scholarly journals Functional Biodiversity of Yeasts Isolated from Colombian Fermented and Dry Cocoa Beans

2020 ◽  
Vol 8 (7) ◽  
pp. 1086 ◽  
Author(s):  
Johannes Delgado-Ospina ◽  
Samantha Triboletti ◽  
Valentina Alessandria ◽  
Annalisa Serio ◽  
Manuel Sergi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fermentation Process ◽  
Yeast Species ◽  
Degradation Products ◽  
Microbial Community Composition ◽  
Functional Biodiversity ◽  
Cocoa Beans ◽  
Pichia Kudriavzevii ◽  
First Time ◽  
Cocoa Fermentation

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

scholarly journals Exploring the Capability of Yeasts Isolated from Colombian Fermented Cocoa Beans to Form and Degrade Biogenic Amines in a Lab-Scale Model System for Cocoa Fermentation

2020 ◽  
Vol 9 (1) ◽  
pp. 28
Author(s):  
Johannes Delgado-Ospina ◽  
Laura Acquaticci ◽  
Junior Bernardo Molina-Hernandez ◽  
Kalliopi Rantsiou ◽  
Maria Martuscelli ◽  
...  
Keyword(s):  
Biogenic Amines ◽  
Yeast Species ◽  
Scale Model ◽  
Cocoa Beans ◽  
Pichia Kudriavzevii ◽  
Toxic Compounds ◽  
Volatile Organic ◽  
Fatty Acids Metabolism ◽  
Cocoa Fermentation

Yeast starters for cocoa fermentation are usually tested according to their enzymatic activities in terms of mucilage degradation and flavor improvement, disregarding their influence on the production or elimination of toxic compounds as biogenic amines (BAs), important for human health. In this work, we tested 145 strains belonging to 12 different yeast species and isolated from the Colombian fermented cocoa beans (CB) for their capability of producing BAs in vitro. Sixty-five strains were able to decarboxylate at least one of the amino acids tested. Pichia kudriavzevii ECA33 (Pk) and Saccharomyces cerevisiae 4 (Sc) were selected to evaluate their potential to modulate BAs, organic acids, and volatile organic compounds (VOCs) accumulation during a simulated cocoa fermentation. The growth of Sc or Pk in the presence of CB caused a significant reduction (p < 0.05) of 2-phenylethylamine (84% and 37%) and cadaverine (58% and 51%), and a significant increase of tryptamine and putrescine with a strong influence of temperature in BA formation and degradation. In addition, our findings pointed out that Pk induced a major production of fatty acid- and amino acid-derived VOCs, while Sc induced more VOCs derived from fatty acids metabolism. Our results suggest the importance of considering BA production in the choice of yeast starters for cocoa fermentation.

scholarly journals Profile of Volatile Compounds of On-Farm Fermented and Dried Cocoa Beans Inoculated with Saccharomyces cerevisiae KY794742 and Pichia kudriavzevii KY794725

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 344
Author(s):  
Gilson Celso Albuquerque Chagas Junior ◽  
Nelson Rosa Ferreira ◽  
Eloisa Helena de Aguiar Andrade ◽  
Lidiane Diniz do Nascimento ◽  
Francilia Campos de Siqueira ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Volatile Compounds ◽  
Multivariate Analyses ◽  
High Capacity ◽  
Starter Cultures ◽  
Cocoa Beans ◽  
Pichia Kudriavzevii ◽  
Fermentative Capacity ◽  
Product Identity ◽  
On Farm

This study aimed to identify the volatile compounds in the fermented and dried cocoa beans conducted with three distinct inoculants of yeast species due to their high fermentative capacity: Saccharomyces cerevisiae, Pichia kudriavzevii, the mixture in equal proportions 1:1 of both species, and a control fermentation (with no inoculum application). Three starter cultures of yeasts, previously isolated and identified in cocoa fermentation in the municipality of Tomé-Açu, Pará state, Brazil. The seeds with pulp were removed manually and placed in wooden boxes for the fermentation process that lasted from 6 to 7 days. On the last day of fermentation, the almonds were packaged properly and placed to dry (36 °C), followed by preparation for the analysis of volatile compounds by GC-MS technique. In addition to the control fermentation, a high capacity for the formation of desirable compounds in chocolate by the inoculants with P. kudriavzevii was observed, which was confirmed through multivariate analyses, classifying these almonds with the highest content of aldehydes, esters, ketones and alcohols and low concentration of off-flavours. We conclude that the addition of mixed culture starter can be an excellent alternative for cocoa producers, suggesting obtaining cocoa beans with desirable characteristics for chocolate production, as well as creating a product identity for the producing region.

scholarly journals Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
A. Espinel-Ingroff ◽  
J. Turnidge ◽  
A. Alastruey-Izquierdo ◽  
F. Botterel ◽  
E. Canton ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Species Complex ◽  
Susceptibility Testing ◽  
Yeast Species ◽  
Sensu Stricto ◽  
Wild Type ◽  
Pichia Kudriavzevii ◽  
Cutoff Value ◽  
Content Type ◽  
Clavispora Lusitaniae

ABSTRACT Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

scholarly journals Fermentation Process of Cocoa Based on Optimum Condition of Pulp PectinDepolymerization by Endogenous Pectolityc Enzymes

1970 ◽  
Vol 26 (2) ◽  
Author(s):  
G.P. Ganda-Putra ◽  
L.P. Wrasiati ◽  
N.M. Wartini
Keyword(s):  
Optimum Condition ◽  
Fermentation Process ◽  
Process Condition ◽  
Cocoa Beans ◽  
Fermentation Time ◽  
Natural Fermentation ◽  
Pectolytic Enzymes ◽  
Split Plot ◽  
Fermentation Period ◽  
Cocoa Fermentation

Pulp degradation during cocoa fermentation can be carried out by depolymerization process of pulp pectin using endogenous pectolytic enzymes at optimum condition. The objectives of this research were to study the effect of fermentation process based on optimum condition in terms of temperature and pH of pulp pectin depolymerization using endogenous pectolytic enzymes polygalakturonase (PG) and pectin metyl esterase (PME) and fermentation period in cocoa processing on quality characteristics of cocoa beans produced and to study the role of those fermentation process in reducing fermentation time to produce cocoa beans with standard quality. This research used split plot design, with treatments of process condition of cocoa fermentation as main plot and fermentation period as split plot. Treatment of process condition of cocoa fermentation consisted of optimum condition for pulp pectin depolymerization by PGs (temperature 47.5OC; initial pulp pH 4.6); optimum condition of depolymerization on sequence depolymerization by PGs (temperature 48.5OC; initial pulp pH 8.0 during 1 day; last temperature 47.5OC; initial pulp pH 4.6 during 6 days), and natural fermentation process a control. While treatment of fermentation period consisted of 0, 1, 2, 3, 4, 5, 6 and 7 days. Evaluation of fermentation period was carried out based on pursuant to criteria of unfermented beans content and fermentation index. The results showed that process condition and fermentation time of cocoa affected quality characteristic of cocoa beans produced. Period of cocoa fermentation process based on optimum condition for pulp pectin depolymerization using endogenous pectolytic enzymes was 2 days shorter compared to natural fermentation. Cocoa beans quality of grade I and II were obtained from fermentation time of 4 and 2 days, respectively, using fermentation process based on optimum condition of pulp pectin depolymerization using endogenous pectolytic enzymes, whereas 6 and 4 days, respectively, when using natural fermentation.Key words: cocoa quality, fermentation, depolymerization, pectolytic.

scholarly journals Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia.

2020 ◽  
Vol 36 (3) ◽  
pp. 236-248
Author(s):  
Maria Denis Lozano Tovar ◽  
Geraldine Tibasosa ◽  
Carlos Mario González ◽  
Karen Ballestas Alvarez ◽  
Martha Del Pilar Lopez Hernandez ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Starter Culture ◽  
Cocoa Bean ◽  
Rrna Gene ◽  
Cocoa Beans ◽  
Isolation And Identification ◽  
Pichia Kudriavzevii ◽  
Cocoa Bean Fermentation ◽  
Cocoa Fermentation

Microbial activity involved in the cocoa beans fermentation process is essential to maintain and improve the organoleptic and nutritional qualities of chocolate; therefore, the aim of this investigation was to search and select microbial isolates with the potential to improve the quality of cocoa beans. Fermentation experimentswere conducted on farms located in Maceo (Antioquia), San Vicente de Chucurí (Santander), and Rivera and Algeciras (Huila), Colombia. Yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and mesophilic aerobic microorganisms were obtained from different fermentation batches. The growth of these microorganismswas tested in six treatments as follows: 50% cocoa pulp agar (CPA), high concentrations of glucose (10%), ethanol (5%), and acetic acid (7%), an acidic pH of 3.0, and a high temperature of 50oC for 24 h. The isolates with the highest growth were identified by 18S and 16S rRNA gene analysis, revealing a high diversity ofspecies associated with cocoa fermentation, including eight species of yeasts (Debaryomyces hansenii, Meyerozyma guillermondii, Wickerhanomyces anomalus, Pichia guillermondii, Pichia kudriavzevii, Trichosporon asahii, Candida parapsilosis, and Pichia manshurica), six species of LAB (Pediococcus acidilactici, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus farraginis, Lactobacillus rhamnosus, and Leuconostoc mesenteroides), four species of AAB (Gluconobacter japonicus, Acetobacter tropicalis, Acetobacter pasteurianus, and Acetobacter malorum/tropicalis), and three species of Bacillus spp. (Bacillusaryabhattai /megaterium, Bacillus subtilis, and Bacillus coagulans). In general, microbial populations increased in cocoa batches after 12 h of fermentation and decreased after 84-96 h. All the yeast isolates grew in 10% glucose and CPA, 85.7% in 5% ethanol, and 95% at a pH of 3.0. All the yeast isolates were affectedby 7% acetic acid and incubation at 50oC for 24 h. Eighty-five percent of the LAB grew in 10% glucose, 100% in 5% ethanol, 42.8% in CPA, 64% at a pH of 3.0, and 35.7% grew after being exposed to 50oC for 24 h; all were affected by 7% acetic acid. As for the AAB, 100% grew in 10% glucose, 71% in 7% ethanol, 100% grew in CPA, in 7% acetic acid, and at a pH of 3.0, while 100% were affected by incubation at 50oC. Three yeast isolates, W. anomalus, D. hansenii and M. guillermondii, three LAB isolates, P. acidilactici, L. brevis, and L. plantarum, and three AAB isolates, A. tropicalis, A. pasteurianus and G. japonicus, were selected as promising strains to be used in a microbial starter culture for cocoa bean fermentation to improve the organoleptic quality of cocoa.

scholarly journals Population Analysis and Evolution of Saccharomyces cerevisiae Mitogenomes

2020 ◽  
Vol 8 (7) ◽  
pp. 1001
Author(s):  
Daniel Vieira ◽  
Soraia Esteves ◽  
Carolina Santiago ◽  
Eduardo Conde-Sousa ◽  
Ticiana Fernandes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Species ◽  
Phenotypic Diversity ◽  
Population Analysis ◽  
Nuclear Genome ◽  
Reference Sequence ◽  
Evolutionary Analysis ◽  
Novel Approach ◽  
History Of ◽  
First Time

The study of mitogenomes allows the unraveling of some paths of yeast evolution that are often not exposed when analyzing the nuclear genome. Although both nuclear and mitochondrial genomes are known to determine phenotypic diversity and fitness, no concordance has yet established between the two, mainly regarding strains’ technological uses and/or geographical distribution. In the current work, we proposed a new method to align and analyze yeast mitogenomes, overcoming current difficulties that make it impossible to obtain comparable mitogenomes for a large number of isolates. To this end, 12,016 mitogenomes were considered, and we developed a novel approach consisting of the design of a reference sequence intended to be comparable between all mitogenomes. Subsequently, the population structure of 6646 Saccharomyces cerevisiae mitogenomes was assessed. Results revealed the existence of particular clusters associated with the technological use of the strains, in particular regarding clinical isolates, laboratory strains, and yeasts used for wine-associated activities. As far as we know, this is the first time that a positive concordance between nuclear and mitogenomes has been reported for S. cerevisiae, in terms of strains’ technological applications. The results obtained highlighted the importance of including the mtDNA genome in evolutionary analysis, in order to clarify the origin and history of yeast species.

scholarly journals Chemical implications and time reduction of on-farm cocoa fermentation by Saccharomyces cerevisiae and Pichia kudriavzevii

2021 ◽  
Vol 338 ◽  
pp. 127834
Author(s):  
Gilson Celso Albuquerque Chagas Junior ◽  
Nelson Rosa Ferreira ◽  
Maria Beatriz A. Gloria ◽  
Luiza Helena da Silva Martins ◽  
Alessandra Santos Lopes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Kudriavzevii ◽  
Time Reduction ◽  
On Farm ◽  
Cocoa Fermentation

scholarly journals Perbaikan Proses Fermentasi Biji Kakao Non Fermentasi dengan Penambahan Biakan Murni Saccharomyces cerevisiae, Lactobacillus lactis dan Acetobacter aceti (Fermentation Process Improvement of Cocoa Beans with Addition of Non Fermentation Inoculum of Saccharomyces cerevisiae, Lactobacillus lactis and Acetobacter aceti)

2017 ◽  
Vol 36 (4) ◽  
pp. 410 ◽  
Author(s):  
Mulono Apriyanto ◽  
Sutardi Sutardi ◽  
Eni Harmayani ◽  
Supriyanto Supriyanto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fermentation Process ◽  
Reducing Sugars ◽  
Acetic Acid Bacteria ◽  
Dry Beans ◽  
Acetobacter Aceti ◽  
Cocoa Beans ◽  
Split Test ◽  
Lactobacillus Lactis

Most cocoa beans produced by Indonesian farmers are non-fermented dry cocoa whose quality can be improved by the fermentation. However, it requires the optimization for fermentation process. This study was conducted to determine the effect of giving a pure culture of Saccharomyces cerevisiae, Lactobacillus lactis and Acetobacter aceti bacteria in fermented dry cocoa beans. Dry beans obtained by drying the wet (fresh) cocoa beans in the cabinet dryer, and subsequently their levels of reduction sugar were determined. The experiments of the fermentation of dry cocoa beans were conducted in a box (p = 120 cm, l = 80 cm, t = 40 cm) with aeration hole (diameter of 1 cm) and the distance between holes was 10 cm. Cocoa beans were incubated for 6 days and without inverted during fermentation. The studied treatments were A1 (without the addition of inoculum (control)), A2 (inocolum was added at the beginning of the incubation), A3 (inoculum was added at the beginning of fermentation (Saccharomyces cerevisiae). After 24 hours of experiment, Lactobacillus lactis was added while Acetobacter aceti was added after 48 hours. Each treatment was repeated three times and observed every two days. The levels of reducing sugars, etanol, acidity, yeasts and acetic acid bacteria population in the fermentation pulp/liquid were observed during the fermentation process. In order to determine the quality of dry beans, several aspects have been measured such as: pH, fermentation index and split test on dry beans after fermentation.ABSTRAKSebagian besar biji kakao yang dihasilkan petani Indonesia merupakan kakao kering non-fermentasi yang kualitasnya masih dapat ditingkatkan dengan metode fermentasi, tetapi dibutuhkan optimasi agar fermentasi dapat berjalan dengan baik. Penelitian ini dilakukan untuk mengetahui pengaruh dari pemberian biakan murni murni Saccharomyces cerevisiae, Lactobacillus lactis dan Acetobacter aceti pada fermentasi biji kakao kering jemur. Biji kakao kering jemur diperoleh dengan mengeringkan biji kakao basah (segar) dalam kabinet dryer, dan ditentukan kadar gula reduksinya. Percobaan fermentasi biji kakao kering jemur dilakukan dalam kotak fermentasi (p = 120 cm, l = 80 cm, t = 40 cm) yang diberi lubang aerasi berdiameter 1 cm dan jarak antar lubang 10 cm. Biji kakao difermentasi selama 6 hari dan tanpa dibalik selama fermentasi. Perlakuan dalam penelitian ini adalah A1 (tanpa penambahan biakan murni murni (kontrol)), A2 (pemberian biakan murni murni diawal fermenatasi), A3 (pemberian biakan murni murni secara bertahap selama fermentasi (Saccharomyces cerevisiae), setelah jam ke 24 diberikan Lactobacillus lactis dan setelah jam ke 48 diberikan Acetobacter aceti. Setiap perlakuan diulangi tiga kali dan diamati tiap dua hari sekali. Kadar gula reduksi, kadar etanol, kadar asam tertitrasi, populasi khamir, dan bakteri asam asetat dalam pulp/cairan fermentasi diamati selama proses fermentasi. Untuk mengetahui kualitas biji kakao kering jemur dilakukan pengukuran pH, indeks fermentasi dan uji belah pada biji kakao kering jemur setelah fermentasi.

scholarly journals Efek Biokonversi Pulp Kakao menjadi Bioetanol Sebagai Sumber Energi Alternatif melalui Fermentasi Aspergillus niger dan Saccharomyces cerevisiae dalam Fermentor Wadah Plastik dan Stainless Steel

2021 ◽  
Vol 5 (1) ◽  
pp. 73
Author(s):  
Desi Riana Saputri ◽  
Fenzy Putri Liewenti ◽  
Stanislaus Dimas Indra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stainless Steel ◽  
Aspergillus Niger ◽  
Fermentation Process ◽  
Test Method ◽  
Cocoa Beans ◽  
Alcohol Fermentation ◽  
Sensory Test ◽  
Steel Material ◽  
Stainless Steel Material

Cocoa beans from Indonesian farmers are not optimal to utilize dan has low quality because they have not done fermentation processing. The aim of this study was to process cocoa beans through fermentation by using Aspergillus niger and Saccharomyces cerevisiae by developing cocoa-pulp byproduct that can be converted to be alcohol. Fermentation process had done in two type of containers such as plastic and stainless steel material. It was observed to know the presence of olygodinamic effect from stainless steel material. The results showed that cacao pulp can be fermented by using Aspergillus niger and Saccharomyces cerevisiae in sensory test method with hyphae growth and strong aromatic from alcohol observation. Stainless steel material in fermentor tub did not give the oligodynamic effect for Aspergillus niger fungus and Saccharomyces cerevisiae growth. The Fermentation process produced 0.66 mL of alcohol from one kilogram fresh cocoa beans. The maximum bioethanol product is that happened in 4th day fermentation process about 5.30 %.

scholarly journals Curing of Cocoa Beans: Fine-Scale Monitoring of the Starter Cultures Applied and Metabolomics of the Fermentation and Drying Steps

2021 ◽  
Vol 11 ◽  
Author(s):  
Cristian Díaz-Muñoz ◽  
Dario Van de Voorde ◽  
Andrea Comasio ◽  
Marko Verce ◽  
Carlos Eduardo Hernandez ◽  
...  
Keyword(s):  
Starter Culture ◽  
Agar Plate ◽  
Starter Cultures ◽  
Cocoa Bean ◽  
Sequence Variant ◽  
Cocoa Beans ◽  
Pichia Kudriavzevii ◽  
Fermentation Processes ◽  
Wide Range ◽  
Cocoa Fermentation

Starter culture-initiated cocoa fermentation processes can be applied to improve the quality of cured cocoa beans. However, an accurate monitoring of the microbial strains inoculated in fresh cocoa pulp-bean mass to assess their contribution to the cocoa bean curing process is still lacking. In the present study, eight different cocoa fermentation processes were carried out with Trinitario cocoa in vessels in Costa Rica to assess the contribution of two candidate yeast starter culture strains, namely Saccharomyces cerevisiae IMDO 050523 and Pichia kudriavzevii IMDO 020508, inoculated in combination with Limosilactobacillus fermentum IMDO 0611222 and Acetobacter pasteurianus IMDO 0506386. A multiphasic approach, consisting of culture-dependent selective plating and incubation, rRNA-PCR-DGGE community profiling of agar plate washes, and culture-independent high-throughput amplicon sequencing, combined with a metabolite target analysis of non-volatile and volatile organic compounds (VOCs), was performed on samples from the fermentation and/or drying steps. The different starter culture mixtures applied effectively steered the cocoa fermentation processes performed. Moreover, the use of an amplicon sequence variant (ASV) approach, aligning these ASVs to the whole-genome sequences of the inoculated strains, allowed the monitoring of these inoculated strains and their differentiation from very closely related variants naturally present in the background or spontaneous fermentation processes. Further, traits such as malolactic fermentation during the fermentation step and acetoin and tetramethylpyrazine formation during the drying step could be unraveled. Finally, the yeast strains inoculated influenced the substrate consumption and metabolite production during all starter culture-initiated fermentation processes. This had an impact on the VOC profiles of the cured cocoa beans. Whereas the P. kudriavzevii strain produced a wide range of VOCs in the cocoa pulp, the S. cerevisiae strain mostly influenced the VOC composition of the cured cocoa beans.

Sign in / Sign up

Export Citation Format

Share Document