Process for obtaining a gluten-free bakery product based on legumes, pseudocereals, and metabiotics

Abstract

The invention provides a process elaboration to obtain a gluten-free bakery product based on legumes (chickpea), pseudocereals (quinoa and buckwheat), and a bioingredient made by fermentation in controlled conditions (sourdough), with improved technological and bioactive properties. The beneficial effects related to the bioactive properties result by the substitution of the gluten-free fours' mixture with gluten-free sourdough obtained by fermentation in controlled biotechnological conditions, with selected and potentially probiotic lactic acid bacteria strains. The sourdough added in a concentration of 10% and 25% contributed to the gluten-free bread product's shelf-life extension with 2...4 days, following storage conditions at a temperature of 25°C, the obtained result depends on the used bioingredient's concentration. It was observed, as well, an improvement on the textural parameters, on the resistant and slowly digested starch content with prebiotic effect, respectively an improvement on the polyphenolic and flavonoids content with antioxidant potential, after the simulated in vitro gastrointestinal digestion.

Description

[0001] PROCESS FOR OBTAINING A GLUTEN-FREE BAKERY PRODUCT BASED ON LEGUMES, PSEUDOCEREALS, AND METABIOTICS

[0002] The aim of the invention herein is represented by developing a process to obtain a gluten-free bakery product, based on legumes, pseudocereals, and sourdough (bioingredient with metabiotic properties, obtained in controlled fermentation conditions), with superior functionality.

[0003] Celiac disease is characterized by the incapacity of the human body to properly metabolize the gluten, a specific storage protein, found mainly in cereals. The gluten’s ingestion results in small intestine’s villi inflammation along with an abnormal absorption of the nutrients and minerals (Aguiar et al., 2023). The celiac disease treatment involves a gluten-free diet that fulfills the dietary fibers, vitamins, Calcium, Iron, and Magnesium requirements, considering also a balanced ratio between carbohydrates, fats, and salt within the gluten-free food product (Pessarelli et al., 2022).

[0004] The obtainment of the gluten-free baked goods it’s challenging for both researchers and food producers because the lack of gluten from the food products affects the textural and sensorial properties. Therefore, using alternative raw materials to overcome such disadvantages and improve the technological and functional benefits is a sustainable solution (Tuna et al., 2023). Chickpea is a legume rich in proteins, dietary fibers, and B-group vitamins, whereas quinoa and buckwheat, included among pseudocereals, are known for their balanced composition in amino acids and saturated fats (Martinez-Villaluenga et al., 2020; Sofi et al., 2023). Fermentation, by the selected lactic acid bacteria strains’ metabolic activity, in controlled and optimized biotechnological conditions of the flours resulted from these raw materials, can increase the nutritional and functional value of the obtained fermented products (bioingredients; sourdoughs), that can be included further in the manufacturing process of the gluten-free baked goods. Thus, the spontaneous fermentation of the mixture made of chickpea and rice flour, at 37°C for 18 hours, and the utilization of the resulting sourdough in a gluten- free bread making process modified the textural parameters, increasing of the total titratable acidity and decreasing the pH (Gidari - Gounaridou et al., 2023). Statistical optimizations to identify the optimal ratio between the gluten-free flours’ mixture and water, as well as the dough’s proofing time were carried out by Ammar et al. (2022) to obtain gluten-free bakery products with high acceptability.

[0005] Metabiotic products can be obtained by fermentation of different substrates with potentially probiotic microorganisms. They contain prebiotics, probiotics, postbiotics, and paraprobiotics. (Sharma et al., 2020). Such products with multifunctional properties represent valuable bioingredients to be used in foods’ formulas with a technological impact upon the microbiological stability as well as on the functional properties.

[0006] In the scientific literature there were identified some patents that provide alternative solutions to obtain gluten-free baked goods with enhanced biotic properties, as following:

[0007] KR20230096773 (A), Method for manufacturing gluten-free rice bread using non-glutinous rice flour and glutinous rice flour, and gluten-free rice bread manufactured thereby. The invention refers to the utilization of a combination of glutenous and non-glutenous rice flour aiming to obtain bakery products with improved aroma and chewiness. The obtainment process described according to the above-mentioned invention involves the utilization of baking powder and almond powder, eggs and milk, to increase the acceptability of the resulting product.

[0008] KR20230095411 (A), How to make gluten-free rice bread without gluten substitute artificial additives. The invention describes a gluten-free bakery product based on rice flour without gluten or its replacers addition into the product’s composition. The technical solution given in the mentioned invention refers to the utilization of purified water, rice flour, along with olive oil and dry yeast to obtain a fermented dough that is further used for gluten-free bread making.

[0009] RO137271 (A2), Gluten-free bread based on rice flour, gram flour and chufa flour. The invention describes a gluten-free bakery product making process with sourdough made of rice, chickpea, and chufa flours, fermented by a Lactobacillus sanfranciscensis strain. To make the gluten- free bakery product, the same mixture of flours is used, including baker’s yeast to ensure dough’s fermentation.

[0010] CN115624045 (A), Preparation method of gluten-free bread capable of controlling blood sugar and bread. The invention provides a production process to make a gluten-free bakery product using rice, quinoa, and wheat flours. Moreover, com starch and cassava starch, gluconic acid delta-lactone, sodium bicarbonate, and guar gum are part of the dough’s composition. The designed composition ensures a low glycemic index and a high polyphenolic content, the product being especially made for the patients suffering from diabetes.

[0011] RO133655 (A2), Gluten-free bread based on rice flour with chickpeas flour and carob powder and process for preparing the same. The invention refers to the obtainment process for a gluten-free bakery product fermented by baker’s yeast by using rice and chickpea flours, and carob powder, potato starch, pectin, xanthan gum, sodium tartrate, and emulsifier (DATEM). The thermal treatment used to make the baked product was carried out at 200°C, for 35 minutes, and the resulting product is characterized by a superior nutritional value, being an alternative in special diets to overcome obesity, diabetes, and cardiovascular diseases.

[0012] JP2022134813 (A), Gluten free bread mix flour, gluten free bread dough composition, and method of producing gluten free bread. The invention refers to the utilization of gelatinized buckwheat flour to partially substitute the native flour from the gluten- free flour mixture made of rice and buckwheat. The gelatinization of the buckwheat flour in the product’s making process contributes to the improvement of the textural and sensorial features.

[0013] The technical problem solved by this invention refers to the particularization of a gluten-free bakery product’s manufacturing process which involves the utilization of legume (chickpea), and pseudocereals (quinoa and buckwheat) flours along with Psyllium bran and a sourdough obtained after fermentation, in optimized biotechnological conditions, with selected and potentially probiotic lactic acid bacteria strains (Pacularu-Burada et al., 2021). In the gluten- free product’s composition according to the invention variable amounts of sourdough (10% and 25%) are used, depending on the substitution level of the flours’ mixture comprising chickpea, quinoa, and buckwheat with the bioingredient used. The gluten-free bakery product obtained can be characterized by an extended shelf-life that ensures resistance to the molds’ spoilage, in storage conditions at 25 °C. Moreover, the metabiotic product obtained has multifunctional potential due to the bioactive contribution of the polyphenols, flavonoids, and other compounds with antioxidant potential from the raw materials’ composition, as well as due to the metabolites (postbiotics) synthesized during the fermentation process, respectively by the slowly digested and resistant starch contribution, which can exert a prebiotic effect inducing the proliferation of the beneficial bacterial populations in the colon. By destroying the probiotic cells during baking, paraprobiotics (non-viable cells, lysed or intact) with high bioactive potential are obtained.

[0014] Parameters of the invention

[0015] 1. Obtainment of the gluten-free bakery product with metabiotic characteristics

[0016] To obtain the gluten-free baking product according to the invention, legume (chickpea) and pseudocereals (quinoa and buckwheat) flours were used, along with com starch, salt, sugar, Psyllium bran, dry baker’s yeast (Dr. Oetker, Romania), tap water, and sunflower oil.

[0017] For the bakery product made, the gluten-free flours’ mixture was substituted with sourdough obtained by fermentation with selected lactic acid bacteria strains, at 32°C for 66 hours, in a solid-state fermentation system of the mixture made of chickpea, quinoa, and buckwheat flours in equal ratios. The lactic acid bacteria strains used, included into the Microorganisms Collection of “Dunarea de Jos” University of Galati (MIUG) were classified based on the biochemical preliminary tests as species of Lactobacillus genus, specifically Lactobacillus paracasei ssp. paracasei (MIUG BL 21) and Lactobacillus parabuchneri (MIUG BL 24). The genetic identification using bacterial RNA demonstrated that the strains belong to the species of Lactiplantibacillus plantarum (MIUG BL 21) and Lactiplantibacillus pentosus (MIUG BL 24) as Cotarlet et al. (2023) described. The probiotic potential of these strains was demonstrated after specific biochemical assessments (Pacularu-Burada, 2022). In the fermentation process aiming at obtaining the gluten-free sourdough, it was used a multiple inoculum (activated cultures; optical density of the suspension ca. 2.0 at the wavelength of 600 nm) in a concentration of 2.90% for the strain MIUG BL 21 and 5% for the strain MIUG BL 24. After fermentation in the established conditions, the resulting sourdough was preserved by freeze- drying, the obtained powder being stored in the dark, in airtight, sterile containers (Pacularu- Burada et al., 2021), until its utilization to obtain the sourdough gluten-free bakery product according to the invention.

[0018] The obtainment process of the gluten-free bakery product with metabiotic characteristics It was formulated the gluten-free flour mixture based on chickpea flour, quinoa flour, and buckwheat flour, used in different concentrations depending on the tested experimental variant. The com starch (20%), salt (4%), sugar (4%), Psyllium bran (2%), baker’s yeast (4%), sunflower oil (6%) and tap water (150%) were included in the manufacturing process, using the amount according to the invention. The gluten-free flour mixture used varied based on its substitution degree with sourdough.

[0019] There were made three experimental variants: Pl - gluten-free bread without sourdough added, with 45% chickpea flour, 45% quinoa flour, and 10% buckwheat flour; P2 - gluten- free bread with 10% sourdough added, with 40.50% chickpea flour, 40.50% quinoa flour, and 9% buckwheat flour; P3 - gluten-free bread with 25% sourdough added, with 33.75% chickpea flour, 33.75% quinoa flour, and 7.50% buckwheat flour. For the rest of the ingredients the concentration was the same for all three experimental variants.

[0020] The gluten-free dough was obtained using a bread making apparatus which ensured the homogenization, kneading, and proofing after 1 hour and 50 minutes. Afterwards, the resulting batter was divided in individual pieces and thermally treated at 200°C for 1 hour, using an electric oven with ventilation. After preparation, the obtained products (gluten-free bread) in the experimental variants tested, were used fresh or freeze-dried (-42°C, 0.10 mbar) to evaluate specific properties.

[0021] 2. Technological and functional characteristics of the gluten-free bakery product with metabiotic characteristics

[0022] 2.1. Total titratable acidity and pH

[0023] The total titratable acidity and pH were determined for the fresh experimental variants of gluten-free bread, as Montemurro et al. (2023) described, the total titratable acidity was expressed in milliliters (mL) solution NaOH 0.10 N to reach a pH value of 8.50, while the pH was determined using a digital pH-meter (Mettler Toledo, FiveEasy F20, Greifensee, Switzerland), results being expressed based on pH units. Results highlighted a variation of the titratable acidity values based on the sourdough amount added in the gluten-free bakery product. Therefore, for the control sample without sourdough added (Pl) it was determined a total titratable acidity of 3.65±0.23 mL NaOH 0.10 N, the gluten-free flours’ substitution with sourdough in a ratio of 25% to obtain the sample P3 determined a decrease of the total titratable acidity value to 3.05±0.27 mL NaOH 0.10 N, the lowest value for this parameter being determined for the sample P2 with 10% sourdough, respectively 2.51±0.00 mL NaOH 0.10 N. On the other hand, the pH values varied between 5.25, for sample P3 and 5.80, for sample Pl. The obtained data can be justified by the presence of the organic acids (postbiotics) in the experimental variants of gluten-free bread, which directly impacts on the analyzed parameters.

[0024] 2.2. Textural characteristics

[0025] To assess the textural characteristics of the gluten-free experimental variants’ crumb, it was used the texture profile analysis proposed by Siepmann et al. (2019). The parameters of interest were evaluated after a double compression of the samples at 1 mm / s, a cell load of 9.80 N, and a sensitivity limit of 0.067 N. The results presented in Table 1 show the modifications determined by the composition of the gluten-free bread dough on specific parameters. The experimental variant P3 was characterized by a high value of hardness (2.22±0.66 N) correlated with lower values for the cohesiveness (0.40±0.08), elasticity (3.44±0.39 mm), and masticability (3.20±l .53 mJ). The substitution of the flours’ mixture made of chickpea, quinoa, and buckwheat, with sourdough in a ratio of 25% for the experimental variant P3 contributed to the modification of the proteins and starch structure and composition, such components being as well affected by the thermal treatment of the gluten-free bread, which impacts the texture of the analyzed product. Table 1. Values for the analyzed textural parameters of the gluten-free bread experimental variants

[0026] 2.3. Microbiological stability

[0027] To evaluate the microbiological stability, the obtained gluten-free bread variants were cooled to room temperature right after baking and packed in aseptic conditions in sterile polyethylene bags. The microbiological stability against molds was analyzed after incubation at 25 °C and daily observation to highlight the visible mold colonies on the surface of the analyzed products. Based on the difference between the number of days necessary for the molds to grow on the control sample (Pl) and the other samples (P2 and P3) it was established an average shelf-life for the gluten-free bakery products (Nami et al., 2019). After 2 days of storage following the mentioned conditions, it was observed the contamination of the control sample (Pl), whereas the addition of sourdough in a ratio of 10% to obtain the experimental variant P2 determined a shelf-life extension with 2 days, the same effect being observed after 4 days for the experimental variant P3, with 25% sourdough added. The antifungal effect is correlated with the high content of postbiotics ensured by supplementation with sourdough.

[0028] 2.4. Bioactive content after simulated in vitro digestion

[0029] The bioactive composition assessment for the experimental variants of gluten-free bread according to the invention was carried out using freeze-dried bread samples, during the simulated in vitro digestion considering the protocols described by Minekus et al. (2014) and Lin et al. (2019). The simulated gastrointestinal digestion was realized using different enzymes as following: alpha-amylase for the oral phase, pepsin for the gastric phase, while for the intestinal phase were used pancreatin and bile salts. At the beginning of the simulated digestion protocol, in the mouth, and at the end of the intestinal digestion, it was aimed the release of some target bioactive compounds. Thus, the total polyphenolic content (TPC) and flavonoids (TFC) were evaluated (Tian et al., 2021), results being expressed in milligrams equivalents gallic acid / 100 g DW (mg GAE / 100 g DW) and milligrams catechin equivalents / 100 g DW (mg CE / 100 g DW), respectively. In the same in vitro simulated system there were evaluated, also, the antioxidant properties of the gluten-free bread experimental variants, obtained according to the invention, using the DPPH (Olojede et al., 2020) and ABTS methods (Robles- Ramirez et al., 2020), results being expressed in millimoles Trolox equivalents / 100 g DW (mM TE / lOO g DW).

[0030] Table 2. Bioactive properties of the gluten-free bread experimental variants n.d. - not determined.

[0031] The data reported in Table 2 suggest that the substitution of the gluten- free flours’ mixture with sourdough, according to the invention, determines a decrease of TPC from 177.15±40.84 mg GAE / 100 g DW, for the control sample (Pl), up to 100.77±5.76 mg GAE / 100 g DW, for the sample with 10% sourdough (P2), at the beginning of the in vitro digestion protocol. However, at the end of the simulated intestinal digestion, a superior concentration of total polyphenols, of 86.25±3.71 mg GAE / 100 g DW was determined for sample P3. On the other hand, the experimental variant P3 allowed a higher release of total flavonoids, with an average value of 7.76±0.18 mg CE / 100 g DW at the end of the simulated intestinal digestion. The values determined for the antioxidant activity varied depending on the analyzed free radical and gluten-free bread sample composition, a superior antiradical activity against ABTS being associated to the experimental variant P3, with a scavenging activity of 2.90±0.04 mM TE / 100 g DW.

[0032] Moreover, the gluten-free bread experimental variants were analyzed during the simulated intestinal in vitro digestion aiming at the starch content and its fractions with functional effect, namely rapidly digested starch (RDS) released in the first 20 minutes of intestinal digestion, slowly digested starch (SDS) released after 120 minutes of intestinal digestion, and resistant starch (RS) released after 180 minutes of in vitro intestinal digestion (Xiao et al., 2021). The results, showed in Table 3, were obtained using spectrophotometric methods and the DNS reagent (3,5 dinitrosalicylic acid), being expressed as percent related to the total starch content (Liu and Liu, 2020).

[0033] Table 3. Starch fractions determined for the experimental variants of gluten-free bread

[0034] The obtaining process for the gluten-free bread and the substitution of the mixture of legumes’ and pseudocereals’ flours with sourdough fermented in optimized biotechnological conditions, with potentially probiotic lactic acid bacteria strains, modified the starch fractions’ ratio of the analyzed experimental variants. Thus, for the gluten-free bread experimental variant P3, obtained by the substitution of the gluten-free flours from the bread making recipe with 25% sourdough, it was determined that 66.63% of total starch was digested after 120 minutes of simulated intestinal digestion, this being included in the slowly digested starch. For this sample it was determined a percent of 10.69% resistant starch, released after 180 minutes of in vitro intestinal digestion. The slowly digested starch, along with the resistant starch, offer to the gluten-free bakery product experimental variant P3 improved functionality determined by the prebiotic effect which allows the selective development and multiplication of the beneficial potentially probiotic bacteria existing in the colon.

[0035] Conclusions

[0036] Conclusively, considering the technological and functional characteristics of the resulted gluten-free bread experimental variants, it is considered that the obtaining process of the gluten-free bread in the experimental variant P3 (Figure 1) leads to a shelf-life extension of the product with 4 days and determines as well the maximization of the beneficial bioactive effects for consumer’s health. The proposed obtainment process of the gluten-free bread with metabiotic characteristics contributes to the diversification of the bakery products’ assortments for special nutrition, with metabiotic characteristics (pre-, pro-, post-, and paraprobiotics).

[0037] References:

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Claims

Claims1. Obtainment process for a gluten-free bakery product (bread) characterized by the fact that it comprises a mixture of chickpea, quinoa, and buckwheat flour, respectively 25% sourdough fermented with selected, potentially probiotic, lactic acid bacteria strains, obtained in optimized biotechnological conditions, that offers technological properties and metab iotic characteristics.

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