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Agrociencia
versão On-line ISSN 2521-9766versão impressa ISSN 1405-3195
Agrociencia vol.50 no.2 Texcoco Fev./Mar. 2016
Food science
Evaluation of pulque sediment (xaxtle) as a starter culture in order to obtain a low glycemic index baked product
1 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica, Plan de Ayala y Carpio, Colonia Casco Santo Tomás. 11340. Delegación Miguel Hidalgo, México D. F. (alimentoselena@hotmail.com), (msanchezp@ipn. com.mx).
2 Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico Nacional Unidad Querétaro, Cerro Blanco 141, Colonia Colinas del Cimatario, 76090. Querétaro, México. (pedrovl70@hotmail.com).
3 Universidad Autónoma Metropolitana Unidad Xochimilco, Departamento de Sistemas Biológicos. Calzada del Hueso 1100, Colonia Villa Quietud, 04960. Delegación Coyoacán, México D. F. (lmayorga@correo.xoc.uam.mx).
Pulque bread is a traditional Mexican product that results from the fermentation by lactic acid bacteria (LAB) present in pulque or in its sediments (xaxtle). The objective of this study was to evaluate xaxtle as a starter culture in the fermentation of bread dough in order to obtain a low glycemic index baked product. Phenotypic and genotypic tests are used to identify bacteria of the genus Lactobacillus and yeasts of the genus of Saccharomyces as predominant organisms in xaxtle. Surface response methodology was used to obtain the optimum amount of lyophilized xaxtle, 3 g (~108 UFC·g-1) and the optimum time (90 min) of fermentation of the dough, considering the value D-optimum of the software DesignExpert. The microstructure of the bread dough fermented with xaxtle showed a partial hydrolysis of the protein matrix, conformed by gliadins and glutenins. Furthermore, the total starch content and available starch of the bread with xaxtle decreased by 9.8 % with respect to the bread made with S. cerevisiae (p=0.03) and the resistant starch was similar in both products (p=0.073). Fermentation with xaxtle reduced the hydrolyzed starch content in bread by approximately 4 %, thus classifying the product as low glycemic index bread. Therefore, the use of xaxtle as a starter culture in the fermentation of bread dough favored the reduction of the glycemic index due to the action of the microorganisms present.
Keywords: Lactobacillus; yeasts; bread; glycemic index
El pan de pulque es un alimento tradicional mexicano que resulta de la fermentación por bacterias ácido lácticas (BAL) presentes en el pulque o en sus sedimentos xaxtle. El objetivo de este estudio fue evaluar el xaxtle como cultivo iniciador en la fermentación de masas para panificación con el objetivo de obtener pan con índice glucémico bajo. Mediante pruebas fenotípicas y genotípicas se identificaron bacterias del género Lactobacillus y levaduras del género Saccharomyces, como microorganismos predominantes en el xaxtle. La metodología de superficie de respuesta se usó para obtener la cantidad óptima de xaxtle liofilizado, 3 g (~108 UFC·g-1) y el tiempo óptimo (90 min) de fermentación de la masa, considerando el valor D-óptimo del software Design Expert. La microestructura de la miga del pan fermentado con xaxtle mostró hidrólisis parcial de la matriz proteínica, conformada por gliadinas y gluteninas. Además, el contenido de almidón total y almidón disponible del pan con xaxtle disminuyó 9.8 % con respecto al pan elaborado con S. cerevisiae (p=0.03) y el almidón resistente fue similar en ambos productos (p=0.073). La fermentación con xaxtle redujo el contenido de almidón hidrolizado en el pan aproximadamente 4 %, clasificándose como un pan de índice glucémico bajo. Por lo tanto, el uso de xaxtle como cultivo iniciador en la fermentación de masas para panificación, favoreció la reducción del índice glucémico debido a la acción de los microorganismos presentes en el mismo.
Palabras clave: Lactobacillus; levaduras; panificación; índice glucémico
Introduction
In Mexico there are artisanally fermented products such as pulque, a traditional drink, which contain diverse microbial groups (Escalante et al., 2004, 2008; Lappe et al., 2008; Torres et al., 2014). Furthermore, Torres-Maravilla et al. (2015) identified and characterized bacteria of the genus Lactobacillus in xaxtle with probiotic properties. Xaxtle consists of the sediments accumulated during fermentation of the pulque and is used as a partial substitute for the yeast and water in artisanal bread-making in Mexico (Barros and Buenrostro, 2004). According to Escalante et al. (2004), lactic acid bacteria (LAB) are one of the most representative microorganism genera present in pulque. However, there is no data with respect to the effect of pulque or xaxtle, or a combination of both as a starter culture for the fermentation of bread dough. Sourdough technology is the use of the LABs in the fermentation of bread dough, which is a common practice for making Pannetone, Pandoro and Colomba of Italy (Corsetti and Settanni, 2007; Sanz-Penella et al., 2012); the type of French bread made in San Francisco (U.S.A.) and chemically acidified cookies in the U.S.A. (Arendt et al., 2007).
Liljeberg et al. (1996) indicated that the organic acids produced by the microorganisms of the fermented dough delay the gastric emptying process and reduce the glycemic index. Furthermore, in baked products fermented with sourdough (at fermentation times between 6 and 10 h), the concentration of resistant starch and lactic acid increased, whereas the concentration of available starch and the glucemic index decreased (Scazzina et al., 2009). The use of xaxtle as a starter culture in dough fermentation could be integrated to the sourdough technology for breadmaking, due to the beneficial effects of the LABs and yeasts present, according to Torres-Maravlla et al. (2015). Therefore, the objective of this study was to evaluate the effect of pulque sediments or xaxtle as a starter culture for obtaining low glucemic index baked products.
Materials and Methods
Raw material
In the region of Huiloapan, Tlaxcala, Mexico, 10 L of xaxtle were collected in March and April of 2013. The samples were placed in sterile jars and maintained between 4 and 7 °C during their transport to the laboratory (~6 h) to be lyophilized (Frezer-dryer SMH-50, Usifroid, Maurepas, France). The cell count was made before making each baking lot to verify the number of viable cells, using a Neubauer camera with staining for exclusion of the blue tripane stain (0.4 %), in order to differentiate live cells from dead cells (Baena et al., 2006).
Phytochemical characterization of xaxtle
The phytochemical characteristics were evaluated in fresh xaxtle prior to its lyophilization. The moisture content was measured using the reduced pressure method (Method 44- 15.02); total ash by the calcinations method (08-03.01); total protein by Kjeldahl (46-13.01); all of which are official methods of the AACC (2001). The values of pH were evaluated with a potentiometer (Denver Instrument pH meter AP5, Westminster, California, U.S.), and the reductive sugar content was determined according to the ICUMMSA manual (1964).
Isolation
For the count of colony forming units (CFU), the isolation of the LABs and of the yeasts, seried dilutions from 10-1 to 10-6 were used from 10 g of lyophilized xaxtle (gXL) in a phosphate regulating solution 0.1 M (pH 7.4). For the LABs, Man-Rogosa- Sharpe agar (MRS, Difco) was used, and for the yeasts, potato dextrose agar (PDA, Difco) and Sabourand dextrose agar (DS, Difco). The plates were incubated 48 h at 37 °C. The isolation was made with successive steps in the same culture media. The presumptive colonies were selected using Gram stain, and the catalase test for the case of the lactic acid bacteria, while malachite green stain was used for the yeasts.
Phenotypic and genotypic identification of the bacteria isolated from the xaxtle
A carbohydrate fermentation profile was used for the identification with API-50 CHL strips (bioMérieux, France. The biomass of the isolated bacteria was obtained from 2 mL of a culture of 12 h in MRS broth (Difco), which was used for the extraction of its DNA (Martín et al., 2007). For the DNA extraction of the yeasts, the biomass was directly harvested from the plates with agar using the phenol-chloroform method (Blin and Stafford, 1976).
The polymerase chain reaction (PCR) was carried out for the amplification of the gene 16S RNAr of the bacteria present in the xaxtle, using the following indicators: forward (5’-AGAGTTT-GATCCTGGCTCAG-3’) equivalent position 9-28 in Escherichia coli 16S RNAr) and reverse (5’GTTGCGCTCGTTGCGGGACT-3’), position 1109-1090 in E. coli 16S RNAr) (Ramírez-Chavarín et al., 2010). The PCR reaction was made with the following mixture of reagents: buffer 1X PCR (200 mM Tris-HCl, pH 8.0, 500 mn KCl); 1.5 mM MgCl2 pH 8.0; 200 mM of each dNTP; Taq polymerase (1.5U·mL-1); 20 pmol of oligos, 10 ng mL-1 of mold DNA; and distilled water was added to the mixture until 50 μL was obtained. The protocol consisted of an initial denaturalization phase at 94 °C for 1 min, alignment of the starter at 65 °C for 90 s, and lengthening at 72 °C for 2 min, as well as 15 additional min to finalize the reaction cycle. For yeast identification, we used the pair of universal starters ITS 5(5’-GGAAGT AAA AGT CGT AAC AAG G-3’) to LR6 (5’- CGC CAG TTC TGC TTA CC-3 ‘) (Fell et al., 2000; Sampaio and Gonçalves, 2008) under conventional conditions of PCR.
The reactions of PCR were carried out in BiometraTGradient thermocycler (Whatman, Biometra, Göttingen, Germany). The amplification products of DNA were confirmed in electrophoresis (0.8 % agarose) and were visualized through ultraviolet fluorescence after being stained with ethidium bromide. The purification of the PCR products was made with the QIAGEN gene cleanup kit and sequenced with the Applied Biosystems (ABI) Prism 3100 GeneticAnalyzer (Vernon Hills, Illinois, U.S.). The homologies were made in the nucleotides base BLAST (Basic Local AlignmentSearchTool) of the NCBI (National Center for Biotechnology Information, National Library of Medicine, U.S.A.).
Baking process
A bread model was made for all of the analyses with the following: 300 g of commercial wheat flour; 3.6 g of NaCl; 24 g of powdered whole milk; 60 g of fresh eggs; 16 g of fatty matter and 100 mL of sterile distilled water. The xaxtle content was added according to the design of surface response methodology (SRM). For the control bread, commercial yeast (Saccharomyces cerevisiae) was used. The ingredients were mixed for 10 min in a mixer (Kitchen-Aid Heavy-Duty, Model K5SS, U.S.A.); the dough was incubated at 37 °C with the time indicated in the experimental design. Next, the dough was divided in portions of 60 g and was fermented 30 min at 37 °C; and to obtain the bread, the doughs were baked 30 min at 180 °C in a swing oven (Henry SimonLimited, Cheshire, U.K.).
The count of colony forming units (CFU) of LAB and yeasts in the optimum dough (surface response methodology) was carried out according to what was described in the previous section. The pH and total titratable acidity (TTA) were determined in the optimum doughs, 10 g of dough were homogenized in 90 mL of distilled water; pH was registered with a potentiometer (AP5, Denver instrument, U.S.A.). The TTA was determined using NaOH 0.1 N until a final pH of 8.5 was reached, and was expressed in mL of NaOH 0.1 N necessary for titrating 10 g of dough (Sanz-Penella et al., 2012).
Surface response methodology (SRM)
SRM was used to determine the optimum conditions of amount of lyophilized xaxtle (gXL) and fermentation time (min) for making the bread. Two variables were used in this investigation: the amount of lyophilized xaxtle (0 to 10 g of xaxtle per 100 g of wheat flour) and the fermentation time (30 to 90 min) (Table 1).
Table 1 Independent variables with their variation level for fermentation using pulque sediment (xaxtle) during the bread making process.
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gt1.gif)
The upper and lower limits were obtained from preliminary studies. The design and analysis of SRM were carried out with the program DesignExpertPlot 7.0 (Stat-Ease Inc., Minneapolis, MN, U.S.A.). The program was based on 13 experiments with five replicates of the central point, four factorial points and four axial points. The response variables evaluated were as follows: specific volume (SV) of the bread with xaxtle, texture (Tx) of the cortex and luminosity (L) of the crumb. The significance of the models (SV, Tx, L) was made with an ANOVA to identify the coefficient of correlation (r) of each response. In addition, the optimization tool D-optimum of DesignExpertPlot was used to obtain the grams of lyophilized xaxtle and fermentation time for making the bread. The value of the D-optimum is the result of the combination of three equations of surface response (specific volume, texture, and luminosity).
Specific volume (SV)
Specific volume of the bread was obtained through the displacement of turnip seed, according to the official method (10-05.01) of the AACC (2001) and with the formula:
Specific volume (cm3 g-1) = Bread volume (cm3) x bread weight (g)
Texture (Tx)
Texture was evaluated with a penetrometer Model 327 Pressure Tester (EFFEGI, Italy), and the measurements (kgf) were made in the upper central part of the bread crust.
Luminosity (L)
Luminosity was measured in the bread crumb with the equipment Color Made HDS (model 347805, Milton Roy Company, Diano Color Products, U.S.A.).
Electronic scan microscopy (ESM)
The bread crumb with xaxtle and the control bread was cut in cubes of 4 mm with stainless steel sheets; the samples were placed introduced in a solution of glutaraldehyde at 2 % (pH 7) for 2 h at 25 °C, then were washed with a solution of saccharose at 1 % in a phosphate buffing solution 0.05 M pH 7.0 and were fixed with a solution of OsO4 at 1 % for 2 h. They were washed again with the sodium phosphate solution at 1 % (Bozzola and Russell, 1999). The samples were dehydrated in a series using solutions of ethanol at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 %, and three times with absolute ethanol (10 min of exposure for each concentration). The ethanol was totally substituted by CO2, using a dryer to critical point (Samdri 780 B, Rockville, MA, U.S.A.). The samples were covered with ionized gold particles (Desk II, Denton Vacuum, MA, U.S.A.) and observed in an electronic scan microscope (JEOL 5410LV, Peabody, MA, U.S.A.).
Determination of in vitro starch hydrolysis
Total starch concentration (TS) was determined with the method of Goñi et al. (1997), utilizing a conversion factor of glucose to glucane of 0.9 and the concentration of resistant starch (RS) was quantified by the method of Goñi et al. (1996). The potentially available starch (AS) was obtained from the difference between the TS and the RS.
The rates of starch hydrolysis in bread with xaxtle and the control bread were determined according to Granfeldt et al. (1992), using 1 g of potentially available starch (AS). The commercial bread was used as reference. The samples were incubated with saliva α-amylase (EC 3.2.1.1) from six healthy and fasted individuals (chewing them for 15 s), dissolved in 15 mL of phosphate regulating solution, 0.05 M, pH 6.9 and were incubated with pepsin (EC 3.4.23.1) at pH 1.5 (P-7012, Sigma-Aldrich, Saint Louis Missouri 6331 03, U.S.) at 37 °C for 30 min with agitation. The pH was adjusted to 6.9 for a second incubation (37 °C from 0 to 180 min) using a-amylase (EC 3.2.1.1) (A-3107; Sigma-Aldrich, Saint Louis, Missouri 6331 03, U.S.A.) in dialysis bags. The hydrolyzed and dialized products were quantified in equivalent of maltose with 3,5-dinitrosalisilic acid (D0550 Sigma Chemical, Perth, Western Australia). The prediction of glucemic index (pGI) was calculated using data of the hydrolysis index with the formula:
pGI = (0.862 x HI) + 8.19
Statistical analysis
All of the results are the average of at least three replicates. The groups of data were analyzed with ANOVA and t-student tests. The Tukey test (p≤0.05) was used to compare means with the control using the software Minitab Version 16.1.0 (Philadelphia State College, U.S.A., 2010). In addition, the software DesignExpertPlot 7.0 (Stat-Ease Inc., Minneapolis, MN, U.S.A.) was used as an optimization tool to define the optimum amount of xaxtle and fermentation time for bread making.
Results and Discussion
Phytochemical characterization of xaxtle
The xaxtle presented 95 g of water, 2.2 g of proteins, 0.6 g of ash, 546.8 g of glucose (reductive sugars), 1.1 g of lactic acid (shown by 100 g of sample) and pH of 4.1. These chemical components promote the growth of microorganisms such as the LABs, which are microorganisms that improve the characteristics of aroma and flavor in various fermented foods (Paramithiotis et al., 2006).
Identification of the microorganisms of the xaxtle
The quantification in 1 g of lyophilized xaxtle (gXL) was ~1x108 viable microbial cells, which were added to the doughs as culture starter, of which 7x106 UFC·g-1 were Gram positive bacteria-negative Catalase (MRS agar) and the remaining 4 x 105 UFC·g-1 yeast forming strains (PDA and SD agar), indicating that the lactic acid bacteria (Gram-positive and Catalase-negative) are in higher proportion in the xaxtle. The analysis of the amplified sequences of the gene 16S RNAr showed that the bacteria in the xaxtle had high similarity to the genus Lactobacillus, as well as presence of the yeasts S. paradox MESP1 and S. cerevisiae MESP2, whose sequences are in the GenBank with the access numbers KY954183 and KT954184. Escalante et al. (2004, 2008) indicated that the main microorganisms present in the final stage of fermentation of pulque are the Lactobacillus (88.1 % of the total clones). The LABs are important microorganisms in pulque because they can ferment glucose, fructose and saccharose, present in aguamiel, (fresh unfermented maguey sap) producing lactic acid (D or L). Furthermore, these microorganisms can resist high concentration of ethanol and grow in acid environments (pH 4.5) characteristic of pulque (Carr et al., 2002). Although the pH of the xaxtle was 4.1, the bacteria counts indicated a viability of 7x106 UFC·gXL-1. These results are similar to those obtained by Valadez-Blanco et al. (2012), with 7.1x107 UFC·mL-1 for pulque samples from Oaxaca.
Surface response methodology
Specific volume (SV)
Specific volume (SV) is an important characteristic in bread because it is a parameter of quality. The coefficient of correlation (r=0.82) of the model showed an adjustment of the experimental data and a p=0.36 (Tukey). The amount of xaxtle and fermentation time are closely correlated with the surface response represented by the multiple regression equation (Equation 1) of the specific bread volume, which indicates as much as 82 % association of the two factors (Figure 1).
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gf6.gif)
Figure 1 Analysis of surface response of specific volume (cm3·g-1) of bread fermented at different concentrations of lyophilized xaxtle and fermentation times.
SV = 1.23 + 9.37x10-3 gXL - 0.06x10-3t + 4.75x103 gXL2 + 1.09x10-4t2 -1.03x10-3gXLt
The use of 3 gXL as inoculum to ferment the dough from 30 to 90 min increased the specific volume (at 90 min, SV=1.31 cm3·g-1), but this was lower as compared to the control bread (SV=2.73 cm3·g-1). The above is related to the report of Rollán et al. (2005) that a pH of 3.5 to 4.0 in bread dough can activate the proteolytic enzymes, modify the gluten structure and produce a lower volume.
The dough inoculated with xaxtle presented 106 UFC per g of dough (108 UFC in 300 g of flour), of which 7x104 UFC·g-1 corresponded to the LABs and 4x103 UFC·g-1 to the yeasts. The count of UFC in the dough after 90 min of fermentation was 5.18x109 UFC·g-1 for the LABs and 1.3x105 UFC·g-1 for the yeasts. The amount of the LABs in the doughs fermented with xaxtle (3 gXL, 90 min fermentation) reduced (p=0.00) the pH from 6.1 to 4.5; whereas in the doughs inoculated with S. cerevisiae the pH was maintained constant at 6.1. Together, these results indicated that the LABs of the xaxtle can adapt to bread-making ingredients during fermentation. The lactic bacteria use various metabolic pathways to utilize the starch as carbon source, using the pathway of the pentoses, hexoses and pathway 6 phosphate gluconate/ketolase, producing lactic acid, acetic acid, in some cases CO2 and ethanol, as final products of the fermentation (Corsetti and Settanni, 2007). Also, there is a close relationship between the reduction of pH in the mother doughs and the increment of the UFCs of LAB, with a ratio of 1:100 of yeasts with respect to the LABs. The prevalence of the lactic bacteria over the yeasts is due to the competition for substrates, such as maltose, disaccharide that forms part of the chemical composition of the wheat flour. Paramithiotis et al. (2006) pointed out that in fermentation in mother doughs, L. brevis ACA-DC 3407 predominates over S. cerevisiae ACA-YC 5065, due to the fact that they present antagonism for maltose, reducing the growth of the yeasts and influencing the reduction of the pH by the production of organic acids (lactic acid and acetic acid). Furthermore, Gänzle et al. (2008) and Katina et al. (2005) indicated that the Lactobacillus have a negative influence on the production of CO2 by yeasts, giving as a result a low development of the alveolus (porosity) in the baked products, and therefore, low specific volume.
Texture (Tx)
In the surface response of texture (Figure 2), it was observed that the factor of the lyophilized xaxtle content has a greater influence on the texture of the product. The data were fitted in a quadratic equation (2), the coefficient of correlation (r=0.823) and the ANOVA of the model indicate that the experimental fit has a p=0.014.
Tx = 0.07 + 0.27 gXL + 0.02 t + 2.32x10-3 gXL-2 - 9.79x10-5t2 - 2.73x10-3gXL
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gf7.gif)
Figure 2 Analysis of surface response of texture (kgf ) of bread at different concentrations of lyophilized xaxtle and fermentation times.
In Figure 2 it is observed that with 3 g of lyophilized xaxtle for every 100 g of wheat flour and with a fermentation time of 90 min, the bread texture was 1.48 kgf, and for the bread made with 3 g of s. cerevisiae and 90 min of fermentation, the texture was 0.9 kgf.
The results are highly correlated with the values of specific volume: a bread with greater volume is due to the development of gluten (protein network), the formation of alveoli in the dough and to the CO2 produced during fermentation, making it less compact and therefore with a lower penetration force, which is related with a lower value of texture (Cauvain and Young, 2007). These results agree with what was described by Sanz-Penella et al. (2012), who show differences in the increase of penetration force (higher value of texture) in breads of dough fermented with Bifidobacterium pseudocatenulatum ATCC27919 at concentrations of 5, 10, 15 and 20 %, with respect to S. cerevisiae.
Crumb luminosity (L)
The bread crumb luminosity evaluated the degree of light reflected by this complex, porous and viscoelastic structure. The regression of the data was fitted to a quadratic model (Equation 3) and the coefficient of correlation was 0.92 (p=0.001). The variables had an influence of as much as 91 % on the behavior of parameter L (Figure 3).
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gf8.gif)
Figure 3 Analysis of surface response for luminosity (R.R.U.) of bread fermented at different concentrations of lyophilized xaxtle and fermentation times.
The quadratic equation is as follows:
L = 84.6 - 4.24 gXL - 0.22t + 0.17x10-3gX2 L + 9.58x10-1t2 - 0.19 gXLt
The content of xaxtle influenced the luminosity of the bread crumbs, from 55.6 to 71.2 R.R.U. (Relative Reflectance Units). That is, the higher the concentration of xaxtle, the lower the bread luminosity due to the color of the inoculum (brown) and to the lower moisture content, which is directly related to the parameter L. The results in our study were similar to those of fermented breads employing B. pseudocatenulatum ATCC 27919 with a luminosity interval of 55.5 to 57.8 R.R.U. (Sanz-Penella et al., 2012).
Optimum conditions for making xaxtle fermented bread
The optimum zone for baking bread with xaxtle corresponded to the superposition of the graphs of contour and the multiple regression equations of specific volume of the bread (cm3 g-1), texture (kgf ) and luminosity (L). The solutions proposed by DesignExpertPlot are shown in Table 2.
Table 2 Solutions to know the optimization of concentration of lyophilized xaxtle (gXL 100g-1 wheat flour) and fermentation time (min) of the dough.
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gt2.gif)
For specific volume of the bread with xaxtle, the optimum zone varied from 1.23 to 1.51 cm3·g-1, the texture between 1.73 and 1.85 kgf and crumb luminosity of 66 R.R.U. from 84 to 99 min of fermentation time and a lyophilized xaxtle concentration of 2.37 to 3.4 g per 100 g of wheat flour. According to the D-optimum of the program, the optimum conditions were 3 g of lyophilized xaxtle and 90 min of fermentation.
For specific volume of the bread with xaxtle, the optimum zone varied from 1.23 to 1.51 cm3·g-1, the texture 1.73 and 1.85 kgf and the luminosity of the crumb of 66 R.R.U. of 84 to 99 min of fermentation and a concentration of lyophilized xaxtle of 2.37 to 3.4 g per 100 g of wheat flour. According to the D-optimum of the program, the optimum conditions were 3 g of lyophilized xaxtle and 90 min of fermentation.
Microstructure of the bread crumb
In the microstructures of the bread with xaxtle and bread with S. cerevisiae, starch granules (SG) of different sizes were observed (Figure 4). In the bread with xaxtle there was a scant film of gluten (GF), probably due to the fermentation produced by the LABs present in the xaxtle (Figure 4a and 4b). The LABs can modify the structure of the baked products due to their proteolytic activity (Gänzle et al., 2008). The bread crumb with xaxtle was more compact than the bread crumb elaborated with S. cerevisiae, and according to Kusunose et al. (1999), in the interaction between yeasts and lactic acid bacteria, there is a reduction in CO2 due to the competition for substrates.
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gf9.gif)
Figure 4 Micrographs of the crumb of A) and B) bread with xaxtle; C) and D) bread with Saccaromyces cerevisiae. SG starch granules. GFB gluten fibrils and GF gluten film. 1000x (A, C), 2000x (B, D). Bar 10 μm.
Sanz-Penella et al. (2012) observed that during the fermentation of doughs with LAB, the protein matrix and the starch granules are degraded, significantly reducing the specific volume of the baked products. The doughs inoculated with xaxtle had a significant production (p=0.00) of TTA (organic acids) of 11.3 mL NaOH 0.1 N higher than the doughs fermented with S. cerevisiae with a TTA of 2.3 mL NaOH 0.1 N. The total titratable acid of the doughs with xaxtle influenced the formation of the structures during kneading, given that the development of the gluten is affected by this acidity, which is shown in the microstructure of the bread crumb (Figure 4). The development of the gluten and the corresponding formation of the structure are due to the fact that during the incubation of the doughs, biochemical changes take place, such as lactic, acetic and alcoholic fermentation, in addition to a proteolysis due to the microbial endogenous enzymes that are activated by the drop in pH (Corsetti and Settanni, 2007). The proteolytic system of the LABs hydrolyzes the proteins to small peptides and amino acids, which are important for the growth of these bacteria. According to Zotta et al. (2007), the gliadins, one of the proteins of gluten, can be fragmented to a size of 20 to 27 kDa.
The structure of the bread crumb elaborated with S. cerevisiae (Figure 4C and 4D) was less dense than that of the bread elaborated with xaxtle (Figure 4A and 4B). Furthermore, in the control bread a well developed film of gluten was observed with some empty spherical spaces that correspond to alveoli of gas formed by the production of CO2, and it was found that the starch granules (SG) are covered by the protein network. The protein network was totally developed in the bread with S. cerevisiae forming PG in the structure, while the bread with xaxtle was not very well developed, which is related to the effect of the proteolytic system of the LABs (Corsetti and Settanni, 2007).
In vitro hydrolysis of the starch
The total starch content of the bread with xaxtle was 9.8 %, significantly (p=0.003) lower than the bread with S. cerevisiae (Table 3). This may be explained by the fermentation of the starch by LAB, and this starch is used as substrate to produce organic acids (Liljeberg et al., 1996). The total resistant starch content in the bread with xaxtle was 3.57 %, similar to the bread with S. cerevisiae (p=0.067). The bread with xaxtle presented an available starch content of 3.93 %, significantly lower (p=0.003) than the bread with S. cerevisiae. Liljeberg et al. (1996) reported an increment of resistant starch and lactic acid in products made with sourdough (more than 5 h of fermentation).
Table 3 Total starch content (TS), total resistant starch (TRS) and available starch (AS) of bread (g per 100 g of sample) fermented with xaxtle and bread fermented with S. cerevisiae.
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gt3.gif)
aART-AT = AD. Different letters in a row indicate significant difference (p≤0.05).
The hydrolysis curves are expressed in percentage of hydrolyzed starch (%H) during the in vitro hydrolysis (Figure 5). The white sliced bread of a commercial brand was used as reference in this analysis, with a percentage of hydrolysis of 25.8 %. Osorio-Díaz et al. (2005) indicated that the white bread (of reference) had a similar hydrolysis after 180 min of incubation (28 %). The behavior of the curves shows that after minute 60, the hydrolysis of the bread with xaxtle presented significant difference (p=0.025) with respect to the bread with S. cerevisiae.
![](/img/revistas/agro/v50n2//1405-3195-agro-50-02-00183-gf10.gif)
Figure 5 Percentages of hydrolysis of the starch determined after incubation with pepsin (EC3.4.23.1) and with α-amylase (EC3.2.1.1) in white commercial bread (reference), bread made with xaxtle, bread made with commercial yeast (Saccharomyces cerevisiae).
The behavior of the degree of hydrolysis of the bread with xaxtle was influenced by the reduction of the pH and the hydrolysis of the starch granules by the microorganisms of the xaxtle. In some cases, according to Scazzina et al. (2009), the use of sourdough improved the response of glucose in blood, where the organic acids delayed the gastric emptying, even without affecting the accessibility of the starch.
The areas below the curve of the hydrolysis of the starch for the bread with xaxtle and for the bread with S. cerevisiae were correlated with the area of the white commercial bread (reference) and the prediction of glucemic index (pGI) was calculated. The pGI of the bread with xaxtle was significantly lower (p=0.03) with 51.8 than the bread with S. cerevisiae with 56.2. The reduction of the pGI may be due to the metabolic action of the lactic acid bacteria of the xaxtle. the content of organic acids in the doughs with xaxtle favored a reduction of the pGI (Åkerberg et al., 1998; Scazzina et al., 2009). The TTA of the dough inoculated with xaxtle was higher (11.78 ± 0.2 mL of NaOH), the lactic acid content was higher for the dough with xaxtle (0.11 ± 0.002 g 100 g-1 of dough), than for the dough with S. cerevisiae (0.02 ± 0.003 g·100 g-1 of dough). The same behavior was observed for the acetic acid with 0.07 ±0.001 g 100 g-1 and 0.016 ± 0.002 g 100 g-1 of dough, respectively.
According to the values of the pGI and the classification of Bjorck et al. (1994), the bread made with xaxtle has a low glucemic index. Besides, a food of low GI produces a higher degree of satiety, due principally to the fact that the hydrolysis and the assimilation of the carbon hydrates is lower than in the foods with higher GI Noriega et al. (2001).
Conclusions
The optimum amount of xaxtle added and the fermentation time of the dough during bread making favored the reduction of pH and the increase in acidity from the lactic acid bacteria, thus influencing the structural changes, the available starch content and the reduction of the glucemic index of the bread. The use of xaxtle as inoculum in bread making could be utilized in the fermentation of dough to obtain low glucemic index baked products.
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Received: October 2014; Accepted: November 2015