Auricularia polytricha was bought from Yutai, China. The dried Auricularia polytricha were washed thoroughly and soaked with water, then drained at room temperature and oven dried to a constant weight. After that, the dried samples were grinded into small particles by grinder and sifted through 100-mesh sieve to eliminate the residues, getting the fine APP for further process. The wheat flour (Shandong King Group Co., Ltd.) was purchased from Hualian Supermarket. A small-scale straight dough steaming test was conducted. APP at 0%, 2.5%, 5%, 7.5%, and 10% (APP adding percentage was the total quality of APP and wheat flour amount) are mixed with wheat flour, respectively. For dough preparation, flour, APP, water (50% on flour basis) and dried instant yeasts (equivalent to 0.3% of dry matter) were mixed in a spiral mixer for 6 min. Then, the prepared dough was kept in a constant-temperature fermentation box for 60 min at 35˚C and relative humidity of 75%. The fermented dough was divided into smooth loaves with uniform sizes. The loaves were placed for 20 min in a constant-temperature fermentation box for second fermentation and then steamed at 100˚C for 30 min in a laboratory oven. Steaming tests were performed in six replicates. Steamed bread samples were cooled for 1 h at room temperature (25˚C) and then wrapped in polyethylene bags.

Slices (approximately 1.5 cm thick) of steamed bread samples enriched with different percentages of APP were subjected to sensory evaluation. The steamed breads were number coded and served to consumers. The evaluation panel consisted of 25 consumers (20 - 40 years old, 13 females and 12 males) who evaluated the overall acceptability of steamed bread. The degree of the overall consumer preference for different types of steamed bread was determined in accordance with a nine-point hedonic scale (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely). The testers rinsed their mouths with tap water before and after testing each sample.

Bread samples were subjected to textural profile analysis 1 h after steaming. Steamed bread was sliced mechanically, and slices were cut from the middle portion of steamed bread (30 mm × 30 mm × 25 mm). The texture meter was TA.XP Plus (Lotun Science Co., Ltd.), using probe SMSP/36R. TPA test condition was followed: compression rate 55%, time interval between two compression 5.0 s, speed before test 2.0 mm/s, test speed 1.0 mm/s, speed i after test 1.0 mm/s, induction force 5 g. Tests were conducted with 12 replicates. In a two-bite test, the samples were compressed twice (curves 1 and 2), to obtain the following textural parameters: hardness, elasticity, cohesiveness, chewiness, gumminess, and resilience.

The quality of steamed bread (g) was measured with an electronic balance. The volume of steamed bread (mL) was measured through steamed bread volume-meter rapeseed displacement. The specific volume of steamed bread (mL/g) was calculated as the ratio of the steamed bread volume and quantity. The pH of the steamed bread was measured using a pH meter (TESTO 206-pH2, Pruszków, Poland) with penetration probe for semi-solid substances.

Digestion of steamed bread in vitro was determined in accordance with the method of Gawlik-Dziki with slight modifications [10] . Buffer extracts (BE) were prepared by extracting powdered samples of steamed breads (1 g) with 20 mL of phosphate-buffered salinebuffer (PBS, pH 7.4) for 1 h. Extracts were separated by decantation, and residues were extracted again with 20 mL of PBS buffer. Extracts were combined and stored at −20˚C. For the preparation of extracts after simulated digestion (GD), simulated saliva was prepared by dissolving 2.38 g of Na₂HPO₄, 0.19 g of KH₂PO₄, 8 g of NaCl, and 100 mg of mucin in 1 L of distilled water at pH 6.75. α-amylase was added to obtain the enzyme activity of 200 U/mL. For the simulation of gastric digestion, 300 U/mL of pepsin in 0.03 M NaCl at pH 1.2 was prepared. Simulated intestinal juice was prepared by dissolving 0.05 g of pancreatin and 0.3 g of bile extract in 35 mL of 0.1 M NaHCO₃. Samples were subjected to simulated digestion as follows: 1 g powdered sample was homogenized in a stomacher laboratory blender for 1 min to simulate mastication with 15 mL of simulated salivary fluid. Samples were then shaken for 10 min at 37˚C. The pH of the samples was adjusted to pH 1.2 using 5 M HCl. Subsequently, 15 mL of simulated gastric fluid was added to the samples. The samples were shaken for 60 min at 37˚C. After digestion with gastric fluid, the pH values of the samples were adjusted to pH 6 with 0.1 M NaHCO₃. Then, 15 mL of intestinal juice was added to the samples. The pH of the extracts was adjusted to pH 7 with 1 M NaOH. Finally, 5 mL of 120 mM NaCl and 5 mL of 120 mM KCl were added to each sample. The prepared samples were subjected to in vitro digestion for 120 min at 37˚C in the dark. Samples were then centrifuged and supernatants were further analyzed. Fluids obtained after in vitro digestion was transferred to dialysis sacks (D9777-100FT, Sigma-Aldrich), placed in an Erlenmeyer flask containing 50 mL of PBS buffer, and incubated in a rotary shaker (twice per 2 h, 37˚C). PBS buffer together with compounds that passed through the membrane served as an equivalent of the raw material absorbed by the intestine after digestion (GDA).

Reducing power was determined in accordance with the method of Zhang with slight modifications [11] . The reaction system contained 1.0 mL of extracts, 2.5 mL of PBS (pH 6.6, 0.2 M), and 1.0 mL of potassium ferricyanide solution (1%, w/v) and was incubated for 20 min at 50˚C. The reaction was terminated by adding 2.0 mL trichloroacetic acid (10%, w/v). The mixture was centrifuged at 3000 × g for 10 min. Then, the supernatant (6.5 mL) was collected and mixed with 1.2 mL of ferric chloride (0.1%, w/v). Absorbance was measured at 700 nm using a spectrophotometer.

Free-radical scavenging activity was measured on the basis of DPPH scavenging activity in accordance with the method of Xu [12] with minor modifications. The reaction mixture contained 2 mL of extracts and 2.0 mL of DPPH solution (0.2 mM) or ethanol (95%, w/v). The reaction mixture was allowed to stand for 30 min in the dark. Then, absorbance was determined at 517 nm, and scavenging ability was calculated using following formula:

S c a v e n g i n g   a b i l i t i e s   ( % ) = ( 1 − A / A 0 ) × 1 00 %

where A is the absorbance of the mixture containing samples and DPPH, and A₀ is the absorbance of the mixture containing samples and ethanol.

Hydroxyl radical scavenging abilities were measured following the method of Zhang [13] with slight modifications. The reaction mixture, which contained 1.0 mL of ferrous sulfate (9 mM), 1 mL of salicylic acid (9 mM), 1 mL of hydrogen peroxide (8.8 mM, v/v), and 1 mL of sample, was incubated at 37˚C for 30 min. Absorbance was measured at 510 nm, and the hydroxyl radical scavenging ability was calculated using the following formula,

S c a v e n g i n g   a b i l i t i e s   ( % ) = ( 1 − A / A 0 ) × 1 00 %

where A is the absorbance of the samples, and A₀ is the absorbance of the blank.

All data were expressed as the means ± standard deviations (SD) of three replicated values. SPSS 17.0 software (SPSS, USA) was used for data analysis. Data were analyzed by one-way ANOVA, followed by Duncan’s multiple-range tests to show intergroup differences. A P-value of <0.05 was considered statistically significant.

The steamed wheat breads with different volume fractions of APP were made and the overall view of different types of steamed bread was exhibit in Figure 1.

Steamed bread enriched with high amounts of APP exhibited increased color scores and decreased loaf volume. The organoleptic properties of APP-enriched steamed bread were determined as shown in Table 1. APP addition significantly influenced the sensory characteristics of the bread. The overall sensory score increased with the added amount of APP. However, the overall sensory scores decreased when more than 5% APP was added to the bread. Increasing the added amount of APP significantly decreased color value, indicating that APP addition influenced the brightness of steamed bread. This result can be attributed to the specific black color of A. polytricha. Steamed bread enriched with 2.5% and 5% APP exhibited acceptable colors. The aroma and taste of steamed bread without APP addition were considered pronounced and aromatic, respectively, and APP-enriched steamed bread had a distinctive fragrance conferred by A. polytricha. The aroma and taste of steamed bread enriched with 5% APP were highly rated by the sensory panel. Moreover, the volume of APP-enriched steamed bread decreased given the presence of small and compact pores. Breads enriched with 2.5% and 5% APP had high texture scores. Breads enriched with 5% APP exhibited the highest sensory evaluation scores.

The basic properties, including pH and specific volume (Table 1), of APP-enriched steamed wheat bread were determined. The pH value of steamed bread negligibly increased with increasing volume fractions of APP. The pH of plain steamed wheat bread was 5.70, whereas that of steamed bread enriched with 10% APP was 5.86. This pH value was considered typical and acceptable. In addition, APP enrichment decreased the volume of steamed bread, consequently decreasing specific volume.

The changes in the textural properties of different types of APP-enriched steamed bread are shown in Table 2. The hardness, chewiness, and gumminess of steamed bread linearly increased as APP content increased from 0% to 10%. Changes in these textural parameters are attributable to reduced bread volume. Therefore, the addition of APP reduced the elasticity, cohesiveness, and resilience of steamed bread.

Reducing power and DPPH and hydroxyl radical scavenging abilities were

investigated to analyze the in vitro antioxidant capacities of steamed bread. The reducing power of steamed bread enriched with 0% - 10% APP is depicted in Figure 2(a). The results showed that the reducing power of APP-enriched steamed bread is a dose-dependent response. In BE stage, the reducing power of plain steamed bread was relative lower. Steamed bread enriched with 10% APP had a highest reducing power. During the GD stage, the reducing power of all kinds of steamed bread is approximately 10% - 20% higher than that in BE, and all the tested steamed bread exhibited dose-dependent reducing power. The reducing power of APP-enriched steamed bread at the GDA stage was lower than that in BE and GD stage. The DPPH scavenging abilities of the breads are shown in Figure 2(b). DPPH scavenging abilities increased in a dose-dependent manner with APP content. During the GD stage, DPPH radical scavenging abilities were considerably higher than those at BE. All tested steamed bread had detectable but low DPPH radical scavenging abilities during the GDA stage. As shown in Figure 2(c), steamed bread enriched with 1% - 10% APP had stronger scavenging hydroxyl scavenging activity than the control bread. Similar to reducing power and DPPH scavenging abilities, hydroxyl scavenging abilities were higher during the GD stage and lower during the GDA stage.