梨果采后病害新型拮抗菌分离、筛选及安全性研究
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摘要
水果在采摘、运销、以及储藏过程中因腐烂造成的损失是巨大的,也是全球关注的问题。在发达国家有10%-30%的新鲜产品由于采后病害导致腐烂,而在缺乏贮运冷链设备的发展中国家,果实采后腐烂率则高达40%-50%。
目前,最常用的有效控制水果腐烂的方法是低温和化学杀菌剂处理。近年来虽然冷藏技术在我国得到了广泛的发展,但是在低温贮藏情况下,仍有相当数量的病原菌可以在水果上面生长,从而引起腐烂。化学杀菌剂处理,虽然效果显著,但是在杀死病原菌的同时也杀死了有益微生物,且药物残留对人体健康和环境的危害很大。化学农药对环境的污染和由此带来的食品安全问题已成为当今世界各国关注的热点,用生物拮抗菌控制水果采后病害是替代或减少化学药剂使用的有效途径和发展方向。
本研究从多种途径分离筛选拮抗菌并确定其生物学分类地位,筛选到生防效果性能优良的菌种。研究了生物防治拮抗菌株的生物安全性,并对生防拮抗菌商品化制剂的生产工艺和保藏方法进行了初步的探讨。得到了如下的结论：
(1)通过体外试验和活体试验筛选,酵母菌Y20和Y21,对梨果灰霉病菌(Botrytis cinerea)、桃果青霉病菌(Penicillium expansum)两种病原菌表现出较好的抑菌效果。对Y20和Y21三株菌株进行形态特征、营养特征和生理特性试验以及DNA分子序列测定,鉴定Y20为胶红酵母(Rhodotorula mucilaginosa),Y21为接合酵母(Zygoascus hellenicus)。目前,尚未见到Y20和Y21两种酵母菌在采后病害生物防治应用中的报道。
(2)酵母菌Y20和Y21两株菌株的生长速度很快,没有明显的迟滞期。Y20和Y21两种拮抗酵母菌都能显著地抑制梨果的灰霉病和青霉病,在1×107CFU/mL、1×108CFU/mL和1×109CFU/mL的浓度下都能显著降低梨果的腐烂率,且酵母浓度越高效果越好。拮抗酵母在1×108CFU/mL浓度下对梨果灰霉病和青霉病具有稳定的生防效果。拮抗酵母Y20、Y21(浓度1×108CFU/mL)处理的梨果,在常温下(20℃)放置45d后,梨果实的发病率显著低于对照组,并且不会对梨果的贮藏品质造成不良的影响。
(3)急性经口毒性试验研究表明,胶红酵母Y20和接合酵母Y21经口急性毒性的MTD>21.5g/kg,根据GB15193.3-2003附录急性毒性剂量分级表的规定,均属无毒级。Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠精子畸形试验等遗传毒性试验表明,胶红酵母Y20和接合酵母Y21对小鼠无致畸、致突变作用。在30d喂养试验中,没有发现拮抗酵母对小鼠生长,血液常规指标、理化指标、生理功能以及主要器官组织(肝脏、脾脏、肾脏等)产生不良影响。
(4)用廉价的糖蜜为原料对Y20进行培养基的优化,均匀试验的结果表明以最优的培养基组合：糖蜜16.58%,硫酸镁0.05%,磷酸二氢钾0.05%,尿素0.3%培养酵母,可以得到最大生物量1.402g/100mL
(5)对Y20和Y21的冻干保藏制剂的研究表明,Y20和Y21在6个月内的活性可以保持在储藏前的75.6%以上。两株酵母冻干粉贮藏在4℃的条件下,可以在6个月内保持良好的生物活性。对Y20和Y21液体保藏制剂的研究表明,在室温20℃、4℃保存条件下的Y20菌悬液保存效果最好组为：10%海藻糖、0.0176%Vc和PBS混合保存液体。在室温20℃、4℃保存条件下的Y21菌悬液保存效果最好组为：5%海藻糖、0.0176%Vc和PBS混合保存液体。
(6)Y20和Y21保藏期内对梨果腐烂发病率的研究结果表明,无论采用冻干保藏还是液体保藏的菌体制剂,对于梨果的病害仍然具有良好的生防效果。
综上所述,拮抗酵母Y20和Y21作为梨果采后病害生物防治拮抗菌,为梨果采后病害生物防治提供了两株生防性能优良的菌株。通过对拮抗酵母Y20和Y21生防效果研究、安全性研究、商品化工艺的研究表明两株酵母的生物防治效果显著,安全无毒,具备商品化应用的潜质,为拮抗酵母Y20和Y21在水果采后生物保鲜技术的研究、菌株安全性研究以及商品化应用奠定了基础。
A huge loss has been made by spoilage in the course of fruit picking, distribution and storage, which is also globally concerned. In developed countries,10-30% fresh fruits perished caused by postharvest disease, while this number was up to 40-50% in developing countries, where the freezing storage and transportation facilities are limited.
At present, effective control methods of low temperature and synthetic fungicide treatment are most commonly used. Although refrigeration technology was widely developed in our country recent years, there was still a considerable number of pathogens could grow on fruits and result in decay under low temperature storage. Synthetic fungicide treatment was significantly effective, but it killed both of the pathogens and the beneficial microorganisms. Moreover, fungicide residues do great harm to human health and the environment. Environmental pollution caused by chemical pesticides and the resulting food safety problem has become the focus of attention around the world. To control postharvest diseases of the fruits and vegetables, antagonistic bacteria and yeasts has became an effective way to substitute or reduce the using of synthetic agents and has been proved to be the new developing trend.
In this research, the antagonistic bacteria and yeasts were isolated, screened and identified with various methods. The research has discovered strains with excellent biocontrol effect. The biological safety of the antagonistic strains was investigated and the production processes of their commercial preparation as well as the preservation method were preliminary discussed. Main conclusions of the research are as follows:
(1) The antagonistic yeast Y20 and Y21 were sieved by in virto and in vivo experiments which were tested to show preferable antibacterial effect to pathagens of Botrytis cinerea in pears and Penicillium expansum in peachs. Experiments on morphological characteristics, nutritional characteristics and physiological characteristics as well as the DNA molecule sequencing analysis of Y20 and Y21 were conducted. The results identified that Y20 is Rhodotorula mucilaginosa and Y21 is Zygoascus hellenicus. There is no report on these two kinds of yeasts (Y20, Y21) used in postharvest diseases control currently.
(2) The two strains of Y20 and Y21 grow up fast without apparent lagging period. The gray mold and blue mold in pome fruit were significantly inhibited by antagonistic yeasts Y20 and Y21. The fruits'disease incidence could be prominently reduced at the concentration of 1×107CFU/mL, 1×108CFU/mL and 1×109CFU/mL, and the higher the yeast concentration was the better the effect was. Antagonistic yeast B19 at the concentration of 1×108CFU/mL had a stable biocontrol effect on gray mold and blue mold in pome fruit. Treated by antagonistic yeasts Y20 and Y21(1×108CFU/mL), then stored at room temperature(20℃) for 45 days, the desease incidence of pome fruit was remarkablely lower than that of the control fruit, and had no adverse effect was found on storage quality of the fruits.
(3) Acute oral toxicity test indicated that the acute peroral toxicity MTD of yeastsY20 and Y21>21.5g/kg. Based on acute toxicity dose classification of the GB15193.3-2003 appendix, Y20 and Y21 both belonged to non-toxic level. Ames test, mouse bone marrow cells, micronucleus test, mouse sperm abnormality test and other genetic toxicity tests showed that Y20 and Y21 yeasts had no teratogenic and mutagenic effect to mice. In the 30-day feeding trial, no harmful effects of antagonistic yeas were found on mice's growth, normal blood index, physicochemical index, physiological functions and major organs and tissues (liver, spleen, kidney, etc.).
(4) Optimizeing Y20 medium using cheap molasses, uniform experimental result showed that using the optimal medium combination:16.58% of molasses, 0.05% of magnesium sulfate,0.05% of potassium dihydrogen phosphate and 0.3% of urea to conduct yeast culture could get the maximum biomass 1.402g/100mL.
(5) Research on freeze-dried agent of Y20 and Y21 indicated that the activity of Y20 and Y21 could be maintained over 75.6% within 6 months. Stored at 4℃, the biological activity of the two yeasts'freeze-dried powder could be well kept within 6 months. Research on Y20 and Y21's liquid agent showed that under the condition of 20℃and 4℃, the best storage group of Y20 was 10% of trehalose,0.0176% of Vc and PBS mixing liquid, and the best storage group of Y21 was 5% of trehalose, 0.0176% of Vc and PBS mixing liquid.
(6) During the storage period of Y20 and Y21, research on the rotten incidence of pome fruit indicated that both of the freeze-dried and liquid storage bacterial agents had a good effect on the diseases of pome fruit.
In summary, as postharvest disease biocontrol antagonists of pome fruit, antagonistic yeast Y20 and Y21 have significant and safe control effect. Research on biocontrol effect, safety, and commercialization technology indicated that the two antagonistic yests of Y20 and Y21 were effective, security and has the potential of commercial application. This research lay a good foundation for studies of postharvest biocontrol preservation, security of strains and commercialization of antagonistic yests of Y20 and Y21.
目前,最常用的有效控制水果腐烂的方法是低温和化学杀菌剂处理。近年来虽然冷藏技术在我国得到了广泛的发展,但是在低温贮藏情况下,仍有相当数量的病原菌可以在水果上面生长,从而引起腐烂。化学杀菌剂处理,虽然效果显著,但是在杀死病原菌的同时也杀死了有益微生物,且药物残留对人体健康和环境的危害很大。化学农药对环境的污染和由此带来的食品安全问题已成为当今世界各国关注的热点,用生物拮抗菌控制水果采后病害是替代或减少化学药剂使用的有效途径和发展方向。
本研究从多种途径分离筛选拮抗菌并确定其生物学分类地位,筛选到生防效果性能优良的菌种。研究了生物防治拮抗菌株的生物安全性,并对生防拮抗菌商品化制剂的生产工艺和保藏方法进行了初步的探讨。得到了如下的结论：
(1)通过体外试验和活体试验筛选,酵母菌Y20和Y21,对梨果灰霉病菌(Botrytis cinerea)、桃果青霉病菌(Penicillium expansum)两种病原菌表现出较好的抑菌效果。对Y20和Y21三株菌株进行形态特征、营养特征和生理特性试验以及DNA分子序列测定,鉴定Y20为胶红酵母(Rhodotorula mucilaginosa),Y21为接合酵母(Zygoascus hellenicus)。目前,尚未见到Y20和Y21两种酵母菌在采后病害生物防治应用中的报道。
(2)酵母菌Y20和Y21两株菌株的生长速度很快,没有明显的迟滞期。Y20和Y21两种拮抗酵母菌都能显著地抑制梨果的灰霉病和青霉病,在1×107CFU/mL、1×108CFU/mL和1×109CFU/mL的浓度下都能显著降低梨果的腐烂率,且酵母浓度越高效果越好。拮抗酵母在1×108CFU/mL浓度下对梨果灰霉病和青霉病具有稳定的生防效果。拮抗酵母Y20、Y21(浓度1×108CFU/mL)处理的梨果,在常温下(20℃)放置45d后,梨果实的发病率显著低于对照组,并且不会对梨果的贮藏品质造成不良的影响。
(3)急性经口毒性试验研究表明,胶红酵母Y20和接合酵母Y21经口急性毒性的MTD>21.5g/kg,根据GB15193.3-2003附录急性毒性剂量分级表的规定,均属无毒级。Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠精子畸形试验等遗传毒性试验表明,胶红酵母Y20和接合酵母Y21对小鼠无致畸、致突变作用。在30d喂养试验中,没有发现拮抗酵母对小鼠生长,血液常规指标、理化指标、生理功能以及主要器官组织(肝脏、脾脏、肾脏等)产生不良影响。
(4)用廉价的糖蜜为原料对Y20进行培养基的优化,均匀试验的结果表明以最优的培养基组合：糖蜜16.58%,硫酸镁0.05%,磷酸二氢钾0.05%,尿素0.3%培养酵母,可以得到最大生物量1.402g/100mL
(5)对Y20和Y21的冻干保藏制剂的研究表明,Y20和Y21在6个月内的活性可以保持在储藏前的75.6%以上。两株酵母冻干粉贮藏在4℃的条件下,可以在6个月内保持良好的生物活性。对Y20和Y21液体保藏制剂的研究表明,在室温20℃、4℃保存条件下的Y20菌悬液保存效果最好组为：10%海藻糖、0.0176%Vc和PBS混合保存液体。在室温20℃、4℃保存条件下的Y21菌悬液保存效果最好组为：5%海藻糖、0.0176%Vc和PBS混合保存液体。
(6)Y20和Y21保藏期内对梨果腐烂发病率的研究结果表明,无论采用冻干保藏还是液体保藏的菌体制剂,对于梨果的病害仍然具有良好的生防效果。
综上所述,拮抗酵母Y20和Y21作为梨果采后病害生物防治拮抗菌,为梨果采后病害生物防治提供了两株生防性能优良的菌株。通过对拮抗酵母Y20和Y21生防效果研究、安全性研究、商品化工艺的研究表明两株酵母的生物防治效果显著,安全无毒,具备商品化应用的潜质,为拮抗酵母Y20和Y21在水果采后生物保鲜技术的研究、菌株安全性研究以及商品化应用奠定了基础。
A huge loss has been made by spoilage in the course of fruit picking, distribution and storage, which is also globally concerned. In developed countries,10-30% fresh fruits perished caused by postharvest disease, while this number was up to 40-50% in developing countries, where the freezing storage and transportation facilities are limited.
At present, effective control methods of low temperature and synthetic fungicide treatment are most commonly used. Although refrigeration technology was widely developed in our country recent years, there was still a considerable number of pathogens could grow on fruits and result in decay under low temperature storage. Synthetic fungicide treatment was significantly effective, but it killed both of the pathogens and the beneficial microorganisms. Moreover, fungicide residues do great harm to human health and the environment. Environmental pollution caused by chemical pesticides and the resulting food safety problem has become the focus of attention around the world. To control postharvest diseases of the fruits and vegetables, antagonistic bacteria and yeasts has became an effective way to substitute or reduce the using of synthetic agents and has been proved to be the new developing trend.
In this research, the antagonistic bacteria and yeasts were isolated, screened and identified with various methods. The research has discovered strains with excellent biocontrol effect. The biological safety of the antagonistic strains was investigated and the production processes of their commercial preparation as well as the preservation method were preliminary discussed. Main conclusions of the research are as follows:
(1) The antagonistic yeast Y20 and Y21 were sieved by in virto and in vivo experiments which were tested to show preferable antibacterial effect to pathagens of Botrytis cinerea in pears and Penicillium expansum in peachs. Experiments on morphological characteristics, nutritional characteristics and physiological characteristics as well as the DNA molecule sequencing analysis of Y20 and Y21 were conducted. The results identified that Y20 is Rhodotorula mucilaginosa and Y21 is Zygoascus hellenicus. There is no report on these two kinds of yeasts (Y20, Y21) used in postharvest diseases control currently.
(2) The two strains of Y20 and Y21 grow up fast without apparent lagging period. The gray mold and blue mold in pome fruit were significantly inhibited by antagonistic yeasts Y20 and Y21. The fruits'disease incidence could be prominently reduced at the concentration of 1×107CFU/mL, 1×108CFU/mL and 1×109CFU/mL, and the higher the yeast concentration was the better the effect was. Antagonistic yeast B19 at the concentration of 1×108CFU/mL had a stable biocontrol effect on gray mold and blue mold in pome fruit. Treated by antagonistic yeasts Y20 and Y21(1×108CFU/mL), then stored at room temperature(20℃) for 45 days, the desease incidence of pome fruit was remarkablely lower than that of the control fruit, and had no adverse effect was found on storage quality of the fruits.
(3) Acute oral toxicity test indicated that the acute peroral toxicity MTD of yeastsY20 and Y21>21.5g/kg. Based on acute toxicity dose classification of the GB15193.3-2003 appendix, Y20 and Y21 both belonged to non-toxic level. Ames test, mouse bone marrow cells, micronucleus test, mouse sperm abnormality test and other genetic toxicity tests showed that Y20 and Y21 yeasts had no teratogenic and mutagenic effect to mice. In the 30-day feeding trial, no harmful effects of antagonistic yeas were found on mice's growth, normal blood index, physicochemical index, physiological functions and major organs and tissues (liver, spleen, kidney, etc.).
(4) Optimizeing Y20 medium using cheap molasses, uniform experimental result showed that using the optimal medium combination:16.58% of molasses, 0.05% of magnesium sulfate,0.05% of potassium dihydrogen phosphate and 0.3% of urea to conduct yeast culture could get the maximum biomass 1.402g/100mL.
(5) Research on freeze-dried agent of Y20 and Y21 indicated that the activity of Y20 and Y21 could be maintained over 75.6% within 6 months. Stored at 4℃, the biological activity of the two yeasts'freeze-dried powder could be well kept within 6 months. Research on Y20 and Y21's liquid agent showed that under the condition of 20℃and 4℃, the best storage group of Y20 was 10% of trehalose,0.0176% of Vc and PBS mixing liquid, and the best storage group of Y21 was 5% of trehalose, 0.0176% of Vc and PBS mixing liquid.
(6) During the storage period of Y20 and Y21, research on the rotten incidence of pome fruit indicated that both of the freeze-dried and liquid storage bacterial agents had a good effect on the diseases of pome fruit.
In summary, as postharvest disease biocontrol antagonists of pome fruit, antagonistic yeast Y20 and Y21 have significant and safe control effect. Research on biocontrol effect, safety, and commercialization technology indicated that the two antagonistic yests of Y20 and Y21 were effective, security and has the potential of commercial application. This research lay a good foundation for studies of postharvest biocontrol preservation, security of strains and commercialization of antagonistic yests of Y20 and Y21.
引文
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