Optimization of Production Process of Alcohol Vinegar Macerated With Acai (Euterpe Precatoria) Using RSM to Add Colour and Functional Compounds to Vinegar

The vinegar is one of the food products elaborated worldwide. It can be produced from a variety of different raw materials which, together with the acetification system and, in some cases, the time it is aged in wood, influence its chemical and organoleptic properties [1]. In Brazil, the alcohol vinegar is the cheapest vinegar found in the market. Regarding the production of alcohol vinegar, sugar cane alcohol is used by the vast majority of Brazilian vinegar manufacturers due to economic reasons, although it is considered to have the lowest nutritional quality [2]. 

Nowadays, the dietary concerns have moved to foods that offer long-term prevention of non-communicable diseases and, therefore, research around such product development has been performed to deliver the bioactive compounds in finished foods [3]. Through the mapping of scientific studies, it was reported that research studies have sought a diversification of acetic fermentation products and the use of different raw materials and fermentation processes to obtain products with greater added value and chemical and sensory quality. Although grapes and apples are fruits that are traditionally used in the process of making acetic fermentation products, exotic/native fruits have increasingly gained visibility [4,5]. 

The acai (Euterpe precatoria Mart.) palm is a native tree of the Amazon forest. Brazil is the main producer, consumer and exporter of acai. Both fruit and pulp are highly perishable, requiring a conservation process immediately after their extraction. It is traditionally consumed in Brazil and has gained popularity abroad due to its nutritional value and functional properties, being classified as a “superfruit”. The superfruits are deemed “super’’ by food scientists as they are packed with extremely high levels of antioxidants, fibers, vitamins, minerals, and other nutrients that improve health [6]. There is an overwhelming literature related to the acai phytochemical composition and potential health benefits such as antioxidant, anti-inflammatory, anti-proliferative and cardio protective activities [6-8]. The amount of anthocyanins in acai is so high that this fruit has been used to obtain isolated standards of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside [9]. The anthocyanins belong to the class of flavonoids, compounds responsible for the natural colour of innumerous fruits, vegetables, and flowers such as of the shiny orange, pink, red, violet, and blue colours. These pigments besides being nontoxic and nonmutagenic also have positive biological activities such as antioxidant, antiinflammatory, anticarcinogenic, antiviral, and antibacterial properties. In the food industry, there is an enormous potential to substitute synthetic colourants by the natural anthocyanins through their incorporation into a variety of food matrices [10,11]. 

In the interior of the Amazonas state, there is a great demand for technological alternatives to add value to regional and native superfruits and a tremendous potential for the development of new products and innovation. The acetic fermentation is a relatively efficient process of preservation, increases the shelf life of a food product and reduces the need for refrigeration or other form of preservation technology. Therefore, this process could be considered an important contribution to the livelihoods of rural and peri-urban dwellers of Amazon region, through enhanced food security and income generation via a valuable enterprise option. This rationale leads to start the research to evaluate the maceration of acai in nature into the alcohol vinegar produced by a local manufacturer in other to add colour, phytochemicals and functional properties of acai to alcohol vinegar and to elaborate an innovative food product from an Amazon superfruit. 

In our previous works, James et al. [12], determined total dry extract content, ashes content and colour parameters of alcohol vinegars macerated with acai using different concentrations of acai, 10% (proportion alcohol: vinegar 9:1) and 50% (proportion 1:1) and maceration times (7, 14, and 21 days) and Boeira et al. [13], determined the colour parameters, antioxidant capacity measured by DPPH and ABTS assays, total phenolics contents and volatiles compounds of alcohol vinegar macerated with acai using different concentrations of acai, 20% (proportion alcohol: vinegar 8:2) and 50% (proportion 1:1) and the same maceration time (7, 14, and 21 days). In both published papers, it was demonstrated a marked difference in the results depending on the concentration of acai used and the maceration period (7, 14, and 21 days). The most attractive product regarding the parameters studied was obtained from the use of the proportion of 1:1 of alcohol vinegar: acai and maceration period of 14 days. Thus, in this paper, and considering the results obtained in our previous works, it is reported the study for optimization of production process of alcohol vinegar macerated with native acai through an experimental design using response surface methodology and acai concentration, maceration time and temperature as independent variables and colour parameters, anthocyanins and ashes content as dependent variables.

Alcohol Vinegar and Acai Fruit 

The alcohol vinegar employed was produced by a local manufacturer (Virrosas, Manaus, AM) through the Frings rapid submerged method using the alcohol from sugar cane as substrate. The native acai (Euterpe precatoria) fruits were from Anori (03º 46' 22" S and 61º 38' 39" W). The acai in natura was washed with water, sanitized by immersion in 200 ppm sodium hypochlorite solution for 15 min and washed again with water. After, the acai berries were added in the alcohol vinegar for maceration. 

Design of Experiments using Response Surface Methodology 

The Face Centered Design (FCD) was applied to investigate the effect of independent variables, X₁, X₂ and X₃ (Table 1) on response variables (colour, anthocyanins and ashes) determined in the alcohol vinegar macerated with acai. 

Table 1: Levels of the different coded and non-coded independent variables used in the FCD. 

A total of seventeen experiments were derived from FCD through Protimiza Experimental Design software (Campinas, São Paulo, Brazil) using a 2³ full factorial, including 6 centered points and 3 repetitions at the central point (Table 1 - Supplemental Material). 

Maceration of Acai Berries into the Alcohol Vinegar 

The experiments were carried out considering three parameters, the acai concentration (30%, 40% and 50%), the maceration time (8 days, 12 days and 16 days) and the maceration temperature (15°C, 25°C and 35°C) following the experimental design demonstrated in table 1 (Supplemental material). 

All tests were performed in glass Erlenmeyer flasks with screw caps previously sterilized in an autoclave at 121°C for 15 minutes and they were carried out in a BOD incubator (Elo Scientif, Presidente prudente, São Paulo) to maintain the maceration temperature. At the end of the test, the acai berries were removed from the alcohol vinegar, the vinegar was vacuum filtered with earth filtration and it was bottled in a glass bottle and kept at the ambient temperature. 

Physicochemical analysis 

• Instrumental Colour Measurement and Ashes Content 

The colourimetric parameters using the L* a* b* C* h° colour spaces established by the CIE (Commission Internationale de l'Eclairage) were determined using the Delta Vista portable spectrophotometer (Delta Colour, São Leopoldo, RS, Brazil), with illuminant D65 and observer at 10º, after calibration with white, black and green porcelain plate with light trap, coupled to the software i7 (Delta Colour). 

The ashes content was determined by the gravimetric method, which is based on sample incinerated at 550ºC [14]. 

• Determination of Anthocyanins 

The anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, were determined using liquid chromatography coupled with mass spectrometry. The parameter “anthocyanins” corresponds to the combined concentration of these compounds in each sample. A 100μL sample was diluted to 5.00mL or 50.00mL with deionized water acidified with formic acid (0.30%, v/v) to fall within the analytical curve. The samples were filtered through a 0.22 pore-size PTFE filter prior to chromatographic analysis. Analysis was carried out using a UHPLC-MS/MS system model LCMS-8040 (Shimadzu, Kyoto, Japan) equpped with a UV-Vis detector and coupled with a triple quadrupole and an Electrospray Ionization (ESI) source. For the analysis, the mobile phase (A) consisted of deionized water (Milli-Q Gradient System, Millipore Corporation, Massachusetts, USA) with formic acid (3.0%, v/v), while the mobile phase (B) consisted of acetonitrile (HPLC-UV grade). Anthocyanins were separated using a Shimpack XR-ODS III reversed-phase column (150mm long, 2.0mm internal diameter, and 2.2μm particle size) with gradient elution at a flow rate of 0.5mL/min, and an oven temperature of 40°C. The gradient elution program initiated with 10% B for the first 2min, followed by a linear increase to 30% B over 5 min, reaching 90% B by 8min, which was maintained for 2min before reverting to the initial conditions (10% B) by 12min. The system was equilibrated for 3min before the next injection. A 10μL injection volume was used. Standard purity was assessed at 520nm and through total ion chromatogram within the m/z range of 100 to 1000, verifying only a single chromatographic peak with the proper precursor ion for each compound. ESI parameters were optimized as follows: capillary voltage, 4.0 kV; interface temperature, 300°C; desolvation line temperature, 300°C, heating block temperature, 300°C; nebulizer gas, 3L/min, and desolvation gas, 10L/min. Collision gas pressure was mantained at 224kPa, with a collision energy of 35V. Precursor and product ions for each anthocyanin were confirmed in the product ion scan spectrum. Anthocyanin quantification in vinegar samples was achieved through external calibration with analytical standards of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in concentrations ranging from 100 to 1000ng/mL.

Figure 1 shows the alcohol vinegars macerated with acai berries obtained in this study through the use of the RSM. The transfer of compounds from the acai to the alcohol vinegar is clearly visible by the colour of vinegars obtained. In all 17 tests carried out, the alcohol vinegar acquired an attractive colour that resembles the colour of acai, with the exception of the first three tests which showed a more reddish colour.

Figure 1: Alcohol vinegar and alcohol vinegar macerated with acai obtained in the 17 tests (T1 to T17) carried out using the FCD. 

The results obtained for the response variables (colour parameters, anthocyanins and ashes) in the alcohol vinegar used in the experiments and in the 17 alcohol vinegars macerated with acai obtained are shown in table 2. As can be clearly observed, the maceration of acai in the alcohol vinegar promoted huge differences in the results of all analyses performed when compared to the results of alcohol vinegar. 

Table 2: Actual values of the independent variables (not coded) and experimental values obtained for the dependent variables used in the FCD. 

The results of the regression analysis and ANOVA for the significant response variables (α=0.05) after reparameterization of the model are shown in table 3. It can be seen that the variables X₁, X₂ and X₃ significantly influenced at a level of 5% the response obtained for the different dependent variables analyzed and the determination coefficients (R²) for the responses presented a percentage of variation explained by the FCD considered sufficient for the creation of contour curves. 

Table 3: Regression analysis and ANOVA results for significant variables after model reparameterization. 

In the contour curves for the colourimetric parameters, it can be observed that the coordinates L* (Figure 2), a*, b* and C* (Figure 1 - Supplementary Material) followed the same trend in relation to the independent variables and different from the trend obtained for the coordinate h° (Figure 2). 

Figure 2: Contour graphs for the colorimetric parameters L (left) and h° (right) in relation to the independent variables acai concentration (X₁), maceration time (X₂) and maceration temperature (X₃). 

In the tests that were employed the lower acai concentration, lower time and temperature of maceration the alcohol vinegar macerated with acai presented a reddish colour. Through visual observation (Figure 1) it can be easily noticed the reddish colour of vinegars obtained in the tests 1, 2 and 3. The colour of vinegars was significantly affected by almost all independente variables, with exception of the h° coordinate. This coordinate was only significantly affect by the interaction between independente variables X₁.X₂ and X₂.X₃ (Table 3). 

The acai berries are characterized by the presence of high contents of anthocyanins [9,15-17]. Certainly, the attractive colours observed in the alcohol vinegars macerated with acai are due to the transfer of anthocyanins from acai berries to alcohol vinegar through the maceration process. The anthocyanins contents determined in alcohol vinegars macerated with acai are presented in table 4.

Table 4: Determination of cyanidin-3-O-glucoside (mg/100 mL) and cyanidin-3-O-rutinoside (mg/100 mL) in the 17 alcohol vinegars macerated with acai.

Note: * Anthocyanins = cyanidin-3-O-glucoside + cianidina-3-rutinosídeo

** Ratio = cianidina-3-rutinosídeo / cyanidin-3-O-glucoside 

As it was expect and acai characteristic, the concentration of cyanidin-3-O-rutinoside was higher than the concentration of cyanidin-3-O-glucoside, in agreement with data reported in the literature [18,19], and the ratio between them varied from 2.16 to 3.40 (Table 4). The anthocyanins content in the vinegars obtained was significantly affected by all three independent variables (Table 3). The anthocyanins content showed a direct relationship with acai concentration (X₁) and maceration temperature (X₃) and inverse with maceration time (X₂). In figure 3, it can be seen that the highest concentrations of anthocyanins were achieved using acai concentrations of at least 40%, maceration time of a maximum of 12 days and a minimum temperature of 24°C. The highest content of anthocyanins found in this study was 21.59mg/100 mL and obtained in T6 (Table 2), test carried out with 50% acai, 8 days of maceration and temperature of 35°C. The climate in Amazonas is humid tropical and according to statistical data the average annual temperature is 27.4°C, with minimum of 24.0°C and maximum of 32.3°C (INMET). The ambient temperature in Amazonas would be suitable for carrying out the experiments, which would reduce energy costs in the production of alcohol vinegar macerated with acai. Casassa & Harbertson [20], reviewed the maceration effect in red wine production. The diffusion process of anthocyanins results in a peak of extraction within the third to fifth day of maceration, following a drop in concentration. The loss of anthocyanins during the latter stages of maceration has been attributed to a variety of factors, namely, incorporation into polymeric pigments, formation of pyranoanthocyanins, and oxidative cleavage of the heterocyclic C ring leading to direct anthocyanin degradation. In this work the same tendency was observed and the maximum maceration time observed to obtain the highest anthocyanin values was 12 days. After 12 days of maceration, lower values for the anthocyanin content were observed.

Figure 3: Contour graphs for the anthocyanins (left) and ashes (right) content in relation to the independent variables acai concentration (X₁), maceration time (X₂) and maceration temperature (X₃). 

The ashes content of vinegars macerated with acai was significantly influenced by the acai concentration (X₁) and maceration time (X₂), as well as by the interaction between time (X₂) and temperature (X₃) of maceration (Table 3). Maceration time and acai concentration are important variables to obtain higher ashes content in vinegars, that is, the longer the maceration time and the higher acai concentration, the greater the ashes content (Figure 3). Ashes content refers to the amount of inorganic material present in a sample and the increase observed was due to the transfer from acai to vinegar and certainly reflect the inorganic composition found in acai. Acai is a good source of inorganic compounds, such as phosphorus, sodium, zinc, iron, manganese, copper, calcium, magnesium, potassium, chrome and others [17,21].

Considering the experimental conditions employed in this study and the results obtained, it was possible to optimize the production process of alcohol vinegar macerated with acai. The independent variables acai concentration (30 to 40%), maceratime time (8 to 16 days) and temperature (15°C to 35°C) were demonstrated to interfere in the dependent variables (colour, anthocyanins and ashes) employed. It was observed that there is no independent variables that promotes the best results for all dependent variables. Thus, if the anthocyanin parameter is prioritized, the conditions that favored obtaining the highest concentrations of anthocyanins in alcohol vinegar macerated with acai corresponded to a minimum acai concentration of 40%, maceration time of a maximum of 12 days and a minimum temperature of 24°C. The alcohol vinegar, which is usually known as poor in chemical composition and is the cheapest to manufacture in Brazil, was transformed into a product with an attractive colour and with the presence of functional compounds through a very simple operation of maceration with in nature acai berries, a fruit recognized worldwide as a superfruit.

To the Virrosas industry (Manaus, Brazil) for providing the alcohol vinegar.

This work was supported by the PADCIT of the Institute Federal of Education, Science and Technology of Amazonas - IFAM; the Scientific Initiation Program of the Amazonas State Research Support Foundation -FAPEAM.

Lúcia S. Boeira - Project administration, funding acquisition, conceptualization, methodology, writing original draft, writing review and editing

André H. M. Oliveira - Investigation, formal analysis, writing original draft

Sandra V. Cad - Formal analysis, writing review

Giovana A. Bataglion - Formal analysis, resources, writing review.

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Table 1: Experiments performed with coded and non-coded variables derived from FCD.

Figure 1: Contour graphs for the colorimetric parameters a*, b* and C* in relation to the independent variables acai concentration (X₁), maceration time (X₂) and maceration temperature (X₃).