Biocatalysis and Agricultural Biotechnology 23 (2020) 101467

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Fungal detoxification of coffee pulp by solid-state fermentation

~ o-Hernandez a, H�ector A. Ruiz a, T. Cristina Ramírez b, Juan A. Ascacio a,
Liliana London
Raúl Rodríguez-Herrera a, Cristo
�bal N. Aguilar a, *
a
Food Research Dept. School of Chemistry. Universidad Aut� onoma de Coahuila, Saltillo, 25280, Coahuila, Mexico
b
Faculty of Engineering. School of Food Engineering. Universidad del Valle, Cali, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: Biotechnological advances offer potential opportunities for economic utilization of agro-industrial residues such
Solid-state fermentation as coffee pulp. Coffee pulp is a fibrous and mucilaginous residual material obtained during the processing of
Coffee pulp coffee cherries by the wet or dry process. The coffee pulp contains some amount of caffeine and tannins, which
Feed
makes it toxic in nature, resulting in the disposal problem, however, the coffee pulp is characterized by its
Caffeine
nutritional high-value. To increase the use of the coffee pulp (Arabica or Robusta) in animal feed, these com­
Condensed tannins
Detoxification pounds were removed by biodetoxification through solid-state fermentation using Rhizopus oryzae (MUCL
28,168). Temperature, moisture, and inoculum size were those studied parameters that had a significant influ­
ence on the decrement of the antiphysiological compounds. It was found that the highest decrease of caffeine
(52%) and condensed tannins (52%) was achieved at pH 6.0, temperature 30 � C, initial moisture 70%, and
inoculum 1 � 106 spores/g. The results show that with this process a reduction of these compounds can be
achieved, allowing efficient use of the coffee pulp in different industries.

1. Introduction Monteiro et al., 2005). The reactivity of condensed tannins with mole­
cules of biological importance has nutritional and physiological conse­
Coffee is one of the most important crops in the world. Annual coffee quences (Schofield, P., Mbugua, D. M., & Pell, 2001), among which the
production is 9.2 million tons (green coffee equivalents), being Mexico formation of complexes with proteins, carbohydrates and minerals, as
the ninth producer with 303,600 tons/year (equivalent to green coffee) well as their effect on oxidant at high concentrations; they bind strongly
(SAGARPA, 2015). There are two methods for processing the coffee to proteins rich in amino acids such as proline, glycine, glutamic acid,
cherry, the moist one used mainly for the arabica coffee variety and the and peptides; inhibit digestive enzymes, compromising the digestion of
dry one used for the robust coffee. Both processes generate a lot of proteins and other macronutrients; and interact with divalent minerals
residues, principally coffee pulp that represents approximately 42% (Paz such as non-haematic iron, inhibiting the absorption of metals (Va �zquez
et al., 2013). Flores, Alvarez Parrilla, Lo �pez Días, Walle Medrano and De la Rosa,
It is well known, the coffee pulp has a high content of proteins, 2012).
carbohydrates, and minerals so that in recent years’ various uses have Caffeine is a methylxanthine with bitter characteristics. Caffeine
been proposed. These include the production of enzymes, colorants, stimulates the central nervous system as an adenosine receptor antag­
flavors and as raw material for animal feed processing (Ramirez-Coronel onist. A low to moderate consumption of caffeine is associated with
et al., 2004). However, the presence of antiphysiological factors such as greater alertness, learning ability, performance in exercise, and perhaps
caffeine, and condensed tannins limited its use (Ashengroph and Ababaf, better mood, but high doses can produce negative effects in some in­
2014) and due to the amount of waste generated in producing countries, dividuals (Farah, 2012). Among the physiological effects that this
its treatment and disposal of coffee wastes is an important challenge for alkaloid can generate are an increase in motor activity, wasting energy
sustainable development and agriculture. and decreasing weight gain; it increases the thirst of the animal, as well
The condensed tannins comprise a group of oligomers and as increases the urinary evacuation, which generates nitrogen excretion
polyhydroxy-flavan-3-ol polymers linked through the carbon-carbon (Guti�errez-Sa
�nchez et al., 2013).
bond between the subunits of flavanol (Cha �vez-Gonza �lez et al., 2012b; In order to valorize these residues and decrease the antiphysiological

* Corresponding author.
E-mail address: [email protected] (C.N. Aguilar).

https://doi.org/10.1016/j.bcab.2019.101467
Received 25 July 2019; Received in revised form 9 November 2019; Accepted 5 December 2019
Available online 6 December 2019
1878-8181/© 2019 Elsevier Ltd. All rights reserved.
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compounds thermal and mechanical treatments can be used. However, Microbiology and Biotechnology laboratory MIBIA collection – Uni­
these treatments can affect the nutritional characteristics of the prod­ versidad del Valle) growing on potato dextrose agar (PDA) in 250 mL
ucts. An alternative treatment is solid-state fermentation (SSF), a bio­ Erlenmeyer flasks (8 days old) were harvested by homogenization with
process which has become more relevant in recent years due to the distilled water. Then, the methodology described by London ~ o (2015)
beneficial transformations that occur on materials (Jezierny et al., 2010) was applied to obtain dry spores, which were used during all the ex­
achieving a greater reduction of anti-physiological factors (Chelule periments. The spores were counted in a Neubauer cell.
et al., 2010; Elkhier & Abd-alraheem, 2011; Liang et al., 2008).
Among some of the studies carried out, Bhoite & Murthy (2015) used 2.3. Proximate analysis
Penicillium verrucosum to ferment coffee pulp, found that the tannin
content decreases by approximately 66%; Madeira et al. (2011) detox­ Moisture, crude proteins, fat, fiber, ash, cellulose, hemicellulose, and
ified beaver seeds with Paecilomyces variotii, reducing, among other lignin were analyzed using the Association of Official Analytical
compounds, the content of tannins by 85%. Oseni and Akindahunsi Chemists procedures (A.O.A.C, 1980). The moisture was determined
(2011) fermented seeds of Jatropha curcas with Rhizopus oryzae, found using thermobalance (OHAUS MB 23) at 105 � C to dry weight. The crude
that, in addition to increasing the protein content, there was a sub­ protein content (N � 6.25) was estimated using the micro Kjeldahl
stantial decrease in different compounds: tannins, phytates, oxalates, method; the fat was determined by extraction using Soxhlet apparatus,
and trypsin inhibitors. Several studies have been carried out to detoxify and the ash content was analyzed by weight before and after incinera­
biological materials. However, few studies have been carried out on the tion at 550 � C for 24 h. All analyses were performed in triplicate.
biotransformation of coffee pulp with safe microorganisms, used in the
preparation of food for human consumption, and whose metabolites 2.4. Phenolic profile analysis
(organic acids, enzymes, others) generated are recognized for their
biological activities that bring health benefits. Among these microor­ To determine the phenolic content, an extract was obtained
ganisms is R. oryzae is a filamentous fungus classified as a GRAS following the methodology proposed by Pe~ na-Aguilar et al. (2017) with
(Generally Recognized As Safe) by USDA, commonly used for produc­ some modification. During the extraction, 10 g of coffee pulp was
tion of some oriental traditional foods, and other industrial used such as extracted with 40 mL of ethanol (60% v/v) at 78 � C for 40 min. The
acid organic, enzymes, volatile compounds and antioxidant production aqueous ethanol extract was centrifugate at 10.000 rpm for 10 min and
(London ~ o-Herna
�ndez et al., 2017). To increase the use of this microor­ was filtered through a nylon membrane (0.45 μm, Millipore). The
ganism is necessary to determine which variables promote the decrease extract was analyses by HPLC-MS.
of these compounds, and in this way to obtain the minimum allowed to
use the coffee pulp as raw material for animal feed processing. 2.4.1. Analytical RP-HPLC-ESI-MS
During the setting up of whichever industrial processes, including The analyses by Reverse Phase-High Performance Liquid Chroma­
solid-state fermentation processes, the optimization of variables such as tography were performed on a Varian HPLC system including an auto­
physical and chemical parameters is a key tool to increase the profit­ sampler (Varian ProStar 410, USA), a ternary pump (Varian ProStar
ability and viability of the process (Olmedo-Obrero, 2010). An optimi­ 230I, USA) and a PDA detector (Varian ProStar 330, USA). A liquid
zation process can be carried out in two steps. An initial stage where the chromatograph ion trap mass spectrometer (Varian 500-MS IT Mass
most influential variables are determined and another stage where the Spectrometer, USA) equipped with an electrospray ion source also was
region with the greatest influence of the selected variables is deter­ used. Samples (5 μL) were injected onto a Denali C18 column (150 mm
mined. Statistical designs can be used for optimization processes, among � 2.1 mm, 3 μm, Grace, USA). The oven temperature was maintained at
which fractional factorial designs are used for screening, and the 30 � C. The eluents were formic acid (0.2%, v/v; solvent A) and aceto­
Box-Behnken design is used for final optimization (Carrillo-Sanc�en, nitrile (solvent B). The following gradient was applied: initial, 3% B;
2011). 0–5 min, 9% B linear; 5–15 min, 16% B linear; 15–45 min, 50% B linear.
There are several variables, both physical and biological, that The column was then washed and reconditioned. The flow rate was
interfere with the SSF. Some of these include the temperature, initial maintained at 0.2 mL/min and elution was monitored at 245, 280, 320
moisture of the substrate, pH, among others. For a particular process, it and 550 nm. The whole effluent (0.2 mL/min) was injected into the
is necessary to know which variable and how it affects the response source of the mass spectrometer, without splitting. All MS experiments
(Pandey, 2003; Singhania et al., 2009; Thomas et al., 2013). To over­ were carried out in the negative mode [M-H] 1. Nitrogen was used as
come these problems, improving the detoxifying activity of the micro­ nebulizing gas and helium as damping gas. The ion source parameters
bial resources used, the objective of the present study was to study the were: spray voltage 5.0 kV and, capillary voltage and temperature were
main variables that interfere with the process and to find the adequate 90.0 V and 350 � C, respectively. Data were collected and processed
value to guarantee the reduction of the antiphysiological compounds in using MS Workstation software (V 6.9). Samples were firstly analyzed in
the coffee pulp: condensed tannins and caffeine. full scan mode acquired in the m/z range 50–2000. MS/MS analyses
were performed on a series of selected precursor ions.
2. Materials and methods
2.5. Solid-state fermentation (SSF)
2.1. Coffee pulp
The parameters evaluated during the solid-state fermentation pro­
Coffee pulp (Coffee arabica) was supplied by local producers from the cess were: initial moisture content (50%, 70%), pH (5, 6) temperature
Xilitla - San Pedro (San Luis Potosí, Mexico) in February 2016. The (30 � C, 38 � C) and inoculum size (104, 106 spores/g). The experiments
material was sun-dried (21 � C and 76% RH) for 3 days until reaching were carried out in a tray bioreactor, using sterile 5 � 10 cm mesh trays;
average moisture of 11.6% (WB). Then, the material was milled in a disc 10 g of dry solid was taken, which was moistened with water to the
mill and sieved to obtain fractions between 0.8 and 2.0 mm particle size, indicated value. Samples were inoculated with spores prepared ac­
and packed in cellophane bags covered in dark polyethylene and kept at cording to item 2.2 and incubated in an oven at controlled temperature
room temperature until its experimentation. and 90% relative humidity conditions for 6 days. After fermentation, 5 g
of the fermented wet substrate was taken and 15 mL of water-ethanol
2.2. Inoculum preparation solution (1: 1) was added; The material was stirred for 10 min and
then filtered and pressed. The supernatant was used to quantify the
Spores of Rhizopus oryzae (MUCL 28,168, belonging to Applied content of total phenols, condensed tannins, and caffeine. Once the

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Fig. 1. Calibration curve of the caffeine standard at different concentrations.

Medina-Morales et al. (2012). For determination, 800 μL of the diluted

Table 1
sample was placed in a test tube, 800 μL of the Folin-Ciocalteu reagent
Proximal composition of coffee pulp (coffee arabica) of Xilitla, San Luís
was added, mixed and the solution left for 5 min to allow reaction. After
Potosí.
this time, 800 μL of sodium carbonate (0.01 M) was added and again
Nutrients Concentration (%) allowed to stand for 5 min. Finally, the solution was diluted with 5 mL of
Moisture (% m/m) 11.6 � 1.8 distilled water and the absorbance read at 790 nm.
Ash (% m/m) 0.31 � 0.06
Protein (% m/m) 13.40 � 0.30
2.5.2. Condensed tannins
Fat (% m/m) 1.57 � 0.13
Carbohydrates available (% m/m) 57.20 � 0.38 The condensed tannins were quantified using the methodology
Fiber (%m/m) 16.23 � 0.12 described by Medina-Morales et al. (2012). For this, 0.5 mL of sample
Hemicellulose (% m/m) 28.66 � 2.30 was taken, 3 mL of HCl - Terbutanol (1: 9) and 0.1 mL of ferric reagent
Cellulose (% m/m) 32.56 � 2.00 were added. This solution was left for 1 h in a metabolic bath at 100 � C,
Lignin (% m/m) 26.40 � 1.10
cooled to room temperature and the absorbance read at 460 nm.

parameters were selected, fermentation kinetics were measured by 2.5.3. Caffeine

measuring pH, phenol content, condensed tannins, and caffeine every 6 The caffeine analyses were carried out using the methodology pro­
h for 72 h. posed by Guti� errez-Sa
�nchez et al. (2013), with some modifications. The
aqueous ethanol extracts were obtained, following the methodology
2.5.1. Total phenols described in item 2.4, and then the analysis was performed using HPLC,
The content of total phenols was determined by means of a spec­ under the above-mentioned operating conditions. The calibration curve
trophotometric method with the Folin-Ciocalteu reagent, described by shown in Fig. 1 was performed with caffeine standard. The samples were

Table 2
Mass spectrometry (MS) data of (þ/ ) LC-ESI-QTOF-MS spectra and the identification of the coffee pulp aqueous ethanol extract.
Peak TR (min) Ion mode Measured mass Molecular formula Compound identified Group

1 2.25 þ 195.1 C10H10O4 Ferulic acid Hydroxycinnamic acid

2 2.59 þ 138.1 C7H7NO2 Trigonelline Alkaloid
3 2.91 – 195.0 C8H7O3 Hydroxycaffeic acid Hydroxycinnamic acid
4 2.94 – 191.1 C10H8O4 Scopoletin Hydroxycoumarins
5 3.86 – 619.3 Unidentified
6 5.26 – 311.1 C13H12O9 Caffeoyl tartaric acid Hydroxycinnamic acids
7 5.80 – 325.1 C14H14O9 Feruloyl tartaric acid Hydroxycinnamic acids
8 8.73 – 297.0 Unidentified
9 16.02 – 264.0 Unidentified
10 16.37 – 152.9 C7H6O4 Protocatechuic acid Hydroxybenzoic acids
11 16.87 – 383.1 Unidentified
12 25.57 þ 195.0 C8H10N4O2 Caffeine Alkaloid
13 28.82 – 353.0 C16H18O9 5-Caffeoylquinic acid (chlorogenic acid) Hydroxycinnamic acids
14 32.13 – 337.9 C16H18O8 3-p-Coumaroylquinic acid Hydroxycinnamic acids
15 32.13 – 337.0 C16H18O8 3-p-Coumaroylquinic acid Hydroxycinnamic acids
16 32.80 – 863.1 C45H38O18 Procyanidin trimer C1 Flavanols
17 33.40 – 705.1 C36H34O15 ( )-Epicatechin-(2a-7) (4a-8)-epicatechin 3-O-galactoside Flavanols
18 33.40 – 367.0 C17H20O9 3-Feruloylquinic acid Hydroxycinnamic acids
19 33.84 þ 453.4 C21H24O11 (þ)-Catechin 3-O-glucose Flavanols
20 33.84 þ 475.4 C22H18O12 Chicoric acid Hydroxycinnamic acids
21 37.76 þ 340.3 C21H35NO N-methyl arachidonoyl amine Fatty amides
22 37.76 þ 679.6 Unidentified
23 37.76 þ 701.5 Unidentified

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Fig. 2. Pareto Chart of the effects fractional factorial experimental design for: a) caffeine concentration (mg/g), b) condensed tannins (mg/g) and c) phenols
concentration (mg(g) in fermented coffee pulp by Rhizopus oryzae (MUCL 28,168).

diluted in distilled water to obtain a suitable concentration of the cali­ 3.1. Physicochemical characterization of coffee pulp
bration curve.
The results of the proximal composition of the coffee pulp are shown
2.6. Experimental design and data analysis in Table 1. The main macronutrients presented in the material are car­
bohydrates (57.20 � 0.38%), protein (13.40 � 0.30%) and fiber (16.23
To determine the most influential parameters in the SSF processes, a � 0.12%) represented mainly by cellulose, hemicellulose, and lignin.
fractional factorial experimental design was applied. Each of the vari­ These values are slightly higher than those determined by Ulloa Rojas
ables was represented in 2 levels, one high and one low identified as (þ) et al. (2003) in terms of carbohydrates (31.6%) and protein (8.0%).
and ( ) respectively. Then, a composite central design was applied to However, is an expected result because in biological products the
achieve optimization. All analyses were performed in triplicate. composition depends on the variety, geographic location, culture con­
ditions and resources used in the production. The results of the
3. Results and discussion composition show the potential of the coffee pulp to be used in various
industries, mainly in animal feed.
Main variables that interfere in the solid-state fermentation bio­
process to find the adequate value to guarantee the reduction of the
3.2. Phenolic compounds
antiphysiological compounds in the coffee pulp: condensed tannins and
caffeine, were studied.
Table 2 shows the summary of the mass spectrometric analysis of the
ethanol extract of the dehydrated coffee pulp. A total of 23 compounds
were obtained, of which 17 compounds were identified, with the aid of a

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et al., 2012).
According to this classification, a high phenolic acid content was
found in the coffee pulp, whose main representatives were ferulic acid,
protocatechuic acid, 5-Caffeoylquinic acid (chlorogenic acid), 3-p-Cou­
maroylquinic acid and 3-feruloylquinic acid. These compounds have
been reported by Duangjai et al. (2016); Bresciani et al. (2014); Garrett
et al. (2012); Farah and Donangelo (2006); Ramírez Martínez and Clif­
ford (2000); Clifford and Ramirez-Martinez (1991) for pulp, skin, and
seed of coffee. Particularly the ferulic and chlorogenic acids have been
widely studied for high content in this type of products and for the
beneficial properties that can bring to health. These compounds are
mainly attributed antioxidant properties by their chemical nature, as
well as they have shown good hypoglycemic, antiviral, hepatoprotective
and antispasmodic activities (Ou and Kwok, 2004; Torres-Mancera et al.,
2013; Zhao and Moghadasian, 2008).
Some researchers (Duangjai et al., 2016; Ramirez-Coronel et al.,
2004) have indicated that the coffee pulp presents a wide content of
condensed tannins, however, in the aqueous ethanol extract studied only
were found Procyanidin trimer C1, ( )-Epicatechin-(2a-7) (4a-8)-epi­
catechin 3-O-galactoside y (þ)-Catechin 3-O-glucose, possibly due to the
extraction method employed.
Among the alkaloids found, by the nature of the sample, the main
one was the caffeine. Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-
2,6 dione), a purine alkaloid, is a central nervous system and metabolic
stimulant and for this reason, has been used in pharmaceutical prepa­
rations as it enhances the effect of certain analgesics and antipyretic
drugs. Moreover, caffeine is one of the most commercialized alkaloids
(Dash and Gummadi, 2012; Gokulakrishnan et al., 2005). However, due
to these same effects, the use of coffee pulp as animal feed is limited.
Therefore, the amount of caffeine should be reduced if this waste is to be
destined for the food industry. In this study, the approximate content of
caffeine in the coffee pulp was 41.45 mg/g, corresponding to approxi­
mately 3% by weight of the dry coffee pulp.

3.3. Solid-state fermentation

Initially, we determined the variables that have a significant influ­

ence on the content of phenols, condensed tannins and caffeine using a
fractional factorial design with four study factors: pH, temperature,
initial moisture, and inoculum size. The results are summarized in the
Pareto chart for each of the responses (Fig. 2). The data obtained indi­
cate that the significant individual factors at a confidence level of 5%
were temperature and inoculum for the concentration of caffeine; Initial
moisture, inoculum size and temperature for phenols; And inoculum and
moisture size for condensed tannins. The best behaviors were obtained
at pH 6.0, temperature 30 � C, inoculum size 1 � 107 and initial moisture
70%, degrading caffeine in about 52%, condensed tannins 52%, and
Fig. 3. Contour plot of a) concentration condensed tannins, b) concentration phenols 51%.
total phenols and c) concentration caffeine in fermented coffee pulp as a
Several studies have reported that factors such as pH, temperature,
function of the temperature and initial moisture content. Values in mg/g.
and inoculum size are important parameters in the SSF process to ach­
ieve the by-product detoxification of the coffee industry (D�
ebora Brand,
database and published references. Of the compounds identified, most Pandey, Roussos and Soccol, 2000). pH plays a crucial role during the
correspond to polyphenolic compounds, and in general, it was found growth and metabolism of an organism. Most of the molds grow between
that 59% are hydroxycinnamic acids, 17% are flavanols, 12% are al­ pH 5 and 6 (Patra, 2007). pH plays a significant role in the activity of
kaloids, 6% are Hydroxycoumarins and 6% are fatty amides. enzymes required for caffeine degradation (Ibrahim et al., 2016).
Due to the characteristics of the coffee pulp, most of the identified However, during the study, it was shown that pH had no influence on the
compounds correspond to phenolics. Phenolic compounds are a large process, due to the fact that the ranges studied correspond to the opti­
group of phytochemicals found in higher plants (V� azquez Flores et al., mum growth of this microorganism. Bhoite & Murthy (2015) evaluated
2012). These compounds are secondary metabolites of plants, generated the pH, humidity and fermentation time on the reduction of the tannin
to defend against pests or pathogens (Farah and Donangelo, 2006). Due content in the coffee pulp and the production of tannase enzyme. The
to its characteristics, phenolic compounds can be divided into flavo­ evaluated pH was between 4 and 6, they found that it had no influence
noids, phenolic acids, and polyphenols. More than 4000 individual on the process, possibly due to the acidic characteristics of the protein
polyphenolic compounds have been identified, which can be grouped in studied. Conversely, Ibrahim et al. (2016) using the strain Leifsonia sp.
two: flavonoids and non-flavonoids, which when polymerized, generate for caffeine degradation found that pH affects the process by deter­
compounds commonly known as condensed tannins and hydrolyzable mining that the optimum is 6.5. Nanjundaiah et al. (2016) using the
tannins respectively (Cha �vez-Gonza �lez et al., 2012a; Va
�zquez Flores microorganisms Fusarium solani for caffeine degradation. They found

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Fig. 4. Fermentation kinetics for total phenols, caffeine, and condensed tannins.

Fig. 5. Profile HPLC of aqueous ethanol extract of unfermented and fermented coffee pulp.

that pH influenced the process and established that the optimal value is microorganism (Bhargav et al., 2008). In tray bioreactors keeping the
5.8. Similar results found Laskshmi and Das (2013) using Trichosporon temperature in the optimum range is complex, because the bed is kept
asahii. The researchers studied a pH range between 4 and 8, being the fixed during the processes and the heat generated is not removed so that
optimal value of 6.5. the microorganism can suffer alterations (Dalsenter et al., 2005).
The temperature is another of the parameters that had influence Therefore, using this system should consider temperatures close to the
during the process. The experimental results show that, at higher tem­ low optimum range of the microorganism. Ibrahim et al. (2016) studied
peratures, the growth of the microorganism was delayed, decreasing its the growth characteristics of the strain Leifsonia sp and the degradation
metabolic activity and its action on the compounds of interest. The of caffeine by this under different parameters. They found that tem­
filamentous fungi grow in an optimal temperature range, however perature is one of the most relevant factors in the process of caffeine
during the process, due to the metabolic activity, heat is generated, degradation since it affects most biochemical processes. In this case, it
increasing the internal temperature and retarding the growth of the was found that the optimal temperature of the microorganism for the

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Table 3
Mass spectrometry (MS) data of (þ/ ) LC-ESI-QTOF-MS spectra and the identification of the fermented coffee pulp aqueous ethanol extract.
Peak TR (min) Ion mode Measured mass Molecular formula Compound identified Group

1 0,997 – 316,7 C15H10O8 6,8-Dihydroxykaempferol Flavonols

2 1614 þ 352,1 Unidentified
3 1614 þ 195,0 C8H10N4O2 Caffein alkaloids
4 2563 – 545,0 Unidentified
5 2884 þ 137,9 C7H6O3 Protocatechuic aldehyde Hydroxybenzaldehydes
6 3057 – 191,0 C10H8O4 Scopoletin Hydroxycoumarins
7 3547 þ 144,0 Unidentified
8 4053 þ 148,9 C9H8O2 Cinnamic acid Hydroxycinnamic acids
9 4559 – 360,9 p-HPEA-EA Tyrosols
10 4585 – 317,0 C15H10O8 6,8-Dihydroxykaempferol Flavonols
11 5596 þ 275,1 C15H14O5 Phloretin Dihydrochalcones
12 7603 – 383,1 Unidentified
13 8195 þ 352,3 Unidentified
14 10,371 – 656,1 Unidentified
15 12,159 – 152,9 C7H6O4 Protocatechuic acid Hydroxybenzoic acids
16 14,852 – 381,1 Unidentified
17 16,442 – 209,0 Unidentified
18 19,057 – 375,1 C20H24O7 Todolactol A Lignans
19 19,251 þ 376,9 C25H44O2 5-Nonadecylresorcinol Alkylphenols
20 19,602 – 352,9 C16H18O9 5-Caffeoylquinic acid (chlorogenic acid) Hydroxycinnamic acids
21 19,626 þ 195,0 C8H10N4O2 caffein alkaloids
22 20,485 – 298,9 C13H16O8 4-Hydroxybenzoic acid 4-O-glucoside Hydroxybenzoic acids
23 21,964 þ 425,2 Unidentified
24 22,101 – 246,9 Unidentified
25 22,970 – 337,0 C16H18O8 3-p-Coumaroylquinic acid Hydroxycinnamic acids
26 23,850 þ 361,0 C20H24O6 Cyclolariciresinol Lignans
27 24,254 þ 437,0 C21H24O10 Phloridzin Dihydrochalcones
28 24,717 – 792,8 Unidentified
29 24,822 – 384,6 C20H18O8 5-50 -Dehydrodiferulic acid Methoxycinnamic acid dimers
30 24,840 – 366,9 C17H20O9 3-Feruloylquinic acid Methoxycinnamic acids
31 25,032 þ 469,0 Unidentified
32 25,726 þ 391,0 C20H20O9 Resveratrol 3-O-glucoside Stilbenes
33 27,027 – 481,2 Unidentified
34 27,077 – 316,8 C15H10O8 Myricetin Flavonols
35 27,617 þ 549,3 C25H24O14 Chrysoeriol 7-O-(600 -malonyl-glucoside) Methoxyflavones
36 27,649 þ 525,1 C25H32O12 Ligstroside Tyrosols
37 27,918 þ 475,2 C22H18O12 Chicoric acid Hydroxycinnamic acids
38 27,947 – 671,1 Unidentified
39 27,981 – 371,0 C20H20O7 Sinensetin Methoxyflavones
40 28,677 þ 227,0 Unidentified
41 28,692 – 497,3 Unidentified
42 29,064 þ 453,2 C21H24O11 (þ)-Catechin 3-O-glucose Catechins
43 29,929 þ 475,2 C22H18O12 Chicoric acid Hydroxycinnamic acids
44 31,267 – 515,0 C25H24O12 1,3-Dicaffeoylquinic acid Hydroxycinnamic acids
45 32,747 – 186,9 Unidentified
46 33,313 þ 340,1 C16H18O8 3-p-Coumaroylquinic acid Hydroxycinnamic acids

process was 27 � C. Nanjundaiah et al. (2016) established that the oryzae, possibly due to the composition of the material since, although
optimal temperature for the decaffeination of residues from the coffee lignocellulosic substrates absorb a large amount of water, they remain in
industry using the fungus Fusarium solani is 24 � C. Laskshmi and Das the substrate, leaving little water available for the microorganism and its
(2013) using the Trichosporon asahii microorganism for caffeine degra­ development (D� ebora Brand et al., 2000). Therefore, moisture is a
dation, found that the appropriate temperature values were 28 � C. complex parameter that may be related to the substrate, the microor­
Moisture and its relationship to the concept of water activity and ganism, and the fermentation system used.
water available in the environment are important parameters in SSF. Size of the inoculum showed a significant influence, also. In SSF, the
Low moisture can limit the growth and metabolism of the microor­ inoculum must be high enough to allow propagation and predominance
ganism. It can also affect sporulation, enzyme production, and second­ of the strain. In general, it is expected that, by increasing the size of the
ary metabolites. Low water activity can cause low mass transfer and inoculum, the adaptation phase will decrease and increase the growth
poor water availability for the microorganism (Bhargav et al., 2008). rate (Thomas et al., 2013). In this case, a high inoculum favored the
Bhoite and Murthy (2015) found that the moisture suitable for the growth of the fungus, allowing its growth in less time. Similar results
production of enzyme tannase from the coffee pulp was 50% because found Laskshmi and Das (2013), with the microorganism Trichosporon
high moisture produces a reduction in the porosity of the solid matrix asahii, who found that as they increased the inoculum size, the caffeine
decreasing the oxygen transfer. Fermented coffee husks with Aspergillus degradation was greater. Nanjundaiah et al. (2016) found that with an
niger in a packed-bed column bioreactor system Brand et al. (2001) inoculum of 4.8 � 105 spores/ml of fungus Fusarium solani, achieve a
found that at the moisture of 55% they achieve the greatest reduction of complete decaffeination of the caffeine present in the waste industry.
the caffeine content, indicating also that moisture lower than 50% limit Then, the optimization of the factors that were found to be influential
the growth of the fungus and the metabolism of the toxic compounds. in the process was performed: temperature, inoculum size, and initial
However, in previous studies using the same strain in the Erlenmeyer moisture. The results are summarized in the surface plots (Fig. 3). The
system Brand et al. (2000) found that moisture had no influence. best behaviors were obtained at temperature 32 � C, inoculum size 1 �
Although most studies have been carried out at an average moisture 106 spores/g and initial moisture of 72%, degrading caffeine by 68.48%,
content of 50%, in this study this moisture limited the growth of R. condensed tannins by 86.75%, and phenols by 91.16%.

7
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no-Hernandez et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101467

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