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Introduction

Agroindustrial residues are secondary products of industrial and economical activities and stand out as potential raw materials for the generation of high value-added products such as microbial proteins, organic acids, ethanol, enzymes and biologically active secondary metabolites (Alexandrino, de Faria, de Souza, & Peralta, 2007; Ferreira-Leitão et al., 2010; Oliveira, Vendruscolo, Costa, & Araújo, 2016). Advances in industrial biotechnology have offered opportunities for the economic use of agroindustrial residues, and prevents its accumulation, which is of great environmental concern due to its potential for contamination of soils, rivers and underground water (Stroparo, Beitel, de Resende, & Knob, 2012).

The production of enzymes from the microbial culture using lignocellulosic agroindustrial residues (sugarcane bagasse, rice straw, wheat straw, oat bark and wood chips) as substrate is well documented in the literature. Approximately 70% of the dry matter of these wastes consist of carbohydrates (cellulose, hemicellulose and lignin) that can be converted into sugars by the action of microbial enzymes. Among the bacteria, the species of the genus Bacillus are one of the most investigated groups regarding the production of enzymes, especially amylase, under cultivation in agroindustrial residues as substrate (Corrêa, Moutinho, Martins, & Martins, 2011; Barros, Simiqueli, de Andrade, & Pastore, 2013). However, the main biological sources of enzymes are filamentous fungi, especially the genus Aspergillus for which was reported the production of cellulase, pectinase, amylase, lipase and others enzymes under culture in many different agroindustrial residues, such as wheat bran, paddy straw, sugarcane bagasse, manioc peel and coffee husk (Cruz et al., 2011; Gusmão, Ferraz, Rêgo, Assis, & Leal, 2014; Kanimozhi & Nagalakshmi, 2014).

However, peculiarities of the composition of some agroindustrial residues can compromise the microbial activity and consequently the conversion of these materials by the enzymes action. In this way, the cassava liquid residue (manipueira), generated by the processing of cassava for the production of flour, presents in its composition a glycoside (linamarin) potentially hydrolyzable to hydrocyanic acid (Barana & Cereda, 2000). In such cases, there is a need to employ appropriate and metabolically adapted microbial strains to cover the residue in the product of interest, involving the isolation and selection of microorganism from the most diverse environments, including agroindustrial residues (Celestino et al., 2014).

In Brazil, the northeast region is the main producer of cassava, and have hundreds of flour houses dedicated to the production of cassava flour low volumes (Serviço Brasileiro de Apoio às Micro e Pequenas Empresas [Sebrae], 2012). Considering that, in the production of cassava flour, an average of 300 liters of manipueira per ton of processed root is generated, with about 50 g L^-1 of COD (Chemical Oxygen Demand) and 140 ppm of hydrocyanic acid (Chuzel, 2001), the region Northeast is also largest generator of this waste, in Brazil. In order to minimize the environmental impacts resulting from the inappropriate disposal of cassava liquid waste in soils and water sources, studies have been developed to reuse this residue, including the use of this residue as a carbon source for microbial cultivation and obtention of biotechnological products of interest: Leonel and Cereda (1995) verified the biosynthesis of citric acid by Aspergillus niger; Barros, de Quadros, and Pastore (2008) reported biosurfactant production by Bacillus subtilis LB5a; Oliveira et al. (2013) used cassava liquid waste as source of carbon for the production of 2-phenylethanol by Saccharomyces cerevisiae, Kluyveromyces marxianus and Geotrichum fragrans.

The high organic load found in manipueira also makes it interesting as source of carbon for the cultivation of microorganisms producing enzymes of industrial interest (Teixeira et al., 2017). Amylase, carboxymethylcellulose (CMCase), pectinase and xylanase are examples of microbial hydrolytic enzymes utilized in several applications, such as food, textile, paper, pulp and industrial detergents, that can be obtained in the saccharification of agricultural residues (El-Shishtawy et al., 2014).

In this sense, and knowing that microorganisms used in enzyme production tests can be obtained from natural sources, in which, the desired enzymes must act, the objective of this work was to isolate and select filamentous fungi from cassava liquid waste for the production of hemicellulases and amylases using the same residue as induction substrate, and adding value to the secondary products of agroindustry.

Material and methods

Samples of fresh cassava wastewaterfrom the first cassava pressing were collected in flour production houses located in the municipality of Vitória da Conquista/BA (14^o51'58"S; 40^o50'22"W). The sample used as substrate for fermentation, composed of eight sub-samples of cassava liquid residue, was characterized for pH, phosphorus, potassium, calcium, magnesium, sulfur, copper, iron, zinc, sodium, aluminum, nitrogen and free cyanide (Empresa Brasileira de Pesquisa Agropecuária[EMBRAPA] 2009), as shown in Table 1.

The isolation of filamentous fungi from the cassava liquid waste was carried out from serial dilutions (10 -1 to 10 -6) using saline solution 0.85%, followed by the surface plating method, in triplicate, using a Dextrose Potato Agar culture medium (pH 5.6 ± 0.2) plus Tetracycline (1%). Plates were maintained at 30°C, for four days, and the isolates obtained were transferred to tubes containing Dextrose Potato Agar culture medium (pH 5,6 ± 0,2) plus Tetracycline (1%), in order to obtain pure cultures. The Castellani method was used for the preservation and maintenance of fungal cultures.

Pure cultures of fungal strains were submitted to qualitative tests in triplicate to determine the enzymatic potential, using solid culture media in Petri plates (0.5 g L^-1 of MgSO₄; 3.0 g L^-1 of NaNO₃; 1.0 g L^-1 of KH₂ PO₄; 0.5 g L^-1 of KCl; 0.01 g L^-1 of Fe₂ SO₄; 15 g L^-1 of Agar) supplemented with starch, carboxymethyl cellulose, pectin or xylan for induction of the bioproduction of amylases, CMCases, pectinases and xylanases enzymes, respectively (Griebeler et al., 2015). After four days of incubation at 25°C, the formation of translucent halo around the microbial colony was evaluated, indicating the degradation of the specific substrates contained in the culture media. Halos with diameter above 3 mm were considered positive for enzymatic activity.

From the qualitative tests, the selected strains were cultivated on Petri plates containing Dextrose Potato Agar culture medium (pH 5.6 ± 0.2) plus Tetracycline (1%), at 30°C, for seven days. 5 mm diameter discs containing fungal mycelium were removed from each plate and transferred individually into Erlenmeyers flasks of 250 mL, containing 100 mL of autoclaved cassava wastewater sample, then maintained at incubator, with orbital agitation of 130 rpm, at 38^oC, for six days.

Aliquots of 1 mL of crude enzyme extract from the fermentation assays were removed at 24 hours culture intervals and added to 9 mL of distilled water. The mixture was filtered and centrifuged at 5,000 rpm for 15 minutes to obtain a clear crude extract. The obtained supernatants were used as crude extract of the enzymes to determine the enzymatic activities.

The activities of amylase, CMCase, pectinase and xylanase were determined by mixing 100 μL of the enzymatic substrate to 1% p/v (starch, carboxymethylcellulose, pectin or xylan, respectively) with 50 μL of the crude extract and incubated at 50°C in a water bath for 30 minutes (Siqueira, de Siqueira, Jaramillo, & Filho, 2010). After incubation, 300 μL of dinitrosalicylic acid solution (DNS) was added for reaction for 10 minutes in a water bath at 98°C. The reaction was stopped by the addition of 1.5 mL distilled water and the absorbance reading was done in a spectrophotometer at 540 nm (Miller, 1959).

The activity assays of all the enzymes were conducted in triplicate, with the preparation of each reaction control: 1) Replacement of crude enzymatic extract with distilled water; 2) Replacement of the substrate with distilled water. The enzyme activities unit (nKat mL^-1) was defined as the amount of enzyme capable of releasing 1 μmol of product (reducing sugars) per second per gram of sample (polysaccharides) under the reaction conditions using as the standard curve the glucose monomers corresponding to the enzymes.

The averages of enzymatic activities obtained at the end of the fermentation process for each fungal line were submitted to analysis of variance (ANOVA), and differences between the averages were analyzed by the Scott-Knott test, at 1% probability, using the software Assistat. Correlation between the fermentation time and enzyme activity were evaluated using the Pearson coefficient.

Fungal lines with higher yields for enzyme production were identified at the genus level, based on the macroscopic morphological analysis of the colonies and on the study of reproductive structures of the lines using optical microscopy (Seifert, Morgan-Jones, Gams, Kendrick, 2011).

Results and discussion

A total of 65 strains of filamentous fungi were isolated from cassava liquid waste. Qualitative tests on the enzyme production potential showed that 53.84% of the lines produced CMCase, 78.46% amylase, 66.15% xylanase, 73.84% pectinase and 13.84% did not produce any of the enzymes (Table 2). The occurrence frequency of enzyme-producing fungi verified in this study was higher or similar than found by other authors using substrates diverse as source of microorganisms: Saleem and Ebrahim (2014) verified that, among the 46 fungal species isolated in legume seeds, eight isolates (17.4% of all isolates) had amylase activity; Simões, Tauk-Tornisielo, Tapia (2009) reported that 76% of total fungi isolated from Caatinga soil presented xylanase activity; Guimarães et al. (2006) verified that 40% of isolate filamentous fungi from soil and humus, plants and sugar cane bagasse presented pectinolytic activity.

The importance of isolating fungi with a broad enzymatic spectrum is due to the possibility that a single microorganism is sufficient to hydrolyse different substrates of complex composition (Stroparo et al., 2012). In this study, approximately 32% of the fungal strains were able to produce all the enzymes evaluated and therefore, selected for the fermentative trials containing cassava liquid residue as source of carbon and nitrogen.

Unlike the qualitative tests, only three of these strains (FUNB40, FUNB4I and FUNB4O) showed activity for all the enzymes under cassava wastewater fermentation (Figure 1). The pectinolytic activity at the end of 144 hours of fermentation was recorded for all strains and the averages varied between 3.8 and 26.73 nKat mL^-1 (p < 0.01). The average activity of xylanases, amylases and CMCase, in medium containing cassava wastewater as substrate varied (p < 0.01), respectively, from 1.05 to 67.96 nKat mL^-1, between 13 strains; 5.91 to 32.46 nKat mL^-1, between 6 strains; and 2.11 to 21.41 nKat mL^-1 between 4 strains, respectively (Figure 1).

The majority of the studies on filamentous fungi enzyme production using agroindustrial residues as substrate indicate solid state fermentation (SSF) as more advantageous compared to submerged fermentation (Hölker and Lenz, 2005; Hansen, Lübeckb, Frisvada, Lübeckb, & Andersena, 2015). However, the use of cassava wastewateras substrate in submerged fermentation to produce enzymes by filamentous fungi, provided results similar or even higher than those reported by other studies that used solid state fermentation in various types of agroindustrial residues. Stroparo et al. (2012) observed maximum activity of pectin esterase (polygalacturonase) by P. verruculosum and xylanase by A. niger J4 equal to 20.50 and 38.6 nKat g^-1, respectively, under SSF in wheat bran. Cunha, dos Santos, Assis, and Leal (2016) verified maximum activity of amylase and CMCase equal to 3.3 and 7.0 nKat g^-1, respectively, by Penicillium spp. LEMI A8221, under SSF in soybean crop residues.

At the end of 144 hours of cassava wastewater fermentation, the best averages of enzymatic activity in relation to the others (p < 0.01) were identified at the genus level, revealing that 5 of them belonged to the genus Aspergillus sp. (FUNB5C, FUNB40, FUNB2J; FUNBB4I, FUNB4O) and 1 to the genus Penicillium sp. (FUNB3A). This confirms the genera Aspergillus and Penicillium as potential producers of enzymes under fermentative processes in agroindustrial residues, as has been reported in the literature (Hansen et al., 2015).

The maximum activity of pectinase, xylanase, amylase and CMCase was observed at 96 hours of fermentation by the strain Aspergillus sp. B5C (67.4 ± 0.6 nKat mL^-1); at 120 hours (163.6 ± 0.13 nKat mL^-1), by the strain Aspergillus sp. B4I; at 144 hours (998 ± 0.24 nKat mL^-1), by the strain Penicillium sp. B3A; and at 48 hours (55.5 ± 0.21 nKat mL^-1), by the strain Aspergillus sp. B4O, respectively (Figure 2).

From these results, it can be considered that the maximal enzyme activity occurred at satisfactory fermentation times and consistent with other studies. Giese, Dekker, and Barbosa (2008) verified maximum pectinolytic activity (approximately 500 nKat mL-1) after 72 hours of fermentation in orange bagasse by Botryosphaeria rhodina. Kronbauer, Peralta, Osaku, and Kadowaki (2007) found values for maximum xylanase activity (30 nKat mg^-1) at 96 hours of fermentation using corn husk as substrate and Aspergillus casielus as a biological agent. Gusmão et al. (2014) used coffee husks as substrate for fermentation and found maximum amylase activity by Aspergillus spp. (178.37 nKat mL^-1) after 12 days of incubation. Santos, de Melo, do Bonfim, de Barros, and Santos (2015) when using banana leaves as a substrate for fermentation by Trichoderma reesei, it was obtained maximum CMCase activity (47.66 nKat mL^-1) after 96 hours.

The production of pectinase by Aspergillus sp. B5C, from xylanase by Penicillium sp. B3A and Aspergillus sp. B40, and CMCase by Aspergillus sp. B40 presented a weak positive correlation in relation to the fermentation time (r < 0.4) (Table 3). That is, the increase in fermentation time had little influence on the pectinolytic activity of these strains. On the other hand, the production of xylanase by Aspergillus sp. B5C, Aspergillus sp. B4I and Aspergillus sp. B4O, and amylase by Penicillium sp. B3A showed a high positive correlation with the fermentation time (r > 0.8), indicating that the activity of these enzymes raised as the fermentation time increased (p<0,05).

Finally, the production of xylanase by Aspergillus sp. B2J and Aspergillus sp. B40 showed a negative correlation (r < 0), that is, the activity of the enzyme decreased with increasing fermentation time (Table 3). According to Whitaker (1994), high values of enzymatic activity in the first hours of fermentation can be explained by the low availability of reducing sugars of the raw material, necessary for the development of the microorganism. This low availability stimulates the mechanism of expression of the enzymes needed to generate simple sugars. In addition, excessive fermentation time can lead to nutritional depletion of the medium, implying the reduction of microbial growth, making the conversion of biomass into products unviable and, consequently, reducing the production of enzymes (Santos, Muruci, Damaso, Silva, & Santos, 2014).

Conclusion

The cassava wastewater was a source of filamentous fungi producing enzymes and appeared as a promising alternative of substrate for fermentative processes aiming to obtain pectinase, xylanase, amylase and CMCase, especially by the Aspergillus sp. B5C, B40 and B4I strains.

The maximum fermentation time of 144 hours was sufficient for all strains to efficiently convert substrate into product.

Acknowledgements

The authors thanks to the Universidade Federal da Bahia (UFBA) and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) for funding grants and fellowships.

References

Alexandrino, N. A., de Faria, H. G., de Souza, C. G. M., & Peralta, R. M. (2007) Aproveitamento do resíduo de laranja para a produção de enzimas lignocelulolíticas por Pleurotus ostreatus (Jack:Fr). Food Science and Technology, 27(2), 364-368. doi: 10.1590/S0101-20612007000200026

Barana, A. C., & Cereda, M. P. (2000). Cassava wastewater (manipueira) treatment using a two-phase anaerobic biodigestor. Food Science and Technology, 20(2), 183-186. doi: 10.1590/S0101-20612000000200010

Barros, F. F., Simiqueli, A. P., de Andrade, C. J., & Pastore, G. M. (2013). Production of enzymes from agroindustrial wastes by biosurfactant-producing strains of Bacillus subtilis. Biotechnology Research International, 2013(2013), 1-9. doi: 10.1155/2013/103960

Barros, F. F. C., de Quadros, C. P., & Pastore, G. M. (2008). Propriedades emulsificantes e estabilidade do biossurfactante produzido por Bacillus subtilis em manipueira. Food Science and Technology, 28(4), 979-985. doi: 10.1590/S0101-20612008000400034

Celestino, J. D. R., Duarte, A. C., Silva, C. M. M., Sena, H. H., Ferreira, M. P. S. B. C., Mallmann, N. H., ... Souza, J. V. V (2014). Aspergillus 6V4, a strain isolated from manipueira, produces high amylases levels by using wheat bran as a substrate. Enzyme research, 2014(2014), 1-4. doi: 10.1155/2014/725651

Corrêa, T. L. R., Moutinho, S. K. S., Martins, M. L. L., & Martins, M. A. (2011). Simultaneous α-amylase and protease production by the soil bacterium Bacillus sp. SMIA-2 under submerged culture using whey protein concentrate and corn steep liquor: compatibility of enzymes with commercial detergents. Food Science and Technology, 31(4), 843-848. doi: 10.1590/S0101-20612 011000400003

Cruz, E. A., Melo, M. C., Santana, N. B., Franco, M., Santana, R. S. M., Santos, L. S., & Gonçalves, Z. S. (2011). Produção de alfa-Amilase por Aspergillus niger em resíduo de cascas de mandioca. Journal of Health Sciences, 13(4), 245-249. doi: 10.17921/2447-8938.2011 v13n4p%25p

Cunha, J. R. B., dos Santos, F. C. P., Assis, F. G. V., & Leal, P. L. (2016). Cultivo de Penicillium spp. em resíduos da colheita de soja para produção de celulase, protease e amilase. Revista Ceres, 63(5), 597-604. doi: 0.1590/0034-737x201663050002

Chuzel, G. (2001). The cassava processing industry in Brazil: Traditional techniques, technological developments, innovations and new markets. African Journal of Food and Nutritional Security, 1(1), 46-59. doi: 10.4314/fns.v1i1.19233.

El-Shishtawy, R. M., Mohamed, S. A., Asiri, A. M., Gomaa, A. B., Ibrahim, I. H., & Al-Talhi, H. A. (2014). Solid fermentation of wheat bran for hydrolytic enzymes production and saccharification content by a local isolate Bacillus megatherium. BMC Biotechnology, 14(2014). doi: 10.1186/1472-6750-14-29

Empresa Brasileira de Pesquisa Agropecuária [EMBRAPA]. (2009). Manual de análises químicas de solos, plantas e fertilizantes (2nd ed.). Brasília, DF: Embrapa Informação Tecnológica.

Ferreira-Leitão, V., Gottschalk, L. M. F., Ferrara, M. A., Nepomuceno, A. L., Molinari, H. B., & Bom, E. P. S. (2010). Biomass Residues in Brazil: availability and potential uses. Waste Biomass Valor, 1(1), 65-76. doi: 10.1007/s12649-010-9008-8

Giese, E. C., Dekker, R. F. H., Barbosa, A. M. (2008). Orange bagasse as substrate for the production of pectinase and laccase by Botryosphaeriarhodina MAMB-05 in submerged and solid state fermentation. BioResources, 3(2), 335-345.

Griebeler, N. E., de Bortoli, V., Astolfi, A. L., Daronch, N. A., Schumann, A. C., Salazar, L. N., ... Zeni, J. (2015). Seleção de fungos filamentosos produtores de amilases, proteases, celulases e pectinases. Revista Acadêmica: Ciência Animal, 13(2015), 15-24. doi: 10.7213/academica.13.FC.AO01

Guimarães, L. H. S., Peixoto-Nogueira, S. C., Michelin, M., Rizzatti, A. C. S., Sandrim, V. C., Zanoelo, F. F. … Polizeli, M. L. T. M. (2006). Screening of filamentous fungi for production of enzymes of biotechnological interest. Brazilian Journal of Microbiology, 37(4), 474-480. doi: 10.1590/S1517-8382 2006000400014

Gusmão, R. O., Ferraz, L. M., Rêgo, A. P. B., Assis, F. G. V., & Leal, P. L. (2014). Produção de enzimas por Aspergillus spp. sob fermentação em estado sólido em casca de café. Scientia Plena, 10(11), 1-11. Retrieved on August 11, 2017 from www.scientiaplena.org.br/sp/article/view/2052/1083

Hansen, G. H., Lübeckb, M., Frisvada, J. C., Lübeckb, P. S., & Andersena, B. (2015). Production of cellulolytic enzymes from ascomycetes: comparison of solid state and submerged fermentation. Process Biochemistry, 50(9), 1327-1341. doi: 10.1016/j.procbio.2015.05.017.

Hölker, U., & Lenz, J. (2005). Solid-state fermentation - are there any biotechnological advantages? Current Opinion in Microbiology, 8(3), 301-306. doi: 10.10 16/j.mib.2005.04.006

Kanimozhi, K., & Nagalakshmi, P. K. (2014). Xylanase production from Aspergillus niger by solid state fermentation using agricultural waste as substrate. International Journal of Current Microbiology and Applied Sciences, 3(3), 437-446.

Kronbauer, W. A. W., Peralta, R. M., Osaku, C. A., & Kadowaki, M. K. (2007). Produção de xilanase por Aspergillus casielus com diferentes fontes de carbono. Boletim do Centro de Pesquisa de Processamento de Alimentos, 25(2), 207-216. doi: 10.5380/cep.v25i2.

Leonel, M., & Cerada, M. P. (1995). Manipueira como substrato na biossíntese de ácido cítrico por Aspergillus niger. Scientia Agricola, 52(2), 299-304. doi: 10.1590/S01 03-90161995000200016

MILLER, G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-429, 1959. doi: 10.1021/ac60147a 030

Oliveira, C. F. D., Vendruscolo, F., Costa, J. P. V., & Araújo, W. D. B. (2016). Bagaço de malte como substrato para produção de biopigmentos produzidos por Monascus ruber CCT 3802. Revista de Agricultura Neotropical, 3(3), 6-9.

Oliveira, S. M. M., Gomes, S. D., Sene, L., Coelho, S. R. M., Barana, A. C., Cereda, M. P., ... Piechontcoski, J. (2013). Production of 2-phenylethanol by Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus in cassava wastewater. Journal of Food, Agriculture & Environment, 11(2), 158-163. doi: 10.1234/ 4.2013.4229

Saleem, A., & Ebrahim, M. K. H. (2014). Production of amylase by fungi isolated from legume seeds collected in Almadinah Almunawwarah, Saudi Arabia. Journal of Taibah University for Science, 8(2), 90-97. doi: 10.1016/j.j tusci.2013.09.002

Santos, F. A., de Melo. A. L. M., do Bonfim, K. S., de Barros, T. V. F., & Santos, S. F. (2015). Utilização de resíduos agroindustriais na produção de celulases pelo fungo Trichoderma reesei em cultivo semissólido. Revista Saúde & Ciência Online, 3(3), 150-163, 2015.

Santos, R. R., Muruci, L. N. M., Damaso, M. C. T., Silva, J. P. L., & Santos, L. O. (2014). Lipase production by Aspergillus niger 11T53A14 in wheat bran using experimental design methodology. Journal of Food and Nutrition Research, 2(10), 659-663. doi: 10.12691/jfnr-2-10-1.

Serviço Brasileiro de Apoio às Micro e Pequenas Empresas [SEBRAE]. (2012). Mandioca (farinha e fécula) – Série Estudos Mercadológicos. Retrieved on March 22, 2018 from www.bibliotecas.sebrae.com.br/chronus/ARQUIVOS_ CHRONUS/bds/bds.nsf/5936f2d444ba1079c3aca02800150259/$File/4247.pdf

Seifert, K., Morgan-Jones, G., Gams, W., & Kendrick, B. (2011). The genera of hyphomycetes (9nd ed.). Utrecht, NL: CBS Biodiversity Series 9.

Simões, M. L. G., Tauk-Tornisielo, S. M., & Tapia, D. M. T. (2009). Screening of culture condition for xylanase production by filamentous fungi. African Journal of Biotechnology, 8(22), 6317-6326. doi: 10.5897/AJB 09.057

Siqueira, F. G., de Siqueira, E. G., Jaramillo, P. M. D., & Filho, F. X. F. (2010). The potential of agro-industrial residues for production of holocellulase from filamentous fungi. International Biodeterioration & Biodegradation, 64(1), 20-26. doi: 10.1016/j.ibiod.2009. 10.002

Stroparo, E. C., Beitel, S. M., de Resende, J. T. V., & Knob, A. (2012). Seleção de fungos filamentosos e de resíduos agroindustriais para a produção de enzimas de interesse biotecnológico. Semina: Ciências Agrárias, 33(6), 2267-2278. doi: 10.5433/1679-0359.2012v33n 6p2267

Teixeira, I. A. L., Gusmão, R. O., Ferraz, L. M., Oliveira, A. P. C., Assis F. G. V., & Leal, P. L. (2017). Isolamento e seleção de bactérias produtoras de amilase e pectinase sob fermentação submersa. Revista Brasileira de Tecnologia Agroindustrial, 11(1) doi: 10.3895/ rbta.v11n1.2847

Whitaker, J. R. (1994). Principles of Enzymology for the Food Sciences (2nd ed.): New York, USA: Dekker.

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