*2.10. TPA*

Texture profile analysis were measured by a P35 cylindrical flat bottom probe with a texture analyser (LS5, Ametek, Berwyn, PA, USA). Parameter setting: sample surface flatness height of 20 mm; front, middle, and final test speed of 4.0 mm/s, 3.0 mm/s, and 4.0 mm/s; descent distance of 40%; compression time of 5 s; trigger force of 5 g.

#### *2.11. Colour*

The colourimeter was used to measure the colourimetry of fish sausage, which was represented by the L\*, a\*, and b\* values. Whiteness is calculated as follows:

$$\text{Whitness} = 100 - \sqrt{(100 - \text{L}^{\ast})^2 + \text{a}^{\ast 2} + \text{b}^{\ast 2}}\tag{3}$$

#### *2.12. LF-NMR*

Low-field nuclear magnetic resonance (MQC-23, Oxford, UK) was used to determine the water distribution of the fish sausage [24]. The fish sausage was packed into a 20 cm NMR tube, sealed with plastic wrap, and the transverse relaxation time (T) of the protein gel was measured by Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence. The parameters were set as: P90 = 6, P180 = 12, SFI = 23.4 MHz, DW = 1.0, SW = 1,000,000 Hz, SI = 1, NECH = 256, and NS = 16; WinDXP software was used to invert CPMG and save its data. The relaxation time diagram was made by drawing software. The peak area in the atlas was accumulated, and the peak area represented the percentage content of water in the group.

#### *2.13. SEM*

The samples were cut into 3 mm × 2 mm and placed in a covered container. They were soaked in 2.5% pH 7.2 phosphate buffer at 4 ◦C for more than eight hours and then washed three times with 0.1 mol/mL pH 7.2 phosphate buffer for 10 min each. Gradient elution was performed with 50%, 70%, 80%, and 90% ethanol solutions for 10 min each. Elution was repeated three times with 100% anhydrous ethanol for 10 min each. The dehydrated samples were defatted with the proper amount of trichloromethane for one hour, then replaced with a mixture of tert-butyl alcohol and ethanol with a volume ratio of 1:1 for 15 min, and after that replaced with 100% tert-butyl alcohol for 20 min and freeze-dried for 48 h. The surface morphology of the sample was observed by a scanning electron microscope (Gemini300, Zeiss, Oberkochen, Germany) under a 5000× field of vision.

#### *2.14. Sensory Evaluation*

The sensory evaluation team of sausages comprised ten teachers and postgraduates (five males and five females, aged 24–35) from the major of food Science and Engineering at Shaoyang University. All of these people have experience with food sensory evaluation and were familiar with the experimental process and operating norms. Prior to the analysis, all participants (volunteers) in this experiment were informed of the aim of the sensory evaluation, and they provided informed consent to perform it. The group members' scores ranged from 0 (poor sensorial property) to 10 (excellent sensorial property), indicating that their expectations for appearance, smell, taste, texture, and overall acceptability ranged from very low to very high. The scoring criteria are shown in Table 1. Sausages were re-heated in water for two minutes and kept in an oven at 40 ◦C before being served to the panel randomly on white porcelain plates under natural light at room temperature. A glass of water was also provided to cleanse the palate.


**Table 1.** Scoring criteria for sensory evaluation of grass carp sausage.

#### *2.15. Statistical Analysis*

SPSS 25.0 software of IBM company was used for statistical analysis. The statistical differences between the two groups were analysed by one-way analysis of variance

(ANOVA) and Duncan's multiple range test (*p* < 0.05). The original Pro 2021 software was used for drawing.
