WO2016201536A1

WO2016201536A1 - Biotransformation method for transforming phenolic compounds from soy extract into equol and bioactive isoflavones by fermentation and/or enzymatic application, thus obtained composition and use

Info

Publication number: WO2016201536A1
Application number: PCT/BR2016/000024
Authority: WO
Inventors: Gabriela Alves MACEDO; Juliana Alves MACEDO; Lívia Dias DE QUEIRÓS
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2015-06-18
Filing date: 2016-03-03
Publication date: 2016-12-22
Also published as: BR102015014499A2

Abstract

The present invention relates to a biotransformation method for transforming phenolic compounds from soy milk into bioactive isoflavones and equol, with a high yield and production volume, for use in the biotechnological field, mainly in the food industry. The invention allows the glycosylated forms of isoflavones (daidzin and genistin) to be biotransformed into the respective aglycone forms (daldzein and genistein) by fermentation of soy milk with initiator cultures and probiotic lactic bacteria, and/or by treatment of the extract with the enzyme tanase from Paecilomyces variotti. In one of the embodiments, the present invention relates to an in vitro method for producing bioactive equol in the absence of intestinal bacteria, using only enzymatic biotransformation.

Description

"BIO-TRANSFORMATION PROCESS OF PHENOLIC COMPOUNDS FROM EQUOL SOY EXTRACT AND BIOACTIVE ISOFLAVONES THROUGH FERMENTATION AND / OR ENZYMATIC APPLICATION,

SO COMPOSITION OBTAINED AND USE "

FIELD OF INVENTION

[001] The present invention relates to a process of biotransformation of water soluble soybean extract phenolic compounds into high yield, high yield bioactive isoflavones; composition thus obtained and use.

[002] The present process has application in the biotechnology area, mainly for the food industry, as food ingredient, and industry of nutricosmetics and dietary supplements.

BACKGROUND OF THE INVENTION

[003] The main application of the present invention is the biotransformation of water soluble soybean extract (EHS) phenolic compounds to obtain product rich in bioactive compounds. The purpose is to increase the content of bioactive isoflavones (agiicones) and to produce equol in soybean extract through the application of starter cultures and probiotic lactic bacteria in the fermentation of EHS, allied to the action of crude tanase extract obtained from Paecilomyces variotti.

The present invention provides a composition in the form of a food ingredient which contains high bioavailable bioactive isoflavones and equol content.

Another possible use is the bioactivity of water-soluble soybean extract before and after biotransformation with P. variotii semisified tanase extract for anti-inflammatory and chemoprevention models.

[006] In addition, the role of P. variotii tanase semipurified extract in other classes of glycosylated flavonoids in foods with large amounts of flavonoids in glycated form is also interesting.

US8765445B2 relates to a process employing Lactobacillus strains in the process for producing product for direct consumption, it is noted that this process is already in the commercial phase.

WO2012033150 relates to the production of equol by fermentation in complex fermentation medium with addition of daizein in kinda gaseous. The process uses the Streptococcus strain. This document teaches that the use of hydrogen in fermentation increases equol production. The process according to this document is suitable for industrial production, obtaining high yield of isoflavones.

US20120094336 describes the production of equol without racemic mixing by chemical synthesis. Describes synthesis by specific chemical catalysts for the production of the preferred type S isomer.

US201 10189134 describes the production of a fermented soy milk-based beverage, with the addition of live Lactobacillus to produce equol during the shelf life of the product. The process according to the teachings of this document is already on an industrial scale. It is also noted that the addition of ascorbic acid to maintain production is described.

JP201 1083273 describes a process for producing soy isoflavones and equol by fermentation using Lactoccoccus isolated from the Japanese ekuoru product. The process according to this document uses lactic acid bacteria (Lactococcus garviaes).

US2013231291 describes the production of isoflavone extract, equol and lunasil peptide by fermentation using isolated lactic acid bacteria. The process according to this document focuses on the use in cosmetics.

Patent document CN102021 130 describes the use of Clostridium bifermentans zx-7 for the production of equol by fermentation.

Patent document TW20071221 1 discloses the production of equol with a collection of intestinal flora microorganisms found in the human intestinal flora.

Patent document CN104031875 discloses the production of equol by genetically modified bacteria from Lactococcus.

It is noteworthy that the processes described in the prior art are mostly based on fermentative processes using different strains of Lactobacillus (enteric bacteria), isolated or in groups. Some of the documents ferment with synthetic culture media by adding the equol daizein precursor, others formulate the soy milk culture medium, a strategy also used in the present invention. Already the present invention uses different processes which are different from those previously described in the prior art. One uses a mixture of Lactobacillus (a strategy also used in the state of the art), with the advantage of using an isolated fungal enzyme to tanase, a new and inventive technical effect.

In one of the processes according to the present invention only one enzymatic treatment is used, another new and inventive technical effect. It is noteworthy that the enzyme preparation according to the present invention (fungus, other novel aspect effect) is not purified and has both known and unknown enzymes. This is a new process and no such report or suggestion has been found in the prior art.

The enzyme according to the present invention was extracted from GRAS fungus, which can be used in food.

BRIEF DESCRIPTION OF THE INVENTION

[0020] The present invention has been the development of in vitro biotechnological processes capable of biotransforming isoflavones into their bioactive forms and increasing the water soluble soybean extract equol content by fermentation of this substrate by priming bacteria and probiotic lactic acid bacteria and application of the same. tanase enzyme, obtained from Paecilomyces variotti; composition thus obtained and use in the food industry as a food ingredient and in the nutricosmetics and dietary supplements industry.

Following biotransformation processes, a soybean-derived food ingredient was obtained which had a high content of bioactive isoflavones and equol, thus demonstrating that the biotechnological processes developed consisted of efficient strategies to improve the bioactive potential of soybean extract. and demonstrated a potential of equol application as a nutraceutical agent for non-isoflavonoid producing populations.

Soy products are a way to include bioactive substances such as isoflavones in the diet, and water soluble soy extract (EHS) is a substrate that has been shown to have potential for the production of novel foods with healthy appeal. Thus, in order to increase the content of bioactive isoflavones and evaluate the viability of a biotechnological process for equol production in vitro, the process was developed. described in this document. It was investigated the application of probiotic starter cultures and lactic acid bacteria in the fermentation of the EHS, allied to the action of the crude tanase enzyme extract obtained from the fungus Paecilomyces varíotti. In addition, the biotransformation of phenolic compounds and antioxidant activity of the obtained product was also evaluated. Total phenol content was evaluated by Folin-Ciocalteau reagent-based methodology, antioxidant activity by in vitro ORAC and DPPH radical sequestration methods and specificity of semipurified tanase extract against commercial isfolavone standards as well as how changes in the phenolic profile of water soluble soybean extract were evaluated by HPLC-DAD. After the fermentative process and / or enzymatic treatment of soybean water soluble extract, there was a significant increase in the total phenolic content and antioxidant capacity, by both tested methods (ORAC and DPPH), when compared with the EHS control without reaction. In addition, a modification of the polyphenolic profile of the biotransformed EHS samples obtained by HPLC-DAD was observed, resulting in an increase in the concentration of the aglycone forms relative to the glycosylated ones, as well as an increase in the equol concentration after the proposed biotransformation processes. The results indicate that P. variotty tanase extract was able to biotransform the glycosylated forms (daidzine and genistine) of isoflavones into their respective aglycone forms (daidzein and genistein). To the best of our knowledge, hydrolysis of tannase glycosylated isoflavonoids, as well as the formation of equol, is an unpublished report in the literature showing that it is possible to develop an in vitro process for obtaining this bioactive compound without the presence of intestinal bacteria, using only one enzymatic biotransformation.

BRIEF DESCRIPTION OF THE FIGURES.

Figure 1 shows HPLC-DAD chromatograms of the reaction of the glycosylated isoflavone (A) daidzine and (B) genistin standards with Paecilomyces variotii tannase semipurified extract at 40 ^s C for 30 minutes.

Figure 2 shows HPLC-DAD chromatograms of (A) standard EHS, (B) fermented EHS, {C} fermented EHS reacted with 1.8 U tannase and (D) standard EHS reacted with 1.8 U tannase at 40 ° C for 30 minutes.

DETAILED DESCRIPTION OF THE INVENTION [0023] The present invention relates to the process of biotransforming phenolic compounds from soluble soybean extract to obtain equol rich product and bioactive isoflavones by fermentation with starter bacteria and probiotic lactic acid bacteria and application of the tanase enzyme; composition thus obtained and use. To this end, the process can utilize biotransformation forms which comprise applying strains of starter bacteria and probiotic lactic acid bacteria and / or reacting with the Paecilomyces variotti tannase enzyme in soybean water soluble.

The process comprises the following steps:

A. Macerate whole soybeans at room temperature in distilled water (soybean water; 1: 3 w / v);

B. Discard the water used for maceration;

C. Add distilled water;

D. Heat the beans in water between 90 and 1 10 ^Q C, preferably to 100 ° C (soybean / water; 1: 2 w / v) for 3 to 10 minutes, preferably 5 minutes;

E. Add distilled water

F. Crush soybeans with distilled water (soybeans / water; 1: 4 P / v);

G. Centrifuge the obtained mass of 9000-10000 x g, preferably 9630 xg for 10 to 20 minutes, preferably 15 minutes at room temperature (25 ± ⁵ ° C);

H. Warm water extract of soybean (EHS) to boiling between 96 and 100 ⁹ C ⁹ C for 10 to 15 minutes, preferably 0 to minutes;

I. Sterilize by heating at 121 ° C for 15 to 20 minutes, preferably 15 minutes;

The. Keep sterile water extract of soybean under refrigeration at 4 to 12 ^Q C for 10 days, the extract or freeze at -80 ^Q C until their use;

J. Carry out the biotransformation processes of water-soluble soybean extract which may be carried out in any of the following ways:

Process 1 - Fermentative Biotransformation: Starter Bacteria Streptococcus ssp. Thermophilus (YF-L81 1) and Lactobacillus delbruecki ssp. Bulgaricus (LB-340) and Lactic Acid Bacteria probiotic Bifidobacterium animales ssp. Laciis (Bb-12) and Lactobacillus acidophillus (LA-05);

i. Inoculate starter cultures at a concentration of 0.5 - 2% (v / v), preferably 1% (v / v), and probiotic lactic acid bacteria at a concentration of 5 to 15% (v / v), preferably 5% (v / v), and a minimum count of 10 ⁶ - 10 ⁷ log CFU / mL and a maximum of 10 ¹⁴ log CFU / mL in the EHS obtained in (I); and

ii. Incubate the solution obtained in (i) ^and from 30 to 40 C, preferably at 37 ⁹ C, for 10 to 24 hours, preferably 12 hours, under anaerobic conditions;

Process 2 - Fermentative Biotransformation followed by Enzymatic

i. Inoculate starter cultures at a concentration of 0.5 - 2% (v / v), preferably 1% (v / v), and probiotic lactic acid bacteria at a concentration of 5 - 15% (v / v), preferably 5% (v / v), and a minimum count of 10 ⁶ - 10 ⁷ log CFU / mL and a maximum of 10 ¹⁴ log CFU / mL in the EHS obtained in (I);

ii. Incubate the cultures with EHS obtained in (i) from 30 to ^S 40 C, preferably at 37 ⁵ C for 10 to 24 hours, preferably 12 hours, under anaerobic conditions; iii. Inoculate in a solution obtained from (ii) with 1 to 5 U tanase, preferably 1.8 U tanase, per ml fermented EHS (ii);

iv. Incubate the solution of (iii) at 35 to 45 ° C, preferably 40 ^g C, for 20 to 50 minutes, preferably 30 minutes; and v. Incubate the solution of (iv) between -2 to 2 ° C, preferably ^Q 0 C (ice bath) for 10 to 30 minutes, preferably 15 minutes;

Process 3 - Enzymatic Biotransformation

i. EHS inoculum obtained in (I) with 1 to 5 U tanase, preferably 1.8 U tanase, per 1 ml EHS (I); ii. Incubate the solution (i) from 35 to 45 DEG C, preferably 40 ^Q C for 20 to 50 minutes, preferably 30 minutes; and

iii. Incubate the solution of (ii) at -2 to 2 ° C, preferably

0 ⁹ C (in an ice bath) for 10 to 30 min, preferably

15 minutes;

K. Maintain EHS product with bioactive isoflavones and frozen equol at -80 ⁹ C.

The compositions obtained from the described process comprise the characteristics according to the type of biotransformation according to table 1 below and have application in the food industry, as a food ingredient, and the nutricosmetics and dietary supplements industry.

Table 1: Compositions obtained from the process described

Exemplification

Preparation of water soluble soy extract

Water-soluble soy extract was prepared according to the methodology described by Mandarino and Carrão-Panizzi (1999). We used soybeans (Glycíne max) from "Natu's" brand.

[0027] The process can be represented by the following steps:

1. Macerate whole soybeans for 6 hours at room temperature in distilled water in distilled water (soybean / water; 1: 3 w / v).

2. Discard the water used for maceration 3. Boil grains in water (soy beans / water; 1: 2 w / v) for five minutes.

4. Crush the soy beans with the aid of a blender with distilled water (soy beans / water; 1: 4 w / v) for 3 minutes.

5. Centrifuge the mass obtained at 9630 x g for 15 minutes at room temperature.

6. Heat the obtained supernatant, known as water-soluble soy extract, to boiling for 10 minutes.

7. Distribute the soy extract in vials and heat sterilize at 121 ° C for 15 minutes.

8. Keep sterile water-soluble soy extract refrigerated until use for analysis.

Obtaining semipurified extract of tanase

The enzyme tanase (E.C: 3.1.1.20), obtained from the microorganism Paecilomyces variotii, as described by Battestin and Macedo (2007a), was used in this study.

This process can be represented by the following steps:

(a) Add 10 g of wheat bran (250 ml) conical flasks (Natu's, Hortolândia, Brazil) to 10 ml of distilled water and 10% tannic acid (w / w) (Tanal B, Prozyn BioSolution, Sao Paulo, Brazil)

b) sterilizing the culture medium at 121 ^Q C for 15 minutes.

c) Inoculate 2.5 mL (5.0x107 spores / mL) of the pre-inoculum suspension in distilled water

d) Incubate the medium at 30 ° C for 120 hours.

e) Add 80 mL of acetate buffer (20 mM, pH 5.0) to the medium.

f) Shake the method at 200 rpm for 1 hour.

g) filtering the final solution and centrifuge at 9630 xg for 30 minutes at 4 ^Q C (J2-2 Beckman Centrifuge, Beckman-Coulter, Inc. Fullerton, CA, USA). (h) Add to the supernatant ammonium sulphate (Ecibra, Santo Amaro, Sao Paulo, Brazil) at a final concentration of 80% saturation (561 g / l). (i) keep refrigerated for 12 hours at 4 ⁹ C.

j) Collect the precipitate formed by centrifugation at (9630 x g for 30 minutes) and resuspend it in distilled water.

k) Dialysate for 48 hours in distilled water. 1) Freeze at -18 ° C and lyophilize in bench top lyophilizer (Liotop L101, Liobras Ind. Com. Serv. Liofilizantes Ltda., Sao Carlos, Sao Paulo, Brazil) for 24 hours,

m) Maintain the lyophilized semipuritized extract at -18 ^g C.

Determination of Enzyme Activity

[0029] The enzymatic activity measurement of the semipuritized tanase extract was performed according to the methodology proposed by Battestin & Macedo (2007b). The substrate consisted of a 0.7% (w / v) tannic acid solution (Sigma-Aldrich, Steinheim, Germany) in acetate buffer (0.2 M, pH 5.5). To perform the reaction, the following steps were followed:

a) Add 0.3 ml of the substrate solution to 0.5 ml of enzyme extract. b) Incubate at 60 ° C for 10 minutes.

c) Stop the reaction by adding 3 mL of a 1.0 mg / mL bovine serum albumin (BSA) solution prepared in 0.17 M sodium chloride solution in acetate buffer (0.2 M, pH 5 .0).

d) Filter and centrifuge the solution at 10,070 x g for 15 min at 4 ° C.

e) Dissolve the precipitate to 3 mL in SDS-triethanolamine

f) Add 1 ml of FeCl3 reagent.

g) Keep at rest for 15 minutes for color stabilization.

h) Measure absorbance at 530 nm (Mondai, Banerjee, Jana, & Pati, 2001). i) Calculate enzymatic activity by Abs530 = Abscontrol-Abstest.

One unit of tanase activity was defined as the amount of hydrolyzed tannic acid per 1 mL enzyme per minute reaction.

Enzymatic biotransformation of isoflavonoids

The commercial standards of isoflavonoids genistin, daidzine, genistein and daidzein (Sigma-Aldrich, Steinheim, Germany) were used as substrate for tanase enzymatic hydrolysis isolated from Paecilomyces variotti (Battestin & Macedo, 2007a).

[0031] This process can be represented by the following steps:

a) Dissolve 1 mg of isoflavonoid standards in 1 ml phosphate buffer (pH 7.4, 75 mM).

b) incubating the solution of tannase 0,18U for 30 minutes at 4 ° C in thermostated bath ⁹ (B12D model Micronal, Sao Paulo, Brazil).

c) Paralyze the hydrolysis process in an ice bath for 15 minutes. d) Evaluate, by HPLC-DAD, the end products of the isoflavonoid standards.

Water soluble soybean extract biotransformation processes

Process 1 - Fermentative Biotransformation

Lyophilized commercial cultures (Christian Hansen Lab. Ind. And Com. Ltda.) Of starter cultures Streptococcus ssp. Thermophilus (YF-L811) and Lactobacillus delbrueckii ssp. Bulgaricus (LB-340) and probiotic lactic acid bacteria Bifidobacterium animales ssp. Lactis (Bb-12) and Lactobacillus acidophillus (LA-05). All cultures were lyophilized reactivated individually in sterile water extract of soybeans ⁹ to 37 C for 12 hours under anaerobic conditions using an anaerobic jar. Starter cultures were inoculated at a concentration of 1% (v / v) and probiotic lactic acid bacteria at a concentration of 5% (v / v), ensuring a minimum count of 10 ⁶ - 10 ⁷ log CFU / mL (Cruz et al ., 2013).

The fermentation of the water-soluble fermented soybean extract was conducted according to traditional methodology (Tamime and Robinson, 2007) according to the following steps:

(a) Aseptically inoculate sterile water-soluble soybean extract with the total contents of each reactivated bacterial culture.

b) incubating the inoculated EHS 37 ^Q C for 24 hours under anaerobic conditions using an anaerobic jar.

Fermented EHS samples were evaluated for chemical profile (total phenol content and HPLC) and antioxidant capacity (ORAC and DPPH).

Process 2 - Fermentative Biotransformation followed by Enzymatic

[0034] The fermented EHS (1ml) obtained in Process 1 was inoculated with 1, 8 U of tannase at 40 ° C ^and for 30 minutes. The hydrolysis process was stopped in an ice bath for 15 minutes.

Biotransformed EHS samples were evaluated for chemical profile (total phenol content and HPLC) and antioxidant capacity (ORAC and DPPH).

Process 3 - Enzymatic Biotransformation

Unfermented EHS (1 mL) was inoculated with 1.8 U tanase at 40 ° C for 30 minutes. The hydrolysis process was stopped in an ice bath for 15 minutes. Biotransformed EHS samples were evaluated for chemical profile (total phenol content and HPLC) and antioxidant capacity (ORAC and DPPH).

Chemical profile assessment

Total phenolic content

The total phenolic content in the samples was determined with the Folin-Ciocalteu reagent (Contemporary Chemical Dynamics, Brazil) according to the method of Chandler and Doods (1983), with some modifications.

[0039] This process can be represented by the following steps:

a) Dilute 1 ml aliquot 1: 5 with 70% methanol.

b) Add 5 ml of distilled water, followed by 0.5 ml of 1 M Folin-phenol Ciacalteu reagent (Contemporary Chemical Dynamics, Brazil). c) Shake the samples.

d) Keep at rest for 5 minutes.

e) Add 1 ml of 5% (w / v) sodium carbonate solution to the reaction medium of each sample.

f) Shake and incubate samples in the dark at room temperature for 1 hour.

g) Shake the samples again.

h) Take absorbance readings at 725 nm in spectrophotometer (model DU®640, Beckman CoulterTM, USA).

Total phenolic concentrations were determined by comparing the absorbance of the samples with a calibration curve using catechin as standard. Results were expressed in g catechin per ml sample.

HPLC isoflavonoid analysis

Glycosidic isoflavones (daidzine and genistine), aglycones (daidzein and genistein) and equol were identified and quantified in all samples by HPLC-DAD.

The extraction of isoflavonoids from samples of soybean extraction was performed according to the methodology proposed by Aguiar (2003), with modifications according to the following steps: a) Mix 1 mL sample of soymilk biotransformed with 5 ml of 70% methanol at 25 ^Q C for 60 minutes at 100 rpm.

b) Centrifuge the mixture for 10 minutes at 5790 x g.

(c) Filter the supernatant through a 0.45 μιη membrane before being injected on an inverted phase HPLC.

The chromatographic conditions used were previously described by Park et al. (2001). The equipment used was Dionex Ulitmate 3000 (Germany) equipped with a Atlantis® C18 column (Waters, 5 μιη, 4.6 x 150 mm) maintained at 30 C. ⁹ Detection was performed using a UV diode array detector / VIS (DAD-3000). The mobile phases consisted of deionized water (solvent A) and methanol (solvent B). The elution gradient was as follows: 20% B (0-15 min), 20-80% B (15-75min), 80-100% B (75-80min), 100-20% B (80-90 min) ) and 20% B (90-95 min) with a flow rate of 0.5 mL / min. The sample injection volume was 20 μΙ_ and all samples were analyzed in triplicate. The spectra were obtained between 190 and 480 nm and the chromatograms processed at 254 nm.

Isoflavonoids were individually identified by comparing their retention times and the spectrum of their patterns: daidzine, genistine, daidzein, genistein and equol. Individual quantification of isfolavonoids was performed by integrating peak areas using calibration curves that were established within the concentration range of 0.01 -1.0 mg / mL of the isoflavones and equol standards. Results were expressed as of each phenolic compound per mL of sample.

Biotransformed soybean extract polyphenols antioxidant activity

Oxygen radical absorption capacity (ORAC)

The determination of antioxidant capacity by the ORAC method was performed according to the methodology described by Macedo et al. (201 L), using fluorescein (FL) as a flag. The automated ORAC test was performed on a Fluostar Optimal Microplate Reader (Fluostar Optima - Labtech BMG, Germany) with fluorescence filters for an excitation wavelength of 485 nm and emission of 520 nm. Measurements were performed on Costar® opaque black plates (Corning Costar Corporation, Cambridge, USA) with 96 wells.

Prior to the reaction several steps of solution preparation should be followed: a) Decompose at 37 ° C AAPH (2,2'-azinobis (2-amidinopropane) dihydrochloride) in phosphate buffer (75 mM, pH 7.4). Note: this fact occurs due to the sensitivity of FL to pH.

b) Prepare the FL solution (70 nM) in phosphate buffer (PBS) (75 mM, pH 7.4) daily.

c) Store in the dark.

d) Prepare (daily) in PBS a solution of Trolox (6-hydroxy-2,5,7,8-tetramethylchromo-2-carboxylic acid) 75 μΜ (used as reference standard).

e) Construct a standard Trolox curve by diluting the solution by 1.5-1500 pmol / mL.

The reaction was performed according to the following steps:

a) Dilute 1 ml of each sample using 70% methanol 1: 5. (b) Mix 120 µL of FL solution with 20 µL of sample, blank (PBS) or standard solution (Trolox) in each well.

c) Add 60 µl AAPH (12 mM).

d) Perform, in triplicate and every 6 minutes for 87 minutes, the fluorescence measurement immediately after the addition of AAPH

ORAC values were defined as the difference between the area under the FL and blank decay curve (net AUC). Regression equations between net AUC and antioxidant concentration were calculated for all samples. A tanase control was performed as a regular sample, and the obtained ORAC value was subtracted from the samples treated with this enzyme. ORAC-FL values were expressed in pmol equivalent of Trolox / L soybean extract.

DPPH Free Radical Hijacking

The antioxidant activity potential of fermented and unfermented water-soluble soybean extract was also assessed by stable free radical sequestration activity 1,1-diphenyl-2-picrylhydrazyl (DPPH), as described by Macedo et al. (201 l).

a) Prepare various concentrations (0.01 - 0.1 mg / mL in 70% (v / v) methanol) of tested samples.

(b) Mix 50 µL of the test samples with 150 µL of 0.2 mM DPPH in methanol) in 96-well plates (BMG LABTECH 96, Germany). c) Read on a Fluostar Optimal Microplate Reader (BMG Fluostar Optima - Labtech, Germany) with absorption filters for an excitation wavelength of 520 nm.

The color decay process of the samples was recorded after 90 minutes of reaction and compared with a control blank; For color samples and tanase-treated samples, an additional control was performed which contained the extract solution (or tanase solution) and pure methanol instead of DPPH. The solutions were prepared and stored in the dark. Measurements were performed in triplicate and antioxidant activity was calculated from the equation obtained by linear regression determined by tracing the antioxidant activity of standard Trolox solutions with known concentrations. Antioxidant activity was expressed as pmol equivalent of Trolox / L soybean extract.

Statistical analysis

Results were expressed as arithmetic means and standard deviations. Statistical significance of differences between groups was analyzed by Tukey test. Differences were considered significant when p <0.05.

Effects of bioprocesses on total phenolic content and antioxidant activity

The effects of fermentation and / or enzymatic biotransformation on total phenolic content and antioxidant activity of soybean water soluble extract, as measured by ORAC and DPPH methods, are presented in Table 2. Significant differences · (p <0.05) In the total phenolic content, ORAC and DPPH were found in all bioprocesses.

Table 2. Total phenolics content and antioxidant activity (ORAC and DPPH) of water soluble soybean extract samples before and after biotransformation processes.

Bioprocess Phenolic Samples ORAC Equiv. DPPH Equiv.

Equiv. Totals Trolox (pmol / L) Trolox (pmol / L) Catechin

(pg / ml_)

Standard EHS 1 101 ± 6 1022 ± 821 ^to 1584 ± 14 ^the 1 EHS 254 ^b ± 11 25987 ^b ± 977 1729 ^b ± 27 fermented

II EHS soybean 387 ^c ± 9 31801 ° ± 858 4439 ^c ± 39 fermented

+ tanase

III EHS + 847 ^d ± 7 45758 ^d ± 937 6724 ^d ± 36 tanase

* Results shown are means (n = 3) ± SD, and those with different letters in the same column are significantly different, with p <0.05.

The results showed that there was a significant increase in total phenolic content for all biotransformed samples (p <0.05). Following the fermentation process with lactic acid bacteria (process 1), the total phenolic content of the soybean water soluble extract increased significantly from 101 to 254 pg catechin / mL, an increase of about 2.5 times compared to the standard (non-EHS). biotransformed). In process 2, when analyzing the reaction of fermented EHS with the enzyme tanase, a significant increase in the total phenolic content was also observed, where the increase was about 3.8 times (101-387 g catechin / mL) when compared with the default. Process 3 showed the largest increase in total phenolic content (101-847 Mg catechin / mL), an increase of about 8.4-fold after the reaction of EHS with the enzyme tanase. These results suggest that the fermentation process and tanase enzymatic biotransformation improve the release of phenolic compounds from soybean extract. Furthermore, the results seem to indicate a higher catalytic potential of tanase over soybean extract phenolics when compared to the fermentative action of lactic acid bacteria.

The antioxidant activity of the samples, before and after the three EHS biotransformation processes, was analyzed by ORAC and DPPH methods in vitro. Results are described in Table 2 and expressed as equivalents of Trolox®. The results showed that there was a significant increase in the antioxidant activity of biotransformed samples in both methodologies (p <0.05), correlating with the results found for total phenol content.

[0052] The results of Table 2 show that the EHS fermentation process (process 1) resulted in a significant increase of about 2.2 times the antioxidant capacity by the ORAC method and an increase of approximately 2.1 times by the DPPH method. Fermentation followed by enzymatic biotransformation (process 2) led to an increase in antioxidant activity of 2.7 and 5.4 times by ORAC and DPPH assays, respectively. Similarly, in the tanase-catalyzed EHS biotransformation only (process 3), there was a significant increase in antioxidant activity of 4 and 8 fold by ORAC and DPPH, respectively.

Soybeans and soy-based products are nutritionally rich foods and have various phytochemicals (isoflavones, phytosterols, saponins, phenolic acids, phytic acid and trypsin inhibitors) that have functional, antioxidant and free radical scavenging properties ( Wardhani, Vázquez, and Pandiellaa, 2010). Soybean phenolic compounds have free radical scavenging activity (Shon et al., 2007), which may explain the correspondence between the increase in total phenolic content with antioxidant activity shown in table 2.

In summary, the results of the present invention indicate a trend towards increased total phenolic content after the three proposed bioprocesses. This trend was similar to that observed for antioxidant activity by both the ORAC and DPPH methods, supporting the results obtained by the microbial and enzymatic biotransformation of the EHS.

Isoflavones profile

The concentration of genistin, daidzine, genistein, daidzein and equol formation were evaluated before and after the three biotransformation processes using HPLC-DAD (Table 3). Isoflavones and equol were separated and identified according to the methodology proposed by Aguiar (2003).

Table 3. Concentrations of isoflavone and equol isomers (Mg / mL) in water-soluble soybean extract before (standard) and after biotransformation processes.

Isoflavonoid content (pg / mL)

Process Glycosylated Samples Aglicones

s Daidzin Genistin I Daidzein I Genistein

aaaa Standard EHS ^to 313.0 ± 393.8 ^to 25.6 ± 10.2 ^to ^within ± 0.08 ^to ±

2.0 3.2 0.6 0.5 +

0.000 3

1 EHS 194.2 ^b ± 202.5 ± 91.4 ^b ± 52.3 ^b ± 0.31 ^b fermented 5.7 5.9 1, 9 2.0 ±

0 0.000

8 li EHS nd ^* nd 717.2 ° ± 251.4 ° ^C ± 0.63 ^and fermented 11.0 + 6.9 + o + tanase 0.014

2

III EHS + ndnd 944.5 ^d ± 321.6 ^d ± 0.82 ^d tanase 0.9 0.8 +

0.010 4 n.d. = Not detected below detection limit for this methodology and equipment. Results are expressed as mean ± standard deviation (n = 3). The mean in the same column with different letters is significantly different (p <0.05).

The results showed that all bioprocesses developed were able to significantly increase the content of the aglycone (daidzein and genistein) and equol forms in water soluble soybean extract. Due to the hydrolysis of daidzine and genistine, the content of their corresponding aglycone, daidzein and genistein forms in EHS increased after the fermentative and enzymatic process (p <0.05).

As shown in Table 3, glycosidic isoflavones were the predominant forms in standard and fermented EHS. However, the concentrations of fermented EHS genistin and daidzine glycosides (process I) decreased by about 1, 6 and 2 times, respectively, and the daidzein and genistein algicones increased by 3.5 and 5 times, respectively, when compared with standard EHS. Therefore, these results suggest that glycosylated isoflavones were transformed into aglycone forms as a result of the fermentation process. According to Duenas et al. (2012), This may be due to the β-glycosidase activity inherent in the microorganisms used in the fermentation process.

As can be seen from Table 3, after processes II and III, the glycosidic forms (daidzine and genistine) were not identified, showing that these forms were converted to their respective aglycone forms, as shown by a significant increase in levels. of daidzein and genistein after these bioprocesses.

Concentrations of the fermented EHS aglycone forms after process II increased significantly about 28-fold (25.6 - 717.2 mg / mL) for daidzein and 25-fold (10.2-251.4 mg). / mL) for genistein compared to standard EHS. In process ill the aglycone forms increased significantly, compared to standard EHS, by 37-fold (25.6 - 944.5 mg / mL) for daidzein and 32-fold (10.2 - 321.6 mg / mL) for daidzein. the genistein. These results indicate that the enzymatic reaction with tanasse (process III) presented the largest increase of the bioactive forms of isoflavones, which was also observed for the results of total phenolic content and antioxidant capacity by ORAC and DPPH methods.

Isoflavones aglycones have been reported to exhibit greater bioactive function than glycosidic forms (Cederroth, and Nef, 2009; Setchell, 1998). Therefore, aglycones are primarily responsible for the antioxidant activity of soy-based foods, although other soy components such as phenolic compounds may also contribute to antioxidant capacity to some extent (Cheng, Lin, and Liu, 201 1).

Therefore, the bioconversion of glycosylated isoflavones into their aglycone forms after the three proposed biotransformation processes proved to be very effective in increasing the antioxidant capacity of the samples by the tested methods, as demonstrated by the in vitro antioxidant activity tests (ORAC). and DPPH).

[0062] The ability of the enzyme tanase to transform commercial patterns of glycosidic isoflavones (daidzine and genistine) has been proven in in vitro assays. Figure 1 shows the chromatograms obtained after 30 minutes of feed at 40 ° C, in which commercial standards of daidzine (A) and genistein (B) were reacted with P. variotti tannase semipurified extract. also retention times and absorption spectra of reaction products are shown.

As shown in Figure 1, it can be seen that the reaction between tannase and the glycosylated isfolavone patterns (daidzine and genistine) led to the formation of their respective (A) dadizein and (B) genistein forms. This fact can be confirmed from the similarity between the retention time of the reaction products. These results prove that tanase is able to act on the hydrolysis of the isoflavone glycosidic bonds.

As shown in Table 3, the concentration of equol after fermentation of EHS with lactic acid bacteria (process I) led to a significant increase in equol content, about 3.9-fold. In process II, enzymatic treatment of fermented EHS led to a significant increase of about 7.9 times this bioactive compound when compared to standard EHS. In process III, after enzymatic treatment of crude EHS the increase was about 10-fold. These results indicate that P. variotiii tannase showed higher hydrolytic action of glycosylated isoflavones in relation to the fermentation process and was also able to improve the equol content in EHS.

The ability of tanase to biotransform glycosylated isoflavones and increase the equol content in the EHS can be confirmed by the chromatograms shown in Figure 2.

[0066] Figure 2 shows the chromatographic profile of the general composition of standard EHS (A) and lactic acid bacteria fermented EHS (B) as well as fermented EHS (C) and unfermented EHS (D) polyphenols. treated with 1.8 U of semi-purified tannase extract.

In Figure 2, it can be seen that both fermentation and enzymatic treatment processes were able to significantly increase the content of isoflavones aglycones (daidzein and genistein). In addition, these bioprocesses led to the formation of equol.

The results of table 3 demonstrated that P. variotti tannase was able to synthesize equol at a higher level compared to microbial strains used in the fermentation process. To the best of our knowledge, studies in this area are rare and there is little information on the correlation between equol formation and enzyme use. It can also be observed that the action of this enzyme was not limited to isoflavone deglycosylation (figure 1), tannase was also able to increase the equol content. These results make this enzyme an interesting option for application in soy-based foods, with a rich polyphenol composition where tannase can act in different ways in the biotransformation process.

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Claims

1 . Isoflavone enzymatic biotransformation process comprising the fermentation of a substrate composed of water-soluble soybean extract by starter bacteria and probiotic lactic acid bacteria and / or tanase enzyme obtained from Paecílomyces variottí.e the steps:

A. Macerate whole soybeans in distilled water at room temperature;

B. Discard the water used for maceration;

C. Add distilled water;

D. Heat the beans in water at 90 to 110 ^g C for 3 to 10 minutes;

E. Add distilled water;

F. Crush soybeans with distilled water;

G. Centrifuge the mass obtained from 9000 to 100,000 for 10 to 20 minutes, preferably 15 minutes at room temperature;

H. Heat the water soluble soybean extract (EHS) to boiling for 10 to 15 minutes;

I. Sterilize by heating at 121 ° C for 15 to 20 minutes;

J. Perform biotransformation; and

K. Maintain EHS with bioactive isoflavones and preserved equol.

Process according to Claim 1, characterized in that the starter bacteria are Streptococcus ssp. Thermophilus (YF-L81 1) and Lactobacillus delbrueckii ssp. Bulgaricus (LB-340); and probiotic lactic acid bacteria to be Bifidobacterium animales ssp. Lactis (Bb-12) and Lactobacillus acidophillus.

Process according to claim 1, characterized in that in step (A) the proportion of soybean weight and volume of water is 1: 3.

Process according to Claim 1, characterized in that in step (D) the proportion of soybean weight and volume of water is: 2 and the process conditions are 00 ° C for 5 minutes.

Process according to Claim 1, characterized in that in step (F) the ratio of soybean weight to water volume is 1: 4.

6. The method / according to claim 1, wherein in step (G) occur at 9630 xg centrifugation for 15 min at 25 C. ⁹

Process according to Claim 1, characterized in that in step (H) the preferred boiling range is between 96 and 100 ° C and a time of 10 minutes.

Process according to claim 1. characterized in that in step (I) the time is 15 minutes.

Process according to Claim 1, characterized in that in step (I) optionally comprises a sterile EHS storage substep (1a).

10. Process according to claim 9, characterized in that in substep step (la) the storage conditions of refrigeration EHS be between 4 and 12 ^S or C ^and freezing at -80 C.

1 1. Process according to claim 1, characterized in that in step (J) the biotransformation is carried out by fermentative biotransformation, fermentative biotransformation followed by enzymatic or enzymatic biotransformation.

Process according to Claim 11, characterized in that the fermentative biotransformation takes place as follows:

i. Inoculate starter cultures at a concentration of 0.5 - 2% (v / v), preferably 1% (v / v), and probiotic lactic acid bacteria at a concentration of 5 to 15% (v / v), preferably 5 % (v / v), and a minimum count of 106 - 107 log CFU / mL and a maximum of 1014 log CFU / mL in the EHS obtained in step

( ; and

i. Incubate the solution obtained in (i) from 30 to 40 ⁹ C, preferably at 37 ^Q C for 10 to 24 hours, preferably 12 hours under anaerobic conditions.

Process according to Claim 11, characterized in that the fermentative biotransformation followed by enzymatic occurs as follows:

i. Inoculate starter cultures at a concentration of 0.5 - 2% (v / v), preferably 1% (v / v), and probiotic lactic acid bacteria at a concentration of 5 - 15% (v / v), preferably at 5 % (v / v), and a minimum count of 106 - 107 log CFU / mL and a maximum of 1014 log CFU / mL in the EHS obtained in step

(D; ii. Incubate the cultures with EHS obtained in (i) between 30 and 40 ^Q C, preferably ^S to 37 C for 10 to 24 hours, preferably 12 hours, under anaerobic conditions;

iii. Inoculate in a solution obtained from (ii) with 1 to 5 U of tanase, preferably 1.8 U of tanase, per 1 ml of fermented EHS (ii);

iv. Incubate the solution of (iii) from 35 to 45 ° C, ^and preferably 40 C, for 20 to 50 minutes, preferably 30 minutes; and

v. Incubate the solution of (iv) between -2 to 2 ° C, preferably ^Q 0 C (ice bath) for 10 to 30 minutes, preferably 15 minutes;

Process according to Claim 11, characterized in that the enzymatic biotransformation takes place as follows:

i. EHS inoculum obtained in (I) with 1 to 5 U tanase, preferably 1.8 U tanase, per 1 ml EHS (I);

ii. Incubate the solution (i) from 35 to 45 ° C, preferably 40 ⁹ C for 20 to 50 minutes, preferably 30 minutes; and

iii. Incubate the solution of (ii) at -2 to 2 ° C, preferably 0 ^g C (in an ice bath) for 10 to 30 min, preferably 15 minutes;

Process according to claim 1, characterized in that in step (K) the preservation preferably takes place by freezing at -80 ° C.

Composition characterized in that it is obtained according to the process described in claims 1 to 15 and comprises Daidzine in aglycone form in a concentration between 90 and 946 Mg / ml, Genistein in aglycone form in a concentration between 50 and 323 Mg / ml and Equol in concentration between 0.30 and 0.83 Mg / mL.

Use of the composition as defined in claim 16 for use in soybean foods.

Use of the composition as defined in claim 16 characterized in that it is a nutraceutical product component.

Use of the composition as defined in claim 16 for use in the food industry as a food ingredient and in the nutricosmetics and dietary supplements industry.