- Email: [email protected]
Use of coffee pulp and sorghum mixtures in the production of n-demethylases by solid-state fermentation
Bioresource Technology xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Use of coffee pulp and sorghum mixtures in the production of n-demethylases by solid-state fermentation Erick M. Peña-Lucioa, Liliana Londoño-Hernándeza, J.A. Ascacio-Valdesa, ⁎ Mónica L. Chavéz-Gonzáleza, Oluwatosin E. Bankoleb, Cristóbal N. Aguilara, a b
Bioprocesses and Bioproducts Research Group. Food Research Department. School of Chemistry. Universidad Autónoma de Coahuila, Saltillo, 25280 Coahuila, Mexico Department of Chemical Sciences, Faculty of Science and Science Education, Anchor University, P. M .B. 001, Ipaja P. O., Ipaja, Lagos State, Nigeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Solid culture N-demethylase Rhizopus oryzae Biodecaffeination
One of the compounds generally found in the residues of the coffee and tea industries is caffeine, which in high concentration is toxic to various organisms, making it necessary to find an adequate treatment for these residues. Biotechnological treatments using enzymes can be an alternative to valorize and detoxify these residues. However, mixtures of substrates have not been evaluated to improve production. Therefore, the present investigation aimed to study the effect of different proportions of sorghum-coffee pulp mixtures as a substrate in solid-state fermentation with the fungus Rhizopus oryzae (MUCL 28168) for the production of n-demethylases. To evaluate the synergistic and antagonistic effects of coffee pulp and sorghum mixtures on n-demethylase enzyme production, a simplex-centroid design, using four levels: 1 (100%), 1/4 (25%), 1/2 (50%), 3/4 (75%). Results obtained were favorable, achieving a caffeine demethylase activity of 18.762 U/g, and reducing the caffeine content in the coffee pulp.
1. Introduction Annual global food consumption has doubled to 1.9 billion tons (FAO 2002). As a result, waste accumulation has increased to 140 kg per capita per year (Pagés Díaz et al., 2011). Some of these residues have high concentrations of caffeine, an alkaloid considered toxic to different organisms. Potential applications of caffeine-degrading enzymes could be found in food, pharmaceutical, and agro-industry (Roussos and Augur, 2013). Development of bio-decaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods of removing caffeine, which is costly, toxic, and non-specific to caffeine. The conversion of caffeine to its metabolites can be realized by n-demethylases (Gummadi et al., 2011). N-demethylases are possibly involved during the initial steps of caffeine degradation that bring about the sequential demethylation of three N-methyl groups present in caffeine (Dash and Gummadi, 2006a). Some fungus belonging to the genera Aspergillus, Penicillium, Rhizopus, and Stemphyllium can metabolize caffeine by enzymatic conversion (Mohapatra et al., 2006). Rhizopus oryzae is a filamentous fungus classified as GRAS (Generally Recognized as Safe) by the FDA, which has been used in different biotechnological processes, in the production of enzymes and the elaboration of
⁎
traditional fermented foods (Londoño-Hernández et al., 2017). It is necessary to look for alternatives to improve the efficiency of biotechnological processes to produce enzymes that degrade caffeine. One of these alternatives is the use of agro-industrial by-products as substrates in the fermentation process (Bansal et al., 2012). This research aims to evaluate the synergistic or antagonistic effect of the mixture of two agro-industrial by-products (coffee pulp and sorghum), in the production of n-demethylases enzyme by solid-state fermentation (SSF) with the fungus Rhizopus oryzae (MUCL 28168). 2. Materials and methods 2.1. Microorganism Rhizopus oryzae (MUCL 28168) was obtained from the DIA-UADEC collection. The microorganism was maintained on potato dextrose agar; test tubes were prepared with 5 mL of potato dextrose agar (PDA), followed by the inoculation of the spore solution in the PDA tubes for 10 days at 30 °C.
Corresponding author. E-mail address: [email protected] (C.N. Aguilar).
https://doi.org/10.1016/j.biortech.2020.123112 Received 5 January 2020; Received in revised form 26 February 2020; Accepted 28 February 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Erick M. Peña-Lucio, et al., Bioresource Technology, https://doi.org/10.1016/j.biortech.2020.123112
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
phase (Dash and Gummadi, 2006b). The oven temperature was maintained at 30 °C. The flow rate was maintained at 0.2 mL/min, while the elution was monitored at 254 nm.
2.2. Substrates and pretreatment The sorghum and the coffee pulp were collected from Colima, Mexico, and Xilitla, San Luis Potosi. Sorghum was clean through aeration before ground into a particle size of 1 mm. The ground particles were packed into polyethylene bags of 500 g and then stored at 5 °C. The coffee pulp was dried in the sun until the average moisture of 10% was obtained. After the treatment, the pulp was ground into a particle size of 1 mm, packed in polyethylene bags, vacuum-sealed and then stored at room temperature.
2.9. Enzymatic production using Rhyzopus oryzae Rhizopus oryzae cells were thoroughly washed with water, deionized water at pH 7.8. Then, the cells were suspended in lysis buffer (5 mL of phosphate buffer pH 7.8 and 50 nM with DL-dithiothreitol, DTT 5 nM); the cell was broken using mortar. The macerated sample was centrifuged at 15,000 rpm for 45 min at 2 °C. The supernatant was separated and filtered using the Millipore membrane (0.45 μm) (Gutiérrez‐Sánchez, 2000). The activity of n-demethylase was determined according to the methodology described by Gutiérrez‐Sánchez (2000). The unit of the enzyme was defined as the unit of an enzyme capable of degrading 1 μmol of caffeine per mL per minute.
2.3. Chemical composition of the coffee pulp and sorghum Moisture, crude proteins, fiber, cellulose, hemicellulose, and lignin were analyzed using the Association of Official Analytical Chemists procedures (A.O.A.C, 1980). 2.4. Statistical mixture design
3. Results and discussion
It was evaluated a mixture between coffee pulp and sorghum to obtain the optimum n-demethylase production. The statistical analysis was a simplex-centroid design in which each component was studied in four levels, 1 (100%), 1/4 (25%), 1/2 (50%), 3/4 (75%). The data was analyzed with a significance level of 0.05, using the software, Minitab©.
The nutritional content of some agro-industrial residues such as coffee pulp can limit its application in the food industry, due to, its caffeine composition. However, an alternative for change these properties can be its biodecaffeination, it which, is realized by the production of some enzymes. It has been reported that Rhizopus sp. is able to degrade caffeine, (Chen et al., 2018), and this fungus has been used for decaffeination purposes (Gummadi et al., 2011). As above agroindustrial residues represent a potential resource for biotechnological processes; in consequence, hence, it could be used mainly due to their low cost, accessibility and nutrient compositions (Dias et al., 2015).
2.5. Kinetics solid-state fermentation Fermentation kinetics will be carried out under the conditions of moisture, inoculum size, and temperature, established by previous work. The SSF was carried out in mesh trays metallic of 8 cm, 4 cm and 4 cm high; the experiments were incubated with relative moisture of 90%, in an oven at a controlled temperature of 30 °C, with a particle size of 1 mm. The fermentation period took 120 h with measurements taken every 12 h.
3.1. The influence of the chemical composition of the agro-industrial residues on n-demethylase production The production of enzymes is induced by the presence of chemical compounds, such as sugars, tannins, alkaloids, and various sources of nitrogen and carbon (Furlan et al., 2015). Soluble sugars levels and proteins content can influence directly in enzymatic yields, because, these compounds are widely used by fungal metabolism to develop, growth functions and adaptation to the substrate, the mixtures that had the best levels of sugars presented the greatest n-demethylase activity (Table 1) (100, 75 and 50% coffee pulp). However, in the case of protein content, synergistic effects were presented in coffee pulp and sorghum mixtures 1/2 (50%). The content of lignocellulosic and fiber materials have a positive effect on microbial metabolism; In the present study, it was determined that the mixture of coffee pulp 100% had high levels of fiber and lignin, consequently, the mixture coffee pulp 100% showed better yields of enzymatic activity. It has been reported that materials with a higher content of these compounds promote the fungal growth (Viniegra-González, 1997).
2.6. Determination of the water absorption index (WAI) and moisture The water absorption capacity was determined using the technique described by Sanchez Blanco et al. (2016). The weight of 1.25 g of dry ground sample was put in centrifuge tubes, followed by the addition of 30 mL of distilled water and then warmed to 30 °C. The warmed suspension was placed at 30 °C for 30 min and stirred for 10 min. The moisture content was determined using a thermobalance by placing 1 g of the sample at a temperature of 120 °C. 2.7. Determination of the content of soluble protein The determination was tested in accordance with the modified Bradford method. A mixture of the 140 μl of sample and 140 μl of the Coomassie blue reagent was placed in a microplate and stirred for 30 s. Then, it was left to stand for 10 min with subsequent reading taken in the spectrophotometer at 595 nm (Kruger, 1994).
Table 1 Proximal composition and characterization of coffee pulp (coffee arabica) and sorghum.
2.8. Determination of caffeine The caffeine analysis was carried out using the methodology proposed by Gutiérrez‐Sánchez (2000), with some modifications. The aqueous ethanol extracts were obtained, and then the analysis was performed using HPLC. The calibration curve was performed with caffeine standard (0–250 ppm). The analyses by Reverse Phase-High Performance Liquid Chromatography were performed on a Varian HPLC system consisting of an autosampler, a ternary pump and a PDA detector (Varian ProStar 330, USA). Samples (5 µl) were injected onto a Denali C18 column (150 mm × 2.1 mm, 3 µm, Grace, USA) with 10 mM ammonium phosphate (pH 2.5)/acetonitrile (4:1, v/v) as mobile 2
Chemical composition
Coffee pulp
Sorghum
Moisture (% m/m) Protein (% m/m) Cellulose (% m/m) Hemicellulose (% m/m) Caffeine (mg/g) Lignin (% m/m) Fiber (% m/m) Water activity Soluble total sugar (mg glucose/g)
11.6 ± 1.8 11.48 ± 0.98 32.56 ± 2.00 28.66 ± 2.30 19.24 ± 0.33 26.40 ± 1.10 16.23 ± 0.12 0.408 ± 0.001 97.74 ± 8.82
7.4 ± 0.4 13.40 ± 0.30 1.70 ± 1.00 82.30 ± 1.60 – 11.80 ± 1.10 2.12 ± 0.27 0.342 ± 0.010 56.13 ± 3.49
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
3.2. The influence of the soluble protein content on n-demethylase production The highest n-demethylase activity was achieved when the soluble protein content was higher, the mixtures 100, 75 and 50% pulp presented high levels of protein soluble. These values could be associated with the soluble protein content because the protein dissociation improves enzymatic production yields (Beltr et al., 2006). When n-demethylase activity was reduced also the content of soluble protein decreased (24 h), it is due to the availability of the enzymatic activity is related to the solubility of the proteins (Mohapatra et al., 2006). Thus, a decrease in the levels of protein promotes a loss of activity. One of the compounds that were detected after 24 h of the fermentation period was theobromine. Dash and Gummadi (2006a,b), reported that some filamentous fungi can metabolize the bioconversion of caffeine to its methylxanthine derivates (theophylline, theobromine, paraxanthine) promoting the loss of enzymatic activity. Fig. 1. N-demethylase activity obtaining from different mixtures from coffee pulp and sorghum by Aspergillus niger 28A.
3.3. The influence of the moisture content on the n-demethylase production Water is essential for microbial metabolism because most of the enzymatic reactions occur in aqueous environments; for example, the high moisture content in the substrate promotes the absorption of caffeine by the microorganism. A percent of moisture around 70% stimulated the maximum caffeine demethylase activity and it is evident that the activity increased with the increasing moisture content of the substrate. On the other hand, at moisture contents of 40, 45 and 60%, the bio decaffeination of the mixtures suffered a decrease, probably due to the reduction in the solubility of substrate nutrients (Suresh and Radha, 2015).
Santhosh (2010), who monitored the consumption of caffeine in a study conducted in Pseudomonas sp., reported a decrease in n-demethylase activity after 24 h of reaction kinetics. Caffeine had different behavior during solid-state fermentation. In the first hours of the kinetic process, the caffeine content decreased; however, after 12 h, the caffeine content began to decline, obtaining levels of 18.05 mg/g. Finally, the concentration continues decreasing until 96 h of the fermentation kinetic. Brand et al. (2000) found that the R. arrizus LPB-79 strain achieved a decrease in caffeine content of approximately 87%. Synergic and antagonist effects were presented on enzymatic activity, mixtures from coffee pulp and sorghum can induce the expression of n-demethylase, and also inhibit its production. The mixture 50% coffee pulp shows a better yield of n-demethylase production that the mixture of 75% coffee pulp; even it had a less concentration of caffeine, the interaction between coffee pulp 1/2 (50%) and sorghum 1/2 (50%) showed an increase of 53.33% and 80% at 24 h and 48 h, respectively, in relation with the coffee pulp mixture 3/4 (75%) and sorghum 1/4 (25%), in a previous study, it was reported that a mixture consisting of 50% wheat bran and 50% soybean meal showed better production yields of protease enzyme, obtaining an increase of 18.9 and 68.6% (Suresh and Radha, 2015). It has been reported that microorganisms utilize a great diversity of substrates, using first simple sources and then complex sources, due to the formation of enzymatic systems more powerful showing an interesting positive effect over enzymatic production (ElShishtawy et al., 2014). Dias et al. (2015), indicated that Aspergillus niger 02 LBA presented synergistic effects similar to the use of agroindustrial waste in a proportion of 50:50. Coffee pulp mixture 1/4 (25%) and sorghum 3/4 (75%) presented a reduction in n-demethylase activity, compared to the other mixtures (100, 75 and 50%), due to, the concentration of caffeine is lower than other mixtures; however, after of 24 h, it presented an increase in the level of n-demethylase activity, obtaining an increase of 60 and 50%, respectively (Fig. 1). The presence of antagonistic effects in this mixture is attributed to the interactions that may occur between some substrates because the composition of elements such as carbon and nitrogen are present in unequal proportions (Suresh et al., 2015).
3.4. Catabolism of caffeine by Rhizopus oryzae (MUCL 28168) It was presented an increase in the n-demethylase activity (0.565 U/ g), that is, an intracellular enzyme involved in the demethylation of caffeine into dimethyl, mono-methyl xanthine and xanthine present the maximum activity at 24 h of processing but the yield tends to decrease after that. Dash and Gummadi (2006a) reported that the degradation of caffeine by fungus occurs through the theophylline route. It has been reported that n-demethylases catalyzes the conversion of caffeine to theophylline, methyl groups, followed by the enzymatic action that generates mono-methylxanthine, which is eventually converted to xanthine (Dash and Gummadi, 2006a,b). Enzymatic activity was not detected until 120 h after the fermentation. Similar results were reported by Gummadi and Santhosh (2010) on the effect of different concentrations of caffeine on the growth and production of n-demethylase by Pseudomonas sp. in a homogeneous system. They found that the highest activity of n-demethylase (18.762 U/ g cell dry weight−1) was at 24 h of processing when the initial caffeine concentration was 6.5 g l−1. However, at higher concentrations of caffeine, enzyme activities were lower. Gummadi et al. (2012) optimized a process for obtaining n-demethylase in fermentation submerged by Pseudomonas sp. and found that the highest enzyme production was 2214 U/g cell dry weight−1 h−1 at 15 h of the process. 3.5. N-demethylase production on each mixture under solid-state fermentation
4. Conclusions
It has been reported that enzymatic activity is induced by the caffeine content (Gummadi and Bhavya, 2011), in consequence, the enzymatic production by Rhizopus oryzae (MUCL 28168) was greater in the mixtures containing 100, 75 and 50% coffee pulp. The best n-demethylase activity was presented in the mixture of 100% coffee pulp at 24 h, n-demethylase activity was greater if the mixture had more percentage of caffeine (100, 75 and 50%) (Fig. 1). It was observed a notable loss of biological activity after 24 h (Fig. 1). Gummadi and
Solid-state fermentation is an interesting bioprocess to obtain enzymes such as n-demethylases. The mixture containing 100% coffee pulp achieved the greatest n-demethylase activity. Some mixtures had synergistic effects; for example, the mixture of 50% coffee pulp and 50% sorghum improved enzymatic yields; however, in the coffee pulp mixture, 1/4 (25%) and sorghum 3/4 (75%) it was presented a reduction in 3
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
n-demethylase activity. The catabolism of caffeine molecule was achieved by enzymatic route; however, the complexity of the matrices used as support for fermentation in solid-state can decrease the level of degradation of caffeine.
https://doi.org/10.1007/s10529-006-9196-2. Dias, F.F.G., de Castro, R.J.S., Ohara, A., Nishide, T.G., Bagagli, M.P., Sato, H.H., 2015. Simplex centroid mixture design to improve l-asparaginase production in solid-state fermentation using agroindustrial wastes. Biocatal. Agric. Biotechnol. 4 (4), 528–534. https://doi.org/10.1016/j.bcab.2015.09.011. El-Shishtawy, R.M., Mohamed, S.A., Asiri, A.M., Abu-bakr, M.G., 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 Biotechnol. 14 (1), 29. FAO, 2002. Human nutrition in the developing world. Available at: http://www.fao.org/ docrep/006/W0073S/w0073s00.html.Contents. Furlan, F., Dias, G., Janser, R., Castro, S. De, Ohara, A., Nishide, T.G., Sato, H.H., 2015. Biocatalysis and agricultural biotechnology simplex centroid mixture design to improve L-asparaginase production in solid-state fermentation using agroindustrial wastes. Biocatal. Agric. Biotechnol. 4 (4), 528–534. https://doi.org/10.1016/j.bcab. 2015.09.011. Gummadi, Sathyanarayana N., Bhavya, B., 2011. Enhanced degradation of caffeine and caffeine demethylase production by Pseudomonas sp. in bioreactors under fed-batch mode. Appl. Microbiol. Biotechnol. 91 (4), 1007–1017. Gummadi, S.N., Santhosh, D., 2010. Kinetics of growth and caffeine demethylase production of Pseudomonas sp. in bioreactor. J. Ind. Microbiol. Biotechnol. 37 (9), 901–908. https://doi.org/10.1007/s10295-010-0737-2. Gummadi, S.N., Bhavya, B., Ashok, N., 2012. Physiology, biochemistry and possible applications of microbial caffeine degradation. Appl. Microbiol. Biotechnol. https://doi. org/10.1007/s00253-011-3737-x. Gutiérrez‐Sánchez, G., 2000. Degradación de cafeína en presencia de ácido tánico por Penicillium commune V33A25. Tésis de Maestria. Universidad Autónoma Metropolitana, Unidad Iztapalapa, México. Kruger, N., 1994. The bradford method for protein quantitation. Basic Protein Peptide Protocols SE 3 (32), 9–15. https://doi.org/10.1385/0-89603-268-X:9. Londoño-Hernández, L., Ramírez-Toro, C., Ruiz, H.A., Ascacio-Valdés, J.A., AguilarGonzalez, M.A., Rodríguez-Herrera, R., Aguilar, C.N., 2017. Rhizopus oryzae – Ancient microbial resource with importance in modern food industry. Int. J. Food Microbiol. 257, 110–127. https://doi.org/10.1016/j.ijfoodmicro.2017.06.012. Mohapatra, B.R., Harris, N., Nordin, R., Mazumder, A., 2006. Purification and characterization of a novel caffeine oxidase from Alcaligenes species. J. Biotechnol. 125, 319–327. https://doi.org/10.1016/j.jbiotec.2006.03.018. Pagés Díaz, J., Pereda Reyes, I., Lundin, M., Sárvári Horváth, I., 2011. Codigestion of different waste mixtures from agro-industrial activities: kinetic evaluation and synergetic effects. Bioresour. Technol. 102 (23), 10834–10840. https://doi.org/10. 1016/j.biortech.2011.09.031. Roussos, S., Augur, C., 2013. Effect of caffeine concentration on biomass production, caffeine degradation, and morphology of Aspergillus tamari. Folia Microbiol. 195–200. https://doi.org/10.1007/s12223-012-0197-3. Sanchez Blanco, A., Palacios Durive, O., Batista Perez, S., Díaz Montes, Z., Perez Guerra, N., 2016. Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes. Braz. J. Microbiol. 47 (3), 665–674. https://doi.org/10.1016/j.bjm. 2016.04.019. Suresh, S., Radha, K.V., 2015. Effect of a mixed substrate on phytase production by Rhizopus oligosporus MTCC 556 using solid-state fermentation and determination of dephytinization activities in food grains. Food Sci. Biotechnol. 24 (2), 551–559. https://doi.org/10.1007/s10068-015-0072-5. Viniegra-González, G., 1997. Solid-state fermentation: definition, characteristics, limitations, and monitoring. In: Roussos, S., Lonsane, B.K., Raimbault, M., ViniegraGonzález, G., (Eds.). Advances in Solid State Fermentation, Kluwer Acad. Publ., Dordrecht, Chapter 2: pp. 5–22.
CRediT authorship contribution statement Erick M. Peña-Lucio: Writing - review & editing, Experimental activities, Writing. Liliana Londoño-Hernández: Data curation, Writing - original draft. J.A. Ascacio-Valdes: Visualization, Investigation. Mónica L. Chavéz-González: Supervision. Oluwatosin E. Bankole: Software, Validation. Cristóbal N. Aguilar: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration. Acknowledgement This work was financed by the National Council of Science and Technology (CONACYT) Mexico through the institutional program CONACYT-SEP-CB-2017-18 A1-S-42515. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Bansal, Namita, Tewari, Rupinder, Soni, Raman, Soni, Sanjeev Kumar, 2012. Production of cellulases from Aspergillus niger NS-2 in solid-state fermentation on agricultural and kitchen waste residues. Waste Manage. 32 (7), 1341–1346. https://doi.org/10.1016/ j.wasman.2012.03.006. Beltr, J.G., Leask, R.L., Brown, W.A., 2006. Activity and stability of caffeine demethylases found in Pseudomonas putida IF-3. Biochem. Eng. J. 31, 8–13. https://doi.org/10. 1016/j.bej.2006.05.006. Brand, D., Pandey, A., Roussos, S., Soccol, C.R., 2000. Biological detoxification of coffee husk by filamentous fungi using a SSF system. Enzyme Microb. Technol. 27 (1–2), 127–133. https://doi.org/10.1016/S0141-0229(00)00186-1. Chen, Rong, Jiang, Hongyu, Li, Yu-you, 2018. Caffeine degradation by methanogenesis: Efficiency in anaerobic membrane bioreactor and analysis of kinetic behavior. Chem. Eng. J. 334, 444–452. https://doi.org/10.1016/j.cej.2017.10.052. Dash, Sucharita Swati, Gummadi, S.N., 2006a. Biodegradation of caffeine by Pseudomonas sp. NCIM 5235. Res. J. Microbiol. 1, 115–123. https://doi.org/10. 3923/jm.2006.115.123. Dash, Swati Sucharita, Gummadi, S.N., 2006b. Catabolic pathways and biotechnological applications of microbial caffeine degradation. Biotechnol. Lett. 28 (24), 1993–2002.
4
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Use of coffee pulp and sorghum mixtures in the production of n-demethylases by solid-state fermentation Erick M. Peña-Lucioa, Liliana Londoño-Hernándeza, J.A. Ascacio-Valdesa, ⁎ Mónica L. Chavéz-Gonzáleza, Oluwatosin E. Bankoleb, Cristóbal N. Aguilara, a b
Bioprocesses and Bioproducts Research Group. Food Research Department. School of Chemistry. Universidad Autónoma de Coahuila, Saltillo, 25280 Coahuila, Mexico Department of Chemical Sciences, Faculty of Science and Science Education, Anchor University, P. M .B. 001, Ipaja P. O., Ipaja, Lagos State, Nigeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Solid culture N-demethylase Rhizopus oryzae Biodecaffeination
One of the compounds generally found in the residues of the coffee and tea industries is caffeine, which in high concentration is toxic to various organisms, making it necessary to find an adequate treatment for these residues. Biotechnological treatments using enzymes can be an alternative to valorize and detoxify these residues. However, mixtures of substrates have not been evaluated to improve production. Therefore, the present investigation aimed to study the effect of different proportions of sorghum-coffee pulp mixtures as a substrate in solid-state fermentation with the fungus Rhizopus oryzae (MUCL 28168) for the production of n-demethylases. To evaluate the synergistic and antagonistic effects of coffee pulp and sorghum mixtures on n-demethylase enzyme production, a simplex-centroid design, using four levels: 1 (100%), 1/4 (25%), 1/2 (50%), 3/4 (75%). Results obtained were favorable, achieving a caffeine demethylase activity of 18.762 U/g, and reducing the caffeine content in the coffee pulp.
1. Introduction Annual global food consumption has doubled to 1.9 billion tons (FAO 2002). As a result, waste accumulation has increased to 140 kg per capita per year (Pagés Díaz et al., 2011). Some of these residues have high concentrations of caffeine, an alkaloid considered toxic to different organisms. Potential applications of caffeine-degrading enzymes could be found in food, pharmaceutical, and agro-industry (Roussos and Augur, 2013). Development of bio-decaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods of removing caffeine, which is costly, toxic, and non-specific to caffeine. The conversion of caffeine to its metabolites can be realized by n-demethylases (Gummadi et al., 2011). N-demethylases are possibly involved during the initial steps of caffeine degradation that bring about the sequential demethylation of three N-methyl groups present in caffeine (Dash and Gummadi, 2006a). Some fungus belonging to the genera Aspergillus, Penicillium, Rhizopus, and Stemphyllium can metabolize caffeine by enzymatic conversion (Mohapatra et al., 2006). Rhizopus oryzae is a filamentous fungus classified as GRAS (Generally Recognized as Safe) by the FDA, which has been used in different biotechnological processes, in the production of enzymes and the elaboration of
⁎
traditional fermented foods (Londoño-Hernández et al., 2017). It is necessary to look for alternatives to improve the efficiency of biotechnological processes to produce enzymes that degrade caffeine. One of these alternatives is the use of agro-industrial by-products as substrates in the fermentation process (Bansal et al., 2012). This research aims to evaluate the synergistic or antagonistic effect of the mixture of two agro-industrial by-products (coffee pulp and sorghum), in the production of n-demethylases enzyme by solid-state fermentation (SSF) with the fungus Rhizopus oryzae (MUCL 28168). 2. Materials and methods 2.1. Microorganism Rhizopus oryzae (MUCL 28168) was obtained from the DIA-UADEC collection. The microorganism was maintained on potato dextrose agar; test tubes were prepared with 5 mL of potato dextrose agar (PDA), followed by the inoculation of the spore solution in the PDA tubes for 10 days at 30 °C.
Corresponding author. E-mail address: [email protected] (C.N. Aguilar).
https://doi.org/10.1016/j.biortech.2020.123112 Received 5 January 2020; Received in revised form 26 February 2020; Accepted 28 February 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Erick M. Peña-Lucio, et al., Bioresource Technology, https://doi.org/10.1016/j.biortech.2020.123112
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
phase (Dash and Gummadi, 2006b). The oven temperature was maintained at 30 °C. The flow rate was maintained at 0.2 mL/min, while the elution was monitored at 254 nm.
2.2. Substrates and pretreatment The sorghum and the coffee pulp were collected from Colima, Mexico, and Xilitla, San Luis Potosi. Sorghum was clean through aeration before ground into a particle size of 1 mm. The ground particles were packed into polyethylene bags of 500 g and then stored at 5 °C. The coffee pulp was dried in the sun until the average moisture of 10% was obtained. After the treatment, the pulp was ground into a particle size of 1 mm, packed in polyethylene bags, vacuum-sealed and then stored at room temperature.
2.9. Enzymatic production using Rhyzopus oryzae Rhizopus oryzae cells were thoroughly washed with water, deionized water at pH 7.8. Then, the cells were suspended in lysis buffer (5 mL of phosphate buffer pH 7.8 and 50 nM with DL-dithiothreitol, DTT 5 nM); the cell was broken using mortar. The macerated sample was centrifuged at 15,000 rpm for 45 min at 2 °C. The supernatant was separated and filtered using the Millipore membrane (0.45 μm) (Gutiérrez‐Sánchez, 2000). The activity of n-demethylase was determined according to the methodology described by Gutiérrez‐Sánchez (2000). The unit of the enzyme was defined as the unit of an enzyme capable of degrading 1 μmol of caffeine per mL per minute.
2.3. Chemical composition of the coffee pulp and sorghum Moisture, crude proteins, fiber, cellulose, hemicellulose, and lignin were analyzed using the Association of Official Analytical Chemists procedures (A.O.A.C, 1980). 2.4. Statistical mixture design
3. Results and discussion
It was evaluated a mixture between coffee pulp and sorghum to obtain the optimum n-demethylase production. The statistical analysis was a simplex-centroid design in which each component was studied in four levels, 1 (100%), 1/4 (25%), 1/2 (50%), 3/4 (75%). The data was analyzed with a significance level of 0.05, using the software, Minitab©.
The nutritional content of some agro-industrial residues such as coffee pulp can limit its application in the food industry, due to, its caffeine composition. However, an alternative for change these properties can be its biodecaffeination, it which, is realized by the production of some enzymes. It has been reported that Rhizopus sp. is able to degrade caffeine, (Chen et al., 2018), and this fungus has been used for decaffeination purposes (Gummadi et al., 2011). As above agroindustrial residues represent a potential resource for biotechnological processes; in consequence, hence, it could be used mainly due to their low cost, accessibility and nutrient compositions (Dias et al., 2015).
2.5. Kinetics solid-state fermentation Fermentation kinetics will be carried out under the conditions of moisture, inoculum size, and temperature, established by previous work. The SSF was carried out in mesh trays metallic of 8 cm, 4 cm and 4 cm high; the experiments were incubated with relative moisture of 90%, in an oven at a controlled temperature of 30 °C, with a particle size of 1 mm. The fermentation period took 120 h with measurements taken every 12 h.
3.1. The influence of the chemical composition of the agro-industrial residues on n-demethylase production The production of enzymes is induced by the presence of chemical compounds, such as sugars, tannins, alkaloids, and various sources of nitrogen and carbon (Furlan et al., 2015). Soluble sugars levels and proteins content can influence directly in enzymatic yields, because, these compounds are widely used by fungal metabolism to develop, growth functions and adaptation to the substrate, the mixtures that had the best levels of sugars presented the greatest n-demethylase activity (Table 1) (100, 75 and 50% coffee pulp). However, in the case of protein content, synergistic effects were presented in coffee pulp and sorghum mixtures 1/2 (50%). The content of lignocellulosic and fiber materials have a positive effect on microbial metabolism; In the present study, it was determined that the mixture of coffee pulp 100% had high levels of fiber and lignin, consequently, the mixture coffee pulp 100% showed better yields of enzymatic activity. It has been reported that materials with a higher content of these compounds promote the fungal growth (Viniegra-González, 1997).
2.6. Determination of the water absorption index (WAI) and moisture The water absorption capacity was determined using the technique described by Sanchez Blanco et al. (2016). The weight of 1.25 g of dry ground sample was put in centrifuge tubes, followed by the addition of 30 mL of distilled water and then warmed to 30 °C. The warmed suspension was placed at 30 °C for 30 min and stirred for 10 min. The moisture content was determined using a thermobalance by placing 1 g of the sample at a temperature of 120 °C. 2.7. Determination of the content of soluble protein The determination was tested in accordance with the modified Bradford method. A mixture of the 140 μl of sample and 140 μl of the Coomassie blue reagent was placed in a microplate and stirred for 30 s. Then, it was left to stand for 10 min with subsequent reading taken in the spectrophotometer at 595 nm (Kruger, 1994).
Table 1 Proximal composition and characterization of coffee pulp (coffee arabica) and sorghum.
2.8. Determination of caffeine The caffeine analysis was carried out using the methodology proposed by Gutiérrez‐Sánchez (2000), with some modifications. The aqueous ethanol extracts were obtained, and then the analysis was performed using HPLC. The calibration curve was performed with caffeine standard (0–250 ppm). The analyses by Reverse Phase-High Performance Liquid Chromatography were performed on a Varian HPLC system consisting of an autosampler, a ternary pump and a PDA detector (Varian ProStar 330, USA). Samples (5 µl) were injected onto a Denali C18 column (150 mm × 2.1 mm, 3 µm, Grace, USA) with 10 mM ammonium phosphate (pH 2.5)/acetonitrile (4:1, v/v) as mobile 2
Chemical composition
Coffee pulp
Sorghum
Moisture (% m/m) Protein (% m/m) Cellulose (% m/m) Hemicellulose (% m/m) Caffeine (mg/g) Lignin (% m/m) Fiber (% m/m) Water activity Soluble total sugar (mg glucose/g)
11.6 ± 1.8 11.48 ± 0.98 32.56 ± 2.00 28.66 ± 2.30 19.24 ± 0.33 26.40 ± 1.10 16.23 ± 0.12 0.408 ± 0.001 97.74 ± 8.82
7.4 ± 0.4 13.40 ± 0.30 1.70 ± 1.00 82.30 ± 1.60 – 11.80 ± 1.10 2.12 ± 0.27 0.342 ± 0.010 56.13 ± 3.49
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
3.2. The influence of the soluble protein content on n-demethylase production The highest n-demethylase activity was achieved when the soluble protein content was higher, the mixtures 100, 75 and 50% pulp presented high levels of protein soluble. These values could be associated with the soluble protein content because the protein dissociation improves enzymatic production yields (Beltr et al., 2006). When n-demethylase activity was reduced also the content of soluble protein decreased (24 h), it is due to the availability of the enzymatic activity is related to the solubility of the proteins (Mohapatra et al., 2006). Thus, a decrease in the levels of protein promotes a loss of activity. One of the compounds that were detected after 24 h of the fermentation period was theobromine. Dash and Gummadi (2006a,b), reported that some filamentous fungi can metabolize the bioconversion of caffeine to its methylxanthine derivates (theophylline, theobromine, paraxanthine) promoting the loss of enzymatic activity. Fig. 1. N-demethylase activity obtaining from different mixtures from coffee pulp and sorghum by Aspergillus niger 28A.
3.3. The influence of the moisture content on the n-demethylase production Water is essential for microbial metabolism because most of the enzymatic reactions occur in aqueous environments; for example, the high moisture content in the substrate promotes the absorption of caffeine by the microorganism. A percent of moisture around 70% stimulated the maximum caffeine demethylase activity and it is evident that the activity increased with the increasing moisture content of the substrate. On the other hand, at moisture contents of 40, 45 and 60%, the bio decaffeination of the mixtures suffered a decrease, probably due to the reduction in the solubility of substrate nutrients (Suresh and Radha, 2015).
Santhosh (2010), who monitored the consumption of caffeine in a study conducted in Pseudomonas sp., reported a decrease in n-demethylase activity after 24 h of reaction kinetics. Caffeine had different behavior during solid-state fermentation. In the first hours of the kinetic process, the caffeine content decreased; however, after 12 h, the caffeine content began to decline, obtaining levels of 18.05 mg/g. Finally, the concentration continues decreasing until 96 h of the fermentation kinetic. Brand et al. (2000) found that the R. arrizus LPB-79 strain achieved a decrease in caffeine content of approximately 87%. Synergic and antagonist effects were presented on enzymatic activity, mixtures from coffee pulp and sorghum can induce the expression of n-demethylase, and also inhibit its production. The mixture 50% coffee pulp shows a better yield of n-demethylase production that the mixture of 75% coffee pulp; even it had a less concentration of caffeine, the interaction between coffee pulp 1/2 (50%) and sorghum 1/2 (50%) showed an increase of 53.33% and 80% at 24 h and 48 h, respectively, in relation with the coffee pulp mixture 3/4 (75%) and sorghum 1/4 (25%), in a previous study, it was reported that a mixture consisting of 50% wheat bran and 50% soybean meal showed better production yields of protease enzyme, obtaining an increase of 18.9 and 68.6% (Suresh and Radha, 2015). It has been reported that microorganisms utilize a great diversity of substrates, using first simple sources and then complex sources, due to the formation of enzymatic systems more powerful showing an interesting positive effect over enzymatic production (ElShishtawy et al., 2014). Dias et al. (2015), indicated that Aspergillus niger 02 LBA presented synergistic effects similar to the use of agroindustrial waste in a proportion of 50:50. Coffee pulp mixture 1/4 (25%) and sorghum 3/4 (75%) presented a reduction in n-demethylase activity, compared to the other mixtures (100, 75 and 50%), due to, the concentration of caffeine is lower than other mixtures; however, after of 24 h, it presented an increase in the level of n-demethylase activity, obtaining an increase of 60 and 50%, respectively (Fig. 1). The presence of antagonistic effects in this mixture is attributed to the interactions that may occur between some substrates because the composition of elements such as carbon and nitrogen are present in unequal proportions (Suresh et al., 2015).
3.4. Catabolism of caffeine by Rhizopus oryzae (MUCL 28168) It was presented an increase in the n-demethylase activity (0.565 U/ g), that is, an intracellular enzyme involved in the demethylation of caffeine into dimethyl, mono-methyl xanthine and xanthine present the maximum activity at 24 h of processing but the yield tends to decrease after that. Dash and Gummadi (2006a) reported that the degradation of caffeine by fungus occurs through the theophylline route. It has been reported that n-demethylases catalyzes the conversion of caffeine to theophylline, methyl groups, followed by the enzymatic action that generates mono-methylxanthine, which is eventually converted to xanthine (Dash and Gummadi, 2006a,b). Enzymatic activity was not detected until 120 h after the fermentation. Similar results were reported by Gummadi and Santhosh (2010) on the effect of different concentrations of caffeine on the growth and production of n-demethylase by Pseudomonas sp. in a homogeneous system. They found that the highest activity of n-demethylase (18.762 U/ g cell dry weight−1) was at 24 h of processing when the initial caffeine concentration was 6.5 g l−1. However, at higher concentrations of caffeine, enzyme activities were lower. Gummadi et al. (2012) optimized a process for obtaining n-demethylase in fermentation submerged by Pseudomonas sp. and found that the highest enzyme production was 2214 U/g cell dry weight−1 h−1 at 15 h of the process. 3.5. N-demethylase production on each mixture under solid-state fermentation
4. Conclusions
It has been reported that enzymatic activity is induced by the caffeine content (Gummadi and Bhavya, 2011), in consequence, the enzymatic production by Rhizopus oryzae (MUCL 28168) was greater in the mixtures containing 100, 75 and 50% coffee pulp. The best n-demethylase activity was presented in the mixture of 100% coffee pulp at 24 h, n-demethylase activity was greater if the mixture had more percentage of caffeine (100, 75 and 50%) (Fig. 1). It was observed a notable loss of biological activity after 24 h (Fig. 1). Gummadi and
Solid-state fermentation is an interesting bioprocess to obtain enzymes such as n-demethylases. The mixture containing 100% coffee pulp achieved the greatest n-demethylase activity. Some mixtures had synergistic effects; for example, the mixture of 50% coffee pulp and 50% sorghum improved enzymatic yields; however, in the coffee pulp mixture, 1/4 (25%) and sorghum 3/4 (75%) it was presented a reduction in 3
Bioresource Technology xxx (xxxx) xxxx
E.M. Peña-Lucio, et al.
n-demethylase activity. The catabolism of caffeine molecule was achieved by enzymatic route; however, the complexity of the matrices used as support for fermentation in solid-state can decrease the level of degradation of caffeine.
https://doi.org/10.1007/s10529-006-9196-2. Dias, F.F.G., de Castro, R.J.S., Ohara, A., Nishide, T.G., Bagagli, M.P., Sato, H.H., 2015. Simplex centroid mixture design to improve l-asparaginase production in solid-state fermentation using agroindustrial wastes. Biocatal. Agric. Biotechnol. 4 (4), 528–534. https://doi.org/10.1016/j.bcab.2015.09.011. El-Shishtawy, R.M., Mohamed, S.A., Asiri, A.M., Abu-bakr, M.G., 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 Biotechnol. 14 (1), 29. FAO, 2002. Human nutrition in the developing world. Available at: http://www.fao.org/ docrep/006/W0073S/w0073s00.html.Contents. Furlan, F., Dias, G., Janser, R., Castro, S. De, Ohara, A., Nishide, T.G., Sato, H.H., 2015. Biocatalysis and agricultural biotechnology simplex centroid mixture design to improve L-asparaginase production in solid-state fermentation using agroindustrial wastes. Biocatal. Agric. Biotechnol. 4 (4), 528–534. https://doi.org/10.1016/j.bcab. 2015.09.011. Gummadi, Sathyanarayana N., Bhavya, B., 2011. Enhanced degradation of caffeine and caffeine demethylase production by Pseudomonas sp. in bioreactors under fed-batch mode. Appl. Microbiol. Biotechnol. 91 (4), 1007–1017. Gummadi, S.N., Santhosh, D., 2010. Kinetics of growth and caffeine demethylase production of Pseudomonas sp. in bioreactor. J. Ind. Microbiol. Biotechnol. 37 (9), 901–908. https://doi.org/10.1007/s10295-010-0737-2. Gummadi, S.N., Bhavya, B., Ashok, N., 2012. Physiology, biochemistry and possible applications of microbial caffeine degradation. Appl. Microbiol. Biotechnol. https://doi. org/10.1007/s00253-011-3737-x. Gutiérrez‐Sánchez, G., 2000. Degradación de cafeína en presencia de ácido tánico por Penicillium commune V33A25. Tésis de Maestria. Universidad Autónoma Metropolitana, Unidad Iztapalapa, México. Kruger, N., 1994. The bradford method for protein quantitation. Basic Protein Peptide Protocols SE 3 (32), 9–15. https://doi.org/10.1385/0-89603-268-X:9. Londoño-Hernández, L., Ramírez-Toro, C., Ruiz, H.A., Ascacio-Valdés, J.A., AguilarGonzalez, M.A., Rodríguez-Herrera, R., Aguilar, C.N., 2017. Rhizopus oryzae – Ancient microbial resource with importance in modern food industry. Int. J. Food Microbiol. 257, 110–127. https://doi.org/10.1016/j.ijfoodmicro.2017.06.012. Mohapatra, B.R., Harris, N., Nordin, R., Mazumder, A., 2006. Purification and characterization of a novel caffeine oxidase from Alcaligenes species. J. Biotechnol. 125, 319–327. https://doi.org/10.1016/j.jbiotec.2006.03.018. Pagés Díaz, J., Pereda Reyes, I., Lundin, M., Sárvári Horváth, I., 2011. Codigestion of different waste mixtures from agro-industrial activities: kinetic evaluation and synergetic effects. Bioresour. Technol. 102 (23), 10834–10840. https://doi.org/10. 1016/j.biortech.2011.09.031. Roussos, S., Augur, C., 2013. Effect of caffeine concentration on biomass production, caffeine degradation, and morphology of Aspergillus tamari. Folia Microbiol. 195–200. https://doi.org/10.1007/s12223-012-0197-3. Sanchez Blanco, A., Palacios Durive, O., Batista Perez, S., Díaz Montes, Z., Perez Guerra, N., 2016. Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes. Braz. J. Microbiol. 47 (3), 665–674. https://doi.org/10.1016/j.bjm. 2016.04.019. Suresh, S., Radha, K.V., 2015. Effect of a mixed substrate on phytase production by Rhizopus oligosporus MTCC 556 using solid-state fermentation and determination of dephytinization activities in food grains. Food Sci. Biotechnol. 24 (2), 551–559. https://doi.org/10.1007/s10068-015-0072-5. Viniegra-González, G., 1997. Solid-state fermentation: definition, characteristics, limitations, and monitoring. In: Roussos, S., Lonsane, B.K., Raimbault, M., ViniegraGonzález, G., (Eds.). Advances in Solid State Fermentation, Kluwer Acad. Publ., Dordrecht, Chapter 2: pp. 5–22.
CRediT authorship contribution statement Erick M. Peña-Lucio: Writing - review & editing, Experimental activities, Writing. Liliana Londoño-Hernández: Data curation, Writing - original draft. J.A. Ascacio-Valdes: Visualization, Investigation. Mónica L. Chavéz-González: Supervision. Oluwatosin E. Bankole: Software, Validation. Cristóbal N. Aguilar: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration. Acknowledgement This work was financed by the National Council of Science and Technology (CONACYT) Mexico through the institutional program CONACYT-SEP-CB-2017-18 A1-S-42515. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Bansal, Namita, Tewari, Rupinder, Soni, Raman, Soni, Sanjeev Kumar, 2012. Production of cellulases from Aspergillus niger NS-2 in solid-state fermentation on agricultural and kitchen waste residues. Waste Manage. 32 (7), 1341–1346. https://doi.org/10.1016/ j.wasman.2012.03.006. Beltr, J.G., Leask, R.L., Brown, W.A., 2006. Activity and stability of caffeine demethylases found in Pseudomonas putida IF-3. Biochem. Eng. J. 31, 8–13. https://doi.org/10. 1016/j.bej.2006.05.006. Brand, D., Pandey, A., Roussos, S., Soccol, C.R., 2000. Biological detoxification of coffee husk by filamentous fungi using a SSF system. Enzyme Microb. Technol. 27 (1–2), 127–133. https://doi.org/10.1016/S0141-0229(00)00186-1. Chen, Rong, Jiang, Hongyu, Li, Yu-you, 2018. Caffeine degradation by methanogenesis: Efficiency in anaerobic membrane bioreactor and analysis of kinetic behavior. Chem. Eng. J. 334, 444–452. https://doi.org/10.1016/j.cej.2017.10.052. Dash, Sucharita Swati, Gummadi, S.N., 2006a. Biodegradation of caffeine by Pseudomonas sp. NCIM 5235. Res. J. Microbiol. 1, 115–123. https://doi.org/10. 3923/jm.2006.115.123. Dash, Swati Sucharita, Gummadi, S.N., 2006b. Catabolic pathways and biotechnological applications of microbial caffeine degradation. Biotechnol. Lett. 28 (24), 1993–2002.
4