Effects of Pleurotus ostreatus on Physicochemical Properties and Residual Nitrite of the Pork Sausage

Abstract

In this work, a novel sausage incorporated with the Pleurotus ostreatus (PO) puree was successfully developed to reduce the residual nitrite and lipid oxidation during refrigerated storage (4 ± 1 °C) for 20 days. Five recipes with the supplement proportion of 0 wt.%, 10 wt.%, 20 wt.%, 30 wt.%, and 40 wt.% PO were produced and their physicochemical properties, nitrite residue, and sensory characteristics were measured. The results show that the content of moisture and all the essential amino acids (especially lysine and leucine) and the non-essential amino acids (especially aspartic and glutamic), lightness, springiness, and water holding capacity of the sausages were increased. However, the content of protein, fat, ash, pH, redness, hardness, gumminess, and chewiness of the sausages was decreased. For the sensory evaluation, the sausage with 20 wt.% PO had better sensory performance including flavor, aroma, and acceptability compared with other experimental groups and the control group. Moreover, the sausages with PO reduced the residual nitrite and inhibited lipid oxidation during storage. All of these results indicate that adding PO puree into pork sausage is a realizable and effective way to obtain nutritional and healthy pork sausages.

1. Introduction

Sausage is a popular meat product manufactured from different meat species such as pork, beef, chicken, fish, and buffalo [1,2,3]. This kind of meat product has important economic value for the meat-packing industry and is relished by consumers around the world for its delicious taste and high nutrition. In order to produce the sausages with high quality, nitrite is widely used as a preservative that can control foodborne pathogens [4]. Moreover, the additional function of the nitrites is to prevent lipid oxidation and rancidity, facilitate stabilization of the bright red color, and guarantee a typical “cured” flavor [5,6,7]. To fight against lipid oxidation, the nitrites could be associated with the binding of heme and prevent the release of the catalytic iron [8]. For keeping the bright red color of meat products, the nitrites can bind to myoglobin, forming the heat-stable NO-myoglobin. However, due to the reaction between nitrites and protein components in meat resulting in the carcinogen nitrosamine formation, the nitrites have been classified as potentially carcinogenic agents by the International Agency for Cancer Research of World Health Organizations (WHO) [9,10]. Consequently, it is important to obtain healthier meat products with a low content of residual nitrite without compromising the quality of the sausage.

Recently, researchers have focused on finding ways to decrease the additional content of nitrite in meat products, especially replacing it with natural resources such as vegetables, mushroom, and their extracts [11,12,13,14]. The reports showed that a part of plant essential oils exhibited strong antioxidative, antimicrobial, anticarcinogenic, and antimutagenic properties, which could be a compressive solution to substitute the nitrite [12]. For instance, Tang et al. (2021) reported that the combination of Flos Sophorae and chilli pepper can improve redness and reduce lipid oxidation of the meat product to replace nitrite in processed meat [15]. Vegetable powder extracted from radish and beetroot can substitute nitrite in sausage and increase the weight loss of sausages [16]. However, the studies on mushroom sausages have mainly focused on the nutritional components and sensory evaluation of the sausages and less concentrated on reducing the content of nitrite by adding edible mushrooms to sausage [17,18,19].

The mushroom is not only popular for its taste and flavor, but also for its high nutritional value and bioactive compounds. The addition of edible mushrooms to meat products can be used as a substitute for salt and phosphates in the formula of meat products and improve the quality of meat products, protein, dietary fiber, and ash content [20]. For example, the addition of shiitake mushrooms to sausage can improve the antioxidant and antibacterial properties of the product [21]. Replacing pork lean meat with Lentinus edodes can enhance total dietary fiber content, total phenol content, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability of sausage [22].

Pleurotus ostreatus (PO) is rich in amino acids (glutamic acid, aspartic acid, and arginine), polysaccharides (PSPO-1a, PSPO-4a, and branched β-glucans and α-glucans), vitamins (riboflavin and ascorbic acid), and dietary fiber with the function of antioxidant, antitumor, hypoglycemic, lipid-lowering, anti-inflammatory, sterilization, liver protection, improving immunity, and other significant effects [23,24,25,26,27]. Herein, a novel sausage incorporated with the Pleurotus ostreatus (PO) puree was developed to reduce the residual nitrite content in low-meat sausages during storage. Five recipes with the supplement proportion of 0 wt.%, 10 wt.%, 20 wt.%, 30 wt.%, and 40 wt.% PO were produced and their compositions, water activities, textures, colors, water holding capacities (WHC), amino acid compositions, and sensory evaluation were characterized. During storage at 4 °C, several characterizations including residual nitrite analysis, lipid oxidation analysis, and microbiological analysis were employed to characterize the quality and shelf-life of the products on days 0, 5, 10, 15, and 20.

2. Materials and Methods

2.1. Materials

The lean pork meat and back fat were purchased from the local market (WalMart Inc., Bentonville, Arkansas, USA) in China and stored at −20 °C. The fresh Pleurotus ostreatus (PO), salt, sugar, white pepper, cinnamon, and dry starch were also purchased from the local market (WalMart Inc., Bentonville, Arkansas, USA) in China. All other additives and chemical reagents shown in

Table 1. Formulations of Pleurotus ostreatus (PO) pork sausages.

Ingredients (g)	Control	PO10	PO20	PO30	PO40
Pork lean meat	400	400	400	400	400
Pork back fat	100	100	100	100	100
PO	0	50	100	150	200
Salt	10	10	10	10	10
Sugar	5	5	5	5	5
Sodium tripolyphosphate	0.400	0.400	0.400	0.400	0.400
Sodium nitrite	0.050	0.050	0.050	0.050	0.050
White pepper	1	1	1	1	1
Cinnamon (refined)	0.500	0.500	0.500	0.500	0.500
Carrageenan	2	2	2	2	2
Isolated soy protein	20	20	20	20	20
Dry starch	30	30	30	30	30
Monascus	0.075	0.075	0.075	0.075	0.075
Ice	125	125	125	125	125

were purchased from Sichuan Jinshan Pharmaceutical Co., Ltd. (Chengdu, China) and Beijing Beihua Co., Ltd., (Beijing, China).

2.2. Formulations and Processing of Sausage

Table 1 shows the formulations of the PO pork sausages with different PO contents. The sausages with 0 wt.%, 10 wt.%, 20 wt.%, 30 wt.%, and 40 wt.% PO were named as the Control, PO10, PO20, PO30, and PO40, respectively. The content of PO supplement used to process the sausages was based on the total content of pork lean meat and pork back fat.

To process the sausages, the pork lean meat was first sliced into sections and pickled at 4 ± 1 °C for 24 h with salt, sugar, sodium tripolyphosphate, and sodium nitrite. Then, the pork back fat was cut into cubes with a side length of 1 cm. The PO was cleaned with water, cut into small pieces, and mashed with a chopping machine into the puree. According to the formulation of each group, the pickled pork lean meat, PO puree, white pepper, cinnamon, carrageenan, isolated soy protein, dry starch, Monascus, ice, and back fat were mixed homogeneously in a mixer (Busch, Marburg, Germany) for 140 s. After that, the mixture was filled into the artificial collagen casings with a diameter of 2.6 cm by a sausage filler (Guangdong Shunde Fangzhan Electric Industrial Co., Ltd., Foshan, China). The methods of cooking, cooling, and preserving sausages were the same as the method reported by Wang et al. (2013) [28]. Finally, the sausage was stored after vacuum packing (Shandong Xiaokang Machinery Co., Ltd., Weifang, China) at 4 °C, which was counted as day 0.

2.3. Proximate Composition

The proximate compositions including protein, fat, ash, and moisture content were analyzed according to the Association of Official Analytical Chemists (AOAC) [29]. Each measurement was replicated three times for repeatability.

2.4. Water Activity (a_w) and pH

The water activity (a_w) of sausages was measured by a four-channel desktop water activity meter (Hygro Lab 2, Rotronic, Bassesdorf, Switzerland) at 25 °C. The sausage (10 g) was chopped and put into the water activity meter with 75% RH at 25 °C for 20 min, and then the data were recorded.

The pH values of sausages were characterized by a portable digital potentiometer (pH meter, Mettler Toledo, Zurich, Switzerland). The sausage (10 g) was crushed in 90 mL distilled water by a homogenate (Changzhou Magnetar Instrument Co., Ltd., Guangzhou, China). Then, we filled the mixture and measured the pH value of the solution at 25 °C in triplicate.

2.5. Color

The L* (lightness), a* (redness), and b* (yellowness) values of sausages were determined by a colorimeter (HunterLab ColorFlex, Reston, VA, USA) with a standard illuminant D65 light source (Xinlian Creation Electronic Co., Ltd., Shanghai, China) and a standard plate (L* = 94.52, a* = −0.86, b* = 0.68). The color values were determined at room temperature by five different areas on the cross-section of sausages. In particular, the area of fat in the sausage slice was not selected. The color differences (ΔE*) can be calculated by the following equations [28,30]:

$$\{\begin{array}{c}\Delta E*={\left(\Delta a^2+\Delta b^2+\Delta L^2\right)}^{0.5}\\ \Delta a=a{*}_{control}-a{*}_{PO}\\ \Delta b=b{*}_{control}-b{*}_{PO}\\ \Delta L=L{*}_{control}-L{*}_{PO}\end{array}$$

(1)

where ΔL, Δa, and Δb are the differences between the L*, a*, and b* values of the control groups and the groups with PO, respectively.

2.6. Textural Profile Analysis (TPA)

Texture profile analysis was employed by a texture analyzer (CT3-50kg, Brookfield Engineering Labs, New York City, NY, USA) to calculate the hardness, springiness, gumminess, chewiness, and cohesiveness of the sausages. The sausage was cut into cylinders with a diameter of 2 cm and a height of 1 cm. Then, the cylinder was axially compressed twice until reaching 80% of its initial height with a 20 s pause time between the descent, 30 mm probe retraction, 6 cm/min detection speed, and 100 N force induction. The analysis was repeated at 25 °C in triplicate.

2.7. Cooking Loss and Water Holding Capacity (WHC)

Cooking loss was characterized by the method reported by Fu et al. (2016) [30]. Sausage (50 g) was cooked at 80 °C for 50 min then cooled at room temperature to determine the weight difference before and after cooking. The cooking loss can be calculated by:

$$\mathit{Cooking} \mathit{loss}=\left[\right(m_1-m_2)/m_1]\times 100\%$$

(2)

where m₁ and m₂ are the weight before and after cooking, respectively.

The WHC of the sample was measured by the method reported by Jridi et al. (2015) with slight modifications [31]. Sausage (10 g) was centrifuged at 12,000 rpm for 30 min at 4 °C. Finally, the WHC can be calculated by:

$$\mathit{WHC}=(W_2/W_1)\times 100\%$$

(3)

where W₁ and W₂ are the weight of the sample before and after centrifugation, respectively.

2.8. Amino Acids Content

The amino acid content was analyzed according to the method reported by Serrano et al. (2005), which separated the amino acids by cation exchange chromatography via an automatic amino acid analyzer (L8900, Hitachi High-Technologies Corporation, Tokyo, Japan) for measurement [32]. Ninhydrin derivative reagents were used to measure the amino acid content at 570 nm.

2.9. Sensory Evaluation

The sensory evaluation of sausages was carried out according to the research reported by Hu et al. (2014) with modification [33]. The cooked sausages (20 min at 80 °C) were assessed by a panel of 30 members (15 males and 15 females). The team members were chosen from students and the faculties of Jilin Agricultural University (Changchun, China). The samples were cut into 5 mm thick slices at room temperature and identified with a three-digit random code. In addition, the sensory panel were provided with water and salt-free biscuits to clean their taste buds. The appearance, texture, flavor, aroma, and overall acceptability were evaluated using a 9-point Hedonic scale (1 = dislike very much, 9 = like very much).

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

Parameters	Control	PO10	PO20	PO30	PO40
Protein	13.23 ± 0.08 a	13.08 ± 0.02 b	12.98 ± 0.02 b	12.92 ± 0.06 b	12.73 ± 0.05 c
Fat	17.60 ± 0.12 a	17.22 ± 0.04 b	16.53 ± 0.06 c	15.79 ± 0.02 d	14.82 ± 0.07 e
Ash	3.23 ± 0.02 a	3.01 ± 0.12 b	2.95 ± 0.01 b	2.75 ± 0.01 c	2.64 ± 0.02 d
Moisture	56.56 ± 0.01 d	63.83 ± 0.12 c	63.90 ± 0.11 c	64.78 ± 0.21 b	66.26 ± 0.62 a
aw	0.98 ± 0.00 a	0.98 ± 0.00 a	0.98 ± 0.00 a	0.98 ± 0.00 a	0.98 ± 0.00 a
pH	6.86 ± 0.01 a	6.74 ± 0.03 b	6.55 ± 0.04 c	6.21 ± 0.02 d	6.12 ± 0.01 d

^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.

. 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.

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.

3.2. Water Activity and pH

Among all samples, there was no significant difference in the water activity (a_w) of sausages (p > 0.05) (Table 2). The results were close to bologna sausages as reported by Câmara et al. (2021), in which the a_w was 0.97~0.98 [43]. The pH value of the control group was the highest and the pH value gradually decreased with the increase in PO puree content significantly (p < 0.05). The result of pH in this research was close to the research reported by Riel et al. (2017) (pH = 6~7) [44].

3.3. Color

Color is an important parameter for consumers to accept processed meat products. The effects of PO puree on the color traits of sausages are evaluated in

Table 3. Color comparison of Pleurotus ostreatus (PO) pork sausages.

Parameters	Control	PO10	PO20	PO30	PO40
L*	47.46 ± 0.12 e	48.08 ± 0.17 d	51.17 ± 0.24 c	52.50 ± 0.21 b	54.62 ± 0.31 a
a*	17.96 ± 0.01 a	16.60 ± 0.03 b	15.94 ± 0.13 c	14.56 ± 0.31 d	13.41 ± 0.57 e
b*	16.35 ± 0.02 a	16.39 ± 0.16 a	16.45 ± 0.11 a	16.29 ± 0.04 a	16.41 ± 0.11 a
ΔE*	-	44.20 ± 0.04 a	44.37 ± 0.13 a	44.47 ± 0.31 a	44.31 ± 0.59 a

^a–e Means within the same row with different letters differed 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.

. With the increase in PO content, lightness (L*) values of the sausages significantly (p < 0.05) increased. This could be explained by the higher lightness of the PO, which enhanced that of the sausages. For redness (a*), the decreasing trend may be due to the decrease in lean meat content, which contributed to the pink color of the sausage product. For the yellowness (b*) and color difference (ΔE*), there were no significant differences among all groups (p < 0.05).

3.4. Textural Profile Analysis (TPA)

The addition of the PO affected the apparent texture properties of the sausages (

Table 4. Texture parameter analysis (TPA), cooking loss (%), and water holding capacity (WHC) of Pleurotus ostreatus (PO) pork sausages.

Parameters	Control	PO10	PO20	PO30	PO40
Hardness (N)	155.63 ± 15.15 a	137.40 ± 4.12 b	111.93 ± 8.70 c	111.83 ± 6.91 c	81.50 ± 7.04 d
Cohesiveness	0.55 ± 0.04 a	0.51 ± 0.06 a	0.42 ± 0.13 a	0.44 ± 0.08 a	0.48 ± 0.01 a
Springiness (mm)	3.50 ± 0.22 b	5.24 ± 0.93 a	5.16 ± 0.36 a	5.10 ± 0.70 a	5.15 ± 0.94 a
Gumminess (N)	85.23 ± 13.32 a	72.53 ± 8.83 a	46.70 ± 11.61 b	45.77 ± 13.74 b	41.70 ± 2.41 b
Chewiness (N)	296.80 ± 14.51 a	277.68 ± 15.02 b	251.94 ± 11.14 c	232.05 ± 81.70 c	241.14 ± 29.23 c
Cooking loss (%)	4.39 ± 0.20 e	16.54 ± 0.21 d	18.44 ± 0.26 c	19.30 ± 0.26 b	21.25 ± 0.26 a
WHC (%)	69.25 ± 0.44 e	73.90 ± 0.56 d	75.81 ± 0.46 c	77.01 ± 0.40 b	78.66 ± 0.42 a

^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 PO puree, respectively.

). The experimental groups had higher hardness (11.71~47.63%), cohesiveness (7.27~23.63%), gumminess (14.90~51.07%), and chewiness (6.44~21.81%) values, while it had a lower springiness (−45.71~−49.71%) value than those of the control group significantly (p < 0.05), showing that the experimental groups were more conducive and convenient for chewing. In the experimental groups, the PO with high water content prevented the gel production of the pork myofibril protein, which caused lower cohesiveness of sausage fillings and the appearance of more inside gaps. Furthermore, high amounts of dietary fiber in PO was another factor leading PO sausages to show lower degrees of hardness, gumminess, and chewiness [45,46].

3.5. Cooking Loss and Water Holding Capacity (WHC)

The cooking loss and WHC of sausages in each group are shown in Table 4. With the increase in PO content, the cooking loss and WHC were significantly (p < 0.05) increased. The higher cooking loss might be due to the high water content of fresh PO, which evaporated during the cooking process. WHC of the supplement group increased 4.7~13.55% compared with that of the control group. This phenomenon was probably due to the higher water retention ability of the fibers in PO [47]. These results were similar to the research about adding shiitake mushrooms to replace lean pork in sausages reported by Wang et al. (2019), in which the shiitake mushroom enhanced the cooking loss and WHC of the sausages [48].

3.6. Amino Acids Profile

Amino acids have many health benefits. For example, lysine can enhance immunity and improve the central nervous function of the human body [49]. Moreover, glutamic and aspartic are umami amino acids that could contribute to the enhancement of the flavor of meat products [50]. Therefore, the amino acid content is another key factor of the meat products’ nutrients [51]. As shown in

Table 5. Amino acid profile (%) of Pleurotus ostreatus (PO) sausages during storage.

Parameters	Control	PO10	PO20	PO30	PO40
Essential amino acid
Threonine	0.67 ± 0.04 d	0.67 ± 0.03 d	0.81 ± 0.07 c	0.86 ± 0.01 b	0.87 ± 0.01 a
Valine	0.76 ± 0.05 e	0.81 ± 0.04 d	0.93 ± 0.02 c	0.98 ± 0.05 b	1.03 ± 0.05 a
Methionine	0.12 ± 0.01 d	0.12 ± 0.03 d	0.21 ± 0.01 c	0.25 ± 0.01 b	0.29 ± 0.01 a
Isoleucine	0.72 ± 0.01 e	0.77 ± 0.04 d	0.92 ± 0.03 c	0.96 ± 0.03 b	1.01 ± 0.03 a
Phenylalanine	0.69 ± 0.02 e	0.71 ± 0.03 d	0.84 ± 0.02 c	0.89 ± 0.04 b	0.93 ± 0.01 a
Lysine	1.35 ± 0.03 e	1.36 ± 0.01 d	1.69 ± 0.03 c	1.80 ± 0.02 b	1.88 ± 0.05 a
Leucine	1.30 ± 0.02 e	1.34 ± 0.03 d	1.60 ± 0.03 c	1.72 ± 0.03 b	1.82 ± 0.02 a
Non-essential amino acids
Histidine	0.53 ± 0.03 e	0.64 ± 0.03 d	0.65 ± 0.06 c	0.69 ± 0.01 b	0.70 ± 0.07 a
Glycine	0.76 ± 0.03 e	0.81 ± 0.02 d	0.85 ± 0.06 c	0.90 ± 0.04 b	0.97 ± 0.02 a
Aspartic	1.55 ± 0.01 e	1.70 ± 0.05 d	1.85 ± 0.03 c	2.00 ± 0.03 b	2.05 ± 0.04 a
Arginine	1.00 ± 0.03 e	1.05 ± 0.03 d	1.24 ± 0.01 c	1.30 ± 0.03 b	1.40 ± 0.01 a
Alanine	0.88 ± 0.03 e	0.91 ± 0.01 d	1.05 ± 0.03 c	1.12 ± 0.02 b	1.19 ± 0.06 a
Tyrosine	0.39 ± 0.07 e	0.47 ± 0.03 d	0.65 ± 0.06 c	0.66 ± 0.06 b	0.72 ± 0.05 a
Cysteine	0.03 ± 0.04 e	0.04 ± 0.07 d	0.05 ± 0.03 c	0.06 ± 0.02 b	0.11 ± 0.09 a
Serine	0.68 ± 0.06 e	0.70 ± 0.02 d	0.80 ± 0.02 c	0.87 ± 0.04 b	0.90 ± 0.03 a
Glutamic	2.54 ± 0.07 e	2.60 ± 0.03 d	3.08 ± 0.07 c	3.28 ± 0.03 b	3.42 ± 0.04 a
Proline	0.68 ± 0.03 e	0.71 ± 0.03 d	0.74 ± 0.03 c	0.76 ± 0.06 b	0.96 ± 0.03 a

^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 addition of the PO enhanced the contents of all the essential amino acids and non-essential amino acids compared with the control group (p < 0.05). It could be seen that the essential amino acids of the control sausage were mainly lysine, leucine, and valine; the non-essential amino acids were mainly glutamic, aspartic, and arginine. Comparing PO40 with the control group, the contents of lysine, leucine, valine, glutamic, aspartic, and arginine in sausage increased by 39.26%, 40.00%, 35.53%, 34.65%, 32.26%, and 40.00%, respectively. All of these increases might be contributed by the abundance of amino acids in PO [27].

3.7. Sensory Evaluation

The sensory evaluation results of each group are shown in Figure 1. The appearance of all groups had excellent performance (>8). In the PO10 and PO20 groups, an appropriate increase in brightness of the sausages might be more loved by the group members. For the PO30 and PO40 groups, the decrease in the redness might reduce group acceptance of the sausages in appearance. In terms of aroma and flavor, all scores exceeded eight and PO20 had the best aroma and flavor. This might be because PO was rich in free amino acids, especially glutamic and aspartic, which were conducive to enhance the aroma and flavor of the sausage. The texture of the sausages decreased with the increase in PO content. This result might be caused by the soft texture of the sausage and more gaps inside the sausages (Section 3.2.) which were disliked by the group member. Although a difference was found between each group, all of these samples were judged as acceptable (>7), suggesting that PO had an opportunity to in sausages. From what has been discussed above, PO20 can be regarded as the best group in sensory evaluation through comprehensive consideration.

3.8. Residual Nitrite Analysis

The residual nitrate in meat products can convert to nitrosamine compounds in the human body and cause cancer in the lungs, stomach, esophagus, liver, and bladder [52,53]. Therefore, the residual nitrite content in meat products should be as low as possible. The results of residual nitrite analysis of all groups on days 0, 5, 10, 15, and 20 are shown in

Table 6. Residual nitrite analysis of Pleurotus ostreatus (PO) pork sausages during storage (mg/kg).

Days	Control	PO10	PO20	PO30	PO40
0	11.75 ± 0.25 a	10.31 ± 0.01 b	9.74 ± 0.03 c	9.33 ± 0.02 d	8.77 ± 0.01 e
5	11.24 ± 0.17 a	9.90 ± 0.17 b	9.35 ± 0.01 c	9.01 ± 0.02 d	8.52 ± 0.03 e
10	10.95 ± 0.11 a	8.26 ± 0.12 b	7.94 ± 0.13 c	6.95 ± 0.13 d	6.87 ± 0.11 d
15	10.79 ± 0.25 a	8.15 ± 0.01 b	7.37 ± 0.10 c	6.81 ± 0.10 d	6.76 ± 0.10 d
20	2.33 ± 0.03 a	1.28 ± 0.01 b	0.70 ± 0.07 c	0.58 ± 0.02 d	0.42 ± 0.01 e

^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.

and

Table 7. Residual nitrite of Pleurotus ostreatus (PO) pork sausages during storage for every group (mg).

Days	Control	PO10	PO20	PO30	PO40
0	7.80 ± 0.17 a	6.40 ± 0.01 b	6.31 ± 0.02 c	6.35 ± 0.01 bc	6.17 ± 0.01 d
5	7.46 ± 0.11 a	6.15 ± 0.11 b	6.06 ± 0.01 c	6.14 ± 0.01 b	6.00 ± 0.02 d
10	7.27 ± 0.07 a	5.13 ± 0.07 b	5.14 ± 0.08 b	4.73 ± 0.09 c	4.84 ± 0.08 c
15	7.16 ± 0.17 a	5.06 ± 0.01 b	4.77 ± 0.06 c	4.64 ± 0.07 c	4.76 ± 0.07 c
20	1.55 ± 0.02 a	0.79 ± 0.01 b	0.45 ± 0.05 c	0.40 ± 0.01 d	0.30 ± 0.01 e

^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.

. Because the PO was the supplement in the sausage and did not replace the meat in the sausage, the residual nitrite concentrations were not convincing to clarify the true content of nitrite in each group shown in Table 1. To solve this problem, the data in Table 7 were calculated to show the weight of the residual nitrite of each group. With the passage of storage time and increase in PO content, the residual nitrite content of sausage showed a gradually decreasing trend before 15 days. On the 15th day of the storage period, the residual amount of nitrite in the supplement group decreased 24.7~37.35% compared with the control group. After 15 days, the nitrite content in the sausages decreased sharply. On the last day (day 20) of the storage period, the residual amount of nitrite in the supplement group could decrease 45.06~81.97% compared with the control group, which was probably due to the microbial proliferation affecting nitrogen compounds. The low residual nitrite of the supplement group can be explained by the decrease in initial nitrite (day 0). The residual nitrite could react with active substances such as phenolics and terpenes into nitrite acid or nitric oxide. This might be because PO was rich in strong antioxidant activities such as Vitamin C (Vc), which could improve the activity of antioxidant enzymes, interrupt the chain reaction of free radicals, maintain the integrity of cell membrane, and convert methemoglobin to hemoglobin and thereby reduce the nitrite content [54,55,56,57,58]. In a word, the nitrite residue of sausage with PO was lower than that of the control group over the entire storage period.

3.9. TBARs

Lipid oxidation is considered as the main mechanism leading to the rancidity, low quality, and low shelf life of meat products [42,59]. In this work, TBARs was employed for the evaluation of the lipid oxidation of the sausages (

Table 8. The TBARs value of Pleurotus ostreatus (PO) pork sausages during storage (mg/kg).

Days	Control	PO10	PO20	PO30	PO40
0	0.51 ± 0.03 a	0.49 ± 0.01 ab	0.46 ± 0.01 b	0.39 ± 0.02 c	0.35 ± 0.01 d
5	0.61 ± 0.01 a	0.58 ± 0.01 a	0.50 ± 0.04 b	0.47 ± 0.02 b	0.41 ± 0.04 c
10	0.72 ± 0.01 a	0.71 ± 0.03 a	0.69 ± 0.01 a	0.51 ± 0.01 c	0.49 ± 0.03 c
15	0.84 ± 0.02 a	0.80 ± 0.03 a	0.74 ± 0.01 ab	0.65 ± 0.01 bc	0.57 ± 0.12 c
20	0.92 ± 0.01 a	0.89 ± 0.02 a	0.85 ± 0.01 b	0.79 ± 0.02 c	0.60 ± 0.03 d

^a–d 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.

). With the increase in PO content, the TBARs values of the sausages showed a decreasing trend. The TBARs value of the experimental group could decrease 3.92~31.37% and 4.76~32.14% compared with that of the control group on the initial day and 15th day, respectively. This phenomenon could be explained by the good antioxidant biological chemical composition of the PO such as phenols, ascorbic acid, α-tocopherol, β-carotene, and flavonoid compounds (rutin and chrysin), which could effectively delay the lipid oxidation of meat products [55,60,61]. Moreover, Selli et al. (2021) reported that the phenolic substance content of heat-treated mushrooms increased significantly, which could also enhance the antioxidant activity directly [62].

3.10. Microbiological Analysis

During the processing and storage of sausage, microorganisms would lead to the decomposition of protein and fat, leading to rot and rancidity, which is harmful to food safety. Therefore, the total number of bacterial colonies was recognized as an important parameter to evaluate the shelf-life stability [44]. The microbiological analysis of sausages in the control group and supplement groups were tested after 0, 5, 10, 15, and 20 days of storage (

Table 9. Microorganisms of Pleurotus ostreatus (PO) pork sausages (log CFU/g).

Days	Control	PO10	PO20	PO30	PO40
0	0.43 ± 0.30 a	0.44 ± 0.29 a	0.45 ± 0.22 a	0.44 ± 0.20 a	0.42 ± 0.30 a
5	0.86 ± 0.20 a	0.65 ± 0.22 a	0.73 ± 0.11 a	0.87 ± 0.19 a	0.76 ± 0.14 a
10	1.24 ± 0.61 a	1.43 ± 0.41 a	1.54 ± 0.36 a	1.67 ± 0.21 a	1.85 ± 0.62 a
15	3.87 ± 0.20 a	3.94 ± 0.11 a	3.97 ± 0.10 a	3.84 ± 0.10 a	3.76 ± 0.20 a
20	5.12 ± 0.46 a	5.32 ± 0.42 a	5.34 ± 0.41 a	5.36 ± 0.39 a	5.57 ± 0.45 a

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

). The total number of bacterial colonies in each group increased significantly after 20 days of storage (p < 0.05). Compared to the supplement groups and the control group, there was no significant difference in the total number of bacterial colonies of sausage, indicating that PO had no significant effect on the total number of bacterial colonies of sausage. According to the existing national standards of China (1 × 10⁵ CFU/g) (GB2726—2016), the sausages expired after 20 days. The sausage prepared in this study was steamed sausage, which was not Chinese sausage and this kind of product normally has a high water content and short shelf life. Therefore, it is acceptable for the product to expire after 20 days.

4. Conclusions

A novel pork sausage incorporated with Pleurotus ostreatus (PO) puree was produced in this study. The PO improved the content of moisture and amino acids, lightness, springiness, and water holding capacity of the sausage while reducing the content of protein, fat, and ash, pH, redness, hardness, gumminess, and chewiness. For the sensory, the best formulation for the addition of PO was 20 wt.%, which not only enhanced the appearance and overall acceptability, but also gave it a better aroma and flavor. During 15 days of storage, the content of residual nitrite in the sausages decreased by 24.7~37.35%, and the TBARs value of the sausages was decreased by 4.76~32.14% compared with that of the control. Based on these results, the PO pork sausage in this study was successfully developed to enhance the quality and nutritional value and reduce the residual nitrite content and lipid oxidation during the storage of sausage. PO could be a competitive solution to improve flavor, safety, and overall quality of the sausages.