论文部分内容阅读
AbstractThe potential mutagenicity of proteinmodified Enterococcus faecalis was evaluated by the Ames test. The test subject was designed with 0.05, 0.5, 5.0, and 50.0 mg/dish doses, and a control group was set. With Salmonella typhimurium mutant strains TA97, TA98, TA100 and TA102 as test strains, the Ames test was carried out with (+) or without (-) S9, and the number of revertant colonies was counted. The results showed that for the proteinmodified E. faecalis in the case of + S9 and -S9, the average numbers of revertant colonies of the four test strains were less than two times of that of the negative control group, and no doseresponse relationship was observed. The proteinmodified E. faecalis was tested to be negative in the Ames test, indicating that the test substance was not mutagenic in vitro.
Key wordsProteinmodified Enterococcus faecalis; Ames test
Received: June 28, 2018Accepted: October 9, 2018
Supported by Topnotch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015C230).
Lijun DING (1980-), male, P. R. China, lecturer, master, devoted to research about clinical veterinary.
*Corresponding author. Email: 641042228@qq.com.
Enterococcus faecalis, also known as Streptococcus faecalis, belongs to group D Streptococcus and is one of the major flora in the intestinal tract of human and animals［1］. E. faecalis can produce antibacterial substances such as bacteriocin, which inhibit the growth of pathogenic bacteria such as Escherichia coli and Salmonella and improve the intestinal microenvironment［2］, and also have the effects of inhibiting the production of urease bacteria and spoilage bacteria in the intestinal tract, reducing intestinal urease and endotoxin contents and reducing the levels of ammonia and endotoxin in the blood［3］. E. faecalis is widely used as a probiotic in medicine and food engineering fields［4-5］. E. faecalis is one of the feedgrade microbial additive strains announced in Feed Additive Species Catalogue published by the Ministry of Agriculture of China in 2013. Studies have shown that the addition of E. faecalis to the diet has the effects of improving animal performance, improving nutrient metabolism and improving immune function［6-8］. Proteinmodified E. faecalis is a product modified by a specific protein against E. faecalis, in order to enhance the immune response while giving the probiotic. This paper evaluated its invitro mutagenicity by Ames test, so as to accumulate data for safety evaluation of this product. Materials and Methods
Materials
Test substance, experimental animals and strains
The test substance was proteinmodified E. faecalis 5×109 CFU/g (lot number: 20141206), provided by some biotechnology company in Jiangsu Province.
SD rats, with a weight of about 200 g, were purchased from the Comparative Medicine Center, Yangzhou University, under animal production license number: SCXK (Su) 20120004, use license number: SYXK (Su) 20120029. The animals were fed with 60Coirradiated feed, and raised at (24±2) ℃ and humidity of (60%±20%). They ate food and drink water freely, and the drinking water was tap water that meets urban drinking water standards. The animals were adapted to the experimental environment before the test for 3 d.
Test strains: Salmonella typhimurium mutant strains TA97, TA98, TA100 and TA102 were provided by the Jiangsu Provincial Center for Disease Control and Prevention. Before the test, the strains were identified to meet the requirements.
Reagents and preparation
Positive controls included 2aminofluorene (purity>99%, Tokyo Chemical Industry), dexon (purity 98.8%, American DIMA Technology Co., Ltd.), sodium azide (purity>98%, Amresco, USA), and polychlorinated biphenyl (Aroclor 1254, American Crescent Chemical Co., Ltd). Sodium azide was dissolved in water, and other positive controls were prepared using dimethyl sulfoxide as a solvent into a solution with corresponding concentration.
0.5 mmol/L histidine -0.5 mmol/L biotin solution, 20% glucose solution, 0.05 mol/L glucose6phosphate solution, nutrient broth and nutrient broth agar medium, and top layer and bottom agar medium, were all prepared according to the National Food Safety Standards: Ames test (GB 15193.42014).
Induction and preparation of rat liver S9: Polychlorinated biphenyl (Aroclor 1254) was used as an inducer. Polychlorinated biphenyl was dissolved in corn oil at a concentration of 200 mg/ml, and the obtained mixture was injected according to 500 mg/kg b.w. into the abdominal cavity of rats, which were given normal diet and drinking water. Five days after the induction by the injection, the rats were killed by cervical spine dislocation. S9 and S9 mixture were prepared according to the method of GB 15193.42014. After preparation, S9 was subjected to sterility test, protein content determination and biological activity identification, and then stored at -80 ℃ for use. Methods
Enrichment culture
The preserved strains, TA97, TA98, TA100 and TA102, were inoculated into a nutrient broth medium, and cultured at 37 ℃ for 10 h with shaking (100 times/min). The culture of each strain should be no less than 1×109-2×109 viable cells per milliliter.
Ames test by plate incorporation method
A series of aqueous solutions of the test substance were prepared in the concentration range of 0.5-500 mg/ml.
2 ml of the top medium that had been melted and incubated at 45 ℃, was added with 0.1 ml of the test solution (each dish contained 0.05, 0.5, 5.0 and 50.0 mg of the test substance, respectively) and 0.1 ml of the test strain enrichment solution (added with 0.5 ml of 10% S9 mixture when requiring activation). Each mixture was mixed quickly, and poured on the bottom medium, followed by solidification and incubation at 37 ℃ for 48 h. The number of colonies per dish was counted, and the average number of each treatment was calculated.
In addition to each dose group of the test substance, a negative control (sterilized water), a dimethyl sulfoxide solvent control, and a positive control were also set. When the positive mutagen was -S9 mixture, the positive control of TA97, TA98, TA102 strains was dixon, 50.0 μg/dish; and the positive control of TA100 was sodium azide NaN3, 1.5 μg/dish. In the case of the +S9 mixture, the positive control of TA97, TA98, TA100 and TA102 was 2aminofluorene, 10.0 μg/dish. Each treatment included six plates, and the test was carried out in parallel twice.
Judgment of results
If the number of revertant colonies of the test sample is equal to or greater than 2 times of the negative control group, there is a doseresponse relationship; or, if a certain dose exceeds the negative control group by more than 2 times, which is of reproducible and statistical significance, the test substance is considered to be positive for mutagenesis.
Results
The results of the Ames test are shown in Table 1. It could be seen from Table 1 that the positive control group caused the numbers of revertant colonies of S. typhimurium strains TA97, TA98, TA100 and TA102 to increase significantly and far exceed the negative control group by more than 2 times; while the 0.05, 0.5, 5.0 and 50.0 mg/dish groups, whether added or not added with S9 for activation, showed the numbers of revertant colonies all less than 2 times of the number of colonies in the negative control group, and there was no doseresponse relationship. Therefore, the test substance was tested to be negative in the Ames test. Table 1Ames test of proteinmodified E. faecalis
Group
TA97
-S9+S9
TA98
- S9+S9
TA100
- S9+S9
TA102
- S9+S9
Tested substance 0.0596.3±7.194.4±5.631.3±2.435.3±5.1133.3±6.9130.7±17.2227.7±36.4220.0±22.0
(mg/dish)0.592.7±4.399.7±4.933.3±3.836.0±6.7143.7±5.6138.7±19.6237.7±21.8222.7±34.9
5.097.0±5.398.7±5.632.7±3.836.3±5.8133.7±21.57142.7±8.4234.7±42.2236.0±19.3
50.0106.7±9.899.0±7.335.0±3.337.0±4.0172.3±10.9135.0±6.7227.0±17.2230.7±30.4
Negative control99.7±5.899.3±3.829.7±1.433.3±1.8161.7±12.9156.7±7.8239.0±26.0228.3±14.4
Solvent control 98.5±6.296.4±5.834.2±2.335.6±5.2146.8±6.8152.1±6.5232.2±15.8229.3±15.0
Positive control 440.7±27.8244.7±14.476.3±12.479.3±13.1661.0±32.7423.3±24.4854.0±65.0615.7±42.4
Conclusions and Discussion
The proteinmodified E. faecalis was tested to be negative in the Ames test, indicating that the product has no mutagenic effect on the tested S. typhimurium mutant strains.
According to New Feed or Feed Additive Safety Evaluation Guide published by the Ministry of Agriculture, in addition to the Ames test, the mutagenicity tests must include mouse bone marrow micronucleus test and mouse sperm abnormality test. On the basis of these three tests, it is evaluated based on the test results whether other mutagenic tests such as the dominant lethal test are still needed［9］. Although the proteinmodified E. faecalis was tested to be negative by the Ames test in this study, at least the mouse bone marrow micronucleus test and the mouse sperm abnormality test are needed to evaluate its mutagenic effect.
References
［1］ MALIK RK, MONTECALVO MA, REALE MR, et a1. Epidemiology and control of vancomycinresistant enterococci in a regional neonatal intensive care unit［J］. Pediatr Infect Dis J, 1999, 18(4): 352．
［2］ DAVID FM, ARTHUR EB, GARY JN, et al. An investigation of vancomycinresistant enterocicci faecium within the pediatric service of a large urban medical center［J］. Pediatr Infect Dis J, 1998, 17(3): 184.
［3］ TANG XD. Enterococcal infection: a review［J］. Chinese Journal of Infection and Chemotherapy, 2007, 7(3): 221-223. (in Chinese)
［4］ BAO YE, DONG XF, TONG JM, et al. Evaluation of probiotic characteristics of Enterococcus faecalis in vitro［J］. Acta Agriculturae Borealioccidentalis Sinica, 2013, 22(11): 202-207. (in Chinese) ［5］ MA LY, XU SZ, MA JP. Pathogenesis of Enterococcus: A research progress［J］. Chinese Journal of Nosocomiology, 2005, 15(3): 356-360. (in Chinese)
［6］ SHI ZT, YAO YC, JIANG S, et al. Effects of Enterococcus faecalis substitute for antibiotic on growth performance, diarrhea rate, blood biochemical parameters and immune organs of weaner piglets［J］. Acta Zoonutrimenta Sinica, 2015, 27(6): 1832-1840. (in Chinese)
［7］ WEI QT, LI PH, WANG H, et al. Effect of dietary Enterococcus faecalis replacing of antibiotic on growth performance, diarrhea rate, humoral immunity and intestinal microflora of nursery pigs［J］. Journal of Nanjing Agricultural University, 2014, 37(6): 143-148. (in Chinese)
［8］ LIU S, DONG XF, TONG JM, et al. Effects of dietary Enterococcus faecalis on performance, egg quality, lipid metabolism and intestinal microflora numbers of laying hens［J］. Acta Zoonutrimenta Sinica, 2017, 29(1): 202-213. (in Chinese)
［9］ National Feed Judgment Committee Office. New feed or feed additive safety evaluation guide. www.chinafeed.org.cn. (in Chinese)
Key wordsProteinmodified Enterococcus faecalis; Ames test
Received: June 28, 2018Accepted: October 9, 2018
Supported by Topnotch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015C230).
Lijun DING (1980-), male, P. R. China, lecturer, master, devoted to research about clinical veterinary.
*Corresponding author. Email: 641042228@qq.com.
Enterococcus faecalis, also known as Streptococcus faecalis, belongs to group D Streptococcus and is one of the major flora in the intestinal tract of human and animals［1］. E. faecalis can produce antibacterial substances such as bacteriocin, which inhibit the growth of pathogenic bacteria such as Escherichia coli and Salmonella and improve the intestinal microenvironment［2］, and also have the effects of inhibiting the production of urease bacteria and spoilage bacteria in the intestinal tract, reducing intestinal urease and endotoxin contents and reducing the levels of ammonia and endotoxin in the blood［3］. E. faecalis is widely used as a probiotic in medicine and food engineering fields［4-5］. E. faecalis is one of the feedgrade microbial additive strains announced in Feed Additive Species Catalogue published by the Ministry of Agriculture of China in 2013. Studies have shown that the addition of E. faecalis to the diet has the effects of improving animal performance, improving nutrient metabolism and improving immune function［6-8］. Proteinmodified E. faecalis is a product modified by a specific protein against E. faecalis, in order to enhance the immune response while giving the probiotic. This paper evaluated its invitro mutagenicity by Ames test, so as to accumulate data for safety evaluation of this product. Materials and Methods
Materials
Test substance, experimental animals and strains
The test substance was proteinmodified E. faecalis 5×109 CFU/g (lot number: 20141206), provided by some biotechnology company in Jiangsu Province.
SD rats, with a weight of about 200 g, were purchased from the Comparative Medicine Center, Yangzhou University, under animal production license number: SCXK (Su) 20120004, use license number: SYXK (Su) 20120029. The animals were fed with 60Coirradiated feed, and raised at (24±2) ℃ and humidity of (60%±20%). They ate food and drink water freely, and the drinking water was tap water that meets urban drinking water standards. The animals were adapted to the experimental environment before the test for 3 d.
Test strains: Salmonella typhimurium mutant strains TA97, TA98, TA100 and TA102 were provided by the Jiangsu Provincial Center for Disease Control and Prevention. Before the test, the strains were identified to meet the requirements.
Reagents and preparation
Positive controls included 2aminofluorene (purity>99%, Tokyo Chemical Industry), dexon (purity 98.8%, American DIMA Technology Co., Ltd.), sodium azide (purity>98%, Amresco, USA), and polychlorinated biphenyl (Aroclor 1254, American Crescent Chemical Co., Ltd). Sodium azide was dissolved in water, and other positive controls were prepared using dimethyl sulfoxide as a solvent into a solution with corresponding concentration.
0.5 mmol/L histidine -0.5 mmol/L biotin solution, 20% glucose solution, 0.05 mol/L glucose6phosphate solution, nutrient broth and nutrient broth agar medium, and top layer and bottom agar medium, were all prepared according to the National Food Safety Standards: Ames test (GB 15193.42014).
Induction and preparation of rat liver S9: Polychlorinated biphenyl (Aroclor 1254) was used as an inducer. Polychlorinated biphenyl was dissolved in corn oil at a concentration of 200 mg/ml, and the obtained mixture was injected according to 500 mg/kg b.w. into the abdominal cavity of rats, which were given normal diet and drinking water. Five days after the induction by the injection, the rats were killed by cervical spine dislocation. S9 and S9 mixture were prepared according to the method of GB 15193.42014. After preparation, S9 was subjected to sterility test, protein content determination and biological activity identification, and then stored at -80 ℃ for use. Methods
Enrichment culture
The preserved strains, TA97, TA98, TA100 and TA102, were inoculated into a nutrient broth medium, and cultured at 37 ℃ for 10 h with shaking (100 times/min). The culture of each strain should be no less than 1×109-2×109 viable cells per milliliter.
Ames test by plate incorporation method
A series of aqueous solutions of the test substance were prepared in the concentration range of 0.5-500 mg/ml.
2 ml of the top medium that had been melted and incubated at 45 ℃, was added with 0.1 ml of the test solution (each dish contained 0.05, 0.5, 5.0 and 50.0 mg of the test substance, respectively) and 0.1 ml of the test strain enrichment solution (added with 0.5 ml of 10% S9 mixture when requiring activation). Each mixture was mixed quickly, and poured on the bottom medium, followed by solidification and incubation at 37 ℃ for 48 h. The number of colonies per dish was counted, and the average number of each treatment was calculated.
In addition to each dose group of the test substance, a negative control (sterilized water), a dimethyl sulfoxide solvent control, and a positive control were also set. When the positive mutagen was -S9 mixture, the positive control of TA97, TA98, TA102 strains was dixon, 50.0 μg/dish; and the positive control of TA100 was sodium azide NaN3, 1.5 μg/dish. In the case of the +S9 mixture, the positive control of TA97, TA98, TA100 and TA102 was 2aminofluorene, 10.0 μg/dish. Each treatment included six plates, and the test was carried out in parallel twice.
Judgment of results
If the number of revertant colonies of the test sample is equal to or greater than 2 times of the negative control group, there is a doseresponse relationship; or, if a certain dose exceeds the negative control group by more than 2 times, which is of reproducible and statistical significance, the test substance is considered to be positive for mutagenesis.
Results
The results of the Ames test are shown in Table 1. It could be seen from Table 1 that the positive control group caused the numbers of revertant colonies of S. typhimurium strains TA97, TA98, TA100 and TA102 to increase significantly and far exceed the negative control group by more than 2 times; while the 0.05, 0.5, 5.0 and 50.0 mg/dish groups, whether added or not added with S9 for activation, showed the numbers of revertant colonies all less than 2 times of the number of colonies in the negative control group, and there was no doseresponse relationship. Therefore, the test substance was tested to be negative in the Ames test. Table 1Ames test of proteinmodified E. faecalis
Group
TA97
-S9+S9
TA98
- S9+S9
TA100
- S9+S9
TA102
- S9+S9
Tested substance 0.0596.3±7.194.4±5.631.3±2.435.3±5.1133.3±6.9130.7±17.2227.7±36.4220.0±22.0
(mg/dish)0.592.7±4.399.7±4.933.3±3.836.0±6.7143.7±5.6138.7±19.6237.7±21.8222.7±34.9
5.097.0±5.398.7±5.632.7±3.836.3±5.8133.7±21.57142.7±8.4234.7±42.2236.0±19.3
50.0106.7±9.899.0±7.335.0±3.337.0±4.0172.3±10.9135.0±6.7227.0±17.2230.7±30.4
Negative control99.7±5.899.3±3.829.7±1.433.3±1.8161.7±12.9156.7±7.8239.0±26.0228.3±14.4
Solvent control 98.5±6.296.4±5.834.2±2.335.6±5.2146.8±6.8152.1±6.5232.2±15.8229.3±15.0
Positive control 440.7±27.8244.7±14.476.3±12.479.3±13.1661.0±32.7423.3±24.4854.0±65.0615.7±42.4
Conclusions and Discussion
The proteinmodified E. faecalis was tested to be negative in the Ames test, indicating that the product has no mutagenic effect on the tested S. typhimurium mutant strains.
According to New Feed or Feed Additive Safety Evaluation Guide published by the Ministry of Agriculture, in addition to the Ames test, the mutagenicity tests must include mouse bone marrow micronucleus test and mouse sperm abnormality test. On the basis of these three tests, it is evaluated based on the test results whether other mutagenic tests such as the dominant lethal test are still needed［9］. Although the proteinmodified E. faecalis was tested to be negative by the Ames test in this study, at least the mouse bone marrow micronucleus test and the mouse sperm abnormality test are needed to evaluate its mutagenic effect.
References
［1］ MALIK RK, MONTECALVO MA, REALE MR, et a1. Epidemiology and control of vancomycinresistant enterococci in a regional neonatal intensive care unit［J］. Pediatr Infect Dis J, 1999, 18(4): 352．
［2］ DAVID FM, ARTHUR EB, GARY JN, et al. An investigation of vancomycinresistant enterocicci faecium within the pediatric service of a large urban medical center［J］. Pediatr Infect Dis J, 1998, 17(3): 184.
［3］ TANG XD. Enterococcal infection: a review［J］. Chinese Journal of Infection and Chemotherapy, 2007, 7(3): 221-223. (in Chinese)
［4］ BAO YE, DONG XF, TONG JM, et al. Evaluation of probiotic characteristics of Enterococcus faecalis in vitro［J］. Acta Agriculturae Borealioccidentalis Sinica, 2013, 22(11): 202-207. (in Chinese) ［5］ MA LY, XU SZ, MA JP. Pathogenesis of Enterococcus: A research progress［J］. Chinese Journal of Nosocomiology, 2005, 15(3): 356-360. (in Chinese)
［6］ SHI ZT, YAO YC, JIANG S, et al. Effects of Enterococcus faecalis substitute for antibiotic on growth performance, diarrhea rate, blood biochemical parameters and immune organs of weaner piglets［J］. Acta Zoonutrimenta Sinica, 2015, 27(6): 1832-1840. (in Chinese)
［7］ WEI QT, LI PH, WANG H, et al. Effect of dietary Enterococcus faecalis replacing of antibiotic on growth performance, diarrhea rate, humoral immunity and intestinal microflora of nursery pigs［J］. Journal of Nanjing Agricultural University, 2014, 37(6): 143-148. (in Chinese)
［8］ LIU S, DONG XF, TONG JM, et al. Effects of dietary Enterococcus faecalis on performance, egg quality, lipid metabolism and intestinal microflora numbers of laying hens［J］. Acta Zoonutrimenta Sinica, 2017, 29(1): 202-213. (in Chinese)
［9］ National Feed Judgment Committee Office. New feed or feed additive safety evaluation guide. www.chinafeed.org.cn. (in Chinese)