Enrichment of sliced apple fruit with Bacillus coagulans

Abstract

R E S E A R C H A R T I C L E Soto-Caballero, et al. Emir. J. Food Agric ● Vol 33 ● Issue 1 ● 2021 13 matrix for probiotic bacteria. Therefore, the objective of this study was to evaluate feasibility to riches apple slices with B. coagulans through different conditions of vacuum impregnation. MATERIALS AND METHODS Raw material and preparation Apple (Malus domestica Borkh.) fruits cv. Golden delicious were purchased in a local market. Fifty apples of adequate ripening were selected (14% of soluble solids content) (Guan et al., 2015), using a hand-held refractometer (Master Series, 0 30%, Atago). Fruit were conditionated at room temperature and the washed and cut into slices (~ 8 mm thickness) along the horizontal axis. Sucrose isotonic solution (IS) and culture preparation Fruit impregnation techniques use IS with high concentrations of probiotic bacteria for short periods of time (Russo et al., 2014) by vacuum impregnation (Alzamora et al., 2005). For this, an IS was prepared with distilled water and 14% sucrose (Sigma, USA), according to soluble solids content of the apple. Osmotic pressure for application of IS was 9.83 atm. The solution was thermally sterilized at 121 °C during 20 min. Osmotic pressure was also calculated. B. coagulans was obtained from the Culture collection of CIAD (Centro de Investigación en Alimentación y Desarrollo) in Cuauhtemoc, Chihuahua, Mexico. Since the bacterium requires a rich mixture of organic substrates to grow in vitro (Jurenka, 2012), we tested several enriched media. The best culture to grown the bacterium was trypticase soy broth (TSB) (BD Bioxon). After shaking for 48 h at 37 °C, the culture was centrifuged and resuspended in distilled water to obtain a high concentration of 2 x 1010 CFU mL-1, as suggested by Russo et al. (2014). Vacuum impregnation experiment Vacuum impregnation was carried out using a vacuum system comprised a glass vacuum desiccator (Wheaton Dry-Seal, capacity 5 L) and a vacuum pump (Felisa pump, Mod. 1600 L, Mexico) (Alzamora et al., 2005). Four unpeeled apple slices (~ 50 g) were immersed in 100 mL IS in beakers containing 2 x 1010 CFU mL-1, and then placed in a vacuum desiccator. Vacuum impregnation pressures of 22, 29, 36 and 43 cm Hg were applied during 5 min. These intervals are within those recommended by other authors (Mujica-Paz, et al., 2003; Neri et al., 2016; Russo et al., 2014; Schulze et al., 2012). After the vacuum period, atmospheric pressure was restored, and the samples were kept in the solution for five relaxation times (35204 min.). The vacuum impregnation process is represented in the Fig. 1. Slices were drained for 1 min. to remove excess of probiotic solution. Sample weight was recorded before and after treatment to calculate initial and final bacterial concentration impregnated per g of fruit. Variables and conditions were established according to previous studies of vacuum impregnation of apples, which indicate high levels of impregnation (Mujica-Paz, et al., 2003; Neri et al., 2016; Schulze et al., 2012). Since the effect of vacuum pressure on the added Bacillus is not known, we wanted to test low vacuum levels, extending the immersion time after vacuum application (relaxation time). Probiotic bacterial count One g of each impregnated apple was aseptically homogenized in 9 mL of sterile buffered peptone water, using a stomacher blender (IUL Instruments). Serial dilutions of each sample were spread on trypticase soy agar plates and incubated at 37 °C for 48 h, under anaerobic conditions, using the AnaeroGenTM atmospheric generator system (Oxoid Ltd, United Kingdom). After this time, bacteria were counted. All plate counts inoculated from each treatment and replication of Bacillus-impregnated apples were converted to CFU g-1, and the average colony counts of five replicates plated in duplicate were transformed to base-10 logarithms. For the enumeration of B. coagulans, the method of the United States Food and Drug Administration, the International Organization for Standardization (1993) and the Official Methods of Analysis (2000) of the AOAC were used. Impregnated apple morphology The morphology of the impregnated apple with B. coagulans was observed by Scanning Electron Microscopy (SEM, Evo MA 25, Carl Zeiss, Oberkochen, Germany). The sample was frozen at -80 °C and then lyophilized (Virtis FM 25 EL 85, Gardiner, NY, USA). Subsequently, cross-sections of the lyophilized apple were made, placed on carbon tape, coated with gold and observed by SEM using an accelerating voltage of 8.0 kV and a working distance of 8.0 mm. Experimental design and statistical analysis Variables of the vacuum impregnation process were vacuum pressure (22 43 cm Hg) and relaxation time (35204 min.) were subjected to an experimental design and optimization by central-composite design. The propose of this methodology (linear and quadratic regretion and ANOVA) is to optimize the variable of interest by determining the optimal conditions of the factors in the Soto-Caballero, et al. 14 Emir. J. Food Agric ● Vol 33 ● Issue 1 ● 2021 system. Experimental design and statistical analysis were carried out in Minitab 16 software (Statistical Software, USA). RESULTS AND DISCUSSION A significant effect of (p < 0.05) for volume fraction impregnated (X) and B. coagulans concentration were vacuum pressure, relaxation time and vacuum pressurerelaxation time interaction. The quadratic terms of vacuum pressure and relaxation time were significant (p < 0.05) only for the probiotic concentration. Determination coefficient values (R2adj) were > 82%, indicating that the statistical model applied provides an adequate description of the experimental data (Table 1). Volumetric impregnation parameter (X) The X parameter estimated IS impregnated into the pores of the fruit during vacuum impregnation process. The –isoterm refers to the same solute concentration in the apple tissue and in the solution in which it was immersed. Fig. 2 shows the effect of IS conditions on impregnated volume fraction. The X values varied from 3 to 17%, while pressure of 36 and 43 cm Hg led to an X value of 12 17% in all relaxation times tested. Higher incorporation of IS into the apple was obtained at 43 cm Hg and relaxation times of 35 and 60 min., thus, high vacuum pressure and short relaxation times, results in fast and high impregnation of IS into apple slices. The pressure of 43 cm Hg is within the range used by various authors for impregnation of probiotics in apple cv. Golden delicious and other cultivars Fig 1. Vacuum impregnation experiment Table 1: Analysis of variance (ANOVA) of level impregnation and concentration of B. coagulans in apple as a function the different factors evaluated Source Df SS F p R2adj Volumetric impregnation parameter (X) Vacuum pressure 1 0.002 27.10 0.001 82.3 Relaxation time 1 0.001 14.84 0.006 Vacuum pressure vacuum pressure 1 0.000 0.59 0.467 Relaxation time relaxation time 1 0.000 3.12 0.121 Vacuum pressure relaxation time 1 0.001 14.85 0.006