口服异常毕赤酵母AR_(2016)对昆明小鼠生长性能和安全性的影响
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摘要
本试验旨在通过研究异常毕赤酵母AR_(2016)菌株对昆明小鼠生长性能、血清生化指标、血常规和免疫功能的影响,综合评价异常毕赤酵母AR_(2016)菌株的安全性。试验1:选择20只健康的昆明小鼠,随机分为2组,每组10只,雌雄各占1/2。按每10g体重最大灌胃容量0.2 mL进行7 d经口毒性试验,对照组灌胃生理盐水,试验组灌胃异常毕赤酵母AR_(2016)菌株发酵液(菌体浓度2×10~9CFU/mL)。试验2:选择40只健康的昆明小鼠,随机分为4组,每组10只,雌雄各占1/2。每10g体重最大灌胃容量0.2 mL进行28 d经口毒性试验,对照组灌胃生理盐水,低、中、高剂量组灌胃异常毕赤酵母AR_(2016)菌株发酵液,菌体浓度分别为2×10~7、2×10~8、2×10~9CFU/mL。结果表明:1)异常毕赤酵母AR_(2016)菌株处理7或28 d后,小鼠无明显中毒现象,未出现死亡,属"无毒"级(最大耐受剂量>20 000 mg/kg)。2)与对照组相比,异常毕赤酵母AR_(2016)菌株处理7 d后,试验组小鼠平均日采食量(ADFI)显著降低(P<0.05);异常毕赤酵母AR_(2016)菌株处理28 d后,各剂量组小鼠ADFI显著提高(P<0.05)。3)与对照组相比,异常毕赤酵母AR_(2016)菌株处理7 d后,试验组小鼠血清白蛋白、尿素氮含量显著降低(P<0.05),血清肌酐含量和谷草转氨酶活性显著提高(P<0.05);异常毕赤酵母AR_(2016)菌株处理28 d后,各组间血清生化指标无显著变化(P>0.05)。4)异常毕赤酵母AR_(2016)菌株处理7 d后,与对照组相比,试验组小鼠白细胞、红细胞压积及淋巴细胞数量显著提高(P<0.05),试验组雄性小鼠血红蛋白含量显著高于对照组雄性小鼠(P<0.05),试验组雄性小鼠红细胞数量显著高于试验组雌性小鼠(P<0.05),试验组雌性小鼠中性细胞比率显著低于对照组雌性小鼠(P<0.05),试验组雌性小鼠嗜酸性粒细胞数量显著高于对照组雌性小鼠(P<0.05);异常毕赤酵母AR_(2016)菌株处理28 d后,各组间血常规指标无显著变化(P>0.05)。5)与对照组相比,异常毕赤酵母AR_(2016)菌株处理7 d后,试验组小鼠血清中干扰素-γ(INF-γ)、肿瘤坏死因子-α含量显著提高(P<0.05);异常毕赤酵母AR_(2016)菌株处理28 d后,高剂量组雌性小鼠血清INF-γ含量显著提高(P<0.05)。综上,异常毕赤酵母AR_(2016)菌株对昆明小鼠无毒,具较高生物安全性,可提高昆明小鼠ADFI,增强机体免疫功能,具有一定的开发利用价值。
To comprehensively evaluate the bio-safety of Pichia anonmala AR_(2016) strain,this study was carried out to investigate effects of Pichia anonmala AR_(2016) strain on growth performance,serum biochemical indexes,hemogram indexes and immune function in Kunming mice. In experiment 1,a total of 20 Kunming mice were randomly assigned to one of two groups with 10 mice in each group (half male and half female). A 7-day oral toxicity test was performed at a dose of 0.2 mL per 10g body weight; control group supplemented with normal saline,and experimental group supplemented with Pichia anonmala AR_(2016) strain at the concentration of 2 ×10~9 CFU/mL. In experiment 2,a total of 40 Kunming mice were randomly assigned to one of four groups with 10 mice in each group (half male and half female). A 28-day oral toxicity test was performed at a dose of 0.2 mL per 10g body weight; control group supplemented with normal saline,lowdose group,middle dose group and high dose group supplemented with Pichia anonmala AR_(2016) strain at concentrations of 2 × 10~7,2×10~8,2×10~9 CFU/mL,respectively. The results showed as follows: 1) after 7 or 28 d treatment by Pichia anonmala AR_(2016) strain,no poisoning and death were found in mice,it was a"non-toxic"grade,and the maximum tolerated dose was more than 20 000 mg/kg. 2) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,average daily feed intake (ADFI) of mice in experimental group significantly decreased (P<0. 05); after 28 d treatment by Pichia anonmala AR_(2016) strain,ADFI of mice in each dosage group significantly increased (P < 0. 05). 3) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,the contents of albumin and urea nitrogen in serum of mice in experimental group significantly decreased (P < 0. 05),and serum creatinine content and aspartate transaminase activity significantly increased (P<0.05); after 28 d treatment by Pichia anonmala AR_(2016) strain,serum biochemical indexes among groups had no significant difference (P > 0. 05). 4) After 7 d treatment by Pichia anonmala AR_(2016) strain,compared with the control group,white blood cell,hematokrit,lymphocytes count of mice in experimental group significantly increased (P < 0.05),hemoglobin content of male mice in experimental group was significantly higher than that of male mice in control group (P < 0. 05),red blood cell count of male mice in experimental group was significantly higher than that of female mice in experimental group (P<0.05),neutral cell rate of female mice in experimental group was significantly lower than that of female mice in control group (P<0.05),and count of eosinophils of female mice in experimental group was significantly higher than that of female mice in control group (P<0.05); after 28 d treatment by Pichia anonmala AR_(2016) strain,blood routine indexes among groups had no significant difference (P>0.05). 5) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,the content of interferon-γ (INF-γ) and tumor necrosis factor-α in serum of mice in experimental group significantly increased (P< 0. 05); after 28 d treatment by Pichia anonmala AR_(2016) strain,INF-γ content in serum of female mice in high dose group significantly increased (P <0.05). Therefore,the Pichia anomala AR_(2016) strain is non-toxic and high safety to Kunming mice,can increase ADFI,improve immune function,and having the value for further research and development.
To comprehensively evaluate the bio-safety of Pichia anonmala AR_(2016) strain,this study was carried out to investigate effects of Pichia anonmala AR_(2016) strain on growth performance,serum biochemical indexes,hemogram indexes and immune function in Kunming mice. In experiment 1,a total of 20 Kunming mice were randomly assigned to one of two groups with 10 mice in each group (half male and half female). A 7-day oral toxicity test was performed at a dose of 0.2 mL per 10g body weight; control group supplemented with normal saline,and experimental group supplemented with Pichia anonmala AR_(2016) strain at the concentration of 2 ×10~9 CFU/mL. In experiment 2,a total of 40 Kunming mice were randomly assigned to one of four groups with 10 mice in each group (half male and half female). A 28-day oral toxicity test was performed at a dose of 0.2 mL per 10g body weight; control group supplemented with normal saline,lowdose group,middle dose group and high dose group supplemented with Pichia anonmala AR_(2016) strain at concentrations of 2 × 10~7,2×10~8,2×10~9 CFU/mL,respectively. The results showed as follows: 1) after 7 or 28 d treatment by Pichia anonmala AR_(2016) strain,no poisoning and death were found in mice,it was a"non-toxic"grade,and the maximum tolerated dose was more than 20 000 mg/kg. 2) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,average daily feed intake (ADFI) of mice in experimental group significantly decreased (P<0. 05); after 28 d treatment by Pichia anonmala AR_(2016) strain,ADFI of mice in each dosage group significantly increased (P < 0. 05). 3) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,the contents of albumin and urea nitrogen in serum of mice in experimental group significantly decreased (P < 0. 05),and serum creatinine content and aspartate transaminase activity significantly increased (P<0.05); after 28 d treatment by Pichia anonmala AR_(2016) strain,serum biochemical indexes among groups had no significant difference (P > 0. 05). 4) After 7 d treatment by Pichia anonmala AR_(2016) strain,compared with the control group,white blood cell,hematokrit,lymphocytes count of mice in experimental group significantly increased (P < 0.05),hemoglobin content of male mice in experimental group was significantly higher than that of male mice in control group (P < 0. 05),red blood cell count of male mice in experimental group was significantly higher than that of female mice in experimental group (P<0.05),neutral cell rate of female mice in experimental group was significantly lower than that of female mice in control group (P<0.05),and count of eosinophils of female mice in experimental group was significantly higher than that of female mice in control group (P<0.05); after 28 d treatment by Pichia anonmala AR_(2016) strain,blood routine indexes among groups had no significant difference (P>0.05). 5) Compared with the control group,after 7 d treatment by Pichia anonmala AR_(2016) strain,the content of interferon-γ (INF-γ) and tumor necrosis factor-α in serum of mice in experimental group significantly increased (P< 0. 05); after 28 d treatment by Pichia anonmala AR_(2016) strain,INF-γ content in serum of female mice in high dose group significantly increased (P <0.05). Therefore,the Pichia anomala AR_(2016) strain is non-toxic and high safety to Kunming mice,can increase ADFI,improve immune function,and having the value for further research and development.
引文
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[2]IZGF,ALTLNBAY D,ACUN T.Killer toxin of Pichia anomala NCYC 432;purification,characterization and its exo-β-1,3-glucanase activity[J].Enzyme and M icrobial Technology,2006,39(4):669-676.
[3]DE MELO E A,DE LIMA R.MARIANO R,LARANJEIRA D,et al.Efficacy of yeast in the biocontrol of bacterial fruit blotch in melon plants[J].Tropical Plant Pathology,2015,40(1):56-64.
[4]OLSTORPE M,PASSOTH V.Pichia anomala in grain biopreservation[J].Antonie van Leeuw enhoek,2011,99(1):57-62.
[5]孙怀永.异常毕赤酵母(Pichia anomala)HN1-2菌株产单细胞蛋白和嗜杀因子的研究[D].硕士学位论文.青岛:中国海洋大学,2012.
[6]秦艳青,任大勇,李诗语,等.益生菌安全性及风险评估研究进展[J].中国兽药杂志,2012,46(4):51-54.
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[10]中华人民共和国国家质量监督检验疫总局.GB14924.3-2001实验动物小鼠大鼠配合饲料[S].北京:中国标准出版社,2002.
[11]王洪亮.微生态制剂在畜禽饲料中的合理使用[J].畜牧兽医科技信息,2017(5):133.
[12]BALZAN S,DE ALMEIDA QUADROS C,DE CLE-VA R,et al.Bacterial translocation:overview of mechanisms and clinical impact[J].Journal of Gastroenterology and Hepatology,2007,22(4):464-471.
[13]FOUTS D E,TORRALBA M,NELSON K E,et al.Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease[J].Journal of Hepatology,2012,56(6):1283-1292.
[14]石陆娥,应国清,唐振兴,等.酵母的开发利用研究进展[J].中国食品添加剂,2006(5):62-65,104.
[15]VAN HEUGTEN E,FUNDERBURKE D W,DORTON K L.Grow th performance,nutrient digestibility,and fecal microflora in w eanling pigs fed live yeast[J].Journal of Animal Science,2003,81(4):1004-1012.
[16]WALKER G M.Pichia anomala:cell physiology and biotechnology relative to other yeasts[J].Antonie van Leeuw enhoek,2011,99(1):25-34.
[17]王婷婷.微生物饲料添加剂的研究进展[J].养殖技术顾问,2014(10):72-73.
[18]魏娜.肝功能中各种蛋白测定的临床意义[J].医学美学美容,2015(5):183.
[19]CHEN S L,XUE N,WU M T,et al.Influence of preoperative serum aspartate aminotransferase(AST)level on the prognosis of patients w ith non-small cell lung cancer[J].International Journal of M olecular Sciences,2016,17(9):1474.
[20]曹雪涛.医学免疫学[M].6版.北京:人民卫生出版社,2013.
[21]裘纪莹,陈相艳,杨金玉,等.解淀粉芽孢杆菌NCPSJ7对小鼠的急性毒性评价[J].食品科技,2017,42(4):13-16.
[22]李文明,谷医林,王远宏,等.解淀粉芽孢杆菌LJ1对实验鼠的急性毒性研究[J].农药学学报,2013,15(4):434-438.
[23]高林,白子金,冯波,等.微生物饲料添加剂研究与应用进展[J].微生物学杂志,2014,34(2):1-6.
[24]KOTZAMANIDIS C,KOURELIS A,LITOPOULOU-TZANETAKI E,et al.Evaluation of adhesion capacity,cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains[J].International Journal of Food M icrobiology,2010,140(2/3):154-163.
[2]IZGF,ALTLNBAY D,ACUN T.Killer toxin of Pichia anomala NCYC 432;purification,characterization and its exo-β-1,3-glucanase activity[J].Enzyme and M icrobial Technology,2006,39(4):669-676.
[3]DE MELO E A,DE LIMA R.MARIANO R,LARANJEIRA D,et al.Efficacy of yeast in the biocontrol of bacterial fruit blotch in melon plants[J].Tropical Plant Pathology,2015,40(1):56-64.
[4]OLSTORPE M,PASSOTH V.Pichia anomala in grain biopreservation[J].Antonie van Leeuw enhoek,2011,99(1):57-62.
[5]孙怀永.异常毕赤酵母(Pichia anomala)HN1-2菌株产单细胞蛋白和嗜杀因子的研究[D].硕士学位论文.青岛:中国海洋大学,2012.
[6]秦艳青,任大勇,李诗语,等.益生菌安全性及风险评估研究进展[J].中国兽药杂志,2012,46(4):51-54.
[7]SUNDH I,MELIN P.Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain[J].Antonie van Leeuw enhoek,2011,99(1):113-119.
[8]中华人民共和国国家卫生和计划生育委员会.GB15193.3-2014食品安全国家标准急性经口毒性试验[S].北京:中国标准出版社,2015.
[9]中华人民共和国国家卫生和计划生育委员会.GB15193.22-2014食品安全国家标准28天经口毒性试验[S].北京:中国标准出版社,2015.
[10]中华人民共和国国家质量监督检验疫总局.GB14924.3-2001实验动物小鼠大鼠配合饲料[S].北京:中国标准出版社,2002.
[11]王洪亮.微生态制剂在畜禽饲料中的合理使用[J].畜牧兽医科技信息,2017(5):133.
[12]BALZAN S,DE ALMEIDA QUADROS C,DE CLE-VA R,et al.Bacterial translocation:overview of mechanisms and clinical impact[J].Journal of Gastroenterology and Hepatology,2007,22(4):464-471.
[13]FOUTS D E,TORRALBA M,NELSON K E,et al.Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease[J].Journal of Hepatology,2012,56(6):1283-1292.
[14]石陆娥,应国清,唐振兴,等.酵母的开发利用研究进展[J].中国食品添加剂,2006(5):62-65,104.
[15]VAN HEUGTEN E,FUNDERBURKE D W,DORTON K L.Grow th performance,nutrient digestibility,and fecal microflora in w eanling pigs fed live yeast[J].Journal of Animal Science,2003,81(4):1004-1012.
[16]WALKER G M.Pichia anomala:cell physiology and biotechnology relative to other yeasts[J].Antonie van Leeuw enhoek,2011,99(1):25-34.
[17]王婷婷.微生物饲料添加剂的研究进展[J].养殖技术顾问,2014(10):72-73.
[18]魏娜.肝功能中各种蛋白测定的临床意义[J].医学美学美容,2015(5):183.
[19]CHEN S L,XUE N,WU M T,et al.Influence of preoperative serum aspartate aminotransferase(AST)level on the prognosis of patients w ith non-small cell lung cancer[J].International Journal of M olecular Sciences,2016,17(9):1474.
[20]曹雪涛.医学免疫学[M].6版.北京:人民卫生出版社,2013.
[21]裘纪莹,陈相艳,杨金玉,等.解淀粉芽孢杆菌NCPSJ7对小鼠的急性毒性评价[J].食品科技,2017,42(4):13-16.
[22]李文明,谷医林,王远宏,等.解淀粉芽孢杆菌LJ1对实验鼠的急性毒性研究[J].农药学学报,2013,15(4):434-438.
[23]高林,白子金,冯波,等.微生物饲料添加剂研究与应用进展[J].微生物学杂志,2014,34(2):1-6.
[24]KOTZAMANIDIS C,KOURELIS A,LITOPOULOU-TZANETAKI E,et al.Evaluation of adhesion capacity,cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains[J].International Journal of Food M icrobiology,2010,140(2/3):154-163.