The strain of P. dubia (CGMCC No. 20731) used in this study is preserved in the China General Microbiological Culture Collection Center. P. dubia was cultured in different liquid mediums. The control group was cultured in ordinary medium (2% glucose, 1% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O) [16 (link)], while the treatment group was cultured in Zn2+-contaminated medium (2% glucose, 1% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.1% ZnSO4·7H2O). A 250 mL shake flask with 100 mL medium was cultured at 20 °C, 120 rpm. Three biological replicates were conducted per treatment condition. After being cultured for 10 days, mycelia pellets were separated from the fermentation broth containing spores by 200 mesh press cloth. Then, the fermentation broth was centrifuged at 8000 rpm for 10 min to take the precipitate to obtain spore powder. The mycelia and spores were washed three times with deionized water. Then place them in a freeze-drying machine (SCIENTZ-10N, NingBo Scientz Biotechnology, Zhejiang, China) to dry at −25 °C, 12 Pa. The weight of mycelia was recorded as biomass. In addition, mycelia and spores were ground into powder for subsequent experiments.
Scientz 10n
Manufactured by Ningbo Scientz Biotechnology
Sourced in China
The SCIENTZ-10N is a laboratory homogenizer designed for efficient tissue sample preparation. It features a variable speed control and a durable stainless steel housing. The homogenizer is capable of processing a wide range of sample volumes.
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Thomas model 4 wiley mill, by Thomas Scientific (1 mentions)
Su5000, by Hitachi (1 mentions)
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Tm 1000, by Hitachi (1 mentions)
Jem 2100, by JEOL (1 mentions)
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Haake mars 60 rotational rheometer, by Thermo Fisher Scientific (1 mentions)
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Nicolet 6700, by Thermo Fisher Scientific (1 mentions)
Su8100 cold field emission scanning electron microscope, by Hitachi (1 mentions)
No 42 filter paper, by Cytiva (1 mentions)
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33 protocols using scientz 10n
1
Cultivation of P. dubia Spores and Mycelia
2
Sample Drying and Preparation for Analysis
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Immediately after collection, the samples were manually cleaned, separating the leaves and inflorescences. The samples were divided into two equal parts to be dried in an oven or by lyophilization. For the oven-drying procedure, the samples (leaves and inflorescences) were placed in a forced-ventilation oven (FX 1375, Shel Lab, Cornelius, OR, USA) at 40 °C for 48 h. For the lyophilization procedure, the samples were frozen at −80 °C for 24 h, and then placed in a freeze dryer (Scientz-10N, Ningbo Scientz Biotechnology Co., Zhejiang, China) at −60 °C and 1 Pa. Once dried, samples were ground in an electric blender (Thomas Model 4 Wiley Mill®, Thomas Scientific, Swedesboro, NJ, USA) and sieved through a 500 µm mesh. The resulting powders were then placed in sealed bags and stored at −80 °C for further analysis.
3
Freeze-Drying Technique for Sample Preservation
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The samples were frozen at −20 °C for 4 h and transferred to −80 °C for 24 h. The freeze-dryer (SCIENTZ-10 N, Ningbo Scientz Biotechnology Co., LTD., Zhejiang, China) was used for drying the samples. The drying process was conducted under 1 Pa of absolute pressure until the moisture content reached 0.08 ± 0.03 g H2O/g d.w. The cold trap and heating plate were maintained at temperatures of −68.5 °C and 27 °C, respectively. Following drying, the samples were ground into powder using a muller and sifted through an 80-mesh sieve before being stored in a dry location for later analysis.
4
Microstructural Analysis of Surimi Gels
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The surimi gels were cut into 3 mm × 3 mm × 1.5 mm pieces and fixed with glutaraldehyde (2.5%, v/v) for 14 h at 4 °C. The fixed samples were rinsed with 0.1 M phosphoric acid buffer (pH 7.2–7.4) three times. After that, the samples were dehydrated with a serious of ethanol solution (30%, 50%, 70%, 80%, 90%, and 100%) and then replaced with tert-butanol solution (absolute ethanol: tert-butanol = 3:1, 1:1, 1:3, 0:1). The dehydrated samples were dried by using a freeze dryer (SCIENTZ-10N, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) and sputter-coated with gold. The microstructures were analyzed by an SEM instrument (Hitachi SU5000, Hitachi High-Tech Co., Ltd., Shanghai, China) at an acceleration voltage of 5 kV.
5
Characterization of Plant-Based Meat Analogs
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In the present study, a total of 15 plant-based meat analogs were selected based on the types of products which are commonly sold on the market. PBMA in the form of meatballs, ground meat, steaks, burger patties, chicken breasts and sauced beef were purchased over the internet and coded with letters A to O. They were produced by Impossible Foods (Oakland, CA, USA), Beyond Meat (Columbia, MO, USA), V2 Food (Sydney, Australia), Hero Protein (Shanghai, China), Harvest Gourmet (Tianjin, China), PFI Foods Co., Ltd. (Shanghai, China), Protein Meat (Shanghai, China), OmniPork (Hongkong, China), Hong Chang Bio-Tech (Suzhou) Co., Ltd. (Suzhou, China), Whole Perfect Food (Shenzen, China) and Caixinxiang Co., Ltd. (Fuzhou, China). Samples were freeze-dried with a vacuum freeze drier (Scientz-10N, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) and then ground to powder using a grinder (SD-JR05, Sande Electric Appliance Co., Ltd., Foshan, China). After sieving with a 40-mesh sieve, the fine powders were stored at –80 °C until further analysis.
6
Synthesis of Glycidol-Chitosan Conjugate
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As illustrated in Figure 1 , glycidol-chitosan (Gly-CS) was synthesized according to previously reported method with some modifications (Zhou et al., 2010 (link); Zhang & Cao, 2014 (link)). Briefly, 1 g chitosan (containing 4.95 mmol glucosamine residue) was dissolved in 100 mL acetic acid solution (1%, v/v), and filtered by 0.22 μm membrane filter. Then, 657 μL glycidol (9.9 mmol, twice of glucosamine residue) was added dropwise to chitosan solution, and reacted for 24 h at 50 °C. The reaction mixture was dialyzed with a dialysis bag (MWCO 3500 Da) on a magnetic stirrer against Milli-Q deionized water, which was replaced with fresh water every 4 h. The sample was dialyzed for 72 h to ensure the unreacted glycidol was completely removed. At last, it was lyophilized by a freeze-dryer (SCIENTZ-10N, Ningbo Scientz Biotechnology Co., Ltd., Zhejiang, China) to obtain the Gly-CS with the yield of 91.6%.
7
Pickling and Freeze-Drying of Duck Eggs
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Fresh duck eggs were obtained from the Tianyun Company in Jiangxi Province, China. Fresh uncracked duck eggs were selected and soaked in a pickling solution containing NaOH (4.5%, m/v), NaCl (3.0%, m/v), and CuSO4 (0.4%, m/v) (10 (link)) for 35 days. Samples were taken once a week during the marinading period, with 15 eggs divided into three equal portions. The preserved eggs were shelled and the yolks were removed from the egg whites. All samples of egg yolks were freeze-dried (SCIENTZ-10N, Ningbo ScientzBiotechnology Co., Ltd., Ningbo, China) for subsequent experimental determination.
8
Moxidectin-loaded PLGA Microspheres
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Using different PLGA blends, four moxidectin-loaded microspheres (F1, F2, F3 and F4) were prepared using an oil-in-water (O/W) emulsion solvent evaporation technique [41 (link)]. Briefly, 900 mg of PLGA was dissolved in 6 mL of DCM, and 600 mg of moxidectin was dispersed in this solution. This organic phase was added slowly to 120 mL of 1% (w/v) aqueous PVA solution and homogenized at 4000 rpm for 2 min (IKA T25, Staufen, Germany) to form an O/W emulsion. Then, the resulting emulsion was transferred to 480 mL of purified water and stirred at 300 rpm for three hours to remove the solvent. Finally, microspheres were washed with purified water and then freeze-dried (SCIENTZ-10N, Ningbo SCIENTZ Biotechnology Co., Ltd., Ningbo, China). The obtained microspheres were stored at 4 °C in the dark until use.
9
Ultrasound-Assisted Betalains Microencapsulation
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10% (w/v) of maltodextrin was dissolved in water at 60 °C for 3 h by a magnetic stirrer (H05-1, Shanghai MeiyingPu Instrument Manufacturing Co., Ltd, China). The maltodextrin solution was cooled to room temperature and stored at 4 ± 1 °C for 24 h to ensure complete hydration. The betalains extraction was mixed with maltodextrin solution for 1:1 (v/v). Then, the mixed solution was sonicated at selected ultrasound power (100, 200, 300 and 400 W) for 1, 2, 5 and 10 min by ultrasound homogenizer (SCIENTZ-ⅡD, Ningbo Scientz Biotechnology Co., Ltd, China). The detailed sonication treatment was showed in Table 1 . Finally, the sonicated solution was pre-frozen in −60 °C for 6 h, and then dried by a vacuum freeze dryer (SCIENTZ-10 N, Ningbo Scientz Biotechnology Co., Ltd, China) for 48 h. Finally, the betalains microcapsule was collected in desiccator and stored in darkness to avoid light degradation.
Ultrasound-assisted preparation of betalains microencapsulates parameters.
| Samples | Ultrasound power (W) | Ultrasound time (min) |
|---|---|---|
| Control | 0 | 0 |
| BM1/5 | 100 | 5 |
| BM2/5 | 200 | 5 |
| BM3/5 | 300 | 5 |
| BM4/5 | 400 | 5 |
| BM2/1 | 200 | 1 |
| BM2/2 | 200 | 2 |
| BM2/5 | 200 | 5 |
| BM2/10 | 200 | 10 |
10
Microencapsulation of Bioactive Compounds
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GA and WPI powder (wall matrix) were dissolved in deionized water (2%, w/v) while stirring at 40 °C for 1 h. Subsequently, the polymer solutions were cooled to room temperature and kept overnight in a refrigerator to ensure complete hydration. Then, 30 mL of concentrated CH solution was blended with the different volume ratios of the GA and WPI solution according to the volume ratio of 1:2 using a magnetic stirrer at 800 rpm. The blending procedure of the core matrix and wall matrix solution is shown in Table 1 . Finally, the mixed solution was pre-frozen at −80 °C for 6 h and then dried with a vacuum freeze dryer (SCIENTZ-10 N, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) for 48 h. The prepared microcapsules were collected into an aluminum foil bag and kept in a desiccator to avoid degradation.
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