浓缩苹果汁中棒曲霉毒素的降解及苹果多酚抗肿瘤作用的研究
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摘要
我国的苹果产量居世界首位,浓缩苹果汁是其主要加工形式,具有很强的出口创汇能力。但近年来,浓缩苹果汁中棒曲霉毒素含量超标已成为影响我国浓缩苹果汁出口的瓶颈之一。因此,提高棒曲霉毒素的检测能力并进行有效的控制是当前一项非常重要的工作。扩展青霉是浓缩苹果汁生产过程中棒曲霉毒素的主要产生菌,对浓缩苹果汁的质量有直接影响。
苹果多酚是苹果中多元酚类物质的总称,是最新型高效抗氧化剂,也是目前发现的最为强效的自由基清除剂之一。它热稳定性好,在pH2-10范围内,120℃仍不失活,吸收迅速、完全,口服20min即可达到最高血液浓度,代谢半衰期达7h之久。实验证明,其多种生理活性比茶多酚高100倍,主要功能有：预防龋齿,抑制人唾液产生口臭成分(四硫醇),抑制鱼臭(三四基胺)的挥发；抑制酪氨酸酶和黑色素的生成；抑制ACE的活性,具有平稳的降压作用；抗过敏、预防心血管疾病等。
本研究从浓缩苹果汁中棒曲霉毒素的检测入手,研究和改进现有的棒曲霉毒素的检测方法；系统考察与分析了棒曲霉毒素的产生过程,并研究其降解机制；针对苹果多酚的抗氧化性,对其抗肿瘤作用效果及机理进行了较为深入的研究,主要研究结果如下：
(1)采用高效液相色谱法测定浓缩苹果汁中棒曲霉毒素的含量,可操作性强,方法准确度可靠,回收率高达98%-103%以上,结果精密度高,重现性好,RSD为<1.8%,灵敏度高,对于5mL的浓缩苹果汁,其检测极限为10μg／L,可作为广大浓缩苹果汁生产厂测定苹果汁中棒曲霉毒素含量的准确定量方法。
(2)在静置培养和摇床培养中,发酵液中棒曲霉毒素含量均出现先上升后下降的趋势；菌体生长在静置培养中出现明显的二次增长,摇床培养生长趋势不明显。发酵液中的残糖和pH呈整体下降趋势,总酸呈整体上升趋势。培养过程中,糖的消耗主要用于菌体生长和棒曲霉毒素的合成；扩展青霉在生长和产毒过程中能够分泌酸性物质。
(3)静置培养中,当菌体干重进入第一次稳定期,孢子开始形成时,棒曲霉毒素达到一个相对平缓的最高平台；当菌体量进入第二次快速增长期时,棒曲霉毒素开始降解；后期菌体量下降时,棒曲霉毒素迅速下降。摇床培养的菌体量是静置培养的2倍之多,但棒曲霉毒素产量不到静置培养的1／2；菌体量处于稳定期的中期时,棒曲霉毒素开始迅速下降。由此说明,棒曲霉毒素的合成条件与扩展青霉的孢子形成条件有一定的相似性；氧气利于菌丝体的生长,但不利于棒曲霉毒素的合成。
(4)在棒曲霉毒素的生长过程中,添加不同剂量的乳酸菌可使其吸附生长的棒曲霉毒素菌株；在发酵早期,还可控制棒曲霉毒素的生长。研究结果表明,添加500mg／L的乳酸菌对棒曲霉毒素的生长可以起到较好的抑制作用,同时,对浓缩苹果汁的产品品质也不会产生影响。
(5)苹果多酚在体外对H22、HT29瘤株具有直接杀伤作用且具有剂量-效应相关性；苹果多酚可抑制H22细胞增殖；苹果多酚还可通过抑制细胞DNA合成,降低细胞周期中S期百分率,从而使细胞停滞于G2/M期,从而减少分裂期细胞而发挥抗癌作用。
(6)苹果多酚在体内对H22移植性肿瘤具有良好的抑瘤作用,但其剂量-效应关系不明确；对于荷EAC小鼠的生存时间无显著影响；对于荷瘤小鼠的免疫功能影响不大。
China's apple production ranks the first in the world. However, in recent years, our export of the concentrated apple juice has been restricted mainly because the amount of Patulin in the juice exceeds the standard. Therefore, it is rather important to improve the detecting ability and to control effectively the amount of Patulin in the juice as well. As the main producing strain in the production of concentrated apple juice, Penicillium expansum affects the quality of concentrated apple juice directly.
The phenolic substances in the apple are generally called Apple Polyphenols, which is the latest potent antioxidant and one of the most powerful free radical scavengers that have been found. Owing to the high thermal stability, within the pH2-10 at 120℃it can still be alive and absorbed quickly and completely. So that it can reach the maximum blood concentration 20 minutes after taken orally with the help of the 7-hour half-life of metabolism. Experimental results show that many of its physiological activity are 100 times higher than the tea polyphenols. The main functions are to prevent tooth decay, to depress bad breath components in saliva production (4 mercaptan), to inhibit the volatile of fish odor (34 amine); to decrease the production of melanin, to restrain tyrosinase function; to reduce the ACE activity, to reduce blood pressure placidly, to relieve hypersensitivity, and to prevent cardiovascular disease, etc.
The study starts from the detection of Patulin in the concentrated apple juice, conducts a thorough research of the recent detecting method. After the systematical research of the production and degradation of Patulin, and the systematical analysis of the antitumor effect of the Apple Polyphenols, major findings are as follows:
(1) To detect the amount of the Patulin in the concentrated apple juice, High Performance Liquid Chromatography (HPLC) is operational, accurate and reliable, with the recovery rate as high as 98% to 103%, and the results are of high precision, good reproducibility, and high sensitivity, with the RSD < 1.8%. For 5mL of concentrated apple juice, the detection limit was 10μg/L, which can be taken as an accurate quantitative method for the apple juice producer.
(2) In both batchs of static and shaking cultivations, patulin content increased firstly and then decreased. A clear second increase of cell growth appeared in the static cultivation, but not in the shaking cultivation. In total, residue sugar and pH kept decreasing and total titrable acids kept increasing through the whole cultivation in both static and shaking cultivations. During the cultivation, sugar was consumed for the cell growth and sythesis of patulin. Some kinds of acidic substance were produced in the cell growth and patulin sythesis.
(3) In the batch of static cultivation, the period for the highest patulin production was consistent with the first high stable period for cell growth, in which spores of the P. Expansum started to form. Patulin reduced quickly from when the second quick cell growth happened in the static cultivation and in the middle period of the stable cell growth in the shaking cultivation. Patulin reduction also happened at the later cultivation stage when cells appeared decrease. Compared with the static cultivation, the dried cell weight was about 2 times more than, but the patulin production was 1/2 lower than, that in the static cultivation. This indicated conditions for patulin production might be consistent with that for spore formation. Abundant oxygen would be good for the cell growth of P. expansum, but not for the patulin production.
(4) It is founded that lactic acid bacteria can absorb the growth of Aspergillus toxins by adding different doses of lactic acid during the growth of patulin, the growth of patulin can be controlled in the early fermentation. The results showed that it can play a better inhibition by addition of 500mg/L of lactic acid bacteria on the growth of Aspergillus clavatus toxins, at the same time, it is not impact the quality of concentrated apple juice products.
(5) There is tumor-inhibitory effect in vivo of apple polephenols to H22, but no dose-dependent relation in the groups of H22. There is no influence on the survival periods by apple polephenols to EAC-receiving mice. There is no influence on the immunological function in tumor-receiving mice treated with apple polephenols.
(6) There is the direct kill function in vitro of apple polephenols to H22, HT29 lump, and the dose-dependent relation in the groups of H22, HT29 lump. The results showed that Apple polyphenols can inhibit the H22 cell proliferation. Through inhibiting cell DNA synthesis, reduces the S phase percentage in the mitotic cycle, induce G2/M phase arrest, plays the antitumoral effect through the reduced fission time cell.
苹果多酚是苹果中多元酚类物质的总称,是最新型高效抗氧化剂,也是目前发现的最为强效的自由基清除剂之一。它热稳定性好,在pH2-10范围内,120℃仍不失活,吸收迅速、完全,口服20min即可达到最高血液浓度,代谢半衰期达7h之久。实验证明,其多种生理活性比茶多酚高100倍,主要功能有：预防龋齿,抑制人唾液产生口臭成分(四硫醇),抑制鱼臭(三四基胺)的挥发；抑制酪氨酸酶和黑色素的生成；抑制ACE的活性,具有平稳的降压作用；抗过敏、预防心血管疾病等。
本研究从浓缩苹果汁中棒曲霉毒素的检测入手,研究和改进现有的棒曲霉毒素的检测方法；系统考察与分析了棒曲霉毒素的产生过程,并研究其降解机制；针对苹果多酚的抗氧化性,对其抗肿瘤作用效果及机理进行了较为深入的研究,主要研究结果如下：
(1)采用高效液相色谱法测定浓缩苹果汁中棒曲霉毒素的含量,可操作性强,方法准确度可靠,回收率高达98%-103%以上,结果精密度高,重现性好,RSD为<1.8%,灵敏度高,对于5mL的浓缩苹果汁,其检测极限为10μg／L,可作为广大浓缩苹果汁生产厂测定苹果汁中棒曲霉毒素含量的准确定量方法。
(2)在静置培养和摇床培养中,发酵液中棒曲霉毒素含量均出现先上升后下降的趋势；菌体生长在静置培养中出现明显的二次增长,摇床培养生长趋势不明显。发酵液中的残糖和pH呈整体下降趋势,总酸呈整体上升趋势。培养过程中,糖的消耗主要用于菌体生长和棒曲霉毒素的合成；扩展青霉在生长和产毒过程中能够分泌酸性物质。
(3)静置培养中,当菌体干重进入第一次稳定期,孢子开始形成时,棒曲霉毒素达到一个相对平缓的最高平台；当菌体量进入第二次快速增长期时,棒曲霉毒素开始降解；后期菌体量下降时,棒曲霉毒素迅速下降。摇床培养的菌体量是静置培养的2倍之多,但棒曲霉毒素产量不到静置培养的1／2；菌体量处于稳定期的中期时,棒曲霉毒素开始迅速下降。由此说明,棒曲霉毒素的合成条件与扩展青霉的孢子形成条件有一定的相似性；氧气利于菌丝体的生长,但不利于棒曲霉毒素的合成。
(4)在棒曲霉毒素的生长过程中,添加不同剂量的乳酸菌可使其吸附生长的棒曲霉毒素菌株；在发酵早期,还可控制棒曲霉毒素的生长。研究结果表明,添加500mg／L的乳酸菌对棒曲霉毒素的生长可以起到较好的抑制作用,同时,对浓缩苹果汁的产品品质也不会产生影响。
(5)苹果多酚在体外对H22、HT29瘤株具有直接杀伤作用且具有剂量-效应相关性；苹果多酚可抑制H22细胞增殖；苹果多酚还可通过抑制细胞DNA合成,降低细胞周期中S期百分率,从而使细胞停滞于G2/M期,从而减少分裂期细胞而发挥抗癌作用。
(6)苹果多酚在体内对H22移植性肿瘤具有良好的抑瘤作用,但其剂量-效应关系不明确；对于荷EAC小鼠的生存时间无显著影响；对于荷瘤小鼠的免疫功能影响不大。
China's apple production ranks the first in the world. However, in recent years, our export of the concentrated apple juice has been restricted mainly because the amount of Patulin in the juice exceeds the standard. Therefore, it is rather important to improve the detecting ability and to control effectively the amount of Patulin in the juice as well. As the main producing strain in the production of concentrated apple juice, Penicillium expansum affects the quality of concentrated apple juice directly.
The phenolic substances in the apple are generally called Apple Polyphenols, which is the latest potent antioxidant and one of the most powerful free radical scavengers that have been found. Owing to the high thermal stability, within the pH2-10 at 120℃it can still be alive and absorbed quickly and completely. So that it can reach the maximum blood concentration 20 minutes after taken orally with the help of the 7-hour half-life of metabolism. Experimental results show that many of its physiological activity are 100 times higher than the tea polyphenols. The main functions are to prevent tooth decay, to depress bad breath components in saliva production (4 mercaptan), to inhibit the volatile of fish odor (34 amine); to decrease the production of melanin, to restrain tyrosinase function; to reduce the ACE activity, to reduce blood pressure placidly, to relieve hypersensitivity, and to prevent cardiovascular disease, etc.
The study starts from the detection of Patulin in the concentrated apple juice, conducts a thorough research of the recent detecting method. After the systematical research of the production and degradation of Patulin, and the systematical analysis of the antitumor effect of the Apple Polyphenols, major findings are as follows:
(1) To detect the amount of the Patulin in the concentrated apple juice, High Performance Liquid Chromatography (HPLC) is operational, accurate and reliable, with the recovery rate as high as 98% to 103%, and the results are of high precision, good reproducibility, and high sensitivity, with the RSD < 1.8%. For 5mL of concentrated apple juice, the detection limit was 10μg/L, which can be taken as an accurate quantitative method for the apple juice producer.
(2) In both batchs of static and shaking cultivations, patulin content increased firstly and then decreased. A clear second increase of cell growth appeared in the static cultivation, but not in the shaking cultivation. In total, residue sugar and pH kept decreasing and total titrable acids kept increasing through the whole cultivation in both static and shaking cultivations. During the cultivation, sugar was consumed for the cell growth and sythesis of patulin. Some kinds of acidic substance were produced in the cell growth and patulin sythesis.
(3) In the batch of static cultivation, the period for the highest patulin production was consistent with the first high stable period for cell growth, in which spores of the P. Expansum started to form. Patulin reduced quickly from when the second quick cell growth happened in the static cultivation and in the middle period of the stable cell growth in the shaking cultivation. Patulin reduction also happened at the later cultivation stage when cells appeared decrease. Compared with the static cultivation, the dried cell weight was about 2 times more than, but the patulin production was 1/2 lower than, that in the static cultivation. This indicated conditions for patulin production might be consistent with that for spore formation. Abundant oxygen would be good for the cell growth of P. expansum, but not for the patulin production.
(4) It is founded that lactic acid bacteria can absorb the growth of Aspergillus toxins by adding different doses of lactic acid during the growth of patulin, the growth of patulin can be controlled in the early fermentation. The results showed that it can play a better inhibition by addition of 500mg/L of lactic acid bacteria on the growth of Aspergillus clavatus toxins, at the same time, it is not impact the quality of concentrated apple juice products.
(5) There is tumor-inhibitory effect in vivo of apple polephenols to H22, but no dose-dependent relation in the groups of H22. There is no influence on the survival periods by apple polephenols to EAC-receiving mice. There is no influence on the immunological function in tumor-receiving mice treated with apple polephenols.
(6) There is the direct kill function in vitro of apple polephenols to H22, HT29 lump, and the dose-dependent relation in the groups of H22, HT29 lump. The results showed that Apple polyphenols can inhibit the H22 cell proliferation. Through inhibiting cell DNA synthesis, reduces the S phase percentage in the mitotic cycle, induce G2/M phase arrest, plays the antitumoral effect through the reduced fission time cell.
引文
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