Effects of high hydrostatic pressure on the microbial inactivation and extraction of bioactive compounds from açaí (Euterpe oleracea Martius) pulp

Journal Pre-proofs Effects of high hydrostatic pressure on the microbial inactivation and extraction of bioactive compounds from açaí (Euterpe oleracea Martius) pulp Ana Laura Tibério de Jesus, Marcelo Cristianini, Nathalia Medina dos Santos, Mário Roberto Maróstica Júnior PII: DOI: Reference:

S0963-9969(19)30742-2 https://doi.org/10.1016/j.foodres.2019.108856 FRIN 108856

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

11 July 2018 19 November 2019 20 November 2019

Please cite this article as: Laura Tibério de Jesus, A., Cristianini, M., Medina dos Santos, N., Roberto Maróstica Júnior, M., Effects of high hydrostatic pressure on the microbial inactivation and extraction of bioactive compounds from açaí (Euterpe oleracea Martius) pulp, Food Research International (2019), doi: https://doi.org/ 10.1016/j.foodres.2019.108856

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© 2019 Published by Elsevier Ltd.

Effects of high hydrostatic pressure on the microbial inactivation and extraction of bioactive compounds from açaí (Euterpe oleracea Martius) pulp

Ana Laura Tibério de Jesusa; Marcelo Cristianinib*; Nathalia Medina dos Santosc; Mário Roberto Maróstica Júniorc.

a

Department of Food Engineering, Sorocaba Engineering College (FACENS),

Senador José Ermírio de Moraes Road, 1425, 18085-784. Sorocaba, SP, Brazil b

Department of Food Technology (DTA), School of Food Engineering (FEA),

University of Campinas (UNICAMP), Monteiro Lobato, 80. PO Box 6121, 13083-862. Campinas, SP, Brazil c

Department of Food and Nutrition (DEPAN), School of Food Engineering

(FEA), University of Campinas (UNICAMP), Monteiro Lobato, 80. PO Box 6121, 13083-862. Campinas, SP, Brazil

*Corresponding author: School of Food Engineering, University of Campinas, Monteiro Lobato, 80, 13083-862. Campinas, SP, Brazil. E-mail address: [email protected] (M. Cristianini) Abstract The aim of this study was to investigate the effects of high hydrostatic pressure (HHP) on the inactivation of Lactobacillus fructivorans, on the inactivation of Alicyclobacillus acidoterrestris spores and on the extraction of anthocyanins and total phenolics from açaí pulp. The tested conditions comprised pressures of 1

400–600 MPa, treatment times of 5 to 15 min, and temperatures of 25 °C and 65 °C. Results were compared to those of conventional thermal treatments (85 °C/1 min). Regarding A. acidoterrestris spores, applying HHP for 13.5 min, resulted in a value of four-decimal reduction.. L. fructivorans presented considerable sensitivity to HHP treatment, achieving inactivation rates above 6.7 log cycles at process conditions at 600 MPa and 65 °C for 5 min. All samples of açaí pulp processed showed absence of thermotolerant coliforms during the 28 days of refrigerated storage (shelf life study). The açaí pulps processed by HHP (600 MPa/5 min/25 °C) had anthocyanin extraction increased by 37% on average. In contrast, conventional thermal treatment reduced anthocyanin content by 16.3%. For phenolic compounds, the process at 600 MPa /5 min/ 65°C increases extraction by 10.25%.. A combination of HHP treatment and moderate heat (65ºC) was shown to be an alternative to thermal pasteurization, leading to microbiologically safe products while preserving functional compounds.

Keywords:

Alicyclobacillus

acidoterrestris,

anthocyanins,

açaí

pulp,

Lactobacillus fructivorans, high pressure processing, phenolic compounds

1. Introduction Açaí pulps (Euterpe oleracea Martius) are consumed mainly by the Brazilian population, especially in the North and Northeast regions of the country. However, there has been an increased demand for açaí pulp in recent years in the domestic and international markets, and the fruit has attracted the interest of investors and researchers (Rogez, 2000; Yamaguchi, Pereira,

2

Lamarão, Lima, & Da Veiga-Junior, 2015). This occurs mainly due to açaí’s high antioxidant capacity provided by its high anthocyanin and tocopherol contents (Bichara & Rogez, 2011). Polyphenols are the predominant chemical constituents of açaí, notably anthocyanins and flavonoids (Iaderoza, Baldini, Draetta, Bovi , 1992; Pacheco-palencia, Duncan, & Talcott, 2009), which justify its classification as a functional food that helps prevent various degenerative diseases (Pacheco-Palencia & Talcott, 2010; Rosso et al., 2008; Yuyama et al., 2011). The main anthocyanins found in açaí are cyanidin-3-glucoside, with values ranging from 11.1 mg of gallic acid equivalents (GAE)/100 g to 117 mg GAE/100 g, and cyanidin-3-rutinoside, with values between 193 mg GAE/100 mg and 241.8 mg GAE/100 mg (Ribeiro et al., 2010; Schauss et al., 2006). Although data on the antioxidant potential of açaí species are conflicting, PozoInsfran, Brenes, & Talcott, (2004) concluded that anthocyanins were the compounds that most contribute to the antioxidant power of açaí, being present at concentrations greater than those found in several fruits, such as in blueberries, cranberries, plums, and raspberries. Some studies consider that açaí has a lower concentration of total phenolics (13.9 mg/g GAE) than other dark fruits. However, extraction methods are of fundamental importance and any difference in procedure must be taken into account when comparing and evaluating results of different studies (Castrejón, Eichholz, Rohn, Kroh, & Huyskens-Keil, 2008; Ribeiro et al., 2010; Schauss et al., 2006). Açaí pulps are highly perishable and deteriorate quickly at ambient temperatures, requiring the use of heat treatments to prolong shelf life as untreated pulps are fit for consumption for 12 h only, even under refrigeration (Menezes et al., 2008; Rogez, 2000). Açaí is prone to infection by agents such

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as fungi and beetles and, due to post-harvest factors (temperature and relative humidity) the fruit pulp is also subject to contamination. Other causes of contamination include lack of hygiene during harvest, transportation, and processing (Nascimento, Couri, Antoniassi, & Freitas, 2008; Santos, Figueiredo Neto, & Donzeli, 2016). Some recent studies reported the presence of Salmonella sp., Escherichia coli, thermotolerant coliforms, molds and yeasts, and rodent hairs in frozen açaí pulps sold in large cities in Brazil (Fregonesi et al., 2010; Nascimento et al., 2008). In a study carried out by Faria, Oliveira, & Costa, 2012, the authors verified the presence of E. coli in 13.8% of the analyzed açaí pulp samples. In fruits, microbial populations generally vary between 5 and 7 log colony-forming units (CFU)/g, with lactic acid bacteria (LAB) constituting a small part (2–4 log CFU/g) of the autochthonous microbiota (Di Cagno et al., 2010; Di Cagno, Surico, et al., 2011; Di Cagno, Minervini, Rizzello, De Angelis, & Gobbetti, 2011). Bacteria of the genus Lactobacillus are the most common LAB and are often contaminants of various food products, especially fruit products. Alicyclobacillus acidoterrestris (AAT), first identified in the 1980s, is a non-pathogenic spore-forming bacterium (Deinhard, Blanz, Poralla, & Altan, 1987; Wisotzjsey, JR, Fox, Deinhard, & Poralla, 2018) that has been linked to several cases of deterioration in apple and orange juices throughout product shelf life. Economic losses can be substantial because this microorganism does not produce gas, making deterioration difficult to detect prior to product consumption. Deterioration induced by AAT is characterized by an off-flavor that can be sensed at very low amounts, such as concentrations in the order of ppb

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of guaiacol (or bromophenol), which can be produced by relatively low cell densities, 105 to 106 cells/mL (Pettipher, Osmundson, & Murphy, 1997). Pasteurization (80–85 °C/30–60 s) followed by freezing at −18 °C is the most commonly used method for preserving and storing açaí pulps, but hightemperature treatment has drawbacks as it easily degrades anthocyanins and phenolic compounds present in the fruit (Markakis, 1982; Embrapa, 2005). Degradation of anthocyanins is a common effect of thermal treatments. In a pasteurization experiment (82.5 °C/1 min) with açaí, degradation of 5 to 30% of the anthocyanin content was observed. According to Rogez (2000), the loss of anthocyanins in non-pasteurized açaí pulp stored for 60 days at −20 °C ranges from 5 to 60%, as even under freezing conditions the enzymes peroxidase and polyphenoloxidase have a residual activity of 60 to 90%, indicating that oxidation reactions continue to occur in frozen açaí. High

hydrostatic

pressure

(HHP)

is one of

the

non-thermal

juice/beverage preservation technology that meets the high-end requirements of consumers and manufacturers: it is a clean technology that generates highquality, microbiologically safe products with long shelf-life (Gopal, Kalla, & Srikanth, 2017). Without requiring the addition of chemical preservatives, HHP treatment is able to eliminate pathogenic and deteriorating microorganisms in beverages using high pressures (400–600 MPa) while preserving nutritional properties, colors, and flavors, for weeks to months, under refrigerated conditions (Gopal et al., 2017; C.-Y. Wang, Huang, Hsu, & Yang, 2016). As discussed in the literature, heat and freezing treatments present important technological challenges. First, the time–temperature parameter used in pasteurization is a factor of fundamental importance since high temperatures

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and/or excessive times can cause undesirable changes in taste and aroma (Butz & Tauscher, 2002). Therefore, the time–temperature condition chosen for pasteurization must be able to inactivate the target microorganisms while preserving the physical, chemical, nutritional, and sensorial characteristics of the product (Rosenthal, Mackey, & Bird, 2002). To the best of our knowledge there is no research investigating the optimization of parameters to achieve microbial inactivation with minimal compound degradation, especially in relation to anthocyanins in açaí pulp. Furthermore, freezing treatments are expensive and do not effectively guarantee the stability of açaí constituents (Alexandre, Cunha, & Hubinger, 2004; Rogez, 2000). In this context, this study aimed to evaluate the effect of high hydrostatic pressure

treatment

on

the

inactivation

of

deteriorating

(Lactobacillus

fructivorans) and sporulated (Alicyclobacillus acidoterrestris) bacteria and establishes adequate process parameters to guarantee microbiological safety while preserving anthocyanins, phenolic compounds, and the antioxidant capacity of açaí.

2. Material and Methods 2.1. Açaí pulp The açaí pulp used in this study was type B (11.3% of total solids), according to the classification of Normative Instruction no. 01 of January 2000 (Brazil, 2000), with pH 4.7 and 3.2 °Bx, purchased from a pulp industry located in Castanhal, Pará, Brazil. For sample storage and processing, pulps (50 g) were stored in flexible multilayer laminated material with oxygen- and lightbarrier properties, composed of a black pigmented inner layer and low-density

6

polyethylene (LDPE) coextruded with ethylene vinyl alcohol (EVOH) and activated carbon (Dixie-Toga Ltda., São Paulo, SP, Brazil).

2.2. High-pressure processing tests The packaged samples were processed in a high-pressure system (QFP 2L-700, Avure Technologies, OH, USA) with a 2 L-capacity chamber, maximum working pressure of 690 MPa, and controlled temperature range of 10–90 °C. The temperature in the chamber was measured by two type K thermocouples inserted into the chamber, one located in the top and other in the middle. The pressure was captured by a pressure transducer. The compression time to reach 600 MPa was approximately 122.7 ± 6.9 s and the decompression was practically instantaneous (2.2±0.3 s). The temperature in the chamber block was set at 25 °C (processes at room temperature). The initial temperature of the water in the chamber was set at 8–10 °C reaching 27.1 ± 1.3 °C (due to adiabatic heating) at the beginning, and

25.2 ± 1.7 °C at the end of the

process. The

process

conditions

were

chosen

according

to

the

target

microorganism and from the first part of the study where enzymatic inactivation (peroxidase and polyphenol oxidase) and instrumental color tests were performed (Jesus, Leite, & Cristianini, 2018). For Lactobacillus fructivorans, 12 conditions were tested: pressures of 400, 500, and 600 MPa, treatment times of 5 and 15 min, and temperatures of 25 °C and 65 °C. From the preliminary tests, it was observed that for the sporulated microorganism Alicyclobacillus acidoterrestris (AAT), combining HHP treatment with higher temperatures resulted in higher inactivation rates of spores. Thus, the process conditions

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chosen for inactivation of AAT spores were based on the use of temperature (65 °C) and high pressure (600 MPa). To obtain an inactivation curve for this microorganism by HHP, the process parameters chosen were: 600 MPa at 65 ° C for 5, 10, 15, 20 and 25 minutes. All the processes were performed in triplicate.

2.3. Thermal treatment For thermal processing, was used the method proposed by Sulaiman, Soo, Farid, & Silva (2015), with a time–temperature condition of 85 °C/1 minute (treatment traditionally used in the industry for açaí pasteurization). Pulps ( 50 g) were vacuum-packed in the same type of material used for HHP treatments (LDPE/EVOH, 78 μm thick), with a large surface area (105 x 115 mm) so that temperature could be considered relatively uniform within the bags. A thermocouple was placed on the center of the package, and samples were fully submerged in a water bath. An ultra-thermostatic bath at 85 ± 1.5 °C (due to adiabatic heating)

(model 115, FANEM, São Paulo, Brazil) was used, with

continuous temperature monitoring through a type T thermocouple and data logger (Almemo® 2890-9, Ahlborn). The temperature of 85 ° C was reached within 60 seconds and the samples were cooled in an ice bath to 25 ° C.

2.4. Microbiological analyses The strains of microorganisms used in this study were donated by the André Tosello Foundation (Campinas, São Paulo, Brazil) and belonged to the collection of tropical cultures (CCT) of the same foundation. Prior to the tests,

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the vacuum-packed açaí pulp was sterilized (121 °C/15 min) in an autoclave to inactivate the natural microbiota.

2.4.1. Lactobacillus fructivorans In order to determine the best processing conditions for the study of açaí pulp shelf life, microbial inactivation tests were performed with a lactobacillus strain. This type of microorganism was selected because the untreated açaí pulp had 2.3 x 104 CFU/mL of lactic bacteria, which could lead to product deterioration during the storage period. The strain used for the inactivation assays was Lactobacillus fructivorans CCT 0850. A L. fructivorans cell suspension (0.3 mL, 109 CFU/mL) was inoculated into 30 mL of açaí pulp, thus yielding an initial count of 107 CFU/mL. After the samples were treated by HHP or thermal pasteurization, enumeration was performed as follows: 1 mL of sample was added to 9 mL of 0.1% sterile peptone water. Counting was carried out by the pour-plate method using MRS agar with overlay. Plates were incubated at 30 °C for 48 hours (Castro, Rojas, Campos, & Gerschenson, 2009).

2.4.2. Alicyclobacillus acidoterrestris (AAT) Alicyclobacillus acidoterrestris CCT 7547 was grown at 45 °C for 3 days in potato dextrose agar (PDA) medium adjusted to pH 4.0 with 10% (w/v) tartaric acid sterilized by filtration to generate a stock culture. Cells from the stock culture were grown on PDA in Petri dishes incubated at 45 °C for 18 days. After reaching more than 80% sporulation, confirmed by microscopy after staining with malachite green, the spores were collected with a sterile swab and

9

suspended in sterile distilled water. Spores collected from different plates were centrifuged at 4000 × g for 20 min at 4 °C and washed with sterile distilled water; this procedure was repeated twice. Samples were resuspended in phosphate buffer (pH 7.2) and stored at 4 °C until the moment of analysis. AAT spore enumeration followed the method proposed by Silva, Tan, & Farid, (2012). For the inactivation of AAT, 3 mL of the spore suspension (107 CFU/mL) was inoculated into 27 mL of açaí pulp, yielding an initial count of 106 CFU/mL. To determine the spore concentration in the unprocessed and processed açaí pulps, 0.1 mL of the spore suspension/açaí pulp mixture was serially diluted in 0.1% (w/v) peptone water in test tubes. Dilutions were heated at 80 °C for 10 min to remove any remaining vegetative cells, and 0.1 mL of each dilution was inoculated onto acidified PDA plates and incubated at 45 °C for 3 days. After incubation, the colony-forming units were counted for each dilution and the mean counts were calculated. The concentration of spores in açaí pulp (N) was expressed as CFU/mL of pulp. Equation (1) was used to describe the Weibull distribution (Mafart, Couvert, Gaillard, & Leguerinel, 2002). The statistical criteria used to compare the experimental results with the obtained model were the correlation coefficient (R²) and root mean square error (RMSE). 𝛽

𝑁⁄𝑁0 = exp(−(𝑡⁄𝛼) )

(1)

where N0 = initial spore concentration/mL; N = concentration of survivor spores/mL (after treatment for a specific time); t = time (in minutes or seconds); α = scale parameter (minutes or seconds); β = shape parameter of the survival curve, used as a (dimensionless) performance index.

10

The correlation coefficient (R2) is generally applied when comparing models with different numbers of parameters. The root mean square error (RMSE), which measures the mean deviation between observed and predicted values, and it given by Equation (2). RMSE values closer to zero indicate better model performance (Buzrul & Alpas, 2004). 2

∑(𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 − 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑) 𝑅𝑀𝑆𝐸 = √ 𝑛−𝑚

(2)

where n is the number of observations and m is the number of estimated parameters.

2.4.3. Microbiological analysis during storage Analyses of total coliforms, thermotolerant coliforms, and salmonella, as regulated by RDC no. 12 of January 2001 (Brazil, 2001), were carried out during a storage period of 28 days at 5 ± 2 °C. In addition, determination of yeasts and molds, aerobic mesophilic bacteria, psychrotrophic bacteria, and lactic bacteria was performed. All analyses were performed according to Silva et al. (2010) and the methods are described by the American Public Health Association (APHA, 2001). Dilutions were plated in duplicate.

2.5. Extraction and analysis of functional compounds Extractions for determination of anthocyanins, total phenolics, and antioxidant activity were planned in order to verify the effects of HHP treatment and heat treatment. Thus, variations in temperature, time of extraction, and solvents were avoided so that differences among samples could better represent the effect of the different processes. Water was selected as solvent (aqueous extraction) for being part of the production process of açaí pulps and 11

was acidified with formic acid (1%) in order to stabilize the anthocyanins (Batista et al., 2017). After the dilution of pulps in acidified distilled water, samples were centrifuged at 4000 x g and 4 °C for 10 min. For comparison purposes, the extraction method was the same for all analyses. Absorbance and fluorescence readings were performed on a Biotek HT microplate reader (Biotek, Winooski, USA) and analyzed using Gen5™ 2.0 software.

2.5.1. Anthocyanins The determination of monomeric anthocyanins was performed using a spectrophotometric pH differential protocol (Wrolstad, Durst, & Lee, 2005). Açaí extracts were diluted with 0.025 mol L−1 potassium chloride buffer (pH 1.0) and 0.4 mol L−1 sodium acetate buffer (pH 4.5) in the same proportions (1:4 dilution factor) according to sample absorbance (0.4–0.6 at 520 nm). After this, 250 μL of the diluted samples were pipetted into a microplate and absorbance was read at 520 and 700 nm. Absorbance (A) was calculated using Equation (3): 𝐴 = (𝐴520𝑛𝑚 − 𝐴700𝑛𝑚 )𝑝𝐻1.0 − (𝐴520𝑛𝑚 − 𝐴700𝑛𝑚 )𝑝𝐻4.5

(3)

The content of anthocyanins (mg 100 g−1) was expressed in mg of cyanidin-3-glucoside equivalents according to Equation (4): 𝐶 (𝑚𝑔 𝐶3𝐺 100 𝑔−1 ) =

𝐴 𝑥 𝑀𝑊 𝑥 𝐷𝐹 𝑒𝑥𝐿

(4)

where C = concentration; C3G = cyanidin-3-O-glucoside; A = absorbance; MW = molecular weight (449.2 g/mol); DF = dilution factor; e = molar absorptivity of the predominant anthocyanin in the sample (26.900 mol L−1); L = length (cm).

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2.5.2. Total phenolic compounds Total phenolics content was measured by the reducing ability of the sample, determined by the Folin-Ciocalteu method (Singleton & Rossi Jr, 1965). Quantification of phenolic compounds was performed with a solution of deionized water, Folin-Ciocalteu reagent, sodium carbonate (20% w/v), and açaí extract in the proportion of 16:1:2:1 according to Batista et al.( 2016) . The reaction was run for 120 min in the dark at room temperature, and absorbance was read at 765 nm. The results were expressed as mg GAE/100 g of pulp.

2.5.3. Antioxidant activity The antioxidant activity was evaluated by ORAC (oxygen radical absorbance capacity) and FRAP (ferric reducing antioxidant power) assays according to Batista et al. (2016), with some adaptations.

2.5.3.1. ORAC assay For the ORAC assay (Dávalos, Gómez-Cordovés, & Bartolomé, 2004), açaí extracts diluted in phosphate buffer (pH 7.4) or standard solutions were added to fluorescein diluted in phosphate buffer and AAPH [2,2'-Azobis(2amidinopropane) dihydrochloride] in the ratio of 1:6:3, respectively, in black microplates. Trolox was used as standard. The microplate reader with fluorescent filters was set to excitation wavelength of 485 nm and emission wavelength of 520 nm. ORAC values were expressed as μmol of Trolox equivalents (TE)/g of sample.

2.5.3.2. FRAP assay

13

The ferric reducing power of the extracts was determined using the FRAP assay according to Rufino et al. (2010).The FRAP reagent was prepared in the dark using 0.3 M acetate buffer (pH 3.6), TPTZ [2,4,6-tris(2-pyridyl)-striazine] in 40 mM HCl, and 20 mM FeCl3 in a 10:1:1 ratio, respectively. Standard solutions or extract samples, water, and FRAP reagent were mixed and incubated in a water bath for 30 min at 37 °C. The samples and Trolox standard curve were read at 595 nm.

2.6. Statistical analysis The effect of HHP treatment and thermal processing was evaluated using analysis of variance (ANOVA) and Tukey’s test at a 95% confidence level with MinitabTM software version 16.1.1 (Minitab Inc., USA). The L. fructivorans inactivation was analyzed by three-way ANOVA followed by the Tukey's test (p≤0.05). Statistical analyses were performed using XLSTAT 2019 (Adinsoft, Paris,França).

3. Results and Discussion 3.1. Inactivation of Lactobacillus fructivorans Figure 1 and Table 1 shows the results of L. fructivorans inactivation in HHP-treated açaí pulps. Given the p-value of the F statistic computed in the ANOVA table, and given the significance level of 5%, the information brought by the explanatory variables is significantly better than what a basic mean would bring. Based on the sum of squares, the following variables bring significant information to explain the variability of the dependent variable (log): time,

14

temperature and

the presure*time interaction. Among the explanatory

variables, the temperature variable is the most influential (p<0.05). As can be observed, the tested conditions resulted in a reduction of at least 4.8 log cycles of CFU/mL.. When the temperature was increased from 25 °C to 65 °C, better inactivation results were obtained (p<0.05). For the process conditions (600 MPa/5 min/25 °C and 600 MPa/5 min/65 °C) selected for the storage study, 4.8 and 6.7 log reduction cycle were obtained, respectively. These two conditions were selected using higher pressure and shorter process time, and preliminary studies (enzymatic inactivation) were also analyzed for this choice (Jesus, Leite, & Cristianini, 2018). The microorganism L. fructivorans was considerably sensitive to HHP treatment, resulting in inactivation values above 5 log cycles at a pressure of 400 MPa when processed for 5 min at 65 °C. Until now, there were no studies evaluating the inactivation of L. fructivorans in açaí pulp by HHP treatment.

3.2. Inactivation of Alicyclobacillus acidoterrestris spores Figure 2 shows the inactivation kinetics of Alicyclobacillus acidoterrestris in HHP-treated açaí pulp and the parity between predicted and observed results. The Weibull model was the best mathematical model for describe the inactivation profile of AAT by HHP treatment, and was generated with the GInaFIT add-in software version 1.6 for Excel. This model is based on the assumption that the population of cells or spores have different heat resistances and the survival curve is given by a cumulative distribution of lethal events. Kinetic models are commonly used to describe the behavior of a target microorganism throughout the inactivation process.

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In Equation (1), when β < 1, the survival curve is concave upwards, and when β > 1, it is concave downwards. When β = 1, the curve is a straight line on a logarithmic scale. The value of α can be considered a measure of microbial resistance to treatment (kinetic parameter), the higher the value of α, the greater the resistance of the target microorganism. The results found were α = 0.26 min, β = 0.35, and 4 log reduction time or 4D600MPa 65°C of approximately 13.5 min predicted by the model. The R2 value for the model was 0.9888 and the RMSE, 0.2503 (Equation 2). Models with an R2 value closer to 1 and smaller RMSE fit the data statistically better (Daroit, Sant’Anna, & Brandelli, 2011; Rudra, Shivhare, & Basu, 2008). Similarly to our study, Buzrul, Alpas, & Bozoglu (2005) also used the Weibull model to describe the inactivation of AAT by HHP treatment (450 MPa/35 °C, 45 °C, or 50 °C) and obtained an RMSE value of 0.31 and an R2 of 0.96. The β value (0.35) found in our study was <1, thus, the inactivation curve had an upward concavity, indicating what some authors call the tailing phenomenon. This shows that a fraction of the AAT spores was resistant to HHP inactivation. In a work by Evelyn & Silva (2015), evaluating HHP-treated strawberry puree, good inactivation rates of Byssochlamys nivea ascospores were achieved with β values between 0.46 and 0.66 (<1), also with an upward concavity of the survival curve. These results are in agreement with other studies on different microorganisms that reported non-linear inactivation curves and showed that the Weibull model was able to predict the inactivation results (Evelyn & Silva, 2015; Serment-Moreno, Barbosa-Cánovas, Torres, & WeltiChanes, 2014; Van Boekel, 2009; Wang et al., 2009).

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The Weibull model also best described the inactivation of Neosartorya fischeri ascospores in apple juice by HHP treatment (600 MPa) combined with temperatures of 50–75 °C (Evelyn & Silva, 2016). Based on the microbial inactivation results, two conditions were chosen for analyses during refrigerated storage (600 MPa/5 min/25 °C and 600 MPa/5 min/65 °C). Inactivation of AAT at 600 MPa/5 min/25 °C resulted in only 1.2 log spore reduction, whereas processing at 600 MPa/5 min/65 °C resulted in 2.72 log reduction. These results suggest that it is necessary to increase temperature to achieve better rates of AAT spore inactivation when using HHP treatment. These data are in agreement with those reported by Silva et al. (2012) in orange juice. The authors were able to increase the rates of spore inactivation from 1 log cycle to 2 log cycles when temperature was increased from 45 to 65 °C and pressure from 200 to 600 MPa while fixing the process time at 10 min. In relation to thermal resistance, studies reported a D95°C of 1.5– 8.7 min for heat-treated endospores of different A. acidoterrestris strains in fruit juices (Eiroa, Junqueira, & Schmidt, 1999; Evelyn & Silva, 2016; Silva & Gibbs, 2001). The mechanism of spore inactivation by HHP treatment is not completely understood; however, some studies report that spores germinate under certain temperature/pressure conditions, losing their resistance, thus being readily inactivated by HHP. At high pressures (400–800 MPa), spore germination is induced, accompanied by the release of dipicolinic acid (DPA) and calcium (Rendueles et al., 2011; Wuytack, Boven, & Michiels, 1998). According to Black et al. (2007), the release of Ca-DPA leads to lysis of the cell cortex, possibly due to effects on DPA channels of the inner membrane or the spore membrane.

17

After germination, spores are much more sensitive to agents such as heat, pH, and pressure in comparison to their vegetative state (Zhang & Mittal, 2008). To date, no studies have been published on AAT inactivation by HHP treatment in açaí pulp. In a related study conducted by Pavan (2010) on açaí juice, the author detected guaiacol and suggested that deterioration may have occurred in the juice or in the fruit prior to processing because high temperature and relative humidity of the harvest/production area favor microbial growth. In addition, AAT spores are resistant to pasteurization and can grow over a wide range of temperature, producing off-flavors (Perez-Cacho, Mahattanatawee, Smoot, & Rouseff, 2007). HHP treatment (600 MPa/5 min/25°C) reduced spore count to less than 5 log CFU/mL (4.93 CFU/mL). These spore concentrations naturally occur in juices/pulps with no signs of deterioration, as AAT starts to produce guaiacol, a compound that causes off-flavors in deteriorating products, when present in concentrations above 5 log CFU/mL (Gocmen, Elston, Williams, Parish, & Rouseff, 2005) indicating that the results of our study are promising. Prior to the tests, the vacuum-packed açaí pulp was sterilized (121 °C/15 min) in an autoclave to inactivate the natural microbiota, however, this preparation was only used in microbiological inactivation studies to ensure that the final count of the microorganisms was only the inoculated microbiota. For the analysis of bioactive compounds these conditions were not used.

3.3. Microbial count during refrigerated storage Analysis of total coliforms, thermotolerant coliforms, and salmonella was carried out during the refrigerated storage period of açaí pulp treated by the

18

most effective studied conditions (600 MPa/5 min/25 °C and 600 MPa/5 min/65 °C). Table 2 shows the results of microbial counts of açaí pulps stored under refrigeration. The control (untreated) sample had a high count (above 4.74 ± 0.27 log CFU/mL) of aerobic mesophiles, aerobic psychrotrophic, and lactic bacteria. The Brazilian legislation does not define specific limits for these microorganisms in fruit pulps. Quantification is usually performed to verify failures during processing and/or lack of adherence to good manufacturing practices. After HHP treatment (600 MPa/5 min/65 °C), the counts of these three groups of microorganisms remained below 2 log CFU/mL during the 28 days of storage at 5 °C. A complete inactivation of psychrotrophic bacteria, aerobic mesophiles, and molds and yeasts in nectarine puree was reported after treatment at 450 MPa/10 min/10 °C or 600 MPa/5 and 10 min/10 °C (García-Parra et al., 2011). The differences between inactivation rates may be related to the differences between the raw materials used. Resolution RDC no. 12 of January 2001 (Brazil, 2001), which establishes the Technical Regulation of microbiological standards for foods in Brazil, determines the limit of 102/g for thermotolerant coliforms in frozen fruit pulps, whether or not subjected to heat treatment. Even though the limits for total coliforms are not defined by legislation, determination was performed to indicate the hygienic/sanitary conditions of the product, reflecting, in turn, the quality of raw materials. According to the results, none of the analyzed açaí pulp samples exceeded the acceptable limits. The results varied from < 3 to 9.2 MPN/mL for total coliforms (control at day 0) and 100% of the samples were negative for

19

thermotolerant coliforms. Therefore, processed samples and the control sample were in accordance with the microbial limits established by legislation. All samples were negative for Salmonella sp., showing that the analyzed açaí pulp was in accordance with current legal requirements for this microorganism. Normative Instruction no. 01 of January 2000, of the Ministry of Agriculture, Livestock, and Supply (Brazil, 2000), regulates the identity and quality standards of açaí pulps. For molds and yeasts, non-pasteurized pulps, whether frozen or not, cannot exceed 5.0 x 103 CFU/g (3.69 log CFU/mL or g). Chemically preserved and/or heat-treated açaí pulps cannot exceed the limit of 2.0 x 103 CFU/g (3.30 log CFU/mL or g). According to this parameter, all samples, treated and control, met the legal requirements. From the second week of refrigerated storage onwards, the control pulp was considered unfit for consumption for having counts of yeasts and molds of 5.74 ± 1.05 log CFU/mL. For HHP-treated and pasteurized (85 °C/1 min) samples, counts remained below the limits established by legislation during the 28 days of storage. The control sample had a high initial count of LAB (4.74 ± 0.27 log CFU/mL), suggesting that the raw material could be undergoing fermentation, which was intensified with time. This was evidenced by the fruity fermented aroma that exuded from the plates during the analyses. The change in microbial counts of control sample (decreased in the first 7 days but bounced back later) may be justified because lactic bacteria can ferment the açaí and lower the pH which may have inhibited the growth of bacteria (non-acid tolerant) in the first week. By the 14th week the pulps began to show a fruity aroma and the presence of different colonies. The plates were

20

taken for analysis and the presence of a contaminant the fungus Geotrichum candidum (contaminant of fruit processing plants) was verified. This may have increased psychrotrophic counts from the 14th week.

3.4. Analysis of functional compounds In the present study, the extracts used for quantification of functional compounds were prepared using water as solvent. Consequently, the levels of anthocyanins and total phenolics reported herein may not be as high as those reported in the literature; the main goal of their determination was to allow a comparison between control, thermally pasteurized, and HHP-treated açaí pulps. Organic solvents such as ethanol, methanol, ethyl acetate, and their mixtures with water have been widely used for extraction of functional compounds, with ethanol and water being preferred due to their low toxicity and high yields (Franco et al., 2008). The main disadvantage of conventional solvents is the low yield of extraction of antioxidants with low polarity/high liposolubility, e.g., carotenoids (Franco et al., 2008). The solubility of polyphenols depends mainly on the number of hydroxyl groups, molecular size, and length of hydrocarbon chain (Franco et al., 2008). Several extraction methods have been developed to increase extraction yields of lowpolarity/highly liposoluble compounds, such as ultrasound-assisted extraction, supercritical fluid extraction, microwave-assisted extraction, and high-pressure extraction (Poejo, 2011).

3.4.1. Anthocyanins

21

As can be seen in Figure 3, the content of monomeric anthocyanins found in the control sample was 25.74 ± 0.78 mg C3G/100 g of pulp. These data are in accordance with those obtained by Constant (2003), who analyzed the total content of anthocyanins in açaí berries and pulps and found values of 127.86 mg/100 g and 27.0 mg/100 g, respectively, with a predominance of cyanidin-3-glucoside and cyanidin-3-rutinoside. Rogez (2000) reported anthocyanin levels ranging from 34–702 mg/100 g of type B açaí (11 to 14% of total solids). Rufino et al. (2010) found an anthocyanin content of 111.0 mg/100 g wet weight (expressed as cyanidin-3glucoside

or

malvidin-3-glucoside).

However,

the

authors

used

a

methanol/water/acetone mixture for extraction. According to Constant (2003), the amounts of anthocyanins reported in açaí are quite divergent in scientific literature, mainly due to the use of the same terminology when referring to açaí fruit and drink. The use of different methods of extraction and quantification also contributes to the considerable divergence in results, as does the wide variability of raw material, caused by seasonality and region of production. Data presented in Figure 3 shows that the amount of monomeric anthocyanins in pulps treated at 600 MPa/25 °C and 65 °C increased (p < 0.05) by 37.6%±0.1 and 36.5%±0.3, respectively. Although HHP treatment of açaí pulp is not able to completely inactivate enzymes (PPO and POD) that catalyze the oxidation of phenolic compounds (article submitted for publication), we observed that there was an increase in the extraction of anthocyanins. According to Ferrari, Maresca, & Ciccarone (2010), anthocyanins of different liquid foods (red fruit juices) are stable to HHP treatment at moderate temperatures.

22

After thermal treatment (85 °C/1 min), anthocyanin content was reduced (p < 0.05) by 16.3%. This reduction can be explained by the deleterious effect of temperature on anthocyanins during food processing and/or storage. The effect of thermal processing on açaí pulp has been determined by studies that evaluated pre-packaged açaí treated at 90 ºC/10 min or 100 ºC/5 min (Sousa et al., 2006) and with açaí packaged after HTST processing (82.5 °C/60 s) (Rogez, 2000). Under these conditions, thermal processing was able to guarantee adequate microbiological inactivation; however, an intense reduction in anthocyanin concentration and sensorial alteration, with separation of lipid phase from pulp and color change, were observed with the most intense process condition (100 °C/5 min). Regarding anthocyanin content after HHP treatment, the results found in this study were much higher than those found by Barba, Esteve, & Frígola (2012) in HHP-treated blueberry juice (400 MPa/15 min/25 °C). The authors achieved an increase of only 16% in the extraction of anthocyanins. There is still a lack of studies that focus on the impacts of HHP treatment on the functional properties of foods. In a study by Liu et al. (2016) in Lonicera caerulea berry (a native Siberian fruit), the anthocyanin content also increased by 5.80 and 6.84% when the fruits were submitted to HHP treatment at 200 MPa/5 and 10 min, respectively. The authors concluded that HHP treatment made anthocyanins more accessible for extraction. In general, studies report an increase in anthocyanin levels after HHP treatment, attributed to the higher extractability of compounds in treated foods. Integrity of the food matrix, intensity and time under pressurized conditions, and

23

temperature are aspects that must be observed in studies evaluating HHPtreated foods rich in anthocyanins.

3.4.2. Total phenolic compounds Data on total phenolic compounds are shown in Figure 4. The amount of total phenolics found in the control sample was 230.75 ± 4.39 mg GAE/100 g of pulp. These values are below those found by Rufino et al. (2010) (454.0 mg GAE/100 g). Such differences can be explained by the extraction solvent used by the authors, methanol/acetone/water, which probably increased the yield of extraction. As described in some studies, different hydroalcoholic, alcoholic, and organic extracts lead to different results (Giada & Mancini-Filho, 2009; Vidal et al., 2009).The differences can be also explained by other possible reasons from the intrinsic characteristics of açaí berries: variety, harvest year and location. Figure 4 shows that, for total phenolics, both processes at 600 MPa/25 °C and 65 °C increased the content of phenolic compounds in açaí pulp by 11.44% and 10.25%, respectively. These results are in agreement to those of anthocyanins, the principal phenolics in açaí, i.e., HHP-treated samples had a higher extraction of anthocyanins as well as total phenolics. For the pasteurized sample, values were lower for both anthocyanin and phenolics determinations. Some studies report that HHP treatment increases the rate of dissolution of bioactive compounds. Under high pressure, rapid solvent permeation occurs due to the large differential pressure between the inside and outside of the cell membrane (Zhang & Wang, 2005). This phenomenon increases solvent penetration through broken cell membranes or increases the rate of mass transfer due to increased membrane permeability. Therefore, as hydrostatic

24

pressure is increased, higher amounts of solvent enters the cell and higher amounts of compounds can pass through the cell membrane, leading to increased extraction yield (Shouqin, Jun, & Changzheng, 2005). In other words, the extraction capacity of phenolic constituents can be increased by HHP treatment, and pressurized samples yield higher levels of bioactive compounds. Studies on the effects of high-pressure processing on total phenols demonstrated that these compounds were either not affected or, more frequently, increased in concentration and/or extractability after high-pressure treatment (Corrales, Toepfl, Butz, Knorr, & Tauscher, 2008; Prasad, Yang, Zhao, Ruenroengklin, & Jiang, 2009; Shouqin et al., 2005; Tokuşoǧ lu, Alpas, & Bozoǧ lu, 2010). Several authors have reported higher concentrations and stability of bioactive compounds when fruits were submitted to HHP treatment. Barba, Cortés, Esteve, & Frígola (2012) compared the effects of HHP treatment (100– 400 MPa/2–9 min/20–42 °C) and thermal processing (90 °C/15 or 21 s and 98 °C/15 or 21 s) on total phenolics of orange juice. The authors observed that the levels of total phenolics were higher in HHP-treated samples, reaching a maximum increase of 22% at 100 MPa/7 min, whereas thermally processed samples increased by 8–17% in comparison to raw samples. In our study, the increase in the content of phenolic compounds may be related to the higher extraction of some antioxidant components by HHP treatment (Barba, Cortés, et al., 2012; Carbonell-Capella, Barba, Esteve, & Frígola, 2013), which promotes the decompartmentalization of the plant cell matrix where the compounds are housed (Butz & Tauscher, 2002).

3.4.3. Antioxidant activity 25

As can be seen in Figure 5, the antioxidant capacity of the control sample by FRAP and ORAC assays was 31.46 ± 0.29 μmol TE/g and 46.71 ± 3.80 μmol TE/g, respectively. By analyzing both methods, it can be observed that HHP-processed samples had no increase in antioxidant capacity in relation to the control sample (p > 0.05). In the ORAC assay, the sample processed at 600 MPa/5 min/65 °C presented higher activity (49.72 ± 0.99 μmol TE/g) than that of pasteurized sample (43.45 ± 0.55 μmol TE/g). According to Ali, Almagribi, & Al-Rashidi (2016), anthocyanins are good hydrogen donors. As the ORAC assay is based on donation of hydrogen atoms to neutralize radical species, this may explain the higher antioxidant capacity of this sample. Several studies using ORAC and FRAP assays have reported an increase in antioxidant capacity of HHP-treated fruit

products

when

compared

to

thermally

treated

fruit

products

(Apichartsrangkoon, Chattong, & Chunthanom, 2012; Chen et al., 2013, 2015; Hernández-Carrión, Vázquez-Gutiérrez, Hernando, & Quiles, 2014; Tadapaneni et al., 2012). The pasteurized sample (85 °C/1 min) had the lowest antioxidant capacity (26.60 ± 0.54 μmol TE/g) in comparison to the control and pressurized samples only in the FRAP assay, with a reduction of 15.4% in relation to the control. This reduced antioxidant capacity can be justified by the reduction in anthocyanins content after heat treatment, as temperature has a deleterious effect on these compounds. The antioxidant activity of many fruits are derived from the combined synergistic action of many molecules, including phenolic compounds, carotenoids, and vitamins C and E. However, in fruits such as açaí, which contain relatively small amounts of vitamins, phenolic acids, and

26

flavonols, anthocyanins are the main compounds responsible for the antioxidant capacity (Rufino et al., 2010). The fact that the antioxidant capacity of HHP-treated samples was higher only when evaluated by FRAP assay can be explained by the method’s mechanism. Comparing FRAP and ORAC results is difficult due to the complexity and different principles of the reactions involved (Paz et al., 2015). Some antioxidant methods produce different or even contradictory results, being impossible to compare them sometimes (Alonso et al., 2002). Differences may also be due to the presence of other reducing compounds, such as sugars and tocopherols, which are known to interfere with antioxidant capacity tests (Rezaire et al., 2014). The results of the present work suggest that HHP treatment can be considered an alternative conservation method for açaí pulp because, in comparison to thermal treatment, it is able to inactivate microorganisms while preserving the antioxidant capacity of the pulp.

4. Conclusions HHP treatment showed to be effective at inactivating L. fructivorans, and the Weibull model was able to describe the profile of inactivation of Alicyclobacillus acidoterrestris spores, however, the inactivation was obtained by a combination of pressure (600 MPa) and moderate temperature (65ºC).The data presented in this study may provide important contributions to the pulp/fruit juice industry and to industries that use açaí pulp as a raw material, considering the trend towards the development of mixed fruit and vegetable beverages. Knowledge on the prevalence of these microorganisms in fruit matrices can be

27

considered the first step towards methods that guarantee microbial stability during shelf life. Concerning bioactive compounds, the extraction of anthocyanins increased 37% on average for HHP-processed açaí pulps. In contrast, thermal processing reduced this pigment content by 16.3% when compared to the control sample, indicating that the application of HHP process to açaí pulps can be a promising method to increase the extraction of anthocyanins and preserve the antioxidant capacity of the fruit. Thus, it can be concluded that HHP processing could be considered an alternative to thermal pasteurization, generating

a microbiologically

safe

product

with

preserved

nutritional

characteristics in relation to anthocyanins and total phenolics. Further studies are needed to assess whether HHP treatment and subsequent refrigerated storage preserve the levels of phenolic compounds and antioxidant activity of açaí pulps in a more effective manner than thermal pasteurization.

Acknowledgments The authors would like to thank the CAPES (Coordination for Supporting and Development Superior Education Personnel) and FAPESP (São Paulo Research Foundation) for the financial support (project 2015/01570-0).

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42

a

8 7

log (N)

6 5 4 b

3

b

b

b

b

b c

2

de de

1

e

de

cd

0

Figure 1 - Effect of the HHP on the inactivation of Lactobacillus fructivorans CCT 0850 in açaí pulp. Different letters in the bars represent a significant difference (p <0.05). N = concentration of surviving microorganisms (CFU/mL).

7 6

log (N)

5 Measured Identified Predicted

4 3 2 1 0 0

5

10

15

20

25

30

Time (min) Figure 2 - Survival curve of Alicyclobacillus acidoterrestris spores in açaí pulp at 65 °C under high pressure (600 MPa). N = concentration of surviving microorganisms (CFU/mL).

43

mg C3G / 100 g

40 35 30 25 20 15 10 5 0

a

a

Control b

85 °C/1 min c

600 MPa/5 min/25 °C 600 MPa/5 min/65 °C

Figure 3 - Effect of HHP on the content of monomeric anthocyanins in açaí pulp 24 hours after treatment.

a,b,c

Means followed by the same letter are not

significantly different (p > 0.05).

300

a b

mg GAE / 100 g

250

a

c Control

200

85 °C/1 min

150

600 MPa/5 min/25 °C 100

600 MPa/5 min/65 °C

50 0

Figure 4 - Effect of HHP on the content of total phenolics in açaí pulp 24 hours after processing.

a,b,c

Means followed by the same letter are not significantly

different (p > 0.05).

44

FRAP 35

31.3 a

30.5 a60

26.6 b

50

25

µmol TE/g

µmol TE / g

30

31.3 a

ORAC

20 15 10

46.7 ab

47.2 ab 43.4 b

40 30 20 10

5

0

0 Control

85 °C/1 min

600 MPa/5 min/25 °C

600 Control MPa/5 min/65 85 °C/1 °C min

600 MPa/ 25 °C/5 min

Figure 5 - Antioxidant capacity of açaí pulp by FRAP and ORAC assays after processing.

49.7 a

a,b

Means followed by the same letters are not significantly different

(p > 0.05).

45

600 MPa/ 65 °C/5

Table 1: Analysis of Variance (ANOVA) for the inactivation of Lactobacillus fructivorans. Degree of Freedom

Source

Sum of squares

Mean squares

F value

P value

Model

10

113.438

11.344

182.469

< 0.0001

Presure (MPa)

2

0.331

0.166

2.665

0.087

Time(min)

1

0.286

0.286

4.604

0.041

Temperature (°C)

1

22.769

22.769

36.244

< 0.0001

Presure (MPa)*Time(min)

2

0.902

0.451

7.251

0.003

Presure (MPa)*Temperature (°C)

2

0.228

0.114

1.835

0.178

Time(min)*Temperature (°C)

1

0.112

0.112

1.805

0.190

Error

28

1.741

0.062

Corrected Total

38

115.179

R2= 0,985.

Table 2 - Average microbial counts of açaí pulps stored for 28 days at 5 °C. Sample

Day

Psychrotrophic

Mesophilic

Yeasts and

Lactic

Salmonella

of

aerobes

aerobes

molds

bacteria

sp.

storage

(log CFU/mL)

(log CFU/mL)

(log CFU/mL)

(log CFU/mL)

5.21 ±

0.36a

2.83 ±

0.49b

14

5.17 ±

0.34a

21

5.68 ± 0.06a

5.55 ± 0.39ab

6.65 ± 0.08a

7.35 ± 016b

-

28

6.30 ± 0.79a

6.25 ± 0.23a

7.25 ± 0.30a

8.51 ± 0.08a

Absent

0

< 1.00 ± 0.00*c

2.66 ± 0.06b

< 1.00 ± 0.00*b

3.19 ± 0.98a

Absent

7

0.00*c

2.36 ±

0.18b

< 1.00 ±

0.00*b

2.39 ±

0.24a

-

2.33 ±

0.29b

< 1.00 ±

0.00*b

2.34 ±

0.34a

-

3.34 ±

0.10a

3.49 ±

0.23a

-

2.55 ±

0.04b

2.53 ±

0.02a

Absent

2.82 ±

0.08b

2.84 ±

0.01b

Absent

2.72 ±

0.18b

2.77 ±

0.00b

-

2.71 ±

0.02b

2.82 ±

0.09b

-

0 7 Control

85 °C/1 min

14 21 28 0 7

600 MPa/5 min/25 °C

< 1.00 ± 2.00 ±

0.43b

3.13 ±

0.25a

< 1.00 ±

0.00*c

< 1.00 ±

0.00*b

< 1.00 ±

0.00b

1.63ab

4.98 ±

0.40b

3.00 ±

0.00c

4.56 ±

0.16b

3.15 ±

0.21b

4.74 ± 0.27c

2.70 ±

0.00b

5.27 ±

0.50c

-

5.74 ±

1.05a

6.63 ±

0.02b

-

1.15 ±

0.21a

< 1.00 ±

0.00*b

< 1.00 ±

0.00*b

< 1.00 ±

0.00*b

< 1.00 ±

0.00*b

Absent

14

1.15 ±

21

3.77 ± 0.21a

4.18 ± 0.13a

1.76 ± 0.08a

4.14 ± 0.06a

-

28

2.24 ± 0.09ab

2.77 ± 0.02b

< 1.00 ± 0.00*b

2.69 ± 0.41b

Absent

46

600 MPa/5 min/65 °C

0

< 1.00 ± 0.00*a

1.00 ± 0.00*a

< 1.00 ± 0.00*a

1.44 ± 0.37a

Absent

7

< 1.00 ± 0.00*a

1.25 ± 0.32a

< 1.00 ± 0.00*a

1.85 ± 0.21a

-

14

0.00*a

1.35 ±

0.07a

< 1.00 ±

0.00*a

0.95 ±

0.07a

-

1.70 ±

0.99a

< 1.00 ±

0.00*a

1.81 ±

1.14a

-

1.15 ±

0.21a

< 1.00 ±

0.00*a

1.35 ±

0.49a

Absent

21 28

< 1.00 ± 1.80 ±

1.56a

1.85 ±

1.10a

Values expressed as mean ± standard deviation. a,b,c Means

in the same column followed by the same letters within each process

condition are not significantly different (p > 0.05). * Values below the detection limit of the method (1.0 log CFU/mL).

47

CRediT author statement Ana Laura Tibério de Jesus: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Visualization. Marcelo Cristianini: Conceptualization, administration, Funding acquisition.

Supervision,

Resources,

Project

Nathalia Medina dos Santos: Methodology, Validation, Formal analysis. Mário Roberto Maróstica Júnior: Supervision, Resources.

Highlights



The HHP may be an effective alternative to thermal pasteurization of açaí pulp.



The HHP- treatment of açaí pulps preserved their antioxidant activity.



The HHP increases extraction of anthocyanins and phenolics in açaí pulp.



Weibull's model described the inactivation of A. acidoterrestris spores in açaí.

48

High Hydrostatic Pressure

Açaí pulp Açaí pulp

3.2°Brix Açaí pulp

Analyses: Packaging

Thermal processing:

   

Microbiological Anthocyanins Total phenolic compounds Antioxidant activity (ORAC, FRAP)

Survival curve of Alicyclobacillus acidoterrestris spores after HHP treatment

Anthocyanins

Antioxidant activity