Effects of Chitosan on Clostridium perfringens and Application in the Preservation of Pork Sausage

The effects of chitosan with 95% deacetylation degree (DD95) on the spore germination, cell proliferation, and heat resistance of Clostridium perfringens CCRC 10,648 and CCRC 13,019 were investigated, and its application on pork sausage with sodium nitrite reduction was also evaluated. DD95 chitosan can strongly reduce the heat resistance of both strains. The D80 and D100 values for strain CCRC 13,019 decreased from 40.98 and 4.64 min to 39.21 and 3.26 min, respectively, as a result of adding 250 ppm DD95; meanwhile, addition of chitosan decreased the D80 and D100 values for CCRC 10,648 from 41.15 and 6.46 min to 39.52 and 3.78 min, respectively. In pork sausage, addition of 3000 ppm DD95 chitosan considerably slowed down the bacterial proliferation and volatile basic nitrogen production. There were no significant differences in color (L* and b* values), shearing force, and hardness in the pork sausages with or without DD95 chitosan during storage at 4 and 25 °C. However, the addition of DD95 chitosan in pork sausage significantly retarded the decrease of the a* value. Therefore, DD95 chitosan could reduce the concentration of sodium nitrite required in pork sausages for color retention.


Introduction
The meat processing industry regularly faces many serious challenges regarding the safety and hygiene of various products [1][2][3]. The pathways for pathogens' transmission into the product could be from throughout handling processes or from the carcass surface [4,5]; however, decontamination or sterilization of the carcass body is very difficult to perform using currently applied antimicrobial agents. Moreover, most commonly used preservatives and antimicrobial agents have a chemical and synthetic nature, giving them many potential side effects and risks on the health of consumers [6][7][8]. The meat industry continues to seek natural preservatives as antimicrobial agents.
Chitosan, a partially deacetylated chitin (poly-β-(1→4)N-acetyl-d-glucosamine) is found in shrimp shells and fungi [9]. This polysaccharide has attracted attention as a biomedical material because of its biocompatibility and various biological activities, including immune-enhancing [10], anti-inflammatory [11], antibacterial [6], and antitumor activities [12]. Because of its excellent antibacterial activity, chitosan has been widely used for food protection [13][14][15][16]. Several studies have shown that chitosan's molecular weight (MW) is a crucial factor in its antimicrobial properties, although equivocal results in terms of the correlation between the antibacterial properties and MWs of chitosan have also been observed [17,18]. Our previous report [6] concluded that the correlation between chitosan MW and its antibacterial properties was dependent on the pH value of the reaction mixture.

Antibacterial Activity of Chitosan Against C. perfringens
In this study, chitosan with a molecular weight of 220 kDa and 95% degree of deacetylation was prepared from shrimp chitin [6]. The antibacterial effect of DD95 against the vegetative cells of C. perfringens CCRC 13,019 and CCRC 10,648 in liver infusion broth (LIB) at 37 • C anaerobically is shown in Figure 1. DD95 at the dosages of 50 and 100 ppm only retarded the growth of C. perfringens. However, DD95 at 250 ppm had a bactericidal effect against this pathogen. The strain CCRC 13,019 ( Figure 1A) was more susceptible to DD95 compared with the strain CCRC 10,648 ( Figure 1B), as evidenced by the exposure time of 5 h for the former and 24 h for the latter, with no survival being observed at 250 ppm DD95.
Mar. Drugs 2020, 18,70 2 of 15 of chitosan have also been observed [17,18]. Our previous report [6] concluded that the correlation between chitosan MW and its antibacterial properties was dependent on the pH value of the reaction mixture. Clostridium perfringens, an anaerobic spore-forming foodborne pathogen, is widely distributed in various foods, such as meat, seafood, and vegetable products. Due to the heat resistance of C. perfringens spores, spores may survive the heating treatment, and then germinate and proliferate in food. After the contaminated food is ingested, this pathogen secretes an enterotoxin in the intestine, which changes the permeability of the cell membrane and causes diarrheal syndrome [19] or even death [20]. Therefore, methods for controlling C. perfringens, especially its spores, in various meat products are necessary [21]. Addition of nitrite derivatives, including sodium and potassium nitrite, is the most effective method for controlling the growth of vegetative cells and spores of Clostridium species including C. perfringens in processed meats. In addition, nitrite also brings a characteristic red color in processed meats [22].
However, the International Agency for Research on Cancer (IARC) reported that consumption of processed meats including sausages may increase the risk for colorectal cancer, and accordingly classified processed meats as carcinogenic to humans. Nitrates and nitrites used as additives in processed meats are sources of N-nitroso compounds (NOCs), which have long been known as carcinogenic [23]. Several studies have attempted to develop new methods to inactivate the spores of Clostridium as a way to decrease or even avoid the addition of nitrites in processed meats [24].
In this study, we prepared shrimp chitosan with 95% deacetylation (DD95) and demonstrated that DD95 has a strong antibacterial activity against C. perfringens and could reduce the heat resistance of C. perfringens spores. Moreover, DD95 in pork sausage stored at 4 and 25 °C effectively retarded the increase of both total bacterial and C. perfringens counts (CPC), as well as volatile basic nitrogen values. In addition, the synergistic effect of DD95 and sodium nitrite for a favored color development in pork sausage was observed. Thus, the addition of DD95 in pork sausage was able to reduce the amount of sodium nitrite required and extended the shelf life of pork sausage.

Antibacterial Activity of Chitosan Against C. perfringens
In this study, chitosan with a molecular weight of 220 kDa and 95% degree of deacetylation was prepared from shrimp chitin [6]. The antibacterial effect of DD95 against the vegetative cells of C. perfringens CCRC 13,019 and CCRC 10,648 in liver infusion broth (LIB) at 37 °C anaerobically is shown in Figure 1. DD95 at the dosages of 50 and 100 ppm only retarded the growth of C. perfringens. However, DD95 at 250 ppm had a bactericidal effect against this pathogen. The strain CCRC 13,019 ( Figure 1A) was more susceptible to DD95 compared with the strain CCRC 10,648 ( Figure 1B

Effect of Chitosan on Decimal Reduction Time of C. perfringens Spores
The outbreak of C. perfringens poisoning is generally initiated by C. perfringens spores that survive the cooking process and are germinated in food, eventually producing enterotoxins in the intestinal tract after ingestion [16]. Therefore, reduction of heat resistance of the spores to decrease spore survival during heating is a preventative approach for C. perfringens poisoning [16]. The effect of DD95 at 250 ppm on the decimal reduction time (D value) of tested C. perfringens strains during heating are shown in Table 1

Applications of Chitosan in Pork Sausage
Ground pork containing various amounts of sodium nitrite was spiked with C. perfringens CCRC 13,019 spores to have the initial density of approximately 10 4 spore/g for the preparation of pork sausages. For the experimental group, DD95 was added to have the final concentration of 3000 ppm in sausage. The sausages with or without DD95 were stored at 25 and 4 °C. Changes in the total aerobic count (TAC) and C. perfringens count (CPC) of pork sausage samples during storage at 25 and 4 °C are shown in Figures 2 and 3. The changes in TAC of sausage samples stored at 25 °C for 48 h were quite similar among the control sample (without sodium nitrite and DD95) and samples containing sodium nitrite only (40-120 ppm); meanwhile, the addition of 3000 ppm DD95 substantially decreased the increase of TAC, regardless of whether or not sodium nitrite was added ( Figure 2A). Sodium nitrite alone in the sausage stored at 25 °C was able to retard the increase of CPC, and a dose-dependent effect was observed. The CPC for sausages containing DD95 gradually decreased during storage, regardless of whether or not sodium nitrite was added ( Figure 2B).

Effect of Chitosan on Decimal Reduction Time of C. perfringens Spores
The outbreak of C. perfringens poisoning is generally initiated by C. perfringens spores that survive the cooking process and are germinated in food, eventually producing enterotoxins in the intestinal tract after ingestion [16]. Therefore, reduction of heat resistance of the spores to decrease spore survival during heating is a preventative approach for C. perfringens poisoning [16]. The effect of DD95 at 250 ppm on the decimal reduction time (D value) of tested C. perfringens strains during heating are shown in Table 1

Applications of Chitosan in Pork Sausage
Ground pork containing various amounts of sodium nitrite was spiked with C. perfringens CCRC 13,019 spores to have the initial density of approximately 10 4 spore/g for the preparation of pork sausages. For the experimental group, DD95 was added to have the final concentration of 3000 ppm in sausage. The sausages with or without DD95 were stored at 25 and 4 • C. Changes in the total aerobic count (TAC) and C. perfringens count (CPC) of pork sausage samples during storage at 25 and 4 • C are shown in Figures 2 and 3. The changes in TAC of sausage samples stored at 25 • C for 48 h were quite similar among the control sample (without sodium nitrite and DD95) and samples containing sodium nitrite only (40-120 ppm); meanwhile, the addition of 3000 ppm DD95 substantially decreased the increase of TAC, regardless of whether or not sodium nitrite was added ( Figure 2A). Sodium nitrite alone in the sausage stored at 25 • C was able to retard the increase of CPC, and a dose-dependent effect was observed. The CPC for sausages containing DD95 gradually decreased during storage, regardless of whether or not sodium nitrite was added ( Figure 2B). Mar. Drugs 2020, 18 Similar results were observed for sausage samples stored at 4 °C for 10 days ( Figure 3). Sodium nitrite alone did not retard the increase of TAC in the sausage samples, whereas DD95 could effectively decrease the increase of TAC ( Figure 3A). The dose-dependent effect of sodium nitrite alone on decreasing CPC was also observed, and this CPC decreasing effect was further enhanced by DD95 and sodium nitrite ( Figure 3B). , 40 ppm sodium nitrite; and , 80 ppm sodium nitrite; and , 120 ppm sodium nitrite. Empty symbols, with 3000 ppm DD95; solid symbols, without DD95. Similar results were observed for sausage samples stored at 4 • C for 10 days (Figure 3). Sodium nitrite alone did not retard the increase of TAC in the sausage samples, whereas DD95 could effectively decrease the increase of TAC ( Figure 3A). The dose-dependent effect of sodium nitrite alone on decreasing CPC was also observed, and this CPC decreasing effect was further enhanced by DD95 and sodium nitrite ( Figure 3B).
According to Kanner et al. [25], the mechanism of nitrite inhibition in C. perfringens is attributable to the destruction of iron-sulfur enzymes such as ferredoxin by nitric oxide (NO), transferred from nitrite by the ferrous ion in meat, and thus inhibition of the synthesis of adenosine triphosphate (ATP). A similar nitrite inhibition effect on other Clostridium species, such as Clostridium botulinum and Clostridium sporogenes, was also reported [26,27]. However, the nitrite effect on C. perfringens was almost diminished in the presence of DD95 in sausage ( Figures 2B and 3B), probably because of the much stronger activity of DD95 against C. perfringens at 3000 ppm, which overwhelmed the inhibition effect of nitrite at 40-120 ppm. Moreover, the iron absorption activity of chitosan may prevent NO production from nitrite [28]. This merits further investigation in the future. The aerobes seemed more resistant to NO produced from nitrite at the dosages tested, probably because of more antioxidant enzymes or compounds present in aerobes to attenuate the NO effect. Therefore, it was observed that nitrite had only a slight effect on TAC in sausages during storage in this study. According to Kanner, et al. [25], the mechanism of nitrite inhibition in C. perfringens is attributable to the destruction of iron-sulfur enzymes such as ferredoxin by nitric oxide (NO), transferred from nitrite by the ferrous ion in meat, and thus inhibition of the synthesis of adenosine triphosphate (ATP). A similar nitrite inhibition effect on other Clostridium species, such as Clostridium botulinum and Clostridium sporogenes, was also reported [26,27]. However, the nitrite effect on C. perfringens was almost diminished in the presence of DD95 in sausage ( Figures 2B and 3B), probably because of the much stronger activity of DD95 against C. perfringens at 3000 ppm, which overwhelmed the inhibition effect of nitrite at 40-120 ppm. Moreover, the iron absorption activity of chitosan may prevent NO production from nitrite [28]. This merits further investigation in the future. The aerobes seemed more resistant to NO produced from nitrite at the dosages tested, probably because of more antioxidant enzymes or compounds present in aerobes to attenuate the NO effect. Therefore, it was observed that nitrite had only a slight effect on TAC in sausages during storage in this study. , 40 ppm sodium nitrite; and , 80 ppm sodium nitrite; and , 120 ppm sodium nitrite. Empty symbols, with 3000 ppm DD95; solid symbols, without DD95.

Color and Color Difference Measurement
Changes in color (L*, a*, and b* values) of pork sausage samples during storage at 25 • C for 48 h and 4 • C for 10 days are shown in Tables 2 and 3, respectively. In general, both L* and b* values did not significantly change for all tested samples during storage at 25 • C, and there were no significant differences in L* values among all tested sausages, regardless of whether or not sodium nitrite and/or DD95 were/was added ( Table 2). The a* value (representing the red color) for the control sausage (without nitrite and DD95) was substantially decreased with the increase in incubation time at 25 • C, from 7.18 ± 0.45 at the beginning to 2.76 ± 0.53 at the end of storage. At the beginning, the addition of sodium nitrite at 40-120 ppm did not significantly change the a* value in sausage without DD95. The retention of a* value was significantly enhanced with the increasing amount of nitrite added during storage. After 48 h of incubation, the a* values for sausages containing 80 and 120 ppm nitrite were significantly higher than those for sausages containing 0 and 40 ppm nitrite. The a* value was further enhanced by DD95 addition in nitrite-containing sausage. The sausage containing DD95 and 120 ppm nitrite had the highest a* value after incubation for 48 h. In addition, at this moment there was no significant difference in a* value between the sausage containing 120 ppm nitrite only and the sausage containing DD95 and 40 ppm nitrite (Table 2).
Similarly, all L* values were not significantly different for all sausage samples during storage at 4 • C (Table 3). Although there was some variation in the b* value of sausage samples during storage, these b* values were still quite similar (in the range of 8.97-10.45) after incubation for 10 days. For the control sausage (without DD95 and nitrite), the a* value significantly decreased with the increase in incubation time. Nitrite addition, especially at 80 and 120 ppm, substantially increased the a* value after incubation for 10 days. There was no significant difference in a* value between the sausage containing 120 ppm nitrite only and the sausage containing DD95 and 40 ppm nitrite (Table 3).
After storage at 25 • C for 48 h, the appearances of pork sausages containing 0-120 ppm sodium nitrite and with/without 3000 ppm DD95 are shown in Figure 4. The control sausage (without nitrite and DD95) became greenish. The attenuation of green color and increase of red color for sausages with increasing concentration of sodium nitrite were observed ( Figure 4A). Moreover, less green color for sausage with DD95 only (sample no. 5 in Figure 4) was observed in comparison with the control sausage (sample no. 1). The light red color for sausages with DD95 and nitrite (40-120 ppm) was observed ( Figure 4B). In addition, a similar or even better appearance for the sausages containing DD95 and 40 ppm nitrite (sample no. 6) was observed in comparison with that for the sausage containing 120 ppm nitrite only (sample no. 4). The color appearance of sausages shown here correlated well with the a* values shown in Table 2.
In this study, except for sodium nitrite and/or DD95, we did not add any other curing agents such as salt and sugar to eliminate the probable combination effects of these curing agents with nitrite and DD95 on color development in pork sausage. It is well known that the oxidation of deoxymyoglobin (red color) to metmyoglobin (brown color) in meat causes the decrease of a* value. The nitrosomyoglobin formation after nitrite reaction with myoglobin results in red color formation [29]-that is, it increased the a* value. The antioxidant activity of chitosan may retard the oxidation of myoglobin [30]. Accordingly, the higher a* value was obtained in sausages containing DD95 after storage at 25 and 4 • C in this study.
In addition, sulfmyoglobin formation by H 2 S induced by some putrefactive bacteria including Pseudomonas spp. [31] causes the greenish color of meat. This study clearly demonstrates that the addition of DD95 in sausage during storage not only retards the increase of TAC, because of its strong antibacterial activity [18], but also effectively prevents green color formation in sausages.
Owing to the carcinogenic potential of nitrite in sausages, the consumers' interest in "synthetic nitrite-free" meat products is continuously increasing nowadays [31]. As a result, several studies have tried to use natural ingredients, such as potato paste and cured brine, to replace nitrite. However, lower L* and a* values were obtained for these products, compared to sausage added with sodium nitrite [32,33].  In addition, sulfmyoglobin formation by H2S induced by some putrefactive bacteria including Pseudomonas spp. [31] causes the greenish color of meat. This study clearly demonstrates that the addition of DD95 in sausage during storage not only retards the increase of TAC, because of its strong antibacterial activity [18], but also effectively prevents green color formation in sausages.
Owing to the carcinogenic potential of nitrite in sausages, the consumers' interest in "synthetic nitrite-free" meat products is continuously increasing nowadays [31]. As a result, several studies have tried to use natural ingredients, such as potato paste and cured brine, to replace nitrite. However, lower L* and a* values were obtained for these products, compared to sausage added with sodium nitrite [32,33].

Volatile Basic Nitrogen (VBN)
In the meat industry, the VBN value is used to indicate the level of putrefaction. As shown in Figure 5, the VBN values in sausages usually increased during storage at 25 • C ( Figure 5A) and 4 • C ( Figure 5B). Nitrite addition at 40-120 ppm significantly retarded the increase of VBN values, and a dose-dependent response was observed. The VBN values were further decreased by adding both DD95 and nitrite in sausage. We also observed that VBN values between the sausage with DD95 and 40 ppm nitrite and the sausages with 120 ppm nitrite only were quite similar after storage at 25 • C for 48 h ( Figure 5A) and 4 • C for 6 days ( Figure 5B). In addition, the shearing force and hardness of pork sausages with or without DD95 and nitrite during storage at 25 and 4 • C were not significantly different (data not shown).
dose-dependent response was observed. The VBN values were further decreased by adding both DD95 and nitrite in sausage. We also observed that VBN values between the sausage with DD95 and 40 ppm nitrite and the sausages with 120 ppm nitrite only were quite similar after storage at 25 °C for 48 h ( Figure 5A) and 4 °C for 6 days ( Figure 5B). In addition, the shearing force and hardness of pork sausages with or without DD95 and nitrite during storage at 25 and 4 °C were not significantly different (data not shown).

DD95 Chitosan Preparation
On the basis of the method described by Chang, Lin, Wu and Tsai [6], chitosan with 95.0% deacetylation degree, as measured using the colloid titration method [34], was obtained after deacetylation of a shrimp chitin powder suspension in 50% NaOH (1.0 g chitin per 13 mL NaOH) at 140 • C for 1 h. DD95 chitosan MW was 300 kDa, as determined by size-exclusion high-performance liquid chromatography using a column packed with TSKgel G4000 PWXL and G5000 PWXL [35].

Culture Conditions
C. perfringens CCRC 10,648 and CCRC 13,019 were stored in LIB containing 50% sterile glycerol at −80 • C. To prepare the bacteria cultures, the strains stored at −80 • C were inoculated into 50 mL LIB and anaerobically incubated at 37 • C for 18-24 h. All strains were subcultured twice in LIB at 37 • C for 18-24 h, and the cultures were diluted to 1.0 × 10 8 CFU/mL with sterile 0.1% peptone water before use.

Spore Preparation
Briefly, 1 mL of the 18-24 h C. perfringens culture in LIB was added in 9.0 mL of the modified Ducan-Strong (DS) medium [36], and incubated for 18-24 h under anaerobic conditions. Next, 1 mL of the cultivated DS medium broth was added into 9 mL fresh modified DS medium and heated at 70 • C for 20 min, then incubated anaerobically for 20-24 h; the process was repeated twice. Cultivated broths were subsequently placed under centrifugation at 1500× g for 30 min; precipitates were washed under suspension in 10 mL phosphate-buffered saline (PBS) and heated at 70 • C for 20 min to inactivate the cells, and then spores were collected and stored at 4 • C until use.

Antibacterial Test
A 1% chitosan stock solution was prepared by adding 0.2 g of DD95 chitosan to 10 mL distilled water, sterilizing at 121 • C for 15 min, and then adding 10 mL sterile 0.2 N HCl. In 50 mL flasks containing 10 mL of LIB, DD95 chitosan in 0.1 N HCl was added to obtain concentrations of 50, 100, and 250 ppm. Next, 1 mL of C. perfringens spore suspension was added into a flask to an initial spore density of ca. 10 3 CFU/mL, and incubated at 37 • C for 24 h under anaerobic conditions. Then, 0.1 mL aliquots of decimal dilutions of LIB culture were spread onto LIB agar plates, which were incubated under anaerobic conditions at 37 • C for 24 h before colonies were counted. For evaluation of chitosan against spores, spores of two strains were collected into PBS added with 250 ppm DD95 chitosan, and further incubation was performed as described in the above test. The experiments were run in triplicate.

Measurement of Heat Resistance of C. perfringens Spores
On the basis of the method of Tsai, Tsai, Lee and Zhong [16], 1 mL of C. perfringens spore suspension (1 × 10 7 spore/mL) in sterile saline alone or in sterile saline containing 250 ppm chitosan was injected into glass microhematocrit tubes (inside diameter, 5 mm; length, 20 cm; wall thickness, 0.5 mm) and submerged in an oil bath at 90 or 100 • C. Three tubes for each strain were removed at intervals and cooled immediately in ice water. The initial spore concentration and the number of spores that survived heating were determined by spreading 0.1 mL of decimal dilutions of spore suspension on LIB agar. Colonies were counted after incubation at 37 • C for 2 days. The log transformed number of surviving spores was plotted against the corresponding heating time. Data points were fitted using the least squares method, and the reciprocal of the slope was taken as the D value.

Application of DD95 Chitosan to Pork Sausage
Raw pork sausage was added to an equal amount (w/w) of chitosan at the final concentration of 3000 and 6000 ppm. The two sausages with or without DD95 chitosan were added with C. perfringens strain spore cells to a density of ca. 10 4 spore/g, then vacuum-packed and stored at 4 and 25 • C during storage until use. Then, 25 g of vacuum-packed pork sausage was poured into a sterile stomacher bag with 225 mL of 0.1% peptone water and homogenized with a stomacher (Stomacher 400 Lab Blender; Seward Medical, London, UK), and the resulting solution was diluted serially with 0.1% peptone water. The total aerobic bacterial count and the CPC for each sample were determined by spread plating 0.1 mL of sample decimal dilutions onto PCA at 37 • C for 48 h and TSC agar (250 mL TSC agar base +20 mL 50% egg yolk saline +20 mL cycloserine (0.5%)) under anaerobic conditions at 37 • C for 24 h.

Application of DD95 Chitosan and Nitrite for Pork Sausage
Pork sausage was added to an equal amount (w/w) of chitosan and finally to 3000 ppm of DD95 chitosan and 0, 40, 80, and 120 ppm of nitrite substitution for pork sausage. The two sausages with or without DD95 chitosan were added with C. perfringens strain spore cells to a density of ca. 10 4 spore/g, then vacuum-packed and stored at 4 and 25 • C during storage until use. The survival counts were measured as described in Section 3.7.

Color and Color Difference Measurement
On the basis of the method described by [37,38], samples were cut into 2 cm-thick pieces and measured on the plate of the Color Difference Meter (Spectrophotometer CM-3500d; Minolta Co., Ltd., Osaka, Japan). The instrument was calibrated to standard black and white tiles before analysis. Eight pieces per treatment were evaluated, and mean values were used for replication. A medium-size aperture was used, and the measurement was duplicated. The Hunter color L*, a*, and b* values were reported through the computerized system using Spectra Magic software (version 2.11; Minolta Cyberchrom Inc. Osaka, Japan).

Volatile Basic Nitrogen
According to the method described by Saito et al. [39], 5 g of the sample was homogenized with 45 mL 7% trichloroacetic acid (TCA) to obtain a final concentration of 50 mL. A volume of 0.02 N boric acid as a VBN absorber was placed in the inner section of a Conway microdiffusion cell [40]. Then, 1 mL of the homogenized solution was pipetted to the Conway dish containing 1 mL saturated K 2 CO 3 solution and allowed for reaction at 37 • C for 90 min. The boric acid solution containing indicator (methyl red and bromocresol green) was used to absorb volatile nitrogen. The solution was titrated with 0.01 N HCl. The VBN value, expressed as mg %, was calculated as follows: VBN value (mg %) = 0.02 × 14 × (a − b) × F × V/W × 100, where a is the titration volume of sample solution, b is the titration volume of water blank, V is the final volume with TCA of sample after extraction, W is the mass of the sample (g), and F is the unit of 0.02 HCl.

Statistical Analysis
Data were analyzed using the general linear model (GLM) of Statistical Analysis System's Procedures (SAS Institute Inc., Cary, NC, USA) with a 5% level of significance. Means were separated using Duncan's new multiple range test.

Conclusions
Sodium nitrite at 40-120 ppm significantly retarded the proliferation of C. perfringens and VBN values, and increased red color retention in pork sausage during storage. However, sodium nitrite at the tested concentration had little inhibition effect on the growth of the aerobes. DD95 chitosan clearly inhibited the growth of C. perfringens and reduced the heat resistance of the spore of this pathogen. The addition of DD95 chitosan effectively slowed down the increase of TAC and VBN values, preserved the red color, and inhibited the growth of C. perfringens. In effect, DD95 chitosan could serve as a substitute for part of the total amount of sodium nitrite used in pork sausages as a way to reduce