Solid-state fermentation using wheat bran to produce glucose syrup and functional cereal bars

doi:10.21203/rs.3.rs-3616675/v1

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Solid-state fermentation using wheat bran to produce glucose syrup and functional cereal bars

https://doi.org/10.21203/rs.3.rs-3616675/v1

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published 13 May, 2024

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Wheat bran is one of the most abundant by-products from grain milling, which can be applied as substrate for solid-state fermentation to obtain enzymes able to convert this agro-industrial residue into glucose syrup, which in turn can be used for the production of different food products. Present study aimed to determine centesimal composition of wheat bran, obtain enzymatic extract that converts wheat bran into wheat glucose syrup, produce rice flakes cereal bars using glucose syrup, and evaluate their nutritional composition and the presence of functional compounds, as well as their antioxidant potential. Cereal bars were evaluated to determine reducing sugars, soluble proteins, and total polyphenols and flavonoids content. Antioxidant evaluation and enzymatic extract obtained presented amylolytic activity and it was able to produce cereal bar. Produced with wheat glucose syrup proved to be a source of dietary fiber (1.8 g) and soluble protein (7.2 g) while the rice flakes produced with corn glucose syrup did not present these nutritional components. Cereal bar produced with glucose syrup presented phenolic compounds, among them flavonoids. In addition, they exhibited antioxidant activity by radical scavenging (47.46%), reduced ion iron (71.70 µM Trolox equivalent/g) and radical stabilization (711.89 µM Trolox Equivalent/g). Final product maintained organoleptic profile characteristic of cereal bars and it showed a decrease in caloric value and sodium content. Therefore, the present study showed that the bioprocess yields a nutritional, ecological, and functional food product, which might be of great interest for food industry, adding nutritional and functional value to a well-stablished product.

Agro-industrial residue

Bioactive compounds

Cereal bars

Convenience food

Rice flakes

Wheat bran is the main and most abundant by-product of grain milling and it consists of a residue that can be considered a renewable food resource [1, 2]. This waste product is produced in large quantities worldwide, although only 10% of wheat bran generated is used to supplement human food in food industry, while the rest is used for animal feed or as soil fertilizer, which are considered low added-value byproducts [3]. Wheat bran is composed of the outermost layer of the wheat grain, known as pericarp, which surrounds the endosperm and germ [4, 5]. During conventional milling, the bran is separated from the endosperm by milling, sifting, and purification processes that result in the refined wheat flour, which is used to produce a wide range of food products derived from wheat [6, 7].

Microorganisms have been used to produce several products, including industrially important biomolecules, although, they have been applied to bio-recycle agro-industrial residues. The low cost of microorganisms allows the development of economically competitive and eco-friendly processes [8, 9]. In recent years, there has been an increase in the number of studies aimed to identify possible uses of agro-industrial wastes to generate high added value products [10–12]. The findings in this field are highlighted by the attempts to find solutions for environmental problems and to reduce bioprocesses costs [13, 14]. Solid-state fermentation (SSF) is one of the processes in which microorganisms and agro-industrial residues can be used since the physico-chemical nature of these substrates facilitates colonization by microorganisms [15, 16].

SSF is a bioprocess that uses a moist, solid, non-soluble organic material that acts as a support and nutrient source for the growth of microorganisms [17, 18]. This process is considered an important approach for bioconversion of agro-industrial wastes. Several industries are benefited by SSF such as pharmaceutical, chemical, and food industries that apply this process to produce useful industrial products [17, 20, 21]. Currently, industries have been investing in research and new bioprocess technologies capable of using agro-industrial residues to obtain high added value products such as organic acids, antibiotics, bioethanol, enzymes, secondary metabolites, biofuels, and biosurfactants [19, 21, 22–24].

The operation of SSF can be used to produce different enzymes of industrial interest, mainly amylases that are generally used in the degradation and transformation of substrates composed of lignocellulosic biomass into concentrated sugar syrups [25–27]. The lignocellulosic biomass is a rich and chemically complex material whose structure is represented by the physico-chemical interaction of cellulose, hemicellulose, and lignin [28]. In order to convert lignocellulosic biomass into sugar syrups by enzymatic hydrolysis a pretreatment step must be undergone to alter its complex structure [29–31]. The pre-treatment is responsible for exposing the chains of cellulose to enzyme to convert the polysaccharide into glucose molecules [32, 33].

Glucose syrup is a type of sugar widely used in food industry, commonly applied as a raw material in several industries including confectionery, beverage, jelly, pharmaceutical, and chemical industries [34–36]. Industries widely use glucose syrup from corn; however, glucose syrup obtained from other sources are important to diversify and increase the supply of material for industrial production. In addition, these raw materials may add functional compounds, increasing nutritional and economical value. Besides, glucose syrup produced from agro-industrial residues also presents ecological and sustainable solutions [37–39].

Convenience foods refer to foods that have been gaining market popularity due to easy preparation and consumption, reducing the need of time, effort, and cooking skills [40–43]. Cereal bars are bar-shaped convenient food products, usually prepared by pressing whole or flaked cereals with dried fruit, candied fruit, or berries, which are in most cases held together by glucose syrup [44, 45]. They can be produced using several cereals as basis for the other ingredients, among them rice is commonly used in the form of flakes that are prepared with dehusked rice and consequently are easy to digest. Besides, production process of rice flakes involves rice roasting, which increases shelf life of these flakes [46, 47]. Rice flakes can be used in different preparations and they are rich in carbohydrates, proteins, vitamins, minerals (Ca, K, Na, Mg, and Fe) and fiber; however, their composition varies depending on the rice varieties and processing methods [48].

One of the most important ingredients in the production of cereal bars is a glucose syrup, which can bind, soften, and sweeten the final product. Glucose syrup is obtained from the hydrolysis of starch, usually obtained from tubers and cereal grains, such as cassava and corn. However, its composition is basically formed by sugars (glucose and sucrose) and do not attribute functional value to the products. Considering that, other sources of starch to obtain glucose syrup have been considered, including some fruits, seeds and roots [30, 49]. In addition, studies have been developed in order to use by-products and agro-industrial residues as source of starch, which could represent economic and ecological gain by reducing the environmental impact caused by the residues and producing an added value product [50]. Among these residues is the wheat bran, currently used to produce animal feed, although studies have shown that this by-product can be used in other industries, especially food industry [51, 52].

Given the above, consumers have been increasingly concerned about their health, looking for food options that may contribute to health benefits and disease prevention. In recent decades, consumers have demanded “clean label” foods, an eco-friendly movement aimed at improving quality of foods [53]. Considering that, the present study aimed to produce enzymes by SSF bioprocess and subsequently generate wheat glucose syrup (WGS) to incorporate in rice flakes for the production of rice flakes cereal bars (RFCB). It also aimed to determine nutritional composition, and evaluate the presence of functional compounds and their antioxidant potential.

2.1 Obtaining wheat bran

The wheat bran was provided by Moinho Nacional Ltda. (Assis, São Paulo, Brazil), in form of powder (bran) packaged in vacuum bags. The wheat bran obtained by extrusion process, where 86% of the bran was retained on 14 and 24 mesh sieves, which corresponded to particle sizes that varied from 1.18 and 0.71 mm. Wheat bran presented initial moisture content of 55% and it was stored in a cold chamber at -10°C.

2.2 Centesimal characterization of wheat bran

2.2.1 Total starch determination

Starch determination was carried out following colorimetric method described by Freitas et al. [54]. Samples were homogenized at 80°C for 20 min before analysis. 4.8 mL HCl (0.1 M) and 0.1 mL of sample were mixed in reaction tubes and 0.15 mL of iodine solution (0.5 g/L of sublimated iodide and 1 g/L KI) were subsequently added. Absorbances were read at 620 nm using a spectrophotometer (BelPhotonics Brazil) and results of starch quantification were determined by means of a standard curve plotted with starch solution (Sigma Aldrich, Brazil) gelatinized from 0 to 8 mg/mL.

2.2.2 Moisture determination

Moisture was determined by gravimetric method. Samples were placed in pre-weighted dishes, which were heated in an oven at 105°C until a constant weight was reached. The difference of weight before and after heating process was used to determine moisture content.

2.2.3 Ash determination

Ash determination was carried out by gravimetric method. Samples were placed in pre-weighted crucibles, which were incinerated in a muffle furnace (Sppencer, Iperó, Brazil) at 550°C for 5 h. Then, samples were maintained in a desiccator until room temperature was achieved. The difference of weight before and after heating process was used to determine ash content [55].

2.2.4 Lipid determination

Soxhlet flask was dried at 105°C, weighted on an analytical balance and coupled to the extractor with petroleum ether. Three grams of each sample was transferred into folded filter paper that was then placed in the extractor. The apparatus was cooled to 20°C in a water bath for condensation and the flask was heated using a thermal blanket. Samples were kept for 10 h in ether washes. Afterwards, the flask was dried at 105°C and maintained in a desiccator until room temperature was reached. The difference of weight between empty flask and flask with lipids was used to determine lipids content [56].

2.2.5 Protein determination

Protein determination was performed according to AOAC [57]. 0.1 g of sample was mixed with 1 g of catalyst and 3 mL of sulfuric acid in digestion tubes and then the mixture was heated at 350°C. After cooling, 5 drops of phenolphthalein were added, and tubes were connected to distillation apparatus. NaOH (40%, w/v) was added to the digested sample and the distillate was collected in an Erlenmeyer containing 10 mL of H₃BO₃ solution with 2 drops of methyl red and bromocrezol green. The distillate was titrated against 0.02 N HCl solution until a pink color was observed.

2.2.6 Crude fiber determination

The analysis of total crude fiber content was performed according to AOAC [56]. Using acidic and basic solutions for digestion of carbohydrates, lipids, among other compounds for fiber determination.

2.2.7 Carbohydrate content

The carbohydrate content was determined by the calculation of the difference between measured values of moisture, ash, proteins, and lipids subtracted from 100% [58].

2.3 Obtaining amylolytic enzymes

2.3.1 Preparation of microorganism and inoculum

Rhizopus oligosporus (CCT 3762) was acquired from Fundação Tropical de Pesquisas e Tecnologia “André Tosello”, Campinas, São Paulo, Brazil. Fungal strains were cultivated in plates containing potato dextrose agar (PDA) (Difco, USA) for 7 days at 30°C. Viable spores from the culture were harvested under sterile conditions by flooding the plates with Tween 80 in ultrapure water (0.01%, v/v). The spore suspension was used as inoculum for solid-state fermentation (SSF).

2.3.2 Solid-state fermentation (SSF)

SSF was performed in 250 mL erlenmeyer flasks containing 10 g of wheat bran (dry basis), moistened with an aqueous nutritive solution (ammonium sulfate (1.25%, w/w), monobasic potassium phosphate (0.25%, w/w), and urea (1.25%, w/w)) optimized for the microorganism under study [59]. Substrate moisture was adjusted to 55%, followed by sterilization (121°C, 1 atm, 20 min) and aseptic inoculation with 10⁶ spores per gram of substrate. After fermentation, the enzymes were extracted using 10 mL distilled water per gram. The mixture was shaken for 30 min at 28°C in an orbital shaker operating at 180 rpm. Then, enzyme extract was filtered and used to measure enzymatic activity [60]. (Escaramboni et al., 2018).

2.3.3 Amylolytic activity

Amylolytic activity was measured using 5-dinitrosalicylic acid (DNS) method described by Miller [61]. 100 µL of enzyme extract and 650 µL of substrate (soluble starch (0.5%, w/v) in 0.05 M sodium acetate buffer (pH 5.5)) were incubated for 10 min at 60°C. Then, 500 µL of DNS solution were added and the mixture was heated in boiling water bath for 5 min and diluted in 3.75 mL distilled water. Absorbances were measured at 540 nm using glucose as standard. One unit of enzyme activity was defined as the amount of enzyme that releases one µmol of reducing sugars per minute and results were expressed as units per gram of dry substrate (U/g).

2.4 Obtaining WGS

2.4.1 Gelatinization of wheat bran using enzymatic fermentation broth

The gelatinization was carried out with constant agitation and varying time (30, 60, or 90 min), temperature (70, 80, or 90ºC), and wheat bran concentration (5, 10, or 15%) to determine the best condition for gelatinization [62].

2.4.2 Enzymatic hydrolysis and production of WGS

For the analysis of enzymatic hydrolysis of wheat bran, the enzymatic extract produced in SSF was used. The substrate (wheat bran (10%, w/v) in 0.05 M sodium acetate buffer (pH 4.5)) was gelatinized at 70°C for 30 min. After that, temperature was reduced to 50°C for the addition of 15 U/g of enzymatic extract and the mixture was incubated at 24 h. After the reaction, hydrolysate was boiled for enzymatic inactivation and centrifuged at 3000g for 15 min. The supernatant was removed, and the reducing sugar was measured by the DNS method and total reducing sugars was evaluated by the method described by Miller [61]. After this process, the resulting WGS was concentrated in an evaporator.

2.5 Obtaining raw material for the production of RFCB

The ingredients to produce the RFCB were purchased from local specialized stores in Assis, São Paulo, Brazil (-22°39′40″ S and − 50°23′58″ W, at 560 m) except for WGS, which was produced in our laboratory by enzymatic hydrolysis of wheat bran. Rice flakes, white sugar, water, and corn glucose syrup (CGS) or WGS were used to obtain the RFCB. CGS (commercial product) was used as control.

The RFCB were prepared using 100g rice flakes, 100g white sugar, 15 mL water and 30 mL glucose syrup (corn or wheat) (Fig. 1). Sugar and water were mixed and heated to an average temperature of 120°C until caramelization. After that, glucose syrup (corn or wheat) was added and when the mixture reached point of candy, rice flakes were added. The ingredients were incorporated to form a homogeneous caramelized mass that was poured evenly onto a stainless-steel surface coated with non-stick baking paper. After cooling, the prepared mixture was cut into bars of 7 cm × 5 cm × 1 cm. RFCB were wrapped in plastic film and stored at room temperature (Fig. 2).

2.6 Determination of bioactive compounds of WGS, RFCB and rice flakes

2.6.1 Determination of total polyphenols content

Total polyphenols content was evaluated following Folin-Ciocalteau method as reported by Singleton and Rossi [63]. 250 µL samples (10 mg/mL), 2.5 mL distilled water, and 125 µL Folin-Ciocalteau reagent were mixed. Then, 500 µL 10% Na₂CO₃ were added, and the mixture was incubated for 1 h. Absorbance was measured at 765 nm and total polyphenols content was expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/mg DW).

2.6.2 Determination of total flavonoids content

Total flavonoids content was evaluated by aluminum chloride method as reported by Christ and Müller [64]. 250 µL samples (10 mg/mL), 1.25 mL distilled water, 75 µL 5% NaNO₂, and 150 µL AlCl₃ were mixed. Afterwards, 500 µL 1M NaOH was added, and the volume was completed with 2.5 mL distilled water. Absorbance was measured at 510 nm and total flavonoids content was expressed as mg rutin equivalent per gram of dry weight (mg RE/mg DW).

2.6.3 Determination of total soluble proteins content

Total soluble proteins were determined by Bradford method as described by Bradford [65]. 10 µL samples (0.1 mg/mL), 790 µL distilled water, and 200 µL Bradford reagent were mixed. The mixture was incubated at room temperature for 5 min and absorbance was measured at 595 nm. The total soluble proteins content was calculated using a calibration curve plotted with bovine serum albumin (BSA).

2.6.4 Determination of reducing sugar content

Reducing sugar was quantified by DNS method as described by Miller [61]. 1 mL samples and 500 µL DNS solution was added and the mixture was placed in a water bath at 100°C for 5 min. Then, the mixture was diluted in 3.75 mL distilled water and absorbances were read at 540 nm using glucose as standard. An enzymatic unit was defined as the amount of enzyme that releases 1 µmol of glucose per minute of reaction.

2.7 Evaluation of antioxidant activity of WGS, RFCB and rice flakes

2.7.1 DPPH free radical scavenging activity

Antioxidant activity was carried out using DPPH method according to Rufino et al. [66]. 1 mL of acetate buffer (100 mM/pH 5.5), 1.25 mL ethanol, 250 µL DPPH (500 µM) and 50 µL of samples were mixed and kept in dark for 30 min. Absorbances of samples were read at 517 nm using a spectrophotometer (BelPhotonics Brazil). Results were calculated following the formula: Antioxidant activity (%) = [(Control − Samples)/Control] × 100, where Control is the absorbance of control, Samples is the absorbance of samples.

2.7.2 Iron ion reducing potential (FRAP)

Antioxidant evaluation by FRAP test was conducted according to Guimarães et al. [67]. 2.7 mL FRAP reagent (acetate buffer (300 mM/pH 3.6) + TPTZ (10 mM/40 mM HCl) + FeCl₃.6H₂O (20 mM) at 10:1:1 (v/v/v)), 270 µL ultrapure water and 90 µL of samples were mixed in a test tube. This solution was heated for 30 min at 37°C and then absorbances were read using a spectrophotometer at 593 nm. Results were calculated and expressed as µM Trolox Equivalent (TE) per gram of sample.

2.7.3 ABTS radical scavenging test

Antioxidant activity was conducted according to Re et al. [68] by the evaluation of ABTS radical scavenging potential. 7 mM ABTS and 2.45 mM potassium persulphate were mixed for the preparation of ABTS solution, which was incubated 12-16h at 23°C in dark. Afterwards, this solution was mixed with 80% ethanol until it reached absorbance of 0.700 at 734 nm obtained with a UV-Vis spectrophotometer. 0.1 mL of samples were added to 3.9 mL of ABTS solution, which was vigorously vortexed and kept at 23°C for 6 min. Absorbances were measured at 734 nm and results were presented as µM Trolox Equivalent (TE) per gram of sample.

2.8 Design of Nutrition Facts label

Nutrition Facts label was designed according to the Brazilian Food Composition Table [69]. The data obtained from the analyses and nutritional information from the raw materials served as basis to calculate and to indicate micro- and macronutrients content of RFCB to be included in nutrition facts label including calories (Kcal), carbohydrates, proteins, total fat, saturated fat, trans fat, dietary fiber, and sodium.

2.9 Statistical analysis

Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey test using BioEstat 5.0 software.

3.1 Centesimal characterization of wheat bran

Centesimal characterization of wheat bran evaluated in this study demonstrated a starch concentration of 52%, which was lower than values obtained by Onipe et al. [52], Shang et al. [70], Ye et al. [71] and Babu et al. [72] that reported starch concentration greater than 80% in wheat bran. However, Kong et al. [73] and Ye et al. [71] reported lower starch concentration percentage than that observed in the present study. This variation in starch concentration can be attributed to grain processing stage, which may present a higher or lower values according to the desired final product.

Wheat bran presented a moisture content of approximately 14%, which differed from study performed by Shang et al. [74] that showed 8.1% moisture content in wheat bran. Moisture content is a parameter directly correlated to storage, packaging, and transportation of grains; thus, the variation of values may be found considering that wheat bran is an agro-industrial residue exposed to several conditions. Wheat bran showed 3.83% ash content and this value is in accordance with that found by Shang et al. [74], which evaluated the presence of mineral components and reported 3.9% ash content.

Wheat bran presented 2.15% lipid content, which was lower than values observed by studies conducted by Yadav et al. [75] and Ye et al. [71]. According to Babu et al.[76] lipids in wheat bran, even at low concentrations, can be beneficial to animal and human health due to their quality and specificity to consumer lipid metabolism. Wheat bran also showed 12.89% crude fiber content and this value indicates that this agro-industrial residue is an important source of fibers due to the presence of cellulose, hemicellulose, and lignin. However, Babu et al. [76] and Ye et al. [71] reported an approximate percentage of 43.24% fiber content in wheat bran, which demonstrates that this residue may present high percentage of fibers under specific conditions, and it reinforces the possible application of the wheat bran in animal and human nutrition.

Wheat bran presented 18.29% protein content, which was higher than that observed by Papageorgio and Skend [77] and Kaur et al. [78] that evaluated this residue in different conditions and reported 8.14% protein content. The carbohydrate content was determined by the difference between moisture, ash, lipid, and protein content, and it showed 84.85%. This value is in accordance with that reported by Javed et al. [79] and Babu et al. [76] and it indicates that the wheat bran evaluated in this study is a rich source of carbohydrates. The presence of carbohydrates may can favor extraction and gelatinization processes, besides it may present viscosity gain and water binding capacity, highlighting this residue as an important source of nutritional compounds of great interest for food industry.

3.2 Obtaining amylolytic enzymes

3.2.1 Solid-state fermentation (SSF)

The development of an economically viable bioprocess seeks to maximize enzyme production with the lowest cost [80, 81]. The use of agro-industrial residue as substrate and minimal nutrient supplementation leads to a low-cost bioprocess [82]. In this study, enzyme production was significantly influenced by fermentation time, which lasted approximately 120 h at 37°C. The substrate initially presented a brown color before inoculation due to the sterilization process; however, it acquired a darker color during fermentation process because of R. oligosporus growth in the solid matrix. The color and aroma changes in the substrate (wheat bran) may indicate that the fermentation has occurred. It also demonstrates that the presence of nutrients dispersed throughout the fermentation process may have contributed to the growth of the microorganisms and consequently to the conversion of the nutrients from agro-industrial waste into added value product. Therefore, it was observed that the parameters for SSF defined in the present study were ideal for obtaining amylase. Similar studies performed by Andrade [83] and Prado et al. [84] also showed promising results in solid-state fermentation using 5 strains of Rhizopus sp. on different substrates, although, they did not exhibit the same capacity to produce enzyme, which may be related to fermentation parameters such as time and temperature.

3.2.2 Amylolytic activity

Fungal amylase production was conducted on a laboratory scale model suitable to produce amylolytic enzymes, resulting in 279.92 U/g in the present study. This result is in accordance with Bachi et al. [85] that demonstrated that wheat bran is a good source of nutrients to produce amylase by fermentation process. The result is like those obtained by Escaramboni et al. [60] and Yang et al. [86] who used the same microorganism and substrate to obtain amylolytic enzyme. According to Fernández Nuñez et al. [87] and Melnichuk et al. [88] the production of amylase using agro-industrial waste is a promising approach, indicating that enzymes obtained by SSF in this study provide a possible application for the food industry.

3.3 Obtaining WGS

3.3.1 Gelatinization of wheat bran using enzymatic fermentation broth

The gelatinization of wheat bran was monitored through the determination of different parameters, and the best conditions were observed at 70°C for 30 min (Fig. 3) using 15 grams of wheat bran. The efficiency of conditions was evaluated by a standard curve of iodine equation: y = 0.0203x + 0.0985 (R² = 0.9974) and the result is in accordance with study conducted by Freitas [54] that used cassava starch and wheat bran as substrate, R. oligosporus as microorganism, and obtained the best condition at 60 to 80°C for 5 h.

3.3.2 Enzymatic hydrolysis and production of WGS

According to the profiles of enzymatic hydrolysis, a gradual decrease in starch hydrolysis was observed through time (Fig. 4). The highest conversion occurred in the 1st hour where it exhibited 2.87 mg/mL reducing sugar (glucose) and 47% starch hydrolysis. The lowest conversion was observed at the 7th hour presenting 0.53 mg/mL reducing sugar and 13% starch hydrolysis. These results corroborate with studies conducted by Escaramboni et al. [89] and Freitas et al. [54] that demonstrated 96% starch hydrolysis and conversion into glucose using amylase obtained by SSF using R. oligosporus, although, they required a period of 48h.

3.4 Production of RFCB

The RFCB produced with either CGS or WGS exhibited similar organoleptic characteristics such as consistency, flavor, and taste, generating a crunchy and sweet product. However, these cereal bars presented different colors (cereal bars with CGS showed a dark caramel color and cereal bars with WGS showed a light caramel color) (Fig. 5). Studies performed by Vollmuth [90], Kocadağlı and Gökmen [91] and Aydın et al.[92] demonstrated that the caramel color of food products is directly correlated with the amount and type of sugar. In addition, they demonstrated that antioxidant compounds in the product might result in a light caramel color, which was observed in the present study.

3.4 Determination of bioactive compounds in WGS and cereal bars

In the reducing sugars analysis, the RFCB produced with WGS presented 3.43 mg/mL and cereal bars produced with CGS exhibited 5.85 mg/mL. Studies carried out by Srebernich et al. [93] and Klerks et al. [94] showed that RFCB that undergo caramelization have less than 1% reducing sugars, considering that most of sugars in the cereal bar are sucrose, a non-reducing disaccharide. Although, the decrease of reducing sugars content in food products is highly recommended, taking into account that the consumption of these type of sugars cause damage to human and animal health, as demonstrated by Stanner and Spiro [95] and Huang et al. [96]. In the present study, it was possible to observe that RFCB produced with WGS maintained its organoleptic characteristics and exhibited a decrease of 41.6% in reducing sugars content compared to RFCB produced with CGS.

In the total soluble proteins determination, RFCB produced with WGS presented 165.9 mg BSAE/mL of total soluble proteins content, while cereal bars produced with CGS did not present total soluble proteins in their composition. Similarly, rice flakes did not exhibit total soluble proteins content. According to Garcia et al. [97] and Shanmugam et al. [98], total proteins content in products derived from rice grains depends on the industrial processes for which they go through after harvest. Patle et al. [99] demonstrated that species and varieties of rice significantly interfere with protein composition of rice flakes. Thus, it was possible to observe that RFCB produced with WGS exhibit total soluble proteins content, which could be attributed to the presence and incorporation of soluble proteins from WGS.

Table 1 shows total polyphenols and flavonoids content at different concentration of WGS. The syrup presented the highest content at 25% (589.25 mg GAE/mg for polyphenols and 152.36 mg RE/mg for flavonoids) and it showed a dose-dependent behavior. On the other hand, CGS (control) did not show total polyphenol and flavonoids content at the same concentrations evaluated for WGS.

Table 2 shows total polyphenols and flavonoids content of rice flakes and RFCB produced with WGS. Rice flakes did not exhibit total polyphenols and flavonoids content at any evaluated concentration, while the RFCB showed a concentration-dependent manner. The highest total flavonoids content (85.5 mg rutin equivalent/g DW) were observed at 25% and total polyphenols content (43.96 mg gallic acid equivalent/g DW) were observed at the same concentration, although, it did not differ statistically from cereal bars at 10%.

Table 1

Determination of total polyphenols and flavonoids content of wheat glucose syrup (WGS) and corn glucose syrup (CGS).
Concentration (%)	Total polyphenols¹		Total flavonoids²
Concentration (%)	WGS	CGS	WGS	CGS
1	181.88 ± 3.24a	ND	50.20 ± 1.12a	ND
5	363.00 ± 1.19b	ND	71.25 ± 1.10b	ND
10	574.00 ± 2.25c	ND	128.20 ± 3.17c	ND
25	589.25 ± 2.20d	ND	152.36 ± 3.10d	ND

Results presented as mean ± standard deviation. ¹mg gallic acid equivalent (GAE)/mg dry weight (DW), ²mg rutin equivalent (RE)/mg DW, ND: not determined. Same letters within the same column indicate no significant differences by Tukey test (p ≤ 0.05).

Table 2

Total polyphenols and flavonoids content of rice flakes and rice flakes cereal bars (RFCB) produced with wheat glucose syrup (WGS).
Concentration (%)	Total polyphenols¹		Total flavonoids²
Concentration (%)	RFCB/WGS	Rice flakes	RFCB/WGS	Rice flakes
1	39.70 ± 1.10a	ND	53.2 ± 1.20a	ND
5	41.90 ± 1.15ab	ND	58.2 ± 1.24b	ND
10	43.00 ± 1.12b	ND	62.2 ± 1.17c	ND
25	43.96 ± 1.11b	ND	85.5 ± 2.10d	ND

According to total sugars analysis, WGS at 25% presented 41.73% total sugar content of which 29.4% were reducing sugars. CGS at the same concentration exhibited 87.73% total sugar of which 94.35% reducing sugars. Total soluble protein determination showed that WGS exhibited 172.90 mg BSAE/mL, and CGS did not present total soluble proteins in its composition.

Recent studies performed by Yafetto [23] have shown that different agro-industrial residues can represent an important source of free bioactive compounds. In the present study, WGS presented polyphenolic compounds, among them flavonoids, and soluble proteins that can result in a more nutritional and functional food product compared to CGS that did not show the presence of these compounds. In addition, WGS showed a lower content of reducing sugars compared to CGS, although, this difference could be reduced with further studies in industrial production of WGS.

3.5 Evaluation of antioxidant activity of WGS and RFCB

Table 3 shows the percentages of the antioxidant activities of WGS and CGS determined by DPPH, FRAP and ABTS tests. It is possible to observe that DPPH scavenging capacity increased according to the concentration and the greatest antioxidant activity (52.23%) was observed at the highest concentration (25%). Furthermore, all concentrations evaluated differed significantly from each other. In FRAP assay, the highest antioxidant activity (525.58 µM Trolox equivalent/g of syrup) was observed at 25%, although, this result did not differ statistically from the antioxidant activity at 10%. In ABTS test, WGS exhibited concentration-dependent manner and the greatest scavenging activity (981.88 µM Trolox equivalent/g of syrup) was observed at the highest concentration (25%). CGS did not show antioxidant activity evaluated by DPPH, FRAP and ABTS tests.

Table 3

Antioxidant activity of wheat glucose syrup (WGS) and corn glucose syrup (CGS) by DPPH free radical scavenging and ferric ion reduction (FRAP) tests.
Concentration (%)	DPPH¹		FRAP²		ABTS³
Concentration (%)	WGS	CGS	WGS	CGS	WGS	CGS
1	15.52 ± 1.12a	ND	ND	ND	84.11 ± 0.01a	ND
5	26.26 ± 1.24b	ND	40.36 ± 2.71a	ND	488.55 ± 0.00b	ND
10	38.80 ± 0.21c	ND	521.30 ± 3.12b	ND	899.66 ± 0.05c	ND
25	52.23 ± 1.21d	ND	525.58 ± 3.01b	ND	981.88 ± 0.01d	ND

Results presented as mean ± standard deviation. ¹%Antioxidant activity, ^2,3µM Trolox equivalent/g, ND: not determined. Same letters within the same column indicate no significant differences by Tukey test (p ≤ 0.05).

The antioxidant activity of rice flakes and RFCB produced with WGS evaluated by DPPH, FRAP and ABTS tests are presented in Table 4. Rice flakes did not present antioxidant activity in DPPH, FRAP and ABTS tests; however, RFCB exhibited antioxidant activity at all concentrations and tests. Nevertheless, the highest antioxidant activity in DPPH test (47.46%) was observed at 25%, although this value did not differ statistically from all concentrations. These cereal bars presented 71.70 µM Trolox equivalent/g at 25% in FRAP test and this value was significant different from all concentrations. For ABTS test, RFCB produced with WGS exhibited concentration-dependent manner and the greatest scavenging activity (711.89 µM Trolox equivalent/g) was observed at the highest concentration (25%).

Table 4

Antioxidant activity of rice flakes and rice flakes cereal bars (RFCB) produced with wheat glucose syrup (WGS) by DPPH free radical scavenging and ferric ion reduction (FRAP) tests.
Concentration (%)	DPPH¹		FRAP²		ABTS³
Concentration (%)	RFCB/WGS	Rice flakes	RFCB/WGS	Rice flakes	RFCB/WGS	Rice flakes
1	45.67 ± 1.11a	ND	51.70 ± 1.02a	ND	103.00 ± 0.00a	ND
5	45.78 ± 1.12a	ND	64.70 ± 0.25b	ND	441.89 ± 0.00b	ND
10	46.98 ± 1.10a	ND	65.00 ± 1.12b	ND	599.67 ± 0.00c	ND
25	47.46 ± 1.16a	ND	71.70 ± 1.70c	ND	711.89 ± 0.03d	ND

Results presented as mean ± standard deviation. ¹% Antioxidant activity, ^2,3µM Trolox equivalent/g, ND: not determined. Same letters within the same column indicate no significant difference by Tukey test (p ≤ 0.05).

In recent decades, the products and by-products of wheat have been widely studied for their nutritional composition and the possible use as a source of functional compounds. Studies conducted by Janiak et al. [100], López-Perea et al. [101], Mao et al. [102], Jimenez-Pulido et al. [103] and Wayenbergh et al. [104] have shown that in natura or low processed wheat bran presents compounds with antioxidant activity, important for human and animal health, which strongly contributes to studies of application of this residue in food industry. Moreover, the present study shows for the first time that WGS produced by enzymes obtained from SSF of wheat bran with R. oligosporus, maintained antioxidant compounds, generating a nutritional and functional final product.

The present study demonstrated that RFCB produced with WGS present polyphenols and flavonoids content. In addition, these cereal bars exhibit antioxidant activity in DPPH and ABTS scavenging, and iron ion reduction, indicating that the product may be identified as functional food and this characteristic is possibly attributed due to the presence of WGS. Studies carried out by Deepa and Singh [46], Bagchi et al. [105], and Amrinola et al. [106] have shown that the phenolic composition and antioxidant activity of rice bran is only observed when rice is obtained in the form of flakes, and it is enriched with pigmented compounds. This report corroborates with results obtained in the present study, which showed values only for RFCB produced with WGS.

3.5 Nutritional Facts label

Figure 6 shows the nutrition facts label of RFCB produced with WGS (Fig. 6A) or CGS (Fig. 6B). For a serving size of 50 g, RFCB produced with WGS presented higher nutritional value, providing 7.2 g of soluble protein and 1.8 g of dietary fiber, and exhibiting less calories (108 kcal), carbohydrate (15 g) and sodium (17 mg). On the other hand, RFCB produced with CGS did not present soluble protein and dietary fiber and it showed high values of calories, carbohydrate and sodium.

According to studies conducted by Ryland et al. [107], Machín et al. [108], and Ballco and Garcia [109], the nutritional information provided in Nutrition Facts labels must guarantee the composition, nutritional characteristic, and validity of products. This information can be complemented when functional compounds are incorporated in the composition, benefiting the product and the consumer, and adding economic and nutritional value to the final products. In the present study, it was demonstrated that RFCB produced with WGS showed superior quality compared to those produced with CGS, presenting a better quality and nutritional value with soluble proteins, dietary fibers, and functional compounds, besides reduced caloric value and sodium content. Moreover, it also presented functional compounds including phenolic compounds, among them flavonoids.

The results obtained in the present study indicate that the application of solid-state fermentation using wheat bran (agro-industrial residue) produced enzymatic extract that were able to convert this residue into wheat glucose syrup (WGS), which were incorporated in rice flakes to produce rice flakes cereal bars (RFCB). Besides, this product proved to be a source of soluble proteins, dietary fibers, and bioactive compounds with antioxidant capacity. In addition, RFCB produced with WGS maintained organoleptic characteristics compared to RFCB produced with industrialized product (corn glucose syrup) and it also showed a decrease in caloric value and sodium content. Therefore, this study demonstrated that the preparation of the food product (RFCB produced with WGS) might be of great interest for food industry, adding nutritional and functional value to a well-stablished product.

CRediT authorship contribution statement

CCMF: Conceptualization, Methodology, Investigation, Writing; FOG: Investigation, Writing - Review & Editing; LPS and IFAN: Formal analysis; JFS, BE and PON: Formal analysis and Investigation; RMGS: Conceptualization, Methodology, Investigation, Writing - Original Draft, Supervision and Project administration.

Funding

This research did not receive any specifc grant from funding agencies.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and there is no hidden or supplementary data.

Acknowledgments

This study was supported by the National Council for Scientific and Technological Development (CNPq) - Process number: 142384/2019-4

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Solid-state fermentation using wheat bran to produce glucose syrup and functional cereal bars

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1 Obtaining wheat bran

2.2 Centesimal characterization of wheat bran

2.2.1 Total starch determination

2.2.2 Moisture determination

2.2.3 Ash determination

2.2.4 Lipid determination

2.2.5 Protein determination

2.2.6 Crude fiber determination

2.2.7 Carbohydrate content

2.3 Obtaining amylolytic enzymes

2.3.1 Preparation of microorganism and inoculum

2.3.2 Solid-state fermentation (SSF)

2.3.3 Amylolytic activity

2.4 Obtaining WGS

2.4.1 Gelatinization of wheat bran using enzymatic fermentation broth

2.4.2 Enzymatic hydrolysis and production of WGS

2.5 Obtaining raw material for the production of RFCB

2.6 Determination of bioactive compounds of WGS, RFCB and rice flakes

2.6.1 Determination of total polyphenols content

2.6.2 Determination of total flavonoids content

2.6.3 Determination of total soluble proteins content

2.6.4 Determination of reducing sugar content

2.7 Evaluation of antioxidant activity of WGS, RFCB and rice flakes

2.7.1 DPPH free radical scavenging activity

2.7.2 Iron ion reducing potential (FRAP)

2.7.3 ABTS radical scavenging test

2.8 Design of Nutrition Facts label

2.9 Statistical analysis

3. Results and Discussion

3.1 Centesimal characterization of wheat bran

3.2 Obtaining amylolytic enzymes

3.2.1 Solid-state fermentation (SSF)

3.2.2 Amylolytic activity

3.3 Obtaining WGS

3.3.1 Gelatinization of wheat bran using enzymatic fermentation broth

3.3.2 Enzymatic hydrolysis and production of WGS

3.4 Production of RFCB

3.4 Determination of bioactive compounds in WGS and cereal bars

3.5 Evaluation of antioxidant activity of WGS and RFCB

3.5 Nutritional Facts label

Conclusion

Declarations

References

Additional Declarations

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