Structural Alterations of Cocoa Shell After Solid State Fermentation With Penicillium Roqueforti

doi:10.21203/rs.3.rs-1598943/v1

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Structural Alterations of Cocoa Shell After Solid State Fermentation With Penicillium Roqueforti

https://doi.org/10.21203/rs.3.rs-1598943/v1

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Lignocellulosic residues, such as cocoa bean shell (FI), are generated in large quantities during agro-industrial activities. Proper management of residual biomass through solid state fermentation (SSF) can be effective in obtaining value-added products. The hypothesis of the present work is that the bioprocess promoted by P. roqueforti can lead to structural changes in the cocoa bean shell fibers that confer characteristics of industrial interest. To unveil such changes, the techniques of FTIR, SEM, XRD, TGA/TG were used. After SSF, an increase of 36.6% in the crystallinity index was observed, reflecting the reduction of amorphous components such as lignin in the FI residue. Furthermore, an increase in porosity was observed through the reduction of the 2θ angle, which gives the FF a potential candidate for applications of porous products. The FTIR results confirm the reduction in hemicellulose content after SSF. The thermal and thermogravimetric tests showed an increase in the hydrophilicity and thermal stability of FF (15% decomposition) in relation to the in natura by-product FI (40% decomposition). These data provided important information regarding changes in the crystallinity of the residue, existing functional groups and changes in degradation temperatures. The techniques employed contributed to the understanding of the changes in the lignocellulosic matrix after the fungus growth, as well as the mechanism that helps to propose the total use of the residual solid after fermentation processes.

Residual biomass

Lignocellulose

Bioprocess

Lignocellulosic fibers.

Lignocellulose, the main part of biomass and a structural component of the plant cell wall, is a three-dimensional structure resulting from the combination of cellulose (35–50%) with hemicellulose (20–35%), lignin (15–20%), ash and other compounds (15–20%) [1, 2, 3]. As a renewable resource, this biomass is used in fermentation processes aimed at obtaining high value products: enzymes [4], protein enrichment [5], xylooligosaccharides [6] and xylitol [7].

Furthermore, lignocellulosic residues are generated in large quantities from agricultural activities [8]. It is estimated that in the cocoa industry (Teobroma Cacao L) approximately six million tons of this residual biomass are produced per year, which makes its management extremely challenging [9]. Recently, there has been an increase in interest in the use of cocoa by-products. The value of exported by-products, such as bark, averaged US$ 244 million in 2017 as a direct consequence of the use of these materials for the development of products on an industrial scale (feed for animal nutrition, fertilizers, biofuels and chemicals) [10] .

From a sustainable point of view, the proper management of residual biomass through cocoa biotransformation seems to be a relevant and effective way to obtain value-added products from the residues of the cocoa production chain [10]. Therefore, the residues from the cocoa bean shell emerge as a potential source of carbon and nitrogen for fermentation processes. In this sense, researches have demonstrated the feasibility of using this by-product to obtain lipase by the fungus Penicillium roqueforti [11] and lignocellulolytic enzymes by the fungus Aspergillus niger [12]. This approach, in addition to reusing and transforming waste into products with high added value (upcycling), reduces the amount of material to be handled for sanitary-environmental treatment [10]. Thus, its implementation presents a better cost-benefit when compared to incineration technology, in which there is a high energy expenditure and environmental impact arising from the release of gases that aggravate the greenhouse effect [13].

However, the reuse of these substrates in bioprocesses depends on the reduction of their recalcitrant properties resulting from the three-dimensional arrangement between lignin, cellulose and hemicellulose [14, 15]. One of the approaches to reduce this recalcitrance is the use of bioprocesses mediated by organisms that produce lignocellulolytic enzymes, a process called biological pretreatment [14].

Among the bioprocesses, solid state fermentation (SSF) has been intensively investigated, given the undisputed potential of fungi regarding the production of high added value metabolites [4, 16, 17, 18]. As examples of successful application of this technology, there is the production of enzymes such as xylanase by Trichoderma stromaticum [19], xylanase and lipase by P. roqueforti [20], endoglucanase [16], exoglucanase [21] antioxidant compounds [22], vitamins and antibiotics [23] and nutraceutical compounds [24].

Among the main fungi used for SSF, the genera Aspergillus [11], Rhizopus [4] and Penicillium [25] stand out. The latter, with the promising representative of the species P. roqueforti, which is classified as GRAS (Generally Recognized As Safe) by the Food and Drugs Administration (FDA). For this reason, the availability and use of products from it becomes faster and more acceptable. Therefore, SSF by P. roqueforti emerges as a fruitful object of study, with considerable direction towards the feasibility of using abundant and low-value substrates.

In order to verify the possible modifications promoted by SSF, techniques such as Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) were successfully applied to identify the main structural transformations that occurred in Eucalyptus grandis [1]. In addition, other studies have successfully used the techniques of FTIR, X-ray diffraction (XRD) and SEM to evaluate the effect of biological treatment promoted by different microorganisms on the structure and morphology of rice straw [26], bamboo (Phyllostachys edulis) [27, 28] and maize [29].

The present study aimed to investigate the use of the agroindustrial by-product cocoa shell bran (Theobroma cacao L.) as a substrate for SSF promoted by the fungus Penicilium roqueforti. It is verified in the scientific literature a greater direction of researches for the optimization of the enzyme production, being evident the lack of studies that investigate the structural modifications that occur in the biomass during such processes. The hypothesis of the present work is that the bioprocess promoted by P. roqueforti can lead to structural changes in the cocoa bean shell fibers that confer characteristics of industrial interest. To unveil such changes, Fourier Transform Infrared Spectrometry (FTIR), Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD) and thermogravimetric analysis (TGA/TG) were employed.

2.1 Cocoa Shell Residue

The cocoa shell was kindly donated by a cocoa processing company located in southern Bahia/Brazil. The residue was crushed in a Wiley knife mill (ACB LABOR®) to particles equal to or less than 0.74 mm and then stored in a glass container until ready for use.

Physicochemical analyzes were carried out according to the methodology of the Association of Official Analytical Chemists – AOAC (2006) to determine the values of Dry Matter (DM), Mineral Matter (MM), Crude Protein (PB), Crude Fiber (FB), Fiber in Neutral Detergent (FDN), Fiber in Acid Detergent (FDA), Ethereal Extract (EE), Lignin, Cellulose, Hemicellulose, Non-Protein Nitrogen (NNP), Neutral Detergent in Non-soluble Nitrogen (NIDN), Acid Detergent in Nitrogen Non-Soluble (NIDA), Non-Fibrous Carbohydrate and Total Carbohydrate, Neutral Insoluble Ash and Acid Insoluble Ash. The values obtained were expressed in % of dry matter. In order to determine the macro and micro minerals present in the cocoa bean shell, the isolated mineral extract was obtained by wet digestion with nitric acid and perchloric acid [30]. For the quantification of Calcium (Ca), Magnesium (Mg), Iron (Fe), Zinc (Zn), Copper (Cu) and Manganese (Mn), a flame atomic absorption spectrometer (Thermo© Ice 3000, series AA, Cambridge-UK) was used. Potassium (K) was determined using a flame photometer (Model FP910 – PG Instruments). Phosphorus (P) was determined by the molybdate method and the reading was made in a spectrophotometer at 725 nm [31]. The results were expressed in mg.kg^− 1 and g.kg^− 1 of cocoa bean shell bran.

2.2 Microorganism and inoculum

The microorganism used for the fermentation tests was P. roqueforti ATCC 10110, which was obtained from the collection of microorganisms of the National Institute for Quality Control in Health (INCQS) of the Osvaldo Cruz Foundation (Fiocruz, Manguinhos, RJ, Brazil) under registration number 40074 and lot 041140074. This fungus was preserved in silica and glycerol medium at -80 ºC in an ultrafreezer. The subculture of the strains was carried out in agar-dextrose-potato agar (PDA) medium in Petri dishes, previously sterilized in a vertical autoclave (CS30 Prismatec, Itu, Brazil) at 121 ºC for 15 min. The inoculated plates were incubated in an incubator (SL 222, Solab) at a controlled temperature of 25 ºC for 7 days and then stored at 4 ºC for later use.

The preparation of the spore suspension was carried out using the microorganism cultivated in PDA medium for 7 days at 27°C. After the incubation period, the sporulated culture was subjected to scraping with the aid of glass beads and suspended in a Tween 80 solution (0.01%, v/v), both previously sterilized in a vertical autoclave (CS30 Prismatec, Itu, Brazil ) at 121°C for 15 min. The suspension was collected in an Erlenmeyer flask and an aliquot of 0.1 mL was diluted in a test tube to count the number of spores in a binocular microscope using a Neubauer chamber.

2.3 Solid State Fermentation

250 mL Erlenmeyers containing 20 g of cocoa shell bran were used as bioreactors. Sterilization took place in a vertical autoclave (CS30 Prismatec, Itu, Brazil) and the medium humidity was adjusted to 50%. The microbial inoculum was carried out with 107 conidia/g of cocoa shell bran and the fermentations carried out at 27ºC for seven days [32]. After this period, the fermented cocoa shell bran was sterilized in an autoclave and oven dried to 7% humidity.

2.4 Characterization

2.4.1 Scanning Electron Microscopy (SEM)

The analysis by scanning electron microscopy was performed at the Center for Electron Microscopy of the State University of Santa Cruz (UESC-Bahia) with the aid of the Quanta250 scanning electron microscope (FEI Company), with a maximum operating voltage of 30 kV and nominal resolution of 1.2 nm at a voltage of 20 kV. The in natura residues, fermented and fragmented in approximately 5 cm, were fixed to a “Stub” support. The readings were carried out in ambient mode, used to investigate samples incompatible with high vacuum and, therefore, impossible to be analyzed by other traditional scanning electron microscopy methods. Electromicrographs were taken with magnification of 1000 and 3000x.

2.4.2 Fourier transform infrared spectrometry (FTIR)

The FTIR of the residues was analyzed using a Cary 630 FTIR Medium Infrared instrument (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with an attenuated total reflectance cell (ATR) and deuterated triglycine sulfate detector (DTGS) with software Agilent microlab PC. The samples were evaluated in the spectral region with wavelength from 4000 cm-¹ to 650 cm-¹, triangular apodization and 64 scans for each spectrum, which were obtained in transmittance mode. All spectra were collected with a resolution of 4 cm-1 and a temperature of 17°C.

2.4.3 X-ray diffraction (XRD)

The XRD of the by-products was performed in a Shimadzu XRD-6000 equipment, with Cu.Kα radiation = 0.15418 nm (40 kV, 30 mA). The analyzed range was 2θ = 2° to 50°, with a step of 0.05° and integration time of 2s at room temperature (25°C). Finally, the crystallinity was calculated based on the ratio of the height of the crystalline peak intensity (I200 – Iam) and the total intensity (I200) after subtracting the measured background signal without the cellulosic sample [33].

2.4.4 Thermogravimetric analysis (TGA / TG)

Thermogravimetric analysis was performed using a TGA equipment – Q series – model Q 500 (TA Instruments, New Castle, Delaware, USA). The samples were heated in the range of ambient temperature (± 25°C) up to 700°C, in an inert atmosphere, with a heating ratio of 10°C. min^− 1.

3.1 Chemical and mineral composition of cocoa shell

The presence of significant levels of carbohydrates, lipids, proteins, macro and micronutrients in the cocoa bean bran make this residue a valuable substrate for bioprocesses such as SSF [34]. The characterization of cocoa bean shell meal showed a composition mainly represented by carbohydrates, proteins and mineral matter (Table 1). It is believed that the considerable proportion of non-fibrous carbohydrates (17.21%) and proteins (17.85%) may support the premature growth of P. roqueforti [35], since the substrate acts as a source of carbon and nitrogen, necessary for the growth of microorganisms. Thus, it can be seen that cocoa shell bran has sufficient potential to be used as a matrix for SSF.

Table 1

Chemical Composition of Cocoa Almond Shell
Component	% dry bottle
Non-Fibrous Carbohydrate	17,21 ± 0,13
Total Carbohydrates	72,8 ± 0,4
Mineral Material	8,4 ± 0,17
Ash	0,14 ± 0,02
Ethereal extract	0,97 ± 0,03
Total Protein	17,85 ± 0,11
NNP	0,23 ± 0,01
FDN	55,58 ± 0,3
FDA	49 ± 0,34
Lignin	21,8 ± 0,26
Cellulose	27,15 ± 0,23
Hemicellulose	6,6 ± 0,14
NIDN	2,08 ± 0,1
NIDA	0,72 ± 0,05
NNP = Non-Protein Nitrogen; FDN = Soluble Fiber in Neutral Detergent; FDA = Soluble Fiber in Acid Detergent; NIDN = Neutral detergent in non-soluble nitrogen; NIDA = Non-Soluble Nitrogen Acid Detergent.

[Insert Table 1 about here]

Furthermore, the quantification of macronutrients in almond shell bran cocoa (FI) (Table 2) indicated the presence of Potassium, Calcium and Phosphorus, important macroelements for the cultivation of fungi. Furthermore, there are reports of increased enzyme production when supplementing salts such as MgCl, CaCO3, Na2CO3 [36], showing the feasibility of using FI as a substrate for biomass production by filamentous fungi.

Table 2

Quantification of macrominerals and microminerals from Cocoa Almond Shell
Macrominerals	Concentration (mg/kg of FI)	Microminerals	Concentration (mg/kg of FI)
Potassium	25,6	Iron	1300
Magnesium	5	Zinc	284
Calcium	4,4	Manganese	73
Phosphorus	3,3	Copper	23,8

[Insert Table 2 about here]

In addition, the micronutrient determination of IF (Table 2) listed Magnesium, Zinc, Manganese and Copper, which are known enzymatic cofactors present in synthetic media for the production of ligninolytic enzymes [20, 37]. Thus, it can be stated that CAC is an adequate substrate for the bioprocess by P. roqueforti, supplying its nutritional demands and also promoting favorable conditions for enzyme expression.

3.2 Scanning Electron Microscopy (SEM)

Figure 1 shows the electromicrographs of the residues. The residue is shown to be a material without preferential organization and without long-range order (Fig. 1a). FI is characterized by being an amorphous material with a smooth porous structure from components such as polysaccharides, hemicellulose and lignin present on the surface [38], and in addition there is an irregular formation of cellulosic fiber blocks in the cocoa bran [39].

[Insert Fig. 1 about here]

After 7 days of SSF (Fig. 1b), colonization of P. roqueforti in the solid matrix of cocoa bran was observed, which was completely covered with spores. Colonization of the mycelium on the matrix is an important characteristic related to the generation of different biotechnological products [40], given that the fungus can degrade macromolecules and natural polymers during and after their “adhesion” to the substrate [41, 42]. On the other hand, in SSF the microorganism attaches itself to the solid substrate and, more specifically in the case of filamentous fungi, the mycelia penetrate the solid particles, making effective separation difficult, requiring the sterilization of the product from the fungal metabolism before its application [34].

When analyzing by SEM the effect of SSF on rice straw, the authors found that the structural properties of biomass are the main obstacles to cellulose hydrolysis and it was observed that the bioprocess promoted a visible destruction in the cell wall structure, alteration in morphology and formation of holes and cracks on biomass surfaces, which culminated in intrinsic structural changes that can increase biomass reactivity and increase cellulose accessibility for enzymatic hydrolysis [26].

3.3 X-ray diffraction (XRD)

The intensity of the crystalline peaks and the amorphous halo of the FI and FF samples (Fig. 2), indicate that the crystalline peak is located between angles 22°≤2θ ≥ 23°, being characteristic of type I cellulose, which is a structure composed of units β-(1 → 4) -D-glucopyranose repeats with parallel-chain glucan building blocks [43]. Furthermore, it appears that the crystallinity index of the residue increased from 19.7 to 27.5 after SSF. This result shows the removal of amorphous components of the residue, such as lignin. This 36.6% increase in the crystallinity index is quite promising, as some works used pure enzymes to perform this function and did not obtain the aforementioned results [44].

[Insert Fig. 2 about here]

It is known that this hydrolysis of amorphous holocellulose (hemicelluloses + amorphous cellulose) occurs as a consequence of enzymatic action [45], which suggests the use of fungi of the species P. roqueforti as biological catalysts for the production of cellulosic nanofibrils. In this sense, it was possible to obtain cellulose nanofibril from licorice residues through an enzymatic pre-treatment [46], as well as the application of xylanase to obtain nanocellulose from sugarcane bagasse [47].

When applying Bragg's Law and Scherrer's Law to calculate the interplanar distance (d) and the crystallite size (Lc), respectively, it is observed that the residue fermented by the fungus P. roqueforti presented a reduction in the 2θ angle for angles minors (21.65). This evidences an increase in the distance between the crystalline planes and in the size of the crystallites by up to 3 times, with direct consequences on the porosity of the product (Table 3).

Table 3

Structural modifications observed by XRD in cocoa bran in natura (FI) and after fermentation (FF)
Sample	2θ	FWHM	d (nm)	Lc (nm)	Ic (%)
FI	22,17	1,8855	0,40	4,30	19,7
FF	21,65	0,5500	0,41	14,72	27,5
2θ = Bragg angle; FWHM: width half height; d (nm) = interplanar distance; Lc = crystallite size; Ic = crystallinity index.

[Insert Table 3 about here]

The measurement of the crystallinity index has received a lot of attention due to commercial applications [48] and due to the applications that fibrous materials have in the most diverse areas: packaging, drug distribution, food, cosmetics and the biomedical industry [49].

3.4 Fourier transform infrared spectrometry (FTIR)

The FTIR spectra obtained from CAC (Fig. 3) showed a broad absorption peak close to 2918 cm^− 1 which corresponds to the C-H stretch of lignocellulose [50], and which was less intense after SSF (FF). Interestingly, the peak at 1742 cm^− 1, arising from the symmetrical stretching of the C = O group of the hemicellulose carboxylic acid [1], was only present in the unfermented substrate (FI).

[Insert Fig. 3 about here]

Furthermore, peaks ~ 1610 and ~ 1507 cm^− 1 were also observed in both samples, due to C-C stretch in the phenyl group of lignin [49] and the position ~ 1460 cm-1 due to the asymmetric deformation of C-H and due to the benzene present in lignin or due to the C-C vibrations of the aromatic rings of lignin [47], as well as the peak at ~ 1425 cm^− 1, referring to the C-H asymmetric deformation of lignocellulose, which were less evident after SSF [27].

CAC before SSF (FI) peaked at position ~ 1320 cm^− 1, associated with typical cellulose values (51). However, after SSF, the presence of ~ 1373 cm^− 1 and ~ 1238 cm^− 1 peaks was verified, related to the symmetrical deformation of H in lignocellulose and the elongation of the C-O group in lignin phenol [2, 3], respectively. On the other hand, the peak at ~ 1150 cm^− 1 (C-O-C stretch vibration in cellulose and hemicellulose) [52, 53, 34] was observed only in CAC before SSF.

Additionally, the broad peak close to 850 cm^− 1, referring to C-H glycosidic deformation with vibration of the O-H deflection ring confirms the presence of β-glycosidic bonds between the cellulose an hydroglucose units [50] After SSF, to two others peaks appeared: 880 and 820 cm^− 1, referring to C-H-type glycosidic bonds and also to deformation caused by O-H vibration associated with glycosidic bonds between an hydroglucose units in cellulose molecules. All results mentioned above allude to the reduction in the hemicellulose content of CAC after SSF, demonstrating the success of the fungus P. roqueforti in producing hemicellulolytic enzymes and, consequently, biotransforming this molecule into modified biomass.

Finally, it was possible to observe a series of peaks indicative of groups with antioxidant properties: phenol group comprised between 3562 − 3322 cm^− 1 and attributed to the O-H stretching and 1244 − 1064 cm^− 1 attributed to the C-H stretching; as well as the aromatic ring in the sign between 2925 − 2854 cm^− 1 and attributed to the stretching of C-H, 992 − 680 cm^− 1 alluding to the angular deformation of C-H; and 1645 − 1544 cm^− 1 assigned to the C-C stretch [54, 55].

These findings suggest the use of fermented biomass as a substrate for several applications, such as the development of corrosion inhibitors and biocatalysts for dye degradation. In this sense, the application of fermented biomass as an ecological process to protect carbon steel was successfully investigated, in which 93% of corrosion inhibition in acidic medium was obtained [56]; as well as its use as support for the production of fungal lipases, with significant cost reduction and fewer steps to obtain the enzyme [57]. Another result reported in the literature was the degradation of methylene blue, in which a maximum amount of 51.4 mg/g of dye adsorption was obtained with the application of fermented residues [58], strengthening the hypothesis of application of fermented products by P. roqueforti as antioxidant agents.

3.5 Thermogravimetric analysis (TGA / TG)

A representative DSC curve of the analyzed residues, in nitrogen atmosphere, is presented in Fig. 4. The endotherm observed at approximately 50°C is attributed to the evaporation of moisture present in the samples.

[Insert Fig. 4 about here]

In the in natura by-product, the water loss occurs at 37 ºC while in the by-product after SSF at 52 ºC. The bioprocess provides the disruption of the cellulose structure through enzymatic action, making the cellulose microfibrils more exposed and, therefore, promoting greater moisture retention on its surface, given the hydrophilic character of cellulose.

The thermal decomposition mechanism of FI (Fig. 5a) and after SSF (Fig. 5b) showed two thermal mass reduction events. The first of them occurred below 220°C and is associated with the evaporation of chemisorbed water [59], corresponding to a reduction of about 40% and a reduction peak at 44°C for FI and 15% of the FF mass with a reduction peak at 63.4°C (Fig. 5).

[Insert Fig. 5 about here]

Then, the second thermal event is observed, obtaining a greater reduction in the FI mass due to the chemical decomposition of hemicellulose components during heating [60]. In this thermal event, the percentage of mass reduction of FF (54.5%) was 18.5% lower when compared to FI (73%). Given that FF has a lower content of cellulosic components, it cannot be said that its higher thermostability comes from them. Consequently, in accordance with the XRD findings, it is concluded that SSF promoted an increase in FF crystallinity which can be explained by the lack of lignin and hemicellulose or the partial reduction of the lignin and hemicellulose portions (in the amorphous part) during the SSF, resulting in fibers more resistant to thermal decomposition, an aspect with prospects for industrial application [61]. In this sense, studies evaluating sisal fibers showed an initial thermal degradation temperature of 250 ºC and suggested that materials in this decomposition range can be used for processing with most polymers in the production of polymeric composites, showing the innovative properties of FF after SSF [62].

The present study demonstrated the changes in cocoa bean shell residue after SSF promoted by P. roqueforti. XRD results indicated a 36.6% increase in the crystallinity index and a reduction in the lignin content of the FI residue. Furthermore, the reduction of the 2θ angle showed an increase in porosity after SSF. The spectra obtained by FTIR confirmed the above-mentioned results, showing a clear modification of the chemical profile, a reduction in the hemicellulose content and an increase in the content of antioxidant compounds after SSF. Furthermore, it was observed greater hydrophilicity and thermostability (15% decomposition for FI versus 40% decomposition for FF). All these findings show the innovative characteristics given to cocoa bean shell residue through the SSF bioprocess. It is concluded that the approach studied in this work has prospects for industrial application in the field of porous products and antioxidant materials, as well as being an innovative green alternative with economic aggregation for the management of cocoa bean shell residue.

SSF, Solid State Fermentation; GRAS, generally regarded as safe; FDA, Food and Drugs Administration; SEM, Scanning Electron Microscopy; FTIR, Fourier transform infrared spectroscopy; XRD, X-ray diffraction; TGA, Thermogravimetric analysis; AOAC, Association of Official Analytical Chemists; MS, Dry Matter; MM, Mineral Matter; PB, Crude Protein; FB, Raw Fiber; FDN, Fiber in Neutral Detergent; FDA, Fiber in Acid Detergent; EE, Ethereal extract; NNP, Non-Protein Nitrogen; NIDN, Neutral Detergent in Non-Soluble itrogen; NIDA, Non-soluble Nitrogen Acid Detergent; K, Potassium; Ca, Calcium; Mg, Magnesium Fe, Iron; Zn, Zinc; Cu, Copper; Mn, Manganese; P, Phosphorus; NIQCH, National Institute of Quality Control in Health; PDA, Potato Dextrose Agar; ATR, Attenuated Total Reflection; DTGS, deuterated triglycine sulfate; FI, Cocoa bran in natura; FF, Cocoa bran after solid state fermentation.

ACKNOWLEDGMENTS

The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), the National Council for Scientific and Technological Development (Productivity in Research Grants 302259 / 2018-0, CNPq, Brazil), and the Research Support Foundation of the State of Bahia (FAPESB, Brazil) and the State University of Southwest Bahia (UESB) for their financial assistance, the State University of Santa Cruz (UESC) for the use of equipment and facilities.

Conflicts of interest The authors declare that they have no conflict of interest

Availability of data and material Not applicable

Code availability Not applicable

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Structural Alterations of Cocoa Shell After Solid State Fermentation With Penicillium Roqueforti

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cocoa Shell Residue

2.2 Microorganism and inoculum

2.3 Solid State Fermentation

2.4 Characterization

2.4.1 Scanning Electron Microscopy (SEM)

2.4.2 Fourier transform infrared spectrometry (FTIR)

2.4.3 X-ray diffraction (XRD)

2.4.4 Thermogravimetric analysis (TGA / TG)

3. Results And Discussion

3.1 Chemical and mineral composition of cocoa shell

3.2 Scanning Electron Microscopy (SEM)

3.3 X-ray diffraction (XRD)

3.4 Fourier transform infrared spectrometry (FTIR)

3.5 Thermogravimetric analysis (TGA / TG)

4. Conclusion

Abbreviations

Declarations

References

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