*2.10. Residual Nitrite Analysis*

GB5009.33-2016 was used to measure the residual nitrite in sausage samples on days 0, 5, 10, 15, and 20 [34]. The sausage (5 g) was mixed in 12.5 mL saturated borax solution (50 g/L) and 150 mL water. Then, the solution was heated in a boiling water bath for 15 min. After cooling, potassium ferrocyanide solution (5 mL, 106 g/L) and zinc acetate solution (5 mL, 220 g/L) were mixed homogeneously and stood for 30 min. Finally, the pink dye formed by the coupling of sulfonamide and naphthalene ethylenediamine hydrochloride was determined by spectrophotometry to obtain the residual nitrite content. Each sample was analyzed in triplicate.

### *2.11. Thiobarbituric Acid Reactive Species (TBARs)*

TBARs of the sausage was analyzed by spectrophotometry according to GB5009.181- 2016 on days 0, 5, 10, 15, and 20 [35]. The sausage (5 g) was mixed into a solution with trichloroacetic acid (75 g/L) and disodium EDTA (1 g/L) by a thermostatic oscillator at 50 ◦C for 30 min. Then, the filtrate (5 mL) was added into 2.88 g/L thiobarbituric acid solution and mixed at 90 ◦C for 30 min. Then, the solution was cooled down to room temperature and the absorbance was measured at 532 nm.

### *2.12. Microbiological Analysis*

The total number of bacterial colonies in sausage was determined by the method in GB4789.2-2016 on days 0, 5, 10, 15, and 20. The total number of germs, yeasts, and molds was analyzed and identified with the same method in GB4789.2-2016 [36].

### *2.13. Statistical Analysis*

All determinations were designed three times and the values were shown as means ± standard deviations. The difference between factors and levels were submitted to the analysis of variance (ANOVA). Duncan's multiple range tests were used to determine the differences among mean values (*p* < 0.05). The analysis was taken by SPSS software version 19.

### **3. Results**

### *3.1. Proximate Composition*

The proximate composition contents of sausage are presented in Table 2. According to the reference, the chemical composition of the PO included 20~25% protein, 2.5~2.9% fat, 5.9~6.7% ash, and 88.0~90% moisture [37]. Obviously, the protein and fat of the PO were lower than those of the meat. For the protein content, the control group was at the highest level (13.23%) and significantly different from other groups (*p* < 0.05). This result was comparable with that of the sausages reported by Lee et al. (2016) and Silva et al. (2019) [38,39]. With the increase in PO content, the protein level of PO40 decreased from 13.08% (the control group) to 12.73% (PO40), which was because the fresh PO contained less protein compared to the meat [40]. The fat content in the sausages decreased from 17.60% to 14.82% with the increase in PO content, which was also because the PO had less fat than the meat.

**Table 2.** Proximate composition (%), water activity, and pH of *Pleurotus ostreatus* (PO) pork sausages.


a–e Means within the same row with different letters differ significantly among the treatments (*p* < 0.05). Values are given as mean ± standard deviations. Control, PO10, PO20, PO30, and PO40 were 0 wt.%, 10 wt.%, 20 wt.%, 30 wt.%, and 40 wt.% addition of PO puree, respectively.

The ash content was significantly reduced from 3.23% to 2.64%, which was comparable with that of the beetroot sausages and Toscana sausages reported by Sucu et al. (2018) and Monteiro et al. (2017) [41,42]. The moisture was a significant difference between the control group and the experimental groups (*p* < 0.05). This is because the moisture content of PO (>80%) was much richer than the meat.
