Rheological and microstructural properties of wheat dough supplemented with Flammulina velutipes (mushroom) powder and soluble polysaccharides

ABSTRACT Partial substitution of wheat flour with Flammulina velutipes powder (FVP) or soluble polysaccharide (SPFV) at different addition levels, and the effects of which on the rheological and microstructural properties of dough were investigated. FVP significantly (P < 0.05) increased the water absorption but decreased the development time and stability of dough significantly (P < 0.05). Furthermore, it was capable of providing a weaker extension and harder dough with the increasing of FVP addition levels. FVP increased the storage (G′) and loss (G″) moduli, while the tan δ decreased with the increasing of FVP addition levels. However, SPFV addition had inconsistent viscoelastic results with that of FVP addition. The microstructure of dough showed that the continuity of gluten networks had been disrupted by FVP and SPFV at a higher addition level. This research could provide a foundation for the application of FVP in wheat-flour foods, and FVP addition levels of 2.5% to 5.0% are recommended.


Introduction
Flammulina velutipes, also known as golden needle mushroom or winter mushroom, is one of edible fungus with the largest production and consumption in China (Shi et al., 2018). As the fourth most popular edible mushroom in the world, this mushroom has an attractive taste and high nutritional values with low calorie and fat contents and high proportions of essential amino acids, fiber, and vitamins (Fang et al., 2016;Jing et al., 2014). It has also been highly valued as a functional food owing to its good antioxidant, anti-inflammatory, immunomodulatory, antitumour, and cholesterol-lowering effects (Fukushima, Ohashi, Fujiwara, Sonoyama, & Nakano, 2001;Leung, Fung, & Choy, 1997;Wu, Duan, Liu, & Cen, 2010;Wu et al., 2014;Xia, 2015). In recent years, many factories have been set up for the large-scale cultivation of F. velutipes in China and Japan (Cai, Liu, Chen, Liao, & Zou, 2013). The annual output of F. velutipes in China has exceeded 4,000,000 tons, and it has become the first largest mushroom variety under factory cultivation. However, there are few deep-processing technologies and products for F. velutipes at present in China, and the main consumption pattern is fresh eating. As a result, the inconsistency between production and marketing is becoming increasingly acute with the fast growth of F. velutipes output. Therefore, developing deep-processing technologies and products is necessary for the healthy and sustainable development of the F. velutipes industry.
Wheat-flour foods have a huge amount of consumption in China and around the world. In recent years, many exogenous materials such as wheat bran, black tea, purple sweet potato powder, banana pulp and peel, seed powder, fiber, non-starch polysaccharides and soy protein, have been added into noodles, bread and steamed bread to improve their nutritional and functional properties (Du et al., 2016;Fu, Chang, & Shiau, 2015;Gökşen & Ekiz, 2016;Li, Zhu, Guo, Brijs, & Zhou, 2014;Ramli, Alkarkhi, Shin, Min-Tze, & Easa, 2009;Santiago et al., 2016;Sim, Aziah, & Cheng, 2015;Song, Zhu, Pei, Ai, & Chen, 2013;Wandee et al., 2015;Zhu, Sakulnak, & Wang, 2016). Dough modulation is the basis for preparing most wheat-flour foods. Dough is an integrated network composed by wheat flour, water, and other ingredients (Peng, Li, Ding, & Yang, 2017), in which starch granules fill within a continuous matrix formed by gluten (Edwards, Dexter, & Scanlon, 2002). Gluten is the major protein source in wheat flour, which is extremely important to form the viscoelastic structure of dough (Mirsaeedghazi, Emam-Djomeh, & Mousavi, 2008). Rheological properties are closely associated with the viscoelasticity and mixing tolerance of dough, which also provide an important reference for the processing properties of flour (Xu, Hu, Liu, Dai, & Zhang, 2017). The rheological properties of dough not only determine the processing characteristics during the manufacture of dough-based products but also affect the quality of final products (Mccann, Gall, & Li, 2016). Exogenous additives, like proteins, polysaccharides, and fibers, can produce significant effects on the rheological behavior of doughs, which is likely due to the interactions between exogenous additives and wheat proteins, and further affect the integrity and stability of the gluten network (Mirsaeedghazi et al., 2008;Morris & Morris, 2012;Rubel, Pérez, Manrique, & Genovese, 2015).
Given its excellent nutritional value and bioactivity, adding F. velutipes into wheat-flour foods can improve the nutritional properties of products. In view of the huge consumption of wheat-flour foods, replacing part of the wheat flour with F. velutipes can also consume a large number of mushrooms, and thereby resolve the inconsistency between the production and marketing of F. velutipes. F. velutipes contains polysaccharides, dietary fiber, proteins, and other low-molecular-weight components, which would affect the rheological properties of dough and therefore influence the qualities of dough-based products. However, little information is available for the properties of wheat dough supplemented with F. velutipes. Thus, the objective of this work was to investigate the effects of F. velutipes powder and soluble polysaccharide on the rheological properties and microstructure of dough, so as to provide the basis for the processing of dough-based foods containing F. velutipes.

Preparation of F. velutipes powder (FVP)
After removal of the root, the shredded fresh F. velutipes were sun-cured outside and dehydrated to about 70%. Semi-dried F. velutipes were then dried in a 75°C draught drying cabinet for 5-6 h. The dried mushrooms were milled using a laboratoryscale pulverizer (Beijing Zhongxingweiye Instrument Co., Ltd., Beijing, China) and then screened (>80 mesh).
Preparation of soluble polysaccharide of F. velutipes (SPFV) FVP was extracted with petroleum ether at 80°C for 2 h to remove lipids and pigments. The residue was then extracted with hot distilled water (ratio of water to the material of 20 mL/g) at 90°C for 1 h and then centrifuged at 5,000 r/min for 15 min to remove the precipitate. Samples were extracted in this manner twice, and the two supernatants were pooled together. Three-fold volume anhydrous ethanol was added into the extraction solution and then kept at 4°C for 24 h. After centrifugation at 5,000 r/min for 15 min, the precipitate was washed with anhydrous ethanol and acetone, respectively, and lyophilized as crude SPFV.

Mixing properties of dough
Mixing properties of dough was measured by the AACCI approved method 54-21 (AACC, 2000) using a Farinograph (Brabender, Duisburg, Germany) with a mixing bowl for 300 g flour. The farinograph parameters measured were water absorption, development time, and stability.

Extension properties of dough
Extension properties of dough were measured by the AACCI approved method 54-10 (AACC, 2000) using an Extensograph (Brabender, Duisburg, Germany). The extensograph parameters measured were area (energy), maximum resistance, extensibility, and ratio between resistance and extensibility.

Dynamic rheological properties of dough
Dough for dynamic rheological assays was kneaded for 5 min using a 50 g Farinograph (Brabender, Duisburg, Germany), and water absorption was optimized to make the consistency at the end of mixing centered in the range of 480 B.U. to 520 B.U. The dough was then covered with a plastic film immediately to avoid water losses, and cylindrical pieces of 3 cm diameter and 2 mm height were obtained from dough. Dynamic oscillatory tests were carried out in a Haake MARS III controlled stress oscillatory rheometer (Haake, Germany) at 25 ± 0.1°C using a 1 mm gap plate-plate sensor system. Test parameters referenced the method of Correa, Añón, Pérez, and Ferrero (2010). Storage modulus (G′), loss modulus (G″), and tan δ (G″/G′) were obtained as a function of frequency.

Scanning electron microscope (SEM) observation of dough
Microstructures of doughs prepared with different levels of FVP and SPFV were performed using a Quanta 200 environmental scanning electron microscope (FEI, Hitachi, USA). Dough samples were cut into 5 mm height and 3 cm diameter cylindrical pieces, which were freeze-dried. The dried dough slices with natural fracture surfaces were fixed by conducting resin and coated with gold and then observed at a voltage of 20 kV and high vacuum condition.

Statistical analysis
All experiments were performed at least in duplicate. Statistical evaluations were performed by SPSS version 17.0 software for Windows (SPSS Inc, Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to test the significant differences between means, and a post-hoc test (Dunnett's T3) was used to perform multiple comparisons between means at a P < .05 significance level.

Results and discussion
The chemical composition of FVP The moisture content of fresh F. velutipes was 89.0%. As shown in Table 1, the primary components in FVP were polysaccharide, dietary fiber, and protein. The potassium content of FVP was high, and the ratio of potassium to sodium was 147. The diet of high potassium and low sodium is conducive to prevent hypertension and cardiovascular disease (Oliver, Cohen, & Neel, 1975) and cardiovascular disease (Chang et al., 2006). Zinc content of FVP was high, which was helpful for human growth (Ploysangam, Falciglia, & Brehm, 1997).
The composition of amino acids in FVP is shown in Table 2. FVP contained 17.82% of amino acids, and E/T value (ratio of essential amino acids to total amino acids) was 37.21%. Lysine is the first limiting amino acid for cereal foods, but its content in FVP was relatively high.
Monosaccharide composition of SPFV was glucose (73.51%), galactose (19.21%), fucose (4.68%) and mannose (2.60%). Polysaccharides are the major bioactive component in F. velutipes, and many studies have proved that they have many biological activities such as immunomodulatory, antitumor, antioxidant, anti-inflammatory, etc (Leung et al., 1997;Wu et al., 2010Wu et al., , 2014. The analytical results suggested that adding a moderate amount of FVP could improve the deficiencies of wheat-flour foods on nutrients such as dietary fiber, lysine, and potassium.

Mixing properties of composite dough
We observed that the water absorption of composite flour supplemented with 2.5% FVP increased significantly (P < .05) compared to the control wheat flour, then the increase tended to be smooth till the addition of FVP was 15% (Table 3). As previously reported, the huge number of hydroxyl groups, such as polysaccharides and dietary fiber, caused the larger water absorption values (Goldstein, Ashrafi, & Seetharaman, 2010;Zhou et al., 2009). Correa et al. (2010) reported that modified cellulose addition increased the water absorption of wheat flour. Therefore, the increased water absorption of composite flour could be attributed to a large number of polysaccharides (14.88%) and dietary fiber (37.27%) existed in FVP.
The development time and stability of the dough for composite flour are shown in Table 3. The development time significantly (P < .05) decreased to the minimum with increasing the level of FVP to 12.5%, then had a small increase at 15.0% level. The farinograph stability decreased significantly (P < .05) when the level of FVP addition was 2.5%, but there was no significant (P >.05) difference among different addition levels from 2.5% to 15.0%. The stability value is closely related to flour strength, with a higher value representing stronger dough. Generally, the addition of exogenetic ingredients containing no gluten will weaken the gluten networks and lead to lower development time and stability of dough (Petitot, Boyer, Minier, & Micard, 2010).

Extension properties of composite dough
The extensographic properties of dough samples containing FVP are shown in Figure 1. At rest period of 45 min, both area (energy) and extensibility of the dough with 2.5% and 5.0% of  The values are mean ± SD of three independent determinations. Los valores representan la media ± desviación estándar de tres determinaciones independientes.
FVP supplementation were higher than the control, but decreased with increasing levels of FVP addition from 7.5% to 15.0% (Figure 1(a,c)), which indicates that a small amount (<5.0%) of FVP addition had a positive impact on the formation of gluten network, while a larger amount (>5.0%) of FVP addition had a negative impact. This result was consistent with Ahmed, Almusallam, Al-Salman, Abdulrahman, and Al-Salem (2013), who reported that fiber addition decreased the area (energy) and extensibility values of dough. At 90 and 135 min of rest period, area and extensibility of the dough decreased with increasing levels of FVP (Figure 1(a,c)), which was in agreement with the results of Wang et al. (2014). All composite doughs containing FVP had lower maximum resistance and ratio number than the control dough, and the troughs appeared at 7.5%-10.0% of FVP addition level. According to Ribotta, Ausar, Beltramo, and León (2005), higher values of maximum and extensibility indicate greater dough strength. This suggests that the addition of FVP weakened the extensographic properties of dough, overall. A possible reason is that partial wheat flour were substituted by FVP, which relatively decreased the wheat gluten content (dilution effect). It is well known that proteins in FVP cannot participate in the dough formation, which disrupts the well-defined protein-starch complex in wheat-flour dough, resulting in a weakening of dough (Sun, Zhang, Hu, Xing, & Zhuo, 2015). In addition, there were huge number of hydroxyl groups existing in FVP, such as polysaccharide and dietary fiber, which had a strong water absorption and would compete for water with starch and proteins in wheat flour (water absorption effect). Furthermore, FVP could fill into the gluten network and thus affect the extension properties of the dough (filling effect).

Viscoelastic properties of composite dough
The viscoelastic properties of dough with FVP addition are presented in Figure 2. The results showed that G' and G'' of all doughs increased with the increase of frequency ( Figure  2(a,b)). All dough samples displayed higher G' than G'' in the range of frequencies swept in this study, indicating that all doughs exhibited more elastic behavior compared to viscous behavior with or without FVP addition (Shi, Wang, Li, & Adhikari, 2013). Meanwhile, G' and G'' of dough roughly increased with the increase of FVP addition level (Figure 2(a,b)). As shown in Figure 2c, the dough at 2.5% of FVP supplementation behaved much more liquid-like than the control dough because its tan δ was higher than the control. This was probably caused by the dietary fiber in FVP, which could strengthen the water absorption ability of dough. The significantly increased water promoted swelling of wheat flour starch and proteins at smaller amount (<2.5%) of FVP addition, and could increase the viscosity of the dough. However, with the FVP addition level increased (>2.5%), the tan δ of dough roughly decreased (Figure 2c), indicating that the elasticity prevailed over the viscosity in the doughs with a larger amount (>2.5%) of FVP addition. This might be attributed to the low solubility and strong water-trapping capacity of the dietary fiber in FVP. According to the research of Blanco Canalis, Steffolani, León, and Ribotta (2017), β-glucans in oat fiber have a high proportion of β-(1→4) linkages which could promote hydrogen bond formation between polymer chains and with Table 3. Mixing properties of composite flour added with FVP at various levels.
Tabla 3. Propiedades de mezcla de la harina compuesta adicionada con FVP en varios niveles. The values are mean ± SD of three independent determinations. Means followed by different alphabetic superscripts within a column are significantly (P <0.05) different. Las medias seguidas por diferentes superíndices alfabéticos dentro de una columna son significativamente (P < 0.05) diferentes. Los valores representan la media ± desviación estándar de tres determinaciones independientes. water molecules as well. Furthermore, because of the low solubility, non-starch polysaccharides cannot enter the aqueous phase of dough, which resulted in the water distribution and dough viscosity changed. Consequently, the increased FVP (>2.5%) imparted greater water-holding characteristics than wheat-flour starch and proteins, which ultimately resulted in an elastic and firmer, but less viscous, dough. The variational tendency in the tan δ of dough was consistent with that in the extensographic properties of area and extensibility. Polysaccharides are one of the primary components and the most important bioactive substance of F. velutipes. Therefore, water-soluble polysaccharides of F. velutipes were prepared to investigate its effect on the viscoelastic properties of dough. SPFV addition levels were designed according to its content in FVP. The viscoelastic properties of dough with SPFV addition are presented in Figure 3. The results showed that G' and G'' roughly decreased (Figure 3(a,b)) and tan δ of doughs roughly increased as the SPFV addition level increased (Figure 3c), which were inconsistent with the results of FVP addition. These results revealed that SPFV addition had weakened the gluten network structure of dough, and the weakening effect was more significant than that of the equivalent addition level of FVP. This might be attributed to the easy formation of hydrogen bonds between SPFV and water molecules (Lerbret et al., 2005), which destroys the formation of gluten network in the dough (Peng et al., 2017). Blanco Canalis et al. (2017) reported an analogous result suggesting that inulin can partially enter the aqueous phase of dough, which lead to an increase of volume and reduce the firmer consistency of    dough. In addition, SPFV has good water solubility and swells sufficiently in dough, which results in more viscous properties of dough but less elastic and firmer consistency. Furthermore, compared to SPFV, there were large number of other components in FVP, such as IDF (35.01%), protein (20.64%), crude fat (5.51%) and other small molecular substances, which might react with wheat-flour starch and proteins and thereby also affect the viscoelastic properties of dough. These factors probably resulted in the more significant effects of SPFV on G′, G″, and tan δ compared to those of equivalent levels of FVP.

Microstructure of composite dough
The microstructures of the doughs with different addition levels of FVP were observed by SEM, as shown in Figure 4. In the micrographs of the control sample (wheat-flour dough), the starch granules (SG) were tightly embedded in the integrated and continuous gluten network composed of gluten strand (GS) and gluten film (GF). While with the increase of FVP addition level, the integrity and continuity of the gluten network became increasingly weaker. At a FVP addition level of 5%, the GS became thinner than that of the control. When the FVP addition level reached 10%, few GS could be found, and GF also became exiguous. When the FVP addition level was 20%, some fiber-like structures could be evidently observed, and the number of SG decreased accordingly; meanwhile, the gluten network was difficult to find.
To further investigate the effect of polysaccharides in F. velutipes on the structure of the dough, part of the wheat flour was replaced with SPFV to make dough. The microstructures of the doughs with different addition levels of SPFV had analogous results to those with FVP ( Figure 5). The addition of FVP and SPFV both disrupted the continuity of the gluten network and enlarged the hollows and voids in the dough. Although some portions of the GF could be observed in the dough with SPFV addition, they could not completely wrapped the SG, which appeared less embedded in the gluten network than those in the control sample. The SEM results showed that FVP or SPFV addition destroyed the integrity and continuity of the gluten network, and thus resulted in a dough with weak extensibility, which was consistent with the rheological results as previously discussed.

Conclusion
This study demonstrated that different addition levels of FVP and SPFV affected the rheological properties and microstructures of dough systems. Farinographic assay results suggested that FVP could increase the water absorption and decrease the development time and stability of dough, while extensographic assays indicated that it was capable of providing weak extension characteristics and harder dough. The dynamic oscillatory tests revealed that the FVP addition increased G' and G'' but decreased tan δ in the entire frequency range, while the addition of SPFV had results inconsistent with those of FVP addition. The microstructure of dough samples showed that the FVP and SPFV addition both decreased the integrity and continuity of gluten network in the dough. Overall, adding FVP produced a significant effect on the quality of dough, and relatively   Figura 5. Micrografías electrónicas de barrido de masa compuesta suplementada con SPFV en varios niveles. SG: gránulo de almidón; GF: película de gluten. low addition levels of 2.5% to 5.0% are recommended. This research could provide a foundation for the application of FVP in wheat-flour foods. Further researches will be conducted to investigate the influence of FVP addition on dough-based foods like steamed bread, noodles, and biscuit, and products with high nutritional and edible qualities will be developed.

Disclosure statement
No potential conflict of interest was reported by the authors.