Yellow mombin and jackfruit seeds residues applied in the production of reducing sugars by a

crude multi-enzymatic extract produced by Penicillium roqueforti ATCC 101110

Short title: Yellow mombin and jackfruit seeds for bioprocess

George Lima MARQUES1, Elizama AGUIAR-OLIVEIRA1,*

1
Dept. of Exact Sciences and Technologies (DCET), State University of Santa Cruz (UESC), Ilhéus,

Bahia, Brazil

* Corresponding author: eaoliveira@uesc.br

ABSTRACT

BACKGROUND

As an alternative to the use of widely investigated agro-industrial residues, this work aimed to promote

the valorization of two selected residues – yellow mombin seed (YS) and jackfruit seed (JS) - due to

their enhanced performance.

RESULTS

YS was applied as a solid state substrate for Penicillium roqueforti ATCC 101110 cultivation (25 °C, Aw

= 0.963, 107 spores g-1 and 142 h) to produce a crude multi-enzymatic extract (CE-YS) containing

activities of CMCase = 31.95 U g-1, xylanase = 56.85 U g-1, exoglucanase = 5.55 U g-1 and FPase =

24.60 U g-1. CE-YS was then applied to six different residues saccharification and the best performance

was obtained with jackfruit seed residue (JS) which was selected for enzymatic saccharification. The

highest productivity of reducing sugars (RS) expressed as glucose (6.26 mg g-1 h-1) was obtained under

the following conditions: 40.7 g L-1 JS, 5 mM MgCl2, 65 °C, 120 rpm, pH 3.0 (citrate buffer 50 mM) and

18 h.

CONCLUSIONS

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jsfa.10377.

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The residues, YS and JS, can be used satisfactorily for the production of bioproducts of great industrial

applicability such as crude extracts (containing cellulolytic enzymes) and RS (which can be converted

into bioethanol, for example).

Keywords: Artocarpus intergrifolia L.; enzymatic saccharification; solid state fermentation; Spondias

mombin L.

1. INTRODUCTION

The use of agro-industrial residues should be stimulated since these low value compounds with

limited applicability can become a new substrate for valuable products. Thus, the bioconversion of these

agro-industrial residues generally requires (prior fermentation) some pre-treatment steps

(saccharification) in such a way as to turn them into more easily fermentable substrates.1,2 However, it

is important to remember that the execution of saccharification should not be highly complex nor demand

excessive amounts of chemical reagents and energy. For each type of residue, different pre-treatments

are suggested; for rigid residues (higher lignin content) and/or complex composition, techniques such

as higher heat or pressure, light acids and bases may be applied.3,4 The fruits of Spondias mombin L.

(yellow mombin) and Artocarpus heterophyllus (jackfruit) are well appreciated in tropical regions.5,6 Their

seeds, and consequently other seeds, are proposed in this present work to have a potential for different

applications such as fermentation and saccharification, rather than just being discharged.

There are several reports of solid-state fermentations conducted with agro-industrial residues

by filamentous fungi such as Aspergillus7,8, Penicillium9,10 and Rhizopus11,12. During fermentation, the

fungi produce different enzymes that can be recovered as a "multi-enzymatic crude extract" by washing

and/or pressing the fermented substrates.13-15 The application of enzymes in different processes may

become more widespread with the application of unpurified extracts, which contain significant activity of

certain enzymes of interest.

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 Thus, in view of previous results on the cultivation of Penicillium roqueforti ATCC 10110 on

different abundant agro-industrial residues in the southern and southwestern regions of Bahia (Brazil),

rice straw and yellow mombin seed were evaluated as substrates for cultivation in order to produce

crude multi-enzymatic extracts, which were then applied in the saccharification of other six agro-

industrial residues.14-18 Based on the best performances, the yellow mombin seed was selected for the

production of an extract that was, in turn, applied to saccharification of the jackfruit seed. Finally,

saccharification conditions were evaluated in order to increase the release of reducing sugars by more

than 60 %.

2. MATERIAL AND METHODS

2.1. Microorganism preparation

Penicillium roqueforti ATCC 10110 belongs to the Laboratory of Biotransformation and Organic

Biocatalysis (LABIOCAT) of the State University of Santa Cruz (UESC, Ilhéus, Bahia, Brazil) and was

originally acquired from the Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil) and it is

registered under the number 40075 (lot 079840075) at INCQS (Fiocruz, Rio de Janeiro, Brazil). The

activation was performed in Petri dishes (Agar Agar and Potato Dextrose Agar) and incubated at 27.5

°C for seven days in a B.O.D. (SOLAB SL 101, Piracicaba - SP - Brazil).14 The inoculum was prepared

with a suspension of cells from the Petri dishes in 0.1 mL L-1 Tween 80 aqueous solution and counting

was performed in a Neubauer chamber using a binocular microscope (Medilux MDL 150 BAI/BPI) with

magnification adjusted to 40x.

2.2. Agro-industrial residues

Agro-industrial residues - acerola bagasse (Malpighia emarginata), peanut shell (Arachis

hypogaea), rice straw (Oryza sativa L.), yellow mombin seed (Spondias mombin L.), jackfruit seed

(Artocarpus intergrifolia L.), mango seed (Mangifera indica) and coffee straw (Coffea arabica) - were

purchased from producers in the southern and southwestern regions of Bahia (Brazil). After removal of

dirt, the residues were minced with a knife and oven-dried (TECNAL model TE-393/1) at 70 °C for 24 h

and ground in a Willey knife mill (ACB LABOR). The meal from each residue was obtained by sieving

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(2 mm grain size or 10 mesh) and named, respectively, AB, PS, RI, YS, JS, MS and CS. These residues

were characterized in triplicate for their cellulose, hemicellulose and lignin content (g kg-1) according to

standard procedures.18

2.3. Cultivating and obtaining crude multi-enzymatic extracts

The residues RI and YS were applied individually as substrate for solid-state cultivation of P.

roqueforti ATCC 10110, which conditions were previously defined for RI as 82 h, 32 °C and Aw = 0.984

and for YS as 142 h, 25 °C and Aw = 0.963, both with 10 g of substrate and an inoculum of 107 spores

g-1 dry residue.14,16 Crude multi-enzymatic extracts (CE) were obtained by pressing the fermented

substrate with citrate buffer (pH 4.8 / 50 mM) at a ratio of 5:1 (mL:g), followed by centrifugation at 1,250

g for 10 min; the extracts were identified as CE-RI and CE-YS.

2.4. Enzymatic saccharification of residues

The two crude extracts obtained, CE-RI and CE-YS, were applied to saccharification of six agro-

industrial residues: AB, PS, RI, JS, MS and CS. The reaction occurred with the addition of: 20 mL of

CE-RI or CE-YS solution (0.01 L L-1) in citrate buffer (50 mM / pH 4.8), 0.4 g residue and 0.1 mL sodium

azide solution (0.05 g L-1) to prevent microbial contamination.14,18 Saccharification was performed in an

orbital shaker (QUIMIS Q816M20, Diadema, Brazil) at 40 °C and 120 rpm for 24 h and aliquots were

removed at 1, 2, 6, 18 and 24 h intervals. The control was performed by replacing the CE with sodium

citrate buffer (pH 4.8 / 50 mM) in the mixing reaction. The medium was then centrifuged at 1,250 g / 10

min and the supernatant was used to estimate the amount of reducing sugars (RS). For the continuity

of the study, it was chosen YS as a substrate for fungi cultivation and production of a multienzimatic

crude extract and JS as a substrate for enzymatic saccharification.

2.5. Effect of ions on enzymatic saccharification of jackfruit seed

The effect of the individual addition of certain salts - NaCl, MgCl2, CuSO4, MnSO4 and ZnCOOH

- to the enzymatic saccharification of JS was investigated, in triplicate, under the same conditions

described above. Subsequently, 0.2 mL of 5 mM saline solution was added to the reactive medium,

including the reaction blank.14,18 The concentration of reducing sugars obtained (RSsalt) after 18 h of

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reaction was compared to the value obtained without the addition of any salt (RSo = 65.12 ± 4.29 mg g-
1
) according to the ratio: [(RSsalt/RSo)·100%]. Based on the results, the salt MgCl2 was selected for its

activation (ratio > 100%) and it was used in the next step of the study.

2.6. Study of enzymatic saccharification conditions of jackfruit seed

The study of the best conditions of enzymatic saccharification of JS by CE-YS was carried out

with the statistical tool of experimental design.19 The effects of combinations of different factor levels,

namely orbital agitation (A = 75 - 225 rpm), temperature (T = 35 - 65 ºC), pH (pH = 3.0 - 9.0), JS

concentration (JS = 5.0 - 45.0 g L-1) and activating salt (MgCl2) concentration (Mg = 1.0 - 9.0 mM), were

investigated on the reducing sugars response obtained per gram of JS (RS, mg g-1) after 18 h of reaction.

For this, a 25-1 Fractional Factorial Desing (FF) matrix was applied and the Effects Analysis was

performed with the help of the statistical software Protimiza Experimental Design (https://experimental-

design.protimiza.com.br/) with 95 % of confidence (α = 0.05). According to the results, the conditions for

saccharification were defined as T = 65 °C, pH = 3.0, Mg = 5.0 mM and t = 18 h.

Next, a new matrix was applied, a 22 Central Composite Rotatable Design (CCRD) for further

analysis of the new levels of factors: A = 75 - 150 rpm and JS = 25 - 75 g L-1; the response RS was

determined after 18 h of reaction. The regression coefficient analysis was performed and the term

statistically not significant at 90 % of confidence (α = 0.10) was removed; therefore, the analysis of

variance (ANOVA) of the reduced quadratic model was performed.19 Finally, the best experimental

conditions were defined as JS = 40.7 g L-1; T = 65 °C, pH = 3.0, Mg = 5.0 mM, A = 120 rpm and t = 18

h and the saccharification was performed in triplicate and RS was quantified.

2.7. Determination of enzymatic activities and reducing sugars

One unit of an enzymatic activity (U) was defined as the amount of enzyme required to release

1 µmol of reducing sugar (RS) release from a given substrate per minute. The dinitro-salicylic acid (DNS)

methodology was used for RS determination; the reaction occurred with 0.5 mL of the solution or crude

extract with 0.5 mL of DNS reagent, boiled for 5 min, then 4.0 mL of distilled water was added, and

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absorbance was read on spectrophotometer at 540 nm (BEL PHOTONICS SF200DM – UV Vis – 1000

nm).18

Enzyme activities (U mL-1) were calculated according to Equation 1 from the values of absorbance

obtained after the DNS reaction (abs), the slope of the DNS calibration curve made with glucose [a =

0.680 μmol abs mL-1, R2 = 0.99], the enzymatic reaction time (t, min) and the dilution factors of the DNS

reaction (fdns = 10.0), the enzymatic saccharification (fsac = 100.5) and the crude extract dilution (fext),

when necessary.

(𝑎𝑎𝑎𝑎𝑎𝑎)∙(𝑎𝑎)
(𝑈𝑈 𝑚𝑚𝑚𝑚−1 ) = ∙ (𝑓𝑓𝑒𝑒𝑒𝑒𝑒𝑒 ) ∙ (𝑓𝑓𝑠𝑠𝑠𝑠𝑠𝑠 ) ∙ (𝑓𝑓𝑑𝑑𝑑𝑑𝑑𝑑 ) (01)
(𝑡𝑡)

Previous works were used to define the reaction conditions for each enzyme.14,18,20

Exoglucanase activity was determined at 70 °C and 10 min, using a 10 g L-1 Avicel® solution in Tris-HCl

buffer (pH 8.5 / 50 mM) as substrate. CMCase and xylanase activities were performed at 50 °C and 10

min with 10 g L-1 solutions of carboxymethylcellulose (CMC, Cromoline) and xylan (Sigma-Aldrich),

respectively, in sodium citrate buffer (pH 4.8 / 50 mM). For filter paper activity (FPase or total cellulases),

a Whatman nº 1 filter paper strip (1.0 cm x 6.0 cm) was used and the reaction occurred in a sodium

citrate buffer at 50 °C and 60 min.

To express the reducing sugars per gram of jackfruit seed residue (RS, mg g-1), the reaction of

DNS was performed, as described above, after saccharification and calculation followed Equation 2 in

which is necessary the amount of JS used (mJS = 0.4 g) and the volume of saccarification (Vsac = 20.1

mL). The RS was also expressed as productivity (mg g-1 h-1) considering the time of saccharification.

(𝑉𝑉𝑠𝑠𝑠𝑠𝑠𝑠 )∙(103 )
𝑅𝑅𝑅𝑅 (𝑚𝑚𝑚𝑚 𝑔𝑔−1 ) = (𝑎𝑎𝑎𝑎𝑎𝑎) ∙ (𝑎𝑎) ∙ (𝑓𝑓𝑑𝑑𝑑𝑑𝑑𝑑 ) ∙ (02)
�𝑚𝑚𝐽𝐽𝐽𝐽 �

3. RESULTS AND DISCUSSION

The two crude multienzyme extracts, CE-YS and CE-RI, obtained respectively, from the

fermentation of yellow mombin seed meal and rice straw meal, by P. roqueforti ATCC 10110, resulted

in the following enzymatic activity profiles (CMCase, xylanase, exoglucanase and FPase): for CE-YS,

6.39 Ucmc mL-1, 11.37 Uxyl mL-1, 1.11 Uexo mL-1 and 4.92 Ufpa mL-1; and for CE-RI, 6.89 Ucmc mL-1, 2.89

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Uxyl mL-1 and 0.89 Uexo mL-1, with no FPase activity detected for this extract, as already observed in a

previous study.14

3.1. Enzymatic saccharification using the extracts CE-YS and CE-RI

The saccharification performance of six different agro-industrial residues - AB, PS, RI, JS, MS

and CS - for both multi-enzyme crude extracts (CE-YS and CE-RI) was evaluated over a period of 24

h. With 6 h of saccharification with CE-RI the best RS values were around 7 – 8 mg g-1 with the residues

AB, JS and MS and, after 24 h, these values increased only to 8 – 10 mg g-1; the other residues

presented the worst RS values (< 3 mg g-1). Considering the saccharification with CE-YS, the two best

results were obtained with JS and AB which presented, at 6 h, RS values more than two times higher

than with CE-RI and after 24 h these values reached around 70 - 80 mg g-1, while for the other residues,

only about 10.0 mg g-1 was obtained. Regarding the yield of reducing sugars, JS saccharification by CE-

YS for 18 h was the best result obtained (3.83 mg g-1 h-1); therefore, this extract and residue were

selected for the next study steps.

Considering that the best results were obtained with AB and JS, for both extracts, the analysis

of lignocellulosic content demonstrated in Table 1 (considering the variability observed between the

fruits sampled) indicates that these residues had the highest amount of hemicellulose. As mentioned

previously, xylanase activity in CE-YS was higher than in CE-RI, and since this enzyme is responsible

for xylan degradation (which is one of the main components of hemicellulose) this could be suggested

for the best performance of CE-YS in these two residues. However, the RI residue had the third highest

hemicellulose content among the analyzed residues (Table 1) but it was not sufficiently saccharified by

both extracts at any time evaluated. The differences in composition of each residue, which is intrinsic to

each plant species,21,22 and the differences of the enzymes produced from different substrates may

result in different saccharification performances, as observed.

As mentioned before, the presence of xylanase increases the hydrolysis of lignocellulosic

materials because xylanase degrades xylan, which, like lignin, acts as a limiting factor for cellulose

access.23 As an important enzyme, xylanase has proven to be essential for obtaining high levels of

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fermentable sugar concentrate, especially in residues with high levels of hemicellulose (xylan type). Xu

et al.,24 while investigating eight agro-industrial residues for solid-state cultivation of a white rot fungus,

obtained different profiles for the enzymes of interest, for example, the highest CMCase activity (~18 U

g-1) was obtained with wheat meal, while the highest FPase activity (~5 U g-1) was obtained with

sugarcane bagasse.

3.2. Effect of ions on saccharification of JS by CE-YS

Many enzymes may require25 or have their catalytic properties enhanced26,27 in the presence of

certain ions. Thus, it was observed the following ratios (RSsalt/RSo, %): Na+ = 91.70 ± 2.33 %; Mg2+ =

146.43 ± 2.24 %; Cu2+ = 110.37 ± 3.92 %; Mn2+ = 103.45 ± 1.06 % e Zn+ = 137.90 ± 2.24 %. As noted,

the addition of 5 mM of MgCl2 (and the consequent presence of the ion Mg2+) resulted in the largest

increase (~ 46 %) in RS values in relation to the medium without the addition of salt, however, it is not

possible to affirm that the presence of Mg2+ acted as a cofactor of enzymatic activity.

As reported elsewhere, cellulolytic enzymes can be adsorbed to lignin in a non-productively

way, but certain ions such as Mg2+ may inhibit this unproductive adsorption.28 According to this present

work, the second-best result was obtained in the presence of Zn+ (~ 38 %), while with Cu2+, Mn2+ and

Na+, the effects were less expressive and varied (positively and negatively) around 10 %. Thus, the

addition of the salt MgCl2 was selected for the next step of study of the synthesis conditions.

Considering another previous work with an extract from P. roqueforti ATCC 10110 also

cultivated in YS,16 it was identified that the addition of 2 mM of MnSO4 was able to specifically elevate

xylanase activity by about 40 % of its initial value, while the addition of the same amount of the salt

MgCl2 resulted in a 16 % reduction in the activity of this enzyme. When the effect of the addition of 10

mM MgCl2 on corncob saccharification was investigated18, it was observed that the RS concentration

increased by 86 %. This indicates that, in isolation, the effect of certain ions may vary on each enzymatic

activity analyzed, but since the extract is used in its crude (unpurified) form, it is more interesting to

evaluate the effect of ions on overall performance.

3.3. Study of enzymatic saccharification conditions

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3.3.a. 25-1 Fractional Factorial Desing (FF)

Table 2 shows the RS values obtained, in this first step of analysis, after 18 h of JS

saccharification by CE-YS. Among the RS values obtained, it can be observed (Table 2) that

experiments 2, 6 and 9 presented the lowest performance (< 10.0 mg g-1). Moreover, these experiments

had the highest level of agitation (225 rpm) besides the smallest amount of saccharification residue (5

g L-1). The highest RS value (> 100 mg g-1) was obtained with experiment 11 (Tab. 1) but the central

points (experiments 17, 18 and 19) also resulted in a close RS value (93.33 ± 0.88 mg g-1).

The Analysis of Effects (Table 2) revealed that the statistically significant (p < 0.05) factors with

positive effects were JS and Mg, which means that changing from the lowest level to the highest levels,

the average RS response is increased. The negative effect of A (Tab. 2) indicates an excessive agitation

can be detrimental to saccharification, probably because it interferes with the contact between enzyme

and substrate. Additionally, the curvature analysis (Tab. 2) indicated that the central conditions were

also favorable to saccharification.

As the factors T and pH were not statistically significant in the analyzed ranges (Table 2), it was

decided to set T at 65 ºC (especially due to technical equipment limitations) and pH at 3.0. The choice

could have been made as a function of the lower temperature (35 °C) aiming at lower energy expenditure

or a pH closer to neutrality; however, the choice of values was made based on the performance of

experiment 11, which was the highest RS, and the fact that pH has a negative effect, even though it is

not significant (Table 2). It has been reported that a crude extract produced by Inonotus obliquus

(cultivated in solid state in wheat meal supplemented with a mineral solution), presented better activities

(CMCase, FPase and β-glycosidase) at pH of 3.0 to 4.5 and a range of temperature of 40 - 60 °C, noting

that, above 70 °C, CMCase and FPase activities were significantly reduced.32 These observations are

in agreement with the results observed in this present work.

Regarding the addition of the salt MgCl2, the choice for the 5 mM concentration (central

condition) was preferred based on the results previously discussed, but the lower concentration (1 mM)

could have also been chosen to reduce reagent expenditures. Thus, only A and JS factors were selected

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for a second stage of experimental design (Table 3) in which a lower agitation (150 rpm) and a higher

JS value (75 g L-1) were considered in the investigation, however, lower agitation and higher residue

concentrations may affect the ideal mixture of a medium.24,29

3.3.b. 22 Central rotational composite design (CRCD)

Table 3 presents the results of this step of the study and shows that the highest RS value

obtained (119.35 mg g-1) occurred under the conditions of experiment 26, which compared to experiment

11 (Table 2), took place at higher agitation and lower residue concentration and resulted in a 16.30 %

increase in RS. The central points (experiments 28 - 30) resulted in the second highest RS value (110.50

± 0.96 mg g-1) which, compared to experiment 11, represents an increase of 7.7 %.

The regression coefficient analysis (Table 3) revealed that only the linear interaction term

between the factors agitation and residue concentration [(A)·(JSM)] was not statistically significant. The

removal of this term from the model and its inclusion in the residues resulted in an increase in the
2
coefficients of determination (𝑅𝑅 2 ) and of adjusted determination (𝑅𝑅𝑎𝑎𝑎𝑎𝑎𝑎 ) values from 0.9074 and 0.8150,

respectively, to 0.9075 and 0.8457 and it was considered a valid decision. The ANOVA (data not shown),

performed with the reduced model, resulted the following: for the Regression, Fcal = 14.7 (where Ftab =

4.53 < Fcal) and p = 0.0030 and for the Lack of Adjustment, Fcal = 190.5 (where Ftab = 19.25 <Fcal) and p

= 0.0052. Thus, the Regression was not statistically significant at 95 % of confidence but the F-value

and the Lack of Adjustment was statistically significant for both, the F-value and the p-value. Figure 1

presents the experimental and predicted values and shows a clearer view of the deviations between

these values.

3.3.c Definition of the best saccharification conditions

Although not approved by the statistical analysis, the reduced quadratic model obtained was

used in order to select the best levels of A and JS; thus, the maximum (theoretical) point were calculated

(data not shown) where the first derivative equals zero. The maximum concentration of RS predicted by

the model was 115.79 mg g-1 for the conditions A = 119.91 rpm and JS = 40.91 g L-1. For practical

reasons, these values were adjusted to A = 120 rpm and JS = 40.7 g L-1 and the mean experimental RS

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value obtained was 112.63 ± 1.87 mg g-1. The deviation between the experimental value and the

theoretical value provided by the model (115.78 mg g-1) considering the new coded A and JS values,

was less than 3 %. This could be an argument in favor of the approval of the obtained mathematical

model; however, the decision was maintained to not approve the model.

Since the model to be fitted to a data series is not approved by ANOVA and/or experimental

validation, the defined conditions that lead to a satisfactory response should be referred to as “best

conditions” rather than “optimal conditions”. Thus, the best conditions for saccharification were defined

as JS = 40.7 g L-1; T = 65 °C, pH = 3.0, Mg = 5.0 mM, A = 120 rpm and t = 18 h; the equivalent productivity

of RS was 6.26 mg g-1 h-1. Compared to experiment 11 (Table 2), this value represents a 9.82 % increase

in yield and compared to the first saccharification presented at the beginning of this work, this value

represents a 63.45 % increase in yield.

Considering other fungi and agro-industrial residues, Zhang and Sang29, for example, obtained

a higher yield of reducing sugars (23.08 mg g-1 h-1 with 24 h) with a crude extract produced by Penicillium

chrysogenum QML-2 applied to the saccharification of pre-hydrolyzed corncob. In this case, solid-state

cultivation was performed on a more nutritionally complex substrate – a 1:1 (w:w) mixture of corn straw

and wheat straw supplemented with ammonium sulfate and yeast extract – than YS, resulting in a

different enzymatic composition, such as xylanase = 19,613.25 U g-1, endoglucanase = 195.13 U g-1

and FPase = 41.87 U g-1. Also according to these authors, saccharification was performed in two steps,

one of which was thermo-chemical with 10 g L-1 NaOH and autoclaving (120 °C) for 1 h, followed by the

enzymatic step that was performed with 20 g L-1 of the pre-hydrolyzed residue at 50 °C and pH 4.8 with

gentle stirring (value not reported) and for 24 h, resulting in a greater amount of released RS. In another

example29, the saccharification of rice straw by a crude extract containing 132.51 ± 3.27 U mL-1 of

xylanase (produced by the fungus Thermomyces lanuginosus VAPS-24 cultivated in rice straw

supplemented with peptone) presented a yield of reducing sugars of 5.07 mg g-1 when pre-treated with

H2O2 and 1.08 mg g-1 without pre-treatment. In this particular case, it is worth to mention that it was

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applied an extract with 10 times more xylanase activity and a residue chemically pre-treated and, even

though, the yields were lower than the one reported in this present work.

As demonstrated elsewhere, the practice of chemical or thermo-chemical pre-treatments of

residue has been widely applied, but in some situations, it may not be feasible.29,30 In this present work

it is proposed the direct use of YM and JS without any pre-treatment; only the addition of MgCl2 was

suggested. Other researchers, argue that the results of saccharification performed by non-enzymatic

pathways are as satisfactory as the results of enzymatic pathways;31 however, it is important to

emphasize that the combination of techniques is always valid when trying to propose the use of residues

in a less impactful way and as efficiently as possible.

The crude extract of P. roqueforti ATCC 101110 (also cultivated in YS) was previously applied

to the saccharification of sugarcane bagasse (without pre-treatment) with about half the residue

concentration (22 g L-1), a similar temperature (62 °C), a different salt (10 mM MnSO4), a higher pH

(4.8), the same agitation (120 rpm) and a much shorter reaction time (4 h).26 Under these conditions,

these researchers obtained better saccharification performances (RS > 660 mg g-1), which is possibly a

reflection of the type of residue and reinforces the potential of this multi-enzymatic crude extract. Another

crude extract (from Inonotus obliquus) resulted in lower equivalent productivities (2.6 - 2.7 mg g-1 h-1)

than the results obtained in this study, probably due to the longer reaction time (48 h) and the type of

residues applied (rice and wheat straws).24

CONCLUSIONS

Agro-industrial residues can (and should) be applied satisfactorily in the production of different

bioproducts since this practice does not endanger food production. These residues can be successfully

employed as a substrate for microbiological cultivation due to their complex chemical compositions

which stimulate the production of different enzymes. These obtained enzymes are an important

biocatalyst for the most varied applications, such as the production of reducing sugars that can be

directed, for example, for bioethanol production. The use of residues should be stimulated on small

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scales, based on local availability of a particular residue, not just considered for specific and widespread

wastes. It is crucial to reduce the impacts caused by our industrial activities no matter in which scale.

ACKNOWLEDGMENT

Authors are greatfull to CAPES, CNPq and FAPESB (Brazil) for the finantial support and also to the

invaluable support of Professors Marcelo Franco and Julieta Rangel and the team of the Laboratory of

Biotransformation and Organic Biocatalysis (LABIOCAT, UESC, Ilhéus, Bahia, Brazil).

REFERENCES

1. Zhang W, Sathitsuksanoh N, Barone JR and Renneckar S. Enhanced enzymatic saccharification of

pretreated biomass using glycerol thermal processing (GTP). Biores Technol 199:148-154 (2016).

doi:10.1016/j.biortech.2015.08.141.

2. Wang Y, Cai D, He M, Wang Z, Qin P and Tan T. Open fermentative production of l-lactic acid using

white rice bran by simultaneous saccharification and fermentation. Biores Technol 198:664-672

(2015). doi:10.1016/j.biortech.2015.09.010.

3. Chin DWK, Lim S, Pang YL, Lim CH and Lee KM. Two-staged acid hydrolysis on ethylene glycol

pretreated degraded oil palm empty fruit bunch for sugar based substrate recovery. Biores Technol

292:121967 (2019). doi:10.1016/j.biortech.2019.121967.

4. Sirohi R and Pandey JP. Dilute acid hydrolysis of spoiled wheat grains: Analysis of chemical,

rheological and spectral characteristics. Biores Technol 283:53-58 (2019).

doi:10.1016/j.biortech.2019.03.068.

5. Tiburski JH, Rosenthal A, Deliza R, Godoy RLO and Pacheco S. Nutritional properties of yellow

mombin (Spondias mombin L.) pulp. Food Res Int 44(7):2326-2331 (2011).

doi:10.1016/j.foodres.2011.03.037.

6. Zhang Y, Zhang Y, Xu F, Li S and Tan L. Structural characterization of starches from Chinese jackfruit

seeds (Artocarpus heterophyllus Lam). Food Hydrocol 80:141-148 (2018). doi:

10.1016/j.foodhyd.2018.02.015.

This article is protected by copyright. All rights reserved.

7. Cai S, Wang O, Wu W, Zhu S, Zhou F, Ji B, Gao F, Zhang D, Liu J and Cheng Q. Comparative study

of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content

(TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.). J Agricult

Food Chem 60(1):507-13 (2012). doi:10.1021/jf204163a.

8. Rodríguez-Zúñiga UF, Farinas CS, Bertucci Neto V, Couri S and Crestana S. Aspergillus niger

production of cellulases by solid-state fermentation. Pesq Agropec Bras 46(8):912-919 (2011).

doi:10.1590/S0100-204X2011000800018.

9. Cunha JRB, Santos FCP, Assis FGV and Leal PL. Cultivation of Penicillium spp. in soy bean crop

residues for production of cellulase, protease and amylase. Rev Ceres 63(5):597-604 (2016).

doi:10.1590/0034-737x201663050002.

10. Patel G, Patil MD, Soni S, Khobragade TP, Chisti Y and Banerjee UC. Production of mycophenolic

acid by Penicillium brevicompactum - A comparison of two methods of optimization. Biotechnol

Reports 11:77-85 (2016). doi:10.1016/j.btre.2016.07.003.

11. Dulf FV, Vodnar DC and Socaciu C. Effects of solid-state fermentation with two filamentous fungi on

the total phenolic contents, flavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus

domestica L.) by-products. Food Chem 209:27-36 (2016). doi:10.1016/j.foodchem.2016.04.016.

12. Pérez-Rodríguez N, Oliveira F, Pérez-Bibbins B, Belo I, Agrasar AT and Domínguez JM.

Optimization of xylanase production by filamentous fungi in solid-state fermentation and scale-up to

horizontal tube bioreactor. Appl Biochem Biotechnol 173(3):803-25 (2014). doi:10.1007/s12010-014-

0895-1.

13. Santos OS, Solidade LS, Souza JGB, Lima GS, Baga Jr. ACR, Assis FGV and Leal PL. Solid-state

fermentation in agroindustrial residues for enzyme production: a systematic review. J Eng Exact Sci

4(2):0181-0188 (2018). doi:10.18540/jcecvl4iss2pp0181-0188.

14. Marques GL, Reis NS, Silva TP, Ferreira MLO, Aguiar-Oliveira E, Oliveira JR and Franco M.

Production and characterization of xylanase and endoglucanases produced by Penicillium roqueforti

This article is protected by copyright. All rights reserved.

 ATCC 10110 through the solid-state fermentation of rice husk residue. Waste Biomass Valor 9:2061-

2069 (2018). doi:10.1007/s12649-017-9994-x.

15. Reis NS, Brito AR, Pacheco CSV, Costa LCB, Gross E, Santos TP, Costa AR, Silva EGP, Oliveira

RA, Aguiar-Oliveira E, Oliveira JR and Franco M. Improvement in menthol extraction of fresh leaves

of Mentha arvensis by the application of multi-enzymatic extract of Aspergillus niger. Chem Eng

Communic 206(3):387-397 (2019). doi:10.1080/00986445.2018.1494580.

16. Marques GL, Reis NS, Silva TP, Ferreira MLO, Aguiar-Oliveira E, Oliveira JR, Franco M. Production

and Characterisation of Xylanase and Endoglucanases Produced by Penicillium roqueforti ATCC

10110 Through the Solid-State Fermentation of Rice Husk Residue. Waste Biomass Valor

9(11):2061–2069 (2018). doi:10.1007/s12649-017-9994-x.

17. Ferraz JLAA, Souza LO, Fernandes AGA, Oliveira MLF, Oliveira JR and Franco M. Optimization of

the solid-state fermentation conditions and characterization of xylanase produced by Penicillium

roqueforti ATCC 10110 using yellow mombin residue (Spondias mombin L.). Chem Eng Communic

207(1):31–42 (2020). doi:10.1080/00986445.2019.1572000

18. Ferraz JLAA, Souza LO, Soares GA, Coutinho JP, Oliveira JR, Aguiar-Oliveira E and Franco M.

Enzymatic saccharification of lignocellulosic residues using cellulolytic enzyme extract produced by

Penicillium roqueforti ATCC 10110 cultivated on residue of yellow mombin fruit. Bioresour Technol

248:214-220 (2018). doi:10.1016/j.biortech.2017.06.048.

19. Rodrigues MI and Iemma AF. Experimental Design and Process Optimization. CRC Press, Boca

Raton (2015).

20. Santos TC, Gomes DPP, Bonomo RCF and Franco M. Optimisation of solid state fermentation of

potato peel for the production of cellulolytic enzymes. Food Chem 133(4):1299–1304 (2012).

doi:10.1016/j.foodchem.2011.11.115

21. Álvarez C, Reyes-Sosa FM and Díez B. Enzymatic hydrolysis of biomass from wood. Microb

Biotechnol 9(2):149–156 (2016). doi:10.1111/1751-7915.12346.

This article is protected by copyright. All rights reserved.

22. Arantes V and Milacles AMF. Relevance of low molecular weight compounds produced by fungi and

involved in wood biodegradation. Quim Nova 32(6):1586–1595 (2009). doi:10.1590/S0100-

40422009000600043.

23. Hu J, Arantes V and Saddler JN. The enhancement of enzymatic hydrolysis of lignocellulosic

substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic

effect? Biotechnol Biofuels 4:36 (2011). doi:10.1186/1754-6834-4-36.

24. Xu X, Lin M, Zang Q and Shi S. Solid state bioconversion of lignocellulosic residues by Inonotus

obliquus for production of cellulolytic enzymes and saccharification. Biores Technol 247:88–95

(2018). doi:10.1016/j.biortech.2017.08.192.

25. Valasatava Y, Rosato A, Furnham N, Thorton JM, Andreini C. To what extent do structural changes

in catalytic metal sites affect enzyme function? J Inorg Biochem 179:40-53 (2018).

doi:10.1016/j.jinorgbio.2017.11.002.

26. Silva TP, Souza LO, Reis NS, Assis SA, Ferreira MLO, Oliveira JR, Aguiar-Oliveira E and Franco

M. Cultivation of Penicillium roqueforti in cocoa shell to produce and characterize its lipase extract.

Rev Mex Ing Quim 16(3):745-756 (2017).

27. Wang G, Zhang X, Wang L, Wang K, Peng F and Wang L. The activity and kinetic properties of

cellulases in substrates containing metal ions and acid radicals. Adv Biol Chem 2:390–395 (2012).

doi:10.4236/abc.2012.24048.

28. Liu H, Zhu JY and Fu SY. Effects of Lignin-Metal Complexation on Enzymatic Hydrolysis of

Cellulose. J Agric Food Chem 58(12):7233–7238 (2010). doi:10.1021/jf1001588.

29. Zhang H and Sang Q. Production and extraction optimization of xylanase and β-mannanase by

Penicillium chrysogenum QML-2 and primary application in saccharification of corncob. Biochem

Eng J 97:101–110 (2015). doi:10.1016/j.bej.2015.02.014.

30. Kumar V, Chhabra D and Shukla P. Xylanase production from Thermomyces lanuginosus VAPS-24

using low cost agro-industrial residues via hybrid optimization tools and its potential use for

saccharification. Bioresour Technol 243:1009–1019 (2017). doi:10.1016/j.biortech.2017.07.094.

This article is protected by copyright. All rights reserved.

31. Zahoor, Tu Y, Wang L, Xia T, Sun D, Zhou S, Wang Y, Li Y, Zhang H, Zhang T, Madadi M and Peng

L. Mild chemical pretreatments are sufficient for complete saccharification of steam-exploded

residues and high ethanol production in desirable wheat accessions. Biores Technol 243:319-326

(2017). doi:10.1016/j.biortech.2017.06.111.

This article is protected by copyright. All rights reserved.

Table 1: Composition (g kg-1) of cellulose, hemicellulose and lignin of agro-industrial residues evaluated

in the saccharification process.

Cellulose Hemicellulose Lignin

Agro-industrial residues
(g kg-1) (g kg-1) (g kg-1)

Acerola bagasse (AB) 488.7 ± 59.3 261.6 ± 46.7 101.0 ± 1.7

Peanut shell (PS) 745.8 ± 7.5 40.4 ± 4.5 44.8 ± 14.4

Rice straw (RI) 631.8 ± 95.1 143.6 ± 23.1 56.5 ± 3.81

Coffe straw (CS) 541.0 ± 48.8 82.0 ± 23.4 121.7 ± 3.1

Jackfruit seed (JS) 127.9 ± 24.7 293.7 ± 48.2 54.6 ± 1.7

Mango seed (MS) 274.3 ± 1.8 32.4 ± 17.1 25.0 ± 12.0

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Table 2: Coded matrix for the 25-1 Fractional Factorial Design (FF) - real values in parentheses - and

Effect Analysis for the factors: agitation (A, rpm), temperature (T, ° C), pH, jackfruit seed meal

concentration (JS, g L-1) and MgCl2 salt concentration (Mg, mM) over the response: reducing

sugar (glucose) per gram of residue (RS, mg g-1).

Matrix 25-1 FF

Factors Response

A T JS Mg RS
Runs pH
(rpm) (°C) (g L-1) (mM) (mg g-1)

1 -1 (75) -1 (35) -1 (3) -1 (5) 1 (9) 19.99

2 1 (225) -1 (35) -1 (3) -1 (5) -1 (1) 4.00
3 -1 (75) 1 (65) -1 (3) -1 (5) -1 (1) 21.99
4 1 (225) 1 (65) -1 (3) -1 (5) 1 (9) 10.49
5 -1 (75) -1 (35) 1 (9) -1 (5) 1 (9) 19.49
6 1 (225) -1 (35) 1 (9) -1 (5) -1 (1) 7.00
7 -1 (75) 1 (65) 1 (9) -1 (5) -1 (1) 20.49
8 1 (225) 1 (65) 1 (9) -1 (5) 1 (9) 8.49
9 -1 (75) -1 (35) -1 (3) 1 (45) 1 (9) 39.26
10 1 (225) -1 (35) -1 (3) 1 (45) -1 (1) 59.32
11 -1 (75) 1 (65) -1 (3) 1 (45) -1 (1) 102.62
12 1 (225) 1 (65) -1 (3) 1 (45) 1 (9) 52.04
13 -1 (75) -1 (35) 1 (9) 1 (45) 1 (9) 89.42
14 1 (225) -1 (35) 1 (9) 1 (45) -1 (1) 23.42
15 -1 (75) 1 (65) 1 (9) 1 (45) -1 (1) 49.01
16 1 (225) 1 (65) 1 (9) 1 (45) 1 (9) 53.41
17 0 (150) 0 (50) 0 (6) 0 (25) 0 (5) 94.33
18 0 (150) 0 (50) 0 (6) 0 (25) 0 (5) 92.70
19 0 (150) 0 (50) 0 (6) 0 (25) 0 (5) 92.96
Effect Analysis
Term Effect Std.Error tcal p
Mean 35.65 3.64 9.79 < 0.0001*
Curvat; 115.36 18.32 6.30 < 0.0001*
A -16.76 7.28 -2.30 0.0400*
T 8.33 7.28 1.14 0.2750
pH -3.62 7.28 -0.50 0.6278
JS 43.32 7.28 5.95 < 0.0001*
Mg 19.38 7.28 2.66 0.0207*
* statistically significant values with 95 % of confidence (p < 0.05)

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Table 3: Coded matrix for the 22 Rotational Composite Central Design (RCCD) - real values in

parentheses - and Regression Coefficients Analysis for the factors: agitation (A, rpm), jackfruit

seeds meal concentration (JS, g L-1) and the response: reducing sugar (glucose) per gram of

residue (RS, mg g-1). Reactions were performed under the fixed conditions of 65 °C, pH 3.0

and 5 mM MgCl2.

Matrix 22 RCCD

Factors Response

A JS RS
Runs
(rpm) (g L-1) (mg g-1)
20 -1 (85.9) -1 (32.27) 54.61
21 +1 (139.09) +1 (32.37) 89.32
22 -1 (85.9) -1 (67.73) 43.92
23 +1 (139.09) +1 (67.73) 77.24
24 -1.41 (75) 0 (50) 47.03
25 +1.41 (150) 0 (50) 81.14
26 0 (112.5) -1.41 (25) 119.35
27 0 (112.5) +1.41 (75) 63.45
28 0 (112.5) 0 (50) 109.40
29 0 (112.5) 0 (50) 111.20
30 0 (112.5) 0 (50) 110.90
Regression Coeffcients Analysis
Term Coefficient Std.Error tcal p
Mean 110.50 6.88 16.06 < 0.0001*
(A) 14.53 4.21 3.45 0.0182*
(A)² -26.08 5.01 -5.20 0.0035*
(FSJ) -12.73 4.21 -3.02 0.0294*
(FSJ)² -12.42 5.01 -2.48 0.0561*
(A)·(FSJ) -0.35 5.96 -0.06 0.9557
* statistically significant values with 90 % of confidence (p < 0.10)

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Accepted Article

Fo
rP
ee

Figure 1 - Experimental data (from the 22 DCCR) and the predicted values given by the reduced coded
model [RS = 110.50 + 14.53·(A) – 26.08·(A)2 – 12.73·(JS) – 12.42·(JS)2; R2 = 0.9075] for the reducing
rR

sugars (RS, mg g-1) and the factors agitation (A, rpm) and jackfruit seeds meal concentration (JS, g L-1).
ev
iew

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