Effect of in vitro gastrointestinal digestion on the antioxidant potential of yogurt added with probiotic culture containing Bacillus subtilis Efeito da digestão gastrointestinal in vitro no potencial antioxidante de iogurte adicionado de cultura probiótica contendo Bacillus subtilis

The addition of probiotic culture confers potential bio functional to food because of the ability to promote health, and the beneficial effects in the body. The objective was to produce with lactose and lactose-free yogurt with Bacillus subtilis addition and to evaluate the antioxidant potential of watersoluble peptides during the storage period. For yogurt production, a simple factorial arrangement was used to evaluate two types of milk, adding probiotic culture containing B. subtilis UFPEDA 86. The fractions were evaluated (pre-digestion and post-digestion) concerning the antioxidant elimination potential of the radical acid 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2'-diphenyl 1-picrylhydrazyl (DPPH), chelation of copper and iron. In the evaluation of the results, the fractions obtained from the different combinations of milk, probiotic culture and different days presented antioxidant potential and chelating capacity, activities showed a significant increase after the digestion process. In addition, the results demonstrated that the B. subtilis could support the production of bioyogurt with antioxidant potential.


INTRODUÇÃO
The addition of ingredients in foods with the function of potentializing their nutritional actions has been growing over the last few years (MOUMITA et al., 2018).
This addition confers potential bio functional to food because of the ability to promote  In this context, this work aimed to evaluate the scavenging capacity of oxidative radicals' compounds and the chelation of metallic ions of the fractions of probiotic yogurt produced through the interactions between the presence and absence of lactose and addition of Bacillus subtilis in its composition, in different days, before and after simulation of in vitro digestion.

YOGURT PREPARATION
Ultra-high temperature (UHT) milk of bovine origin with lactose and lactose-free, pasteurized were subjected to an additional heat treatment (91 ± 1 °C / 10 min). Then, the milk was cooled to 45 °C and the lyophilized starter culture consisting of  (Table 1) and the final point of the yogurt fermentation was based on the clot firmness check. Subsequently, the product was cooled to 4 ± 1 °C and the clot was broken by manual shaking with glass a rod.

Eq.1
Where, Asample is the absorbance of the samples, and Acontrol is the absorbance of the negative control contains phosphate buffer.

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Where Asample is the absorbance of the samples, Awhite sample is the absorbance of the sample without DPPH • , and Acontrol is the absorbance of the control sampled DPPH solution.

PEPTIDES
The iron chelating activity (CA) was performed with modifications, using 125 μL For both activities, the negative control was performed using water in substitution of the sample; the positive control was performed with 0.045% EDTA solution, the percentage of inhibition was determined according to the equation 3: Eq. 3

DIGESTION IN VITRO
The digestion experiment was performed simulating the physiological conditions of the human organism in the stomach and intestine, adapted from a method already described by Gião et al. (2012). The volume of each infusion was divided equally by 3

STATISTICAL ANALYSIS
The parameters were analyzed in triplicate and the data expressed as mean and standard deviation. The experimental design was a factorial (2 x 2), totaling 4 systems, according to Frame 1. Significant differences between the yogurts submitted to different additions of probiotic culture were obtained using unidirectional analysis of variance followed by evaluation of differences between the means using the Tukey multiple comparison test in SISVAR version 5.6 (FERREIRA, 2008).

RESULTS AND DISCUSSION
The bio-yogurt showed the potential of elimination of the ABTS + • and DPPH + • radicals in all systems throughout the storage period ( Table 2). The S3 system (lactose- Tests carried out in the absence of lactose in the presence or absence of Bacillus subtilis showed a higher elimination potential of the radical, S2 (63.52%) and S1 (58.58%), respectively (  It is known that high levels of iron and copper ions in the body lead to oxidative stress and consequently cause cellular damage and disease (PRESA et al., 2018).
Regarding the iron chelating activity, a lower elimination potential was observed in the first storage period with an increase during the storage time. There was no significant difference between the times and the systems after 7, 14 and 21 days of storage with activity greater than 80%.
The chelation of copper remained above 85% in almost all the tests, with no significant difference between the times and the systems. Presa  applications in the industrial sectors are under specific legislation that the limit their use, therefore, it is necessary to prospect for new natural antioxidant agents, such as bioactive peptides. After bio-yogurt submission to the simulation of the in vitro digestion process, it was observed that the antioxidant and chelating potential of metal ions was maintained in most of the trials (Table 3).  The iron chelation capacity in the periods of 7, 14 and 21 days of storage remained statistically the same and did not present post-digestion difference. There was a decrease in the ability to chelate copper after the digestion simulation, highlighting the S1 (between 13.45 and 38.53%) and S3 (between 18.06 and 29.81%) trials that presented the greatest activities between trials.

CONCLUSION
The prospecting of bioactive peptides from yogurt produced using milk with lactose and lactose-free in the presence or not of probiotic (Bacillus subtilis) demonstrates efficacy against the ability to scavenge radicals' oxidants (ABTS + • and DPPH + • ) and potential chelating of copper and iron metal ions. The lactose-free systems show peptides with higher potential to eliminate the radicals ABTS + • , DPPH + • , and chelating iron and copper. Bacillus subtilis is a promising probiotic in the production of yogurt with antioxidant potential and shelf life. The antioxidant potential of the yogurt produced in vitro using B. subtilis was reported. However, for future use in the industry, studies about toxins production must be evaluated.