3.1. Drying Process
Drying is crucial in the post-harvest management of plant material to recover phenolic compounds; it allows rapid protection against microbial attacks and chemical alteration due to inner processes, such as oxidation and enzymatic reactions [
60,
61]. Although the effects of the drying process on health-promoting compounds in foods have been extensively studied, little is known about the impact on agro-industrial waste, such as agave bagasse. Freeze-drying methods are widely accepted as methods that allow for the greater preservation of high-value phytochemicals [
62].
However, implementing the technology to treat the 150,000 tons of
A. lechuguilla bagasse produced annually is not viable due to the high operating costs. That is why oven-drying and sun-drying procedures were compared to freeze-drying to study the impact of such processes on the chemical and biological properties of the
A. lechuguilla agro-waste. Sun-drying is commonly used for medicinal plants, although, because the parameters cannot be controlled, the heterogeneity in the quality of derived products has been highlighted [
63]. Oven-drying was chosen as a scalable method allowing fast drying at a controlled temperature [
64] set at 40 °C to ensure flavonoid stability [
62].
The results showed that oven-dried bagasse presented similar extraction yields to the freeze-dried biomass. The extraction yield from oven-dehydrated and sun-dried material was not significantly different (
Figure 1). In contrast to the extraction yields, exposure to light affected the total phenolic content, which was lower for sun-dried and freeze-dried with light. In addition, oven dehydration ensured the same range of TPC as the freeze-drying process (
Figure 2). A higher TPC in air-dried than in freeze-dried material was previously attributed to the release of phenolic acid and flavonoids from the plant matrix due to heat [
61].
In contrast, freeze-drying in the dark obtained the highest total flavonoid concentration, and no difference could be found among the three other treatments (
Figure 2). The effect of light on TFC could not be demonstrated, although the impact of sunlight was inferred by the twice-higher TFC in the extracts obtained from LD compared with S dried bagasse (
Figure 2), which concurs with the UV-sensitivity of flavonoids reported in other plant material, such as berries [
62] and medicinal herbs [
65].
This fact is further supported by the verified impact of UV-C on the flavonoid content of
Agave tequilana extracts. A previous study reported that the 85 °C temperature had more impact on the TFC of
A. tequilana extracts than the light exposure [
49]. In comparison, the lower temperature used for oven dehydration (40 °C) appears to preserve thermal-sensitive flavonoids in the
A. lechuguilla bagasse. The similar TPC and TFC exhibited by ethanolic extracts of
Agave fourcroydes oven-dried at a higher temperature (60 °C) [
47] support this statement.
The different drying methods significantly influenced the content of individual flavonoids. Light exposure drastically decreased the anthocyanins content (
Table 1), likely due to their particular vulnerability to chemical reactions involving enzymes, light, and oxygen, leading to leakage of components [
60,
62,
64]. In this respect, Leong et al. [
52] reported the use of dim light to properly preserve the betacyanins from red-purple pitaya. Similarly, the lowest glycosyl flavonol and flavanone concentrations were found in the sun-dried biomass (
Table 1).
This fact is coherent with the decrease of glycoside flavonoid content observed in full sunlight exposed leaves [
65]. In contrast, the constant flavanol and flavanone contents in
A. lechuguilla bagasse, according to the drying procedure (
Table 1), reflected their stability as previously observed in air-dried berries [
62]. Finally, the variation of the free-radical scavenging capacity of the extracts was similar to the TPC variation, and the highest inhibition values were obtained from bagasse dried in the dark (
Figure 3).
These results contrast the usual conclusions about lower AA with freeze-drying than oven-drying [
62,
64]. To sum up, TPC, the individual concentration of glycosyl flavonoids and anthocyanins, and the antioxidant capacity of the extracts were increased in the
A. lechuguilla bagasse dried in an oven at 40 °C in the dark compared to freeze-drying. Therefore, oven-drying appeared to be the most efficient process to preserve the chemical and biological properties of
A. lechuguilla bagasse.
3.2. Enzymatic Pretreatment
The recovery of the phytochemicals from plant material is generally limited by physical barriers, e.g., cell walls and membranes. The lignocellulosic matrix of
A. lechuguilla is composed of 18.3 ± 1.1 to 20.18 ± 1.71% cellulose, 9.7 ± 0.5 to 11.08 ± 0.84% hemicellulose, and 18.9 ± 0.8 to 19.36 ± 0.49% lignin on an oven-dry basis [
19,
21] depending on the development stage and collection site [
66]. The extractive fraction represents from 26.2 to 37.21% [
19,
21,
41], and must contain around 12.28% ± 2.99% of phenolic compounds [
38,
39], which was corroborated by the current results of extraction yield (38.98 ± 0.64 %DW), and TPC (14.41 ± 1.81 mg GAE/g FW) obtained from oven-dry bagasse (
Figure 1 and
Figure 2).
Enzymatic hydrolysis is an efficient pretreatment of the lignocellulosic biomass to enhance the release of bioactive phenolic compounds [
57,
67]. Uses of lignocellulolytic enzymes and microorganisms for the bioconversion of
A. lechuguilla bagasse into bioenergy products have been previously studied [
18,
19,
20,
21], although the impact of such processes on phytochemicals has not been considered.
Among the enzymes commonly required to degrade the plant matrix and used in both the biorefinery process and active molecule extraction [
25,
54,
55,
66,
68,
69,
70,
71,
72,
73], cellulase, pectinase, and laccase were screened to formulate an enzymatic cocktail. The variability of extraction yield explained by the factor pH according to the Taguchi analysis (
Figure A2) suggested better activity of the enzymes at pH range from 4 to 5 (
Figure A2). In addition, the temperature factor evaluated in the second DOE further induced variability of extraction yields (
Figure 4), suggesting an effect of temperature on enzymatic activity.
This finding is in accordance with the physicochemical characteristic of the purchased enzymes. Cellulase and pectinase acquired from Sigma present optimal activity at pH 5.0 and 37 °C and pH 4.0 at 50 °C, respectively. The fungal laccase provided by the workgroup presents optimal activity at pH 4.0, and is stable at a range of temperatures from 25 to 70 °C with the highest relative activity from 40 to 60 °C [
74]. Hence, a pH of around 4 to 5 and temperature of 37–50 °C are recommended for the hydrolysis process using the formulated mix to ensure enzyme activity in the optimal range and facilitate the recovery of phytochemicals from lignocellulosic materials.
Screening of the enzyme amount (5–15 IU) in the formulation revealed that only laccase increased the extraction yield, whereas an increasing concentration of cellulase and pectinase maintained the yield as in the control treatment (
Table A4). In addition, the highest TPC and TFC values were obtained with the lowest levels of enzyme concentration (
Figure A1 and
Figure A2). The Taguchi modeling suggested a positive effect of the pectinase concentration from 5 to 10 IU on the phenolic and flavonoid contents in the extracts (
Table A4,
Figure A1 and
Figure A2). The use of 5 IU (= 2 mg/mL) of cellulase and pectinase previously increased the TFC in
Ginkgo biloba leave hydrolysates [
66]. Similarly, cellulase enhanced the recovery of soluble phenolic compounds from
Psidium guajava leaves [
69].
In contrast, increasing the laccase concentration in the formulation showed a negative impact on TPC and TFC (
Figure A1 and
Figure A2). Laccases are interesting for waste valorization processes in the biorefinery context because they avoid sugar degradation by acting on the phenolic units of lignin polymers [
66]. However, the phenoloxidase activity of the laccases led to the oxidation of phenolic acids [
75]. Thereby, the alteration of other phenolic compounds, such as flavonoids, is probable and could explain the present results. In general, oxidation of polyphenols is not recommended because it reduces the antioxidant potential [
61]. Low phenol reduction was observed when using bacterial laccases instead of fungal laccases [
66]. This option should be considered in the treatment of plant biomass for the procurement of phenolic compounds as added-value co-products.
Based on these preliminary results, three enzyme mixtures were formulated with laccase, cellulase, and pectinase at the respective proportions of: 1:1:1 (LCP), 1:1:2 (LCPP), and 0:1:2 (CPP). According to the second DOE, the prediction of a positive effect of pectinase amount was refuted since it did not significantly increase the concentrations of phenols and flavonoids in the extracts (
Table A4,
Figure 4 and
Figure 5). On the other hand, a minimum amount of laccase in the formulation improved the extraction yield, as previously obtained in DOE I, compared to treatment without laccase. Surprisingly, phenolics and flavonoids presented the highest concentrations in the presence of laccase in the cocktail (
Figure 5 and
Figure 6). This could be due to the release of a high free-sugar content allowed by laccase since reducing-sugars are known to interfere with the TPC detection method [
76].
In addition to the enzyme ratio, the pH, temperature, and treatment time were evaluated in the second DOE because of their effect on enzymatic activity and phenolic compound retrieval. Optimal activity at pH 4, suggested in the first DOE (
Figure A1 and
Figure A2), was confirmed by the results of DOE II. Therefore, the highest yield (
Figure 4a), TPC (
Figure 5a), and TFC (
Figure 6a) were obtained for pH 4 (
Figure 4a,
Figure 5a, and
Figure 6a). Likewise, the total phenolic and flavonoid recovery were improved with incubation time (
Figure 5c and
Figure 6c), whereas the tested temperature range had no significant impact (
Figure 5b and
Figure 6b). The PCA confirmed this result (
Figure 7), which concluded that enzyme mix composition was the main factor influencing the phenolic and flavonoid recovery from
A. lechuguilla hydrolyzed bagasse.
The formulated mixture’s efficiency toward flavonoid extraction was compared to a commercial enzymatic cocktail. A third DOE was performed to evaluate the impact of pH, temperature, and incubation time on the hydrolysis of A. lechuguilla bagasse with 2 mg/mL of Ultraflo© and Viscozyme© purchased from Novozymes®. The Ultraflo© cocktail is characterized by cellulase and xylanase activity. Viscozyme© contains a wide range of carbohydrases, including arabanase, cellulase, β-glucanase, hemicellulase, and xylanase. Optimum conditions for the enzymatic activity of Ultraflo© are pH 6.0 and temperature of 50–60 °C, and for Viscozyme© are pH 3.3–5.5 and temperature 40–50 °C.
In the present study, the pH tested from 4 to 6 did not impact the TPC and TFC in the extracts, whereas reducing the temperature from 50 to 30 °C, and increasing the incubation time from 0.5 to 2.5 h showed a positive effect on the TPC and TFC (
Figure A4 and
Figure 5 and
Figure 6). Similarly, Viscozyme© used at 0.2% was previously found to allow higher phenolic recovery from
Brassica oleracea leaves in comparison with control, and the authors reported no effect of pH (3–6) and temperature range (30–50 °C) [
58]. Likewise, increasing the time enhanced the phenolic recovery from
Rosmarinus officinalis leaves hydrolyzed with Viscozyme©, although longer than a 3 h incubation time was found to have a negative effect [
76]. In contrast, time does not affect the phenolic recovery using Ultraflo© at pH 5.0 and 50 °C for 6 h [
77] and pH 4.0 and 40 °C for 12 h [
78].
In comparison to the formulated enzyme mixtures, the two commercial cocktails were more efficient for the recovery of phenolic and flavonoid content, and the highest TPC and TFC were obtained with Ultraflo© compared to Viscozyme© (
Figure 5,
Figure 6 and
Figure 7). Ultraflo© and Viscozyme© enhance the TPC in
Opuntia humifusa hydrolysate [
72], and the Viscozyme© efficiency was confirmed for the recovery of specific flavonoids from
Opuntia ficus-indica [
67], which could be attributed to the presence of xylanases in both cocktails, which allows for a better degradation of the hemicellulose of
A. lechuguilla composed at 63% of xylose [
21]. Thus, xylanase should be considered in future mix formulations, such as those proposed by Wang et al. [
69]. In addition, if Viscozyme© is preferred for cactus mucilage digestion to retrieve phenolics [
67,
77], the Ultraflo© appeared to be more specific and efficient to release phenolic compounds from the lignocellulosic matrix of
A. lechuguilla.
In general, enzymatic hydrolysis enhanced the TPC and TFC, which increased the radical scavenging capacity of the extracts compared to the control treatment (
Figure 8 and
Figure 9). Likewise, enzymatic-assisted extraction improves the DPPH scavenging capacity of
R. officinalis extracts due to a higher TPC than conventional extraction methods [
76], and enzymatic pretreatment of
P. guajava leaves enhances the DPPH values by 2.3 fold [
69]. Both the highest and lowest DPPH radical scavenging values were observed with laccase depending on the pH, temperature, and hydrolysis time (
Figure 8).
The release of reducing sugar and phenolic acids by the action of laccase likely participates in the AA [
72], explaining the large deviation. Lower variation and 1.5-fold increased AA results were obtained from
A. lechuguilla bagasse hydrolyzed by Ultraflo© and Viscozyme© for 2.5 h, at 40 °C and pH 4 (
Figure 8). Similarly, the DPPH radical scavenging capacity of
O. humifusa hydrolysates increases using Ultraflo© and Viscozyme© [
77]. The use of enzyme-assisted extraction for the obtention of bioactive flavonoids from diverse biomass is globally accepted. However, it has been reported that enzymes significantly modulate the flavonoid profile of the extracts, which impacts the antioxidant capacity [
67,
79]. In addition, the anti-cancer activity of
A. lechuguilla decreases in acid hydrolyzed extracts [
32]. Hence, a specific flavonoids profile must be characterized to obtain further conclusions regarding the optimal enzymatic pretreatment.
In this study, 10 flavonoids previously described in
A. lechuguilla-untreated material were quantified in the extract obtained after enzymatic hydrolysis executed for 2.5 h at pH 4 with Ultraflo© (26), Viscozyme© (22), LCP (13), CP (14), and control (C1). The alteration of flavonoid profile by laccase, as previously suggested, was confirmed by the reduced concentration of all quantified flavonoids, except quercetin, compared to the other treatments (
Figure 10). In particular, cyanidin decreased by 43.5%, delphinidin by 21.2%, and flavanone by 1.5%. In addition, drastic decreases in the kaempferol, apigenin, naringenin, and hesperidin contents were observed, which went below the detection limits (
Figure 10).
In contrast, Ultraflo© increased the content of hesperidin by 2.5 fold, cyanidin by 1.5 fold, delphinidin by 1.4 fold, isorhamnetin by 1.3 fold, and flavanone by 1.2 fold in the extracts compared to the control, and the obtained concentrations were higher when compared with Viscozyme© and CP. The quercetin concentration in the extract was reduced by using Ultraflo© and increased by the use of Viscozyme©. Furthermore, the apigenin, kaempferol, and naringenin concentrations remained at the same concentration as untreated bagasse (
Figure 10).
The anthocyanins, flavanone, isorhamnetin, quercetin, and kaempferol quantified fractions contain glycoside derivatives [
40,
41]. Hence, a decrease of these fractions agrees with the previously reported bioconversion of phenolic glycosides into their aglycones caused by the use of Viscozyme©. Wang et al. [
69] described a loss of glycosyl quercetin derivatives, and aglycone quercetin and kaempferol gain. Similarly, Antunes-Ricardo et al. [
67] demonstrated the breakdown of the sugar moiety from original triglycosylated forms of isorhamnetin and quercetin. Kim et al. [
77] reported a decrease in the quercitrin concentration and an increase in the quercetin and isorhamnetin contents after enzymatic hydrolysis.
In contrast, the Ultraflo© and CP mix likely preserved glycoside forms and even enhanced their recovery in the case of Ultraflo©. The efficiency of cellulase and pectinase for recovering flavonol aglycones, such as quercetin, kaempferol, and isorhamnetin, from
G. biloba leaves has been demonstrated [
70]. Likewise, using a mix prepared with pectinase and cellulase (1:2,
w/
w), it was possible to extract 16 flavonoids, including glycoside conjugates with higher yields than without enzymatic pretreatment [
73]. Transglycosylation activity of the enzymes can explain the lowest loss of glycosyl flavonoids in the Ultraflo© and CP treatment [
70].
On another hand, pH and temperature could affect the flavonoid profile not only in the hydrolysis process but also in the extraction process. Tran et al. [
56] demonstrated that temperature and time particularly impact the extraction of isoflavones and reported that pH may affected the isoflavone structure. These modifications on the flavonoid profile are relevant due to the well-studied structure–activity relationship. The release of aglycone flavonols is particularly interesting for pharmaceutical applications [
58,
67,
69,
79]. At the same time, enhanced glycosyl flavonoids, particularly the anthocyanidin content, suggest applications in cosmetics and nutraceuticals [
80].
Therefore, Ultraflo© at pH 4.0, at 40 °C, and for 2.5 h were considered in this study to be the best hydrolysis parameters to enhance bioactive flavonoid recovery from
A. lechuguilla bagasse. Experimental validation was conducted, and the results confirmed the increase of the extraction yield, total phenolic recovery, DPPH radical scavenging, and anthocyanidin concentrations (
Table 2). The highest concentration of anthocyanins supports the use of enzymatic treatment for the procurement of high added-value co-products. Thus, despite the fact that the high cost of enzymes is usually considered a limitation for scaling-up [
53], a techno-economic evaluation should be performed to reconsider the enzyme-assisted extraction within biorefinery schemes.