Amino acid‐montmorillonite nanocomposites: preparation, characteristics and biological effects on ruminal fermentation and greenhouse gas emission potential in diets exposed to aflatoxin B1 in vitro

Abstract

BACKGROUND

Modified clays demonstrate superior modulation of ruminal fermentation compared to natural clays and exhibit effects distinct from monensin, suggesting novel modes of action for methane (CH₄) mitigation. The objective of this study was to develop and evaluate the effectiveness of amino acid–montmorillonite nanocomposites (AA‐MNC) in modulating ruminal fermentation and mitigating methane production in diets contaminated with aflatoxin B1 (AFB1) by the in vitro gas production technique. Two AA‐MNC clays were developed by modifying natural montmorillonite (NM) with methionine and threonine, resulting in Met‐MNC and Thr‐MNC clays, respectively. The treatments consisted of –AFB1diet (basal substrate without supplementation), +AFB1 diet (‐AFB1diet at a final concentration of 20 ppb AFB1), NM diet [+AFB1 diet supplemented with NM at 5 g kg⁻¹ dry matter (DM)], monensin (+AFB1 diet supplemented with monensin at 40 mg kg⁻¹ DM), and Met‐MNC and Thr‐MNC supplemented to the +AFB1 diet at two levels: 0.5 g kg⁻¹ DM (low) and 1 g kg⁻¹ DM (high).

RESULTS

Both AA‐MNC clays showed increased surface area, cation exchange capacity, and narrow particle size distribution compared to NM clay. The +AFB1 diet decreased (P < 0.05) acid detergent fiber degradation compared with the other treatments. Both low and high levels of Met‐MNC and Thr‐MNC resulted in comparable reductions in CH₄ production. These reductions were similar to those observed with the monensin diet when compared with the –AFB1 and NM diets (P = 0.058). High levels of AA‐MNC decreased (P < 0.001) total short‐chain fatty acid concentrations relative to the NM and –AFB1 diets. By contrast, low levels of AA‐MNC increased (P = 0.001) acetate production compared with high supplementation levels.

CONCLUSION

These findings highlight the potential of supplementing AA‐MNC at low dose as a promising feed additive for sustainable livestock production through CH₄ mitigation at the same time as maintaining rumen nutrient degradability. © 2026 Society of Chemical Industry.

INTRODUCTION

Mitigation of greenhouse gas (GHG) emissions has become a global priority because of their significant contribution to climate change. Among agricultural sources, enteric CH₄ emissions from ruminal fiber degradation represent an environmental challenge, accounting for a substantial portion of GHG emissions from livestock.^{1, 2} In addition, as global temperatures increase, the range and prevalence of mycotoxigenic fungi are likely to change, potentially resulting in elevated mycotoxins levels.^{3, 4} Aflatoxin B1 (AFB1) is a mycotoxin that occurs naturally and is produced by Aspergillus species, poses additional challenges in ruminant nutrition. AFB1 contamination in feed negatively impacts ruminal fermentation, inhibiting nutrient degradability and overall animal health.^{4, 5} This inefficiency in digestion can increase CH₄ emissions per unit of degraded fiber because more undigested material passes through the digestive system, resulting in higher CH₄ output. Thus, developing solutions that can simultaneously address GHG emissions and AFB1 contamination is essential for sustainable livestock production.

Natural montmorillonite (NM) is a globally available and affordable clay widely used as a feed additive for binding aflatoxin. It is characterized by a negatively charged surface, high buffering capacity and elevated cation exchange capacity (CEC); however, it exhibits lower efficiency in inhibiting CH₄ formation than kaolinite clays. ⁶ Recently, organically modified montmorillonite clays in the nano form have shown improved physicochemical properties compared to its natural form. These properties include increases in the CEC, negative charged surfaces, specific surface area and effective functional groups in the modified clays that enable them to providing more opportunities for van der Waals and π‐π interactions with AFB1 molecules and adsorption of the positively charged ions such as hydrogen.^{7, 8, 9, 10} Because hydrogen is a significant factor in CH₄ emissions in the rumen, it is proposed that these modified clays could help to mitigate CH₄. ¹ These modifications have also positively influenced ruminal fermentation, acted as a pH buffer, and reduced methane (CH₄) emissions compared to the natural form. Recently, modified montmorillonite clays by different surfactants were reported to reduce CH₄ formation in vitro by 57% and 38%, whereas NM decreased CH₄ by only 6.8%. ¹¹ Furthermore, gum Arabic–modified nano‐montmorillonite enhanced short‐chain fatty acids (SCFA) production and fiber digestibility at the same time as lowering estimated CH₄ emissions in dairy cows, offering a safer alternative to ionophore‐based strategies. ¹²

Other natural compounds, such as amino acids, offer a promising alternative to conventional chemical modifiers (e.g. surfactants) for tailoring NM properties. Unlike surfactant‐modified clays, which often rely on synthetic organic cations and may raise concerns regarding toxicity and environmental persistence, amino acids are biodegradable, biocompatible and nutritionally relevant.^{13, 14} In the present study, methionine and threonine were selected as bio‐based modifiers to synthesize amino acid‐montmorillonite nanocomposites (AA‐MNC), namely methionine–montmorillonite nanocomposite (Met‐MNC) and threonine–montmorillonite nanocomposite (Thr‐MNC). Structurally, the functional groups of these amino acids (e.g. hydroxyl (–OH) and carboxyl (–COO⁻)), may enable multiple interaction pathways with the clay surface, including electrostatic interactions, hydrogen bonding and coordination with silicate layers. ¹⁴ These interactions facilitate intercalation into the clay interlayer space and modification of surface functionality.^{14, 15}

To the best of our knowledge, AA‐MNC have not been previously investigated at the nanoscale as feed additives for ruminant nutrition. The nanoscale structure of AA‐MNC is expected to enhance their reactivity because of an increased surface area; greater exposure of active sites and improved CEC compared to bulk or conventionally modified clays. Such nanoscale features may improve adsorption behavior and interaction with ruminal metabolites beyond that typically reported for traditional surfactant‐modified montmorillonite. Moreover, the use of essential amino acids as modifiers introduces functional groups while maintaining compatibility with animal nutrition, representing a safer and more sustainable alternative to synthetic modifiers.^{14, 15} To the best of our knowledge, this is the first study to develop and evaluate amino acid‐modified montmorillonite nanocomposites in nanoscale form as feed additives for ruminants, with a focus on their potential to modulate ruminal fermentation and mitigate enteric methane emissions. Therefore, the aims of this study are: (i) to develop and analyze the physicochemical properties of Met‐MNC and Thr‐MNC and (ii) evaluate the efficacy of both modified AA‐MNC clays on ruminal CH₄ production and fermentation of AFB1‐contaminated diets in comparison with NM and the ionophore antibiotic monensin.

MATERIALS AND METHODS

The Met‐MNC and Thr‐MNC clays were developed and evaluated in vitro at the Nanotechnology and Greenhouse Gases Laboratory, Faculty of Agriculture, Alexandria University.

Preparation of AA‐MNC feed additives

Two types of AA‐MNC were prepared using a modified version of the methods of Bujdakova et al. ⁷ and Soltan et al. ⁴ Specifically, dl‐methionine (Met; ≥ 99% purity; Sigma‐Aldrich, St Louis, MO, USA) and l‐threonine (Thr; ≥ 98% purity; Sigma‐Aldrich) were used to modify natural montmorillonite clay [NM; Ca‐montmorillonite (95%), Sigma‐Aldrich Co., Irvine, UK], resulting in the formation of Met‐MNC and Thr‐MNC, respectively. Five grams of NM clay were mixed in 300 mL of distilled water for 24 h at room temperature with a magnetic stirrer (508 Hotplate Magnetic; Globe Scientific, Mahwah, NJ, USA) and then the desired amounts of the Met and Thr [depending on the CEC and molecular weight) were added. The reaction mixture was stirred for 5 h at 80 °C. The resulting suspensions were stirred for 12 h at room temperature, filtered, washed three times with distilled water to remove the remains of the amino acids that did not interact with the NM, and then dried at 50 °C for 72 h. The dried AA‐MNC materials were ground using a high‐energy planetary ball miller (Photon, PH‐BML 912; Photon Scientific Co., Qalyub, Egypt) using zirconia balls as described by Adawi et al. ¹² to obtain the final Met‐MNC and Thr‐MNC clays.

Physicochemical properties of clay

All physicochemical analyses of the experimental clays were performed in triplicate. The analysis of the CEC of the AA‐MNC and NM clays was assessed using 1 m sodium acetate and 0.1 m sodium chloride solutions. ¹⁶ pH values were measured with a pH meter (GLP2; Crison Instruments, Barcelona, Spain). The particle size distribution and specific surface area (SSA) of the experimental clays were analyzed using a laser particle analyzer (Better Size 2600 Particle Analyzer; Dandong Better Size Scientific Ltd, Dandong, China) equipped with an automatic laser centering feature. Particle size distribution measurements were recorded at D ₁₀, D ₅₀ and D ₉₀. ¹² The morphology of the clay nanoparticles was examined using a scanning electron microscope (SEM) (JSM‐6360 LA; Jeol, Tokyo, Japan).

Energy‐dispersive X‐ray analysis (EDX) was used in conjunction with the SEM to identify and quantify the elements present on the clays surface, thereby providing a detailed specimen map.

The functional groups of the modified clay samples were characterized using Fourier transform infrared (FTIR) spectroscopy with an infrared spectrometer (model 8400S; Shimadzu, Osaka, Japan). Measurements were conducted employing a deuterated triglycine sulfate detector and a KBr beam splitter and spectra were recorded at a scanning rate of 45 scans per 60 s. ⁶

The basal substrate diet and treatments

A basal substrate diet designed to meet the nutrient requirements of dairy cattle NRC ¹⁷ was used as a substrate for the in vitro evaluation. The basal diet was analyzed according to AOAC ¹⁸ standards included dry matter (DM), ether extract and crude protein. The sequential determination of neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin (ADL), excluding residual ash, was performed ¹⁹ using filter bags (F57; ANKOM Technology, Macedon, NY, USA) and a semi‐automatic fiber analyzer unit (Smart Fiber analyzer, FA‐12; Smart Lab, Cairo, Egypt). Table 1 provides a detailed overview of the composition and chemical constituents of the basal substrate diet.

Table 1.

Ingredients and chemical composition based on dry matter (DM) of the experimental basal substrate

Item	Experimental diet (g kg−1 DM)
Ingredients
Trifolium alexandrinum clover	450
Wheat straw	50
Ground maize	100
Wheat bran	85
Soybean meal	40
Barley	212
Brocken rice	45
Calcium carbonate	8
Sodium chloride	5
Vitamins and minerals mixture 1	5
Chemical composition
Organic matter	911
Crude protein	165
Neutral detergent fiber	470
Acid detergent fiber	127
Acid detergent lignin	138
Cellulose	117
Hemicelluloses	205
Ether extract	15.8

The basal diet was analyzed for AFB1 and its background levels were measured before spiking; the experimental AFB1 dose was then adjusted to reach the target concentration. All of the feed additives, including NM, AA‐MNC and monensin, were supplemented to the +AFB1 diet (the basal substrate contaminated with 20 ppb AFB1 (98% purity; Sigma‐Aldrich). Total aflatoxins in the basal diet were extracted and purified in duplicate using VICAM immune affinity columns (Aflatest; VICAM, Milford, MA, USA), as described by Hafez et al., ²⁰ and quantified with the VICAM fluorometry method (Series 4EX Fluorometer; VICAM) in accordance with the manufacturer's instructions. The concentration of total aflatoxins in the +AFB1 diet was 29 ppb. These levels exceed the maximum permissible concentrations for AFB1 and total aflatoxins in dairy animal feeds in Egypt, which are set at 10 ppb and 20 ppb, respectively. ²¹

The treatments were: –AFB1 (basal substrate without supplementations), +AFB1 diet, NM diet (+AFB1 diet supplemented with NM at 5 g kg⁻¹ DM), monensin (+AFB1 diet supplemented at 40 mg kg⁻¹ DM; Elanco Animal Health, Greenfield, IN, USA)nd the prepared AA‐MNC clays (Met‐MNC and Thr‐MNC), which were supplemented to the +AFB1 diet at two levels: 0.5 g kg⁻¹ DM (low) and 1 g kg⁻¹ DM (high). These levels were selected based on previous studies showing effective rumen modulation and methane mitigation at similar doses using other modified clays.^{4, 11}

The in vitro gas production (GP) procedure

All the treatments were assessed by the in vitro semi‐automatic system of GP. ²² Ruminal contents were collected individually from three fasted, slaughtered crossbred buffalo calves (average body weight of 410 ± 14.2 kg) at the Alexandria University slaughterhouse. Buffalo rumen fluid was used as a model for ruminants as it provides a representative microbial ecosystem that reliably simulates rumen fermentation, including CH₄ production. The calves were allowed ad libitum access to a traditional local diet for beef production in Egypt. The collected ruminal contents were promptly transferred to pre‐warmed thermo‐containers (39 °C) treated with CO₂ and quickly transported to the laboratory. Once there, the contents were mixed in equal proportions for 10 s, strained through three layers of cheesecloth and held in a water bath (39 °C) under CO₂ infusion to prepare the inoculum for the GP assay.

Six incubation flasks were prepared for each treatment, along with blank flasks (n = 6; containing inoculum and buffer solution) used to obtain net GP values. Substrate treatments (1 g) were carefully weighed into pre‐numbered Ankom filter bags (F57; 50 mm × 40 mm; pore size 25 ± 10 μm; ANKOM Technology) that had been pre‐rinsed with acetone and pre‐weighed. The bags were then placed in the incubation flasks and were incubated with 30 mL of Menke's buffered incubation medium and 15 mL of ruminal inoculum, allowing for a 75‐mL headspace. ²² The incubation flasks were sealed using 20 mm butyl rubber stoppers, secured with aluminum seals and incubated at 39 °C for 24 h.

The gas pressure of the headspace was measured at 4, 8, 12 and 24 h from the beginning of incubation (Pressure Logger, model PSI‐V2; Smart Lab).

Greenhouse gas production

For CH₄ and CO₂ measurements, 5 mL of gas was collected at each pressure measurement hour using a syringe and transferred into 5‐mL vacutainer tubes. The CH₄ and CO₂ concentrations were determined using a laser gas Pro detector (Gas‐Pro‐IR‐W368334/01‐001; Crowcon Detection Instruments Ltd, Abingdon, UK). Before gases measurements, the detector was calibrated according to the LRQA‐validated ISO9001 quality standards, using a standard gas mixture prepared in accordance with BS EN ISO 6145‐1‐2008.

Ruminal nutrient degradability

After incubation, the bags containing the non‐degraded matter were removed from the flasks and placed on ice to halt microbial fermentation. The bags were then treated sequentially for 1 h at 90 °C in an ANKOM220 fiber analyzer unit with neutral detergent solution, washed, dried and weighed then treated with the acid detergent solution again for 1 h, and then washed, dried and weighed. All bags were treated with H₂SO₄ (72%) to determine the lignin contents, and all bags were subsequently incinerated. The difference between the incubated organic matter (OM) amounts and the remaining non‐degraded amounts was considered the truly degraded organic matter (TDOM) as per Blümmel et al. ²³ The difference between the amounts of either NDF or ADF that were incubated and those that remained non‐degraded was regarded as either truly degraded NDF (DNDF) or truly degraded ADF (DADF), respectively. The cellulose and hemicellulose contents were calculated by subtracting ADL from NDF and ADF from NDF, respectively. The fiber fractions were expressed exclusively in the residual ash.

Rumen fermentation characteristics

Ruminal pH was measured by a pH meter (GLP2; Crison Instruments). The contents of the incubation flasks were centrifuged at 10 000 × g for 15 min to determine the SCFAs and ammonia (NH₃‐N) concentrations. For SCFA analysis, an aliquot of 1.6 mL of the supernatant was combined with 0.4 mL of 25% metaphosphoric acid (4:1 ratio) and centrifuged at 22000 × g for 20 min at 4 °C using a K1015 Micro Prime centrifuge (Centurion Scientific Ltd, Stoughton, Chichester, UK). The SCFAs concentrations were measured according to De Baere et al. ²⁴ with some modifications using HPLC (Agilent 1260 Infinity II with Quaternary Pump (Agilent Technologies, Inc., Santa Clara, CA, USA). The detector was an Agilent 1260 Infinity Multiple Wavelength Detector with a Detection type: 1024‐element photodiode array. The instrument was supplied by an auto sampler and a separation column (Eclipse XDB‐C18. 4.6 × 150, 3.5 u Rapid Resol; Agilent Technologies, Inc.) and the SCFA standards were obtained from Sigma Chemie GmbH (Steinheim, Germany). The ruminal NH₃‐N concentration was measured calorimetrically in another part of the supernatant by a spectrophotometer (E‐2100 V; PEAK Instruments, Houston, TX, USA) using commercial lab kits (Biodiagnostic Inc., Giza, Egypt).

Statistical analysis

Each incubation flask represented an experimental unit, with six replicates for each treatment. Data were analyzed using one‐way analysis of variance with the MIXED procedure in SAS, version 9.0 (SAS Institute Inc., Cary, NC, USA). The experimental design followed a completely randomized structure with repeated measures. P < 0.05 was considered statistically significant, with trends identified at P ≤ 0.10.

RESULTS

Physicochemical properties of clay

The results of the physicochemical properties of the experimental clays are shown in Table 2. The physicochemical properties of the resulting AA‐MNC were distinct from those of NM. Variations in pH were observed among all clays. The AA‐MNC clays resulted in higher pH conjugated with increases in the CEC compared to NM. The results of particle size distribution showed a successful transformation from a broad distribution in NM clay (range 89–285 nm) to a narrower nanoscale distribution for both AA‐MNC clays. The Met‐MNC had the lowest particle size (range 20–21 nm) among the experimental clays.

Table 2.

Physicochemical properties of the natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites (AA‐MNC)

Item	NM	AA‐MNC
		Met‐MNC	Thr‐MNC
pH	6.72	7.02	7.05
Cation exchange capacity [cmol(+) kg−1]	13.6	14.2	15.7
Zeta potential (mV)	−21.5	−11.80	−31.10
Particle size distribution
D 10 (nm)	89	20	78
D 50 (nm)	131	21	82
D 90 (nm)	285	21	86
Specific surface area (m2 g−1)	14.67	109.9	72.15

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite.

The results provided in Table 3 show that the element quantity of the experimental clays differed. Sulfur was only detected by the Met‐MNC, whereas the C and O elements were higher in the Thr‐MNC compared to other clays.

Table 3.

Quantity of elements of the natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites (AA‐MNC) by the energy‐dispersive X‐ray analysis

		AA‐MNC
Element (% mass)	NM	Met‐MNC	Thr‐MNC
C	6.46 ± 0.25	ND	7.74 ± 0.25
O	53.14 ± 0.52	52.39 ± 0.51	55.25 ± 0.49
Mg	0.84 ± 0.06	0.65 ± 0.06	0.68 ± 0.05
Al	5.96 ± 0.15	7.59 ± 0.17	6.41 ± 0.14
Si	30.64 ± 0.33	35.11 ± 0.36	27.72 ± 0.29
K	1.09 ± 0.08	1.96 ± 0.10	1.16 ± 0.07
Fe	1.88 ± 0.13	1.99 ± 0.13	1.04 ± 0.09
S	ND	0.30 ± 0.04	ND
Total	100	100	100

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite; ND, not detected.

Figure 1 illustrates the SEM micrographs of the clays. The SEM analysis revealed a cracked and rough texture of both AA‐MNC clays, in contrast to the smooth and tightly layered edges of the NM flakes.

Figure 1.

The surface morphology of the experimental natural montmorillonite (NM), methionine‐montmorillonite nanocomposite (Met‐MNC) and threonine montmorillonite nanocomposite (Thr‐MNC) via scanning electron microscopy.

The results of the FTIR spectra of the AA‐MNC showed clear deviations from the NM clay (Table 4). The FTIR spectra of NM exhibited 13 distinct absorption bands, whereas Met‐MNC and Thr‐MNC displayed 31 and 21 bands, respectively. The FTIR spectra of NM and AA‐MNC displayed the characteristic structural –OH bands at ~3654–3606 cm⁻¹ and water‐related vibrations at ~3437–1636 cm⁻¹. Compared with NM, both modified clays showed additional or shifted absorption bands, indicating the presence of amino acid functional groups. In Met‐MNC, a band near 1636 cm⁻¹ appeared, consistent with C=O stretching of carboxyl groups, and distinct bands in the 600–700 cm⁻¹ region were observed, which may correspond to C—S stretching. These bands were not detected in NM or Thr‐MNC. Thr‐MNC showed minor shifts in the –OH and water vibration regions but did not exhibit bands in the C—S region as Met‐MNC.

Table 4.

Fourier transform infrared spectroscopy analysis of the natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites

Peak number	Maxima (cm−1)
	NM	Met‐MNC	Thr‐MNC
1	3654.84	3654.84	3653.91
2	3635.09	3437.66	3645.21
3	3626.47	2004.00	3625.63
4	3617.35	1871.66	3616.16
5	3606.24	1636.08	3605.74
6	3584.85	796.42	3584.12
7	3439.97	700.32	3563.53
8	1634.03	695.09	3443.62
9	1111.77	619.16	1993.23
10	798.28	610.27	1870.22
11	695.17	600.32	1658.09
12	560.01	573.11	1650.74
13	485.11	566.44	1643.80
14	–	559.16	1633.40
15	–	553.01	1233.03
16	–	548.95	796.74
17	–	546.11	693.43
18	–	537.24	580.75
19	–	532.96	550.92
20	–	527.02	489.60
21	–	519.73	474.15
22	–	515.36	–
23	–	509.95	–
24	–	507.33	–
25	–	499.58	–
26	–	495.07	–
27	–	492.09	–
28	–	484.66	–
29	–	480.83	–
30	–	475.00	–
31	–	470.89	–

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite.

Ruminal gas and greenhouse gas production

The results presented in Table 5 present the effects of supplementation with monensin, NM, and AA‐MNC clays on ruminal GP, as well as CH₄ and CO₂ emissions. The +AFB1 diet exhibited lower GP values (P = 0.09) compared to the other treatments. Both low and high levels of Met‐MNC and Thr‐MNC presented similar significant reductions (P = 0.058) in CH₄ (mL g⁻¹ DNDF) as the monensin diet compared to the –AFB1 and NM diets. The +AFB1 diet showed lower (P = 0.058) CH₄ (mL g⁻¹ DNDF) compared to the –AFB1 diet, whereas this effect did not appear (P > 0.05) for CH₄ related to TDOM, OM and DADF. For CO₂ production, all of the experimental feed additives showed reduced CO₂ values related to TDOM, DNDF and DADF compared to the –AFB1 and + AFB1 diets.

Table 5.

Supplementation effects of monensin, natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites (AA‐MNC) on ruminal gas production (GP), and methane (CH₄) and carbon dioxide (CO₂) production

	Treatments (T)									P value
					AA‐MNC
	Control		NM	Monensin	Met‐MNC		Thr‐MNC		SEM
	−AFB1	+AFB1			Low	High	Low	High
GP (mL g−1 DM)	137	153	157	153	166	165	183	165	16.21	0.09
Methane
CH4 (mL g−1 OM)	7.220 a	6.539 ab	6.238 ab	5.929 b	6.315 ab	7.52 a	5.708 b	6.045 b	0.237	0.036
CH4 (mL g−1 TDOM)	9.876	9.18	9.38	8.547	8.054	9.022	8.361	8.054	0.296	0.102
CH4 (mL g−1 DNDF)	22.78 a	17.05 b	22.54 a	17.46 b	17.92 b	17.26 b	17.64 b	17.26 b	0.900	0.058
CH4 (mL g−1 DADF)	35.29 ab	26.37 b	43.80 a	24.40 b	33.32 ab	27.58 b	37.1 ab	34.19 ab	1.946	0.0004
Carbon dioxide
CO2 (mL g−1 OM)	22.27	23.0	22.9	22.26	24.99	23.99	24.45	24.8	0.656	0.303
CO2 (mL g−1 TDOM)	87.50 a	93.5 a	41.498 b	31.66 b	33.82 b	32.2 b	32.24 b	32.99 b	0.925	< 0.0001
CO2 (mL g−1 DNDF)	143 a	138 a	81.4 b	63.18 b	64.3 b	60.9 b	65.4 b	66.0 b	13.135	< 0.0001
CO2 (mL g−1 DADF)	484 a	437 a	109 b	105 b	101 b	95.1 b	112 b	112 b	12.29	< 0.0001

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite; SEM, standard error of the mean. Different lowercase letters within a row indicate a significant difference at P < 0.05.

Nutrient degradability

The results provided in Table 6 showed that the low dose of Met‐MNC supplemented diets had higher (P < 0.05) values of TDOM and DNDF than the NM and monensin diet. All of the AA‐MNC diets had higher (P < 0.001) values of DADF than the +AFB1 diet. Similarly, all AA‐MNC diets enhanced (P < 0.001) the degraded cellulose compared to both +AFB1 and monensin diets. No significant differences were detected for hemicelluloses degradation.

Table 6.

Supplementation effects of monensin, natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites (AA‐MNC) on ruminal nutrient degradability

	Treatments (T)									P value
					AA‐MNC
	Control		NM	Monensin	Met‐MNC		Thr‐MNC		SEM
	−AFB1	+AFB1			Low	High	Low	High
Nutrient degradability (g kg−1 DM)
TDOM	691.7 ab	683 ab	678 b	666 b	696 ab	730 a	710 ab	693 ab	14.63	0.055
DNDF	343 ab	324 ab	314 b	294 b	352 ab	425 a	383 ab	347 ab	19.05	0.063
DADF	192 abc	123 c	170 bc	138 bc	202 ab	262 a	248 a	245 a	15.20	< 0.0001
Degraded cellulose	213 ab	91.2 d	118 bc	107 dc	200 ab	277 a	257 ab	254 ab	13.23	< 0.0001
Degraded hemicelluloses	470	494	436	426	480	563	497	434	28.27	0.340

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite; SEM, standard error of the mean; TDOM, truly degraded organic matter; DNDF, degraded natural detergent fiber; DADF, degraded acid detergent fiber. Different lowercase letters within a row indicate a significant difference at P < 0.05.

Ruminal fermentation

The impacts of NM, monensin and AA‐MNC on ruminal fermentation parameters and protozoal counts are presented in Table 7. The results showed that both AA‐MNC clays can modulate the ruminal fermentation pathways in a different way than monensin did. All of the the AA‐MNC diets significantly enhanced (P = 0.0007) the ruminal pH compared to the other feed additives and the +AFB1 diets. The +AFB1 diet significantly reduced (P = 0.0001) ammonia production compared to the –AFB1 diet, whereas both levels of Thr‐MNC and the high Met‐MNC treatments increased (P = 0.0001) ammonia production compared to the –AFB1, NM and monensin diets.

Table 7.

Supplementation effects of monensin, natural montmorillonite (NM) and amino acid‐montmorillonite nanocomposites (AA‐MNC) on ruminal fermentation parameters, and short chain fatty acids (SCFAs)

	Treatments (T)									P value
					AA‐MNC
	Control		NM	Monensin	Met‐MNC		Thr‐MNC		SEM
	−AFB1	+AFB1			Low	High	Low	High
Ruminal pH	6.106 abc	6.036 bc	6.090 bc	6.050 bc	6.043 bc	6.026 c	6.150 ab	6.216 a	0.025	0.0007
Ammonia (mg dL −1)	27.80 a	17.88 bc	14.59 c	17.40 bc	20.6 abc	22.2 ab	26.2 a	25.5 a	1.526	0.0001
SCFAs (mm)
Total	118.8 a	113 bc	118 a	116 ab	116 ab	107 cd	109 c	101 d	1.673	< 0.0001
Acetate	79.07 ab	74.65 bc	79.20 ab	78.7 ab	80.8 a	73.7 bc	77.05 ab	70.57 c	1.121	0.0001
Propionate	19.09 abc	19.87 ab	19.52 ab	19.33 ab	20.05 a	17.87 c	19.27 abc	18.53 bc	0.296	0.0019
Butyrate	11.43 abc	11.06 bc	11.78 ab	12.02 a	10.6 c	10.92 c	11.25 abc	9.66 d	0.192	< 0.0001
Isobutyrate	1.544 a	0.935 b	0.897 b	0.502 c	0.958 b	1.010 b	0.844 bc	0.933 b	0.073	< 0.0001
Valerate	6.32 a	5.26 ab	6.31 a	4.52 ab	0.93 b	0.85 b	0.501 b	0.507 b	0.984	0.0005
Isovalerate	1.330 ab	1.371 ab	0.685 b	1.211 ab	2.741 ab	3.503 a	0.998 ab	1.049 ab	0.541	0.0235

Abbreviations: Met‐MNC, methionine‐montmorillonite nanocomposite; Thr‐MNC, threonine montmorillonite nanocomposite; SEM, standard error of the mean. Different lowercase letters within a row indicate a significant difference at P < 0.05.

High levels of Met‐MNC and Thr‐MNC decreased (P < 0.001) the total SCFA compared to the NM and –AFB1 diets, whereas their low levels enhanced (P = 0.001) the acetate production compared to their high levels. Low Met‐MNC enhanced (P = 0.001) the propionate compared to high Met‐MNC. Among the experimental diets, high Thr‐MNC diet presented the lowest (P < 0.001) butyrate concentration. Monensin diet had the lowest (P < 0.001) isobutyrate and highest (P < 0.001) butyrate concentrations whereas high Met‐MNC increased (P = 0.023) the isovalerate values compared to the NM diet. High Thr‐MNC and +AFB1 treatments tended to have reductions in the acetate to propionate ratio compared to other treatments (except for the low Thr‐MNC).

DISCUSSION

The modified clays of AA‐MNC showed differences in the physicochemical properties compared to the unmodified montmorillonite NM clay because both AA‐MNC clays resulted in higher pH conjugated with increases in the CEC compared to NM. The physicochemical changes observed in the AA‐MNC are consistent with interactions between the clay surface and the incorporated functional groups. FTIR analysis confirmed the presence of carboxyl groups in both Met‐MNC and Thr‐MNC, whereas EDX detected sulfur‐containing moieties in Met‐MNC, supporting the successful incorporation of methionine.^{4, 11} The introduction of amino acid functionalities likely altered the clay surface chemistry. ¹³ Such modifications may influence the surface charge and CEC of the clays. Specifically, the higher CEC of AA‐MNC compared to NM could facilitate the exchange of protons (H⁺) with other cations (e.g. Mg²⁺, K⁺ and Si⁴⁺) in solution, contributing to the observed increase in pH. ¹⁵ These interpretations remain hypothetical and are supported by the detected functional groups in the FTIR analysis, although direct measurements of proton interactions were not performed in this study.

The nanoparticle size ranges of both AA‐MNC clays indicate successful modification and nanoscale structuring. The reduction and narrowing of the particle size distribution suggest effective modification techniques, likely a result of the incorporation of threonine or methionine functional groups and montmorillonite. The narrow distribution range reflects the high quality of the synthesis method used to produce similar nanoparticles in AA‐MNC clays. These transformations are important because they increase the SSA of AA‐MNC, consequently enhancing its reactivity, which may impact its properties and biological effects. ⁹ The present findings show that the smaller particle size of Met‐MNC compared to Thr‐MNC led to an increase in SSA, despite both being derived from the same clay minerals with identical structural characteristics. This also suggests that Met‐MNC exhibits higher reactivity than Thr‐MNC. ²⁵ Generally, the increases in the SSA of both AA‐MNC clays compared to NM may enhance its hydration and swelling capacity, making it easier for cations to diffuse into the clay's structure and bind to the exchange sites. This swelling effect increases the number of cations that the modified nano‐clay can exchange, contributing to a higher CEC compared to NM, which has a more limited swelling capacity. ²⁵ In NM, many of the layers are tightly packed, limiting the accessible surface area, whereas the nano‐modification process by amino acids can open up these layers, making more sites available for cation adsorption and exchange and thus increase the CEC values. ¹⁵ In this study, the experimental NM showed a high CEC (136 mmol per 100 g), which further increased after modification with amino acids. This increase in CEC for both types of AA‐MNC indicates a greater capacity for metal hydrolysates and ions to intercalate into the clay's interlayer spaces, thereby enhancing the clay's activity compared to NM clay. Similar results were observed in our previous study, where the modified NM by anionic and cationic surfactants demonstrated a higher CEC compared to the natural clay. ¹¹

The characterization of the AA‐MNC using FTIR and EDX confirms the successful incorporation of functional groups from methionine and threonine into the montmorillonite structure. FTIR spectra of Met‐MNC and Thr‐MNC showed additional absorption bands compared to NM, particularly in the 1636 cm⁻¹ region, consistent with C=O stretching of carboxyl groups. In Met‐MNC, subtle bands in the 600–700 cm⁻¹ region were observed, which may correspond to C—S stretching, whereas Thr‐MNC did not show these features. Distinct amine bands were not observed in the AA‐MNC clays, possibly as a result of overlapping with –OH and water‐related vibrations. EDX analysis further supports these modifications. ²⁵ Sulfur was detected only in Met‐MNC (0.30%), confirming the presence of methionine‐specific sulfur‐containing moieties, whereas Thr‐MNC and NM lacked detectable sulfur. Oxygen levels increased in Thr‐MNC (55.25%) compared to NM (53.14%), consistent with the incorporation of oxygen‐rich hydroxyl and carboxyl groups from threonine. ¹⁴ Both AA‐MNC clays also showed altered elemental compositions, including changes in Al, Si and K contents, suggesting that the introduction of amino acids may influence cation distribution in the clay interlayers or exchangeable sites.^{14, 26}

Together, the FTIR and EDX results indicate that methionine and threonine were successfully incorporated into the clay matrix, introducing carboxyl and, in the case of methionine, sulfur‐containing functionalities.^{15, 26} These chemical modifications likely contribute to the observed differences in physicochemical properties, such as CEC and pH, relative to NM, although the exact functional impacts on clay behavior remain to be further investigated.

The literature shows NM clay is characterized by soft, tightly bound layer edges.^{26, 27} In this study, SEM analysis showed that the AA‐MNC flakes developed cracked, rough edges. This change is mainly a result of the separation of the clay layers, with methionine or threonine functional groups settling between the octahedral alumina and silica tetrahedral layers. These findings support our first hypothesis that the physicochemical properties of montmorillonite can be enhanced through nano‐modification by both amino acids. As a result, we assessed their impact on rumen fermentation characteristics in the in vitro assay.

The results of the GP experiment revealed that the +AFB1 diet tended to have low GP values compared to the other diets. Similarly, Soltan et al. ⁴ observed the inhibition effect of AFB1 on GP using the same source of AFB1; in addition, Mojtahedi et al. ⁵ observed that increasing levels of AFB1 (from 0 to 900 ng mL⁻¹ ruminal fluid) resulted in a reduction in GP from 196 to 166 mL g⁻¹ DM. It appears that the presence of AFB1 in the diet can disrupt the normal microbial fermentation process, leading to lower overall GP. However, other studies reported that supplementation of AFB1 up to1.5 μg mL⁻¹ cannot affect the total GP. ²⁸ Many of the previous studies did not specify the baseline total aflatoxins concentration in the diets prior to AFB1 supplementation, which could influence the results. Therefore, variations in the source of aflatoxin, animal diet, ruminal communities and the specific experimental doses of AFB1 used may affect the GP results.

It appears that both AA‐MNC reduced CH4₄ concentrations more than NM. This observation may be associated with the altered physicochemical properties of the AA‐MNC, including increased pH, SSA, CEC and nanoscale structure. FTIR and EDX analyses confirmed the incorporation of carboxyl groups in both Met‐MNC and Thr‐MNC, and sulfur‐containing moieties in Met‐MNC, which might hypothetically influence proton availability and cation exchange within the clay. ² These modifications may enhance the clay's activity and adsorption properties, potentially affecting hydrogen availability in the rumen. ² Given that hydrogen ions are key intermediates in microbial fiber and organic matter degradation, and that methanogens use them to reduce CO₂ to CH₄, it is plausible that the observed CH₄ reduction reflects altered hydrogen dynamics mediated by AA‐MNC. However, these mechanisms remain speculative because direct measurements of hydrogen capture, electron sinks or methanogen activity were not performed in this study. The reduction in CH₄ measured per unit of DNDF and DADF suggest that the reduction in CH₄ observed with both AA‐MNC is not the result of a broad inhibition of fiber degradation. Although this remains to be confirmed, the presences of S detected by EDX analysis and spectra of sulfide groups detected by FTIR, coupled with the higher CEC and pH of Met‐MNC compared to NM, might suggest a greater ability to bind H+ because sulfur containing compounds can utilize H⁺ at low concentrations to produce hydrogen sulfide (H₂S), which may compete with methanogens for H⁺, thereby reducing CH₄ production.^{4, 22, 29} The reduction of sulfite to H₂S involves the consumption of eight electrons, making it a potential alternative electron acceptor. ³⁰ However, it should be noted that H₂ capture, methanogen abundance or activity and microbial community shifts were not directly measured in this study; therefore, these proposed mechanisms remain speculative and should be considered as hypotheses for future investigation.

The reductions in CO₂ observed with the prepared AA‐MNC clays may be attributed to their capacity to capture CO₂ (the primary component of gas production) through a reaction between CO₂ molecules and the interlayer OH functional groups of NM, resulting in the formation of –HCO3. This compound can subsequently react with additional interlayer cations.^{4, 31}

The nutrient degradability results indicated that all AA‐MNC treatments exhibited higher DADF values than the +AFB1 diet, reflecting improved cellulose degradation relative to both +AFB1 and monensin diets. These results suggest that the mode of action of reducing CH₄ by the prepared AA‐MNC is different from the monensin because the CH₄ reduction by monensin was conjugated with the reduction in cellulose degradability. Monensin acts as a CH₄ inhibitor as a result of its antibacterial activity against H⁺ producing bacteria, including methanogens and cellulolytic bacteria. It disrupts bacterial cell membranes by facilitating ion transport of H⁺/K⁺ and Na⁺/H⁺ cations or by inhibition of ruminal fungi and protozoa involved in fiber degradation,^{2, 32} and this partly explains the reduction in the degradability of cellulose alongside the decrease in CH₄ caused by monensin. By contrast, the use of AA‐MNC, particularly Met‐MNC, enhanced cellulose degradation.

The findings of our study also indicate that both AA‐MNC clays influence ruminal fermentation pathways in a manner distinct from monensin because the high dose of Thr‐MNC feed additives significantly enhanced the ruminal pH compared to monensin or NM. The reduction in free hydrogen ion concentration in the medium is associated with an increase in pH ² ; thus, it can be speculated that the Thr‐MNC may influence proton dynamics, potentially through interactions between the modified clay surface and hydrogen ions, thereby reducing the concentration of free hydrogen ions in the medium and contributing to the observed increase in pH. When amino acids are bound to the clay, their basic amine groups (NH₂) may neutralize the acidic components present in the clay; this process elevates the pH because the amino groups are capable of binding with hydrogen ions in the solution.^{2, 13, 15} By decreasing the concentration of these hydrogen ions, the overall acidity of the environment is decreased, making the system more alkaline and contributing to a higher ruminal pH.^{13, 15} The higher pH observed in Thr‐MNC may be related to its elevated oxygen content, as indicated by EDX analysis, which showed a higher oxygen content than the other clays. This increase in oxygen is consistent with the incorporation of oxygen‐rich functional groups, such as hydroxyl and carboxyl groups from threonine, as supported by the FTIR results. These functional groups may enhance the interaction between the clay surface and hydrogen ions, potentially reducing the concentration of free protons in the medium and thereby contributing to the observed increase in pH.^{2, 15} By contrast, Met‐MNC, which contains sulfur‐bearing functional groups, may exhibit different interaction pathways that do not directly influence proton dynamics to the same extent. However, these interpretations remain hypothetical and require further investigation to confirm the underlying mechanisms. These results may add importance of AA‐MNC for the dairy animals because they may suffer from the low ruminal pH at early lactation periods. ³³

Reductions in the OM and fiber degradability by NM, monensin and +AFB1 were associated with decreases in ammonia concentration, whereas the increases in fiber degradability observed in the AA‐MNC treatments were accompanied by higher ammonia levels. These results may also contribute to the increases in the ruminal pH caused by AA‐MNC clays. Increases in ruminal ammonia concentration were not observed in our previous studies with other nano modified montmorillonite using anionic and cationic surfactants.^{4, 11} It appears that the modifier can not only affect the properties of the clay but also its biological effects. The clay's pore structure may be responsible for enhancing ammonia production. The final concentration of ruminal ammonia may also be influenced because of the balance between its release and uptake by specific microbes. Generally, the increases in ammonia production may be in favor of microbial growth, and consequently animal productivity. ²⁹

Generally, the +AFB1 diet showed lower cellulose degradability combined with low fermentation characteristics as indicated by a low total SCFA concentration compared to the –AFB1 diet. These findings suggest that the AFB1 may inhibit fiber degradation, particularly cellulose, leading to reduced fermentation activity. Similar results were observed in our previous studies.^{6, 34} Although the exact mechanism remains unclear, it appears that AFB1 likely interferes with microbial processes involved in cellulose breakdown, resulting in diminished SCFA production, which is a key indicator of fermentation efficiency in the rumen. Low levels of AA‐MNC clays enhanced the acetate production compared to the high supplementation levels. Increases in OM and fiber degradability caused by low Met‐MNC may partly explain such results. These findings suggest that careful consideration is needed when selecting the appropriate dose of AA‐MNC to maximize its nutritional benefits. Low Met‐MNC as well as monensin increased propionate production compared to a high level, although they differed with respect to the butyrate pattern. However, this study revealed marked differences between the effects of AA‐MNC and monensin on the butyrate pattern, despite similar overall CH₄ inhibition. Both Met‐MNC levels and high Thr‐MNC decreased the butyrate concentration compared to monensin. This suggests that AA‐MNC clays and monensin likely reduce CH₄ emissions through different mechanisms. The reduction of CH₄ by nano‐modified montmorillonite with cationic and anionic surfactants (cetyl trimethylammonium bromide and sodium dodecyl sulfate) was linked to an increase in acetate production, ¹¹ whereas the nanocomposite of gum Arabic extract and NM led to increased total SCFA, acetate and butyrate in lactating cows. ¹² It appears that the choice of modifier influences the ruminal pathways involved in SCFA production. It should be noted that AFB₁ binding efficacy by these modified clays was not directly measured in this study, and any potential mitigation is inferred from fermentation responses. Direct assessment of AFB₁ binding remains a priority for follow‐up studies.

CONCLUSIONS

The developed AA‐MNC clays showed improved physicochemical properties, including a higher surface area, CEC and narrower particle size than NM. The control diet contaminated with AFB1 reduced cellulose degradability and SCFA concentration compared to the control without AFB1, whereas AA‐MNC clays showed potential to modulate rumen fermentation and reduce CH₄ production under in vitro conditions. A low Met‐MNC level and NM more effectively promoted acetate production and fiber degradation than the control diet contaminated with AFB1. These findings indicate that low levels of Met‐MNC may have the potential to modulate ruminal fermentation and reduce CH₄ production in AFB₁‐contaminated diets under in vitro conditions. However, further research, including in vivo studies and microbial community analyses, is required to confirm the mode of action, long‐term safety and practical applicability of AA‐MNC as a feed additive for sustainable ruminant production.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

ETHICAL STATEMENT

The experimental protocol was conducted according to the guidelines for the care and use of animals in research at Alexandria University (Protocol Approval no. AU 082210254114).

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.