Influence of pulp on the microbial diversity during cupuassu fermentation

Journal Pre-proof Influence of pulp on the microbial diversity during cupuassu fermentation

Simone Ramos, Marcela Salazar, Leandro Nascimento, Marcelo Carazzolle, Gonçalo Pereira, Tiago Delforno, Maristela Nascimento, Tiago de Aleluia, Renata Celeghini, Priscilla Efraim PII:

S0168-1605(19)30396-4

DOI:

https://doi.org/10.1016/j.ijfoodmicro.2019.108465

Reference:

FOOD 108465

To appear in:

International Journal of Food Microbiology

Received date:

9 February 2019

Revised date:

31 October 2019

Accepted date:

26 November 2019

Please cite this article as: S. Ramos, M. Salazar, L. Nascimento, et al., Influence of pulp on the microbial diversity during cupuassu fermentation, International Journal of Food Microbiology (2019), https://doi.org/10.1016/j.ijfoodmicro.2019.108465

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© 2019 Published by Elsevier.

Journal Pre-proof INFLUENCE OF PULP ON THE MICROBIAL DIVERSITY DURING CUPUASSU FERMENTATION Simone Ramosa, Marcela Salazarb, Leandro Nascimentob, Marcelo Carazzolleb, Gonçalo Pereirab, Tiago Delfornoc, Maristela Nascimentoa, Tiago de Aleluiaa, Renata Celeghinia, Priscilla Efraima* a Department of Food Technology. University of Campinas, Campinas, São Paulo, Brazil. b

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Laboratory of Genomic and Expression, Institute of Biology, University of Campinas, São Paulo,

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Brazil. c Microbial Resources Division, Research Center for Chemistry, Biology and Agriculture

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(CPQBA), University of Campinas – UNICAMP, São Paulo, Brazil. *Corresponding author e-mail: [email protected]

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Abstract

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Cupuassu (Theobroma grandiflorum Schum) is a fruit belonging to the same genus as cocoa and, through seed fermentation, a chocolate-like product called “the

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cupulate” is obtained. The pulp is removed from the seeds before fermentation because its abundance hinders the process. Unlike cocoa, little is known about the

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microbial diversity involved in cupuassu fermentation. The goal of this study was to explore the use of next-generation sequencing to identify the yeasts and bacteria

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communities involved in cupuassu seed fermentation on three different pulp concentrations (0, 7.5, and 15%) as well as two turning schemes on the microbial growth. In order to do that, a massive sequencing of the 16S and ITS4 rRNA region (S) using the Illumina MiSeq Platform identified some genera of bacteria and yeasts, respectively, in the fermentation environment. Taxonomic analyses of both communities, especially at the genus level, revealed a predominance of yeasts such as Pichia and Hanseniaspora, and bacteria such as Acetobacter and Lactobacillus. A predominance of bacteria over yeasts diversity was observed in the experiments with higher pulp concentrations (15%). The physicochemical analysis showed that fermentation of samples with 15% pulp exhibited longer fermentation times, the highest temperatures, and elevated production of organic 1

Journal Pre-proof acids such as acetic acid, a precursor of flavor. In addition, the turning applied every 24 hours to the mass slightly favored the formation of flavor precursors. It seems that the abundance and composition of cupuassu pulp, rich in organic compounds, can influence the diversity of some populations of yeasts. Some of those compounds identified in previous studies are terpenes with antimicrobial activity. More studies will be necessary to confirm if the presence of terpenes compounds in the cupuassu pulp exert some inhibitory action on microorganism diversity.

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Keywords: cupuassu, fermentation, bacteria, yeasts, next-generation sequencing

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1. Introduction

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Cupuassu (Theobroma grandiflorum Schum) is a native fruit of the Amazon

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region belonging to the same genus of cocoa, widely distributed in the Brazilian

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Legal Amazon and South America (Ecuador, Guyana, Martinique, Costa Rica, Sao Tome, Trinidad and Ghana) (Venturieri, 1993). The fruit presents a strong and

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pleasant fragrance due to the volatile compounds present, such as esters (ethyl

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acetate, ethyl butanoate, ethyl propanoate, ethyl hexanoate) (Quijano and Pino,

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2007). Cupuassu fruit exhibits different shapes (oblong, oval, elliptical, obovoid or round) and weight between 200 and 4000 g. Each fruit has from 15 to 50 seeds surrounded by a pulp, which represents 38 – 43 % of the fruit (Matos et. al., 2008; Souza and Souza, 2002). The pulp presents a pH of 3.5 ± 0.2, while the seeds have a pH of 6.35 (Canuto et al., 2010). Among the macronutrients present in the pulp, carbohydrates stand out, with sucrose being the predominant sugar (34.6% of dry matter). Concerning fatty acids, the pulp has palmitic, linoleic and α-linolenic acids in higher concentration than any other acids. The pulp has an appreciable amount of micronutrients such as K, Mg and P (34.27; 13.07 and 15.73 mg / 100 g, 2

Journal Pre-proof respectively) in relation to the other minerals. In the seed mineral K is also predominant (26.21 mg / 100 g). The seeds also have a high protein and lipid content (64.8%). The stearic and oleic acids in stand out. Cupuassu pulp contains almost all essential amino acids (tryptophan was not detected) and some nonessential amino acids (asparagine and glutamine are 15.77 and 16.25 g / 100 g protein, respectively) and considerable levels of ascorbic acid (~ 96-111 mg / g)

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(Rogez et al., 2004).

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All these characteristics make cupuassu stand out as a fruit with high

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nutritional value (Canuto et. al, 2010; Carvalho et. al, 2005; Carvalho et. al, 2008;

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Naozuka, 2008; Pugliese et. al, 2013; Rogez et. al., 2004). Cupuassu pulp also

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presents pectin, which makes it suitable to prepare jams and jellies, for example (Gondim, 2001). Studies indicated that the pectin fraction of cupuassu pulp

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presented higher yield (7%) when compared to Citrus depressa (4.1%) and yellow passion fruit (2.9%) (Vriesman et al., 2010). Despite all this, the seeds are still

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considered by-products (Cohen et al., 2004) and the butter is extracted in small

2006).

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scale from them only to be used by the cosmetic industry (Luccas and Kieckbusch,

Cupuassu seeds must be fermented in order to obtain the cupulate, a product similar to chocolate (Cohen et al., 2009). The producers perform total or partial seed depulping (5%) (Cohen and Jackix, 2005; Matos et al., 2008), since the higher quantities naturally slow down or prevent fermentation, for unknown reasons. As occurs with cocoa fermentation, the pulp represents an important source of substrates in the consortium of microorganisms (yeasts, lactic acid and 3

Journal Pre-proof acetic acid bacteria) for the process (Ardhana and Fleet, 2003; Guehi et al., 2010b; Schwan and Wheals, 2004). Many factors can contribute to spontaneous contamination, such as the surface of the pods, workers’ hands, utensils, soil, plant leaves, among other factors (Schwan and Wheals, 2004). The fermentation process is a decisive step in the formation of the flavor precursors, through the transformation of substrates in the pulp, mediated by

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microorganisms (Ardhana and Fleet, 2003; Schwan and Wheals, 2004). In the

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beginning, the environment is anaerobic with a predominance of yeasts that are

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important for the sugars conversion into ethanol and pulp liquefaction (Fowler,

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2009; Schwan and Wheals, 2004). Lactic acid bacteria (LAB), which are also

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present, use citric acid and sugars from the pulp to form lactic acid, a non-volatile organic compound that may give an undesirable acidity to the final product

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(Afoakwa, 2011; Schwan, 1998; Schwan and Wheals, 2004). However, studies have indicated that some heterofermentative LAB strains are important producers

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of flavor precursor compounds (Lefeber et al., 2011). Furthermore, some species

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of LAB isolated from the fermentation of cupuassu seeds exhibited probiotic properties (Ornellas et al, 2017). As the fermentation progresses, the environment becomes more aerated, favoring acetic acid bacteria (AAB) which oxidize ethanol into acetic acid, a flavor precursor, through a highly exothermic reaction, culminating with the seed death (Jinap et al., 1994). In order to obtain well-fermented beans, turning the mass is required for oxygenation and, consequently, temperature elevation in the environment (Guehi et al., 2010a). This procedure also stimulates an increase in 4

Journal Pre-proof the number of AAB and acetic acid production (Camu et al., 2008). It is believed that all these events also occur during cupuassu fermentation, considering that previous studies identified genera of microorganisms similar to those found in cocoa fermentation (Oliveira, 2001). Bacillus species could be present during fermentation at a higher rate during advanced stages, but their role is unknown (Ouattara et al., 2008). Studies indicate

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pectinolytic activity of some species (Ouattara et al., 2008; Ouattara et al., 2011;

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Yao et al., 2017). However, Bacillus can be implicated in the formation of off flavors

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during fermentation (Schwan and Wheals, 2004).

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In the last decade, culture-independent methods have emerged as an

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approach that allows the large-scale study of microorganism diversity in different environments where they cannot be recovered only by using culture-dependent

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methods (Guazzaroni et al., 2009; Simon and Daniel, 2011). High throughput

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metabarcoding, for example, is an important tool in sequencing well-conserved DNA regions, such as the ITS (Internal Transcribed Spacer) and the 16S, fungi and

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bacteria region, respectively. Sequencers such as Pyrosequencing 454 (Illeghems et al., 2012) and Illumina MiSeq (Schmidt et al., 2013) are commonly used for identifying microorganisms in cocoa fermentation. Culture-dependent methods, besides requiring a lot of time to carry out, are quite limiting, and do not allow the identification of all populations that are present (Romero-Cortes, 2012). After extracting and sequencing DNA samples from any environment, it is doubtful that the collected sample amount could accurately represent the microorganism diversity from that community. Thus, mathematical models are 5

Journal Pre-proof adopted to understand the microorganisms' ecology and evolution, in addition to the quantity of communities found in the environment. These quantifications, made through estimates, are very important to understand the correlation between the microorganisms and their environments (Haegeman et al., 2013). Studies on cupuassu fermentation are still scarce. So far, no methodology has

been

used

through

culture-independent

methods

to

identify

the

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microorganisms involved in the cupuassu fermentation. Thus, the goal was to carry

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out fermentation with three conditions of pulp (0, 7.5, and 15%) to verify the

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influence on the microorganisms and production of important precursors of flavor,

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considering the difficulty to carry out the fermentation of cupuassu seeds with the

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whole pulp. Two turning schemes were adopted during fermentation and their impact on microbial communities. Sequencing of the ITS4 and 16S rRNA region

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was performed in order to verify the dominant populations of yeasts and bacteria, respectively, as well as their richness and diversity. The communities of yeasts and

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bacteria were enumerated by culture-dependent methods. Moreover, correlations

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between the identified microorganisms and abiotic factor changes such as pH, temperature, water activity, moisture, titratable acidity, total nitrogen, and organic compounds were evaluated. 2. Material and Methods 2.1. Cupuassu seed fermentation trials and cutting test To define the pulp concentrations that would be studied, preliminary fermentation tests were conducted for the cupuassu seeds with 0, 20, 30, 40, and

6

Journal Pre-proof 100% of pulp in 4 Kg batches. A cutting test was adopted to define the ideal degree of fermentation through coloration (browning) and partitioning inside the beans. 2.2. Cupuassu seed fermentation About one ton of ripe and healthy cupuassu fruits were collected up to three

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days after their fall at the Peri Farm, in Presidente Figueiredo, Amazonas State, Brazil. After opening the fruits, the seeds were depulped in a depulper (HEER,

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Brazil) in 10 Kg batches in order to obtain seed samples with 0, 7.5, and 15%

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concentrations of pulp, through a controlled depulping time, based on the initial

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weight of the seeds with pulp. After that, fermentation was carried out in triplicate in

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20 L new styrofoam boxes of 8 kg for each experiment. The boxes had holes of 2 cm in diameter at the bottom and sides for the flow of the liquefied pulp from

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fermentation. Chopped banana leaves were mixed to the fermentation mass, which

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act as a natural inoculum, and then the mass was covered with the same material. Two types of turning were applied within the same boxes: R1, with the first turning

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done 48 hours after the start of fermentation and then after that every 24 hours; and R2, where the first turning occurred once the temperature had doubled inside the fermenting mass and the following turns occurred when the average fermentation temperature dropped. The two types of turning were applied to the experiments with three conditions of pulp being previously indicated, 0R1 (depulped and turning each 24 hours), 0R2 (depulped and turning when temperature drops), 7.5R1 (7.5% of pulp and turning each 24 hours), 7.5R2 (7.5%

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Journal Pre-proof of pulp and turning when temperature drops), 15R1 (15% of pulp and turning each 24 hours), and 15R2 (15% of pulp and turning when temperature drops). 2.3. Physical and Chemical Determinations 2.3.1. Temperature and pH of the Fermenting Mass The first measurements related to the pH and temperature of the mass

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occurred at 12h and after that every four hours during fermentation, in order to

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determine the moment when an inflection was observed in the curve of temperature versus time of fermentation in experiment R2. Afterwards and during

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the same experiment, the following turnings were performed when the temperature

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of fermenting mass dropped. Measurements were made with a digital thermometer

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(Test Mod. 0526) and a portable digital pH meter (Digimed Mod. DM20).

fermentation

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2.3.2. Water Activity (Aw), Moisture, Titratable Acidity, and pH of seeds during

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Samples collected daily from fermentation were crushed and submitted to

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determination of the Aw with a hygrometer (Decagon-Aqualab Mod. CX-2), at a resolution of 0.01 coupled with a thermostatic bath (Brookfield Mod. TC 500), at a resolution of 0.1 °C at 25 ± 0.3 °C. The determination of moisture and titratable acidity were done according to AOAC (2005), methods 931.04 and 942.15, respectively. Measurements of pH on the crushed samples were made with a portable digital pH meter (Digimed Mod. DM20). 2.3.3. Organic Acids and Total Nitrogen

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Journal Pre-proof The organic acids were extracted from 5 g of the sample and mixed with 25 mL of deionized water. The solution was subjected to vortex stirring and centrifugation (centrifuge Fanem BABY I 206 BL, BRAZIL) at 3000 rpm for 45 minutes at room temperature (Jinap, Dimick, 1990; Rodriguez-Campos et al., 2011). After that, some modifications of the methodology were necessary: an aliquot of 500 µL of the sample was extracted from 10 mL of supernatant. The

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aliquot was mixed with 500 µL of MilliQ® water (Millipore Corporation MA, USA)

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and centrifuged at 15000 rpm for 10 minutes at 4 oC. The supernatant was filtered

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through a syringe filter with 0.45 µm membrane Millipore®. The separation for

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identification of organic acids was carried out using an Aminex HPX-87H column (300 x 7.8mmx9 µm) (Bio-Rad, USA) and a modular Shimadzu LC-10 system

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(Columbia, MD) comprised of a LC-10AT VP pump, a CTO-10AS VP column oven

Class VP Workstation.

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at 30 °C, a SPD-M20A VP diode array detector (DAD), a SCL-10A interface, and a The DAD was operated between 200 and 800 nm.

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Chromatograms for quantitative analysis were extracted at 210 nm. The samples

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were eluted at 0.6 mL with isocratic elution in a mobile phase of H2SO4 0.004 M. The injection volume for all samples was fixed at 20 µL. The organic acids were quantified by an external standard method. Standard solutions of known concentrations of citric, malic, lactic, and acetic acids (Sigma Aldrich, São Paulo, Brazil) were used. The standards were injected in triplicate and the corresponding chromatograms were obtained for each one. The graphs were obtained with at least 6 concentration points. The areas obtained were matched to their respective concentrations. The concentrations of the organic acids were calculated for each

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Journal Pre-proof treatment by interpolation of the areas and expressed in mg/g. The organic acid peaks were identified by a comparison of the retention times (RT) and confirmed by a comparison of the UV spectra with those of the reference materials. Regarding total nitrogen, 0.2 g of each sample dried in an oven with air circulation (TECNAL Mod. TE-394/2) at 105 oC were submitted to determine the total nitrogen

(AOAC, 2005).

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2.4. Culture-dependent microbiological analysis

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in Nitrogen distiller (TECNAL Mod. TE-036/1), using the Kjeldahl method, 955.04C

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Fresh samples (~ 200 g) were collected on the first day (time 0) and then

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every 24 hours from the fermentation mass of each experiment. The samples were

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contained in a sterile package and transported to the laboratory for immediate plating on selective agar media after appropriate dilution which enabled cell count

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enumeration [expressed as colony forming units (CFU) per gram]. The media used

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were the following: Malt Extract Agar (MEA, Merck®) supplemented with oxytetracycline (100 mg / L) for yeasts; Man Rogosa Sharp Agar (MRS, Merck®)

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supplemented with cycloheximide (400 mg / L) (Camu et al., 2007) for LAB; a modified formulation for counting of AAB according Spinosa (2002): yeasts extract (20 g / L), agar (20 g / L), bromocresol green (0,02g / L), ethanol (2% v/v), final pH 5.5; Tryptone Glucose Extract Agar (TGE, Merck®) for enumeration of mesophilic bacteria (Stevenson and Segner, 2001); and Dextrose Tryptone Agar (DTA, Merck®) for enumeration of thermophilic bacteria (Olson and Sorrells, 2001). All agar media were incubated at 35 oC for up 2 days, except for AAB medium which was incubated at 42oC for up 3 days. 10

Journal Pre-proof 2.5. Cupuassu seeds sample collection and DNA extraction Samples weighing approximately 30 g were collected on the first day (time 0) and then every 24 hours from the fermentation mass of each experiment in triplicate and held in a sterile package. Aliquots for taxonomic analysis were kept under the freezing point (-70 oC) until the beginning of the DNA extractions in the Laboratory of Genomics and Expression (LGE) of the University of Campinas, São

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Paulo.

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About 12 g of fermented seeds were macerated in liquid nitrogen. Then,

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pulverized samples were submitted to fat removal with petroleum ether. Samples

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were submitted to DNA extraction using an extraction buffer (2% CTAB, 2%

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polyvinylpyrrolidone, 100mM TRIS-HCL (pH 8.0), 25mM EDTA, 2.0 M NaCl, 10mg RNAse, 10% ß-mercaptoethanol). After steps of treatment with NaCl solutions,

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chloroform, isoamyl alcohol, and CTAB, a centrifugation process and isopropanol

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precipitation were performed. Sample pellets were washed with 70% ethanol, dried, and resuspended in 40µL of DNAse-free water. Purification steps were

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performed using the DNeasy Plant Minikit (Qiagen). DNA concentration and quality were verified with a Nanodrop 2000 instrument (Thermo Scientific). PCR with universal 16S and ITS primers and with GoTaq polymerase (Promega) were performed as follows: 1X GoTaq Buffer, 10mM MgCl2, 40uM dNTPs, 5mM primers, 10u GoTaq, 100ng DNA. PCR was carried out with an initial denaturation step at 94° C, 2 minutes followed by 30 cycles of denaturation (94°C, 40 seconds), annealing (50°C, 30 seconds) and elongation (72°C, 1 and a half minutes) and a

11

Journal Pre-proof final step of elongation for 4 minutes (Ramos et al., 2014). Purified DNA samples (> 50 ng/ μL) were stocked at -20 oC until they were shipped for sequencing. 2.5.1. Sequencing 16S and ITS4 regions Sequencing of amplicons (ITS4 and 16S regions) extracted from yeasts and bacteria DNA, respectively, was performed on a large-scale Illumina MiSeq

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sequencer at the University of North Carolina, USA. The 16S regions from each sample were amplified by targeting the V4 region. Paired-end type reads with 300

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bp fragments were generated and amplified using the primers pair 515F: 5'

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GTGCCAGCMGCCGCGGTAA 3' and 806R: 5' GGACTACHVGGGTWTCTAAT

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3' (Caporaso et al., 2011).

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2.5.2. Bioinformatics analysis of the 16S and ITS4 rRNA regions

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In the analysis of 16S rRNA sequences, OTUs (operational taxonomic units) were identified with 97% identity equality between reads, with the most common

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representative from each OTU being aligned with the SILVA database release119

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(Pruesse et al., 2007). Mothur software (Schloss et al., 2009) was used to analyze the data sequences. After removing reads belonging to chloroplasts, mitochondria, and plant origin structures, OTUs were normalized and grouped. OTUs that did not generate sequences known by BLAST analysis were excluded. In the analysis of ITS4 rRNA sequences, the reads were grouped into OTUs using the cd-hit-est module of the Cd-hit program (Li and Godzik, 2006) requiring 97% similarity between the sequences. OTUs with a representative sequence less than 200 bp and having a read count of less than five in all the libraries were 12

Journal Pre-proof discarded. Using a PERL script, the FASTA and Genbank files of 790,365 fungal ITS sequences were obtained from the NCBI (http://www.ncbi.nlm.nih.gov/). The filtered OTUs were compared to the ITS sequences using BLASTn (Altschul et al., 1997). For BLAST, only hits with e-value <= 1e-10 were accepted, and these covered at least 80% of OTUs. The genus relative to each OTU was identified based on the hit sequence of BLASTn. The raw reads have been deposited in the database

under

BioSample

numbers

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between SAMN10228731 and SAMN10228808.

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NCBI

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2.5.3. Diversity parameters analysis

analyzed

using

the

PAST

software

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were

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Alpha (Shannon and Chao1) and beta (Bray-Curtis) diversity parameters

(http://folk.uio.no/ohammer/past/index_old.html) to measure the diversity present in

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the environment in relation to the number of taxa in the community, and to estimate

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the total species richness, respectively (Hughes et al., 2001), as well as coverage

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estimate (Hamer et al., 2001).

2.5.4. Bacteria and yeasts taxonomic analysis In order to achieve a more precise attribution relative to the obtained OTUs, taxonomic analyses were performed for family and genus levels in the bacterial and yeasts communities. For the sequence identity, the cut off level was 97% for genus (bacteria) and 80% for phylum (yeasts). The identification did not extend to the species level, since in other studies it was verified that using the ITS region (fungi) for identification at that level is disadvantageous. The reason for this is that

13

Journal Pre-proof some microorganisms of different species which also belong to the same genus have sequences with high similarity in the ITS region (Arroyo-López et al., 2016). 2.5.5. Statistical Analyses The results of the physical and chemical analyses and yeasts’ and bacteria’s OTUs obtained in each experiment were statistically evaluated with the software SAS (Statistical Analysis System) version 9.0 USA using the analysis of variance

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(ANOVA) and Tukey’s test (p≤0.05).

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3. Results

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3.1. Cupuassu seed fermentation trials

Figure 1 shows the fermentation trials with cupuassu seeds to define the

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ideal concentration of pulp, as well as evaluation of the degree of fermentation

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through the cutting test of beans to stop the process. In the experiment with 100% of pulp, fermentation did not occur, even after

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five days, presenting neither temperature increase nor pulp liquefaction. The

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experiment with 20% of pulp was the only one that presented the highest temperature increase (38.3 oC), thus demonstrating that this level of pulp was the highest possible at which fermentation could occur, even after five days (Figure 1). Besides that, through cut test, cupuassu seeds were considered well fermented by exhibiting darker surface and deep partitioning formation (Figure 1). Despite this result, 15% was set as the maximum concentration of pulp, in addition to the concentrations of 0 and 7.5% for the fermentation. 3.2. Temperature and pH of the fermenting mass 14

Journal Pre-proof Figure 2 shows the evolution of the temperature and pH during fermentation. Experiments 0R1 and 0R2 showed higher temperatures in the first hours, while experiments 15R1 and 15R2 presented later high peaks of 41.5 oC and 42.0 oC, respectively, within 72 h of fermentation (Figure 2). All experiments showed progressive increases of pH, while in experiments 15R1 and 15R2, this increase

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happened only in the last hours (Figure 2). The cut test and the marked rise in pH

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(>8.0) were used as criteria to interrupt the process and to prevent the formation of

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off-flavors compounds. For the cutting test, the beans were sampled from the

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fermentation mass in the last hours to verify the degree of fermentation (data not

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shown).The total fermentation time was 60 h for 0R1; 84 h for 0R2, 7.5R1 and 7.5R2; and 108 h for 15R1 and 15R2 experiments. The R2 experiments with more

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pulp (7.5 and 15%) also received more turnings (Figure 2).

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3.3. Physical and chemical characterization of the seeds during fermentation

different

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The physical and chemical characterization of the cupuassu seeds from the experiments

are

presented

in

Table

1.

3.3.1. Moisture content and Water Activity Experiments with higher pulp content (15R and 15R2) presented the highest initial values of moisture which influenced the time required for moisture loss. Water activity values fluctuated during fermentation for all experiments and, at the end of the process, the values were around 0.98 (Table 1). 3.3.2. Titratable acidity (TA) and pH of crushed seeds 15

Journal Pre-proof In the first 12 h of fermentation, 0R1, 0R2, and 7.5R1 showed a rapid increase in TA. Thereafter, a constant decrease in TA occurred for all experiments until the end, except for 15R1 and 15R2, which alternated moments of decrease and increase of TA (Table 1). During fermentation, cupuassu seeds showed increased pH from 4.0-4.2 to 5.5-6.0. The shell exhibits a more acidic profile as it is in direct contact with the fermentation mass. Thus, this could explain the low initial

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3.3.3. Organic Acids and Total Nitrogen

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acidity (Table 1).

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The quantity of citric and malic acids was significantly higher in experiments

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with higher concentrations of pulp, with a linear decrease during fermentation,

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except for 15R1 that presented an increase in the level of citric acid after 84 hours of fermentation. Higher concentrations, particularly of acetic acid, were observed in

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the experiments with pulp, and the highest peaks occurred within 36 h of

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fermentation in experiments 7.5R1 and 7.5R2 and within 60 h in experiments 15R1 and 15R2. In all the experiments, the lactic acid production profile was similar to

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the acetic acid, although at much lower concentrations, with a decrease in the last hours for both acids. Concerning the concentration of total nitrogen, no marked changes were observed during fermentation across the experiments (Table 1). In the present work it was only possible to determine sugars in the samples collected from the experiments using 0% pulp and 15% pulp before fermentation (0R1 and 15 R1). In samples without pulp the concentration of sucrose, glucose and fructose was 3.4 mg / g; 4.9 mg / g; and 4.64 mg / g, respectively. For samples with 15%

16

Journal Pre-proof pulp the concentrations were 3.9 mg / g; 5.66 mg / g; and 5.91 mg / g, respectively (data not shown). 3.4. Culture-dependent microbiological analysis The evolution of colony forming units (CFU) of yeasts, LAB, AAB, mesophilic and thermophilic bacteria throughout fermentation for all experiments is

counts

increased since

the beginning of

the

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The microorganism

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shown in Figure 3.

fermentation process for all experiments, except for thermophilic bacteria which

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reached their highest count only in experiment 15R2. In all experiments, the

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highest counts of most of microorganisms appear during the last hours of

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fermentation (Figure 3).

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3.5. Sequencing – yeasts and bacterial OTUs from each experiment Table 2 shows the results of the OTUs triplicate averages obtained from

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yeasts and bacteria for each experiment, as well as the coverage.

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Comparing the results of OTUs between the two communities, bacteria were present in higher number than yeasts (Table 2), especially in experiments with pulp (7.5R1, 7.5R2, 15R1, and 15R2). All experiments were not significantly different at the 5% level for both microorganism communities. 3.6. Bacterial and yeast population richness Figure 4 shows the results of beta-diversity (Bray-Curtis) for each experiment concerning to bacteria (A) and yeasts (B). Beta-diversity corresponds to the degree of differentiation between two or more samples. 17

Journal Pre-proof A grouping of bacteria was observed as a function of the pulp concentration. In the experiments with total depulping (0%), the microorganisms for both turnings (R1 and R2) had the highest similar percentage (80%) when compared to the other experiments and their respective concentrations (Fig. 4A). In the case of yeasts, similar grouping (almost 90%) occurred as a function of the turning scheme (R1) in the experiments with the highest pulp concentration (7.5 and 15%) (Fig. 4B).

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Table 3 shows the results of the diversity index and richness estimator

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(Shannon and Chao1 index, respectively) in the six experiments throughout

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fermentation.

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Values of the alpha-diversity (Shannon) in all the experiments were higher

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for bacteria (between 5.90 and 6.02) than for the yeasts (between 1.70 and 2.42). Comparing the two communities, results also indicate low yeasts richness (Chao-1)

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in relation to bacteria in all experiments, especially those with higher pulp (Table

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3).

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3.7. Composition of the taxonomic profile of yeasts and bacteria communities Figure 5 shows the number of yeasts and bacteria genera identified in each experiment.

Results showed that both yeasts and bacteria diversity varied according to the pulp concentration, and the two communities were inversely proportional (Fig. 5), evidencing that cupuassu pulp influences microorganisms’ diversity. Fig. 6, 7, and 8 show the genera of yeasts and bacteria identified in the six experiments. 18

Journal Pre-proof Among the yeasts populations, genus Pichia was present until the end of fermentation with greater abundance in the last hours in all of the experiments. Hanseniaspora genus predominated in the six experiments, especially those with pulp (7.5 and 15%) and in both turning schemes. The presence of other genera of yeasts identified in the experiments were variable but did not become dominant as occurred with Pichia and Hanseniaspora.

of

For the lactic acid bacteria (LAB), there was an abundance of the genus

ro

Weissella at the beginning of the fermentation in all experiments and an expressive

-p

presence of the genus Lactobacillus after the beginning with a slight decline in the

re

final stage (Fig. 6, 7 and 8). For most of the experiments, but especially 15R1 and

lP

15R2, there was an increase of Lactobacillus within 36 hours of fermentation (Fig. 6, 7 and 8).

na

In the group with acetic acid bacteria (AAB), genus Gluconobacter was

ur

abundant in the first days of fermentation for all the experiments, whereas genus

Jo

Acetobacter was expressive in the last hours (Fig. 6, 7 and 8). Genotypes of the Enterobacteriaceae family (Escherichia, Shigella) were also identified in all experiments (Fig. 6, 7 and 8). Concerning the sporulated bacteria, in the experiments with the highest amount of pulp (15R1 and 15R2) (Fig. 8) an emergence of the genus Bacillus was observed at the end of fermentation. Only Clostridiaceae family manifested in the experiments 0R2 and 15R2 in the last hours of fermentation (Fig. 6 and 8). 4. Discussion

19

Journal Pre-proof The presence of organic acids (citric, malic, oxalic, etc.) in cupuassu pulp contribute to the low pH (approximately 3.30) (Gondim et al. 2001), thus explaining the higher values of TA found in experiments with pulp (7.5R1, 7.5R2, 15R1, and 15R2). The content of citric acid for experiments with 15% pulp was higher during the fermentation, differing significantly from the other experiments. These conditions (higher acidity and higher amount of pulp) seem to have prolonged the

the

physicochemical

analysis,

ro

Concerning

of

fermentation period.

some

results

between

-p

experiments did not differ significantly. Apparently, the turning scheme adopted in

re

15R1 experiment favored the production of acetic acid, in this case differing

lP

significantly from 15R2. The turning applied to the fermentation mass is important for the homogenization of the temperature and aeration. Those conditions favor the

na

development of acetic acid bacteria (AAB), important for the formation of flavor precursor compounds such as acetic acid from ethanol (Fowler, 2009; Lopes et al.,

ur

2003). Thus, the production of flavor compounds is strongly tied to the application

Jo

of turning to the mass during fermentation (Hamdouche et al., 2019). In the fermentation of cupuassu, the maximum temperature usually reached is between 47 oC and 49 oC (Cohen and Jackix, 2005). This range was not achieved in these experiments, maybe because of the small scale (8 kg/batch) performed in styrofoam boxes. Fermentation usually takes place in wooden boxes in the range of tons of seeds. Higher temperatures were observed in the experiments with higher amounts of pulp (15R1 and 15R2) and longer periods

20

Journal Pre-proof (between 60 and 72 hours of fermentation) when compared to the other experiments (between 24 and 32 hours). Experiment 7.5R1 showed a huge drop of citric acid throughout fermentation. Comparing 7.5R1 and 7.5R2, it is possible to notice that in the total LAB count, 7.5R1 showed a higher number along the fermentation, which could explain the intense metabolism of citric acid by these microorganisms. Also, in the

of

first hours of fermentation, an increase in the production of lactic acid was

ro

observed, in a higher quantity than the other experiments, which would justify the

-p

abrupt drop in the citric acid content for lactic acid production by LAB in experiment

re

7.5R1. But it is not clear why the same process did not happen with the other

lP

experiments.

The changes in the abiotic factors occurred late for experiments 15R1 and

na

15R2, in the last hours of fermentation (108 hours), except for acetic acid which

ur

was elevated after 60 hours. In those experiments it was observed that the peak temperatures coincided with the higher quantity of acetic acid, which in higher

Jo

concentrations can give undesirable acidity to the beans. Studies carried out with volatile compounds in depulped and partially depulped cupuassu seeds demonstrated that the latter presented higher levels and diversity of compounds such as aldehydes, ketones, and alcohols during fermentation than the former (Ramos et al., 2016). In the same study, a higher amount of linalool was found in the partially depulped beans. Linalool is an aromatic terpene present in the pulp that confers floral and green odor.

21

Journal Pre-proof Experiments 0R1 and 0R2 exhibited a lower production of acetic acid than others experiment with pulp. The removal of the pulp certainly contributed to a reduction in the availability of sugars, resulting in less ethanol for production of acetic acid. Preliminary determinations of alcohols were done by GC-MS only for experiments 0R1 and 15R2, having as a criterion of choice their better results in

of

acceptance test in the sensory analysis performed in the samples of cupulate (data

ro

not shown here). As expected, the sample 15R2 showed more variety and higher

-p

concentration of alcohols during fermentation, specially ethanol, with a 10-fold

re

increase of that compound in relation to 0R1 at 60 h of the process.

lP

When the yeasts, LAB, and AAB counts are observed using the culturedependent method, the three populations show continuous growth throughout the

na

fermentation. The development of yeasts did not appear to be by the pulp content

ur

and composition. However, in the culture-independent method the pulp seems to influence the diversity of yeasts, which was higher in the depulped experiments. A

Jo

stable LAB count was higher than AAB in all experiments at the end of the fermentation. The presence of higher amount of pulp prolonged the fermentation time and exposed more clearly the competition that exists between the microorganisms. Higher amount of pulp favored bacteria diversity, although it had caused a delay in the fermentation time, in the liquefaction of the pulp, and the later increase of the temperature of the mass, especially in the experiments with 15% of pulp. Apparently, yeasts required more time to adapt to the adverse conditions and to promote the metabolization of sugars for ethanol production, 22

Journal Pre-proof which could explain the later temperature elevation for the experiments with more pulp. Only in the experiments with 15% of pulp showed an expressive increase in the population of thermophilic bacteria. These microorganisms are bacteria that sporulate under stressful conditions with reduced availability of water and nutrients, in addition to thermal shock. These observations in the experiments with higher pulp contents (15%) were also observed in studies with cocoa in the most

of

advanced phase of fermentation (Hamdouche et al., 2019).

ro

Next-generation sequencing via Illumina Platform was carried out in the

-p

ITS4 (yeast) and 16S (bacteria) regions of all samples to identify which

re

microorganisms were present in the fermentation environment. Considering the

lP

complexity of the samples (high moisture content, organic acids, pectin, phenolic compounds, etc.), which could have interfered in the quality of the DNA obtained

na

from the samples, a DNA extraction method was developed (Ramos et al., 2014). Results of sequencing showed that the experiments with pulp (especially 15R1 and

ur

7.5R1 and 7.5R2) presented higher number of bacteria OTUs, whereas the

Jo

experiments without pulp (0R1 and 0R2) recorded the highest number of yeasts, suggesting that pulp influences their diversity. Cupuassu has in its composition important volatile compounds such as myrcene, ocimene, caryophyllene, geraniol, eugenol, linalool (Quijano and Pino, 2007). Such compounds are terpenes that play an important role on the microbial inhibition (Padalia et al., 2017), especially linalool, which is present in the cupuassu pulp in a higher concentration (986 µg / kg) than these other compounds (Quijano and Pino, 2007). Linalool has in vitro antimicrobial activity against Candida species and some bacteria species (Padalia 23

Journal Pre-proof et al., 2017). It seems that besides the abundance of pulp, its composition also contributes to hinder the diversity of yeasts. Perhaps this explains why fermentation does not occur in cupuassu seeds with whole pulp. More studies will be necessary to prove that the terpenes present in the cupuassu pulp could have some minimum inhibitory concentration on microorganisms. In cupuassu studies, an increase in temperature during fermentation favors

of

the activity of pectinolytic enzymes at pH levels above 4 (Garcia, 2006). The

ro

experiments that were performed with cupuassu seeds with 100% pulp showed an

-p

initial pH at 3.5, which, together with the abundance of pulp and presence of

re

terpenes, could have contributed to the process’s failure.

lP

Regarding the bacteria, the results of beta-diversity (Bray-Curtis) were similar in the experiments without pulp (0R1 and 0R2). The turning scheme (R1 or

na

R2) did not seem to interfere in the development of the population. An environment

ur

with reduced pulp concentration apparently restricted the increase in diversity among the groups, possibly due to the lower supply of substrates. However, for

Jo

yeasts, the grouping occurred as a function of the turning scheme adopted in the experiments with pulp (7.5 and 15%). In contrast to the bacteria, the experiments without pulp (0R1 and 0R2) favor the yeasts’ diversification and/or its maintenance. Results of alpha-diversity and richness showed an advantage for bacteria in relation to yeasts in all experiments. This characteristic can be better observed through the taxonomic profile for both communities and their distribution in the family and genus level.

24

Journal Pre-proof Sequencing allowed for the identification of both bacteria and yeasts in the cupuassu fermentation only up to the genus level. Regarding yeast diversity, genus Pichia in experiments 0R1 and 0R2 was predominant since the start of fermentation. In cocoa seed fermentation, the development of Pichia seems to be more pronounced from the middle until the final moments (Hamdouche et al., 2014; Nielsen et al., 2007; Pereira et al., 2017; Schwan, 1998). Some of yeast also

of

metabolize citric acid, leading to a rise in pH and a favorable environment for

ro

bacteria (Lagunes-Gálvez et al., 2007; Schwan and Wheals, 2004). In fact, the

-p

abundance of the genus Pichia in all experiments became more significant at 36

re

hours for experiments with higher pulp concentration (15R1 and 15R2), the same period at which the ethanol content was high, as evidenced by studies with

lP

cupuassu seed fermentation (Ramos et al., 2016). The population of genus Pichia

na

also increased in the last hours. Recent studies have demonstrated that Pichia species are potent producers of flavor compounds, being more efficient in the

ur

conversion of sugar to ethanol (Pereira et al., 2017).

Jo

The genus Saccharomyces did not stand out in the present study. This genus, as well as other yeasts, is related to the saccharolytic action exerted on the pulp sugars (Ardhana and Fleet, 2003; Lagunes-Gávez et al., 2007; Schwan and Wheals, 2004). Genus Hanseniaspora predominated in the six experiments, especially those with pulp (7.5 and 15%). Hanseniaspora and Saccharomyces species have been identified as the largest producers of volatile compounds in cocoa fermentation (Schwan and Wheals, 2004).

25

Journal Pre-proof Brown et al. (2010) and Cadez and Smith (2011), cited by Papalexandratou et al. (2013), in a description of yeasts metabolism, demonstrate that Hanseniaspora is a microorganism that does not ferment maltose, unlike the genus Saccharomyces. The latter usually develops in culture-dependent methods, such as those using malt extract agar (MEA), due to the presence of the sugar mentioned in its composition. In the present study with cupuassu, the results

of

showed that the genus Saccharomyces appeared secondarily in the process, with

ro

Hanseniaspora being quite predominant, especially in the experiments with higher

-p

pulp concentration.

re

Genera such as Pichia, Saccharomyces, and Candida have also been

lP

described as microorganisms commonly present in the fermentation environment which contribute to the production of important aromatic compounds and to flavor

na

enhancement (Arroyo-López et al., 2012; Arroyo-López et al., 2016). In the

times.

ur

cupuassu fermentation, the abundance of some populations occurred at different

Jo

Regarding the LAB, the results of sequencing showed a stable growth until the end. The same was observed in the culture-dependent method. The genus Weissella was present specially in the beginning of process. The decrease of Weissella throughout fermentation is associated with the generation of adverse conditions, different from the genus Lactobacillus, whose development emerged after 24 hours. Studies indicate that the genus Weissella is not able to stand an environment with high temperatures and ethanol concentrations, while some species of Lactobacillus can grow in these conditions (Pereira et al., 2012). This 26

Journal Pre-proof would explain the predominance of Lactobacillus and the decrease of Weissella during cupuassu fermentation when the temperature of mass and ethanol levels were high. The real role of LAB in cocoa fermentation is still questionable. Some studies suggest that success in the process does not depend essentially on these microorganisms (Miguel et al., 2017). It has been demonstrated, for example, that

of

LAB do not impact the sensory characteristics of chocolate (Ho et al., 2015).

ro

However, there are studies demonstrating that their presence allows the production

-p

of well-fermented beans (Mai et al., 2014). Moreover, some species present have

re

potent heterofermentative action (Ouattara et al., 2014). LAB species also have the

lP

property of using citric acid as a carbon source, through the action of citrate lyase in the early stages under favorable acidity, producing acetic acid besides lactic acid

na

(Droux and Bernard, 2017).

ur

The participation of AAB increases at a later stage of cupuassu (Oliveira, 2001) stimulated by the environment that becomes more aerated because of the

Jo

pulp liquefaction and the increase of ethanol concentration, an important substrate used by them to produce acetic acid (Schwan and Wheals, 2004). The experiments with higher amounts of pulp (15R1 and 15R2) showed, in the last hours, higher concentrations of acetic acid. Thus, the removal of the pulp affects the production of important flavor compounds, as demonstrated by Ramos et al. (2016). Sequencing results showed the abundance of the Acetobacter genus in experiments 15R1 and 15R2, especially at 60 and 84 hours of fermentation, when the temperature was already high, as well as the quantity of acetic acid formed 27

Journal Pre-proof from ethanol. AAB are also capable of converting alcohols by dehydrogenation to other types of acids (propanoic, butanoic, 2-methylpropanoic, 2-methylbutanoic, 3methylbutanoic), which are potent precursors of chocolate flavor compounds (Schrader, 2007). Results of the study of volatile compounds in cupuassu beans showed high production of the aforementioned alcohols and acids in the same periods (half and final stages of fermentation) (Ramos et al., 2016) when the AAB

of

population increased, as demonstrated in the present study. These findings

ro

indicate an intense metabolic activity by those microorganisms. In cocoa

-p

fermentation, Acetobacter seems to be more frequent than Gluconobacter from the

re

beginning of the fermentation process (Papalexandratou et al., 2013; Schwan and Wheals, 2004). The latter can oxidize glucose to gluconic acid, whereas

lP

Acetobacter oxidizes ethanol to acetic acid (Yamada and Yukphan, 2008). Thus,

na

the abundance of Gluconobacter observed in the beginning of cupuassu

period.

ur

fermentation may be related to the higher availability of glucose in the pulp in that

Jo

During fermentation, there was a sudden increase in the moisture content at 60 hours in all experiments. There is the argument that AAB promotes a superoxidation of the acetic acid with a release of CO2 and water in the process (Schwan and Wheals, 2004). The presence of some genera of Enterobacteriaceae (Escherichia, Shigella) in the experiments seems to be a contamination from several sources: soil (where fruits were deposited), cutting tools (cutlass, machete), workers' hands, fermentation boxes, and banana leaves (inserted into the mass as a natural 28

Journal Pre-proof inoculum). In this study, neither Tatumella nor Pantoea were detected. These bacteria belong to the genera of enterobacteria normally present at the initial stage of cocoa fermentation, although in low concentrations (De Vuyst and Weckx, 2016; Papalexandratou et al., 2011a; Papalexandratou et al., 2013). In the same manner as AAB Gluconobacter, Tatumella also produces gluconic acid from the glucose in the initial stages, giving an undesirable acidity and providing less glucose to LAB

of

and yeasts (Illeghems et al., 2015; Papalexandratou et al., 2011b). In the present

ro

study, its presence was not detected, perhaps because of the influence of the

-p

cupuassu pulp composition. The same may have happened to genus Pantoea.

re

At the last hours of fermentation an increase in the number of thermophilic

lP

bacteria was observed in the experiments 15R1 and 15R2, suggesting the presence of spore forming microorganisms such as Bacillus, identified in the

na

sequencing. This microorganism is usually observed at an advanced stage of cocoa fermentation (Miguel et al., 2017) and some species produce fatty acids of

ur

low molecular weight, with degradation of amino acids, leading to over

Jo

fermentation (Biehl and Ziegleder, 2003). However, recent studies have indicated that Bacillus strains isolated from the cocoa fermentation environment have pectinolytic and citrate metabolizing properties (Yao et al., 2017), thus contributing to the formation of flavor compounds. The reduction of nutrients in the environment would be the reason to cause the sporulation of this microorganism. Studies indicate that a long period of fermentation has a negative influence on the concentration of some bioactive substances like catechins, naturally present in the cupuassu beans not being advisable a fermentation for more than six days 29

Journal Pre-proof (Álvarez et al., 2017). In the present study the process was interrupted due to the higher pH (>8,0) to prevent the formation of undesirable flavor compounds (off flavors). Only experiments with higher amount of pulp had longer times of fermentation (up to five days). 5. Conclusion

of

Sequencing by a culture-independent method allowed for the identification of the composition of the yeasts and bacteria communities in an environment of

ro

fermentation of cupuassu seeds for the first time. Although the fermentations were

-p

carried out in styrofoam boxes, it is important to note that in the present study the

re

results were quite satisfactory, allowing for the observation of the predominant

lP

populations throughout the process. The use of next-generation sequencing (Illumina MiSeq Platform) has proven to be an important tool to identify the

na

microorganism communities. In the case of cupuassu, results also revealed the

ur

importance of the pulp concentration for the process, especially for bacteria populations. Some amount provides important substrates that can undergo

Jo

biochemical transformations through the consortium of microorganisms, essential for the formation of desirable flavor precursor compounds. However, pulp composition seems to have hindered yeasts diversity, possibly by some organic compounds naturally present in cupuassu, such as terpenes that have some antimicrobial activity, but more studies are necessary to confirm if there is some correlation. The application of the turning scheme at each 24 hours in the experiments had a slight advantage to the temperature of the mass. Thus, maintaining some amount of pulp adhered to the cupuassu seeds ensures the 30

Journal Pre-proof development of essential microorganisms, such as yeasts (Pichia, Hanseniaspora) and bacteria (Acetobacter, Lactobacillus, Bacillus), which can assure production of flavor compounds to confer desirable sensory characteristics to the final product. The study also opens perspectives for further studies regarding the use of wooden boxes, as well as the increase of the batches, to verify the influence on the dynamics of the microbial growth involved in the process.

of

Acknowledgements

ro

To the National Council for Scientific and Technological Development –

-p

CNPq (Process Number 485287/2011-0) and São Paulo Research Foundation –

re

FAPESP (Process 2012/00296-4) for the granted resources for the development of

lP

this research. To the Amazonas Research Foundation – FAPEAM, for granting the

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stress conditions. St. Cerc. St. CICBIA, Food Industry, 18(2), 201–211.

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Journal Pre-proof Fig 1. Fermentation trials with experiments to define the ideal concentration of pulp. (1) cupuassu fruit; (2) seeds with pulp of cupuassu; (3) experiment with 20% of pulp at five days of fermentation; (4) experiment with 100% of pulp at five days of fermentation. A. poorly fermented seed: without partitioning and of beige color; B. partially fermented seed: with partial partitioning and of brown color; C. well fermented seed: with partitioning and of dark brown color.

Fig 2. Evolution of the temperature and pH during the fermentation of experiments 0R1, 0R2, 7.5R1, 7.5R2, 15R1 and 15R2.

of

Turning (R2) applied during fermentation after a decrease in the temperature of the mass.

-p

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Fig 3. Evolution of development of yeasts, acetic acid bacteria (AAB), lactic acid bacteria (LAB), thermophilic bacteria and mesophilic bacteria during fermentation of cupuassu seeds in the experiments 0R1, 0R2, 7,5R1, 7.5R2, 15R1 and 15R2.

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Fig. 4. Similarity of bacteria (A) and yeasts (B) populations among the six experiments of cupuassu seeds during fermentation.

lP

Fig 5. Number of yeast and bacterial genera identified in each experiment.

na

Fig 6. Taxonomy at the genus level of yeast and bacteria identified during fermentation (h) and relative abundance (%) for the experiments 0R1 e 0R2: <1, grey; 1-10, green; 10-50, blue;

Dipodascaceae;

(4)

ur

50-80, red; 80-100, black. Family (Yeasts): (1) Candidaceae; (2) Debaryomycetaceae; (3) Metschnikowiaceae;

Family

(Bacteria):

(8)

Phaffomycetaceae; Acetobacteraceae;

(9)

(6)

Pichiaceae;

(7)

Clostridiaceae;

(10)

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Saccharomycetaceae;

(5)

Enterobacteriaceae; (11) Lactobacillaceae; (12) Leuconostocaceae; (13) Sphingobacteriaceae..

Fig 7. Taxonomy at the genus level of yeast and bacteria identified during fermentation (h) and relative abundance (%) for the experiments 7.5R1 e 7.5R2: <1, grey; 1-10, green; 10-50, blue; 50-80, red; 80-100, black. Family (Yeasts): (1) Candidaceae; (2) Cystobasidiaceae; (3) Debaryomycetaceae; (4) Debaryomycetaceae; (5) Dipodascaceae; (6) Malasseziaceae; (7) Phaffomycetaceae;

(8)

Pichiaceae;

(9)

Saccharomycetaceae.

Family

(Bacteria):

(10)

Acetobacteraceae; (11) Bacteriovoracaceae; (12) Carnobacteriaceae; (13) Enterobacteriaceae; (14) Flavobacteriaceae; (15) Hydrogenophilaceae; (16) Lactobacillaceae; (17) Leuconostocaceae; (18) Moraxellaceae; (19) Pseudomonadaceae; (20) Sphingomonadaceae; (21) Staphylococcaceae; (22) Streptococcaceae.

45

Journal Pre-proof

Fig 8. Taxonomy at the genus level of yeast and bacteria identified during fermentation (h) and relative abundance (%) for the experiments 15R1 e 15R2: <1, grey; 1-10, green; 10-50, blue; 5080, red; 80-100, black. Family (Yeasts): (1) Candidaceae; (2) Cystobasidiaceae; (3) Debaryomycetaceae;

(4)

Dipodascaceae;

Saccharomycetaceae;

Family

(Bacteria):

(5) (8)

Phaffomycetaceae; Acetobacteriaceae;

(6) (9)

Pichiaceae; Bacillaceae;

(7) (10)

Burkholderiaceae; (11) Caulobacteraceae; (12) Clostridiaceae; (13) Enterobacteriaceae; (14) Enterococcaceae; (15) Flavobacteriaceae; (16) Lactobacillaceae; (17) Leuconostocaceae; (18) Nocardiopsaceae; (22)

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Methylobacteriaceae; (19) Moraxellaceae;(20) Nocardiaceae; (21)

Paenibacillaceae; (23) Planococcaceae; (24) Pseudomonadaceae; (25) Sphingobacteriaceae; (26)

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na

lP

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-p

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Staphylococcaceae; (27) Streptococcaceae; (28) Xanthomonadaceae.

46

Journal Pre-proof Table 1. Physical and chemical characterization of cupuassu seeds from the different experiments during fermentation

15 R2 0 R1 0 R2 7.5 R1 12 7.5 R2 15 R1 15 R2 0 R1 0 R2 7.5 R1 36 7.5 R2

15 R2 0 R1 0 R2 7.5 R1 60 7.5 R2 15 R1 15 R2 0 R2 7.5 R1 7.5 R2

55.2 ± 0.00

11.4c ± 0.01

55.2c ± 0.00

13.3b ± 0.01

59.7b ± 0,00

13.3b ± 0.01

59.7b ± 0,00

15.4a ± 0.01

64.5a ± 0,00

15.4a ± 0.01

64.5a ± 0,00

11.6a ± 1.26

53.7c ± 0.39

12.5a ± 2.63

54.0c ± 0.61

9.3a ± 1.12

55.9c ± 1,38

8.4a ± 1.30

59.0b ± 0,14

13.6a ± 1.92

61.4a.b ± 0,73

11.3a ± 0.79

63.8a ± 1,04

7.1a ± 1.01

51.7b.c ± 0.45

8.0a ± 2.36

47.3c ± 0.62

11.2a ± 0.90

51.7b.c ± 3,67

7.8a ± 2.67

58.4a ± 0,62

10.3a ± 0.31

57.3a.b ± 1,93

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15 R1

11.4 ± 0.01

84

0.983c ± 0.00 0.983c ± 0.00 0.985b ± 0.00 0.985b ± 0.00 0.991a ± 0.00 0.991a ± 0.00 0.990a ± 0.00 0.985a ± 0.00 0.986a ± 0.00 0.985a ± 0.00 0.987a± 0.00 0.979a ± 0.00 0.980c ± 0.00 0.980b.c ± 0.00 0.984a ± 0.00 0.983a.b ± 0.00 0.982a.b.c ± 0.00 0.981a.b.c ± 0.00 0.986a ± 0.00 0.982a ± 0.00 0.983a ± 0.00 0.983a ± 0.00 0.982a ± 0.00 0.984a ± 0.00 0.984a ± 0.00 0.984a ± 0.00 0.985a ± 0.00

7.7a ± 0.76

60.7a ± 2,33

2.4b ± 0.38

54.3c ± 092

2.8b ± 0.35

53.3c ± 048

4.1b ± 1.37

56.1b.c ± 0,85

3.3b ± 0.67

58.5a.b ± 2,46

15.3a ± 0.41

62.5a ± 1,29

17.3a ± 2.84

62.2a ± 1,20

1.7b ± 0.22

47.8b ± 1.06

2.5b ± 0.33

47.8b ± 0,46

2.7b ± 0.17

51.5b ± 1,38

citric

malic

lactic

acetic

9.0c ± 0,45 9.0c ± 0,45 11.7b ± 0,10 11.7b ± 0,10 13.4a ± 0,11 13.4a ± 0,11 7.8c ± 0,05 7.6d ± 0,10 1.4f ± 0,05 6.4e ± 0,25 11.5a ± 0,46 9.9b ± 0,27 2.5d ± 0,01 2.5d ± 0,05 0.9e ± 0,00 4.9c ± 0,30 9.2a± 0,04 8.4b ± 0,49 1.4d ± 0,05 1.6c ± 0,03 0.9f ± 0,00 1.1e± 0,05 3.2b ± 0,02 4.5a± 0,38 1.7b± 0,05 0.8e± 0,03 1.0d± 0,03

2.7b ± 0,19 2.7b ± 0,19 3.2c ± 0,05 3.2c ± 0,05 4.1a ± 0,02 4.1a ± 0,02 1.0d ± 0,01 1.1c ± 0,02 0.5e ± 0,00 1.0d ± 0,04 2.8a ± 0,11 2.1b ± 0,01 0.3c ± 0,01 0.3c ± 0,01 0.2d ± 0,01 0.4b ± 0,02 0.9a ± 0,00 0.9a ± 0,03 0.2c ± 0,00 0.4a ± 0,01 0.3b ± 0,01 0.2c ± 0,01 0.2c ± 0,01 0.2c ± 0,01 0.4a ± 0,00 0.2b ± 0,01 0.1c ± 0,01

0.8b ± 0,01 0.8b ± 0,01 1.0a ± 0,04 1.0a ± 0,04 1.0a ± 0,03 1.0a ± 0,03 1.9c ± 0,02 1.4d ± 0,01 2.1a ± 0,09 2.0b ± 0,18 1.1e ± 0,04 1.4d ± 0,07 1.5e ± 0,01 1.9d ± 0,03 2.5b ± 0,10 2.6a ± 0,18 2.5b ± 0,00 2.2c ± 0,10 0.9e ± 0,00 1.3d ± 0,02 1.6c ± 0,01 0.7f ± 0,05 2.5b ± 0,05 2.6a ± 0,06 1.3c ± 0,03 0.5d ± 0,04 0.4e ± 0,02

2.1c ± 0,08 2.1c ± 0,08 2.5b ± 0,01 2.5b ± 0,01 2.8a ± 0,03 2.8a ± 0,03 2.3c ± 0,00 2.6b ± 0,03 4.1a ± 0,09 2.1d ± 0,15 0.1e ± 0,02 0.1e ± 0,03 3.8d ± 0,01 4.3c ± 0,09 10.2b ± 0,06 10.8a ± 0,41 1.8e ± 0,02 4.3c ± 0,89 4.3e ± 0,38 3.8f ± 0,06 7.9c ± 0,03 5.2d ± 0,26 14.3a ± 0,17 12.7b ± 0,25 3.4d ± 0,66 3.4d ± 0,19 4.5c ± 0,03

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15 R1

c

ro

0 7.5 R2

c

Aw

-p

7.5 R1

Moisture***

re

0 R2

4.2a ± 0.13 4.2a ± 0.13 4.1a ± 0.14 4.1a ± 0.14 4.0a ± 0.01 4.0a ± 0.01 4.4a.b ± 0.12 4.5a.b ± 0.21 4.6a ± 0.06 4.6a ± 0.06 4.1b ± 0.06 4.1b ± 0.12 4.7a ± 0.16 4.7a ± 0.21 4.6a ± 0.05 4.6a ± 0.07 4.1b ± 0.15 4.5a.b ± 0.15 5.7a ± 0.19 5.6a.b ± 0.20 5.3b ± 0.21 5.4a.b ± 0.08 4.3c ± 0.10 4.3c ± 0.15 6.0a ± 0.21 5.7a ± 0.20 5.6a ± 0.11

Titratable acidity**

lP

0 R1

pH*

na

Fermentation Time (h)

ur

EXP

Total Nitrogen *****

Organic Acids ****

47

9.7c ± 0,00 9.7c ± 0,00 10.1b ± 0,00 10.1b ± 0,00 10.2a ± 0,00 10.2a ± 0,00 9.9b.c ± 0,30 9.8c ± 0,10 10.8a ± 0,50 10.4a.b.c ± 0,20 10.6a.b ± 0,40 10.7a.b ± 0,10 10.7a ± 0,10 10.3a.b ± 0,10 10.4a.b ± 0,40 10.3a.b ± 0,20 9.8b ± 0,20 10.4a.b ± 0,20 10.4a.b ± 0,10 10.1b ± 0,20 10.7a ± 0,40 10.7a ± 0,20 10.4a.b ± 0,10 10.5a.b ± 0,10 10.9a.b ± 0,20 10.9a.b ± 0,10 10.3b.c ± 0,20

Journal Pre-proof 4.5b ± 0.979a ± 3.7a ± 0.2b ± 2.3a ± 12.6a ± 1.57 57.4a ± 3,19 0.12 0.00 0,29 0,00 0,05 4.7b ± 0.981a ± 1.4c ± 0.2b ± 2.0b ± 15 R2 11.2a ± 0.78 48.1b ± 0,20 0.38 0.00 0,08 0,01 0,08 5.5a ± 0.981a ± 0.8a ± 0.2a ± 1.4a ± a a 15 R1 4.2 ± 1.00 54.2 ± 0,06 0.30 0.00 0,00 0,01 0,01 108 a 5.7 ± 0.983a ± 0.8a ± 0.2a ± 1.0b ± a b 15 R2 3.2 ± 0.68 52.5 ± 0,46 0.32 0.00 0,01 0,02 0,02 Values are expressed as Mean (SD). Samples with the same letters in the same column are not significantly different at the 5% level (Tukey’s test) * Whole seeds (pulp + cotyledon) **meqNaOH/100g ***(%) **** (mg/g) ***** (g/100g)

10.9a ± 0,66 10.4b ± 0,28 5.4a ± 0,07 3.3b ± 0,05

10.2c ± 0,20 11.0a ± 0,30 10.4a ± 0,00 10.5a ± 0,20

re

-p

ro

of

15 R1

lP

Table 2. Number of sequences and coverage index obtained for yeasts and bacteria in each experiment

0R1

Yeasts

1,387a

Bacteria

9,919a

0R2

Coverage

7.5R1

7.5R2

15R1

15R2

1,901a

1,571a

1,484a

2,771a

1,846a

99.9%

9,636a

10,280a

11,374a

10,353a

10,988a

99.9%

Jo

ur

COMMUNITIES

na

Number of Sequences

Samples with the same letters in the same row are not significantly different at the 5% level (Tukey’s test)

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Table 3. Analysis of the diversity index and richness estimator for bacteria and yeast from cupuassu seed fermentation

Yeasts

Shannon_H 5.98 ± 0.04 5.99 ± 0.03 5.90 ± 0.04 6.00 ± 0.03 6.02 ± 0.03 6.01 ± 0.03

Chao-1 479 ± 21 478 ± 21 451 ± 21 501 ± 25 499 ± 23 499 ± 22

ro

of

Chao-1 447 ± 43 368 ± 38 269 ± 30 294 ± 31 351 ± 35 262 ± 30

ur

na

lP

re

-p

Shannon_H 2.42 ± 0.01 2.39 ± 0.01 1.70 ± 0.01 1.89 ± 0.01 2.02 ± 0.01 2.20 ± 0.01

Jo

EXP 0R1 0R2 7.5R1 7.5R2 15R1 15R2

Bacteria

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Highlights

of

ro

-p re lP



na



ur



Culture-independent method allows to identify bacteria and yeast genera, richness, and diversity in environment of fermentation of cupuassu seeds. Main populations of yeasts (Hanseniaspora and Pichia) and bacteria (Lactobacillus and Acetobacter) and their predominance are identified throughout the fermentation process. Pulp concentration of cupuassu seeds and its composition influence the diversity of bacteria and yeasts. Higher pulp concentration (15%) promotes longer fermentation time, highest temperature, and more organic acids production during the process.

Jo



50

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8