Materials

In the design of the preparation, the following ingredients were used: sweet potato (Ipomoea batatas L.), zucchini (Cucurbita pepo L.), dried tomatoes, eggs, powdered milk, cornstarch and Danbo cheese. All the ingredients were bought at the local market. The vegetables were selected considering their freshness, firmness and absence of microbial or mechanical damage. They were sanitized with 0.1% sodium hypochlorite (1 mL/L) and chopped. Sweet potato slices 3 mm thick and 50 mm in diameter were vacuum impregnated (VI) with polyphenols following the technique proposed by Abalos et al.¹⁶. The polyphenol solution used was Vitisol® from the national company Nialtec S.A. This polyphenol extract contains 95% oligomeric proanthocyanidins. The formulation of the 3.5% VI solution was made based on the study carried out by Rózek et al.¹⁷. For the VI experiments, the Gastrovac® vacuum cooking equipment was used. The sweet potatoes were immersed in the VI solution in the equipment and subjected to vacuum for 25 minutes at 20 °C. Pressure was restored at the same time intervals as that of the LV.

Millefeuille preparation

To prepare the millefeuille, the following proportion of ingredients was used: sweet potatoes vacuum impregnated with polyphenols (35%) and Zucchini (24%) in slices 3 mm in thickness and 40 mm in diameter, dried tomatoes (11%), Danbo cheese (21%), and a binder -milk, cornstarch, and egg- (9%). For the assembly, the ingredients were interspersed in layers (Figure 2), resulting in 285 g servings of millefeuille. The preparation was vacuum packed (VACPACK, ICC, 80016™, Spain) in polyamide-polyethylene bags (O2 permeability, 25 to 30 cm³/m²/day; water vapor permeability of 5 g/m²/day). The heat treatment was by sous vide cooking in a constant water-circulation bath with temperature and time regulation (RONER COMPACT, ICC, 80060, Spain) at 80 °C for 45 minutes. It was subsequently rapidly cooled in a water bath at 0 °C for 5 minutes. The cooking temperature was monitored with a thermocouple (HANNA Instruments, HY 93530N, Italy). The cooked samples were refrigerated at 2 °C, until further study.

Figure 2. Ready-to-eat vegetable-based dish enriched with polyphenols preparation. 

Physicochemical characterization

The ready-to-eat millefeuille with the addition of polyphenols was characterized physicochemically, in triplicate, as follows, the compositional properties of the SRB were determined according to AOAC (Association of Official Analytical Chemist)¹⁸. Moisture content was determined after heating in an infrared drying oven (RADWAG, Mag. 50/WH, Poland) according to 7.003-8421. Protein was estimated by the Kjeldahl method described in AOAC 928.08, with a conversion factor of 6.25 for protein. Total fat was determined by the Soxhlet solvent extraction gravimetric method, following method official AOAC 965.33. Total carbohydrates were estimated by subtraction of the percentage of protein, lipid, ash from the total dry weight.

For polyphenols, total phenol content was determined by the reagent Folin-Ciocalteu method, using the test Heimer, Vignolini, Dini and Romani¹⁹. Samples of sweet potatoes of 16 g were processed in a blender with 50 mL of methanol (Sintorgan, Lot. Nº 38931). The homogenates obtained were placed in a beaker and heated at 40 °C with continuous stirring (Barnstead Thermoline) for 15 minutes before centrifugation (Rolco, CM 2036.4, Argentina) for 5 minutes at 11.200 g force and filtration. A 0.5-mL aliquot was added to a test tube together with 1 mL of Folin-Ciocalteau reagent (Biokar) and 10 mL of distilled water, left to stand for 2 minutes. Then, 4 mL of a 20% (w/v) sodium carbonate solution (Merck, Argentina) was added, brought up to a final volume of 25 mL, and heated at 50 °C for 5 minutes in a thermostatic bath (Vicking SRL, Masson, Argentina). The optical density was finally measured in a spectrophotometer (Jenway Mod. 6505 UV/VIS™) at 765 nm¹⁶.

The pH was measured using a pH-meter with a combined Ag/AgCl electrode (ORION, S.A. 720, United States). The pH measurement was performed by immersing the electrode in a homogenate of 10 g of millefeuille in 100 mL of distilled water. Soluble solids (°Bx) were estimated using a table refractometer (A. KrussOptronic, Germany) according to AOAC 932.1221. Acidity was determined by the volumetric method. Water activity was measured with a hygrometer (Rotronic, Hygrolab C1, Switzerland) according to AOAC method 978.18.

Sensory evaluation

To determine the consumer acceptability of the product, the smell, color, texture, and taste parameters were evaluated. The millefeuille, cooked the day before tasting and kept refrigerated, was heated to the usual temperature for consumption (65 °C), cut into 5 cm/5 cm portions, and served on white plates. The sensory evaluation was carried out by a panel of 105 people, selected for being regular consumers of ready-to-eat products, in the laboratory facilities of the Faculty of Bromatology, following the ISO 8589 standard²⁰. Consumers were asked to indicate their level of appreciation for the sensory attributes and the overall acceptability of the sample on a 9-point hedonic scale ranging from 1 = "I don’t like it very much" to 9 = "I like it very much." The study was approved by an Institutional review board of the National University of Entre Ríos (RESOLUTION "C.D." Nº 017/17).

Evolution of physicochemical and microbiological parameters during refrigerated storage

Twelve servings of sous vide cooked millefeuille were prepared and stored in a refrigerator (2 °C). The temperature was monitored throughout the storage period with a digital thermometer (Elitech, D-T1, China). The samples were analyzed physicochemically and microbiologically at different times (0, 7, 14, and 21 days) by the determinations reported below.

Physicochemical analysis: Polyphenols and pH were determined following the techniques previously mentioned. Weight variation: samples were weighed using a precision balance (OHAUS, ADVENTURES, Canada), and the weight variation (ΔP) was calculated as the percentage of variation between the weight of the samples on days 7, 14, and 21 with respect to the weight of samples on day 0.

Color was determined using a colorimeter (HUNTER ASSOCIATES LABORATORY, Inc., MiniScan EZ Model, EE UU) in the CIE L* a* b* color space. The variation was evaluated in the parameters L* (black 0, white 100), a* (red-green), and b* (yellow-blue). The color parameters were determined from triplicate measurements in each sample. Color intensity C*ab (Equation 1) and color change ∆E (Equation 2) were calculated, where L0, a0, and b0 represent the readings of the non-impregnated samples.

Texture was determined by a compression test using a texturometer (Instron 3342™, Massachusetts, USA). The values obtained for the maximum compression force were expressed in Newtons (N). The tests were carried out in quintuplicate, with 30% deformation and at a test speed of 0.5 mm/s. The firmness (strength) of the material was calculated from the relationship between the maximum force (N) and the maximum deformation (mm). The elastic modulus was calculated from the relationship between the maximum force (N) and the initial module (mm).

Microbiological analysis: To perform the microbiological analysis of the millefeuille, a 10-g sample was weighed in a sterile bag and diluted with 90 mL of buffered peptone water (Acumedia. Neogen Corporation, Michigan, USA). A 1/10 dilution was obtained after homogenization. Counts were done in duplicate.

To estimate molds and yeasts, a culture medium composed of oxytetracycline, gentamicin, yeast extract, and glucose (OGY) (Merck KGaA, Germany) was used. The inoculated plates were incubated at 25 °C for 5 days, according to ISO 17410²¹.

To determine total aerobic psychrotrophic bacteria, the Plate Count Agar (PCA) culture medium (Britania, Argentina) was used. The inoculated plates were incubated for 7 days in a cold room at 4 °C.

Determination of total anaerobic psychrotrophic bacteria was carried out in Tryptone Soya Agar (TSA) medium (Oxoid, Basingstoke, United Kingdom). The inoculated plates were placed in an anaerobic jar with a capacity of 2.5 L and incubated in a cold room at 4 °C for 7 days according to ISO 21527-2²².

At the end of the incubation period, agar plates with dilutions that allowed the best Colony Forming Unit (CFU) counts were selected. The counts were expressed as log CFU/g of product for the different groups of microorganisms analyzed.

Statistical analysis

The data of the test results are expressed as means ± standard deviations, as computed by the software XLSTAT (New York, USA) program for Windows, version 2018.7.525²³. The significance of differences between the means was determined by the analysis of variance ANOVA followed by the Tukey multiple-comparison test with a limit of significance at p<0.05.

Multiple regression was performed to analyze the overall acceptability by considering the influence of the specific sensory attributes (color, taste, texture, and smell), using the Minitab 18 Statistical Software (LLC, United States).

Ready-to-eat millefeuille enriched with polyphenols

This work was based on the design of a ready-to-eat vegetable-based preparation using vacuum impregnation (VI) to incorporate polyphenols and sous vide cooking as the heat treatment (Figure 3).

Figure 3. Ready-to-eat vegetable-based dish enriched with polyphenols. 

The physicochemical characterization of the designed culinary preparation is shown in Table 1. The ready-to-eat product obtained provides 200.23 mg GAE/100 g per serving, through the VI.

Sensory evaluation

For the sensory evaluation, a group of 105 consumers participated in the study, of which 65% were women and 35% men. The age ranges were 18-30 years (54%), 31-59 years (35%), and 60 years or older (12%).

The results presented in Table 2 show the level of satisfaction of each parameter studied. It was observed that the general acceptability, taste, smell and color were indicated in the "I like" category, with color obtaining the highest score. The texture was identified as different from the rest corresponding to the category "I am indifferent about it".

Table 3 shows that texture and taste significantly influence the other sensory characteristics (smell and color) as indicated by the significant differences between the two means (p<0.05). The main sensory characteristics that influence the consumer in the global acceptability was the texture and flavor of the product.

Physicochemical and microbiological parameters during refrigerated storage

These physicochemical and microbiological parameters were analyzed over 21 days, estimated useful lifetime for vacuum-packed vegetables cooked by sous vide. The parameters evaluated are presented in Table 4.

It was observed that the polyphenol content remained constant until day 14. It was observed that the studied product did not experience changes in pH or weight (p>0.05) during the storage period. Regarding the color parameters, it was observed that the values of a* and b* remained constant until day 7. The color intensity and luminosity during storage did not show a significant difference (p>0.05) in the evaluated time. The ∆E* remained constant until day 14. Taking into account the evolution of the values, color was observed to decrease over time.

The uniaxial compression test, it was observed that the values of firmness and maximum force remained constant until day 14 and then began to decrease.

The microbiological parameters studied showed that there was no proliferation of the groups of microorganisms evaluated, possibly due to the absence of oxygen, the low refrigeration temperature, and the appropriate conditions of the sous vide cooking process. In this way, the product could be considered suitable for human consumption during the period studied.