Effects of Water Activity and Temperature on Growth and Toxin-producing of Aspergillus flavus MI18-S1 on Tartary Buckwheat Grains
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摘要:
目的 探究不同水活度和温度对苦荞成熟种子中黄曲霉菌株生长及产毒的影响，明确荞麦中黄曲霉生长及产毒的适宜条件，为荞麦在收获、加工、运输和储藏各环节的科学管理提供数据支撑。 方法 从贵米苦荞18-1号成熟种子中分离出黄曲霉菌株MI18-S1，通过添加不同量的甘油调节培养基水活度（0.87、0.90、0.93、0.95、0.99），分别置于不同温度（15、21、28、35、42℃）下培养7 d。测量菌落直径，用血球计数板计算产孢量，用高效液相色谱（HPLC）法测定菌株产毒量。 结果 黄曲霉菌株MI18-S1能产毒B1、B2，以B1为主，但不产毒G1和G2；当温度为28℃、水活度为0.93时，该菌株的产孢量和产毒量均最大；当温度高于28℃时，菌落在高水活度下生长较好；当温度低于15℃或高于42℃时生长速率很缓慢甚至不生长、也不产毒；在15～42℃内，产毒量随着温度的升高呈先上升后下降趋势。多元线性回归分析结果表明，菌落直径对产毒量的线性影响不显著，产毒量主要受水活度、温度和产孢量这3个因素影响，并且产孢量对产毒量的影响最大；产孢量主要受水活度、温度、产毒量和菌落直径这4个因素影响，并且产毒量对产孢量影响最大。 结论 当水活度为0.93、温度28 ℃时黄曲霉产孢产毒量均达到最大，因此在荞麦生产链的很多环节，尤其是储藏阶段，应尽量避免此最适条件，从而控制荞麦的产孢产毒量，保障食品健康。本研究可为制定荞麦收获、加工、运输和储藏时黄曲霉污染的防控措施提供理论依据和数据支撑。 Abstract:Objective Effects of water activity (aw) and temperature on the growth and toxicity of Aspergillus flavus on mature grains of tartary buckwheat were studied to determine the safety conditions for harvest, processing, transportation, and storage of the crop. Method Varied medium aw (i.e., 0.87, 0.90, 0.93, 0.95 and 0.99) were created by additions of glycerol to culture A. flavus MI18-S1 isolated from the infected grains for a 7-day test at 15, 21, 28, 35 or 42 ℃. Colony diameter on petri dish was measured, spore count calculated from the blood cell counter board, and aflatoxin content determined by HPLC. Result The fungus was found to produce aflatoxins B1 and B2, predominately B1, but not G1 or G2. The highest spore count and toxicity were generated at 28 ℃ and aw=0.93. When the temperature was above 28 ℃, the colony could grow better under high water activity. At a temperature either below 15℃ or above 42 ℃, the fungal growth and toxin-production slowed or ceased. Within the range between these temperatures, the aflatoxin content increased at first and followed by a decline. A multivariate linear regression analysis showed no significant correlation between the colony diameter and the toxin production. Instead, the amount of aflatoxin generated in the culture was mainly affected by the aw and temperature of the environment, and most critically, sporulation of the fungus. And, sporulation of the fungus was significantly affected by the aw, temperature of the environment, the amount of aflatoxin generated in the culture and the colony diameter. Conclusion The growth and aflatoxin production of A. flavus on buckwheat grains were at peak when stored at 28 ℃ on a medium with aw of 0.93. Therefore, in many aspects of the buckwheat production chain, especially during the storage stage, it is advisable to avoid these optimal conditions in order to control the spore and toxin production of buckwheat, ensuring food safety. This study can provide a theoretical basis and data support for the development of preventive and control measures against Aspergillus flavus contamination during buckwheat harvesting, processing, transportation, and storage. -
Key words:
- Buckwheat /
- aflatoxin /
- temperature /
- water activity /
- sporulation quantity /
- toxin production /
- HPLC
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图 2 不同水活度和温度对黄曲霉菌落生长的影响
Figure 2. Effect of medium aw and culture temperature on diameter of A. flavus colonies
图 3 温度与产孢量的曲线拟合
Figure 3. Curve fitting for culture temperature and sporulation of A. flavus
表 1 黄曲霉毒素混合标准溶液的线性回归方程
Table 1. Linear regression equation of aflatoxin mixed standard solution
黄曲霉毒素混合标准品
Aflatoxin mixed
standard线性回归方程
Equation of linear
regression相关系数
Correlation
coefficent线性范围
Linearity
rangeAFB1 y=24 739.5x−939.92 0.999 6 0～4.0 AFB2 y=54 916.41x−469.89 0.999 9 0～4.0 AFG1 y=14 664.22x−691.64 0.999 8 0～4.0 AFG2 y=51 585.69x−49.80 0.999 9 0～4.0 AFB1、AFB2、AFG1、AFG2分别为黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2。
AFB1, AFB2, AFG1, AFG2 are aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, respectively.
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表 2 不同温度及水活度对荞麦中黄曲霉菌株产孢的影响
Table 2. Effect of different temperatures and water activity on sporulation of Aspergillus flavus in buckwheat
温度
Temperature/℃产孢量 Sporulation quantity/(×106个·mL−1) aw=0.87 aw=0.90 aw=0.93 aw=0.95 aw=0.99 15 0 Dd 0 Dc 0.20±0.03 Cc 0.98±0.05 Be 1.92±0.02 Ae 21 0 Dd 0.42±0.07 Bc 0.43±0.02 Bc 1.70±0.06 Cd 2.40±0.07 Ad 28 2.40±0.05 Eb 3.23±0.16 Db 14.80±0.13 Aa 10.80±0.22 Ba 8.25±0.08 Cc 35 1.13±0.07 Ec 3.80±0.07 Db 5.80±0.03 Cb 7.40±0.03 Bb 10.00±0.02 Aa 42 4.10±0.05 Ea 4.39±0.07 Da 5.70±0.12 Bb 5.23±0.07 Cc 9.22±0.06 Ab 同行数据后不同大写字母表示相同温度不同水活度之间产孢量差异显著（P<0.05）；同列数据后不同小写字母表示相同水活度不同温度下产孢量差异显著（P<0.05）。表3同。
Different uppercase letters at the same line indicate significant differences between different water activities at the same temperature (P<0.05)；Different lowercase letters in the same column indicate significant differences in sporulation between different temperatures at the same water activity (P<0.05). Same for Table 3.
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表 3 不同温度水活度对荞麦中黄曲霉菌株产毒的影响
Table 3. Effects of different temperatures and water activity on the toxicity of Aspergillus flavus strains in buckwheat
毒素
Toxin温度
Temperature产毒量 Toxigenic quantity/(µg·kg−1) aw=0.87 aw=0.90 aw=0.93 aw=0.95 aw=0.99 B1 15 0 c 0 c 0 d 0 e 0 e 21 0 Dc 0.27±0.01 Bb 0.25±0.04 Cb 0.33±0.01 Ab 0.27±0.01 Bb 28 0.40±0.02 Db 0.40±0.01 Da 2.71±0.09 Aa 0.71±0.04 Ba 0.56±0.03 Ca 35 0.82±0.04 Aa 0.27±0.01 Bb 0.19±0.01 Dc 0.18±0.02 Dc 0.21±0.01 Cc 42 0 Cc 0 Cc 0 Cd 0.09±0.01 Bd 0.12±0.02 Ad
B215 0 b 0 c 0 c 0 c 0 c 21 0 Cb 0.04±0.01 Aa 0.03±0.01 Bb 0 Cc 0.03±0.01 Ba 28 0.03±0.01 Ba 0.03±0.01 Bb 0.04±0.02 Aa 0.01±0.01 Cb 0 Dc 35 0 Bb 0.03±0.01 Ab 0.03±0.01 Ab 0 Bc 0.03±0.01 Aa 42 0 Cb 0 Cc 0 Cc 0.03±0.01 Aa 0.02±0.01 Bb
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