[關(guān)鍵詞]
[摘要]
目的 探討淡豆豉炮制過程中環(huán)境因素對黃曲霉毒素(AFs)含量的影響。方法 依據(jù)《中國藥典》2020年版記載的淡豆豉制法,設置不同的溫度、濕度、是否水封“再悶”環(huán)境條件下的6個組進行自然炮制,檢測各組炮制至不同時間豆粒的溫度、水分活度(AW)、含氧量;采用超高效液相色譜-串聯(lián)質(zhì)譜法(UPLC-MS/MS)檢測各組在“黃衣上遍”第3天(F3)、第6天(F6)和“再悶”第6天(Z6)、第12天(Z12)樣品中AFB1、AFB2、AFG1、AFG2含量,分析淡豆豉自然炮制中影響AFs產(chǎn)生的主要環(huán)境因素;模擬淡豆豉自然炮制,通過純培養(yǎng)考察炮制原料、“再悶”環(huán)境(低含氧量)對產(chǎn)毒黃曲霉菌標準株(簡稱:產(chǎn)毒株)產(chǎn)AFB1的影響,實時熒光定量PCR(qRT-PCR)法檢測產(chǎn)毒株產(chǎn)毒關(guān)鍵基因(aflR、aflD、aflM、aflO、aflQ) mRNA相對表達量。結(jié)果 自然炮制過程中濕度與AFs含量呈顯著性相關(guān)(r=0.435,P<0.05),且組6(濕度為90%)中F6、Z6、Z12的AFs含量均最高(分別為159.11、45.43、8.78 μg·kg-1);純培養(yǎng)實驗中,產(chǎn)毒株在黑豆基質(zhì)上比馬鈴薯葡萄糖肉湯(PDB)培養(yǎng)基上的產(chǎn)毒量減少95.31%及以上,在密封罐內(nèi)培養(yǎng)比罐外培養(yǎng)的產(chǎn)毒量減少99.93%及以上,均以aflM、aflO、aflQ mRNA表達下調(diào)更顯著(P<0.001)。結(jié)論 淡豆豉自然炮制過程中濕度是影響AFs含量的主要因素,炮制原料、“再悶”環(huán)境可顯著降低產(chǎn)毒株的產(chǎn)毒量。
[Key word]
[Abstract]
Objective The effects of environmental factors on the content of aflatoxins (AFs) in the processing of Sojae Semen Preaparatum (SSP) were investigated. Methods According to the 2020 edition of Chinese Pharmacopoeia, six groups were naturally processed under different temperature, humidity, and whether they were sealed with water or not in the process of the “secondary fermentation” stage. Conventional methods were used to detect the temperature, water activity (aw), and oxygen content of each group at different processing times. The contents of AFs (AFB1, AFB2, AFG1, AFG2) in the samples of each group on the 3rd day (F3) and the 6th day (F6) of the "yellow cladding" stage, the 6th day (Z6) and the 12th day (Z12) of the "secondary fermentation" stage were detected by method of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The main environmental factors affecting the production of AFs in natural processing of SSP were analyzed. Simulating the natural processing of SSP, the effects of processing raw materials and the environment of the "secondary fermentation" stage on the production of AFB1 by the standard strain of Aspergillus flavus (abbreviated as toxigenic strain) were investigated through pure culture. Real -time quantitative PCR (RTqPCR) was used to detect the mRNA expression levels of the key genes aflR, aflD, aflM, aflO and aflQ of toxigenic strains. Results There was a significantly positive correlation between humidity and AFs content during natural processing (r = 0.435, P < 0.05), and the AFs content of F6, Z6 and Z12 in group 6 (humidity of 90%) was the highest (159.11, 45.43 and 8.78 μg·kg-1, respectively). In pure culture experiments, the toxin amount produced by toxigenic strain was reduced by 95.31% or more on black bean substrate compared to PDB medium; and by 99.93% or more in sealed jar compared to out of jar culture. The mRNA expression of aflM, aflO, and aflQ was significantly inhibited (P < 0.001). Conclusion Humidity was the main factor affecting the content of AFs during the natural processing of SSP. Processing raw material and the environment of the "secondary fermentation" stage can significantly reduce the amount of toxic production.
[中圖分類號]
R283.3
[基金項目]
國家自然科學基金資助項目(82360772);國家自然科學基金資助項目(82060709);國家自然科學基金資助項目(82060699);南昌大學香樟育才計劃;江西省自然科學基金資助項目(20242BAB26173);江西省自然科學基金資助項目(20232BAB206161)