterhouse for permission to take the rumen fluid.

Novelty Statements

This study introduces a novel valorization strategy by converting fruit and vegetable waste into a metabolically characterized fermentation broth (FB) and systematically evaluating its synergistic interaction with ammoniated rice straw on rumen fermentation in vitro. Unlike previous studies that primarily assess either chemical pretreatment or additive supplementation independently, this research integrates LC–MS–based metabolite profiling with a factorial fermentation approach to elucidate how bioactive compound classes (e.g., flavonoids, oligosaccharides, and amino acids) modulate microbial activity, digestibility, and VFA production. The demonstrated interaction effect between FB and ammoniated straw provides new mechanistic insight into synchronized carbon–nitrogen fermentation and offers a sustainable strategy to enhance the nutritive value of low-quality crop residues.

Authors Contribution

WN: conducting experiments, collecting, analyzing, interpreting data, and writing the original manuscript draft. MB and SR: designed the study and supervised the project. EAR and CHP: reviewing and editing the manuscript.

Generative AI and AI-assisted technology statement

Generative AI tools were used solely to assist in language refinement, structural editing, and improvement of clarity in the manuscript. All experimental design, data analysis, interpretation of results, and scientific conclusions were developed independently by the authors. The authors take full responsibility for the accuracy, integrity, and originality of the content.

Conflict of interest

The authors have declared no conflict of interest.

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Supplementary Table 1: List of bioactive compounds found in fermentation broth.

Peak	Bioactive compound	%	RT min	Group	Peak	Bioactive compound	%	RT_min	Group
1	Acetic acid	1.219	1,039	Organic acid	62	Quercetin	1.778	11,427	Flavonoid
2	Propionic acid	0.987	1,04	Organic acid	63	Epigallocatechin	1.567	11,503	Catechins
3	Glycine	0.599	1,049	Amino acid	64	Myricetin	2.485	11,514	Flavonoid
4	Alanine	0.756	1,158	Amino acid	65	Glutamyl tyrosine	0.099	11,55	Peptide
5	Pyruvic acid	0.519	1,161	Organic acid	66	Rhamnetin	1.208	12,001	Flavonoid
6	Butyric acid	0.640	1,165	Organic acid	67	Melibiose	0.539	12,291	Oligosaccharides
7	Lactic acid	1.053	1,192	Organic acid	68	Trehalose	0.628	12,293	Oligosaccharides
8	Serine	0.309	1,204	Amino acid	69	Biflorin	1.086	12,417	Other
9	Valeric acid	0.308	1,206	Organic acid	70	Isobiflorin	0.836	12,42	Other
10	Proline	0.480	1,232	Amino acid	71	Chlorogenic acid	1.232	12,421	Phenolic acid
11	Fumaric acid	0.454	1,238	Organic acid	72	Stellasterol	0.243	14,402	Sterol
12	Valine	0.658	1,241	Amino acid	73	Β-sitosterol	0.381	17,163	Sterol
13	Succinic acid	0.404	1,246	Organic acid	74	Epiafzelechin -3-O-gallate	1.586	19,211	Catechins
14	Threonine	0.605	1,248	Amino acid	75	Friedelan -3-one	0.825	19,341	Other
15	α-aminobutyric acid	0.427	1,283	Organic acid	76	Lupeol	1.267	19,614	Triterpenoid
16	Cystein	0.147	1,286	Amino acid	77	Friedelinol	0.284	19,739	Triterpenoid
17	Pyroglutamic acid	0.069	1,466	Organic acid	78	Isovitexin	1.205	21,385	Flavonoid
18	Malic acid	0.232	1,473	Organic acid	79	Kaempferol -3-O-rhamnoside	1.395	21,429	Flavonoid
19	α -ketoglutaric acid	0.138	1,535	Organic acid	80	Quercetin -3-arabinoside	0.696	21,436	Flavonoid
20	Arabinose	0.766	1,602	Oligosaccharides	81	Dihydrokaempferol -3-O-α-L-	1.198	21,453	Flavonoid
21	Methyl salicylate	0.177	1,606	Other	82	Epicatechin gallate	1.148	22,183	Catechins
22	3,4-dihydroxybenzoic acid	1.086	1,664	Phenolic acid	83	Betulin	0.827	22,284	Triterpenoid
23	Isoleucine	0.725	1,752	Amino acid	84	Quercetin -3-O-rhamnoside	1.706	22,616	Flavonoid
24	Leucine	1.041	1,766	Amino acid	85	Luteolin -7-glucoside	0.830	22,628	Flavonoid
25	Asparagine	0.629	1,773	Amino acid	86	Cyanidin -3-O-glucoside	0.658	22,747	Anthocyanin
26	Aspartic acid	0.450	1,794	Organic acid	87	Betulinic acid	0.298	23,517	Triterpenoid
27	Glutamine	0.105	2,204	Amino acid	88	Epigallocatechin gallate	1.210	23,705	Catechins
28	Lysine	0.705	2,228	Amino acid	89	Myricitrin	1.019	24,119	Flavonoid
29	Glutamic acid	1.330	2,276	Organic acid	90	Delphinidin -3 glucoside	0.557	24,121	Anthocyanin
30	Methionine	0.423	2,538	Amino acid	91	Β-amyrin acetate	0.323	24,131	Triterpenoid
31	Xylose	0.829	2,539	Oligosaccharides	92	Quercituron	0.535	25,835	Other
32	Histidine	0.641	2,598	Amino acid	93	Cyanidin 3 -(6''-	0.692	25,942	Anthocyanin
33	Rhamnose	0.630	2,649	Oligosaccharides	94	Malvidin -3-glucoside	0.482	25,953	Anthocyanin
34	Phenylalanine	0.512	2,687	Amino acid	95	Maltotriose	0.630	26,302	Oligosaccharides
35	Gallic acid	1.815	3,042	Phenolic acid	96	1-kestose	1.277	26,303	Oligosaccharides
36	Arginine	1.310	3,094	Amino acid	97	Raffinose	0.827	26,305	Oligosaccharides
37	Ascorbic acid	0.198	3,166	Organic acid	98	Inulotriose	1.496	26,309	Oligosaccharides
38	Caffeic acid	1.630	4,643	Phenolic acid	99	Kaempferol -3-O-(6-	0.744	30,865	Flavonoid
39	Fructose	2.066	4,647	Oligosaccharides	100	Malvidin -3-(6''-	0.183	30,884	Anthocyanin
40	Glucose	1.488	4,709	Oligosaccharides	101	Quercetin -3-O-(6-	0.996	31,822	Flavonoid
41	Myo inositol	0.493	4,73	Poliol	102	Schaftoside	0.378	33,031	Other
						Table continues on next pages................
Peak	Bioactive compound	%	RT min	Group	Peak	Bioactive compound	%	RT_min	Group
42	Tyrosine	0.557	4,745	Amino acid	103	Stigmasterol -3-O-β-D-	0.271	33,422	Sterol
43	Citric acid	0.976	5,008	Organic acid	104	Β-sitosterol -D-glucoside	0.161	33,428	Sterol
44	Ferulic acid	1.373	5,043	Phenolic acid	105	Pelargonidin -3-p-	0.305	33,513	Anthocyanin
45	Tryptophan	0.204	5,485	Amino acid	106	Naringin	0.403	33,563	Other
46	β-caryophyllene	0.373	5,494	Terpenoid	107	Kaempferol -7-rhamnoside -4'-	1.209	34,003	Flavonoid
47	Benzyl benzoate	1.120	6,408	Terpenoid	108	Campesterol glucoside	0.433	34,013	Sterol
48	Γ-glutamyl alanine	0.405	6,702	Peptide	109	Cyanidin -3-(6-O-p-	0.814	34,037	Anthocyanin
49	Spathulenol	0.230	6,883	Terpenoid	110	Peonidin -3-(6''-p-	0.080	35,489	Anthocyanin
50	Β-caryophyllene epoxide	0.765	6,889	Terpenoid	111	Quercetin -3-glucoside -7-	0.744	35,511	Flavonoid
51	Epiafzelechin	1.109	9,783	Catechins	112	Rutin	0.310	35,517	Flavonoid
52	Luteolin	1.448	10,265	Flavonoid	113	Isorhamnetin -3-O-rutinoside	0.535	36,852	Flavonoid
53	Kaempferol	2.603	10,322	Flavonoid	114	Quercetin -3,7-diglucoside	0.859	36,872	Flavonoid
54	2',4' -dihydroxy -6'-methoxy -3'-	1.521	10,336	Other	115	Malvidin -3-(6''-p-	0.179	37,089	Anthocyanin
55	Glutathione	0.068	10,511	Peptide	116	Isorhamnetin -3,4' diglucoside	0.744	38,017	Flavonoid
56	4',6' -dihydroxy -3',5'-dimethyl -	1.655	10,517	Other	117	Inulotetraose	1.246	43,21	Oligosaccharides
57	7-hydroxy -5-methoxy -6,8-	0.971	10,519	Other	118	Nystose	1.128	43,213	Oligosaccharides
58	2',4' -dihydroxy -6'-methoxy -	1.086	11,017	Other	119	Stachyose	0.894	46,176	Oligosaccharides
59	Kaempferide	1.205	11,021	Flavonoid	120	1(F)-α-D-galactosylraffinose	1.491	46,18	Oligosaccharides
60	Rhamnocitrin	1.138	11,024	Flavonoid	121	Quercetin -3,7,4' -triglucoside	0.650	49,892	Flavonoid
61	Ellagic acid	1.488	11,401	Phenolic acid	122	1F-fructofuranosylnystose	0.839	49,954	Oligosaccharides