| Spices are highly susceptible to microbial contamination from the environment and people during the harvest until consumption,and often lack effective sterilization treatments.When highly contaminated dried spices are added to high-moisture foods as raw materials,the increase in moisture content can promote the rapid growth and reproduction of microorganisms in spices,and may produce metabolic toxins,which pose a serious threat to human health.Therefore,the microbial safety of spices is important.In this study,200 samples of dried spices（100 of pepper and 100 chili samples,respectively）collected from 39 regions in Sichuan Province were tested for microorganisms to understand the status of microbial contamination of commercially available samples and judge their pass rate.The high microbial contamination samples were analyzed for microbial heat resistance to judge the ability of heat sterilization to control the microbial flora in the spice samples,and provide a reference for the design of the spice sterilization process;the construction of a Whole-genome database for the highly heat-resistant strains provided reliable risk assessment data for the microbial safety of spices,and also provided support for the production and application of the highly heat-resistant strains.Below are key research findings:（1）The microbial contamination of commercially available peppers and chilies was serious.Compared with the Agricultural Industry Standard NY/T 901-2021"Green Food Spices and Their Products",the unqualified rate of powdered pepper samples is81.43%,and the unqualified rate of granular pepper samples is 60.00%.The main reason for the unqualified samples is that the number of molds exceeds the standard.Compared with the Agricultural Industry Standard NY/T 1711-2020"Green Food Chili Products",the unqualified rate of powdered chili samples is 62.50%,and unqualified rate of granular chili samples is 41.67%.The main reason for the unqualified samples was that the number of coliform bacteria exceeded the standard.（2）According to the test results,12 samples（6 samples of pepper and chili,respectively）with high pollution degree were selected for microbial heat resistance analysis.The samples were loaded into aluminum TDT heating units and placed in oil baths at 95℃,100℃and 105℃for treatment.Through the analysis of the survival number of microorganisms,it was found that heat treatment is difficult to control the number of microorganisms in dried spices.The aroma of the samples after high-temperature treatment was seriously lost.If the heating temperature is forcibly increased to meet the demand of reducing the number of microorganisms below the detection limit,it will lead to the loss of flavor and nutrients of spices and loss of commercial value.（3）After heating 6 pepper and 6 chili samples used for microbial heat resistance analysis at 100℃for 100 minutes,six strains with high heat resistance were screened.Survival experiments were carried out on six highly heat-resistant strains,and it was found that they all had good viability:after storage for 240 days in an environment with a water activity of 0.53,the number of the six strains only decreased by 0.067±0.031～0.463±0.128 log.Antibiotic resistance experiments on six highly heat-resistant strains found that all strains were sensitive to most of the antibiotics tested in the experiment.The six strains all had strong heat resistance,and the D values ranged from 57.56±1.36 to 141.51±4.89 min in the environment of aw,100℃0.53.（4）The whole-genomes of the six highly heat-resistant strains were sequenced by the Illumina Hiseq 550 sequencing platform,and the results showed that they were:one strain of Bacillus subtilis,one strain of Bacillus halotolerant,two strains of Bacillus safensis,one strain of Bacillus licheniformis and one strain of Bacillus velezensis.The genome size of these six strains ranged from 3,682,190 to 4,294,294 bp,the GC percentage ranged from 41.35%to 46.01%,and 4,052,4,218,4,050,3,722,4,383 and3987 coding genes were annotated,respectively.The length of the coding genes ranged from 3,264,681 to 3,771,000 bp,and the average gene length is 860.37 to915.83 bp,accounting for 87.81 to 89.35%of the genome.The six highly heat-resistant strains had 72-88 t RNAs,9-14 r RNAs,and 81-97 s RNAs.The genome also contained 5 to 8 gene island groups,1 to 7 prophages,and 2 to 13 CRISPR sequences.Functional gene annotation of the genome,23 COG classifications were annotated in the COG database,42 GO functional classifications were annotated in the GO database,and 2,649～2,822 genes were involved in six major KEGG functions（including metabolism,genetic information processing,environmental information processing,cell functional annotations,organism system and human disease）.In addition,fewer virulence factors and antibiotic resistance genes were annotated in the six strains,as well as two secretion pathways consistent with the characteristics of Gram-positive bacteria.In conclusion,two kinds of dried spices—pepper and chili were seriously polluted by microorganisms,and the unqualified rates of samples were high.It was difficult to control the number of microorganisms carried by high-temperature heat treatment without changing the original state of dried pepper and pepper,and high-temperature treatment would cause the two spices to lose their unique flavor and commercial value.The highly heat-resistant strains in dried pepper and capsicum were all Bacillus,which had good survival ability in the environment of low water activity,strong heat resistance and low antibiotic resistance.At the same time,fewer virulence factors and antibiotic resistance genes were annotated in each strain,which has certain food-borne risks.Conclusions of this study provided some data support for understanding the microbial contamination of commercial spices,provided a reference for the design of their sterilization process,provided reliable risk assessment data for the microbial safety of high heat-resistant strains derived from spices,and provided basic data and strong support for Bacillus’production and application. |
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