Materials
Sorghum and Daqu (market procurement); Total Starch Assay Kit (Shanghai Huicheng Biotechnology Co., Ltd. Shanghai, China); Amylose Assay Kit (Hefei Laier Biotechnology Co., Ltd. Anhui, China); All reagents and chemicals used were of analytical grade (Chengdu Kelong Chemical Reagent Factory, Sichuan, China).
Technological process of gelatinization
The technological process of gelatinization is shown in Fig S1. Sorghum was used as raw material for multiple cycles of gelatinization and fermentation. Samples were taken after each gelatinization and the first, fourth, and seventh day of each fermentation cycle, and Baijiu distillers' grains were used as controls. All samples were pretreated by wet screening, water washing, and centrifugation to remove rice hulls and water-soluble substances.
Starch separation
The starch separation according to the method of Rittenauer et al. with modifications (Rittenauer et al., 2016). The samples of Zaopei with different gelatinization and fermentation cycles were taken, mixed with an appropriate amount of distilled water, screened with 20 mesh, then centrifuged three times (5000r / min, 5min), and freeze-dried for 24h. The dried sample was put into an extraction flask with 5.0g and extracted with 2/3 of petroleum ether for 2 h. After that, the sample was taken out and placed for several hours until the petroleum ether volatilizes completely. Added ethanol (90%) (solid-liquid ratio: 1g: 10ml) to the above samples and stirred evenly at room temperature, and centrifuged three times (5000r / min, 10min) after keeping in water at 50 ℃ for 2h, and collected the supernatant. Ethanol (90%) (the ratio of material to liquid is 1g:10ml) was added to the sediment again. After a water bath for 2h (5 ℃), centrifuging three times (5000r / min, 10min), the supernatant was combined, and the starch content was measured. The two sediments were collected and then freeze-dried.
Starch content
The total starch content of sorghum was determined by the Total Starch Assay Kit. Weighed 100mg of sample (35 mesh sieve) and mixed with 0.2ml of ethanol solution (80%), immediately added 2ml of DMSO (dimethyl sulfoxide), boiling water bath for 5min (shaking the tube), then put the tube in 50℃ water bath and added 0.1ml of amyloglucosidase (3300U/ml) and kept for 30min. The above solution was diluted to 100ml, part of the solution was centrifuged (3000rpm, 10min), and the supernatant was taken. The 0.1ml supernatant was mixed with 3ml GOPOD (including glucose oxidase > 12000U, peroxidase > 650U, 4-amino antipyrine 80mg) solution and then water bath (50℃) for 20min, and the absorbance of the sample was measured at 510nm. The glucose control included 0.1mL glucose standard solution (1mg/mL), 3.0mL GOPOD solution. The blank reagent control included 0.1mL distilled water and 3.0mL GOPOD solution. The total starch content was calculated according to the following formula.
$$\begin{array}{c}Total starch \left(\text{\%}\right)=\varDelta A\times \frac{\text{F}}{\text{W}}\times FV\times 0.9\#\left(1\right)\end{array}$$
Where: ΔA = Absorbance of sample
F = 100 (µg of D-glucose) / Absorbance for 100 µg of glucose
FV = Total volume
W = Mass of sample
The amylose content of sorghum was determined by Amylose Assay Kit. Weighed 0.1 mg of sample and mixed with 1 ml of DMSO (to ensure no lumps), and boiling water bath for 15 min. After cooling to room temperature, added 6ml of ethanol (95%), placed for 15 min and then centrifuged (2000rpm, 5min), collected the sediment and placed until the ethanol volatilizes. Then added 2ml of DMSO to the sediment. After boiling water bath (15min), 4ml of ConA solution was added immediately and then diluted to 25ml with ConA. The 1ml of the above sample solution was mixed with 3ml of sodium acetate solution (100mM,pH4.5), After boiling water bath (5min) and then held in a water bath at 40℃ for 5min, and 1ml of a mixture of starch transglucosidase and α-amylase was added and reacted for 30min and then centrifuged ( 2000rpm, 5min). The absorbance of samples and glucose control was measured at 510nm after taking 1ml of supernatant and mixed with 4ml of GOPOD reagent at 40℃ for 20min. The amylose content was calculated according to the following formula.
$$\begin{array}{c}Amylose \left(\text{\%}\right)=\frac{2\times \varDelta {A}_{1}}{\text{W}\times \varDelta {A}_{2}}\#\left(2\right)\end{array}$$
Where: ΔA1 = Absorbance of sample
ΔA2 = Absorbance of standard glucose solution
W = Mass of sample
Starch gel properties
Light transmittance
The light transmittance was determined using a method described by Faiza Shaikh et al. (Shaikh et al., 2019) with modifications. The 0.50 g starch (dry basis) was prepared into starch milk (1%) with distilled water. The starch milk was cold to room temperature after a boiling water bath. Starch milk was poured into a 1 cm cuvette and determined light transmittance (620 nm, distilled water as control) with an ultraviolet spectrophotometer (UV2400, Beijing Purkinje General Instrument Co., Ltd., China).
Water-holding capacity
According to a method of Abhari et al.(Abhari et al., 2017) with modifications. The 1.0 g starch (dry basis) and 15 mL distilled water were put in a centrifuge tube and stirred evenly. The starch solution was centrifuged (3000 x g) (Allegra 64R, Beckmancoulter Co., Ltd., USA) for 10 min after shaking at room temperature for 1 h. The centrifuge tube was tilted slightly after discarding the supernatant. The droplets on the inner and outer tube walls and supernatant were absorbed after standing for 10 min. The water holding capacity was the mass of water absorbed per gram of sample.
$$\begin{array}{c}Water holding capacity\left(g/g\right)=\frac{{m}_{2}-{m}_{1}-m}{m}\#\left(3\right)\end{array}$$
Where:m = Mass of sample (dry basis)
m1 = Mass of centrifuge tube (with lid)
m2 = Sum of m1 and mass of sediment
Freeze-thaw stability
The freeze-thaw stability of starch was determined using the Zhang et al.(Zhang et al., 2019) method with modifications. The 3.0 g starch sample (dry basis) and the distilled water were prepared into starch solution (6%). The appropriate amount of starch solution was transferred into a centrifuge tube (50 mL) and a boiling water bath for 30 min. The starch solution was placed in the refrigerator (-18°C) for 12 hrs after cooling to room temperature. The frozen starch solution was thawed for 6 hrs at room temperature and centrifuged (3000 x g) for 20 min. The centrifuge tube was weighed after discarding the supernatant, and starch samples were freeze-thawed three times. The syneresis rate expresses the freeze-thaw stability.
$$\begin{array}{c}syneresis rate\left(\text{\%}\right)=\frac{\left({M}_{1}-{M}_{2}\right)\times 100}{\left({M}_{1}-{M}_{0}\right)}\#\left(4\right)\end{array}$$
Where: M0 = Mass of the centrifuge tube
M1 = Mass of the centrifuge tube and starch solution before freezing
M2 = Mass of centrifuge tube and sediments after discarding the supernatant
Solubility and swelling power
According to a method of Farrag et al.(Farrag et al., 2018) with modifications. The 1.0 g starch sample (dry basis) and the distilled water were prepared into starch solution (2%). The starch solution (25 mL) was transferred into the two centrifuge tubes (50 mL) and put into water bath pots (30°C and 70°C) for 30 min (stirring continuously during the water bath). The starch solution was centrifuged (5000 x g) for 10 min after cooling to room temperature. The supernatant was poured into two glass dishes, respectively, and most of the water was evaporated in a 90°C water bath. The two glass dishes were dried to constant weight in the electric thermostatic drying oven (105°C).
$$\begin{array}{c}Solubility\left(\text{\%}\right)=\frac{A\times 100}{M}\#\left(5\right)\end{array}$$
$$\begin{array}{c}Swelling power\left(\text{\%}\right)=\frac{P\times 100}{M\times \left(1-S\right)}\#\left(6\right)\end{array}$$
Where: A = mass of starch on the glass dish
P = mass of sediments
M = mass of the starch sample (dry basis)
S = Solubility
Sorghum starch structure
Scanning electron microscope (SEM)
Scanning electron micrographs were taken by a VEGA 3SBU Scanning Electron Microscope (VEGA 3SBU, TESCAN Ltd., The Czech Republic) at an accelerating voltage of 3 kV. The dried starch samples were evenly applied on the double-sided adhesive tape and coated with gold for 120 s.
Fourier transform infrared reflection (FTIR)
The 3 mg of starch sample dried to constant weight, and 300 mg of potassium bromide powder were mixed and ground for 10 min, and then passed 100 mesh sieve. The mixed powder was pressed into tablets and used for FT-IR measurement (Nicolet6700, Thermofisher Ltd., USA). The potassium bromide was used as blank control. The spectra were recorded from 400 to 4000 cm− 1, and the resolution is 4 cm− 1. Each sample was determined three times.
X-ray diffraction
XRD patterns of starch were prepared using an X-ray diffractometer (D2PHASER, Bruker AXS Ltd., Germany). Measurements were obtained at 40 kV of tube voltage with a scanning rate of 0.02º/s and a diffraction angle range of 5º to 90º. The relative crystallinity was calculated using peak fitting software.
Differential scanning calorimetry (DSC)
The 3 mg sorghum starch and 10 µL ultrapure water were mixed and sealed and then placed at room temperature for 3 hrs and used for DSC measurement (DSC200F3, Netzsch Ltd., Germany). The sealed, empty aluminum box is the blank control. Measurements were obtained with a scanning rate of 10°C/min and a scanning range of 30°C to 150°C. The parameters in the gelatinization process of sorghum starch were analyzed and compared, including onset gelatinization temperatures (TO), peak gelatinization temperatures (TP), conclusion gelatinization temperatures (TC), and gelatinization enthalpy (△H).
Statistical analysis
All the experiments were performed at least in triplicate. One-way analysis of variance (ANOVA) by SPSS 24.0 (SPSS Inc., Chicago) was used to establish differences among data. Data of experiments were analyzed by Origin 2021 (Origin Lab Company, USA).