Effect of a compound starter cultures inoculation on bacterial profile and biogenic amine accumulation in Chinese Sichuan sausages

Hongyng Ren, Yunpeng Deng, Xinhui Wng

a School of Chemistry and Chemical Engineering, Southwest Petroleum University, Chengdu 610500, China

b Meat-processing Application Key Laboratory of Sichuan Province, College of Food and Bioengineering, Chengdu University, Chengdu 610106, China

Keywords:

Bacterial community

Biogenic amine

Chinese sausages

High-throughput sequencing technology

Starter cultures

A B S T R A C T

Effects of a starter culture composed of Lactobacillus sakei, Pediococcus pentosaceus, Staphylococcus xylosus and S. carnosus at the ratios (m/m) of 1:1:1:1 on bacterial profile and biogenic amine accumulation in Chinese Sichuan sausages were demonstrated. In starter culture-inoculated sausages, Lactobacillus spp., Pediococcus spp. and Staphylococcus spp. were predominant bacterial genus and their relative abundances ranged from 70.14% to 25.98% , 57.66% to 14.08% , and 15.71% to 13.40% during fermentation, respectively. Accordingly, Lactobacillus spp. and Weissella spp. were predominant bacterial genus and their relative abundances ranged from 63.14% to 24.70% and 49.40% to 11.96% during spontaneous fermentation, respectively. Furthermore, the abundances of undesirable microorganisms such as Yersinia spp., Enterobacter spp., Acietobacter spp. and Psychrobacter spp. were lower than that of the control. The levels of histamine, putrescine, tyramine and cadaverine in Chinese Sichuan sausages with starter cultures inoculation were significantly lower (P ＜ 0.05) than that of the control, and were decreased by 83.09% , 69.38% , 51.87% and 57.20% , respectively, at the end of the ripening. These results revealed that the starter cultures inoculation was a better alternative for microbial quality improvement and biogenic amine reduction of Chinese Sichuan sausages with good sensory attributes.

1. Introduction

Chinese Sichuan sausages as a Chinese typical fermented meat product are well preferred by most Chinese consumers, especially in Sichuan province in China, for their mellow and delicious flavor and their strong characteristics of beautiful appearance, bright color, thin skin and tender meat [1]. The traditional Chinese Sichuan sausages are usually made with raw pork meat, salt, sucrose, red chili powder, Baijiu,colorless soy sauce, white pepper, fresh ginger juice and nitrite [1,2],and then were fermented spontaneously for 25–30 days at 9–14 °C [3,4].Apart from sausage formula, the bacterial diversity involved in sausages fermentations is one of the most important factors shaping the final characteristics of sausages. Traditional Chinese Sichuan sausages are generally fermented spontaneously which mainly relies on native bacterial population from raw materials, resulting that sausages are highly susceptible to grow undesirable microorganisms and a uniform product quality and their safety standards are not guaranteed [5]. It has been reported that a suitable starter culture inoculation could enhance flavor, prolong shelf life and improve quality and safety of production [6]. In European countries, lactic acid bacteria (LAB) was considered as probiotics and have been used in fermented sausages, such asLactobacillus sakeiandPediococcus pentosansin Spanish fermented sausages [7],L. plantarumin Italian fermented sausages, which can metabolize carbohydrates to produce organic acids and volatile compounds [8,9], and have an ability to produce bacteriocins to inhibit pathogenic bacteria [10].Staphylococcusspp. such asS. carnosusandS. xylosuswith nitrite reducatase activity and protolysis ability have been inoculated in Italian-style salami, which is conducive to the color improvement,unique flavor formation and delay rancidity of sausages [11,12].Moreover, various biogenic amines, such as histamine, putrescine,tyramine and cadaverine, have been detected in dry fermented sausages, resulting in increasing attention among consumers [6,13,14].

The formation of biogenic amines is mainly attributed to the presence of decarboxylase-producing microoganisms, which may be introduced by contamination in dry fermented sausages [15,16].Therefore, the growth of the potential biogenic amines producers should be controlled. A number of researchers have investigated that certain starter culture inoculation was conducive to inhibiting the growth of biogenic amines producers, resulting in a decrease in biogenic amines levels in dry fermented sausages [1,17,18].

Although the value of these starter culture is well-known,their contributions to bacterial communities and biogenic amine reduction have not been fully described. Therefore, it is interest to further characterize the microbial community and biogenic amines decrease of sausages fermented by starter cultures inoculation. Highthroughput sequencing technology (HTS) as a culture-independent molecular biology-based technique not only can reveal the dominant members present in the samples, but also uncover the rarer bacterial species. Hence, HTS technology has successfully been widely applied to monitor the microbial structure and dynamics in various fermented foods,such as fermented vegetables, fermented meat [19], and cheese [20,21].

In this study, a starter culture composed ofL. sakei,P. pentosaceus,S. xylosusandS. carnosuswas inoculated in Chinese Sichuan sausages. Comparative analysis of bacterial communities by HTS technology and contribution to decrease in biogenic amines levels between starter culture inoculation and spontaneous fermentation in Chinese Sichuan sausages were performed to gain insight about improvement by starter culture inoculation.

2. Materials and methods

2.1 Starter culture

The starter culture used in this study was successfully isolated and purified from Chinese fermented sausages by Meat-Processing Application Key Lab of Sichuan Province, China. The starter culture consists of 4 strains at the ratios (m/m) of 1:1:1:1, namelyP. pentosaceus,L. sakei,S. xylosusandS. carnosus, which were cultured to 108–109CFU/mL and then immediately prepared into freeze-drying powder, respectively.

2.2 Chinese Sichuan sausages preparation and sampling

Fresh lean pork and back fat accounted for 70% and 30% ,respectively, was cut into pieces (3 cm × 3 cm × 0.5 cm) and maintained in refrigeration at 4 °C for 24 h. Then ground pork was mixed thoroughly with salt, sugar, Chinese prickly ash powder,chili powder, white pepper powder, gourmet powder, five spice powders and rice wine at the ratios (m/m) of 100:5:2:1:3:0.3:1:0.2:3 and was inoculated compound starter cultures in a mount of 0.5% (m/m)labelled as CWS. After mixing, the mixture was stuffed into natural casings which were previously soaked in water with a diameter of 10 cm.Accordingly, a non-inoculated sample was taken as the control labelled as CWC. Then the environmental conditions of Chinese Sichuan sausages fermentation and ripening were performed according to the methods as described by Wang et al. [1]. The sausages (100 g)were sampled in triplicate from fermentation on day 0, 10, 20 and 30 for HTS analyses, and the analysis for physicochemical properties and biogenic amine accumulation were performed on day 0, 5, 10, 15, 20,25 and 30.

2.3 Biogenic amine measurement

The biogenic amine content was analyzed by high performance liquid chromatography (HPLC) as described by Lu et al. [6]. The biogenic amine extraction of sausages sample was performed based on the method proposed by Wang et al. [1]for HPLC analyses (Agilent 1100 series, Agilent Technologies, Inc., Palo Alto, CA) to detect the content of histamine, putrescine, tyramine and cadaverine. Biogenic amine standard solution includes 80 mg histamine, 90 mg putrescine,120 mg spermidine and 90 mg cadaverine, which was purchased from Sigma (San Francisco, CA). A gradient elution program was used with a mixture of ammonium acetate and acetonitrile. Separation was carried out on Hypersil BDS C18(4 mm × 125 mm) column (Thermo Fisher Scientific, Waltham, MA) and the peak was detected at 254 nm with a diode array detector. The column temperature was 40 °C and the flow rate was 1.2 mL/min.

2.4 pH, water activity (aw) and total volatile bases nitrogen(TVB-N) measurement

The values of pH andawwere measured according to the methods as described by Wang et al. [12]. The pH was determined using a pH meter (NKW K21, Germany) with automatic temperature compensation (NTC) electrode. Theawwas measured by a Fast-lab water activity meter (Gbx, Romans sur Isère Cédex, France) at 25 °C.The TVB-N content was detected according to the steam distillation method as described by Wang et al. [13].

2.5 Color measurement and texture properties analysis (TPA)

The color measurement and TPA was performed according to the methods as described by Wang et al. [1]. Color evaluation was made through the CIEL* (lightness),a* (redness) andb*(yellowness) space obtained from five different cut areas of each sample using a colorimeter Spectro-pen (Dr. Lange, Dusseldorf,Germany) with D65 illuminant (light source: polychromatic with tungsten lamp). TPA was performed using a texture analyzer (TA.XT2i, Stable Micro Systems, Vienna Court, UK). Sausage pieces(1 cm × 1 cm × 1 cm) were compressed twice to 50% of their original height at a crosshead speed of 120 mm/min and compression interval was 2 s. Hardness and springiness were obtained using computer software (Texture Exponent 32, Stable Micro Systems).

2.6 Microbiological analysis

The culture-independent method (high throughput sequencing)was employed to evaluate the bacterial communities in sausages.Total bacterial DNA in sausages was extracted as described by Wanget al. [3]. The genomic DNA of samples was extracted using the E.Z.N.ATM Mag-Bind Soil DNA Kit (OMEGA, USA) following the manufacturer’s protocol and purified using ethanol precipitation.The DNA concentration were measured by absorbance at 260,280 and 230 nm to guarantee values of OD260nm/OD280nmand OD260nm/OD230nmabove 1.8 and 2.0, respectively, using Nanodrop 2000 (Thermo Fisher Scientific, USA) [3]. The DNA quality was checked on a 1.5% agarose gel based on the methods as reported by Wanget al. [3]. Extracted DNA was used as the template and the barcoded primers 515F and 805R with Illumina barcoded adapters for PCR amplifications of the bacterial V4 16S rRNA region [3,4]. The two step-PCR technology was preformed and the amplification system and reaction conditions based on the methods as reported by Wang et al. [4]were used. Subsequently, the sequencing reaction was performed with a MiSeq Illumina instrument (Illumina, USA). The operational taxonomic unit (OTU) at an identity threshold of 97% was assigned using the uclust method. Set 97% sequence similarity to use RDP calculate estimator values of Shannon index and Chao1 (http://pyro.cme.msu.edu/). Alpha diversity was evaluated by Good’s coverage,Shannon diversity indices and Chao 1 richness [19].

2.7 Sensory evaluation

Sensory analysis was performed on 3 mm thick slices of Chinese Sichuan sausages as described by Cavalheiro et al. [22]. All samples were coded with three-digit numbers and presented to 11 assessors in a random order. The color, odor, hardness, flavor, texture and overall acceptability on hedonic scale from 1 (dislike extremely) to 10 (like extremely) was evaluated by assessors.

2.8 Statistical analysis

Data were presented as mean values accompanied with the standard deviation. Duncan’s multiple range test (P＜ 0.05) was employed for the independence of error terms using the SPSS Statistics software (IBM, Chicago, Ill., USA).

3. Results and discussion

3.1 Effect of the starter culture inoculation on pH, aw and TVB-N content

The initial pH was 5.94 and 5.84 in sample with starter culture inoculated (CWS) and control sample without starter culture inoculation (CWC), respectively (Fig. 1a). Then a decrease in pH was observed in both CWS and CWC samples during ripening, and the decrease was more intense in CWS sample (P＜ 0.05), probably due to the lactic acid accumulation derived fromP. pentosaceusandL. sakeiwhich was more intense in CWS sample. These results are in agreement with microorganisms’ outcomes. This lower pH value is conducive to improving microbiological safety. A slight increase in pH was found in both CWS and CWC samples at the last phase of ripening, probably because proteolytic activity has a buffering effect on the organic acids [23]. This behavior was also reported by Cavalheiro et al. [22]and Wang et al. [1].

Fig. 1 Effects of starter cultures inoculation on pH (a), water activity (b),TVB-N content (c) in Chinese Sichuan sausages. *P ＜ 0.05, **P ＜ 0.01.

Theawdecreased up to end of the ripening in both CWS and CWC samples because of dehydration (Fig. 1b). Theawin CWS sample was lower than that of the control sample (P＜ 0.05), which might be related to the differences in texture of sausages caused by starter culture inoculation. The fermented sausages with starter culture inoculation was less hardness than that of the spontaneous fermentation based on the analysis of texture data (Table 1), which is more conducive to the water migration from the inside to the outside of the sausages,resulting in more water loss. Similar results were found in fermented sausages withLactobacillusspp. inoculation [22,24].

Table 1Texture properties analysis of Chinese Sichuan sausages at the end of ripening.

The TVB-N is generally considered as an indicator of freshness and protein degradation of meat [13,17]. The levels of TVB-N in CWS and CWC samples were present in Fig. 1c. The TVB-N values increased substantially in both CWS and CWC samples, and a significant lower TVB-N content was observed in CWS sample(P＜ 0.05). This result agrees with those reported by Xiao et al. [25],who found that starter culture inoculation was conducive to reducing TVB-N production.

3.2 Effect of the starter culture inoculation on biogenic amines accumulation in Chinese Sichuan sausages

The biogenic amines, namely histamine, putrescine, tyramine and cadaverine accumulation in CWS and CWC samples are shown in Fig. 2.The 4 kinds of biogenic amines were all detected in both CWS and CWC samples and tyramine accumulation was the highest followed by cadaverine, putrescine and histamine. Substantial increases in putrescine, tyramine and cadaverine accumulation were observed in both CWS and CWC samples during ripening. The maximum concentration of histamine was present on day 20 and then the levels of histamine dropped rapidly from 7.85 mg/kg to 3.25 mg/kg,and from 27.32 mg/kg to 19.23 mg/kg in CWS and CWC samples,respectively. These results revealed that the levels of histamine,putrescine, tyramine and cadaverine in starter culture-inoculated sausages were significantly lower (P＜ 0.05) than that of the control.At the end of the ripening, the concentrations of histamine, putrescine,tyramine and cadaverine in starter culture-inoculated sausages were decreased by 83.09% , 69.38% , 51.87% and 57.20% , respectively.

Fig. 2 Effects of starter cultures inoculation on histamine (a), putrescine (b),cadaverine (c) and tyramine (d) accumulation in Chinese Sichuan sausages.*P ＜ 0.05, **P ＜ 0.01.

The biogenic amines, especially for histamine, putrescine,tyramine and cadaverine, have undesirable physiological effects such as stimulating the nerves and blood vessels in humans and have been thought to pose a hazard in human health [26,27]. Recently, the biogenic amines were frequently detected in Chinese sausages from different regions and manufactures [6]which mainly derived from microbial decarboxylation of amino acids during ripening processes,food spoilage or transamination of aldehydes and ketones by amino acid transaminases. Enterobacteriaceae andPseudomonasspp.,which were normally observed in spontaneous fermented sausages,have been revealed to be involved in the production of histamine,putrescine and cadaverine. Hence, the growth of microorganisms responsible for the decarboxylation reactions should be controlled to reduce the probability of biogenic amine production. Recently,the effects of starter culture inoculation on the reduction of biogenic amines accumulation in fermented sausages were studied and compared with spontaneous fermentation [1,26,28].L. sakeiandS. carnosushave been observed to exhibit an obvious inhibitory effect on histamine, putrescine, cadaverine, tyramine and phenylethylamine accumulation in fermented sausages [1,6]. In the study, the levels of biogenic amines in starter culture-inoculated sausages were significantly lower (P＜ 0.05) than that of the control, which suggests that the starter culture inoculation is conducive to decreasing the biogenic amines accumulation in Chinese Sichuan sausages.Subsequently, the bacterial communities involved in biogenic amines formation were determined to elucidate the inhibitory effect on biogenic amines accumulation of the starter culture. According to the results of bacterial communities, the starter culture inoculation contributes to increase the relative abundance of lactic acid bacteria and reduce the relative abundance of spoilage bacteria compared with spontaneous fermentation, which might be beneficial to control the biogenic amines accumulation.

3.3 Effect of the starter culture inoculation on bacterial diversity and microbiological quality

The bacterial relative abundance of 8 examined samples, namely CWS1 (0 days), CWS2 (10 days), CWS3 (20 days), CWS4 (30 days),CWC1 (0 days), CWC2 (10 days), CWC3 (20 days) and CWC4(30 days), was analyzed by HTS technology. After the quality control,a total of 294 449 and 259 423 high quality bacterial sequences were obtained from GWS and GWC samples, respectively (Table 2).Furthermore, the bacterial sequence reads from CWS sample were clustered into 1 148 OTUs at a 97% similarity level. Accordingly,the bacterial sequence reads from CWC sample were clustered into 1 363 OTUs at a 97% similarity level. Additionally, the average read lengths were between 271.2 and 292.6 bases, and Q20 values ranged from 97.25 to 97.43, indicating that both quantities and qualities of the sequences were sufficient.

Table 2Bacterial community diversity indices (at 97% sequence similarity) of Chinese Sichuan sausages based on 16S rRNA gene.

A total of 4 bacterial phyla were classified from CWS samples at 97% similarity level (Fig. 3), in which Firmicutes was the primary dominant bacterial phylum and its relative abundance ranged from 99.67% to 99.85% . Accordingly, a total of 4 bacterial phyla, including Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, was also detected in CWC samples, in which Firmicutes and Proteobacteria were predominant bacterial phyla and their relative abundances ranged from 85.65% to 93.96% , and 5.59% to 13.95% , respectively.

Fig. 3 Comparative analysis of bacterial relative abundance at phylum level between starter culture inoculation and spontaneous fermentation in Chinese Sichuan sausages.

These findings showed that a significant (P＜ 0.05) effect on the bacterial relative abundance at phylum level by starter culture inoculation was present in Chinese Sichuan sausages, especially for Firmicutes and Proteobacteria. Briefly, compared with spontaneous fermentation, the relative abundance of Firmicutes in sausages treated with starter culture inoculation increased by 10.00% , 14.02% , 5.89% and 6.85% on fermentation 0, 10, 20 and 30 days, respectively. In contrast, Proteobacteria remained rare in sausages treated with starter culture inoculation, and by which time it had become a dominant phylum in spontaneous fermentation. Its relative abundance was significant lower than that of spontaneous fermentation, and it decreased by 9.56% , 13.76% , 5.47% and 6.18% on fermentation 0,10, 20 and 30 days, respectively. These findings revealed that starter culture inoculation could promote the dominance of Firmicutes to be more prominent. The principal reason for increase of Firmicutes might be thatP. pentosaceus,L. sakei,S. xylosus,S. carnosusfrom starter culture belong to Firmicutes at phylum level.

In total, 30 bacterial genera were detected in CWS samples on 0, 10, 20 and 30 days sampling sets, in whichLactobacillusspp.,Pediococcusspp. andStaphylococcusspp. were predominant bacterial genus and their relative abundances ranged from 70.14% to 25.98% , 57.66% to 14.08% , and 15.71% to 13.40% , respectively.Accordingly, a total of 30 bacterial genera was also identified from CWC samples on 0, 10, 20 and 30 days sampling sets at genus level,in whichLactobacillusspp. andWeissellaspp. were predominant bacterial genus and their relative abundances ranged from 63.14% to 24.70% and 49.40% to 11.96% , respectively. These results revealed that a conspicuous effect on the bacterial relative abundance of dominant bacteria genera by starter culture inoculation was found in Chinese Sichuan sausages as shown in Fig. 4.Pediococcusspp.andStaphylococcusspp. with a minimal relative abundance (＜ 3% )in spontaneous fermentation, while they became dominant bacterial genera in fermentation treated with starter culture inoculation with a much higher relative abundance than that of spontaneous fermentation, and increased by 55.7% and 13.15% at the end of fermentation, respectively. Interestingly,Weissellaspp. had a high relative abundance, ranging from 49.40% to 11.96% in spontaneous fermentation, whileWeissellaspp. (＜ 0.5% ) was barely detectable in fermentation treated with starter culture inoculation. Moreover,Lactobacillusspp. with a high relative abundance was determined both in spontaneous fermentation and fermentation treated with starter culture inoculation. Apart from the dominant bacterial genera,the starter culture inoculation also had a certain impact on some bacterial genera with minimal relative abundance, such asYersiniaspp.,Enterobacterspp.,Acietobacterspp. andPsychrobacterspp.,resulting in an extreme low relative abundance (＜ 0.05% ) (Table 3).

Fig. 4 Comparative analysis of bacterial relative abundance of dominant bacterial genera between starter culture inoculation and spontaneous fermentation in Chinese Sichuan sausages.

Table 3Comparative analysis of bacterial relative abundance (% ) of minimal bacterial genera between starter culture inoculation and spontaneous fermentation in Chinese Sichuan sausages.

The bacterial community involved in Chinese Sichuan sausages fermentation is one of the most important factors shaping the final characteristics of sausages. It has been reported that the core functional bacteria involved in Chinese sausages fermentations mainly includeMicrococcusspp.,Staphylococcusspp. and lactic acid bacteria such asLactobacillusspp.,Pediococcusspp. andWeissellaspp. [29].In this study,Lactobacillusspp. (ranged from 24.7% to 63.14% ) andWeissellaspp. (ranged from 49.4% to 11.96% ) with a high relative abundance was determined in spontaneous fermentation. WhilePediococcusspp. (ranged from 0.09% to 2.74% ) andStaphylococcusspp. (ranged from 0.06% to 1.89% ) with a minimal relative abundance were present in spontaneous fermentation. Accordingly,Lactobacillusspp. (ranged from 31.90% to 70.11% ),Pediococcusspp. (ranged from 14.81% to 57.66% ), andStaphylococcusspp. (ranged from 12.83% to 15.71% ) were enriched in CWS sample due to inoculationofP. pentosaceus,L. sakei,S. xylosusandS. carnosus. The members ofLactobacillusspp. andPediococcusspp. have strong abilities of proteolysis and acidification which not only could be conducive to development of the distinctive sour taste and rich flavor, but also could inhibit the growth of pathogens in sausages.S. xylosusandS. carnosushave strong protease and lipase activities which contribute to texture softening and generation of meat flavor of sausages [30]. In additionally,S. xylosusandS. carnosuscan contribute to the redness improvement of sausages due to their nitrate-reductase activity [29,31].Moreover,S. xylosuscan degrade effectively biogenic amine due to its amine oxidase activity [1]. Thus, starter culture inoculation can contribute to flavor and food safety improvement by enriching functional bacteria.

Pork sausages are highly susceptible to the growth of undesirable pathogenic and spoilage bacteria from raw meat, especially for spontaneous fermentation, which generally leads to the development of off- flavors, oxidative rancidity, discoloration, gas production and biogenic amines formation, and makes it unacceptable to the consumer from a sensory point of view [32,33]. In this study,Yersiniaspp.(2.42% ),Enterobacterspp. (1.07% ) andPsychrobacterspp. (0.86% )with a minimal relative abundance was detected in spontaneous fermentation at the end of fermentation. Correspondingly, a trace ofYersiniaspp.,Enterobacterspp. andPsychrobacterspp. with 0.01% relative abundance was present in starter culture inoculation at the end of fermentation. It has been reported that the members ofPsychrobacterspp. such asP.immobilishave identified as spoilage bacteria which might cause incidental infections. The members ofYersiniaspp. such asY. enterocolyticahave been reported as foodborne pathogens, andEnterobacterspp. is an indicator of fecal contamination in foods. These results revealed that there is a good sanitary hygienic quality and a low occurrence of potential pathogenic and spoilage bacteria in sausages fermented by starter culture inoculation, probably due to the interspecific competition and acidification by starter culture. Thus, it is strongly recommended to apply starter culture inoculation instead of spontaneous fermentation to acquire good sanitary hygienic quality and shelf-life extension of sausages produced in industry.

3.4 Effect of the starter culture inoculation on color and texture

After ripening, the evaluation of lightness (L*), redness (a*), and yellowness (b*) of CWS and CWC samples was present in Fig. 5.A significant higher (P＜ 0.05)a* value was observed in CWS sample, compared with CWC sample, which probably related to the nitrosomyoglobin formation and moisture loss leading to promote an increase in haemepigment concentration. These results revealed that the starter culture inoculation was conducive to the improvement of color characteristics, and this was confirmed by assessors in the sensory evaluation (Fig. 6).

Fig. 5 Effects of starter cultures inoculation on color values in Chinese Sichuan sausages. *P ＜ 0.05, **P ＜ 0.01.

Fig. 6 Comparative sensory characteristics of Chinese Sichuan sausages with starter culture inoculation and without starter culture inoculation as the control.

The TPA analysis of sausages at the end of ripening are shown in Table 1. Hardness and chewiness values of starter culture-inoculated sausages were lower than that of the control, while springiness value of starter culture inoculated sausages was higher than that of the control. These results suggested that starter culture inoculation could give a better taste, and this was confirmed by assessors in the sensory evaluation.

3.5 Sensory evaluation

To better understand the effect of starter culture inoculation on the sensory attributes of Chinese Sichuan sausages, a preliminary sensory evaluation test was conducted. After ripening, the sensory evaluations of CWS and CWC samples based on the average scores for color,flavor, odor, appearance, hardness, texture and overall acceptability are shown in Fig. 6. The starter culture-inoculated sausages received significant higher scores (P＜ 0.05) for odor and color, which contributed to its acceptance. Compared with the control, the starter culture-inoculated sausages were reflected in a higher score (+0.5 points) in relation to the parameters of flavor, appearance and texture,though this was not statistically significant. The hardness of starter culture-inoculated sausages was evaluated as a significant lower score(P＜ 0.01) than that of the control, which was in line with the result of TPA analysis. Similar results were reported by Sidira et al. [24]and Chorianopoulos et al. [34]who revealed that the probiotic fermented sausages were given a higher score in overall sensory quality including color, flavor, taste and appearance.

4. Conclusions

The pH andawvalues in starter culture-inoculated samples were lower than that of spontaneous fermentation, as well as TVB-N level. In starter culture-inoculated samples, the relative abundances of functional bacteria such asLactobacillusspp.,Pediococcusspp.andStaphylococcusspp. were higher than that of the control, while the relative abundance of spoilage bacteria such asYersiniaspp.,Enterobacterspp. andPsychrobacterspp. was quite low. Moreover,the levels of histamine, putrescine, tyramine and cadaverine were lower in starter culture-inoculated samples than that of spontaneous fermentation, and its physicochemical characteristics and sensory evaluation exhibited good values. These results suggest that the inoculation of a compound starter culture composed ofL. sakei,P. pentosaceus,S. xylosusandS. carnosusis a better alternative for microbial quality improvement and biogenic amine reduction of Chinese Sichuan sausages with good sensory attributes.

Acknowledgements

The research was supported by National Natural Science Foundation of China (31772093), Key innovation projects of Chengdu Science and Technology (2019-YF05-00066-SN) and Key Laboratory of Collaborative Control and Remediation of Soil and Water Pollution(GHBK-003). The authors thank Sangon Biotech for technical assistance in carrying out the analyses.

Conflict of Interest

The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could be have influenced its outcome.