Recovery of amylolytic enzymes from triticale malt (X. Triticosecale Wittmack) using two-phase aqueous systems

doi:10.21203/rs.3.rs-2468244/v1

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Recovery of amylolytic enzymes from triticale malt (X. Triticosecale Wittmack) using two-phase aqueous systems

https://doi.org/10.21203/rs.3.rs-2468244/v1

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Introduction: Triticale malt has shown higher amylolytic activity than other cereal malts, a characteristic of great importance for the brewing and starch industry. The scope of this work was to obtain concentrated enzymatic aqueous extracts containing β-amylase, α-amylase, and amyloglucosidase from triticale malts of Bicentenario and Siglo-XXI varieties, using aqueous two-phase systems (ATPS) for enzymes recovery.

Methodology: The malts produced had 5 days of germination and were dried at 50°C. The extracts were prepared by mixing ground malt with deionized water (1:10), stirred at 180 rpm, testing three stirring times (30, 120, and 270 min) and two temperatures (30 and 40°C) (12 treatments). The extracts were centrifugated, freeze-dried and purified, varying the concentrations of alcohol and Na₃C₆H₅O₇. Enzyme quantification was performed using: BETAMYL-3^®(β-amylase); AOAC 2002.0I, AACC22-02.0I (α-amylase), and McCleary et al., (1991) (amyloglucosidase).

Results: In relation to the malt, an increase in extract enzyme quantification was achieved, mainly α-amylase for Siglo-XXI malt, initially with 99.56 CU/g to 1,268.89 CU/mL (507.5 CU/g dry matter) in 30 min/30°C extract. For the same treatment, the best ATPS for enzymatic recovery was 30% alcohol/18% Na₃C₆H₅O₇, α-amylase predominantly at the inferior phase 1,514.03 CU/mL (605.6 CU/g dry matter) and β-amylase 51.43 BU/mL (10.2 BU/g dry matter) at the upper phase.

Conclusion: Aqueous amylase extraction from triticale Siglo-XXI malt in 30 min/30°C conditions is a suitable option for production of β-amylase and α-amylase in combination with the use of ethanol and Na₃C₆H₅O₇ ATPS to recovery amylolytic enzymes.

Malt

triticale

amylolytic enzymes

ATPS

α-amylase

Aqueous two-phase extraction systems (ATPS) are a technique applied to separate and recover biomolecules. These systems are composed of two different immiscible polymers (polyethylene glycol, dextran) or a polymer with salt (NaCl, KH₂PO₄), where both substances are soluble in water at specific concentrations (Hong Yang, 2013; Xu et al., 2003). ATPS involves the transfer of biomolecules from the sample to one phase or another, as a function of variables such as polymer concentration, size, molecular weight, as well as the ionic strength of the salt. Other factors such as the pH or the affinity of the polymer to a certain receptor of the molecule of interest can influence molecule partitioning. These characteristics make ATPS a highly efficient option for proteins, genetic material, or nanoparticle recovery. These systems can be adapted to fit continuous operation programs and are labeled as environmentally friendly, making ATPS a viable purification technique for industrial scale (Asenjo & Andrews, 2011).

In recent years, the application of protein extraction through liquid-liquid systems based on ATPS has increased due to its great efficiency (Hong Yang, 2013). The most outstanding studies are on the recovery of glutenin from wheat flour, human recombinant protein from alfalfa green tissue (Barberino do Nascimento et al., 2010; Ibarra-herrera et al., 2011) and enzymes, such as α-amylase and β- amylase from malted corn seed extracts. Previous research shows that alpha and beta amylases with elevated enzymatic activity can be extracted from malting process surplus, using PEG/CaCl2 systems (Biazus et al., 2007; Ferreira et al., 2007).

Triticale (X. Triticosecale Wittmack) is an anthropogenic cereal designed to incorporate the functionality and high yield of wheat (Triticum spp. Linnaeus 1753) and the durability of rye (Secale cereale Linnaeus 1753). This cereal is known for being resistant to pests and diseases, as well as tolerant to droughts and poor soils. This cultivar has a nutritional value similar to wheat, making it a suitable option for human consumption but has yet to consolidate its place in the market, leaving its applications unexplored (Ayalew et al., 2018; Mcgoverin et al., 2011; Zhu, 2018). In the last fifteen years, different Triticale varieties have been evaluated as alternatives to substitute barley in the brewing-wort industry, either as an adjunct or as a malt source (Glatthar et al., 2005; Grujić & Pejin, 2007; J. Pejin et al., 2013). The results have been unfavourable for Triticale. The physicochemical and sensory characteristics of Triticale wort differ from those displayed by barley wort, and when the latter is substituted by Triticale viscosity and fermentable extract are negatively impacted. The addition of α-amylase, β-amylase, α-glucosidase, or limit dextrinase to ensure the complete transformation of the starchy material (malt and adjuncts) into sugars (maltose, glucose and dextrins), is a recurrent practice inside the brewing industry since the malt does not have enough enzymes to achieve high sugar yields. These enzymatic complexes open the opportunity to finding new starch sources that could lead to light or gluten-free beers (Guerra et al., 2009).

Previous work has shown that triticale seed and malt can produce more than double the units per gram of β-amylase and α-amylase produced by barley and wheat (Glatthar et al., 2005; Pejin et al., 2009; Peña, 2004). Biazus et al (2007) work has shown that is possible to extract and purify enzymes with elevated enzymatic activity from malted grains using aqueous two-phase systems (Biazus et al., 2007). Triticale could be a suitable alternative source to produce highly demanded enzymatic cocktails for the brewing industry. The objective of this research was to obtain enzymatic aqueous extracts containing a high amount of β-amylase, α-amylase and amyloglucosidase from triticale malts of Bicentenario and Siglo-XXI varieties, using an ATPS for enzymatic recovery, to generate a suitable enzymatic product for the starch industry and brewing.

2.1 Raw material

The malts were prepared from triticale seeds (X. Triticosecale Wittmack) of the Bicentenario and Siglo-XXI varieties. Both varieties had a 95% germination percentage with a protein percentage of 13.5% and 14.1%, respectively. These materials were obtained from the Instituto de Investigación y Capacitación Agropecuaria, Acuícola y Forestal of the México State (ICAMEX), harvested in 2017.

2.2 Experimental design

A randomized block design with three repetitions was applied, respectively, to produce the malts and crude aqueous extracts (treatments). Triticale seed with two levels (varieties) was considered as a factor for the malts and the response variables are presented in section 2.5 "Malt analysis" in triplicate. For crude aqueous extracts, two factors were considered, the extraction temperature (30°C and 40°C) and the stirring time (30 min, 120 min, and 270 min). The response variables analysis is presented on section 2.6.2 “Analysis on crude aqueous extracts” in triplicate.

The ATPSs were prepared in triplicate. The two treatments with highest enzyme activity were selected (30 min/30°C Siglo-XXI and 30 min/40°C Bicentenario). A completely random design was applied for each treatment, taking three absolute ethanol (Merck brand) and sodium citrate (Na₃C₆H₅O₇·2H₂O, Merck Brand) (SC) concentrations as a factor, as follows 45% ethanol/10% SC, 35% ethanol/13% SC and 30% ethanol/18% SC. The measured response variables were the activity quantification of α-amylase, β-amylase and amyloglucosidase, by triplicate.

2.3 Malt production

Triticales seeds, 100 g, were immersed in distilled water for 24 h by triplicate, until reaching a moisture percentage of 45%. The seeds were removed from the water and drained for 1 h and germinated for 5 days at 17°C and 100% relative moisture (Farzaneh et al., 2017), in a germination chamber (Seedburo model SEARS 255.94299110). Once germinated, the seeds were dried at 50°C/24 h in an oven with warm air circulation (Riosa model HCF-62D) (Ijasan et al. 2011). The malts were cleaned, removing plumules and rootlets, and then ground in a disc mill (model SM4, Brabender GmbH & Co. KG) according to the coarse grind of the Malt-4 method (ASBC, 2004). The malts were stored in airtight bags for later analysis.

2.5 Malt analysis

Moisture and protein analyses were performed according to Malt-3 and Malt-8 methods (ASBC, 2004); reducing sugars by the DNS method (Bello et al., 2006). Amylase quantification was measured following Megazyme Kit manual, α-amylase by AOAC 2002.0I, AACC22-02.0I and ICC Standard No. 303 Methods, β-amylase by BETAMYL-3® Method and amyloglucosidase by McCleary et al. (1991) in malt, in triplicate.

2.6 Malt crude aqueous extracts

2.6.1 Obtaining

The aqueous extracts were prepared in triplicate for three stirring times (30, 120, and 270 min) and two temperatures (30 and 40°C). 1,000 g of ground malt and 10 mL of distilled water were placed in 20 mL polypropylene tubes with screw caps, stirred at maximum power in a vortex (Ika Vortex3) for 10 min until homogeneously dissolved (Sagu et al., 2015). They were incubated with shaking at 180 rpm, to obtain 18 samples (3*2*3). The samples were centrifuged at 10,000 gx, the supernatant was labelled as “crude extract” and filtered using Whatman number 41 filter paper, a vacuum pump with a Kitasato flask and Buchner funnel. The samples were stored in 10 mL propylene tubes at 0°C for subsequent analysis.

2.6.2 Analysis

The total solids analysis was performed using the weight difference method (Ndife et al., 2019). The protein quantification followed the BCA method (García & Vázquez, 1998). The reducing sugars analysis and the activity quantification of α-amylase, β-amylase and amyloglucosidase according to section 2.5 in triplicate.

2.7 ATPS enzyme recovery

Two treatments were selected based on the highest enzyme activity yield for α-amylase, β-amylase ad amyloglucasidase, for Siglo-XXI the 30 min/ 30°C treatment and for Bicentenario 30 min/ 40°C. The treatments were frozen at − 80°C for 36 h and then freeze-dried in a Huanghua Faithful FSF-18N equipment to obtain concentrated enzymes solid samples. Both freeze-dried systems were treated through three ATPS systems: 45% ethanol/10% SC, 35% ethanol/13% SC, 30% ethanol/18% SC. Each ATPS was completed with distilled water and 1 g of sample from each treatment (González, 2018). Each freeze-dried treatment and separation system (2*3) was quantified for α-amylase, β-amylase and amyloglucosidase according to section 2.5 in triplicate.

2.8 Statistic analysis

Data that did not pass the parametric tests were analysed using the Kruskal Wallis test, otherwise, simple ANOVA was used (P ≤ 0.05). When the results of the simple ANOVA presented significant differences (P ≤ 0.05), a Fisher's test was applied. Additionally, a multivariable regression analysis was performed (P ≤ 0.05), to observe correlations between variables.

3.1 Malts analysis

The results of the moisture, reducing sugars, protein, β-amylase, α-amylase and amyloglucosidase activities of the malts obtained from the two varieties of triticale are shown in Table 1.

3.1.1 Moisture

The tested malts displayed moisture levels with values of 6.48 ± 0.23% (Siglo-XXI) and 6.57 ± 0.13% (Bicentenario) with no significant differences. These values match the moisture levels expected in standard base malts, such as "lager" which are dried at temperatures below 100°C to guarantee preservation of all the enzymes produced during malting (O´Rourke, 2002; Stewart, 2012). Triticale malts are characterized by their elevated enzymatic content in comparison with other low moisture malts (< 5%) dried at temperatures above 100°C, such as Munich, brown, amber, and chocolate. The elevated enzymatic concentration exhibited by Triticale malts levels allows their combination with low moisture malts to produce different types of beer (Davies, 2016; Edney & Izydorczyk, 2003). The obtained malts were dried at 50°C, until the samples reached malt base humidity level (5%-6%) to halt enzymatic activity. Elevated temperatures have effect of denaturation on amylolytic enzymes, especially on purified form, values near or above 70°C can lead to enzymatic denaturation, however its impact during malting process is usually lesser (Gebremariam et al., 2013).

3.1.2 Reducing sugars

Siglo-XXI variety yielded 7.66 ± 0.04 g/Kg while Bicentenario reached 8.95 ± 0.05 g/Kg (Table 1). Despite the existence of significant differences (P ≤ 0.05), both values were lower than those obtained by Balcerek et al. (2016). These authors reported values in between 16.5 g/Kg and 18.8 g/Kg for commercial pale barley, wheat, and rye malts used to produce alcoholic distillates. In both cases, the analysed malts were labelled as “base malts”, but the yield of α-amylase (14.98–35.04 CU/g) and β-amylase (3.06–9.55 BU/g) was lower than those exhibited in the present work (Table 1). Every cereal can produce different levels of relative enzyme-sugar relationship, enzymatic activity, and yield of reducing sugars (Guerra et al., 2009). In the first 2 to 3 days of germination, most of the amylolytic enzymes are synthesized, creating a positive relationship with the enzymatic activity while starch is minimally hydrolysed (Durand et al., 2009; Skendi & Papageorgiou, 2018; Stewart, 2012). This may explain the low concentration of reducing sugars reported in the present work for both Triticale varieties, compared to Balcerek et al. (2016), as well as the average reports for barley malts (20 g/L- 21 g/L) (Skendi & Papageorgiou, 2018). However, it does not represent a limitation since the enzymatic units found are larger (Table 1) and they can increase their activity in the presence of substrate as well as Ca²⁺ and in optimal pH ad temperature conditions.

3.1.3 Protein content

The protein content displayed by both malts showed significant differences, Siglo-XXI malt had the highest value (12.30%±0.10%) close to the highest standard percentage for lager malts (10.8%-12.3%). Protein is important for optimal yeast nutrition during the fermentation process, as well as providing stability for beer foam (O´Rourke, 2002; Stewart, 2012). Cereal malts with elevated protein and proteolytic activity values can lead to the disproportionate generation of higher alcohols, producing a strong solvent flavor in beer (Loviso & Libkind, 2019). (Loviso & Libkind, 2019). Malts from other cereals showed that protein concentration values vary between varieties, with numbers in between 8.4%-10%, but their combination with barley or craft malt gives a suitable brewing product (Balcerek et al., 2016). For the present research, the interest was on malts for amylolytic enzyme production with elevated protein content, regardless of its suitability for brewing.

3.1.4 Amylases activity.

The β-amylase activity quantification for Siglo-XXI (37.16 ± 0.10 BU/g) and Bicentenario (28.63 ± 0.10 BU/g) triticale malts (Table 1) was higher than those reported for commercial pale malts of wheat (9.55 BU/g), rye (2.61 BU/g − 8.61 BU/g), and barley (2.98 BU/g-3.06 BU/g) proposed for production of alcoholic distillates by Balcerek et al. (2016a, b). Dziedzoave et al. (2010) reported higher enzyme values for dried-rice malt following 5 days of germination (60 BU/g) with the maximum yield of approximately 170 BU/g, at day 9. However, lower values were observed for corn and millet malts, approximately 20 BU/g under the similar malting conditions. These results suggest that Triticale malt are suitable to yield higher β-amylase levels than wheat, rye, corn, millet, or barley, but lower than whole rice. In the present work, standard germination time for barley malt (5 days) was used as a baseline but no time variations were tested. However, the enzymatic production yield by Triticale varieties could be increased if the germination time is prolonged.

Both tested Triticale malts displayed α-amylase activity levels (Table 1) above those reported by Traoré et al. (2004) in red sorghum malts (58.0 CU/g), millet (37.6 CU/g) and corn (13.7 CU/g) intended to reduce the viscosity of porridge for infants. In a similar work by Dziedzoave et al. (2010), malts were dried after a 5day germination period, similar to current research, where the tested sorghum and millet malts yielded approximately 5 CU/g, while corn and rice reached roughly 25 CU/g. Balcerek et al. (2016) reported 35.04 CU/g in wheat malt, 26.60 CU/g in rye and 9.38 CU/g in barley, and Balcerek et al. (2016) 46.89 CU/g in rye malt and 86.83 CU/g in barley malt. In the present work, Bicentenario triticale malt yielded 113.10 ± 0.53 CU/g and the Siglo-XXI obtained 99.56 ± 1.06 CU/g. Yu & Liu, (2019) and Oza et al. (2020) agree that Tricale malts produce elevated levels of α-amylase following the malting process, greater than other cereals, hinting at a possible use for production of an enzymatic conjugate with biotechnological applications in the starch processing industry.

In the case of amyloglucosidase activity (Table 1), the values obtained were similar to those reported by Dziedzoave et al. (2010) for sorghum, millet and corn malts (≈ 0.05–0.09 U/g) but lower than rice malt (≈ 0.27 U/g). These low values are expected since amyloglucosidase is a heat-sensitive enzyme that denatures during the malting process, the activity analysis can only measure the residual activity. Martin et al. (2019) state that the role of this enzyme is to convert dextrins into glucose by hydrolyzing the alpha (1–6) and alpha (1–4) bonds at the non-reducing end of the chains, a key step in starch processing, and proposed solid or submerged phase fermentation using microorganisms as suitable process for commercial scale production. Supporting the previous idea, Oza et al. (2020) and Singh & Kumar, (2018) indicate that light beers have been produced under extraction conditions similar to this research with excellent results. Guerra et al. (2009) indicate that 99% of the diastatic power of a brewing malt depends on the joint activity of β-amylase and α-amylase; enzymes present in considerable quantities in Triticale malt (Table 1). The low levels of amyloglucosidase can be improved by adding exogenous amylases.

Table 1

Average values for the variables of moisture, reducing sugars, protein, β-amylase, α-amylase and amyloglucosidase, of Bicentenario and Siglo-XXI triticale malts.¹
Malt	Moisture (%)	Reducing sugars (mg/mL)	Protein (%)	β-amylase (BU/g)²	α-amylase (CU/g)³	Amyloglucosidase (U/g)⁴
Bicentenario	6.57 ± 0.13 a	8.95 ± 0.05 a	11.33 ± 0.01 b	28.63 ± 0.10 b	113.10 ± 0.53 a	0.09 ± 0.01 a
Siglo XXI	6.48 ± 0.23 a	7.66 ± 0.04 b	12.30 ± 0.10 a	37.16 ± 0.10 a	99.56 ± 1.06 b	0.08 ± 0.01 a

¹ Different letters between each column indicate significant differences (p ≤ 0.05).

² BU/g: It is defined as the amount of enzyme, in the presence of an excess of thermostable β-glucosidase, required to convert one micromole of p-nitrophenol to PNPβ-G3 in one minute under defined assay conditions.

³ CU/g: It is defined as the amount of enzyme, in the presence of an excess of thermostable α-glucosidase, required to convert one micromole of p-nitrophenol to BPNPG7 in one minute under defined assay conditions.

⁴ (U/mL: One unit is defined as the amount of enzyme needed to convert one µmol of p-nitophenol from the substrate, per minute under defined temperature and pH conditions.

3.2 Analysis of aqueous extracts

Tables 2 and 3 show the results of total solids, reducing sugars, protein, β-amylase, α-amylase and amyloglucosidase.

3.2.1 Total solids

For Bicentenario malt, an increase in the percentage of total solids was observed at both temperatures (40°C and 30°C) as extraction time increased (Table 2), with the highest percentages at 270 minutes, 5.27 ± 0.79% at 30°C, and 6.68 ± 0.12% at 40°C. A multivariable regression analysis showed a positive correlation for time, 30% (P = 0.34), and temperature, 28% (P = 0.36). However, for Siglo-XXI malt, an increasing trend was observed over time without significant differences (P ≤ 0.05), except for the 270 min/40°C treatment, which obtained the highest percentage of total solids with 6.90 ± 0.92%. The correlation with time and temperature was positive, 30% (P = 0.33) and 60% (P = 0.03), respectively. This suggests that for both extract varieties, a significant amount of malt solids was dissolved in the water as a function of stirring time as well as temperature and were retained after the filtration and centrifugation process. These solids are associated to the presence of starchy sugars derived from enzymatic activity and non-starchy (Durand et al., 2009). This could explain the elevated content of total solids in Siglo-XXI triticale malt, considering that the β-amylase results for the malt are higher than those obtained for Bicentenario (37.16 ± 0.10 and 28.63 ± 0.10 BU/ g, respectively), which suggests that it could promote greater degradation of the endosperm, and acting synergistically with temperature to help dissolve more total solids in the extracts.

Duan et al. (2019), indicated in their research on the production of enzymes by solid-state fermentation using microbial strains, that the dry biomass produced by microbes does not necessarily have to be the highest to obtain the highest enzymatic activity. This supports the results obtained in the present work, the extracts at 270 min were not the ones with the highest amount of α-amylase and β-amylase. The extract at 30 min/40°C for the Bicentenario variety (4.35 ± 0.48%) and the extract at 30 min/30°C for the Siglo-XXI malt (4.99 ± 0.44%), showed the lowest total solids percentage, but the highest enzymes yield (Tables 2 and 3).

3.2.2 Reducing sugars

Bicentenario malt showed similar behaviour for both tested extraction temperatures, yielding their highest values at 120 min, 31.21 ± 0.02 mg/mL at 30°C, and 26.78 ± 0.16 mg/mL at 40°C. Most likely, temperature and extraction time favoured an optimal enzymatic hydrolysis reaction generating the highest concentration of reducing sugars. Extracts stirred for 30 min, at both temperatures, had the lowest reducing sugars concentration with the highest yield of amylases (Tables 2 and 3). In contrast, 270 min treatment showed lower reducing sugars concentration than 120 min treatment, for both temperatures (Table 2). This might indicate that the agitation time has a slight inhibition effect. Multivariate regression analysis showed that the correlation between reducing sugars and stirring time was positive (30%, P = 0.34) and a weak negative (-0.34%, P = 0.27) regarding temperature, confirming that at constant temperature, an increment in stirring time (120 min to 270 min) negatively influences the enzymatic activity of malt extracts.

Regarding Siglo-XXI malt, an increase in reducing sugars concentration was observed as extraction time increased, for both temperatures. Multivariate analysis revealed a positive correlation of 68% (P = 0.01) for stirring time and 52% (P = 0.07) for temperature. The treatments with the highest concentration were 270 min/30°C and 270 min/40°C (Table 3), but these treatments did not yield the highest activity quantification for β-amylase and α-amylase, suggesting loss of enzyme activity due to substrate inhibition. Treatments at 30 min stirring time did not show significant differences; these samples had the lowest reducing sugars concentration with the highest yield for β-amylase, α-amylase and amyloglucosidase. Treatment 30 min/ 40°C of Bicentenario malt displayed similar behaviour, however, treatment 30 min/30°C of Siglo-XXI malt yielded the highest enzyme concentration, suggesting that a short extraction period can produce the highest enzyme concentration. However, the time is not sufficient to transform starch into reducing sugars, requiring a prolonged extraction time that leads to loss of enzymatic activity.

Recent studies have estimated the amylolytic enzyme yield produced by microbial strains, measuring the concentration of sugars these enzymes can release in a solid or aqueous medium. Taking the enzymatic unit as "U/mL", which corresponds to the amount of enzyme required to produce 1 µMol/min/maltose, usually determined by the DNS method (Sagu et al., 2015). It has been suggested that a high concentration of sugars derived from enzymatic activity on substrates such as starch results in a high quantification of enzymes, in enzymatic measurement tests (Almanaa et al., 2020; Sagu et al., 2015). In the present research, an opposite behaviour is observed regarding the amylase production process from microbial strains. The highest enzyme concentrations were obtained in treatments with short stirring times and the lowest reducing sugar concentration, suggesting that for enzyme extraction from Triticale seeds, temperature and time variables have an important technological role in aqueous extraction.

3.2.3 Protein

Bicentenario malt treatments at 30°C did not show a significant difference in protein content between the 30 min (P ≤ 0.05; 12.52 ± 0.06 mg/mL) and the 270 min (11.0 ± 0.62 mg/mL) extraction time. Opposite to 40°C treatments, where the increase in extraction time led to increase in protein yield (Table 2). Multivariate regression analysis showed positive correlations of 26% (P = 0.40) for temperature and 51% (P = 0.08) for stirring time, indicating that as temperature and time increased, protein concentration also increased. However, this trend was not showed by the enzymes in the present research work (Table 2). Negative "protein-enzyme" correlations indicated that protein concentration can increase as enzyme concentration decreases, such is the case for α-amylase − 48% (P = 0.11), β-amylase − 63% (P = 0.02) and amyloglucosidase − 10% (P = 0.74). According to Kamal et al. (2021), the behaviour described could be related to the activity degree of other enzymes, such as proteolytic enzymes, which contribute to amylases synthesis and the solubilization of grain reserve protein fractions during malting that can be extracted in aqueous suspension. In addition, the temperature and extraction time variations can lead to deviations in the activity quantification of amylases.

For Siglo-XXI malt treatments, an increasing trend was observed due to the stirring time. For both tested temperatures, the 270 min treatment had the highest protein levels with low amylase activity quantification. The 30 min/ 30°C had the highest amylase activity quantification (9.35 ± 0.40 mg/mL). Contrary to Bicentenario malts, Siglo-XXI malts had no statistically significant correlation (P = 0.57) with respect to temperature and had a positive correlation of 85% (P = 0.01) with respect to extraction time. This suggests that stirring time is a key parameter for protein extraction regardless of the nature of the protein. Interestingly, protein extraction time was negatively correlated with β-amylase extraction by -50% (P = 0.09) and α-amylase by -85% (P = 0.01); showing that the enzymatic protein fraction of the Siglo-XXI malt extracts was achieved with the lowest energy expenditure.

Recent work has proposed that protein concentration in plant materials, such as sweet potato, fluctuates between varieties and it is related to the plant’s ability to produce amylases. However, a small concentration of amylase (3.2 mg/mL) can derive in the production of 55.69 (U/mL) of β-amylase (Hesam et al., 2015). Another research team proposed that with plant materials such as Abrus Precatorius (American regaliz), it is convenient to obtain rich protein extracts in order to ensure the highest β-amylase activity (Sagu et al., 2015). In the present work, a strong correlation between protein concentration and enzymatic activity was not found. It is necessary to further identify and characterize the protein fractions corresponding to amylolytic enzymes.

3.2.4 β-amylase from triticale malts aqueous extracts

For Bicentenario tricale malts, β-amylase yields ranged from 201.13 ± 0.34 BU/mL to 160.79 ± 0.20 BU/mL for 30°C treatments, and in between 223.66 ± 0.27 BU/mL at 149.21 ± 0.27 BU/mL for 40°C treatments. The highest quantification of 223.66 ± 0.27 BU/mL was obtained at 40°C/30 min treatment followed by 30°C/ 270 min treatment with 201.13 ± 0.34 BU/mL (Table 2). Multivariate regression analysis showed weak negative correlations. For temperature, it was − 0.21% (P = 0.99), indicating that as temperature increases, β-amylase decreases from 223.66 ± 0.27 BU/mL to 149.21 ± 0.27 BU/mL. Regarding extraction time, the correlation was − 16% (P = 0.60), displaying a similar behaviour as temperature (Table 2). However, the activity of β-amylase in the extracts could be conditioned by the availability of total solids (substrate), which was negatively correlated (-41%, P = 0.17).

For Siglo-XXI triticale malt treatments, the 30°C/ 30 min treatment (Table 3) showed the highest activity quantification (255.71 ± 0.12 BU/mL) followed by 30°C/120 min (254.09 ± 0.07 BU/mL) with significant differences (P ≤ 0.05). This suggests that increasing the stirring time or temperature has a negative impact on β-amylase activity, which was confirmed by multivariate regression analysis, showing a negative correlation of -50% (P = 0.09). The temperature variable also had a negative correlation (-81%, P = 0.01) and showed a similar behaviour, increase in temperature led to a decrease in β-amylase yield, from 255.7 BU/mL (30°C treatment) to 207.35 BU/mL (40°C treatment).

The β-amylase activity values displayed by both malt treatments (Bicentenario and Siglo-XXI) observed in this study (Tables 2 and 3) were higher than those reported by previous literature. Sagu et al. (2015), obtained 20.42 BU/mL yield in aqueous extracts of Abrus precatorius (American regaliz) stems, while Hesam et al. (2015), reported 55.69 BU/mL yield in aqueous extracts of sweet potato. Recent reports indicate that β-amylase production by microbial strains tends to be more efficient. Duan et al. (2019) reported a yield of 128.9 BU/mL produced by Bacillus Choshinensis fermentation in a polypeptone, glycerol and hydrated salts enriched medium. The addition of carbon sources (glucose), nitrogen (pig bone peptone) and metal ions (Mg2+) to the fermentation medium increased the production to 5,371.8 BU/mL (Duan et al, 2019). The values obtained in the present research are above those reported for similar plant materials by Sagu et al. (2015) and Hesam et al. (2015) but well below those reported for microbial fermentations by Duan et al. (2019).

3.2.5 α-amylase from triticale malts aqueous extracts

For Bicentenario malt treatments, α-amylase yields ranged from 1,222.25 ± 3.32 to 847.18 ± 2.65 CU/mL for 30°C treatments, and from 1,212.07 ± 3.32 to 776.27 ± 1.99 CU/mL for 40°C, with significant differences (P ≤ 0.05) between all treatments (Table 2).The highest α-amylase activity quantification of 1,222.25 ± 3.32 CU/mL was obtained at 30 min/30°C treatment, followed by 30 min/40°C treatment with 1,212.07 ± 3.32 CU/mL. The results showed that the increase in stirring time and temperature had a negative impact on the α-amylase presence. This was confirmed by multivariate regression analysis (P ≤ 0.05), which showed negative correlations of -51% (P = 0.08) for stirring time and of -28% (P = 0.36) for temperature. Indicating that as stirring time increases, α-amylase activity decreases, exhibiting a similar behaviour as the temperature variable.

The α-amylase results for Siglo-XXI malt showed a similar behaviour than those of Bicentenario malts. Treatment 30 min/30°C had the highest activity quantification, 1,268.89 ± 1.32 CU/mL (Table 3). The multivariable regression analysis also showed negative correlations for stirring time (-85%, P = 0.01) and temperature (-41%, P = 0.17), indicating that as the variable increases there is a decrease in α-amylase (Table 3). This suggests that to obtain enzyme with the elevated activity quantification, from Siglo-XXI and Bicentenario triticale malts, the extraction process must have a short stirring time and low temperature. These conditions make the production of α-amylase concentrated a viable option at industrial scale.

Previous studies have shown that microbial α-amylase is suitable for use in industrial processes (Oza et al., 2020). Simair et al. (2017) focused on the use of agroindustrial residues as carbon/nitrogen source for microbial α-amylase production and reported similar and higher values than those found in the present research. The authors indicated that bacillus sp. BCC 01–50 strain produced near 600 CU/mL using a fermentation medium with glucose, yeast extract, magnesium sulphate heptahydrate and monopotassium phosphate, at 37°C and agitation at 150 rpm for 48 h. This value is much lower than Siglo-XXI triticale malt yield under 30°C/30 min treatment,1,268.89 ± 1.32 CU/mL. The authors also evaluated incorporating molasses and meat extract as carbon/nitrogen source, fermentation at 50°C for 60 h reported the production of 4,500 CU/ mL α-amylase, a higher yield than the previous fermentation treatments. Based on the present work, 30 min/ 30°C treatment delivered elevated α-amylase yields for both malt varieties, however, these values are lower than those reported for several microbial fermentations. Aqueous extraction is a suitable process that requires less time and energy expenditure than fermentations. It is simple in its preparation, susceptible to optimization and environmentally friendly, the only compound required for their extraction is distilled water and undissolved malt can function as a material for enriching livestock feed or composting for plant nutrition.

3.2.6 Amyloglucosidase from triticale malts aqueous extracts

Bicentario malt yielded the highest amyloglucosidase values in the 30°C treatments under 30 min (0.67 ± 0.03 U/g) and 120 min (0.64 ± 0.02 U/g) stirring time, with no significant differences (P ≤ 0.05). For 40°C treatments, the highest activity was reported for 30 min (0.52 ± 0.01 U/g) stirring time. For both temperatures, a decrease in activity quantification was observed as the stirring time increased. A multivariate regression analysis showed negative correlations for temperature (-26%; P = 0.40), indicating that as temperature increases the enzyme yield, decreases. Stirring time also had a negative correlation (-70%, P = 0.01), displaying the similar negative impact on enzyme activity as time increases.

For Siglo-XXI malt, both temperature treatments yielded the same highest activity quantification (0.48 U/g) under 120 min (30°C) and 270 min (40°C) (Table 3). Temperature showed a positive correlation (22%, P = 0.48) while stirring time had a negative correlation, close to zero (-0.008%, P = 0.98). The results show that Siglo-XXI malt tends to produce less amyloglucosidase than Bicentenario malt, but higher α-amylase and β amylase activity under lower energy expenditure treatment (30 min/30°C).

In recent works, microbial strains have proven to be superior sources of amyloglucosidase. Aspergillus isolated from different soils types are capable of producing enzymes with activities ranging from 83.92 U/g (A. niger OTF ) to 886.25 U/g (A. niger NRRL 3122) through solid-phase fermentation, using agroindustrial residues under adjusted moisture, temperature and pH conditions (Pervez et al., 2015; Colla et al., 2017; Osho & Solomon, 2020). A different group reported that Leohumicola embedata have produced 2.17 U/g (Adeoyo et al., 2018), these values are notably higher than those reported for the triticale Bicentenario and Siglo-XXI malts in this work, which range from 0.36 to 0.67 U/g and are not a representative value but shows the presence of the enzyme.

Based on the β-amylase and α-amylase activity yields displayed by tested triticale malt aqueous extracts, treatments 30 min/40°C for Bicentenario variety, and 30 min/30°C for Siglo-XXI were selected to test enzymatic recovery using ATPS systems composed of ethanol and SC.

Table 2

Average values for the variables of total solids, reducing sugars, protein, β-amylase, α-amylase and amyloglucosidase of the aqueous extracts of bicentenario variety triticale malt.^{1, 2}
Treatment		Total solids (%)	Reducing sugars (mg/mL)	Protein (mg/mL)	β-amylase (BU/mL)^{2. 3}	α-amylase (CU/mL)⁴	Amyloglucosidase (U/g)⁵
Time (min)	Temperature (°C)	Total solids (%)	Reducing sugars (mg/mL)	Protein (mg/mL)	β-amylase (BU/mL)^{2. 3}	α-amylase (CU/mL)⁴	Amyloglucosidase (U/g)⁵
30	30	3.42 ± 0.61 c	18.47 ± 0.37 e	12.52 ± 0.06 b	160.79 ± 0.20	1,222.40 ± 3.32 a	0.67 ± 0.03 a
120	30	4.55 ± 0.06 b	31.21 ± 0.02 a	10.00 ± 0.40 c	162.27 ± 0.76	847.18 ± 2.65 e	0.64 ± 0.02 a
270	30	5.27 ± 0.79 b	24.85 ± 0.20 c	11.00 ± 0.62 b	201.13 ± 0.34	1,133.17 ± 3.32 c	0.30 ± 0.01 d
30	40	4.35 ± 0.48 b	16.72 ± 0.06 f	9.21 ± 0.71 d	223.66 ± 0.27	1,212.07 ± 3.32 b	0.52 ± 0.01 b
120	40	5.40 ± 0.14 b	26,78 ± 0.16 b	11.87 ± 1.01 b	149.21 ± 0.27	902.12 ± 1.99 d	0.43 ± 0.01 c
270	40	6.68 ± 0.12 a	20.78 ± 0.20 d	16.03 ± 0.54 a	151.03 ± 7.32	776.27 ± 1.99 f	0.46 ± 0.01 b

¹ Different letters between each column indicate significant differences (p ≤ 0.05).

² The average values that do not present literals were analyzed using a Kruskal Wallis test, since the data did not present normality or homoscedasticity.

³ BU/mL: It is defined as the amount of enzyme, in the presence of an excess of thermostable β-glucosidase, required to convert one micromole of p-nitrophenol to PNPβ-G3 in one minute under defined assay conditions.

⁴ CU/mL: It is defined as the amount of enzyme, in the presence of an excess of thermostable α-glucosidase, required to convert one micromole of p-nitrophenol to BPNPG7 in one minute under defined assay conditions.

⁵ U/g: One unit is defined as the amount of enzyme needed to convert one µmol of p-nitophenol from the substrate, per minute under defined temperature and pH conditions.

Table 3

Average values for the variables of total solids, reducing sugars, protein, β-amylase, α-amylase and amyloglucosidase, of aqueous extracts of siglo XXI variety triticale malt.^{1, 2}
Treatment		Total solids (%)	Reducing sugars (mg/mL)	Protein (mg/mL)²	β-amylase (BU/mL)³	α-amylase (CU/mL)⁴	Amyloglucosidase (U/g)⁵
Time (min)	Temperature (°C)	Total solids (%)	Reducing sugars (mg/mL)	Protein (mg/mL)²	β-amylase (BU/mL)³	α-amylase (CU/mL)⁴	Amyloglucosidase (U/g)⁵
30	30	4.99 ± 0.44 b	26.44 ± 0.18 e	9.35 ± 0.40	255.71 ± 0.12 a	1,268.89 ± 1.32 a	0.40 ± 0.01 b
120	30	5.39 ± 0.76 b	30.91 ± 0.11 d	10.53 ± 0.18	254.09 ± 0.07 b	1,103.59 ± 1.32 c	0.48 ± 0.01 a
270	30	5.01 ± 0.60 b	33.99 ± 0.39 c	12.11 ± 0.05	246.89 ± 0.07 c	1,041.13 ± 0.66 d	0.37 ± 0.01 b
30	40	5.60 ± 0.59 b	26.29 ± 0.30 e	9.04 ± 0.01	238.21 ± 0.20 d	1,173.09 ± 2.65 b	0.47 ± 0.02 a
120	40	5.94 ± 1.12 b	43.29 ± 0.09 b	8.37 ± 0.52	230.81 ± 0.07 e	1,032.21 ± 1.32 e	0.36 ± 0.01 b
270	40	6.90 ± 0.92 a	46.79 ± 0.13 a	12.83 ± 1.10	207.39 ± 0.13 f	947.68 ± 5.31 f	0.48 ± 0.02 a

¹ Different letters between each column indicate significant differences (p ≤ 0.05).

² The average values that do not present literals were analyzed using a Kruskal Wallis test, since the data did not present normality or homoscedasticity.

⁵ U/g: One unit is defined as the amount of enzyme needed to convert one µmol of p-nitophenol from the substrate, per minute under defined temperature and pH conditions.

3.3 Recovery of enzymes present in aqueous extracts by ATPS

Tables 4 and 5 show the results of activity quantification of enzymes recovered by ATPS alcohol/SC, applied to previously selected aqueous extracts for Bicentenario (30 min/40°C) and Siglo-XXI (30 min/30°C) malts.

Table 4

Average values of the content of enzymes (β-amylase, α-amylase, Amyloglucosidase) recovered in each phase of the Alcohol-Sodium citrate systems, applied to the aqueous extract of bicentenario triticale malt (40°C for 30 min).¹
System		β-amylase (BU/mL)²	α-amylase (CU/mL)³	Amyloglucosidase (U/g)⁴
Alcohol (%)	Sodium citrate (%)	β-amylase (BU/mL)²	α-amylase (CU/mL)³	Amyloglucosidase (U/g)⁴
Upper phase
45	10	26.87 ± 0.63 a	426.88 ± 1.99 c	nd
35	13	39.25 ± 0.42 b	40.86 ± 1.99 a	0.06 ± 0.01 a
30	18	46.99 ± 0.07 c	346.57 ± 1.33 b	0.13 ± 0.01 a
Lower phase
45	10	8.88 ± 0.42 b	956.13 ± 1.33 b	0.09 ± 0.01 a
35	13	5.52 ± 0.14 a	182.68 ± 4.65 a	nd
30	18	9.22 ± 0.35 b	996.99 ± 0.66 c	0.11 ± 0.02 a

¹ Different letters between each column indicate significant differences (p ≤ 0.05).

² BU/mL: It is defined as the amount of enzyme, in the presence of an excess of thermostable β-glucosidase, required to convert one micromole of p-nitrophenol to PNPβ-G3 in one minute under defined assay conditions.

³ CU/mL: It is defined as the amount of enzyme, in the presence of an excess of thermostable α-glucosidase, required to convert one micromole of p-nitrophenol to BPNPG7 in one minute under defined assay conditions.

⁴ U/g: One unit is defined as the amount of enzyme needed to convert one µmol of p-nitophenol from the substrate, per minute under defined temperature and pH conditions.

Table 5

Average values of the content of enzymes (β-amylase, α-amylase, Amyloglucosidase) recovered in each phase of the Alcohol-Sodium Citrate systems, applied to the aqueous extract of siglo XXI triticale malt (30°C for 30 min).¹
System		β-amylase (BU/mL)²	α-amylase (CU/mL)³	Amyloglucosidase (U/g)⁴
Alcohol (%)	Sodium citrate (%)	β-amylase (BU/mL)²	α-amylase (CU/mL)³	Amyloglucosidase (U/g)⁴
Upper phase
45	10	15.38 ± 0.42 a	111.30 ± 0.66 a	nd
35	13	52.81 ± 0.63 b	406.22 ± 1.99 c	0.06 ± 0.01 a
30	18	51.43 ± 0.35 b	228.70 ± 1.99 b	0.20 ± 0.02 b
Lower phase
45	10	11.93 ± 0.14 a	284.59 ± 1.33 a	0.10 ± 0.01 a
35	13	13.12 ± 0.14 b	398.70 ± 1.99 b	nd
30	18	12.48 ± 0.35 b	1514.03 ± 3.98 c	nd

¹ Different letters between each column indicate significant differences (p ≤ 0.05).

⁴ U/g: One unit is defined as the amount of enzyme needed to convert one µmol of p-nitophenol from the substrate, per minute under defined temperature and pH conditions.

The three tested ATPS systems showed partition behaviour variations for each enzyme. Considering the highest activity quantification values of β-amylase, α-amylase and amyloglucosidase, the most effective ATPS system was 30% Alcohol/18% SC for both triticale malts. Bicentenario malt, 30 min/40°C aqueous extract showed a partition of 46.99 ± 0.07 BU/mL on the upper level and 996.99 ± 0.66 CU/mL on the lower level, while the recovery of amyloglucosidase was similar in both phases (Table 4). Siglo-XXI malt, 30 min/30°C aqueous extract reported a partition of 51.43 ± 0.35 BU/mL and 1,514.03 ± 3.98 CU/mL in the upper and lower phases, respectively, and 0.20 ± 0.02 (U/g) of amyloglucosidase in the upper phase. The tested ATPS systems showed a tendency, in all the systems β-amylase migrated to the upper phase, enriched with alcohol, while α-amylase migrate towards the lower phase supplemented with sodium citrate. A definite trend was not detected for amyloglucosidase.

It was observed that increasing the percentage of sodium citrate improved α-amylase and β-amylase recovery. This agrees with reported research stating that ATPS systems prepared with varying MW polyethyleneglycol and salts, such as sodium sulphate, monopotassium phosphate and sodium citrate, significantly affected the partitioning coefficient of amylases between phases. β-amylase and amyloglucosidase showed a tendency to migrate and remain in the salt-rich phase, when salt concentration was increased to an optimum point, however, higher than this concentration, the enzyme precipitated. A similar effect manifest when the molecular weight of the selected polymer increases, this change increases hydrophobicity and the exclusion volume in the polymer-rich phase (Shahriari et al., 2010).

For Siglo-XXI variety, the 30% alcohol/18% SC system yielded the highest α-amylase quantification at the lower phase enriched with soudium citrate, however, this partitioning behaviour could be influenced by the pH of the system. Nascimento et al. (2018) reported that pH had the greatest influence on the formation of upper, lower and interphase phases, on the tendency of α-amylase to migrate to the lower salt-rich phase at pH 5.0 or to the polymer-rich upper phase at pH 4.0 and affected the relationship with intermolecular interactions in enzyme partitioning. Recently, the partial purification of amylases produced by Bacillus Subtillis C10, Aureobasidium pullulans and Aspergillus niger using a polymer/salt ATPS has been investigated. The authors reported α-amylase tendency to migrate to the upper phase when systems are composed of PEG 20%-48% with molecular weights between 4000 g/mol and 6000 g/mol and salts such as potassium phosphate, sodium citrate, sodium chloride, lithium sulfate or sodium sulfate in concentrations of 7.5%-18%. These systems generate yields in between 59.37%-88% based on test results and experimental designs carried out by the authors. Different authors have also reported that cations present in the salts, such as Na+, influence the formation and size of the biphasic region, which could lead to a means to improve key parameters such as purification factor and the partitioning coefficient (Ademakinwa et al., 2019; dos Santos et al., 2020; Loc et al., 2010).

Results similar to those presented in this research have been reported for different plant matrices employing similar ATPS systems. Sagu et al. (2015) applied aqueous triphasic partitioning systems to yield β-amylase from aqueous extracts of Abrus precatorius (American regaliz) stems. In these systems they varied the pH and the crude extract ratios of β-amylase/t-butanol, and ammonium sulfate. It was observed that a high saturation of ammonium sulfate, in combination with a low crude extract/t-butanol ratio, significantly improved parameters such as enzymatic activity recovery and purification factor. Shad et al. (2018) used ATPS systems with PEG of varying concentrations (10–18%) and molecular weight, sodium citrate (12–20%) and NaCl, to partially purify α-amylase from extracts made of white pitahaya (Hylocereus spp.) peel. Comparing the different systems, increasing the components concentration considerably improved the partition coefficient and purification factor of system 14% PEG (6000g/mol)/16% sodium citrate/5% NaCl. The increase in concentration facilitates adequate electrostatic potential among the phases, leading to hydrophobic interactions between proteins and components. However, increasing salt concentration can lead to “salting-out” effect, where enzymes migrate towards the phase with the lowest salt concentration, commonly the upper phase, and losing enzyme in the interface. Additionally, the precipitation effect can lead to enzyme concentrating in the lower phase and hindering their recovery. In this research, the tested systems displayed varying levels of salting-out, leading to low quantification of the three studied enzymes where β-amylase was the most affected. The 30% alcohol/18% SC system use on Siglo-XXI malt treatment 30 min/ 30°C, displayed the highest β-amylase and α-amylase quantification. These results could indicate that these are the optimal extraction conditions and the suitable combination of salt and organic solvents that allows optimal functioning (Sagu et al., 2015). In a different study, Aurélien et al. (2019) used a polyethylene glycol/ammonium sulfate ATPS to purify α-amylase from Burnatia enneandra Micheli extracts and reported 12% as the optimal concentration of ammonium sulfate that can yield the maximum purification factor. Increasing the concentration above this range produced a decrease in enzyme since the protein precipitated to the lower phase.

In the present research, the lower phase yielded the highest α-amylase quantification and this value increased as sodium citrate concentration increased to 18%. If protein precipitation took place, it was minimal, especially in ATPS system 30% alcohol/18% SC applied to 30min/30°C Siglo-XXI malt compared against other systems tested for the same extract and those tested for extract 30 min/40°C of the Bicentenario malt. Figure 1 and Fig. 2 compares the quantification values obtained from the spent malt generated from the extraction treatment, from the clarified aqueous extract as well as the total enzyme yield, the sum of the values obtained for the three enzymes, against the values obtained from the ATPS treatments for both malt varieties. The purification yields achieved by ATPS systems were significantly lower for β-amylase and amyloglucosidase, the result could be attributed to the retention of enzymes at the interface. This effect was less noticeable in the 30% alcohol/18% SC system used with the 30 min/30°C treatment for Siglo-XXI malt

Our research showed that the most profitable amylase extraction treatment for Siglo-XXI triticale variety was 30 min/ 30°C, yielding the highest quantifications of enzymatic activity for β-amylase, α-amylase and amyloglucosidase. For the Bicentenario variety, treatment 30 min/ 40°C showed the best enzymatic yield. Both treatments have the shortest stirring time tested and use “low temperatures”, however Siglo-XXI does require a smaller energy expenditure. The results show Siglo-XXI malt as a suitable choice for amylases production combined with ATPS recovery system integrated with ethanol and sodium citrate (Na₃C₆H₅O₇) that helps enzymes retaining its highest activity. Interestingly, the ATPS system with the highest salt concentration with respect to ethanol had the highest recovery yield. Regarding the correlations between protein, total solids, and enzymatic activity, it is necessary to further characterize the protein fractions of the extracts to determine the fraction corresponding to the enzymes as well as their relationship with the production of sugars.

Ethical Approval and Consent to Participate. This is not applicable.

Consent for publication. We, the authors, give consent for the information and data to be published in the Journal Plant Foods for Human Nutrition.

Authors' contributions. We declare the participation and contribution of each author as follows:

Diego Girón Orozco: experimental design, methodology, statistical analysis, laboratory analysis, data curation, and writing the manuscript.

María Dolores Mariezcurrena Berasain: funding acquisition, laboratory analysis, and writing the manuscript.

Oscar Aguilar: experimental methodology, experimental support guidance, and editing of the manuscript.

José Francisco Ramírez Dávila: funding acquisition, provided the triticale seeds, and data curation.

Erick Heredia Olea: conceptualization, methodology, writing and editing of the manuscript.

Funding. This research was founded by UAEMéx project whit number 6169/2020CIB, and CONACYT, grant number 2020-000026-02NACF-26828.

Availability of data and materials. The data of this research paper can be provided by the corresponding author via email ([email protected]).

Conflicts of interest/Competing interest: Not applicable

Code availability: Not applicable

Consent to participate: Not applicable

Acknowledgments: We thank the Consejo Nacional de Ciencia y Tecnología CONACYT for the scholarship with registration number 2020-000026-02NACF-26828 granted to Diego Girón Orozco to carry out the doctorate in Ciencias Agropecuarias y Recursos Naturales of the Universidad Autónoma del Estado de México registered in the Programa Nacional de Posgrados de Calidad. We also thank the Universidad Autónoma del Estado de México for the economic resource of the project with registration 6169/2020CIB and for providing access to Facultad de Ciencias Agrícolas laboratories to carry out this research. Lastly, we thank the Tecnológico de Monterrey for providing access to Centro de Biotecnología FEMSA laboratories and the authors of this article.

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Recovery of amylolytic enzymes from triticale malt (X. Triticosecale Wittmack) using two-phase aqueous systems

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Raw material

2.2 Experimental design

2.3 Malt production

2.5 Malt analysis

2.6 Malt crude aqueous extracts

2.6.1 Obtaining

2.6.2 Analysis

2.7 ATPS enzyme recovery

2.8 Statistic analysis

3. Results And Discussion

3.1 Malts analysis

3.1.1 Moisture

3.1.2 Reducing sugars

3.1.3 Protein content

3.1.4 Amylases activity.

3.2 Analysis of aqueous extracts

3.2.1 Total solids

3.2.2 Reducing sugars

3.2.3 Protein

3.2.4 β-amylase from triticale malts aqueous extracts

3.2.5 α-amylase from triticale malts aqueous extracts

3.2.6 Amyloglucosidase from triticale malts aqueous extracts

3.3 Recovery of enzymes present in aqueous extracts by ATPS

4. Conclusion

Declarations

References

Additional Declarations

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