利用低次烟叶蛋白制备生物活性肽及烟用香精的研究
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摘要
本文利用碱溶酸沉法从低次烟叶中提取出可溶性蛋白质,研究了低次烟叶蛋白酶解物抗氧化和抑菌活性的变化规律,并通过超滤膜分离,确定了活性最强的肽段,同时以低次烟叶蛋白酶解物为原料,通过Maillard反应制备高质量的烟用香精。研究得到如下结论:
烟叶蛋白提取过程中磨浆工艺的最佳水平组合为固液比1:17,提取液温度60℃,提取液pH8.0,磨浆两次;碱溶最佳工艺:温度60℃,pH8.0,时间60min,搅拌条件下提取三次;酸沉过程中,pH3.0,4℃静置8hr效果最佳。蛋白酸沉提取率最高为86.71%,产品中蛋白质含量为70.66%。B1K级烟叶蛋白中必需氨基酸含量最高(41.80%)。
低次烟叶蛋白利用不同蛋白酶(Papain、ProtamexTM和Alcalase2.4L)进行酶解,以水解度和肽提取率为指标,以Papain酶解效果最好,通过单因素和正交试验表明:Papain酶解低次烟叶蛋白最优条件:酶用量1.25%、pH6.5、60℃、底物浓度8.00%、搅拌下酶解6hr,肽提取率相对最高为37.42%。游离氨基酸含量随酶解时间的延长而增加,酶解24hr后水解产物中游离氨基酸总量为未酶解样品中的15倍左右。
建立了木瓜蛋白酶酶解低次烟叶蛋白的动力学模型:
酶失活动力学常数k d=0.0077 min-1;对酶解动力学模型验证表明,该酶解动力学模型在0-300min范围内与实际结果基本相吻合。
酶解液体外抗氧化活性以5kDa组分最高。Papain酶解液对超氧阴离子自由基的抑制率最高(28.41%),接近VE(28.56%),ProtamexTM酶解液的抑制率次之(25.48%),Alcalase2.4L酶解液抑制率最低(24.55%)。清除DPPH自由基的能力以Papain酶解液最高为83.82%,ProtamexTM酶解液的清除率次之为80.08%,Alcalase2.4L酶解液清除率最低为71.93%,但均比VE差(93.24%)。三种酶解液清除羟自由基清除的能力依次为:Alcalase2.4L酶解液(67.18%)>ProtamexTM酶解液(65.42%)>Papain酶解液(64.17%)。酶解液抗氧化活性组分分子量集中在3-5kDa。
酶解液(酶解6hr)中,色氨酸、组氨酸、蛋氨酸、半胱氨酸含量,在经Papain酶解后的原液和经5kDa超滤膜处理的透过液中均最高,分别为45.87mg/mL酶解液和81.44mg/mL酶解液,而经Alcalase2.4L酶解后,四种氨基酸的总含量均最低,分别为42.89mg/mL酶解液和79.18mg/mL酶解液。酶解液和经5kDa超滤膜处理的透过液的氨基酸组成有明显差异。
低次烟叶蛋白经Papain酶解后的酶解液具有抑菌活性,且只抑制大肠杆菌的生长,酶解6hr的酶解液抑菌效果最好,其中以5kDa酶解液抑制效果最佳,对大肠杆菌的最低抑菌浓度为0.40g/L,高于尼泊金丙酯对大肠杆菌的最低抑菌浓度(0.35g/L)。pH值对5kDa酶解液抑制大肠杆菌活性的影响不明显,pH3.0-10.0时,5kDa的酶解液能高效抑制大肠杆菌的生长繁殖;高温处理对5kDa酶解液的抑菌能力影响不明显,5kDa酶解液具有良好的耐热性。
液质联用分析结果表明:膜超滤方法能高效实现对低次烟叶蛋白酶解液的初级分离。
复合酶解技术深度酶解低次烟叶,氨基酸转化率最高达36.94%,明显优于单酶酶解效果。
利用低次烟叶深度酶解液,通过Maillard反应制得的烟用香精,给烟草制品增香调味效果明显,评吸评分最高为96分。通过气质联用色谱分析表明:Maillard反应产物的醚溶性成分中酸、醇、酯成分含量较高,特别是具有明显烤烟香味和甜味的成分含量比较高。
Water-soluble protein was extracted from discarded tobacco leaf in this study. The protein was extracted from tobacco at alkaline condition and precipitated at acidic condition. Enzymatic hydrolysis was taken to modify the physico-chemical properties of tobacco protein to produce bio-active peptides. The membrane ultrafiltration was used to separate the bio-active peptides. The antioxidant activity and bacteria growth inhibition activity of the tobacco protein hydrolysates were measured. Extensive enzymatic hydrolysis technique and combined enzymatic hydrolysis technique were taken to hydrolyze tobacco, and the hydrolysates were used to produce cigarette essence by using Maillard reaction. This research had great significance in improving by-product value of tobacco process, favor to protecting enviroment and benefiting tobacco-planting farmer.
The protein extraction process was investigated in this research. Under the optimal conditions for tobacco grinding, protein dissolving and protein precipitation, the protein recovery and the amino acid score were 86.71%, the resulting product included 70.66% of crude protein.
Three proteinases (Papain, Protamex and Alcalase) were chosen to hydrolyze tobacco protein. The highest peptide recovery was obtained at 37.42% when tobacco protein was hydrolyzed by means of Papain. During the hydrolysis, free amino acids increased as hydrolysis time went on. The free amino acid content was 15 times higher than that in unhydrolyzed tobacco protein. The kinetic mathematical model of tobacco protein enzymatic hydrolysis by Papain was built as follows:
Enzymatic deactivitation kinetic constant k d=0.0077 min~(-1);
When the hydrolysis time ranged from 0-300 min, the DH value computed by this model had no significant difference from the results determined.
In vitro antioxidant activity determination of tobacco protein hydrolysates showed that peptides with 5 kDa of molecular weight had the highest antioxidant activity. The hydrolysates by Papain had the highest superoxide anion scavenging capacity (28.41%), similar to that of VE (28.56%). They also had the highest DPPH radical scavenging capacity (83.82%), lower than that of VE (93.24%). The hydrolysates by Alcalase had the highest hydroxyl radical scavenging capacity (67.18%), similar to that of VE (67.36%). By comparison of in vitro antioxidant activities of the hydrolysates with different molecular weight, it indicated that the molecular weight of tobacco bio-active peptides should be in the range of 3-5 kDa.
The amino acid composition in tobacco protein showed that the total content of tryptophan, histosine, methine and cysteine in tobacco protein supernatant hydrolyzed by Papain for 6hr and the filtrate through 5 kDa membrane ultrafiltration were 45.87 mg/mL hydrolysate and 80.44 mg/mL hydrolysate, respectively. The content in tobacco protein supernatant hydrolysed by Alcalase for 4 hr and the filtrate through 5kDa ultra-membrane were 42.03 mg/mL hydrolysate and 76.79 mg/mL hydrolysate. Different hydrolysates had different antoxidant activities for their differences in bio-active amino acid content.
The results of bacterial-growth inhibition activity trials showed only the hydrolysate produced by Papain had this activity. This hydrolysate was only effective to E. coli. The hydrolysate produced by Papain with 6 hr hydrolysis had the strongest activity in inhibiting bacterial growth, and the diameter of inhibition circle reached 1.80 cm. After partitioned by ultra-membrane, the hydrolysate with 5 kDa of molecualr weight had the highest inhibition activity with 3.60 cm of the inhibition circle diameter. The lowest effective concentration was 0.40 g/L, little more than that of propyl p-hydroxybenzoate (0.35 g/L); The 5 kDa molecular weight hydrolysate had strong alkaline-endurant and acid-endurant capacity and thermostability. Analysis by high performance liquid chromatography / mass spectrometry suggested ultra-membrane filtration had a good resolution for separation of tobacco protein hydrolysates.
Extensive hydrolysis could improve the conversion rate of amino acid. The highest conversion rate of amino acid of the hydrolysate reached 36.94%.
The cigarette essence made by Maillard using tobacco protein hydrolysate as material had good effect on improving the flavor of cigarette. The gas chromatography / mass spectrometry profile showed high contents of acid, alcohol and ester in ether-soluble product of Maillard reaction, especially those components having significant roasting-cigarette flavor and sweet flavor existed with relative high content. Therefore, this cigarette essence could be used for bad flavor removal and flavor enhancing.
烟叶蛋白提取过程中磨浆工艺的最佳水平组合为固液比1:17,提取液温度60℃,提取液pH8.0,磨浆两次;碱溶最佳工艺:温度60℃,pH8.0,时间60min,搅拌条件下提取三次;酸沉过程中,pH3.0,4℃静置8hr效果最佳。蛋白酸沉提取率最高为86.71%,产品中蛋白质含量为70.66%。B1K级烟叶蛋白中必需氨基酸含量最高(41.80%)。
低次烟叶蛋白利用不同蛋白酶(Papain、ProtamexTM和Alcalase2.4L)进行酶解,以水解度和肽提取率为指标,以Papain酶解效果最好,通过单因素和正交试验表明:Papain酶解低次烟叶蛋白最优条件:酶用量1.25%、pH6.5、60℃、底物浓度8.00%、搅拌下酶解6hr,肽提取率相对最高为37.42%。游离氨基酸含量随酶解时间的延长而增加,酶解24hr后水解产物中游离氨基酸总量为未酶解样品中的15倍左右。
建立了木瓜蛋白酶酶解低次烟叶蛋白的动力学模型:
酶失活动力学常数k d=0.0077 min-1;对酶解动力学模型验证表明,该酶解动力学模型在0-300min范围内与实际结果基本相吻合。
酶解液体外抗氧化活性以5kDa组分最高。Papain酶解液对超氧阴离子自由基的抑制率最高(28.41%),接近VE(28.56%),ProtamexTM酶解液的抑制率次之(25.48%),Alcalase2.4L酶解液抑制率最低(24.55%)。清除DPPH自由基的能力以Papain酶解液最高为83.82%,ProtamexTM酶解液的清除率次之为80.08%,Alcalase2.4L酶解液清除率最低为71.93%,但均比VE差(93.24%)。三种酶解液清除羟自由基清除的能力依次为:Alcalase2.4L酶解液(67.18%)>ProtamexTM酶解液(65.42%)>Papain酶解液(64.17%)。酶解液抗氧化活性组分分子量集中在3-5kDa。
酶解液(酶解6hr)中,色氨酸、组氨酸、蛋氨酸、半胱氨酸含量,在经Papain酶解后的原液和经5kDa超滤膜处理的透过液中均最高,分别为45.87mg/mL酶解液和81.44mg/mL酶解液,而经Alcalase2.4L酶解后,四种氨基酸的总含量均最低,分别为42.89mg/mL酶解液和79.18mg/mL酶解液。酶解液和经5kDa超滤膜处理的透过液的氨基酸组成有明显差异。
低次烟叶蛋白经Papain酶解后的酶解液具有抑菌活性,且只抑制大肠杆菌的生长,酶解6hr的酶解液抑菌效果最好,其中以5kDa酶解液抑制效果最佳,对大肠杆菌的最低抑菌浓度为0.40g/L,高于尼泊金丙酯对大肠杆菌的最低抑菌浓度(0.35g/L)。pH值对5kDa酶解液抑制大肠杆菌活性的影响不明显,pH3.0-10.0时,5kDa的酶解液能高效抑制大肠杆菌的生长繁殖;高温处理对5kDa酶解液的抑菌能力影响不明显,5kDa酶解液具有良好的耐热性。
液质联用分析结果表明:膜超滤方法能高效实现对低次烟叶蛋白酶解液的初级分离。
复合酶解技术深度酶解低次烟叶,氨基酸转化率最高达36.94%,明显优于单酶酶解效果。
利用低次烟叶深度酶解液,通过Maillard反应制得的烟用香精,给烟草制品增香调味效果明显,评吸评分最高为96分。通过气质联用色谱分析表明:Maillard反应产物的醚溶性成分中酸、醇、酯成分含量较高,特别是具有明显烤烟香味和甜味的成分含量比较高。
Water-soluble protein was extracted from discarded tobacco leaf in this study. The protein was extracted from tobacco at alkaline condition and precipitated at acidic condition. Enzymatic hydrolysis was taken to modify the physico-chemical properties of tobacco protein to produce bio-active peptides. The membrane ultrafiltration was used to separate the bio-active peptides. The antioxidant activity and bacteria growth inhibition activity of the tobacco protein hydrolysates were measured. Extensive enzymatic hydrolysis technique and combined enzymatic hydrolysis technique were taken to hydrolyze tobacco, and the hydrolysates were used to produce cigarette essence by using Maillard reaction. This research had great significance in improving by-product value of tobacco process, favor to protecting enviroment and benefiting tobacco-planting farmer.
The protein extraction process was investigated in this research. Under the optimal conditions for tobacco grinding, protein dissolving and protein precipitation, the protein recovery and the amino acid score were 86.71%, the resulting product included 70.66% of crude protein.
Three proteinases (Papain, Protamex and Alcalase) were chosen to hydrolyze tobacco protein. The highest peptide recovery was obtained at 37.42% when tobacco protein was hydrolyzed by means of Papain. During the hydrolysis, free amino acids increased as hydrolysis time went on. The free amino acid content was 15 times higher than that in unhydrolyzed tobacco protein. The kinetic mathematical model of tobacco protein enzymatic hydrolysis by Papain was built as follows:
Enzymatic deactivitation kinetic constant k d=0.0077 min~(-1);
When the hydrolysis time ranged from 0-300 min, the DH value computed by this model had no significant difference from the results determined.
In vitro antioxidant activity determination of tobacco protein hydrolysates showed that peptides with 5 kDa of molecular weight had the highest antioxidant activity. The hydrolysates by Papain had the highest superoxide anion scavenging capacity (28.41%), similar to that of VE (28.56%). They also had the highest DPPH radical scavenging capacity (83.82%), lower than that of VE (93.24%). The hydrolysates by Alcalase had the highest hydroxyl radical scavenging capacity (67.18%), similar to that of VE (67.36%). By comparison of in vitro antioxidant activities of the hydrolysates with different molecular weight, it indicated that the molecular weight of tobacco bio-active peptides should be in the range of 3-5 kDa.
The amino acid composition in tobacco protein showed that the total content of tryptophan, histosine, methine and cysteine in tobacco protein supernatant hydrolyzed by Papain for 6hr and the filtrate through 5 kDa membrane ultrafiltration were 45.87 mg/mL hydrolysate and 80.44 mg/mL hydrolysate, respectively. The content in tobacco protein supernatant hydrolysed by Alcalase for 4 hr and the filtrate through 5kDa ultra-membrane were 42.03 mg/mL hydrolysate and 76.79 mg/mL hydrolysate. Different hydrolysates had different antoxidant activities for their differences in bio-active amino acid content.
The results of bacterial-growth inhibition activity trials showed only the hydrolysate produced by Papain had this activity. This hydrolysate was only effective to E. coli. The hydrolysate produced by Papain with 6 hr hydrolysis had the strongest activity in inhibiting bacterial growth, and the diameter of inhibition circle reached 1.80 cm. After partitioned by ultra-membrane, the hydrolysate with 5 kDa of molecualr weight had the highest inhibition activity with 3.60 cm of the inhibition circle diameter. The lowest effective concentration was 0.40 g/L, little more than that of propyl p-hydroxybenzoate (0.35 g/L); The 5 kDa molecular weight hydrolysate had strong alkaline-endurant and acid-endurant capacity and thermostability. Analysis by high performance liquid chromatography / mass spectrometry suggested ultra-membrane filtration had a good resolution for separation of tobacco protein hydrolysates.
Extensive hydrolysis could improve the conversion rate of amino acid. The highest conversion rate of amino acid of the hydrolysate reached 36.94%.
The cigarette essence made by Maillard using tobacco protein hydrolysate as material had good effect on improving the flavor of cigarette. The gas chromatography / mass spectrometry profile showed high contents of acid, alcohol and ester in ether-soluble product of Maillard reaction, especially those components having significant roasting-cigarette flavor and sweet flavor existed with relative high content. Therefore, this cigarette essence could be used for bad flavor removal and flavor enhancing.
引文
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