锦橙皮中多甲氧基黄酮抑菌抗氧化活性研究
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摘要
锦橙(Citrus sinensis Osbeck)是我国特有的柑橘品种,其果皮中富含多甲氧基黄酮(PMFs),具有较好的生物活性。本研究立足于对柑橘果皮中所特有的PMFs进行系统探讨,在对PMFs分离纯化和结构鉴定的基础上,分析我国长江中下游地区种植柑橘品种果皮中PMFs组分与含量差异。通过研究PMFs提取物对常见食品腐败菌和致病菌的抑制效果,进一步系统探讨PMFs单体对假单胞菌的抑制机制。同时,对PMFs提取物的抗氧化活性进行研究,并从氨基酸和有机酸中筛选PMFs提取物的抗氧化协同增效剂。另外,将PMFs提取物应用于冷却肉保鲜并评价其保鲜效果,为开发新型高效的天然抗氧化剂和食品防腐剂提供理论支持。
主要研究内容和结果如下：
1,采用纤维素酶酶解预处理与醇浸提相结合的提取工艺从锦橙皮中提取黄酮,与传统工艺相比,锦橙皮中黄酮浸出率显著提高。在单因素实验的基础上,通过正交实验得出酶解温度、酶解时间、酶液添加量及pH值对酶解效果的最佳组合。确定了最佳提取条件：pH值为4,酶解温度60℃,酶解时间2h,酶浓度5%,料液比1：20,乙醇浓度80%时,总黄酮浸出率达到2.67±0.06%,较单因素实验最佳得率、提高了14%。
2,锦橙皮总黄酮提取物经过乙醚萃取,凝胶Sephadex LH-20柱层析,甲醇-水梯度洗脱并于340nm检测,收集最大吸收峰组分可得较高纯度的PMFs提取物。采用液相色谱-质谱联用(LC-MS)、核磁共振光谱(NMR)、紫外光谱(UV)等技术对PMFs进行结构鉴定,可推断锦橙皮中主要含有7种PMFs。其中3,5,6,7,3’,4’六甲氧基黄酮(QUE)、川陈皮素(NOB)、七甲氧基黄酮(HEP)、5-去甲基川陈皮素(5-DN)和桔皮素(TAN)通过质谱／电喷雾电离(ESI/MS)在正离子模式下的准分子离子峰和诊断碎片离子,加上UV以及文献报道的HPLC洗脱顺序鉴定。甜橙黄酮(SIN)和5,6,7,4’-四甲氧基黄酮(SCU)经HPLC纯化,通过MS、1H NMR和UV鉴定。另外,首次系统分析了长江中下游地区种植的柑橘品种果皮中PMFs的组分与含量,发现不同柑橘品种中PMFs的含量与组成差异显著。酸橙是NOB和TAN的优质来源,而温州蜜柑含有丰富的HEP,其中大叶尾张含量最高。甜橙中含有7种不同结构的PMFs, SCU和SIN含量较丰富。
3,采用牛津杯法测定PMFs提取物对细菌的抑菌活性,生长速率法测定PMFs提取物对霉菌的抑制效果,并使用琼脂稀释法确定PMFs提取物对各微生物的最小抑菌浓度(MIC)。实验结果表明,PMFs提取物具有较好抑菌效果,对革兰氏阳性细菌的抑制效果强于革兰氏阴性细菌,对霉菌也具有较强抑制效果。同时,MIC结果显示牛津杯法测定抑菌圈直径与在琼脂稀释平板获得的MIC并不相关。PMFs提取物对两种供试霉菌的抑制作用较显著,且对黑曲霉的抑制作用强于桔青霉。PMFs提取物对微生物的抑制作用及药效持续性存在明显的量效关系,随着浓度的降低,抑菌效果和药效持续性逐渐降低。
4,评价了NOB和TAN对假单胞菌的抑菌活性,并从细菌超微机构、胞内物质的泄露、蛋白质的合成和代谢关键酶活性等方面探讨NOB和TAN对假单胞菌的作用机制。结果表明,经过NOB和TAN处理过的菌体上清液中胞内转氨酶的浓度显著高于对照,还原糖也发生大量泄露,超微结构显示NOB和TAN使假单胞菌菌体严重受到损伤,出现质壁分离现象。同时,NOB和TAN处理后假单胞菌其胞内琥珀酸脱氢酶(SDH)、苹果酸脱氢酶(MDH)以及ATP酶的活性较对照明显降低,PMFs对假单胞菌胞内的脱氢酶及ATP酶活性表现出抑制作用。另外,菌体内可溶性蛋白质含量较同期对照组显著降低。因此可认为NOB和TAN可能破坏了菌体细胞膜的选择通透性功能,导致细胞内容物大量流失,破坏了呼吸链,引起代谢功能障碍,菌体内蛋白质的合成受阻,致使细胞质固缩,从而导致细菌生长繁殖受到抑制并死亡。
5,利用化学发光法评价锦橙皮PMFs提取物的体外抗氧化活性及其对DNA损伤的保护作用。分别采用Pyrogallol-Luminol化学发光体系、CuSO4-Phen-Vc-H2O2化学发光体系和H2O2-Luminol化学发光体系测定PMFs提取物对超氧阴离子、羟基自由基及过氧化氢的清除能力。采用CuSO4-Phen-Vc-H2O2-DNA化学发光体系衡量PMFs提取物对DNA损伤的保护作用。结果表明：PMFs提取物可有效清除超氧阴离子、羟基自由基和过氧化氢,其半数抑制率(IC50)分别为6.1、7.8、0.6μg/ml。PMFs提取物对DNA损伤能产生较好的保护作用,其保护机制属于抑制型,IC50为4.0μg/ml。当PMFs提取物浓度增加时,抑制能力增强,表现出明显的浓度依赖关系。同时,从4种氨基酸(丙氨酸、赖氨酸、苏氨酸和丝氨酸)及3种有机酸(草酸、柠檬酸和苹果酸)中筛选PMFs提取物的抗氧化协同增效剂。在PMFs提取物与氨基酸或有机酸复配的混合物中,赖氨酸和柠檬酸对PMFs提取物显示出较好的协同增效作用。当42μg/mL赖氨酸与7μg/mL PMFs提取物复配(1.10,p<0.05)以及120μg/mL柠檬酸与7μg/mLPMFs提取物共混(1.20,p<0.05)时,可得到最佳清除超氧阴离子协同效果。对于其他氨基酸或有机酸与PMFs提取物的复配组合,其测定抑制率(%MIE)与期望抑制率(%EIE)之间不具有显著差异,因而不认为具有协同增效作用。
6,通过将PMFs提取物对鲜猪肉进行涂膜处理,并采用PE有氧包装形式,以色价、pH值、挥发性盐基氮值(TVB-N值)、细菌总数和假单胞菌数的变化为指标,评价PMFs提取物对冷却肉的保鲜效果。结果表明,PMFs提取物对冷却肉具有一定的保鲜效果,与乙醇对照组相比,经PMFs提取物处理后的冷却肉其pH值、TVB-N值、细菌总数和假单胞菌数等指标均变化缓慢,且对肉色无不良影响,可使冷却肉保质期延长至8d。
Jinchen orange is a native sweet crop of Citrus sinensis Osbeck in China, and the peels of fruit is considered as an important source of polymethoxylated flavones (PMFs) of high concentration. PMFs are known to have physiological effects on organ systems. This study was established in the systemic discussion of PMFs from Jinchen orange peels. At the function of isolation and identification of PMFs, the levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were analyzed. The inhibitory activities of PMFs against food spoilage organisms and pathogenic bacterias in vitro were examined and the action mechanism against Pseudomonas was also evaluated. Otherwise, the antioxidant of PMFs extracts was evaluated, and the synergistic effects of PMFs extracts with organic acids and amino acids on superoxide anion were also determined. In addtion, the effect of preservation on chilled meat coated by PMFs extracts was studied. Theae researches would give references for exploitation of potential natural food antioxidant and preservative.
The main research subjects and results are listed as follows:
1, The extraction technique about flavonoids from Jinchen orange peels by means of enzyme hydrolysis and alcohol-extraction was studied. The yield of flavonoids increased distinctly, comparing with the traditional technique of extraction. The influences of extraction time, extraction temperature, pH value and the mass ratio of cellulase to material on the yield of total flavonoids from jinchen orange peels with the enzyme-assisted extraction technique were studied, according to the orthogonal test design based on the single factor experiments. The optimum extraction conditions were obtained as follows:the added ratio of cellulose 5%, the extraction time 2h, pH value 4 and the temperature 60℃, and the yield of flavonoids was up to 2.67±0.06%, which incresed by 14%, comparing with the the yield of flavonoids from the single factor experiments.
2, The solution of total flavonoids from Jinchen orange peels were extracted with diethyl ether, and then loaded on a sephadex LH-20 gel column (2.5 cm×30 cm) with an ultraviolet detector set at 340 nm. The peaks with maximum absorbance were collected to give a purified PMFs mixture. PMFs extracts were characterized by chromatographic and spectroscopic techniques, such as LC-MS、NMR and UV. Seven individual PMF were identified. Quercetogetin (QUE), nobiletin (NOB), heptamethoxyflavone (HEP), 5-demethylnobiletin (5-DN) and tangeretin (TAN) were characterized through electrospray ionization mass spectrometry (ESI-MS) in positive mode of protonated molecular ions [M+H]+, the diagnostic fragment ions, together with the UV-Vis spectra and HPLC elution order from literature data. Sinensetin (SIN) and tetramethyl-o-scutellarein (SCU) were isolated and identified through their MS,1H NMR and UV-Vis spectral studies. The levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were determined for the first time. The results showed that PMFs levels were dependent on the cultivar, C. aurantium'Bitter orange'to be the most valuable citrus variety for isolating NOB and TAN, while C. unshiu'Owari satuma'is the most appropriate variety for HEP. C. sinensis contains seven individual PMFs, with significant levels of SIN and SCU.
3, Antimicrobial activity of PMFs extracts against bacteria and fungi by Oxford plate assay and by growth rate method, respectively. The minimum inhibitory concentration (MIC) were tested by two-fold serial dilution method. The results showed that PMFs extracts had strong inhibition on microorganism with broad-spectrum. Gram positive bacterias were more sensitive to PMFs extracts than gram negative bacterias. It could also inhibit the growth of fungi. The MIC test results illustrated that the higher inhibition diameter did not always correspond to the minor value obtained with the MIC test. The inhibitory effects of PMFs extracts against Aspergillus. niger was stronger than Penicillium. digitatum. PMFs extracts appeared obviously inhibitory effect and efficacy at a dose-dependent manner.
4, The inhibitory activities of NOB and TAN against Pseudomonas in vitro were examined. The effects of NOB and TAN on cell morphology, the release of cell constituents, the synthesis of proteins and activities of key dehydrogenase were evaluated with the aim of elucidating its antibacterial mechanism. The results showed that the concentrations of transaminase and reducing sugar in bacteria solution increased when treated with NOB and TAN. It was observed by electron microscopy that the structure of bacteria cells were destroyed seriously and induced cells plasmolysis. In addition, NOB and TAN could significantly inhibit the activities of succinate dehydrogenase (SDH), malata dehydrogenase (MDH) and ATPase, as well as the proteins synthesis in Pseudomonas. Therefore, the study suggested that NOB and TAN could destroy the permeability of cell membrane with the great release of cell constituents, lead to metabolic function dysfunction and inhibit the synthesis of proteins, result in cell pyknosis. Eventually growth and reproduction of Pseudomonas cells were inhibited and death.
5, The antioxidant of PMFs extracts of Jinchen orange peels was evaluated by using a chemiluminescence (CL) method in vitro. The scavenging ability of PMFs on superoxide anion, hydroxide radial, and hydrogen peroxide were determined by the Pyrogallol-Luminol system, CuSO4-Phen-Vc-H2O2 system, and Luminol-H2O2 system, respectively. DNA damage preventing effect of PMFs was evaluated by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that PMFs extracts exhibited effective antioxidant activity to remove superoxide anion, hydroxide radial, hydrogen peroxide and prevent DNA from being damaged in the tested method. The IC50 was 6.1, 7.8,0.6 and 4.0μg/ml, respectively. PMFs extracts had antioxidative effect in a dose-dependent manner. In addition, the synergistic effects of PMFs extracts with organic acids (citric acid, oxalic acid and malic acid) and amino acids (alanine, lysine, serine, threonine) on superoxide anion were determined by the pyrogallol-luminol system. Mixtures of citric acid and lysine with PMFs extracts had higher synergistic effects than any other tested mixtures, and the optimum concentrations were exhibited by combinations of 7μg/mL PMFs+120μg/mL citric acid (1.2, P<0.05) and 7μg/mL PMFs+42μg/mL lysine (1.1, P< 0.05). For the other amino acids and organic acids, the values of the measured inhibition effect (% MIE) of the mixtures with PMFs extracts were not significantly different from their expected inhibition effect (% EIE), which did not show synergism.
6, The effect of preservation on chilled meat coated by PMFs extracts was studied. The colour, pH value, TVB-N, total plate counts and Pseudomonas number were evaluation target. The result showed that the evaluation targets of chilled meat coated by PMFs extracts was obviously better than control group. Also, there is no bad effect on the colour of chilled meat coated by PMFs extracts. The shelf life was 8d.
主要研究内容和结果如下：
1,采用纤维素酶酶解预处理与醇浸提相结合的提取工艺从锦橙皮中提取黄酮,与传统工艺相比,锦橙皮中黄酮浸出率显著提高。在单因素实验的基础上,通过正交实验得出酶解温度、酶解时间、酶液添加量及pH值对酶解效果的最佳组合。确定了最佳提取条件：pH值为4,酶解温度60℃,酶解时间2h,酶浓度5%,料液比1：20,乙醇浓度80%时,总黄酮浸出率达到2.67±0.06%,较单因素实验最佳得率、提高了14%。
2,锦橙皮总黄酮提取物经过乙醚萃取,凝胶Sephadex LH-20柱层析,甲醇-水梯度洗脱并于340nm检测,收集最大吸收峰组分可得较高纯度的PMFs提取物。采用液相色谱-质谱联用(LC-MS)、核磁共振光谱(NMR)、紫外光谱(UV)等技术对PMFs进行结构鉴定,可推断锦橙皮中主要含有7种PMFs。其中3,5,6,7,3’,4’六甲氧基黄酮(QUE)、川陈皮素(NOB)、七甲氧基黄酮(HEP)、5-去甲基川陈皮素(5-DN)和桔皮素(TAN)通过质谱／电喷雾电离(ESI/MS)在正离子模式下的准分子离子峰和诊断碎片离子,加上UV以及文献报道的HPLC洗脱顺序鉴定。甜橙黄酮(SIN)和5,6,7,4’-四甲氧基黄酮(SCU)经HPLC纯化,通过MS、1H NMR和UV鉴定。另外,首次系统分析了长江中下游地区种植的柑橘品种果皮中PMFs的组分与含量,发现不同柑橘品种中PMFs的含量与组成差异显著。酸橙是NOB和TAN的优质来源,而温州蜜柑含有丰富的HEP,其中大叶尾张含量最高。甜橙中含有7种不同结构的PMFs, SCU和SIN含量较丰富。
3,采用牛津杯法测定PMFs提取物对细菌的抑菌活性,生长速率法测定PMFs提取物对霉菌的抑制效果,并使用琼脂稀释法确定PMFs提取物对各微生物的最小抑菌浓度(MIC)。实验结果表明,PMFs提取物具有较好抑菌效果,对革兰氏阳性细菌的抑制效果强于革兰氏阴性细菌,对霉菌也具有较强抑制效果。同时,MIC结果显示牛津杯法测定抑菌圈直径与在琼脂稀释平板获得的MIC并不相关。PMFs提取物对两种供试霉菌的抑制作用较显著,且对黑曲霉的抑制作用强于桔青霉。PMFs提取物对微生物的抑制作用及药效持续性存在明显的量效关系,随着浓度的降低,抑菌效果和药效持续性逐渐降低。
4,评价了NOB和TAN对假单胞菌的抑菌活性,并从细菌超微机构、胞内物质的泄露、蛋白质的合成和代谢关键酶活性等方面探讨NOB和TAN对假单胞菌的作用机制。结果表明,经过NOB和TAN处理过的菌体上清液中胞内转氨酶的浓度显著高于对照,还原糖也发生大量泄露,超微结构显示NOB和TAN使假单胞菌菌体严重受到损伤,出现质壁分离现象。同时,NOB和TAN处理后假单胞菌其胞内琥珀酸脱氢酶(SDH)、苹果酸脱氢酶(MDH)以及ATP酶的活性较对照明显降低,PMFs对假单胞菌胞内的脱氢酶及ATP酶活性表现出抑制作用。另外,菌体内可溶性蛋白质含量较同期对照组显著降低。因此可认为NOB和TAN可能破坏了菌体细胞膜的选择通透性功能,导致细胞内容物大量流失,破坏了呼吸链,引起代谢功能障碍,菌体内蛋白质的合成受阻,致使细胞质固缩,从而导致细菌生长繁殖受到抑制并死亡。
5,利用化学发光法评价锦橙皮PMFs提取物的体外抗氧化活性及其对DNA损伤的保护作用。分别采用Pyrogallol-Luminol化学发光体系、CuSO4-Phen-Vc-H2O2化学发光体系和H2O2-Luminol化学发光体系测定PMFs提取物对超氧阴离子、羟基自由基及过氧化氢的清除能力。采用CuSO4-Phen-Vc-H2O2-DNA化学发光体系衡量PMFs提取物对DNA损伤的保护作用。结果表明：PMFs提取物可有效清除超氧阴离子、羟基自由基和过氧化氢,其半数抑制率(IC50)分别为6.1、7.8、0.6μg/ml。PMFs提取物对DNA损伤能产生较好的保护作用,其保护机制属于抑制型,IC50为4.0μg/ml。当PMFs提取物浓度增加时,抑制能力增强,表现出明显的浓度依赖关系。同时,从4种氨基酸(丙氨酸、赖氨酸、苏氨酸和丝氨酸)及3种有机酸(草酸、柠檬酸和苹果酸)中筛选PMFs提取物的抗氧化协同增效剂。在PMFs提取物与氨基酸或有机酸复配的混合物中,赖氨酸和柠檬酸对PMFs提取物显示出较好的协同增效作用。当42μg/mL赖氨酸与7μg/mL PMFs提取物复配(1.10,p<0.05)以及120μg/mL柠檬酸与7μg/mLPMFs提取物共混(1.20,p<0.05)时,可得到最佳清除超氧阴离子协同效果。对于其他氨基酸或有机酸与PMFs提取物的复配组合,其测定抑制率(%MIE)与期望抑制率(%EIE)之间不具有显著差异,因而不认为具有协同增效作用。
6,通过将PMFs提取物对鲜猪肉进行涂膜处理,并采用PE有氧包装形式,以色价、pH值、挥发性盐基氮值(TVB-N值)、细菌总数和假单胞菌数的变化为指标,评价PMFs提取物对冷却肉的保鲜效果。结果表明,PMFs提取物对冷却肉具有一定的保鲜效果,与乙醇对照组相比,经PMFs提取物处理后的冷却肉其pH值、TVB-N值、细菌总数和假单胞菌数等指标均变化缓慢,且对肉色无不良影响,可使冷却肉保质期延长至8d。
Jinchen orange is a native sweet crop of Citrus sinensis Osbeck in China, and the peels of fruit is considered as an important source of polymethoxylated flavones (PMFs) of high concentration. PMFs are known to have physiological effects on organ systems. This study was established in the systemic discussion of PMFs from Jinchen orange peels. At the function of isolation and identification of PMFs, the levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were analyzed. The inhibitory activities of PMFs against food spoilage organisms and pathogenic bacterias in vitro were examined and the action mechanism against Pseudomonas was also evaluated. Otherwise, the antioxidant of PMFs extracts was evaluated, and the synergistic effects of PMFs extracts with organic acids and amino acids on superoxide anion were also determined. In addtion, the effect of preservation on chilled meat coated by PMFs extracts was studied. Theae researches would give references for exploitation of potential natural food antioxidant and preservative.
The main research subjects and results are listed as follows:
1, The extraction technique about flavonoids from Jinchen orange peels by means of enzyme hydrolysis and alcohol-extraction was studied. The yield of flavonoids increased distinctly, comparing with the traditional technique of extraction. The influences of extraction time, extraction temperature, pH value and the mass ratio of cellulase to material on the yield of total flavonoids from jinchen orange peels with the enzyme-assisted extraction technique were studied, according to the orthogonal test design based on the single factor experiments. The optimum extraction conditions were obtained as follows:the added ratio of cellulose 5%, the extraction time 2h, pH value 4 and the temperature 60℃, and the yield of flavonoids was up to 2.67±0.06%, which incresed by 14%, comparing with the the yield of flavonoids from the single factor experiments.
2, The solution of total flavonoids from Jinchen orange peels were extracted with diethyl ether, and then loaded on a sephadex LH-20 gel column (2.5 cm×30 cm) with an ultraviolet detector set at 340 nm. The peaks with maximum absorbance were collected to give a purified PMFs mixture. PMFs extracts were characterized by chromatographic and spectroscopic techniques, such as LC-MS、NMR and UV. Seven individual PMF were identified. Quercetogetin (QUE), nobiletin (NOB), heptamethoxyflavone (HEP), 5-demethylnobiletin (5-DN) and tangeretin (TAN) were characterized through electrospray ionization mass spectrometry (ESI-MS) in positive mode of protonated molecular ions [M+H]+, the diagnostic fragment ions, together with the UV-Vis spectra and HPLC elution order from literature data. Sinensetin (SIN) and tetramethyl-o-scutellarein (SCU) were isolated and identified through their MS,1H NMR and UV-Vis spectral studies. The levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were determined for the first time. The results showed that PMFs levels were dependent on the cultivar, C. aurantium'Bitter orange'to be the most valuable citrus variety for isolating NOB and TAN, while C. unshiu'Owari satuma'is the most appropriate variety for HEP. C. sinensis contains seven individual PMFs, with significant levels of SIN and SCU.
3, Antimicrobial activity of PMFs extracts against bacteria and fungi by Oxford plate assay and by growth rate method, respectively. The minimum inhibitory concentration (MIC) were tested by two-fold serial dilution method. The results showed that PMFs extracts had strong inhibition on microorganism with broad-spectrum. Gram positive bacterias were more sensitive to PMFs extracts than gram negative bacterias. It could also inhibit the growth of fungi. The MIC test results illustrated that the higher inhibition diameter did not always correspond to the minor value obtained with the MIC test. The inhibitory effects of PMFs extracts against Aspergillus. niger was stronger than Penicillium. digitatum. PMFs extracts appeared obviously inhibitory effect and efficacy at a dose-dependent manner.
4, The inhibitory activities of NOB and TAN against Pseudomonas in vitro were examined. The effects of NOB and TAN on cell morphology, the release of cell constituents, the synthesis of proteins and activities of key dehydrogenase were evaluated with the aim of elucidating its antibacterial mechanism. The results showed that the concentrations of transaminase and reducing sugar in bacteria solution increased when treated with NOB and TAN. It was observed by electron microscopy that the structure of bacteria cells were destroyed seriously and induced cells plasmolysis. In addition, NOB and TAN could significantly inhibit the activities of succinate dehydrogenase (SDH), malata dehydrogenase (MDH) and ATPase, as well as the proteins synthesis in Pseudomonas. Therefore, the study suggested that NOB and TAN could destroy the permeability of cell membrane with the great release of cell constituents, lead to metabolic function dysfunction and inhibit the synthesis of proteins, result in cell pyknosis. Eventually growth and reproduction of Pseudomonas cells were inhibited and death.
5, The antioxidant of PMFs extracts of Jinchen orange peels was evaluated by using a chemiluminescence (CL) method in vitro. The scavenging ability of PMFs on superoxide anion, hydroxide radial, and hydrogen peroxide were determined by the Pyrogallol-Luminol system, CuSO4-Phen-Vc-H2O2 system, and Luminol-H2O2 system, respectively. DNA damage preventing effect of PMFs was evaluated by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that PMFs extracts exhibited effective antioxidant activity to remove superoxide anion, hydroxide radial, hydrogen peroxide and prevent DNA from being damaged in the tested method. The IC50 was 6.1, 7.8,0.6 and 4.0μg/ml, respectively. PMFs extracts had antioxidative effect in a dose-dependent manner. In addition, the synergistic effects of PMFs extracts with organic acids (citric acid, oxalic acid and malic acid) and amino acids (alanine, lysine, serine, threonine) on superoxide anion were determined by the pyrogallol-luminol system. Mixtures of citric acid and lysine with PMFs extracts had higher synergistic effects than any other tested mixtures, and the optimum concentrations were exhibited by combinations of 7μg/mL PMFs+120μg/mL citric acid (1.2, P<0.05) and 7μg/mL PMFs+42μg/mL lysine (1.1, P< 0.05). For the other amino acids and organic acids, the values of the measured inhibition effect (% MIE) of the mixtures with PMFs extracts were not significantly different from their expected inhibition effect (% EIE), which did not show synergism.
6, The effect of preservation on chilled meat coated by PMFs extracts was studied. The colour, pH value, TVB-N, total plate counts and Pseudomonas number were evaluation target. The result showed that the evaluation targets of chilled meat coated by PMFs extracts was obviously better than control group. Also, there is no bad effect on the colour of chilled meat coated by PMFs extracts. The shelf life was 8d.
引文
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