Rheological and quality characteristics of composite gluten-free dough and biscuits supplemented with fermented and unfermented Agaricus bisporus polysaccharide flour

Accepted Manuscript Rheological and quality characteristics of composite gluten-free dough and biscuits supplemented with fermented and unfermented Agaricus bisporus polysaccharide flour Abdellatief A. Sulieman, Ke-Xue Zhu, Wei Peng, Hayat A. Hassan, Mohammed Obadi, Azhari Siddeeg, Hui-Ming Zhou PII: DOI: Reference:

S0308-8146(18)31346-3 https://doi.org/10.1016/j.foodchem.2018.07.189 FOCH 23304

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

14 January 2018 22 July 2018 25 July 2018

Please cite this article as: Sulieman, A.A., Zhu, K-X., Peng, W., Hassan, H.A., Obadi, M., Siddeeg, A., Zhou, HM., Rheological and quality characteristics of composite gluten-free dough and biscuits supplemented with fermented and unfermented Agaricus bisporus polysaccharide flour, Food Chemistry (2018), doi: https://doi.org/ 10.1016/j.foodchem.2018.07.189

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Rheological and quality characteristics of composite gluten-free dough and biscuits supplemented with fermented and unfermented Agaricus bisporus polysaccharide flour

Abdellatief A. Suliemana, b*, Ke-Xue Zhua, Wei Penga, Hayat A. Hassanb, Mohammed Obadia, Azhari Siddeegc, Hui-Ming Zhoua* a

State Key Laboratory of Food Science and Technology, School of Food Science and

Technology, Jiangnan University, Wuxi, PR China. b

Department of Cereal Science and Technology, National Food Research Center, Ministry of

Agriculture and Forestry, Khartoum North, P.O. Box. 213, Sudan. c

Department of Food Science and Technology, Faculty of Engineering and Technology,

University of Gezira, Wad Medani, P.O. Box 20, Sudan. *Corresponding author: [email protected]; [email protected] Tel: +86 15961799718

Grahical abstract

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Highlights 

Novel CGF biscuits were prepared by added FABP flour and UABP flour.



Rheological properties were improved by both FABP flour and UABP flour.



An increase of fibers, amino acids and minerals of CGF biscuits was observed.



Physical and sensorial properties were enhanced by added polysaccharide flours.

Abstract In this study, functional, rheological and physicochemical characteristics were carried out for composite gluten-free (CGF) flours, dough and biscuits, respectively fortified with fermented and unfermented Agaricus bisporus polysaccharide (FABP and UABP) flours. Addition of both FABP flour and UABP flour improved functional properties, while addition of FABP flour decreased viscosity property. Incorporation of both polysaccharide flours in CGF biscuit dough revealed a significant increase in rheological moduli (G‫ ׳‬and G‫ )״‬and a decrease in tan (δ). Supplementation of UABP flour increased thickness, whereas supplementation of FABP flour increased diameter and spread ratio. All CGF biscuit formulations exhibited lower fracture strength and hardness compared to the control. Furthermore, both UABP flour and FABP flour 2

formulation (F3) contained the highest nutrients in terms of protein, dietary fibers, amino acids and minerals among the CGF biscuit formulations. The sensory evaluation result showed that FABP flour formulation (F1) and UABP flour F1 were most acceptable.

Keywords: Fermented Agaricus bisporus polysaccharide flour; composite gluten-free biscuit; rheology; sensory evaluation; texture; chemical compositions.

1. Introduction The importance of gluten-free products in food industry is increasing annually, taking into account the increase of celiac disease (CD) patients, and diagnosed cases of wheat allergy and gluten sensitivity. CD is an inflammatory disease of the upper small intestine caused by the ingestion of gluten, a protein present in wheat (gliadin), rye (secalin), and barley (hordein) (Capriles & Areas, 2014). It is common worldwide and affects around 1 in 100 to 1 in 300 of the population (Rewers, 2005). Nowadays, people are becoming aware of their health, food, and nutrition. They are seeking for convenient foods with good taste, cheap price, and nutritious. Biscuits are one of the most popular bakery products in human diet, convenient, palatable, ready-

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to-eat, high nutritional quality, inexpensive, and longer shelf-life in comparison to the other bakery products (Nagi, Kaur, Dar & Sharma, 2012). Biscuits are also used as weaning foods for infants and as a snack in schools for children. Long shelf-life of baked products assists on large scale production, storage, and distribution. In addition, low nutritional quality makes bakery products, such as gluten-free biscuits attractive for fortification with protein, dietary fibers, and other nutritional and functional ingredients. From a nutritional point of view, preparation of gluten-free biscuits from composite flour (sweet potato/glutinous rice flour) and fortified with fermented button mushroom polysaccharide flour is wholesome for CD patients. Sweet potato (Ipomoea batatas Lam.) root is known as an important source of energy and carbohydrates, and a staple food in many countries. Sweet potato flour (SPF) is reported to possess high levels of dietary fibers, β-carotene, and minerals, and can improve the color and flavor of the baked products (Bibiana, Grace & Julius, 2014; Krishnan, Menon, Padmaja, Sajeev & Moorthy, 2012). Thus SPF, as well as glutinous rice flour (GRF) can be substituted with wheat flour in the preparation of gluten-free bakery products for their color and taste. Mushrooms are characterized as functional foods due to their low calories, fats, and high contents of proteins, minerals, vitamins, and β-carotene, in addition to their protective role against chronic diseases (Roncero-Ramos & Delgado-Andrade, 2017). Agaricus bisporus is the most consumed mushroom species worldwide, followed by Pleurotus spp., Lentinula edodes and Flammulina velutipes. Mushroom polysaccharides, such as pleuran, erothionine, lentinan, agaritine, ganoderan (Rathore, Prasad & Sharma, 2017) and β-glucans (Khan et al., 2015) have been isolated and characterized from different varieties of mushrooms, which are known as prebiotics, and to possess immense biological properties in curing various degenerative diseases. Several studies have been accomplished the incorporation of different mushroom powders in

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gluten-free doughs and bread to enhance their rheological, sensorial, nutritional, and health characteristics. Different amounts of Pleurotus sajor-caju powder could be added to the biscuit and cake formulations without affecting the pasting, compositional and sensorial properties (Ng, Robert, Ahmad & Ishak, 2017). Ibrahium & Hegazy (2014) stated that the incorporation of mushroom powder in wheat flour could greatly improve the nutritional quality of the cookies. Fermentation is one of the oldest and most economical processes, which is required low energy to food preservation (Fernandez-Orozco et al., 2008). It has been suggested to be a viable method to enhance the nutritional and health quality of the composite gluten-free (CGF) biscuits. Fermentation also leads to improve texture, shelf life, taste, aroma, and digestibility, and it significantly reduces anti-nutrients. According to Hugenholtz & Smid (2002), lactic acid bacteria such as Lactobacillus plantarum are used worldwide in a large variety of industrial food fermentations. Fermentation of mushroom polysaccharide by L. plantarum has been caused significant changes in its chemical composition (Radzki et al., 2016). Due to weakening of gluten-free flour characteristics in the baking process, usage of hydrocolloids such as xanthan gum is highly required to improve its functional, pasting, and rheological properties. To the best of our knowledge, there is limited information on the quality characteristics of the fermented Agaricus bisporus polysaccharide (FABP) flour and its baked products. Therefore, the objective of this research was to evaluate the rheological properties of the gluten-free biscuit dough supplemented with FABP flour and unfermented Agaricus bisporus polysaccharide (UABP) flour, in addition to investigate the quality and nutritional characteristics of the produced CGF biscuits. 2. Materials and Methods 2.1. Materials

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Sweet potato roots (white variety) and glutinous rice flour (8.03 g/100 g protein, 1.06 g/100 g ash and 1.03 g/100 g fat) were purchased from local supermarket. Freeze-dried button mushroom powder (27.91 g/100 g protein, 7.29 g/100 g ash and 2.22 g/100 g fat) was obtained from Longhai Union Food Co., Ltd. (Zhangzhou, China), and xanthan gum from Danisco Co. In addition, De Man Rogosa Sharpe (MRS) broth medium was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All baking ingredients (baking powder, sodium bicarbonate, sugar, margarine and skim milk powder) were purchased from local supermarkets. 2.2. Preparation of sweet potato flour Sweet potato flour was prepared according to the method described by Sulieman et al. (2017). Briefly, the roots after peeling, washing and slicing, the slices were blanched, and then dried at 60 °C for 10 h in convection oven (Dasol Scientific Co. Ltd., Seoul, Korea). The dried slices were milled into flour using a laboratory-scale mill (Tianjin Taisite Instrument Co., Ltd., Tianjin, China), sieved through an 80-mesh sieve, packed, sealed in high density polyethylene bags and analyzed (Shown in supplementary data). 2.3. Extraction of Agaricus bisporus polysaccharide (ABP) flour Using the procedure described by Fan, Zhang, Yu & Ma. (2007), with some modifications, freeze-dried button mushroom powder (200 g) was boiled with 4000 mL distilled water in a vessel for 4 h with stirred regularly. The solid residue was filtered through a 200-mesh nylon cloth, and the residue was re-extracted twice with 2000 mL distilled water to obtain the polysaccharide fraction. All extracted liquid fractions were combined and concentrated at 50 °C using a rotary evaporator under reduced pressure (Wuxi Shenke Instrument, Wuxi, China). The concentrated fraction was precipitated overnight with four portions of 70% ethanol at room temperature and centrifuged at 2080 ×g for 10 min. The resulting pellet was freeze-dried (Free

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Zone, Labconco Co., Ltd., Kansas, USA) at -55 ° C and 0.123 mbar (pressure) for 96 h to obtain ABP flakes, which were then ground in a coffee grinder (Baijie, Wuxi, China). Finally, the ABP flour was stored in sealed bags at 4 °C until further use. 2.4. Fermentation of Agaricus bisporus polysaccharide (ABP) flour MRS broth medium was prepared and dispensed (100 mL) in conical flask (250 mL), sterilized in an autoclave at 121 °C (200 kPa) for 15 min, and then cooled to room temperature (24 ± 1 °C). Lactobacillus plantarum was grown in MRS at 37 °C for 24 h. The cells were harvested and re-suspended in sterilized tryptone (2%) and adjusted to 106 CFU/mL. The cells were then used as an inoculum for ABP flour fermentation. The extracted ABP flour (200 g, wet weight basis) was put tightly into screw-capped plastic vessel and inoculated with 2 mL L. plantarum suspension (106 CFU/mL), without mixing. The fermentation process was carried out at 37 °C for 72 h. The final pH of the fermented product was 3.6. The product was freeze-dried (Free Zone, Labconco Co., Ltd., Kansas, USA) at -55 °C and 0.123 mbar (pressure) for 96 h and then ground in a coffee grinder (Baijie, Wuxi, China) to obtain a FABP flour and stored in sealed bags at 4 °C for further use. 2.5. Preparation of composite gluten-free (CGF) flours and biscuits Three blends were prepared as follows (shown in Table 1, supplementary data): sweet potato flour (SPF) was blended with a constant level of glutinous rice flour (GRF), and then it was enriched with different percentages of FABP flour, and UABP flour (86.5/10/3, 83.5/10/6, and 80.5/10/9). These blends were referred to the formulations (F1, F2, and F3, for both FABP flour and UABP flour). Control sample was composed of SPF (89.5%) and GRF (10%), as well as xanthan gum was added to all flour blends at 0.5% by the total weight. The CGF flours were kept in sealed bags at 4 °C for analyzes.

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CGF biscuits were produced using the method described by Sai Manohar & Haridas Rao (1999), with modifications. The dough was prepared in a laboratory dough mixer. The margarine (120 g) and ground sugar (130 g) were creamed in a Hobart mixer (model SM-5D, Sinmag Machine Co. Ltd., Wuxi, China) with a flat beater for 2 min at 61 rpm and to the cream, water (60 mL) containing sodium bicarbonate (3 g), baking powder (3 g) and sodium chloride (3 g) was added and mixed more for 5 min at 125 rpm to obtain a homogeneous cream. The CGF flour (300 g) and skim milk powder (15 g) were added to the cream and mixed continuously to form the final dough and then sheeted to a thickness 5.0 mm with a rolling pin and using aluminium platform and frame. The biscuits were shaped with a cutter (diameter 55 mm), and baked on aluminium trays at 200 °C for 12 min, cooled for 30 min and stored in air-tight containers for sensory evaluation and further analyzes. 2.6. Functional and pasting properties Functional properties, such as bulk density (BD), water and oil absorption indexes (WAI and OAI), swelling power (SP) and solubility (S), and emulsion activity (EA) were determined using the methods described by Maninder, Kawaljit & Narpinder (2007), Crosbie (1991), and Yasumatsu et al. (1972), respectively, with minor modifications. Briefly, for BD, CGF flour (10 g) was placed into a graduated cylinder (25 mL), tapped the cylinder on a laboratory bench several times, and then based on the weight (g) and volume (mL) the bulk density was calculated. For WAI and OAI, flour sample (1 g) was mixed with distilled water (15 mL), and sunflower oil (10 mL) in a centrifuge tube (25 mL), respectively agitated on a vortex for 2 min, and allowed to stand for 30 min, respectively and then centrifuged at 1850 ×g for 20 min. The adhering drops of water and oil were removed and the tubes re-weighed again. WAI or OAI was examined as gram water or oil bound per gram flour. Distilled water (15 mL) was added to CGF flour (0.5 g) in a

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centrifuge tube, and heated in a water bath at 95 °C for 30 min with stirred regularly. The resulting slurry was cooled to room temperature and centrifuged at 1850 ×g for 15 min. The supernatant was decanted into an evaporating dish, dried in an oven at 105 °C for 4 h. The dried supernatant and the sediment were weighed. The percentage of solubility (S%, dry weight basis) and swelling power (SP g/g) was calculated as follows: Swelling power (SP g/ g) = (sediment weight× 100) ∕ (dry sample weight) × (100 - S%)

(1)

Solubility (S%) = (dry supernatant weight ∕ weight of dry sample) × 100%

(2)

For EA, CGF flour (1 g) was blended with distilled water (10 mL) and sunflower oil (10 mL) in a graduated centrifuge tube, and then the emulsion was centrifuged at 2000 ×g for 5 min. The ratio of the height of the emulsion layer to the total height of the mixture was calculated as the emulsion activity expressed in percentage. Pasting profiles of CGF flour were determined using the RVA 4500 (Perten Instruments, Warriewood, Australia). CGF flour (3.5 g, 14% w.b) was dispersed in distilled water (25.0 ± 0.1 mL) into the RVA canister. The suspensions were manually homogenized using the plastic paddle right before the RVA test, and then the tests were subjected to heating-cooling cycle with a constant stirring speed (160 rpm), it was held at 50 °C for 1 min, heated to 95 °C at 6 °C/min, and held at 95 °C for 5 min, and then it was cooled and held to 50 °C. The viscosity parameters measured were pasting temperature, peak viscosity, trough viscosity, breakdown, setback and final viscosity. 2.7. Rheological properties Rheological properties of CGF biscuit doughs were conducted on a rheometer (DHR3, TA, Instruments, West Sussex, England) according to the procedure described by Ziobro, Witczak, Juszczak & Korus (2013), with some modifications using a parallel plate geometry

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(diameter 20 mm, gap 1 mm) at 25 °C. The prepared doughs (as for CGF biscuit making) were placed in the rheometer measuring system and the edges were removed. Then the doughs were left for 5 min to relax and stabilize the temperature. The test was performed at constant strain amplitude of 0.05% in the angular frequency of 1 to 100 rad/s. The mechanical spectra were determined by recording elastic modulus (G‫)׳‬, viscous modulus (G‫)״‬, and phase shift tangent (tan δ = G‫״‬/G‫)׳‬. 2.8. Physicochemical analyzes 2.8.1. Physical analyzes Weight of CGF biscuits was measured as average value of four individual biscuits with the help of digital balance. Diameter was measured as average value of placing four biscuits edge to edge. Thickness was measured using vernier caliper by taking the average of stacking four biscuits on top of each other. Spread ratio was calculated by dividing the average value of diameter by average value of thickness of biscuits. The color of CGF biscuit was carried out using a chromameter (CR-400, Konica Minolta, Japan). It was calibrated with a white standard plate (L0* = 99.50, a0* = -0.06 and b0* = - 0.19). The values were L*, a* and b*. The L* values (white 100/ black 0), the a* values (red positive/ green negative), and the b* values (yellow positive/blue negative). The colorimetric parameters (L0*, a0* and b0*) were taken as a reference to evaluate total color difference (∆E) according to the following equation: ∆E = [(a* - a0*)2 + (b*- b0*)2 + (L* - L0*)2]-2

(3)

Fracture strength and hardness of CGF biscuits were measured using a texture analyzer TA-XT2i (Stable Micro Systems, London, England). The distance between the two beams was 50 mm. Another identical beam was brought down from above at a pre-test speed of 10 mm/s, test speed of 1 mm/s, post-test speed of 10 mm/s, and distance of 5 mm to contact the biscuit.

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The downward movement was continued till the biscuit breaks. The peak force (g) was reported as fracture strength (Kaur, Sandhu, Arora & Sharma, 2015). 2.8.2. Chemical analyzes Water activity (aw) was determined in CGF biscuit using a Novasina Thermo-constanter model Lab swift-aw (Lucerne, Switzerland) according to the manufacturer’s instructions. The proximate analyzes of the CGF biscuits were determined according to the method of AOAC (2006). Carbohydrate was obtained by difference from moisture, ash, protein and fat. Energy was calculated according to the equation: Energy (kcal/100g) = 4× (g proteins + g carbohydrates) + 9× (g fat). Dietary fibers were determined using enzymatic-gravimetric methods. Briefly, CGF biscuits were digested with heat-stable alpha-amylase, protease and amyloglucosidase. The types of dietary fibers were separated using vacuum filtration. All experiments were performed in triplicate. Amino acids composition of CGF biscuit was analyzed according to the method described by Huang et al. (2011), with modifications using a HPLC in an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) assembly system with a UV detector operated at 338 nm. Powdered CGF biscuit (100 mg) was dissolved in 6N HCL (8 mL) and poured into a hydrolysis tube with screw cap and then hydrolyzed under a nitrogen gas in an oven at 121 °C for 22 h. The extracted sample was dissolved in 4.8 mL 10M NaOH to neutralize for acid hydrolysis. After dilution to a known volume and filtration, the hydrolysate (400 µL) was injected into HPLC column. Minerals composition of CGF biscuit was determined using a wet digestion with closed system according to the method of AOAC (2006). Powdered CGF biscuit (1 g) was digested with concentrated HNO3 (5 mL) and HClO4 (1 mL) in an oven at 100 °C for 6 h. The digested

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samples were transferred to volumetric flasks (100 mL), diluted, filtered through a Whatman filter paper No. 541 and then transferred to plastic bottles (50 mL) for mineral detection using atomic absorption spectrophotometer. 2.9. Sensory evaluation Sensory evaluation was carried out in baking laboratory under normal conditions described by PN-ISO 8589: (2010) . CGF biscuit was evaluated by 61 untrained and semi-trained panelists, male and female (ages 25-42), comprised mainly of students and staff members of School of Food Science and Technology of Convenient Food and Quality Control Laboratory (Wuxi, China). Panelists were used a nine-point hedonic scale for degree of acceptance of color, appearance, aroma, taste, crispiness, and overall acceptability taking 1 extremely dislike, 5 neither like nor dislike and 9 extremely like. 2.10. Statistical analysis All tests were done in triplicate. The data were assessed using analysis of variance (ANOVA), and Duncan’s multiple range (DMART) test was carried out to separate means at a significance level of 95% (p ˂ 0.05). Statistical analyzes and calculations were performed using SPSS software (version 16, SPSS Inc., Chicago. USA). 3. Results and Discussion 3.1. Functional and pasting properties Functional properties are substantial physicochemical characteristics of food materials that reflect the complex interaction among the structure, composition, and other analytical properties of nutrients. Functional properties of UABP flour, FABP flour and CGF flour formulations are provided in Fig. 1a and b. The BD of the UABP flour and FABP flour was 0.45 g/mL and 0.50 g/mL, respectively; while those of CGF flour formulations ranged from 0.56 to

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0.66 g/mL. The highest BD was observed in FABP flour F3 (0.66 g/mL) followed by UABP flour F3 (0.61 g/mL), and the lowest (0.56 g/mL) in the control. There were no significant differences (p ˃ 0.05) in BD among the other formulations. In this study, the fermentation process and particle size of the polysaccharide flour added affect the BD, which is a good physical characteristic in food preparations, as well as highly required in food packaging as a relative volume. The WAI and OAI of UABP flour and FABP flour were 6.08 g/g and 3.69 g/g, and 5.65 g/g and 3.23 g/g, respectively. Moreover, WAI and OAI of CGF flour formulations ranged from 1.88 g/g to 2.69 g/g and 1.85 g/g to 2.49 g/g, respectively. UABP flour F3 had the highest WAI and OAI among the formulations, whereas the control sample had the lowest (Fig. 1a). The lower in WAI and OAI of FABP flour compared to UABP flour may be due to the degradation of carbohydrate, which is a major component of polysaccharide flour known to contribute significantly to water and oil absorption. On the other hand, the addition of both FABP flour and UABP flour increased WAI and OAI in the CGF flour formulations. This might be related to increase in hydrophilic nutrients (e.g. protein and dietary fibers) and effect of fermentation process by L. plantarum. Fermented and malted flours had higher affinity for water, as well as a loss in the structure of the starch polymers, which were degraded during the fermentation and malting caused the flours to absorb more water (Adebiyi, Obadina, Mulaba-Bafubiandi, Adebo & Kayitesi, 2016). Duta & Culetu, (2015) reported a similar result of WAI in preparation of oatbased gluten-free cookies. Oil intake is an important feature since fat is a flavor enhancer and increases the mouth-feel of food products. An increase of WAI and OAI in germinated amaranth flour could be attributed to an increase in protein content and change in the quality of protein upon germination, as well as breakdown of polysaccharide molecules; therefore the sites for

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interaction with holding water would be increased; and its capacity to hold fat globules as the amount of lipophilic nutrient also increased (Chauhan, Saxena & Singh, 2017). UABP flour had high SP (11.72 g/g) and low solubility (36.15 g/g), whereas FABP flour had high solubility (37.94 g/g) and low SP (10.09 g/g), in addition to incorporation of both UABP flour and FABP flour resulted in a decrease in SP and an increase in solubility of CGF flours, but there were no significant differences (p ˃ 0.05) in SP among UABP flour F1, FABP flour F1, and control; and in solubility among UABP flour F2, F3 and FABP flour F2 (Fig. 1b). A possible explanation for the decreased SP and increased solubility attributed to reduced in starch molecules/or content and increment in soluble dietary fibers and protein. SP is ascribed to the capacity of starch constituents to water intake within its structure through bond with hydrogen atom. Chinma, Anuonye, Simon, Ohiare & Danbaba (2015) explained that the reduction in starch content and alters in its structure take place by activities of enzymes during germination process decreased in SP of Nigerian germinated rice flours. The swelling capacity increased by the inclusion of sesame peels flour in wheat flour, which is reported by Zouari, Besbes, Ellouze-Chaabouni & Ghribi-Aydi (2016). On the other hand, Majzoobi, Pashangeh, Farahnaky, Eskandari & Jamalian (2014) found that the fermentation process increased the soluble dietary fiber content of the bran leading to higher water solubility. Emulsion is a complex and thermodynamically unstable system, prepared by two mutually unmixable liquids dispersion into one another through blending, shearing, and homogenization (Zhang, Guo, Zhu, Peng & Zhou, 2015). The emulsion activity of UABP flour was higher (36.81%) than FABP flour (35.50%). The difference in particle size of the polysaccharide flour might influence on the emulsion activity. In addition, the incorporation of UABP flour and FABP flour in the blends contributed to a significant increase in emulsion activity of all samples compared to the control. FABP flour F3 had the highest

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emulsion activity (68.31%) followed by UABP flour F3 (66.33%), and the lowest (59.52%) in the control (Fig. 1b). The type of the protein, as well as its concentration and solubility affect the efficiency of emulsification. In this study, the formation of protein-stabilized emulsion of CGF flour could be stronger than a starch-stabilized emulsion of the control. According to Zhang, Guo, Zhu, Peng & Zhou (2015), oat protein isolate-dextran conjugates had a strong emulsion, which revealed smaller particle size and better storage stability compared to the native oat protein isolate. Fig. 1c shows the pasting parameters of the CGF flour enriched with UABP flour and FABP flour. Addition of UABP flour increased peak viscosity (PV), trough viscosity (TV), breakdown (BD), final viscosity (FV) and pasting temperature (PT), and decreased setback (SB) compared to the control, while incorporation of FABP flour reduced all the mentioned viscosity parameters, with exception of FABP flour F1. PV is an indicative of swelling capacity of flour starch or the blend due to water absorption during heating cycle. UABP flour F1 exhibited the highest PV, TV and FV, while FABP flour F2 exhibited the lowest in these parameters (Fig. 1c). An increase in the mentioned above parameters could be due to an interaction between protein and dietary fiber of polysaccharide flour added and starch molecules of CGF flour. Setback is a retrogradation process of starch molecules upon cooling, resulting in an increase in viscosity. FABP flour F3 had the lowest setback (633.21 cP), whereas control sample had the highest (971.67 cP). From these values, it was observed that there was a significant decrease (p ˂ 0.05) in setback of FABP flour F3, thus less retrogradation of amylose takes place. Sarabhai, Sudha & Prabhasankar (2017) reported that low amylose content of the amaranth flour provided less soluble amylose during cooling an indicating to lower setback and reduce retrogradation. Pasting characteristic of flour starch is affect by swelling of granules during the heating process. The

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increment in pasting temperature of CGF flour formulations after enrichment with UABP flour and FABP flour might be attributed to increment in insoluble dietary fibres and decrement in carbohydrates occurred by these mushroom polysaccharide flours. As interpreted by Sulieman et al. (2017), the pasting temperature of CGF flour increased with increase in dietary fibers, protein and fat contents, and decrease in starch molecules as a result of button mushroom powder incorporation. 3.2. Rheological properties Frequency sweep tests were performed to study the influence of UABP flour and FABP flour on the rheological parameters (such as, elastic modulus (G‫)׳‬, viscous modulus (G‫)״‬, and phase shift tangent (tan δ)) of CGF biscuit doughs. The changes in elastic modulus (G‫)׳‬, viscous modulus (G‫)״‬, and tangent (tan δ) for the control and CGF biscuit dough formulations are given in Fig. 2a and b. In this investigation, G‫ ׳‬was higher than G‫ ״‬in the whole frequency range tested; this shows the solid-like behavior of all the CGF biscuit doughs, it means that elastic characteristics predominated viscous characteristics. This is in line with previous findings regarding rheological properties (e.g., G‫ ׳‬and G‫ )״‬of gluten-free biscuit dough (Sarabhai, Sudha & Prabhasankar, 2017). Both G‫ ׳‬and G‫ ״‬increased with increasing angular frequency (Fig. 2a). According to Ashraf Khan, Gani, Masoodi, Mushtaq & Naik (2017), storage modulus (G‫ )׳‬can be defined as the deformation of the stored energy in the sample after oscillation, which is providing its elastic behavior, in contrast to loss modulus (G‫ )״‬is the energy lost by the sample throughout oscillation, an indicating to its viscous behavior. Compared to control dough, both G‫ ׳‬and G‫ ״‬for all CGF biscuit dough formulations increased with the incorporation of UABP flour and FABP flour. Among the CGF biscuit dough formulations, UABP flour F3 and FABP flour F3 had the highest G‫ ׳‬and G‫״‬, whereas FABP flour F1 was the lowest in these rheological moduli. It can be

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noticed that the addition of UABP flour increased the both G‫ ׳‬and G‫ ״‬of the doughs more than inclusion of FABP flour (Fig. 2a). This could be ascribed to the high viscosity of the UABP flour formulations (shown in Fig. 1c). Moreover, in the case of FABP flour added, formation of some organic acids during the fermentation process may be led to a decrease in the viscosity of its formulations. The acidification of the dough have an impact on structure-forming components especially protein and starch. Raymundo, Fradinho & Nunes (2014) explained that the increase of Psyllium fiber level in biscuit dough resulted in an increase degree of dough structure, which leads to an increase of G‫׳‬. With regard to tangent, control dough had the highest tan (δ), whereas UABP flour F3 and FABP flour F3 had the lowest (Fig. 2b). It seems that the addition of unfermented and fermented polysaccharide flours decreased the tan (δ) of all the dough formulations. In addition, at lower frequencies (1 to 9 rad/s), tan of all dough formulations reduced when frequency was increased, and at higher frequencies (10 to 100 rad/s) increased (Fig. 2b). These differences indicate that the doughs seem more solid and elastic like, when subjected to slow chances in stress, but very fast changes will make the doughs act more liquid and viscous like. Blanco Canalis, Steffolani, Leon & Ribotta (2017) had also observed that tan (δ) of dough supplemented with fiber and polysaccharide powder seemed to be more elastic behavior at lower frequencies (between 0.05 and 10 rad/s), and found to have viscous behavior at higher frequencies. 3.3. Physical analyzes Table 1 shows the results of physical characteristics of control and CGF biscuit formulations. UABP flour F3 presented the highest (20.22 g) and FABP flour F2 the lowest (18.01 g) weight value, compared to the control (19.18 g). This increase may be associated with dietary fibers of UABP flour added have ability to bind with water molecular that prevent

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moisture loss during the baking process. Diameter and spread ratio values ranged from 60.43 to 67.00 mm and 5.70 to 6.56, respectively (Table 1). From the Table, it can be observed that the lowest diameter and spread ratio for UABP flour F 3 and highest for FABP flour F1 and F3 were obtained. This means that addition of FABP flour increased diameter and spread ratio of CGF biscuits, while inclusion of UABP flour gave a reversible result. Decrease in diameter, in this study was in agreement with the result of Srivastava, Genitha & Yadav (2012), who mentioned that due to increment in fibers content of biscuits. FABP flour significantly (p ˂ 0.05) increased spread ratio, indicating better quality parameters of these biscuits, which are more preferable to consumers. UABP flour F1 and F2 had the highest thickness (10.97 mm and 10.83 mm, respectively), whereas FABP flour F3 revealed the lowest thickness (9.93 mm). The hydrophilic nature of the FABP flour added, in addition to the acidification might be influenced on thickness of its formulations. The surface color of CGF biscuit in terms of L*, a*, b* and ∆ E values is shown in Table 1. The L*, a* and b* values reduced with the increase in the levels of both UABP flour and FABP flour in the CGF biscuit formulations; it means control biscuit had the highest lightness (L*), redness (a*) and yellowness (b*) values, compared to the UABP and FABP flours formulations (UABP flour F3 had the darkest color), in contrast to total color difference (∆ E) values, which increased by fortification (Table 1). In the current investigation, the decrease in the lightness (L*) value may be attributed to increase of dietary fibers, protein and some colored pigments that present in the UABP flour and FABP flour added, in addition to some changes in color, which was happened during the fermentation process in the FABP flour, as well as caramelization of sugars upon the baking process. Incorporation of oat bran in cookies increased the protein content and contributed to the increment in Maillard reaction, thus the samples

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became darker as explained by Duta & Culetu (2015), besides low yellowness (b*) values in CGF biscuit formulations an indicative of low lightness (L*) values. Regarding the fracture strength and hardness of CGF biscuits, the lowest values of fracture strength (775.59 g) and hardness (748.11 g) were obtained by UABP flour F2, whereas the highest values (1399.90 g and 1590.59 g, respectively) were gained by the control. In general, supplementation of CGF biscuits with both mushroom polysaccharide flours, especially FABP flour reduced in fracture strength and hardness values (Table 1), due to decrease in carbohydrate content and increase in dietary fibers and some organic acids, a result of fermentation process. Similarly, results were obtained by Raymundo, Fradinho & Nunes (2014) for wheat biscuits enriched with Psyllium fiber showed a significantly decrease in hardness with the increase of fiber and decrease of protein content, and subsequently reduction in the capacity of the gluten development. Substitution of buckwheat flour with starch in gluten-free biscuit led to increase in fracturability values, an indicative of looser matrix formation in the doughs, which contain lower amount of proteins as described by Oksuz & Karakaş (2016). 3.4. Chemical analyzes Chemical compositions of FABP flour, control, and CGF biscuit formulations are listed in Table 2. The water activity (aw) and moisture content of the samples ranged between 0.02 to 0.41 and 2.23 g/100 g to 5.76 g/100 g, respectively, with the FABP flour significantly (p ˂ 0.05) lower than all CGF biscuits. Addition of both UABP flour and FABP flour increased the aw and moisture content of the CGF biscuit formulations, compared to the control (Table 2). This could be associated with the higher WAI of both polysaccharide flours (Fig. 1a). FABP flour had the highest ash, protein, and soluble dietary fiber contents, and the lowest insoluble dietary fiber, compared to UABP flour (Table 2, supplementary data). Among the CGF biscuit samples,

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UABP flour F3 showed the highest contents of ash, fat and total dietary fibers, while FABP flour F3 exhibited the highest protein and soluble dietary fibers and the lowest carbohydrate contents. An increase of ash, dietary fibers and protein in CGF biscuit formulations compared to the control, resulting from high levels of minerals, fibers and protein in both UABP flour and FABP flour added. Moreover, the high fat content of the CGF biscuits, compared to the polysaccharide flour added could be ascribed to fat added during preparation of dough. Furthermore, the observed increment in protein and soluble fibers of FABP flour formulations (Table 2) may be due to creation of some more amino acids in FABP flour from accumulation of proteins as a result of fermentation process. Similar observations were reported by Adebiyi, Obadina, Adebo & Kayitesi (2017) for biscuit prepared from inclusion of fermented and malted pearl millet flour. According to Majzoobi, Pashangeh, Farahnaky, Eskandari & Jamalian (2014), hydrothermal and fermentation of wheat bran increased the amount of total and soluble dietary fibers. Amino acids can be defined as important biological constituents required in the human body for biosynthesis, neuro-transmission and other metabolic activities (Adebiyi, Obadina, Adebo & Kayitesi, 2017), and they are divided into two groups: essential (cannot synthesize by the body and must be obtained from food) and non-essential (can synthesize by the body). Table 3 displays the findings of amino acids of UABP flour, FABP flour and CGF biscuits. In this study, FABP flour was twofold higher in majority of amino acids than UABP flour. This was observed in the CGF biscuit formulations, which FABP flour F3 revealed the highest total amino acids (6127.08 mg/100 g), followed by FABP flour F2 (3812.49 mg/100 g) and then UABP flour F3 (3334.32 mg/100 g). Incorporation of both UABP flour and FABP flour was significantly (p ˂ 0.05) led to increase in all essential and non-essential amino acids of CGF biscuit formulations, due to high amino acids content of both polysaccharide flours added (Table 3). Glutamic acid,

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aspartic acid, glycine and alanine had the highest non-essential amino acids in CGF biscuit, while valine, phenylalanine, isoleucine and leucine were the highest essential amino acids (Table 3). Button mushroom powder has a relatively high amount of essential amino acids, such as arginine, valine, leucine and lysine, and non-essential amino acids (e.g. aspartic and glutamic acids, and proline) compared to sweet potato flour (Sulieman et al., 2017). Aspartic and glutamic acids are monosodium glutamate-like constituents, which give the mushroom and its products palatable taste. Mineral elements are necessary for the growth, development, maintenance and repair of the human body. There are two groups of minerals as follows: macro-minerals, such as Ca, P, K, Mg and Na, and micro-minerals, for example Cu, Zn and Fe. Fig. 3a and b shows the mineral contents of the SPF, UABP flour, FABP flour and CGF biscuits. SPF had the lowest content in all minerals; and UABP flour exhibited higher macro-minerals (Ca, K, P and Na) and lower micro-minerals (Cu, Zn and Fe) contents, whereas lower macro-minerals (except Mg) and higher micro-minerals were detected in FABP flour (Fig. 3a). Sade (2009) reported that the fermentation process reduced Ca, P, and K contents of millet flour. For the CGF biscuit samples, Ca and K contents ranged from 421.02 to 490.57 mg/100 g and 158.00 to 203.13 mg/100 g, respectively, and were significantly (p ˂ 0.05) higher in the UABP flour and FABP flour formulations. Similar results were also recorded for Mg, P, Cu, Zn, and Fe; but lower for Na content was observed in some formulations compared to the control biscuit (Fig. 3b). Incorporation of FABP flour, as well as UABP flour improved minerals composition of CGF biscuits. Besides this, higher mineral contents of CGF biscuit samples could be attributed to Maillard reaction that happened during the baking process. FABP flour F3 exhibited the highest Mg (27.20 mg/100 g), Cu (0.50 mg/100 g) and Zn (2.36 mg/100 g) and the lowest P (135.18

21

mg/100 g), while UABP flour F3 showed the highest K (203.13 mg/100 g). The formation of complexes or separation of compounds, resulting from Maillard reaction could affect solubility and availability of some minerals (Adebiyi, Obadina, Adebo & Kayitesi, 2017). 3.5. Sensory evaluation The CGF biscuits fortified with UABP flour and FABP flour were evaluated for their color, appearance, aroma, taste, crispiness and overall acceptability using 9-point hedonic scale (Fig. 3c). Among the CGF biscuits, control sample recorded the highest color (8.65) and appearance (8.46), but FABP flour F1 and UABP flour F1 obtained the highest aroma (8.15 and 7.50) , taste (8.48 and 7.49), crispiness (6.86 and 7.88) and overall acceptability (7.82 and 7.55, respectively). Panelists found out that the FABP flour F1 was the better in all sensorial characteristics, as well as UABP flour F1 in the second order. While UABP flour F3 recorded the lowest scores (p ˂ 0.05) in all sensorial attributes according to panelists acceptability. This could be ascribed to higher dietary fibers and protein from UABP flour added, leading to the darker color and higher degree of hardness of UABP flour F3. FABP flour formulations were more acceptable for panelists compared with UABP flour formulations, due to softer texture, lighter color and better flavor. Inclusion of moderate amounts of FABP flour elevated the panelist acceptability, whereas increase levels of UABP flour reduced acceptability scores. This study in agreement with an investigation conducted using different amounts (4-12%) of Pleurotus sajorcaju powder in wheat biscuits (Ng, Robert, Ahmad & Ishak, 2017). FABP flour F2 was also acceptable for panelists in aroma, taste, crispiness and overall acceptability, because the fermentation process led to enhance the aroma and crispiness of the CGF biscuits. Wan Rosli, Nurhanan & Aishah (2012) suggested that the replacement of wheat flour with up to 4% oyster mushroom powder improved crispiness and flavor attributes of butter biscuits, as well as

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increased protein, dietary fibers and β-glucan levels. Furthermore, up to 9% Auricularia auricula polysaccharide flour could be incorporated in the baked products without changing the sensory preference of the final product (Fan, Zhang, Yu & Ma, 2007). 4. Conclusions CGF biscuits were developed using SPF/GRF fortified with FABP flour and UABP flour. Incorporation of both polysaccharide flours had positive effects on functional, pasting, and rheological properties of CGF flour and biscuit doughs, respectively. CGF biscuits fortified with FABP flour and UABP flour showed interesting physical characteristics, with softer texture (especially FABP flour formulations), but lower lightness compared to the control. In addition, higher water activity, moisture, ash, protein, fat and total dietary fibers, with lower carbohydrate were observed for CGF biscuit formulations, as well as they contained good amounts of amino acids and minerals. With regard to sensory evaluation, addition of 3-6% FABP flour and 3% UABP flour were acceptable for consumers. Further studies on the antioxidant and antimicrobial activities of the FABP flour and CGF biscuits are needed to understand the effect of the fermentation process on these activities. FABP flour and UABP flour, which represent as novel alternative food ingredients, can be widely applied to developing various FABP and UABP flours-based functional foods for CD patients. Acknowledgements We would like to thank the Priority Academic Program Development of Jiangsu Province, Higher Education Institutes, Wuxi, China. We also extend our sincere gratitude to all co-authors to their contributions in this work, in addition to the staff and students of the Research Center of Convenient Food and Quality Control, especially Dr. Ze-Hua Huang for his valuable assistance. References

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Fig. 1. Functional (a and b) and pasting (cP) (c) properties of composite gluten-free (CGF) flours (UABP flour = unfermented Agaricus bisporus polysaccharide flour, FABP flour = fermented Agaricus bisporus polysaccharide flour, BD = bulk density, WAI = water absorption index, OAI = oil absorption index, SP = swelling power, S = solubility, EA = emulsion activity). Error bars indicate standard deviations; mean values marked with different letters are significantly different. Fig. 2. Mechanical spectra (a) and phase shift tangent (b) of composite gluten-free (CGF) biscuit doughs (UABP flour = unfermented Agaricus bisporus polysaccharide flour, FABP flour = fermented Agaricus bisporus polysaccharide flour); square = control dough, triangle = UABP flour F1, reverse triangle = UABP flour F2, diamond = UABP flour F3, star = FABP flour F1, polygonal = FABP flour F2, circle = FABP flour F3 (G′ = closed symbols, G″ = open symbols). Fig. 3. Minerals composition of SPF, UABP flour, FABP flour and composite gluten-free (CGF) biscuits (a and b) and sensory evaluation results of CGF biscuits (c) enriched with fermented and unfermented Agaricus bisporus polysaccharide flour (SPF = sweet potato flour, UABP flour =

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unfermented Agaricus bisporus polysaccharide flour, FABP flour = fermented Agaricus bisporus polysaccharide flour). Error bars indicate standard deviations; mean values marked with different letters are significantly different.

Table 1. Physical characteristics of composite gluten-free biscuit enriched with fermented and unfermented Agaricus bisporus polysaccharide flour. Parameters Weight (g) Diameter (mm) Thickness (mm) Spread ratio Color L*

Control 19.18 ± 0.37d 65.03 ± 0.58d 10.33 ± 0.36b 6.29 ± 0.23c

U ABP flour F1 18.58 ± 1.78f 64.47 ± 0.58e 10.97 ± 0.28a 5.88 ± 0.20d

F2 19.31 ± 1.03c 63.80 ± 1.73f 10.83 ± 0.06a 5.89 ± 0.14d

50.52 ± 1.01a 9.29 ± 0.55a

47.52 ± 1.49b 7.08 ± 0.29b

44.17 ± 0.41e 6.41 ± 0.18c

F3 20.22 ± 1.73a 60.43 ± 0.63g 10.60 ± 0.26ab 5.70 ± 0.08e

FABP flour F1 19.66 ± 2.07b 67.00 ± 1.89a 10.40 ± 0.46b 6.44 ± 0.14b

F2 18.01 ± 0.69g 65.90 ± 1.05b 10.30 ± 0.50b 6.40 ± 0.24b

41.01 ± 45.62 ± 44.85 ± 0.72g 0.41c 0.68d 6.10 ± 7.01 ± 6.17 ± 0.40cd a* cd b 0.34 0.19 22.21 ± 18.77 ± 16.85 ± 15.15 ± 18.33 ± 16.92 ± b* a b c d b 0.25 0.48 0.20 0.13 0.03 0.52c 54.66 ± 55.79 ± 58.25 ± 60.78 ± 57.41 ± 57.60 ± ∆E 0.72e 1.26d 0.31b 0.43a 0.42c 0.52c 1393.64 ± 775.59 ± 1217.52 ± 1045.30 ± 1225.56 ± Fracture strength 1399.90 ± a a d b c 3.83 4.33 1.70 5.83 2.33 4.32b (g) 1590.59 ± 1490.77 ± 748.11 ± 1409.18 ± 1097.10 ± 1188.99 ± Hardness (g) a b f c e 6.01 4.38 1.50 4.10 1.82 2.43d Mean ± standard deviation (n= 3). Mean values within a raw followed by a different letter are significantly different (p ˂ 0.05). UABP flour = Unfermented Agaricus bisporus polysaccharide flour, FABP flour = Fermented Agaricus bisporus polysaccharide flour.

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F3 19.02 ± 1.60e 65.15 ± 1.08c 9.93 ± 0.23c 6.56 ± 0.28a

41.50 ± 0.51f 5.45 ± 0.31e 15.00 ± 0.25d 60.21 ± 0.53a 1392.95 ± 4.45a 1200.82 ± 3.26d

Table 2. Chemical compositions of fermented Agaricus bisporus polysaccharide flour and composite gluten-free biscuit enriched with fermented and unfermented Agaricus bisporus polysaccharide flour. Parameters (g/100 g)

UABP flour

FABP flour

F3 FABP flour Control F1 F2 F1 F2 0.05 ± 0.35 ± 0.39 ± 0.45 ± 0.39 ± 0.41 ± 0.38 ± aw 0.00c 0.01bc 0.01ab 0.00a 0.02ab 0.02a 0.00b 2.23 ± 3.39 ± 3.94 ± 4.92 ± 4.79 ± 4.81 ± 4.84 ± Moisture 0.05f 0.13e 0.17d 0.09b 0.07c 0.11c 0.15c 4.32 ± 2.23 ± 2.27 ± 2.31 ± 2.42 ± 2.23 ± 2.28 ± Ash a d cd c b d 0.29 0.03 0.02 0.06 0.03 0.04 0.08c 1.09 ± 14.01 ± 14.65 ± 15.09 ± 15.41 ± 14.13 ± 14.62 ± Fat g f d b a e 0.11 0.13 0.10 0.08 0.12 0.09 0.10d 20.05 ± 7.80 ± 8.68 ± 10.67 ± 11.13 ± 8.83 ± 11.16 ± Protein 0.72a 0.09g 0.11f 0.80d 0.10c 0.08e 0.13c 72.57 ± 70.46 ± 67.01 ± 66.25 ± 70.00 ± 67.10 ± Carbohydrate 72.31 ± b a c e f d 0.69 1.01 0.81 0.99 0.63 0.95 0.79e 379.25 ± 447.57 ± 448.41 ± 446.53 ± 448.21 ± 442.49 ± 444.62 ± Energy h c a d b f 1.91 1.02 1.07 1.09 0.89 0.95 0.99e (Kcal/100 g) Dietary fiber 6.61 ± 1.01 ± 2.67 ± 2.98 ± 3.30 ± 2.41 ± 2.74 ± IDF a g e d b f 0.45 0.04 0.05 0.05 0.10 0.09 0.11e 3.01 ± 1.34 ± 1.43 ± 1.76 ± 1.93 ± 1.62 ± 1.98 ± SDF a h g e d f 0.28 0.02 0.02 0.03 0.01 0.05 0.08c 9.62 ± 2.35 ± 4.10 ± 4.74 ± 5.23 ± 4.02 ± 4.63 ± TDF 0.52a 0.07g 0.09e 0.11c 0.18b 0.12f 0.22d Mean ± standard deviation (n= 3). Mean values within a raw followed by a different letter are significantly different (p ˂ 0.05). UABP flour = Unfermented Agaricus bisporus polysaccharide flour, FABP flour = Fermented Agaricus bisporus polysaccharide flour, aw = Water activity, IDF = Insoluble dietary fiber, SDF = Soluble dietary fiber, TDF = Total dietary fiber.

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F3 0.40 ± 0.01ab 5.76 ± 0.10a 2.32 ± 0.04c 14.85 ±0.11c 11.98 ± 0.09b 65.09 ± 0.88g 441.93 ± 1.22g 3.11 ± 0.27c 2.08 ± 0.10b 5.19 ± 0.35b

Table 3. Amino acid compositions of fermented and unfermented Agaricus bisporus polysaccharide flours and composite gluten-free biscuit enriched with fermented and unfermented Agaricus bisporus polysaccharide flours. Amino acids (mg/100 g)

Aspartic acid Glutamic acid Tyrosine Serine Proline Glycine Alanine Cysteine Arginine* Histidine* Threonine* Valine* Methionine* Phenylalanine* Isoleucine* Leucine* Lysine*

UABP flour UABP flour 1670.13 ± 1.47b 1970.24 ± 0.31b 1142.24 ± 0.21a 275.59 ± 0.91b 496.25 ± 0.36a 1341.85 ± 1.24b 1405.62 ± 0.98b 7.74 ± 0.14d 425.96 ± 0.88b 107.88 ± 0.43b 263.62 ± 0.66b 422.57 ± 0.81b 147.55 ± 0.40b 323.25 ± 0.39c 408.00 ± 0.83b 567.96 ± 0.99c 388.67 ±

FABP flour 1914.65 ± 0.97a 2787.90 ± 1.41a 606.55 ± 0.31b 871.74 ± 0.29a 447.42 ± 0.29b 1385.41 ± 0.75a 1721.92 ± 0.99a 1.13 ± 0.02g 1122.10 ± 0.89a 170.13 ± 0.29a 450.42 ± 0.21a 1136.38 ± 0.91a 378.55 ± 0.31a 915.01 ± 0.69a 993.53 ± 0.88a 2345.23 ± 1.33a 281.29 ±

FABP flour

Control

F1

F2

F3

F1

F2

F3

323.11 ± 0.13g 389.34 ± 0.21h 69.31 ± 0.09h 9.16 ± 0.05i 64.32 ± 0.04i 81.37 ± 0.10g 144.45 ± 0.11i 3.32 ± 0.03f 61.66 ± 0.08i 2.57 ± 0.03g 1.63 ± 0.01f 64.75 ± 0.09i 21.08 ± 0.10g 59.46 ± 0.11i 57.88 ± 0.09i 168.81 ± 0.19i 18.45 ±

558.65 ± 0.19d 534.00 ± 0.36g 162.38 ± 0.21g 163.79 ± 0.11e 87.16 ± 0.12h 201.15 ± 0.12d 286.36 ± 0.12d 4.23 ± 0.04e 131.25 ± 0.11e 2.92 ± 0.05g 62.23 ± 0.08d 137.48 ± 0.18h 44.97 ± 0.08d 132.63 ± 0.18h 111.33 ± 0.15h 366.59 ± 0.32e 29.45 ±

541.36 ± 0.15e 782.29 ± 0.19e 182.29 ± 0.14f 131.56 ± 0.13g 103.71 ± 0.10g 125.96 ± 0.12e 195.98 ± 0.07g 38.46 ± 0.07a 109.56 ± 0.11g 8.37 ± 0.06e 61.65 ± 0.19d 232.75 ± 0.25e 29.08 ± 0.10f 189.49 ± 0.15e 163.99 ± 0.13e 330.97 ± 0.30f 30.84 ±

589.75 ± 0.21c 781.78 ± 0.39e 211.92 ± 0.11e 140.53 ± 0.15f 188.80 ± 0.08d 121.37 ± 0.19e 202.23 ± 014f 4.22 ± 0.03e 116.54 ± 0.13f 4.25 ± 0.02f 60.51 ± 0.06d 214.94 ± 0.14f 32.14 ± 0.12e 177.78 ± 0.20g 152.47 ± 0.16g 306.73 ± 0.38h 37.36 ±

244.24 ± 0.11i 758.95 ± 0.50f 179.72 ± 0.14f 124.86 ± 0.09h 119.45 ± 0.13f 123.44 ± 0.0.08e 185.82 ± 0.11h 11.12 ± 0.05c 104.18 ± 0.11h 17.52 ± 0.09d 55.45 ± 0.08e 212.52 ± 0.17g 7.88 ± 0.07h 184.36 ± 0.13f 159.96 ± 0.17f 318.44 ± 0.41g 32.18 ±

311.12 ± 0.16h 927.55 ± 0.41d 223.29 ± 0.12d 177.28 ± 0.08d 182.53 ± 0.15e 185.33 ± 0.09f 254.41 ± 0.99e 5.87 ± 0.04e 156.55 ± 0.15d 16.78 ± 0.09d 83.73 ± 0.10c 297.98 ± 0.19c 47.19 ± 0.10c 246.82 ± 0.22d 217.87 ± 0.28d 434.88 ± 0.38d 43.31 ±

467.82 ± 0.23f 1284.37 ± 1.07c 311.07 ± 0.22c 257.25 ± 0.17c 433.77 ± 0.25c 302.04 ± 0.17c 408.35 ± 0.88c 25.51 ± 0.06b 322.88 ± 0.26c 81.94 ± 0.10c 264..06 ± 0.18b 489.22 ± 0.27d 8.33 ± 0.06h 418.16 ± 0.41b 339.90 ± 0.27c 585.88 ± 0.71b 126.53 ±

32

TNAA TEAA TAA

0.72a 0.17b 0.08h 0.10g 0.05g 0.05e 0.09f 0.04d b a i g f e h 7813.41 9736.72 1084.38 1997.72 2101.61 2240.60 1747.60 2267.38d b a i h e f g 3055.46 7511.35 456.29 1018.85 1156.70 1102.72 1092.49 1545.11d 10868.87b 17248.07a 1540.67i 3016.57g 3258.31f 3343.32e 2840.09h 3812.49d Mean ± standard deviation (n= 3). Mean values within a raw followed by a different letter are significantly different (p ˂ 0.05). UABP flour = Unfermented Agaricus bisporus polysaccharide flour, FABP flour = Fermented Agaricus bisporus polysaccharide flour, *Essential amino acids; TNAA = Total non-essential amino acids, TEAA = Total essential amino acids, TAA = Total amino acids.

33

0.13c 3490.18c 2636.90c 6127.08c