Improving nutritional and functional quality characteristics in bread by using flours obtained from fermentation of kidney beans and oats with Pleurotus ostreatus

ABSTRACT Pleurotus ostreatus is an edible fungus with several functional compounds. Previous studies have shown that solid-state fermentation of Phaseolus vulgaris and Avena sativa with P. ostreatus improves their functional properties. Since bread is a highly consumed foodstuff, the objective of this work was to improve its nutritional and functional properties based on P. vulgaris and A. sativa grains biotransformed by P. ostreatus. Results showed that bread made with flour of solid-state fermented grains, provided a higher antioxidant capacity compared to bread made without fermented grains flour. The antioxidant capacity was maintained even after in vitro simulated digestion assays. Bioaccessible protein and fiber content results showed an increase compared to commercial breads. Fermented-flour bread showed lower hardness in its crumb, changed color, and had a good sensory acceptability. This makes these flours potential ingredients in the development of baking goods, making the bread a vehicle for nutrient delivery.


Introduction
Current prevalence of chronic noncommunicable diseases, as well as the need for a healthier diet, have increased the demand and interest in the development of healthier and functional foodstuff containing natural bioactive compounds (Soler et al., 2017).Bread is a basic foodstuff highly consumed worldwide; the most consumed variety is the one produced using refined wheat flour, making it a product with low protein and fiber content (Wandersleben et al., 2018).Therefore, there is a search for alternative ingredients and processes to increase the nutritional and functional value of bread, particularly its content of bioactive antioxidant compounds.
There is evidence that solid-state fermentation can improve substrates it acts on, and that these fermented products can be used to fortify other foodstuffs.Pleurotus ostreatus is an edible fungus that stands out for its antioxidant compounds and protein content along with its ability to synthesize essential amino acids (Kozarski et al., 2015).
Legumes are highly accessible and widely consumed goods with a distinctively high content of protein and antioxidant compounds (Manonmani et al., 2014).Because these characteristics make them promising ingredients for the development of food products, various studies have sought to use them in bakery products, partially replacing wheat flour.However, there are some limitations like undesired organoleptic characteristics (e.g., they can have a hard texture) that can negatively impact the appearance and/or taste of derived products (Gobbetti et al., 2020;Miñarro et al., 2012;Viswanathan & Ho, 2014).This has permitted the addition of low percentages of legume flour as limited replacement of wheat flour in bread (Pejcz, 2015;Ragaee et al., 2012).Developing alternative baked goods, with good nutritional properties and improved sensory characteristics, is an ongoing task.
Our group has previously evaluated the fermentation of Phaseolus vulgaris and Avena sativa with P. ostreatus, halting the process before the fruit body producing stage (Espinosa et al., 2017).The ingredients obtained (fermented flours) were high in bioaccessible protein and antioxidant compounds, as well as in fiber content.In this work, these ingredients were used in the development of several bread formulations with the objective to evaluate the effect over nutritional, antioxidant, physical, and sensorial properties.These results provide evidence that fermented flours with P. ostreatus improve bread quality.

Raw materials and chemical products
The kidney beans (KB) and Chihuahua oat grain (OG) were obtained from the local food market in Guadalupe, Nuevo León, México.P. ostreatus CS155 strain was obtained from the Enzymology Laboratory from the School of Biological Sciences of Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León, México.This strain was maintained by periodic transfers (2-3 months) in petri dishes with growth medium prepared with yeast extract (0.4%), malt extract (0.1%), glucose (0.4%), and agar (1.5%) all in w/v.

Flour with P. ostreatus production
Four different types of flour were produced using a solidstate fermentation method.Briefly, seeds of fresh kidney beans and oats were washed and sterilized in mason jars at 121°C for 45 min using 1:1 (bean) and 1:1.35 (oat) substrate-to-water (w:v) ratios.The fungus was inoculated in a YMG medium (yeast extract [0.4%], malt extract [0.1%], and glucose [0.4%], all in w/v) and incubated under agitation (150 rpm) for 14 days at room temperature.The culture was homogenized during four periods of 15 s and used as inoculum.Then, 8 mL of homogenate that containing 2.64 mg of biomass (d.w.) per gram of the homogenized culture were added to each pre-treated jar for solid fermentation, ensuring that the inoculum covered the whole sample while affecting the ratio of nutrients as little as possible.Afterwards, looking for sufficient biomass, the jars with the inoculated substrates were incubated for 2 weeks at room temperature in the dark under static conditions.The grains with mycelium were ground using a grinder (Moulinex) and dehydrated in a convection furnace set at 70°C.Flours obtained with fermented and unfermented grains were labeled as kidney beans (KB); kidney bean with P. ostreatus (KBP); oats (O); and oats with P. ostreatus (OP).

Bread development
Flour formulations were combined with fermented oat and kidney bean flours as well as unfermented one in different proportions, see Table 1.Breads obtained with fermented and unfermented flours were labeled as control bread (BC), fermented kidney bean flour, and unfermented oat flour (BBP), fermented oats flour and unfermented kidney bean (BOP).
Upon formulation standardization based on the desired characteristics, a final ingredient composition was achieved.It is shown in Table 1.

Nutritional evaluation of bread made with flours with P. ostreatus
Chemical compositions of bread were analyzed using standard methods of Association of Official Analytical Chemistry (AOAC, 2006).Nitrogen content was determined with the Kjeldahl method (AOAC 930.29).Fat content was measured using the Soxhlet method (AOAC 920.39).Ash content was evaluated gravimetrically (AOAC 14.006), and total dietary fiber (TDF) and available carbohydrates were measured with the gravimetric-enzymatic (AOAC 985.29) and chemical (AOAC 962.09) methods, respectively.For TDF determination, the TDF-100A-KT Sigma-Aldrich kit was used as suggested by the manufacturer.Briefly, TDF was determined by using a combination of enzymatic and gravimetric methods.
Samples of dried, fat-free foods were gelatinized with heat stable α-amylase and then enzymatically digested with protease (Sigma-Aldrich) and amyloglucosidase (Sigma-Aldrich) to remove the protein and starch present in the sample.Ethanol was added to precipitate the soluble dietary fiber that was in turn recovered in a filter and washed with ethanol and acetone.After drying, the residue is weighed.Half of the samples are analyzed for protein and the others are ashed.Total dietary fiber is the weight of the residue minus the weight of the protein and ash.Water activity of the samples (crumb and crust) was measured utilizing an AquaLab 4TE (Decagon devices, U.S.A) using previously established methods (Turkut et al., 2016).Data acquired were by means of three separate replicates for each sample.

SimulatedIn vitro digestion
A simulated digestion protocol based on the use of digestive enzymes was followed (Lamothe et al., 2012) with simulated fluids being prepared according to the protocol proposed by Minekus et al. (2014).Portions of 5 g of each sample were placed into a 50-mL tube.For the oral phase, 5 mL of simulated oral fluid (SOF) was added, incubated for 5 min at 37°C in agitation.Then, 12 mL of simulated gastric fluid (SGF) with 3850 U/mg of gastric porcine pepsin (Sigma-Aldrich) at pH 2.3 was added following incubation for 2 h at 37°C in 55 rpm orbital agitation for the gastric phase.Finally, 20 mL simulated intestinal fluid (SIF) with 1.98 mg of pancreatin porcine pancreas 8 USP (Sigma-Aldrich) and bile bovine pancreas (Sigma-Aldrich) at pH 8 were added and incubated for 2 h at 37°C on orbital agitation for the intestinal phase.This was done to measure protein digestibility and antioxidant capacity after simulated in vitro digestion.

Antioxidant capacity and total polyphenol content
Both antioxidant capacity and total polyphenol content were measured in samples before and after in vitro digestion.Extraction of the antioxidants and polyphenols from undigested samples was carried out with methanol 80% (1-part undigested sample and 5 parts aqueous methanol solution at 80%) while being agitated at 80 rpm overnight at 50°C.Digested samples were centrifuged at 2,500 rpm for 10 min.
No methanol extraction was performed on these samples.To measure antioxidant capacity, samples of 0.1 mL from both undigested and digested samples were taken, mixed with DPPH reagent, and incubated at room temperature for 30 min in darkness.Afterwards, absorbance was measured at 515 nm.The results were expressed in the mg equivalent of Trolox (mg TEAC) (Aruguman & Perumal, 2012;Singh et al., 2017).
To determine total polyphenol content, 1.0 mL of sample was mixed with 0.025 mL of Folin -Ciocalteu reagent (1 N), 2.5 mL of a sodium carbonate aqueous solution (20%), incubated for 40 min in darkness, and then measured at 725 nm absorbance.The results were expressed as equivalents of gallic acid (GAE) (Aruguman & Perumal, 2012).

Protein digestibility
This method is based on establishing the ratio of precipitable products with trichloroacetic acid (TCA) after simulated digestion.Protein digestibility was analyzed in the products of simulated digestion.Briefly, 37 mL of intestinal phase product was obtained.Protein not digested was precipitated with TCA aqueous solution (12%).Samples were centrifuged at 3500 rpm for 15 min and then decanted.The precipitate was washed with distilled water and centrifuged twice.The in vitro protein digestibility was evaluated based on the total soluble protein content and the content of protein determined after digestion in vitro (Couto et al., 2015).

Texture profile analysis
Texture profile analysis (TPA) of bread crumb was performed 24 h after its baking using a Texture Analyzer CT325K, Middleboro, MA.U.S.A, following Turkut et al. ( 2016) methods with slight modifications.For each bread sample, individual slices were cut to 15 mm width and crusts were removed before TPA analysis.A double penetration assay (two cycles of continuous compression) using a cylinder probe of 25.4 mm of diameter (D), 35 mm L (TA11/1000) was performed configuring the assay to TPA mode with an assay speed of 1.00 mm/s and a penetration distance 10 mm with an initial load of 0.07 N.
The quality properties measured were hardness, elasticity, cohesion, and chewiness.Results shown are the average of triplicates for each sample.

Sensory evaluation
Sensory evaluation was carried out in three breads.A hedonic test to measure acceptability level based on four attributes: look, smell, flavor, and texture was conducted using 86 untrained panelists.Panelists evaluated each attribute in breads indicating on a scale of 1-9, where 1 represented "do not like it at all" and 9 "like it a lot".Bread samples were presented in identical containers, using random 3 digits codes in a balanced presentation (Chopra et al., 2018).

Statistical analysis
Data for each assay were obtained on independent samples by triplicates.Each of them was evaluated to determine if the variances were statistically homogeneous, results are expressed as average ± SD.Statistical comparisons were carried out by a one-way ANOVA followed by a Duncan test using IBM SPSS 27 software.The difference is considered significant when p values are under 0.05.

Chemical composition
Table 2 presents the chemical composition of breads made with fermented bean and oat flours with P. ostreatus, and the bread without fermented bean and oat flours.The use of fermented ingredients increased fiber, protein, and antioxidant compounds content as well as the bioaccessibility of the protein of the breads.
The highest protein content was in (BBP) fermented kidney bean flour bread, existing significant difference compared with the other two treatments.The protein content in breads ranged from 22.39 to 24.31 g/100 g, which represent an increase ranging from 150% to 224% compared to commercial breads commonly made with wheat flour.These breads have an average protein content ranging from 10 to 15 g/100 g (US Department of Agriculture [USDA], 2021).Even though protein content is affected by the quantity of egg used in the making of this bread, protein content is more affected by adding fermented flours (Espinosa et al., 2017) where a higher protein content was reported in fermented flours than in wheat flour.Ragaee et al. (2012) reported that protein content of breads made from alternative ingredients such as amaranth, pea, rye, barley, and oatmeal to substitute wheat flour ranged from 14 to 16 g/ 100 g.
In fat content, results show no significant difference between breads made with fermented and unfermented flours.However, fat content increased 3-fold in the breads compared to commercial ones.This can be explained by the ingredients used, such as almond milk and canola oil; however, previous works have shown that P. ostreatus synthesizes a small amount of fats (Espinosa et al., 2017;Mbassi et al., 2018).Mineral content was similar in all treatments ranging from 4.5 to 4.8 g/100 g. being higher than commercial breads which have between 2 to 3 g/100 g.This could be attributed to the whole grain (beans and oats) flours without a refinement process (Oghbaei & Prakash, 2016).Results showed significant difference between the three treatments in fiber content, being BOP 12.37 g/100 g the lowest while the highest one, BBP, was 14.96 g/100 g.P. ostreatus uses several compounds such as cellulose, hemicellulose, and lignin for mycelium growth, as well as it adjusts its enzymes depending on the substrate (Alsanad et al., 2021).
These results show that fiber content in the bread developed in this work is higher; an increase of 134-200% comparing with commercial breads (USDA, 2021) and higher than other breads such as those made of rye, amaranth, barley, oatmeal, and other breads that have added xantham gum (Pejcz, 2015;Ragaee et al., 2012).This highlights the positive effect of the use of flours made with whole grains and without any type of grinding and refining prior to fermentation, allowing a greater contribution of fiber in bread products made from them.

Antioxidant activity and total polyphenol content
Antioxidant properties of foodstuff can be altered during food processing and during digestion.To evaluate this antioxidant capacity, assays were performed before and after in vitro digestion.The results are shown in Table 3. Breads made with fermented flours showed an antioxidant capacity significantly higher compared to control; the highest one been BBP with a 108.55 mg TEAC/g bread.Ikram et al. (2021) showed that there is a higher antioxidant capacity in kidney beans than oats, which could explain the results obtained in this work.Also, there are reports that P. ostreatus produces several antioxidant compounds during fermentation, which could partially explain the increase in antioxidant capacity in breads supplemented with fermented grain flour (Espinosa et al., 2017).
The antioxidant capacity increased significantly after in vitro digestion in samples made with fermented flours compared to control samples.These results agree with those that have been reported previously by our group, where it was shown that the fermentation with P. ostreatus increases antioxidant compounds bioaccessibility (Espinosa et al., 2017).Enzymes play a key role in this effect by hydrolyzing antioxidant compounds (Bouayed et al., 2011).Bouayed et al. (2011) reported that both kidney beans and oats contain several antioxidant compounds such as polyphenols, flavonoids, and anthocyanins: the latter ones evidencing resistance to acidic pH during digestion.
BOP polyphenol content showed to be the highest with 39.60 mg GAE/g bread; significantly higher compared to the control bread.These results can be attributed to oats having a higher phenolic compound content than kidney beans as previously reported (Yang et al., 2020).Likewise, BBP was the sample that showed the lowest content of total polyphenols, which matches with what has been reported previously by our group (Espinosa et al., 2017).This study proved that the effect of the fungus on the content of polyphenols in kidney beans was lower because the phenol oxidase enzyme's activity was affected, thus degrading conjugated phenolic compounds by not having enough access to them.This leads to lower content of phenolic compounds in the flour, which translates into a lower content in the bread made with it.
Similarly, to what was discussed earlier, these phenolic compounds increased after in vitro digestion due to the enzyme's actions increasing polyphenol availability (Bouayed et al., 2011).The baking process could also lead to a higher phenolic compounds content in breads with a high fiber content since Maillard's reactions synthesize antioxidants that reduce free radicals (Ragaee et al., 2012).
The use of ingredients obtained from the fermentation with P. ostreatus can increase phenolic content and the antioxidant capacity of bakery products.Since bread is a highly consumed product and there is a need for more nutritional processed foods, the use of fermented flours rises as an alternative to traditional commercial breads (Prieto et al., 2022).This could cooperate with fulfilling the demand for functional foods that has been discussed (Wandersleben et al., 2018).

Protein digestibility
The current trend in food development focuses on searching for alternative ingredients or vegetable protein sources.However, it has been shown by Viswanathan and Ho (2014) that vegetable protein sources often are of lower biological quality, having decreased bioaccessibility and digestibility when compared to animal protein sources.Figure 1 suggests that breads developed with fermented flours have higher protein digestibility with a significant difference compared to control bread in both intestinal and gastric in vitro digestion.
Bread with fermented oat flour hydrolyzes easier than fermented bean bread and control bread.These results concur with what has been reported previously by our team showing P. ostreatus ability to hydrolyze the inoculated substrates, diminishing antinutrient compounds, synthesizing essential amino acids, and increasing its digestibility (Espinosa et al., 2017).At the same time, this confirms that foods made with these ingredients possess a higher digestible protein content (Espinosa-Páez et al., 2021).Digestibility values obtained ranging between 57.3 and 59.8% are similar to the ones reported by Sciarini et al. (2017) in breads made with wheat flour supplemented with fiber.On the other hand, digestibility results obtained in the present work are higher than those reported for breads made with raw and soaked red kidney bean flour (Viswanathan & Ho, 2014).
This proves that the use of fermented bean and oat ingredients in breadmaking increases protein content as well as its digestibility and bioaccessibility.

Bread crumb texture profile analysis
One of the most appreciated quality attributes for consumers is its texture.Crumb freshness is related to the mechanical properties of the walls of the air cells formed in bread during fermentation and baking (Angioloni & Collar, 2009).TPA results can be seen in Table 4. Hardness is the required force to compress food between the teeth, food freshness is stimulated and perceived in this manner (Turkut et al., 2016); the more force required to compress, the harder the bread is.Bread crumb in BBP showed the lowest instrumental hardness value, whereas BC showed the highest hardness among all samples.Carocho et al. ( 2020) conducted a study comparing different types of breads, including oat bread among them.Their results show that bread made with oat have a lower hardness than all the analyzed samples, which was expected since oat is recognized for its soft doughs.Worked on a bread where there were different substitutions of wheat flour for red bean flour (5% to 25%); the authors mention that increasing the level of substitution of wheat flour with red bean flour, led to an increase in hardness.The three treatments were developed with 50% fermented oat or kidney bean, or without fermentation.
Results showed hardness decrease in fermented treatments, this could be due to a process during P. ostreatus fermentation.Elasticity and cohesiveness values, crumb showed no significant difference between treatments.Chewiness values showed significant difference between fermented breads BBP or BOP, and non-fermented bread BC.Few studies exist that show the effect that P. ostreatus have on oat and kidney bean that can be involved in structural changes in the flours used for breads.Further studies could enlarge the field for the confirmation of a correlation between fermentation and structural change.

Color analysis in bread crumb and crust
The two main chemical reactions that occur during the baking process are Maillard reactions and caramelization; these reactions cause the darkening of baked goods.In addition, bread's color can be influenced by the physicochemical characteristics of the raw dough and the baking conditions (Manonmani et al., 2014).Both raw dough's physico-chemical characteristics and the baking conditions can lead to changes in bread's color, even between breads with the same formulation (Turkut et al., 2016).The effect the different treatments have on both bread crumb and crust can be shown in Table 5. Significant difference between crumb and crust samples between the different treatments can be seen.L* value corresponds to lightness in breads, the higher the value, the lighter the bread.In crumb results, BOP treatment showed the highest value, contrary to crust where BC showed the highest one.This could mean that the lightness differences affect the   The slightly darker color in BBP could be due to the P. ostreatus fermentation process since the other treatments are unfermented; Omarini et al. (2010) showed evidence that during fermentation, P. ostreatus could intensify color to a darker more intense brown color according to the inoculated substrate.Kidney bean caused changes during fermentation in pigmented compounds found mostly in the bean testa, which leads to a flour color change.

Sensory evaluation
In functional food development, along with achieving a high nutritional value and with a measurable contribution of bioactive compounds, assuring sensorial quality is of prime importance.Because of this, all breads obtained in the work were evaluated to determine if they comply with the required sensorial characteristics demanded by consumers.
Average acceptability was obtained by evaluating five different attributes for each sample: appearance, aroma, flavor, texture, and aftertaste.Figure 3 shows these results, where there was no significant difference between appearance and texture between breads with fermented flours and breads without.BOP had a higher value with 7.01 and 6.43, respectively, while BPP had the lowest ones with 6.61 and 5.79.Appearance is perceived through sight and can be influenced by both crumb and crust color, as well as crumb structure which are highly related with the ingredients used in this formulation (Callejo, 2011).In this case, color intensity increased by adding kidney bean and oat flour, which provide the bread with a brown color, along with Maillard reaction producs synthesized during baking.
In addition, the texture provided by crumb's hardness was lower in fermented breads could be having an influence in the fermentation effect in those attributes compared to control.
The use of fermented oat and bean flours has no effect in appearance and texture in bread compared to those with unfermented flours.BOP had the highest flavor value with 5.66, while BBP had the lowest with 4.20 while having no significant difference between treatments for this attribute.This means, fermentation with P. ostreatus has no effect in bread flavor.It should be noted that the values for flavor obtained, are higher than other studies that evaluated breads made with a mix of cereals such as rice, soy, and chia (da Mota Huerta et al., 2019).
For both aroma and aftertaste attributes, BOP showed the highest values with 6.72 and 6.43, respectively.BBP showed the lowest values with 4.74 and 5.79, respectively.Both BOP and BBP showed significant difference.Bread's aroma is one of the most influential attributes for the general acceptability of consumers.Bioproducts of P. ostreatus such as octenols, octanones, aliphatic aldehydes, benzaldehydes (almond smell), benzyl alcohol (spicy sweet aroma), phenethyl alcohol (rose aroma), and monoterpenes provide the commonly known "fungus smell" to P. ostreatus fermented products (Misharina et al., 2009).Results suggest that these bioproducts enhance the attributes evaluated and this has a positive effect in consumer's acceptance of the breads made.
BOP treatment had the highest value for acceptability with 5.91, not being significant difference between breads made with fermented flours and without fermented flours.It can be concluded that the use P. ostreatus fermented flours has no effect in the attributes compared with breads made with flours without fermentation.Cruz et al. (2018) reported that both legumes and P. ostreatus share physicochemical properties such as water absorption capacity and oil absorption capacity that allow them to be better in the development of bakery and meat products.
It should be noted that most evidence of breads made with flours coming from alternative grains such as barley, pea, bean, and rye, do not exceed 30% substitution of wheat flour (Cyran et al., 2021;Pejcz et al., 2015;Viswanathan & Ho, 2014).The results obtained during sensorial evaluation, where 100% of wheat flour was substituted by oat and kidney bean fermented flours, strongly suggests that these ingredients could be used for bakery product development with high acceptance by consumers.

Conclusion
The use of P. ostreatus fermented ingredients enhanced bread's biochemical, functional, and nutritional characteristics.The use of fermented flours of legumes and grains in breads provide functional properties, increases protein content and digestibility, and enhances polyphenol and antioxidant compound content available after in vitro digestion.In addition, consumer acceptance was adequate compared to control breads.All matters discussed, provide evidence that these fermented flours are potential ingredients to be used in bakery goods as a vehicle for several nutrients.This could prove an alternative for consumers that seek a healthier diet and an alternative for the food industry to improve commonly used foods and the development of functional foods that are currently in trend.

Figure 1 .
Figure 1.Protein digestibility percentage (%) of breads developed with ingredients obtained from P. ostreatus fermentation (BOP, BBP) and the control (BC).Data is mean ± SD of triplicates, values with different letters present significant difference (p < .05).
clarity of breads.A* evaluates the range between red and green (values from 0 to + 100 is red, whereas 0 to −100 is green color).The bread crumbs showed values close to 0, BBP showing a slightly higher value.Similarly, in bread crust, values were near 0 where the BOP treatment was slightly higher than the other ones measured.Finally, b* value represents blue (negative values) and yellow (positive values).Although there is significant difference between values, there is no high variability, showing crumb values between 21.91 and 23.54.In crust, values ranged from 22.70 to 26.55.Figure2shows bread images are shown where BBP treatment show a slightly darker color.However, in crust instrumental values obtained BBP show the lowest values, while in crumb show the highest variability.Generally, both in crumb and crust, there are no drastic changes in color due to the proportion between the quantities of flours in treatments.

Figure 2 .Figure 3 .
Figure 2. Bread slice images of crumb (a) and crust (b) of breads developed with ingredients obtained from P. ostreatus fermentation (BOP, BBP) and the control (BC).

Table 2 .
Nutritional components of different obtained bread.
aBreads developed with ingredients obtained from P. ostreatus fermentation (BOP, BBP) and the control (BC).Data is mean ± SD of triplicates, values with different letters present significant difference (p < .05).

Table 3 .
Antioxidant capacity and total polyphenol content in different breads.
dBreads developed with ingredients obtained from P. ostreatus fermentation (BOP, BBP) and the control (BC).Data is mean ± SD of triplicates, values with different letters present significant difference (p < .05).

Table 4 .
Texture profile analysis in bread crumb and crust.

Table 5 .
Color analysis in bread crumb and crust.
bBreads developed with ingredients obtained from P. ostreatus fermentation (BOP, BBP) and the control (BC).Data is mean ± SD of triplicates, a-c different letters in the same column show significant difference between the means (p < .05).