Impact of fermentation conditions on the quality and sensory properties of a probiotic cupuassu (Theobroma grandiflorum) beverage

Accepted Manuscript Impact of fermentation conditions on the quality and sensory properties of a probiotic cupuassu (Theobroma grandiflorum) beverage

Ana Lúcia Fernandes Pereira, Wallaff Sammk Corrêa Feitosa, Virgínia Kelly Gonçalves Abreu, Tatiana de Oliveira Lemos, Wesley Faria Gomes, Narendra Narain, Sueli Rodrigues PII: DOI: Reference:

S0963-9969(17)30395-2 doi: 10.1016/j.foodres.2017.07.055 FRIN 6852

To appear in:

Food Research International

Received date: Revised date: Accepted date:

26 May 2017 23 July 2017 26 July 2017

Please cite this article as: Ana Lúcia Fernandes Pereira, Wallaff Sammk Corrêa Feitosa, Virgínia Kelly Gonçalves Abreu, Tatiana de Oliveira Lemos, Wesley Faria Gomes, Narendra Narain, Sueli Rodrigues , Impact of fermentation conditions on the quality and sensory properties of a probiotic cupuassu (Theobroma grandiflorum) beverage, Food Research International (2017), doi: 10.1016/j.foodres.2017.07.055

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Impact of fermentation conditons on the quality and sensory properties of a probiotic cupuassu (Theobroma grandiflorum) beverage

Ana Lúcia Fernandes Pereiraa*, Wallaff Sammk Corrêa Feitosaa,Virgínia Kelly Gonçalves

Curso de Engenharia de Alimentos, Universidade Federal do Maranhão, Centro de Ciências

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a

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Abreua, Tatiana de Oliveira Lemosa; Wesley Faria Gomesc; Narendra Narainc; Sueli Rodriguesb

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Sociais, Saúde e Tecnologia, 65.900-410, Imperatriz, Maranhão, Brazil, E-mail: [email protected],

[email protected],

Departamento de Engenharia Química, Universidade Federal de Sergipe, Cidade Universitaria,

Jardim

Rosa

Elze,

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b

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[email protected].

[email protected],

49100-000

–

São

Cristovão

–

Sergipe,

Brazil,

E-mail:

Departamento de Tecnologia de Alimentos, Universidade Federal do Ceará, Centro de

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c

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[email protected], [email protected].

Ciências Agrárias, Campus do Pici, Bloco 851, 60455–760, Fortaleza, Ceará, Brazil, E-mail:

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[email protected].

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*Corresponding author: Tel +55-99-981696263; E-mail [email protected].

ABSTRACT

The aim of the study was to evaluate the conditions of fermentation pH and temperature and also the fermentation time of Lactobacillus casei in the cupuassu (tropical fruit native to the Brazilian Amazon) beverage. The sugars, organic acids, and antioxidant activity during the fermentation also were investigated. The sensory characteristics were also evaluated. Moreover, the effect of expectation on the acceptability of probiotic and symbiotic cupuassu beverages 1

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was rated under three conditions. The blind (consumers were informed that the samples were probiotic and symbiotic beverages and they tasted them); expected (only nutritional claims in short text were informed) and informed (consumers were asked to evaluate the product when they had nutritional information). The conditions for probiotic beverage production were initial

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pH 5.8, the temperature of 30 °C, and 18 h of fermentation. L. casei had viability higher than 9.34 Log CFU/mL with 18 h of fermentation. The fructose was the most consumed sugar

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(84.76%), followed by sucrose (62.10%) and glucose (34.52%). The antioxidant activity

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increased during the fermentation. The organic acids present in the cupuassu (citric, ascorbic and quinic acids) also supported L. casei growth, being consumed during the fermentation

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improving the nutritional value of the beverage. The acceptance of the probiotic drink increased

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when the juice was presented to the informed tasters. Therefore, the nutrition claims were effective in increasing the acceptance. The probiotic cupuassu beverage was well accepted as

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an alternative functional food.

Keywords: Fermentation; Lactobacillus casei; viability; exotic fruits.

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1. Introduction

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The rise of vegetarianism and consumer concerns regarding an alternative diet to probiotics dairy products with high nutritional value and free of cholesterol and lactose is of great interest to researchers. As a result of this, new food matrices as probiotic carriers have been tested. Fruit and vegetable juices inoculated with probiotic microorganisms showed promising results (Costa, Fonteles, Jesus, & Rodrigues, 2013; Fonteles, Costa, Jesus, & Rodrigues, 2012; Martins et al., 2013; Pereira, Almeida, Jesus, Costa, & Rodrigues, 2013). The most common probiotic microorganisms used and marketed in food worldwide belong to the genera Lactobacillus and Bifidobacterium. In fruits juices, the Lactobacillus have 2

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shown higher resistance to the acid when compared to Bifidobacterium (Champagne, Ross, Saarela, Hansen, & Charalampopoulos, 2011; Kumar, Vijayandra, & Reddy, 2015; Sheehan, Ross, & Fitzgerald, 2007). Among the strains of Lactobacillus, Lactobacillus casei NRRL B442 showed high survival rates in the gastrointestinal tract, up-regulation of IFNγ levels and

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enhances the resistance against parasite infections (Bautista-Garfias et al., 1999). Thus, studies have used the strain with fruit juices (Fonteles et al., 2013; Pereira, Maciel, & Rodrigues, 2011).

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The production of the fruit juice probiotic can be done by microorganism addition to the

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fruit juice or the fermentation with probiotic microorganisms. The technique of the addition is successful if the strain is acid tolerant or if it uses the microencapsulation (Antunes et al., 2013).

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The fermentation, present some advantages over the addition since the microbial strain growth

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into the juice result in a more adapted microbial strain, which might contribute to higher survival rates. Pereira, Almeida, Jesus, Costa, and Rodrigues (2013) observed viability greater

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than 8.00 log CFU/mL during the storage of the cashew apple juice fermented with L. casei.

antioxidant activity.

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These authors reported that fermentation contributed to the preservation of ascorbic acid and

The cupuassu tree (Theobroma grandiflorum) is naturally distributed in Brazilian

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rainforests. Cupuassu has a high economic potential because of its excellent characteristics such

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as aroma, flavor, and texture (Faber, & Yuyama, 2015). However, because of its distinctive flavor, cupuassu pulp is used as an ingredient in the manufacture of ice cream, juice, liquors, wines, jellies, and other products, such as yogurts, rather than being consumed in natura (Vriesmann; & Petkowicz, 2009; Duarte et al., 2010). The pulp is yellowish-white and has high nutritional value, being a source of the ascorbic acid (96-111 mg/ 100 g) and phenolic compounds (20,5 mg/ 100 g). Health beneficial properties have been proposed for cupuassu, its antioxidant capacity is one of the most studied (Pinent et al., 2015). Fresh pulp presents a considerable antioxidant activity (1,7 – 2,0 µM 3

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Trolox/g) superior to, for example, strawberry and similar to other Brazilian native fruits like “araca̧ -boi (Eugenia stipitata Mc. Vaugh) and jaracatiá (Jaracatia spinosa Aubli) (Kuskoski, Asuero, Troncoso, Mancini-Filho, & Fett, 2005; Pugliese, Tomas-Barberan, Truchado, & Genovese, 2013). The antidiabetic potential was also evaluated, and cupuassu fruit showed the

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most potent inhibition of α-amylase activity among sixteen Brazilian native fruits and six commercial frozen pulps (Gonçalves, Lajolo, & Genovese, 2010).

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Moreover, the cupuassu is a potential source of dietary fiber, mainly soluble fiber

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(Salgado, Rodrigues, Donaldo-Pestana, Dias, & Morzelle, 2011). The cupuassu pulp has a particular chemical composition, rich in fibers, and contains a considerable amount of starch as

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well as pectin polysaccharides (Vriesmann, & Petkowicz, 2009), which can provide a different

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texture than other fruit pulps.

The food can influence growth, viability and survival, acid and bile tolerance, and

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different functionality of probiotics that determine their efficacy in the gastrointestinal tract.

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Thus, careful investigation of the interaction of different probiotics and food components should be considered in developing functional probiotic foods (Ranadheera, Baines, & Adams, 2010). Studies with fruit juices evidenced the increase in antioxidant capacity along the

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fermentation with probiotic microorganisms. According to Mousavi et al. (2013), the fermented

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pomegranate juice presented grown using L. acidophilus. Wang, Ng, Su, Tzeng and Shyu (2009) observed that noni juice fermented with B.longum showed a higher antioxidant capacity compared to the non-fermented juice. The sensory impact that probiotic cultures can cause in foods or beverages to which they are added has been little studied, although it is understood that products to which these functional ingredients have been added can create different flavor profiles when compared to the conventional products (Cruz et al., 2010; Matilla-Sandholm et al., 1999). Moreover, the choice of appropriate technique allows obtaining relevant sensory information that contributes 4

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to the consumer’s acceptance. In this kind of food, the information given through the label can create positive expectative. Therefore, the use of consumer expectation technique, which evaluates the effect of the expectation caused by nutritional information on the acceptability of a new probiotic beverage, plays a major role in the acceptance (Behrens, Villanueva, & Silva,

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2007). Thus, the objective of this paper was to evaluate the conditions of Lactobacillus casei

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cultivation in the cupuassu beverage. Moreover, it was investigated the sugars, organic acids,

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antioxidant, and sensory characteristics during the fermentation. The final product was submitted to sensory evaluation using the consumer’s expectation theory. This study has a

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patent deposited at National Institute of Industrial Property in Brazil with a register number of

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BR10201503045.

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2. Materials and methods

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2.1 Fermentation of the cupuassu beverage Cupuassu pulps obtained from the local market and were diluted in potable water, adjusting the pulp content to 34% (w/v).

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A strain of Lactobacillus casei NRRL B-442 obtained from ARS Culture Bacterial

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Collection (NRRL Culture Collection, United States Department of Agriculture, Peoria, IL, USA) was activated at 37 °C with De Man, Rogosa and Sharpe (MRS) broth (Himedia, India). The fermentation conditions were studied through a central composite rotated experimental design, changing the initial pH and temperature from 4.29 to 7.11 and 10.44 to 41.44 °C, respectively (Table 1). Erlenmeyer containing cupuassu beverage were inoculated with 7.00 log CFU/mL, which is the minimal counts recommended for better efficacy in regulating beneficial effects of probiotic foods (Vinderola, & Reinheimer, 2000). Fermentations were carried out statically in 5

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an incubator oven for 24 h at the different temperatures of the experimental design. After 24 h, the biomass, the viable counts, and pH analyses were carried out. The results were analyzed by Response Surface Methodology to find the optimum pH and temperature for the fermentation

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processing.

2.2 Biomass analysis

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Biomass was measured at 590 nm (triplicate) according to Rodrigues, Lona, and Franco

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(2003). The absorbance was measured after 1:10 dilution of the beverage with distilled water.

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The results were calculated with Eq. (1):

(1)

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m (g/L) = Absorbance 590nm – 0.008180/ 3.395

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2.3 Viable cell counts determination

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Viability was obtained by serial dilution with peptone water. Aliquots of 0.1 mL of dilution were plated (triplicate) in plates containing De Man, Rogosa and Sharpe (MRS) Agar (Himedia, India) using the spread plate method. The plates were incubated under aerobic

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mL.

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conditions for 72 h at 37 °C and the viability was recorded as colony forming units (CFU) per

2.4 pH analysis

The beverage pH was determined (triplicate) by direct measurement in a Biotech mPa210 potentiometer.

2.5 Fermentation time of L. casei in cupuassu beverage

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At the initial pH (5.8) and fermentation temperature (30 °C), the cupuassu beverage was inoculated with 7.00 Log CFU/mL of L. casei, and measurements of biomass, viability, and pH were done each 2 h, during 24 h to determine the proper fermentation time. Every 2 h, the cells were harvested by centrifugation at 1.389 g for 10 min, and the supernatant was evaluated to

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sugars, organic acids, phenolic compounds and antioxidant activity. Treatments were

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performed in triplicate.

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2.6 Sugars and organic acids

High Performance Liquid Chromatographic (HPLC) analysis was used to detect and

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quantify sugars and organic acids. For sugar, the samples were treated with 3 volumes of

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methanol to remove high molecular weight saccharides. Before injection, the samples were cleaned through a C18 SPE cartridge and filtered with 0.45 uM HA membrane cellulose ester

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(13 mm in diameter). The injection volume was 20 µL. It was used a 1260 Infinity Quaternary

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LC System (Agilent Technologies, USA) equipped with a pump system and RI detector. Sugars were separated in Supelcogel Ca column (300 × 7.8 mm) (Supelco Analytical) thermostatted at 80 °C. The isocratic elution was performed with deionized water as a mobile phase for 40 min

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(flow rate 0.5 mL/min). Calibration curves were obtained by mixing these sugars at different

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concentrations to quantify the sucrose, glucose, and fructose. For organic acids, the samples were filtered, using glass fiber pre-filters AP25 13 mm

diameter (Merck Millipore Ltd.) and cleaned using C-18 SPE cartridge, followed by filtration cellulose ester acetate membrane (0.45µM, 13 mm in diameter). HPLC system (Agilent Technologies 1260 Infinity) equipped with a quaternary pump system and UV-DAD detector monitored at 210 nm was used. Organic acids were separated in Aminex HPX-87H column (300 × 7.8 mm) (Bio-Rad) at 50 °C. The isocratic elution was performed with 0.01 M sulfuric acid in deionized water as a mobile phase for 30 min at 0.6 mL/min. 7

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2.7 Total phenolic content Total phenolic content was assayed according to Singleton and Rossi (1965). The method consisted on the ability of phenolic compounds to reduce the mixture of

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phosphomolybdic/phosphotungstic acid complexes in alkaline medium. For the analysis, 0.5 mL of the sample (or water for blank) was mixed with 0.5 mL of the Folin–Ciocalteu solution

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(diluted in water 1:3) and 1 mL of 20% (w/v) sodium carbonate solution. After 30 min of

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reaction, the reading was taken at 765 nm using spectrophotometer (Biospectro, SP-220, Curitiba, Brasil). Gallic acid was the reference standard, and phenolic was expressed as

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equivalents of gallic acid.

2.8 Antioxidant activity determination

The antioxidant activity was assayed according to DPPH and ABTS methods during

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fermentation of the beverage. The DPPH• free radical method was carried out according to Brand-Williams, Cuvelier, and Berset (1995) with modifications. Aliquots of 30 μL of the beverage were added to 1200 μL of DPPH• solution (0.06 mM). The absorbance was taken at

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515 nm at 1 min intervals during 30 min. The antioxidant efficiency was determined as the time

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when the concentration of substrate causes 50% loss in absorbance. For the ABTS method, ABTS radical was produced by reacting of the ABTS stock solution (7 mM) with potassium persulfate (140 mM). The ABTS•+ radical was diluted with ethanol to an absorbance of 0.700 at 734 nm. Aliquots of 30 μL of the cupuassu beverage diluted were mixed to 3000 μL of ABTS•+ radical, and the absorbance was measured after 6 min. Trolox was the antioxidant standard. Results were expressed as Trolox equivalent antioxidant activity per mL (Re et al., 1999).

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2.9 Beverage formulations for sensory analysis After the development of probiotic beverage production, the sensory analysis was performed in two stages. In the first stage, four cupuassu beverages formulations were produced: conventional, prebiotic (with 8 g/L of fructooligosaccharides addition), probiotic

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(fermented with L. casei) and symbiotic (fermented with L. casei and added of 8 g/L fructooligosaccharides after fermentation). The soluble solids content of each formulation was

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standardized to 11 °Brix by sucrose addition. For probiotic and symbiotic beverages, this

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standardization was done after fermentation. The conventional and prebiotic beverage were pasteurized (80 °C/ 1 minute). The probiotic and the symbiotic beverages cannot be submitted

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to thermal processing to assure the microbial viability necessary in probiotic products. On the

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other hand, during the fermentation L. casei produces bacteriocins that avoid contamination and

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2.9.1 Sensory evaluation

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pathogens development (Schnürer, & Magnusson 2005).

Sensory evaluation was performed by 60 untrained panelists (60% female, 40% male) ranging from 19 to 25 years old. The session was conducted in individual booths. Samples of

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40 mL were served one at a time at the 7 °C in glass cups.

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The consumer acceptance tests were conducted for the evaluation of color, appearance, body, flavor, sweetness and overall acceptance. The hedonic scale was used, ranging from 1 to 9. For these attributes, the sum of hedonic values percentages from 1 (dislike extremely) to 4 (dislike slightly) was named “rejection zone.” The hedonic value percentage at score 5 (neither like nor dislike) was called “indifference zone,” and the sum of hedonic values percentages from 6 (like slightly) to 9 (like extremely) of “acceptance region.” In the second stage, the effect of the nutritional information provided by the beverages on the acceptance was evaluated. In this stage, the formulations were standardized to 15 °Brix 9

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by sucrose addition. Subjects performed three different sensory acceptance tests, using 9-point hedonic in just one session following the sequence according to the methodology described by Deliza and MacFie (1996): 1 Blind test (B): Firstly, subjects were only informed that the samples were probiotic and

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symbiotic beverages and they tasted them and rated the overall liking. Purchase intention was also evaluated using a scale ranging from 1 (definitely would not buy) to 9 (definitely would

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buy).

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2 Expected liking (E): in a subsequent test, only nutritional and health claims framed in short text were presented, and subjects were asked to read the frames and then rate the expected liking

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(E).

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3 Informed evaluation (I): consumers were asked to evaluate the products when they also had information about the nutritional and health claims, to study the combined effect of the sensory

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attributes and the nutritional and health claims.

2.10 Statistical analysis

Statistica software version 7.0 (Statsoft, USA) was used to built the experimental design,

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the surface viability graph and optimize the processing.

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For the sensory data of the second stage, ANOVA was used to the blind, expected and informed conditions tests. Paired t-tests were used to examine how expectations on the nutritional information influenced the informed liking scores by investigating differences between expected and blind, and informed and blind conditions. Comparisons of proportions of subjects fitted in assimilation or contrast, as well as those showing no effect, were examined.

3. Results and discussion 3.1 Fermentation of the cupuassu beverage 10

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According to the results of Table 1, there was an increase in viability of L. casei in all assays. It was inoculated 7.00 log CFU/ mL and after 24 h of microbial fermentation, counts higher than 8.00 log CFU/ mL were observed. Pereira, Maciel, and Rodrigues (2011) and Fonteles, Costa, Jesus, and Rodrigues (2012) evaluated the growth conditions of L. casei

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fermentation in cashew apple and cantaloupe juices, respectively. Depending on the cultivation condition, reduction in the microbial viability was reported by these authors. Thus, the result

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obtained herein indicate that cupuassu is a good matrix for the probiotic beverage production

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since no viability loss was observed.

In assays 1, 3 and 7, where the lowest temperature was applied, L. casei growth was

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lower (Table 1). These results confirmed that L. casei is a mesophilic microorganism. On the

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other hand, the assays 4, 9, 10 and 11, that had mesophilic temperatures, presented the higher viability.

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According to the estimated effects, only the temperature had an effect on L. casei

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viability, being statistically significant at 95% of confidence level. For biomass formation, the pH and temperature were not statistically significant. Data presented in Table 1 was fitted to the quadratic model for viability given in Eq.

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(2). The model was statistically significant at 95% of confidence level since the calculated F

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values (15.97) was higher than the listed F value (F5.5=5.05). Good correlation coefficients were also obtained (R2=0.94).

Viability (log CFU/mL) = 0.48 + 2.02 pH – 0.19 pH2 + 0.21 T – 0.004 T2 – 0.004 pH . T (2) where T

temperature (°C)

pH

initial pH values

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Fig. 1 present the surface graph built using Eq. (2). The initial pH did not significantly influence the viability. High viability was observed at mild temperatures. The high microbial viability was obtained at pH 5.8 and fermentation temperature of 30 °C (critical point of Eq. (2), calculated by the statistical program).

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Our results are in agreement to the previously reported for fruit juices. Zheng et al. (2014) reported that L. casei in litchi juice grew rapidly at the same fermentation temperature

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(30 °C). Costa, Fonteles, Jesus, and Rodrigues (2013) evaluating the use of sonicated pineapple

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juice for producing of the probiotic beverage by L. casei observed the maximal microbial viability at pH 5.8.

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The condition (initial pH 5.8 and temperature of 30 °C) for microbial viability was

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chosen for the evaluation of the fermentation time. According to Fig. 2a, the growth of L. casei was slow at the processing beginning. After 8 h of fermentation, L. casei presented fast growth.

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The viability increased until 18 h, afterward some viability loss was observed (Fig. 2a).

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Although no cellular lysis (decrease of optical density), viability losses were observed with the maintenance of the fermentation.

Dimitrovski, Velickova, Langerholc, and Winkelhausen (2015) produced fermented

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apple juice with Lactobacillus plantarum. These authors reported viability of 8.51 log CFU/mL

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after 24 hours of fermentation at 37 °C. Herein, the time required to reach the same viability was lower (8 h), and the cell viability increased until 18 h of fermentation reaching more than 9 log CFU/ mL. In this way, probiotic cupuassu beverage production showed to be advantageous due to the faster microbial growth, requiring lower fermentation periods. The pH dropped along the fermentation time due to the increase in lactic acid content (Fig. 2b). Lactic acid is recognized as the main metabolite of lactic acid bacteria and acidification is one of the most desirable effects of their growth. The pH may drop to as low as 4.5, low enough to inhibit the growth of pathogens and many spoliating microorganisms. 12

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Vodnar, Paucean, Dulf, and Socaciu (2010) examined the capacity of L. casei to produce lactic acid on model MRS media during fermentation (37 °C, 78 hours) using HPLC. These authors obtained 6.16 g/L of lactic acid. In the present study, similar value (6.44 ± 0.00 g/L) was obtained at 20 h of fermentation. Thus, cupuassu beverage showed to be a good substrate for L.

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casei growth, presenting faster lactic acid production than in the model MRS media. The maximal cell viability (9.34 ± 0.06 log CFU /mL) and pH of 4.30 ± 0.01 were

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obtained at 18 h of fermentation. The final pH was below 4.5, which inhibit the pathogenic

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microorganisms' growth. Thus, 18 hours is the better fermentation time of probiotic beverage

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production.

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3.2 Organics acids and sugars in the cupuassu beverage HPLC analysis was performed to evaluate the changes in citric, ascorbic and quinic

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acids concentrations during 18 hours of fermentation. The initial concentration of citric acid

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was 8.45 dropping to 6.02 g/ L at 18 h. Mousavi, Mousavi, Razavi, Emam-Djomeh, and Kiani (2011) also reported a reduction in citric acid in pomegranate juice during the fermentation. According to theses authors, L. plantarum and L. delbrueckii were able to metabolize the citric

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beverages.

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acid as a carbon source, indicating that this juice is a suitable substrate for producing probiotic

The initial concentration of ascorbic acid was 90 mg/L reaching 30 mg/L at 18 h of fermentation. Ascorbic acid was associated with the good survival of lactic acid bacteria, which was attributed to the fact that it acts in the reduction of dissolved oxygen content and to the decreased redox potential. García-García et al. (2015) added ascorbic acid in milk and reported that ascorbic acid increased the viability of L. casei. Therefore, the increase of L. casei viability in the cupuassu beverage also resulted in the protective effect of ascorbic acid present in this matrix. It is important to emphasize that herein, 13

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the ascorbic acid addition was not necessary. The cupuassu beverage is a source of ascorbic acid, presenting, in the fermentation beginning, the recommended dietary value, which is 90 mg/day for adult men and 75 mg/day for women (US National Academy of Sciences, 2000). The cupuassu beverage proved to be more advantageous to probiotic production when

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compared to dairy products due to be a source of acid ascorbic. The initial concentration of quinic acid was 0.63 g/L and dropped to 0.26 g/ L at 18 h of

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fermentation. This reduction could be due to the metabolism of L. casei that produce others

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compounds. The L. plantarum was able to metabolize catechol from quinic acid. Data on the metabolism of this compound in other lactic acid bacteria species are scarce. Studies using cell

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extracts from Lactobacillus paracollinoides indicated that the first stage into the metabolism of

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chlorogenic acid was the hydrolysis to caffeic acid and quinic acids. Both products were further metabolized (Rodríguez et al., 2009).

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Fig. 3 shows the carbohydrate consumption during the fermentation. The sugars in

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cupuassu beverage, at the beginning of fermentation, were fructose (105.97 g/ L), sucrose (53.68 g/ L) and glucose (39.37 g/ L). The fructose was the most consumed sugar during fermentation (84.76%), followed by sucrose (62.10%) and glucose (34.52%).

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The results obtained herein were different from those reported by others researchers.

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Kun, Rezessy-Szabo, Nguyen, and Hoschke (2008) reported that in the carrot juice, glucose and sucrose were the main carbon and energy sources for probiotics growth. According to these authors, Bifidobacteria can utilize only 20% of glucose and 10% of sucrose during fermentation. Mousavi, Mousavi, Razavi, Emam-Djomeh, and Kiani (2011) produced a probiotic pomegranate juice and reported that glucose and fructose were the dominant sugars metabolized by Lactobacillus. These authors observed that the metabolism of carbohydrates by Lactobacillus varies from strain to strain and depends on the substrate and even on the fermentation time. 14

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3.3 Total polyphenolic compounds and antioxidant activity of cupuassu beverage The total polyphenolic and the antioxidant activity measured during fermentation are presented in Fig. 4. The antioxidant capacity was evaluated using ABTS and DPPH free radical

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methods. DPPH did not show any change along the fermentation (results not shown). On the other hand, the antioxidant activity measured by ABTS increased until 18 h of fermentation.

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Therefore, the result indicates that the fermentation increased the antioxidant activity.

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Moreover, the results obtained herein suggest that the ABTS method was more sensitive since there was a reaction between the ABTS radical and the antioxidants compounds of the cupuassu

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beverage not observed with DPPH.

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Mousavi et al. (2013) also found a reduction in the phenolics concentration and the increased in antioxidant activity of pomegranate juice after fermentation by L. plantarum and

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L. acidophilus. According to Rodríguez et al. (2009), the explanation for the contrast detected

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between the decreased concentration of phenolic and the increased antioxidant activity could is that some lactic acid bacteria can to degrade some phenolic compounds producing compounds that increased antioxidant activity. This result corroborates with the results found for quinic

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acid as earlier discussed.

3.4 Sensory evaluation In the first stage of sensory evaluation, for color and appearance attributes, the higher percentages were obtained in the acceptance region for the cupuassu beverages (Fig. 5a and b). It is important to emphasize that color and appearance are the major attributes that are evaluated by consumers. Thus, the presence of prebiotics and probiotics did not affect the color and appearance of the beverage.

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For body attribute, the higher percentages also were obtained in the acceptance region for the beverages. However, the probiotic and symbiotic had lower percentages (Fig. 5c). Cruz et al. (2010) reported that substitution with prebiotic ingredients has a greater influence on texture. Herein, the prebiotic addition did not influence the beverage body negatively, because

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acceptability was similar to the conventional beverage. Ellendersen, Granato, Guergoletto, and Wosiacki (2012) reported that a thick texture

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characterized the probiotic apple beverage. These authors explained that this texture change is

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due to the bacterial biomass addition that provided higher turbidity. Thus, the lower percentages in the acceptance region for the body of probiotic and symbiotic when compared to

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conventional and prebiotic beverages could be related to this change.

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The flavor, sweetness, and overall acceptance of the conventional and prebiotic cupuassu beverages presented higher percentages in the acceptance region. However, the

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probiotic and symbiotic beverage showed higher percentages in the rejection region (Fig. 5d, e,

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and f). Pimentel, Madrona, and Prudencio (2015) also reported lower acceptance on the flavor and overall acceptance in the probiotic clarified apple juice when compared to conventional juice.

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Herein, the panelists mentioned that probiotic and symbiotic beverages were not sweet

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enough. This observation was confirmed by the lower acceptance of the sweetness attribute. Mondragón-Bernal, Rodrigues, Bolini, and Maugeri (2010), optimizing the symbiotic food from hydrosoluble soy extract, reported that higher sucrose content improved the flavor. Thus, an alternative to improve the sensory impact of probiotic and symbiotic cupuassu beverages is the addition of a higher sucrose amount. In the second stage of sensory evaluation, sucrose content was increased to 15 °Brix (2 times more than in the first stage). The results obtained confirmed what these authors earlier

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reported because, in the blind condition, the percentages in the acceptance region of overall acceptance and purchase intent were higher than rejection region (Fig 6a and b). Pereira, Almeida, Jesus, Costa, and Rodrigues (2013) also reported greater overall acceptance in probiotic cashew apple juice containing sucrose compared with the juice with

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lower sucrose content. These authors indicated that this preference might be related to the Brazilian preference for sweet products.

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In the blind, expected and informed conditions, there was not difference among the

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means of probiotic and symbiotic beverages (Table 2). This result confirms those obtained in the first stage sensory analysis (Fig. 5f).

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Comparing the expected and blind conditions (E-B), it was observed a disconfirmation

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of the information for beverages, as the expected liking was significantly higher than blind liking. Tasting with information only had a significant effect on overall liking for the probiotic

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beverage, as expressed by the significant I–B score (Table 2). Thus, the overall acceptance was

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significantly higher when consumers were informed of the nutritional and health claims. Therefore, the result indicates an assimilation effect occurred. A potential explanation for this assimilation effect is that the consumers are more willing to compromise on taste if informed

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about other (non-sensory) benefits associated with the product (Schouteten et al., 2016).

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Behrens, Villanueva, and Silva (2007) evaluated the expectation effect on the acceptance of probiotic yogurt with soy milk. These authors reported that hedonic values increased on the expected when compared with the blind condition. However, in the informed tasting, the health claims did not increase the acceptance, since the average was the same than blind tasting. Thus, the results obtained herein for the probiotic beverage was positive because, on the informed tasting, the nutrition claims were effective in increasing the final acceptance. Herein, the results were also present in percentages of assimilation and contrast models. An assimilation effect of nutritional and health claims information increasing consumers’ liking 17

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in the expected direction could be an advantage for commercial food products. About a contrast effect, the consumer expectations that insufficiently matched by actual product performance generate consumer dissatisfaction, a critical limiting factor for commercial food products. Therefore, it was calculated the individuals percentage of these models to improve the

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viewing of assimilation and contrast effects. The consumer percentages that presented the same average in the three condition was also calculated, indicating that the information did not

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produce an effect (B = E = I) and those that had effects unclear (Table 3).

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Assimilation was the main information effect on the acceptability of the probiotic beverage (40.00%). It was observed the predominance of assimilation model with negative

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disconfirmation (E>B and I>B) (Table 3). This result indicates that the sensory characteristics

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found in the product did not match high expectations generated by the health claims. However, consumers assimilated expectation increasing the final acceptance.

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The assimilation model occurred when any difference between the expectation

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consumer at the purchase moment and what was found in the product was assimilated, causing the perception of the product after tasting it is close to the expectations generated (Behrens, Villanueva, & Silva, 2007). Thus, the results obtained herein are in accordance with these

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authors, indicating the importance of the nutritional information on the acceptance.

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In the symbiotic beverage, the higher percentage of panelists presented unclear effects (41.67%) (Table 3). The unclear effects came up in two situations: (1) being (I = B) and (E > B) or (E < B), or (2) being (E = B) and (I > B) or (I < B). In the first case, both positive and negative disconfirmation seemed to occur, but with no effect on the acceptance (I = B). On the other hand, there was confirmation of the expectation when (E = B), but the difference between (R) and (B) ratings might have occurred at random. This indicates that the behavior exhibited by these consumers do not follow any of the models mentioned. The hypotheses for such effects

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are lack of critical judgment and lack of involvement or interest in health issues (justifying mostly B = E = I ratings) (Behrens, Villanueva, & Silva, 2007). Therefore, according to the results obtained for the consumer's expectations, the nutritional information provided to the consumer increased the acceptance of probiotic

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beverage. The same behavior did not occur with symbiotic beverage. This result for symbiotic may be due to the consumer having expected more of this product which has more nutritional

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advantages.

4. Conclusions

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Although the use of fruit juice as probiotic carriers has high potential, few works have

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been done in this field considering the Amazonian fruits. Herein, it was demonstrated that the use of cupuassu beverage as a substrate for probiotic growth is a good alternative because no

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preservatives neither heat treatment was required prior fermentation. This research

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demonstrated the technological advantage of using cupuassu beverage as a substrate for probiotic fermentation since its composition (natural sugars and organic acids) provides an enabling environment for the development of probiotic. The fermentation provided an increase

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of the antioxidant activity, which confers nutritional benefits to this functional food.

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The sensory analysis measured by the effect of expectation on the acceptability showed that the product was well-accepted. Moreover, the overall acceptance was higher when consumers were informed about the nutritional and health claims of probiotic beverage, showing that the acceptance is dependent on the product information.

Acknowledgments

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Authors thank the National Institute of Tropical Fruits (INCT-FT), National Council for Scientific and Technological Development (CNPq) and Technological Development of Maranhão (FAPEMA) for the financial support and scholarships.

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Figure Captions

Figure 1 - Surface graph of viability (Log CFU/mL) Lactobacillus casei NRRL B-442 in the

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cupuassu beverage as a function of initial pH and temperature (°C).

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Figure 2 - Viability (Log CFU/mL) and biomass (g/L) of Lactobacillus casei (a) and pH and lactic acid (b) in cupuassu beverage during 24 h of fermentation.

Figure 3 - Total sugar (in grams per liter) in cupuassu beverage during 18 h of fermentation.

Figure 4 - Antioxidant activity (AA) (in micromolar Trolox per milliliter) and total polyphenolic compounds (TPC) (in milligrams per 100 milliliters) of the cupuassu beverage during 18 h of fermentation. 25

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Figure 5 - Color (a), appearance (b), body (c), flavor (d), sweetness (e), and overall acceptance (f) of cupuassu beverages (conventional, prebiotic - with 8 g/L of fructooligosaccharides addition, probiotic - fermented with L. casei and symbiotic - beverage fermented with L. casei

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and added of 8 g/L fructooligosaccharides after fermentation).

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Figure 6 - Overall acceptance (a) and purchase intent (b) of probiotic and symbiotic cupuassu

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beverages (probiotic - fermented with L. casei and symbiotic - beverage fermented with L. casei

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Table 1- Experimental design and responses on Lactobacillus casei NRRL B-442 growth in cupuassu beverage after 24 h of fermentation. Fermentation Assay

Initial pH

temperature

Viable cells Biomass (g/L)

CFU/mL)

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(ºC)

counts (log

4.70

15.00

0.67±0.04

8.29±0.10

2

4.70

37.00

2.05±0.01

9.12±0.18

3

6.70

15.00

0.82±0.01

8.27±0.10

4

6.70

37.00

2.03±0.07

9.30±0.12

5

4.29

26.00

1.26±0.11

9.10±0.09

6

7.11

26.00

1.54±0.22

9.11±0.06

7

5.70

10.44

0.95±0.10

8.17±0.21

8

5.70

41.44

0.79±0.01

8.91±0.03

9

5.70

26.00

1.99±0.41

9.56±0.01

10

5.70

26.00

1.67±0.24

9.49±0.04

11

5.70

26.00

1.97±0.11

9.26±0.02

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Table 2 Overall liking mean ratings obtained by the four probiotic and symbiotic cupuassu beverages evaluated under blind (B), expected (E) and informed (I) conditions. Differences between the mean ratings tested through paired t-tests. Beverages

Conditions

Paired t test

Expected

Informed

Probiotic

6.35±1.72ns

8.05±0.89ns

7.00±1.79ns

1.70±1.78*

0.65±1.74*

Symbiotic

6.50±1.85ns

8.03±0.84ns

6.80±1.71ns

1.53±1.70*

0.30±1.57ns

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significant by t test (p> 0.05), * Significant by paired t test (p<0.05).

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nsNo

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Table 3 Observed proportion of consumers showing assimilation, contrast, unclear or no effect of expectation created by information nutritional and health claims of the probiotic and symbiotic cupuassu beverages (n= 60). Effects

Consumers (%) Symbiotic

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Probiotic

40.00

Positive disconfirmation

3.33

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Negative disconfirmation

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35.00 1.67

Negative disconfirmation

11.67

11.67

0.00

0.00

15.00

10.00

30.00

41.67

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Positive disconfirmation

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Contrast

No effect (B = E = I)

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No clear effects

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Graphical abstract

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Highlights

The cupuassu is a good substrate for probiotic growth, with viability higher than 8 log CFU/ mL. The probiotic cupuassu beverage has good sensory acceptance.

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The nutritional informations increase the acceptance of probiotic cupuassu beverage. The fermentation with Lactobacillus casei provide increase of the antioxidant activity in

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cupuassu beverage.

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