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Article

Production of an Extract with β-1,4-Xylanase Activity by Fusarium oxysporum f. sp. melonis on a Sonicated Brewer’s Spent Grain Substrate

by
Irma A. Arreola-Cruz
,
Rosalba Troncoso-Rojas
,
Francisco Vásquez-Lara
,
Nina G. Heredia-Sandoval
and
Alma R. Islas-Rubio
*
Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera Gustavo E. Astiazarán Rosas # 46, Col. La Victoria, Hermosillo 83304, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(1), 42; https://doi.org/10.3390/fermentation11010042
Submission received: 9 December 2024 / Revised: 4 January 2025 / Accepted: 16 January 2025 / Published: 18 January 2025
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
The Fusarium oxysporum species commonly found in soil include plant and human pathogens, and nonpathogenic species. F. oxysporum grown on lignocellulosic substrates under submerged conditions produces an extracellular enzyme profile with hemicellulolytic and cellulolytic activities. Our aim was to produce an extract of Fusarium oxysporum f. sp. melonis with β-1,4-xylanase activity after fermentation on a Brewers’ spent grain (BSG)-containing substrate. We prepared the BSG substrate, with or without sonication, for the submerged fermentation of Fusarium oxysporum previously isolated from local soil and preserved at 4 °C. First, an enriched inoculum was prepared, and later, the production of β-1,4-xylanase using the BSG substrates was monitored for up to 6 or 10 days in the enriched inoculum or in the enzyme extract, respectively. An activity of β-1,4-xylanase 12.0 U/mL (day 3) was obtained in the enriched inoculum with the untreated BSG, remaining constant for 3 days. A significant increase in the activity of this enzyme was observed (day 6), especially in the extract obtained using the sonicated BSG substrate (39 U/mL). Applying ultrasound to the BSG before its use in a submerged fermentation with Fusarium oxysporum f. sp. melonis could be an alternative for producing β-1,4-xylanase.

1. Introduction

Fusarium oxysporum is a facultative pathogenic fungus. It is a ubiquitous species common for diverse soil habitats, with more than 120 special forms (formae speciales). This fungus survives in organic matter for long periods due to the type of spores, called chlamydospores [1,2]. F. oxysporum possesses the ability to bioconvert lignin, cellulose, and hemicellulose using as a carbon source BSG, which is rich in these polymeric structures [3], through the production of degrading enzymes such as xylanase, endoglucanase, β-D-glucosidase, α-L-arabinofuranosidase, cellobiohydrolase, feruloyl esterase, β-D-xylosidase, and acetyl esterase [4]. Among the five different formae speciales of Fusarium oxysporum that have been found, F. oxysporum niveum, melonis, and cucumerinum are globally distributed and more important pathogens from an economic standpoint [5].
F. oxysporum’s extracellular enzyme profile presents hemicellulolytic and cellulolytic activities when the fungus is grown on lignocellulosic substrates under submerged conditions [6]. The submerged fermentation (SmF) technique has gained importance over the last years for the production of enzymes and secondary metabolites on an industrial scale since it is less problematic (much better heat and oxygen mass transfer and superior culture homogeneity), making it more reliable and reproducible and more accessible to monitor and to control critical operational parameters, and it is more flexible [7]. On the other hand, solid-state fermentation (SSF), the bioprocess carried out in the absence or near-absence of free water, is preferred over SmF as it is cost-effective, eco-friendly, and delivers a high yield of enzymes, it presents a lower risk of contamination, and it uses mostly agro-industrial wastes as substrate [8,9,10]. The agro-industrial residues from forestry, agriculture, agroindustry, and food waste, such as brewery spent grain (BSG), can be used as a culture media to produce various enzymes such as cellulases, hemicellulases, proteases, xylanases, amylases, and phytases, among others.
The most commonly used grain for producing beer-type beverages is barley [11]. BSG is the main residue generated by the brewing industry. In 2023, approximately 41.9 million tons of BSG were produced worldwide [12]. BSG has a short shelf life due to its high moisture content and susceptibility to microbial spoilage, with current outputs mainly restricted to low-value animal feed or landfill [13]. Therefore, the wet BSG must be dried to extend its shelf life and facilitate its use as a food ingredient due to its considerable amounts of fiber, protein, cellulose, lignin, starch, lipids, and phenolic compounds [9]. BSG mostly contains lignocellulosic material (30–50%), of which hemicellulose represents between 19 and 42% [14]. Bonifácio-Lopes et al. [15] reported a composition of BSG of 25% hemicellulose, 20% cellulose, 18% lignin, 22% protein, and 15% lipids and other compounds. The hemicellulose fraction of BSG primarily consists of arabinoxylans (AX), a dietary fiber linked with potential health benefits such as prebiotic activity, improved glycemic control, and antioxidant activity [16,17,18,19]. The high crosslinking of this lignocellulosic material prevents the accessibility of bioactive compounds present in BSG. AX are made up of linear chains of β-(1→4)-xylopyranose and the α-l-arabinofuranose units are attached to the main xylose chain at positions two/or three. Arabinose molecules can be esterified with hydroxycinnamic acids, monomeric or dimeric ferulic acid, and p-coumaric acid [17].
AX has been used as an emulsifying agent, film-forming agent, gelling agent, antioxidant, and prebiotic [20]. Among the prebiotics, xylooligosaccharides (XOS) have the potential to work against several gastrointestinal disorders. XOS production relies on lignocellulosic materials, which are unsuitable for human consumption and available worldwide [21]. Chemical, biological, physical, and enzymatic methods have been used to increase the accessibility to the bioactive compound in BSG. The chemical treatments of BSG have been the most widely used method to obtain AX. The biological methods use microorganisms to degrade cell walls by secreting enzymes to hydrolyze polymers into monomers, and they carry out the growth of microorganisms using submerged fermentation and solid-state fermentation [3,21,22]. The use of fungal extracts to release ferulic acid from different lignocellulosic materials has already been reported [23,24]. Faulds et al. [23] reported the release of 65% of ferulic acid from wheat bran by a ferulic acid esterase (Fae-III) from Aspergillus niger and Humicola insolens. Similarly, Xiros et al. [24] pretreated the BSG with NaOH and used Fusarium oxysporum and Trichoderma longibrachiatum, in synergy with a commercial xylanase, to obtain the extract. They reported the release of 40% of the ferulic acid present in the BSG.
Among the physical treatments for BSG are extrusion, ultrasound application [25], and microwave irradiation [26]. Combined methods to treat BSG have been reported. Heredia-Olea et al. [27] used extrusion and hydrolysis with fiber-degrading enzymes to produce C5 and C6 sugars from BSG for ethanol production. Severini et al. [28] used enzymatic and technological treatments of BSG to solubilize AX. Recently, the extrusion of BSG in combination with solid-state fermentation using Fusarium oxysporum f. sp. lycopersi increased the release of AX from BSG as well as the antioxidant capacity of the extracts [29]. The use of physical treatments in combination with commercial enzymes to release ferulic acid from BSG has been reported [30]; however, the high cost of commercial enzymes is a disadvantage for treating BSG to release its bioactive compounds.
The solid-state fermentation process using filamentous fungal cultures has been studied to produce xylanases and feruloyl esterases. This process has several technological and operational advantages over submerged fermentation [31] since there is a greater production of enzymes. The objective of this study was to evaluate the effect of the sonication of BSG on the production of β-1,4-xylanase after submerged fermentation with F. oxysporum f. sp. melonis using a BSG-containing substrate, evaluate the composition and antioxidant capacity of the untreated BSG, and evaluate the ferulic acid content in the solid fractions remaining after fermentation.

2. Materials and Methods

2.1. Material

The wet BSG was provided by Cuauhtemoc-Moctezuma-Heineken®, Navojoa, México. It was dried in an MP-500 dryer (Enviro-Pak, Clackamas, Portland, OR, USA) at 40 °C for 40 h, ground in a Pulvex 200 mill (Pulvex de México, S.A. de CV, Mexico City, México), and stored in polyethylene bags for further analysis. All of the reagents were from Sigma Aldrich Co. (St Louis, MO, USA).

2.2. BSG Composition

The moisture, protein, ash, fat, and dietary fiber (soluble, insoluble, and total) contents of BSG were determined using the approved methods 44-15, 46-13, 08-01, 30-20, and 32-07 of the American Association of Cereal Chemists [32], respectively. The dietary fiber was measured using a Megazyme total dietary analysis kit (Megazyme International, Bray, Ireland). Cellulose, hemicellulose, and holocellulose contents of BSG were determined according to Zobel et al. [33]. Details of these methods were reported by Martinez-Encinas et al. [34]. The lignin content of BSG was determined according to Jay et al. [35].
Total, free, and bound phenolic compounds of BSG without pretreatment were evaluated with the Folin–Ciocalteu colorimetric method described by Chen et al. [36]. The results were expressed as milliequivalents of gallic acid per gram of dry sample. The contents of ferulic, p-coumaric, sinapic, and caffeic acids present in BSG were analyzed by the method described by Mradu et al. [37], and the chromatographic separation was carried out on an RP-HPLC (Agilent 1290 Infinity, Agilent, Inc., Santa Clara, CA, USA), equipped with a photodiode array detector. The analytical column was a C18 110A column, 300 mm × 4.6 mm, with a particle size of 5 µm (Beckman Coulter, Brea, CA, USA). The mobile phase consisted of A (100% acetonitrile) and B (0.1% formic acid in water). The analytes were eluted using an isocratic separation with 8% A and 92% B from the injection time until 35 min. The conditions were set as follows: 24 °C column temperature, 1 mL/min flow rate, and 10 µL injection volume. The phenolic acids were detected at a wavelength of 320 nm and were identified by comparing retention times with those of their respective standards. The contents of phenolic acids were quantified using external calibration curves. The samples were analyzed in triplicate, expressing the results in mg/g of BSG.
The antioxidant capacity was evaluated by the DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) free radical elimination method, as described by Chen et al. [36]. Both techniques are based on measuring the decrease in the absorbance of the DPPH radical and the ABTS radical when the antioxidant is added, in this case, BSG. The results were expressed as mg equivalent of Trolox per gram of sample (mg TE/g) and % inhibition [1-A Sample/A control] × 100. Samples were analyzed in triplicate.

2.3. Pretreatment of BSG

The pretreatment of BSG consisted of sonication at 600 watts for 30 min at room temperature, using an XO-SM50 ultrasonicator (Nanjing Xianou Instruments Manufacture Co., Ltd., Nanjing, China). These conditions were chosen based on the higher release of total phenolic compounds from the sonicated BSG (350 or 600 W, for 30 min). The pretreatment was carried out in 250 mL Erlenmeyer flasks containing 4 g of BSG and 100 mL of the mineral medium, as described by Xiros and Christakopoulos [6]. In the case of the BSG treatment, 50 mL of distilled water was used to dissolve the mineral medium, and the other 50 mL was added to the BSG to apply the pretreatment of sonication, and both were mixed for incubation to obtain the enriched inoculum. The amounts of reagents to prepare 1 L of this medium are shown in Table 1. Both BSG samples were used as carbon sources to inoculate with Fusarium oxysporum f. sp. melonis.

2.4. Origin and Growth of the Microorganism

A strain of the fungus F. oxysporum f. sp. melonis, isolated from melon fruit (collection of the Laboratorio de Biotecnología Vegetal y Poscosecha, CIAD, Hermosillo, México) was used. The strain morphological and microscopic characteristics coincide with those reported by Leslie and Summerell [1], Ramos et al. [38], and Troncoso-Rojas et al. [39]. The fungus was initially grown on potato dextrose agar (PDA) (BD Bioxon, Guadalajara, México) and was incubated (Shel Lab SMI2, Cornelius, OR, USA) at 30 °C for 10 days.

2.5. Preparation of Mineral Medium with BSG

Five hundred mL of mineral medium were prepared in a 1 L Erlenmeyer flask according to the materials and quantities (g) listed in Table 1. Three 500 mL flasks with 150 mL of mineral medium were stirred constantly with a magnet (250 rpm) for 40 min, and the pH was adjusted to 6.0 (HCl 36% purity), and sterilized with gauze stoppers and aluminum foil for 35 min at 121 °C. Each flask was inoculated with 7.5 mL of F. oxysporum (1 × 106 spores) and incubated at 30 °C with constant shaking (150 rpm) for 7 days.

2.6. Enriched Inoculum Preparation

For the inoculum preparation, 15 mL of sterile deionized water with 100 μL of Tween 80 was added to the fungus colony surface. The spore suspension was prepared according to French and Herbert [40]. The colony surface was scrapped, and the spore suspension was adjusted to a concentration of 1 × 106 spores/mL using a Neubauer chamber (Bright-Line, Buffalo, NY, USA) [39]. Aliquots of spore suspension (5 mL) were taken and added to 100 mL of mineral medium, as described by Xiros and Christakopoulos [6]. An amount of 20 g/L sonicated-treated or untreated BSG was added, and the pH adjusted to 6.0 by the addition of hydrochloric acid. The flasks were incubated by triplicate at 30 °C for 6 days in an orbital shaker (125 rpm) for mycelium production. The activity of xylanase was monitored at different time intervals to determine the time at which the maximum enzyme activity was obtained, since the inoculum is essential because this is used to stimulate the production of the enzymatic extract [6,23,41]. Five hundred mL of sterile mineral medium was prepared as described in Section 2.5, but this time 6 g of BSG was put in each flask with 150 mL of medium + 7.5 mL of the mineral medium described in Section 2.5, and incubated at 30 °C with constant shaking (150 rpm) for 6 days. This inoculum was called “enriched inoculum”. Three flasks were removed in each time interval (0, 48, 7, 96, 120, and 144 h), and the xylanase activity of the extract was determined as described in Section 2.7.

2.7. Production of the Enzyme Extract and Xylanase Activity

The SmF assay was performed according to Xiros and Christakopoulos [6] with some modifications. A total of 4 g of BSG (sonicated-pretreated or untreated) was added to the mineral medium (100 mL) as a carbon source (pH 6.0), and 5 mL of the enriched inoculum (Section 2.6) was added. The triplicate of the second mineral medium, incubated at 30 °C with constant shaking (150 rpm) for 10 days, was filtered through a funnel with a cotton fabric. The supernatants were centrifuged at 14,000 rpm for 20 min at 4 °C. Afterward, it was vacuum filtered in ice with a pore size of 0.45 µm. The enzymatic extract was concentrated by ultrafiltration using a membrane of 10 kDa cut-off. Three washes were carried out with Milli Q water shaking for 20 min. The extract was stirred at 150 rpm with a controlled pressure by nitrogen gas (50 psi) until the extract was filtered. Afterward, the membrane was turned (shiny side down), and 50 mL of Milli Q water was added and filtered. At different time intervals (0, 48, 96, 144, 192, and 240 h), triplicate flasks were withdrawn and used to measure xylanase production.
The activity of the β-1,4-D-endoxylanase (1,4-β-D-xylan xylanohydrolase EC 3.2.1.8) was estimated by incubating the extract with 1% birchwood xylan [42]. The release of reducing sugars was measured by the 3,5-dinitrosalicylic acid (DNS) method [43]. The activity of β-1,4- xylanase is defined as the unit of enzyme (U) needed to liberate one µmol of xylose per min. To determine the specific activity of the enzyme, its protein content was measured by Bradford [44]. In the solid fraction remaining after fermentation, the composition of the lignocellulosic material was determined according to Zobel et al. [33]. The free and bound phenolic compounds from BSG and in the solid fraction remaining after fermentation were extracted and analyzed by HPLC (using ferulic, sinapic, p-coumaric, and caffeic acids as standards) according to Chen et al. [36].

2.8. Statical Analyses

Experimental results were reported as mean ± standard deviation. A factorial analysis (2 × 6) was performed for monitoring β-1,4-xylanase activity, with NCSS version 2007 (Keysville, UT, USA). Multiple comparisons of the means were performed using the Tukey–Kramer test when interactions were not significant. The significance level was set at p < 0.05.

3. Results and Discussion

3.1. Chemical Composition of BSG

Table 2 shows the chemical composition of BSG. The moisture content of the wet BSG was 83.0%, and after drying, it reduced to 4.8%, which is within the range reported in dry BSG, which goes from 4 to 10% [45,46,47,48]. Moisture content is an essential parameter since it helps us keep the storage of BSG under favorable conditions [49]. The protein content of BSG was 24.3% (Table 2), in the range (13 and 31%) reported by other authors [50,51]. The dried BSG is considered a protein rich residue, in comparison to other cereals having a protein content at 17.1% (oats), 14.4% (wheat), 7.5% (rice), and 7.5–15.6% (even barley grain). The protein is mainly in the pericarp, the aleurone layer, and the barley grain husk, that is, the main structural constituents of BSG [51]. Therefore, its addition to some food gives it added value since it has been found that around 30% of the amino acid profile present in BSG is essential, which does not happen in cereals. The fat content of BSG was 8.7%, similar to that reported by other authors (6–10.6%) [3,13,47]. On the other hand, the ash content of BSG was 4.1%, representing the minerals present in the BSG, and this coincides with what has been reported in the literature [3,47,52].
A total dietary fiber content of 60.8% was found, of which 59.9% represents insoluble dietary fiber. Regarding the lignocellulosic fraction that makes up most of the insoluble dietary fiber, lignin values of 24.4%, hemicellulose of 22.7%, and cellulose of 15.9% were obtained. The content of dietary fiber, protein, and the rest of the components of the BSG varied when compared to other studies [3,53,54], which could be mainly due to the variety of barley grain and the adjuncts (rice, oats, sorghum, wheat, etc.) used to obtain beer.
The total phenol content (TPC) of BSG was 4.6 mg GAE/g d.b., of which 4.5 mg GAE/g are bound phenolic compounds, and the remaining 0.1 mg GAE/g are free phenolic compounds (Table 2). Meneses et al. [55] and Stefanello et al. [56] reported 4.6 mg/g and 1.4 mg/g of bound and free TPC in BSG, respectively. Most of the phenols present in BSG are not available. On the other hand, Ktenioudaki et al. [57] reported 5.4 mg GAE/g corresponding to bound phenols and 0.5 mg GAE/g to free phenols in BSG. The phenolic acid content of BSG is shown in Table 2. The phenolic acid present in the highest proportion in BSG was ferulic acid (3.1 mg/g), followed by sinapic (1.5 mg/g), p-coumaric (0.1 mg/g), and caffeic (0.02 mg/g) acids. The ferulic acid content coincides with those reported by Swajgier et al. [58] and Niemi et al. [51], who obtained between 3 and 3.4 mg/g of ferulic acid. Sinapic acid had already been reported as the third in abundance in BSG, after ferulic and p-coumaric acids [58]. On the other hand, the p-coumaric acid content was slightly lower than the values reported in the literature (between 0.2 and 0.6 mg/g). The caffeic acid content was found below what was reported (0.06–0.09 mg/g). The content of phenolic compounds can vary depending on the plant, species, environmental conditions, geographical location, and storage conditions, hence the variability in the data compared to other authors. According to Meneses et al. [55], to release the phenolic compounds and take advantage of these natural antioxidants with potential for addition to food, treatments based on saponification (with NaOH solution), enzymatic hydrolysis, or microwave-assisted extraction need to be applied to BSG.

3.2. Antioxidant Capacity of BSG

The antioxidant capacity found in the extract of free and bound phenolic compounds of BSG is shown in Table 3, determined by the DPPH and ABTS methods. The DPPH method is based on the ability of the DPPH radical to react with the hydrogen donor, that is, the phenols present in BSG, and the ABTS cation decolorization test is based on the reduction of the radical by the antioxidants found in the phenolic extract of BSG [55]. Reis and Abu-Ghannam [59] obtained an 80% inhibition of the DPPH radical after 30 min of reaction in the BSG, which is higher than the results obtained in this work (Table 3). On the other hand, Moreira et al. [60] obtained 39 mg TE/g for ABTS, values lower than our results (69.1 mg TE/g). Zhao et al. [61] determined the antioxidant capacity for barley by ABTS, finding around 1.5 mg TE/g and a percentage of inhibition by DPPH of 60%, a value much lower than those found in our samples; this may be mainly due to the sample, since BSG contains a higher concentration of phenolic compounds, which determine the antioxidant capacity.
The composition of the phenolic compounds in the sample influences the free radical scavenging activity. Therefore, the differences found with other authors, where they obtained around 5.2 mg TE/g in total phenols (DPPH), may be due to the content and profile of phenols in the sample, as well as the extraction techniques of the phenols and the determination of antioxidant capacity [57]. On the other hand, when comparing the results obtained by both methods (DPPH and ABTS), a higher percentage of inhibition can be observed when using the ABTS radical, and this could be due to the reaction kinetics between phenols and the radical cation ABTS, or the radical DPPH in a similar range of concentrations, leading to the different results of the two methods.

3.3. Monitoring of the Enzyme Extract

First, an enzyme extract with low β-1,4-xylanase activity was obtained, incubating the untreated BSG with Fusarium oxysporum f. sp. melonis for 6 days in the inoculum and 10 days in the production of the enzyme extract. Therefore, we decided to monitor the activity of the enriched inoculum and the enzyme extract production, comparing the BSG without pretreatment and the BSG treated with ultrasound, to know the behavior of the β-1,4-xylanase activity. According to studies reported in the literature, obtaining the inoculum is essential since this inoculum will be used to stimulate the production of enzymes by fungi and bacteria to release high value products [6,23,41]. However, no data have been reported on the behavior of β-1,4-xylanase production from F. oxysporum f.sp. melonis using untreated or sonicated BSG as a substrate in a submerged fermentation. In the present study, it was found that after 48 h, the activity of β-1,4-xylanase increased in both samples (BSG without pretreatment and BSG treated with ultrasound); however, in the unmodified BSG, the enzymatic activity was maintained without significant changes (p > 0.05) from 48 h to 144 h of cultivation. On the other hand, a significant increase (p < 0.05) was observed in the BSG treated with ultrasound at 72 h (22.7 U/mL), an activity that was maintained during the following 72 h (Figure 1). Therefore, we decided to take the enriched inoculum from the 72 h of cultivation to continue with the enzyme extract production. Hence, the enriched inoculum served as a stimulus to enhance enzymatic activity in the enzyme extract production.
In the enzyme extract production, significant differences (p = 0.0000 *) were found in the interaction treatment and reaction time for β-1,4-xylanase activity (Figure 2). It was observed that at 144 h, using ultrasound-treated BSG as substrate, an activity of β-1,4-xylanase of 38.7 U/mL was obtained. It increased to 41.3 U/mL at 240 h without statistical significance (p > 0.05). Regarding the BSG without pretreatment (control), it was found that at 240 h, a β-1,4-xylanase activity of 23.2 U/mL was obtained, significantly higher than that obtained at 144 h. However, even though the activity of the β-1,4-xylanase in untreated BSG increased at 240 h, the BSG treated with ultrasound showed significantly higher β-1,4-xylanase activity (p < 0.05) in a shorter time (144 h), with an increase in the activity of 15.5 U/mL between samples.
Bartolomé et al. [41] report a β-1,4-xylanase activity of 0.75 U/mL after 24 h of cultivation, using an actinomycete (S. avermilitis CECT 3339). In 1995, Faulds and Williamson [23] used Aspergillus niger and obtained a β-1,4-xylanase activity of 12 U/mL after 96 h of reaction. Xiros and Christakopoulos [6] used Fusarium oxysporum isolated from cumin, obtaining a β-1,4-xylanase activity of 58 U/mL (120 h). Compared with the results obtained in our work, the authors mentioned above used a different source of fungi and/or bacteria to obtain the extract. It is also important to highlight that the source of the BSG and its composition influence the behavior of the microorganism. When comparing the results obtained by Xiros and Christakopoulos [6], it was observed that the hemicellulose content of their substrate (BSG) was higher (40%) than in our study (22.6%). In addition, the lignin content was lower (11.9%) than that of our substrate (24.4%), which could explain a greater stimulation of the enzyme activity in a shorter time.
A mechanism for the production of β-1,4-xylanase has been proposed [4], which explains that there is an initial production in the extracellular medium, induced by the presence of xylo-oligosaccharides, xylobiose, or xylotriose. According to studies published in the literature, the hypothesis has been raised that xylobiose is the true inducer of xylanase synthesis. With the help of active transport (β-xylosidase permease), these oligosaccharides pass over the cell membrane, further activating the gene expression of xylanolytic enzymes [4]. Thus, this may explain the increase in β-1,4-xylanase activity when using the ultrasound method, since this could have released both the AX chains crosslinked with the rest of the lignocellulosic material, as well as having stimulated the hydrolysis of available AX towards xylo-oligosaccharides, xylobiose, xylotriose, etc. [4,62]. These results suggest that the ultrasound method (600 W for 30 min) is a good, harmless pretreatment to apply to BSG used as a substrate in a submerged culture to obtain an enzyme extract with β-1,4-xylanase activity using Fusarium oxysporum f. sp. melonis as the enzyme producer.
Xiros et al. [3] used BSG treated with 10% NaOH in a test carried out by solid-state fermentation, where they obtained a ratio of 11% cellulose, 13% hemicellulose, and 11.9% lignin in the solid fraction and 20% hemicellulose and 3.3% lignin in the liquid fraction. Under such conditions, they reported a maximum β-1,4-xylanase activity of 1,090 U/g (approx. 66 U/mL), at 144 h of cultivation, where they observed a delay in the enzyme activity of 24 h compared to submerged cultivation. If we relate the concentrations of the lignocellulosic material to the concentrations of our substrate, it can be seen that the maximum activity of β-1,4-xylanase obtained by the authors, compared with this work (41.3 U/mL at 240 h), in which ultrasound was used as pretreatment, does not vary drastically. In addition, it is worth mentioning that the enzymatic production can vary between 10 and 15% in the repetition of the tests. Therefore, ultrasound can be a good, harmless alternative as pretreatment in BSG to be used as a substrate in producing an enzyme extract under a submerged culture using Fusarium oxysporum f. sp. melonis. It is important to highlight that the studies mentioned above have been carried out to obtain biofuel or purify and market lignocellulolytic enzymes, so they do not show interest in the toxicity of the solvents and methods used for pretreatment. In this case, the purpose of our study is to use an economical alternative with future applications in the food area [6,23,41]. Ultrasound is a viable option due to its zero toxicity and wide use in the food industry.

3.4. Ferulic Acid Content in the Enriched Inoculum and Enzymatic Extract

The ferulic acid content was monitored by HPLC simultaneously with the β-1,4-xylanase activity in the enriched inoculum and the enzyme extract. On day 0, ferulic acid values close to the free phenols found in the untreated BSG (Table 2, 0.1 mg GAE/g) were obtained and decreased as the incubation time progressed, until reaching a non-detectable value at 144 for the enriched inoculum, and 240 h for the enzyme extract (Table 4). This behavior was probably due to the bioassay being carried out in an incubator where the samples were exposed to light during the day and night, and so the phenols could have been oxidized and not show the accurate concentrations released during the production of the enzyme extract. In work carried out by Faulds et al. [63], where they produced an enzyme extract using wheat bran as carbon source and Aspergillus niger as enzyme producer, they found a decrease in ferulic acid values from 24 to 120 h of reaction, at which time it was no longer possible to detect it due to the detection limit of HPLC; however, such extract presented feruloyl esterase activity (0.24 U/mg). Another study found that by adding free ferulic acid to BSG, feruloyl esterase activity was delayed (optimal time 9 h), until 28 h [24]. Therefore, it would be necessary to measure the activity of feruloyl esterase to determine if the decrease in ferulic acid values was due to the pretreatment of our substrate, since this increased the amount of free phenols in the medium, or if there was simply a degradation of ferulic acid due to exposure to light, without being related to the activity of feruloyl esterase.

3.5. Lignocellulosic Composition in the Solid Fraction of the Enzyme Extract

Table 5 shows the lignocellulosic composition in the solid fraction remaining after the fermentation of the enzymatic extract. A 13% decrease in the hemicellulose present in the residual substrate was obtained, and a 20% decrease in lignin for the BSG without pretreatment; however, an increase of 36% in cellulose was observed. This effect may be because the structure of cellulose directly affects the enzymatic system produced by fungi since the crystallinity and the contact surface area between cellulose and the enzymes present can affect their activity [64]. On the other hand, a repressive mechanism inhibits the production of cellulases in the presence of a more easily assimilated source; in this case, due to the composition of the substrate (BSG), hemicellulose is more readily available [4]. Thus, there may not necessarily have been an increase in the percentage of cellulose, but rather a decrease in the percentage of hemicellulose.
This study focused on the production of an extract with β-1,4-xylanase activity by Fusarium oxysporum f. sp. melonis using a BSG-containing substrate. The activity of this enzyme was monitored at different cultivation times, and the untreated and sonicated BSG substrate was compared. The purpose of sonication (600 W, 30 min) as a pretreatment for BSG was to facilitate the release of phenolic compounds. In preliminary work, we evaluated the sonication of BSG with different solvents, times, and potencies. The higher total phenolic compounds released from the treated or untreated BSG corresponded to the BSG sonicated at 600 W for 30 min. For this reason, we chose these conditions. The effect of ultrasound could be based on a direct interaction with molecular species as well as on the cavitation phenomenon. Furthermore, the mechanical impact produced by the collapse of cavitation bubbles provides an important benefit of opening up the surface of solid substrates to the action of enzymes [65]. Limitations of this study include the need for a composition analysis of the extract to follow up on the products released into the cultivation medium. Further research needs to address this subject.
The co-existence of the cellulose, hemicellulose, and lignin of lignocellulosic biomass makes it difficult to convert into valuable products [66]. In this regard, pretreatment plays a vital role in disrupting the complex network of lignocellulosic biomass for the sustainable production of value-added products [67,68]. The potential of emerging pretreatment methods for the sustainable conversion of biomass was discussed by Haldar and Purkuit [69]. These methods are ultrasound, microwave, the use of deep eutotic solvents (DESs), irradiation, the use of pulsed electric field, and high pressure-assisted pretreatment. The application of ultrasound technology during the pretreatment of biomass has emerged as one of the front runners among the other comparable methods due to the shorter reaction time, and its effectiveness in the biomass conversion process [69]. The impact of ultrasound-based pretreatment methods on different lignocellulosic biomass using different integrated methods and process conditions was also reported [69]. All of these integrated methods involve the usage of harsh chemicals.
The novelty of this work is the production of an extract with β-1,4-xylanase activity using Fusarium oxysporum f. sp. melonis on BSG pretreated only by ultrasound without the use of chemicals commonly used as treatment prior to the use of other methods.

4. Conclusions

The ultrasound treatment (600 W for 30 min) of the BSG stimulated the production of β-1,4-xylanase by 33% in the submerged culture with Fusarium oxysporum f. sp. melonis (144 or 192 h) in comparison with the untreated BSG. The results of this work show the potential use of Fusarium oxysporum f. sp. melonis, a fungus present in the region, to obtain enzymes of high commercial value. The ferulic acid contents in the solid fractions remaining after fermentation decreased in the untreated and sonicated BSG. Additionally, ultrasound offers an alternative treatment to the use of contaminating chemical agents, at a lower cost and with potential use in human nutrition. Finally, is still necessary to evaluate the activity of the rest of enzymes that degrade lignocellulosic material and undertake toxicity testing.

Author Contributions

Conceptualization, A.R.I.-R.; methodology, A.R.I.-R., I.A.A.-C., and R.T.-R.; validation, I.A.A.-C., N.G.H.-S., and F.V.-L.; formal analysis, I.A.A.-C., and N.G.H.-S.; investigation, A.R.I.-R., I.A.A.-C., and R.T.-R.; resources, A.R.I.-R., and R.T.-R.; data curation, N.G.H.-S., and F.V.-L.; writing-original draft preparation, I.A.A.-C., and A.R.I.-R.; writing-review and editing, A.R.I.-R., R.T.-R., and N.G.H.-S.; visualization, N.G.H.-S., and A.R.I.-R.; supervision, A.R.I.-R., and R.T.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within this article.

Acknowledgments

The authors acknowledge M.C. Granados-Nevárez and A. Sánchez-Estrada for technical assistance. The authors thank CONAHCYT (México) for the scholarship granted to I.A. Arreola-Cruz.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00042 g001
Figure 1. Monitoring of β-1,4-xylanase (enriched inoculum) activity in untreated BSG and sonicated BSG at 30 °C and 150 rpm. Values (mean ± standard deviation) with different superscript letters are significantly different (p < 0.05).
Figure 1. Monitoring of β-1,4-xylanase (enriched inoculum) activity in untreated BSG and sonicated BSG at 30 °C and 150 rpm. Values (mean ± standard deviation) with different superscript letters are significantly different (p < 0.05).
Fermentation 11 00042 g001
Fermentation 11 00042 g002
Figure 2. Monitoring of β-1,4-xylanase (enzyme extract production) activity in untreated BSG and ultrasound-treated BSG at 30 °C and 150 rpm. Values (mean ± standard deviation) with different superscript letters are significantly different (p < 0.05).
Figure 2. Monitoring of β-1,4-xylanase (enzyme extract production) activity in untreated BSG and ultrasound-treated BSG at 30 °C and 150 rpm. Values (mean ± standard deviation) with different superscript letters are significantly different (p < 0.05).
Fermentation 11 00042 g002
Table 1. Mineral medium composition with Brewers’ spent grain (BSG) in 1 L.
Table 1. Mineral medium composition with Brewers’ spent grain (BSG) in 1 L.
ReagentWeight (g)
KH2PO41.0
CaCl2.2H2O0.3
MgSO4.7H2O0.3
(NH4)2HPO410.0
NaH2PO4.2H2O6.94
Na2HPO4.2H2O9.52
BSG20.0
Table 2. Chemical composition of BSG *.
Table 2. Chemical composition of BSG *.
Compound%
 Moisture4.8 ± 0.4
 Protein24.3 ± 0.0
 Fat8.7 ± 0.2
 Ash4.1 ± 0.0
 Total dietary fiber60.8 ± 0.8
 Insoluble dietary fiber59.9 ± 0.7
 Soluble dietary fiber0.9 ± 0.1
Total Phenolicsmg GAE/g d.b.
 Total4.6 ± 0.1
 Free0.1 ± 0.0
 Bound4.5 ± 0.1
Hydroxycinnamic Acidsmg/g d.b.
 Total4.7 ± 0.1
 Ferulic3.1 ± 0.0
 Sinapic1.5 ± 0.0
 p-Coumaric0. 1 ± 0.0
 Caffeic0.02 ± 0.0
* Values are the mean of triplicate ± standard deviation.
Table 3. Antioxidant capacity of the phenolic extract of BSG *.
Table 3. Antioxidant capacity of the phenolic extract of BSG *.
SampleDPPHABTS
mg TE/g% Inhibitionmg TE/g% Inhibition
Free phenolics14.7 ± 0.838.4 ± 2.00.9 ± 0.059.9 ± 0.8
Bound phenolics56.3 ± 1.272.3 ± 1.569.1 ± 0.997.2 ± 1.5
* Values are the mean of triplicate ± standard deviation.
Table 4. Ferulic acid content of the solid fractions remaining after fermentation from the enriched inoculum and the enzyme extract production *.
Table 4. Ferulic acid content of the solid fractions remaining after fermentation from the enriched inoculum and the enzyme extract production *.
Ferulic Acid (mg/g)
Cultivation Time (h)
Residual Sonicated BSG7296120144240
Ferulic acid, enriched inoculum0.05 ± 0.000.03 ± 0.000.03 ± 0.00ND ---
Ferulic acid, enzyme extract production---0.01 ± 0.000.02 ± 0.000.01 ± 0.00ND
* Values are means of triplicate ± standard deviation. ND: No Detected.
Table 5. Lignocellulosic composition in the solid fraction remaining after fermentation of the enzymatic extract *.
Table 5. Lignocellulosic composition in the solid fraction remaining after fermentation of the enzymatic extract *.
Lignocellulosic Material%
Hemicellulose 19.7 ± 0.5
Cellulose21.6 ± 0.5
Lignin19.5 ± 1.7
* Values are means of triplicate ± standard deviation.
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Arreola-Cruz, I.A.; Troncoso-Rojas, R.; Vásquez-Lara, F.; Heredia-Sandoval, N.G.; Islas-Rubio, A.R. Production of an Extract with β-1,4-Xylanase Activity by Fusarium oxysporum f. sp. melonis on a Sonicated Brewer’s Spent Grain Substrate. Fermentation 2025, 11, 42. https://doi.org/10.3390/fermentation11010042

AMA Style

Arreola-Cruz IA, Troncoso-Rojas R, Vásquez-Lara F, Heredia-Sandoval NG, Islas-Rubio AR. Production of an Extract with β-1,4-Xylanase Activity by Fusarium oxysporum f. sp. melonis on a Sonicated Brewer’s Spent Grain Substrate. Fermentation. 2025; 11(1):42. https://doi.org/10.3390/fermentation11010042

Chicago/Turabian Style

Arreola-Cruz, Irma A., Rosalba Troncoso-Rojas, Francisco Vásquez-Lara, Nina G. Heredia-Sandoval, and Alma R. Islas-Rubio. 2025. "Production of an Extract with β-1,4-Xylanase Activity by Fusarium oxysporum f. sp. melonis on a Sonicated Brewer’s Spent Grain Substrate" Fermentation 11, no. 1: 42. https://doi.org/10.3390/fermentation11010042

APA Style

Arreola-Cruz, I. A., Troncoso-Rojas, R., Vásquez-Lara, F., Heredia-Sandoval, N. G., & Islas-Rubio, A. R. (2025). Production of an Extract with β-1,4-Xylanase Activity by Fusarium oxysporum f. sp. melonis on a Sonicated Brewer’s Spent Grain Substrate. Fermentation, 11(1), 42. https://doi.org/10.3390/fermentation11010042

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