发酵小米多肽的抗氧化与抗菌活性的研究
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摘要
小米是世界上干旱和半干旱地区的主要的食物来源,可为人类提供蛋白质、脂肪酸、矿物质、维生素、膳食纤维以及多酚类物质。典型小米蛋白含有大量的人体必需氨基酸尤其是含硫氨基酸(如蛋氨酸和胱氨酸)。小米加工过程中除去了富含纤维素和植物素的麸皮层和胚芽层,因此造成了小米的营养流失。另外,小米也是酚酸类和黄酮类抗氧化物质的主要来源。小米食品可作为一种潜在的益生元,它能够显著增强益生菌的活性和生物学功能。许多传统发酵食品和饮料来自小米或小米与其他谷物(玉米和高粱)混合物,包括Koko(小米稀饭)、Fura、Mangishi、 Jandh、宇治、Burukutu、库努崎、奥吉和Bushera而Dambu♀Masvusvu和罗迪是典型的非发酵小米食品的代表。用副干酪乳酸杆菌Fn032对小米进行固态发酵是一种典型有效的生物技术处理方法。
本文研究了副干酪乳杆菌Fn032发酵和湿热处理对小米粉理化性质的影响。结果表明,发酵和湿热处理方法能显著增强小米粉中功能性化学成分的含量(P<0.05)差示扫描量热分析显示湿热处理,小米粉出现了较高的分解温度(Td),温度范围在180.59oC到189.82oC之间,而热焓量(△H)显著下降。发酵和湿热处理处理后,慢消化淀粉和抗性淀粉含量分别为6.83-18.42%,以及7.61-22.68%。另外,x射线衍射对糊化特性的分析及荧光分光光度计结果显示湿热处理对小米粉的组成有很大的影响。扫描电镜分析结果显示了小米粉样品微观结构的差异性。
对副干酪乳杆菌Fn032发酵以及蛋白酶处理的小米粉及其米糠水提物的抗氧化、抗菌性能及营养价值评价结果表明。发酵小米粉中添加蛋白酶后的水解提取物与发酵小米面粉和发酵米糠提取物相比有较高的DPPH自由基清除能力。小米发酵物中添加或不添加蛋白酶,与发酵米糠提取物相比,表现较强的抑菌能力。氨基酸图谱分析表明发酵小米中添加蛋白酶组,具有较强的抗氧化、抗菌活性,其总疏水性氨基酸含量达到最高(51.39%),其疏水性指数为(8.47日/moL氨基酸残基)。此外,蛋白酶水解的小米发酵物组蛋白质含量达到最高、有效提高蛋白比和蛋白质消化率。提取物的分子量分布范围为180-5000Da。反相液相色谱纯化副干酪乳杆菌Fn032发酵小米肽,并通过液质联用测定分离产物的氨基酸序列。富含酪氨酸／亮氨酸的小米肽(FFMp)(FFMp4=756.84,FFMp6=678.74和FFMp10=678.87Da)有较强的(P<0.05)DPPH和超氧阴离子自由基的清除能力。合成的FFMp多肽能够显著抑制大肠杆菌ATCC8099的生长。此外,FFMp4和FFMp6显示了抗胰蛋白酶水解能力,而FFMp10有部分被水解。化学合成短肽(含有6个氨基酸残基),在大肠杆菌ATCC8099细胞表面疏水性,微生物增长,与DNA的结合水平等方面,与小米发酵肽(FFMp10)不同。通过高效液相色谱法也对多肽的蛋白水解能力进行了分析。FFMp10的氨基酸序列是在Mp1、Mp2、Mp3、Mp4多肽序列的基础上用Arg和Lys氨基酸替换。结果表明所有多肽对胃蛋白酶水解1h均有抗性。而Mp2、Mp3和Mp4高效液相色谱洗脱分析表明其对胰蛋白酶水解具有抗性。与Mp3和Mp4相比,Mp10、Mp1和Mp2处理大肠杆菌2小时,表现出较高的抑菌活性。与正常对照组相比,所有肽处理均能降低细菌增长。对Mp1和Mp2于大肠杆菌共培养分析的电泳图分析表明,肽能够与大肠杆菌ATCC8099的DNA结合。
发酵及湿热处理方法对小米理化性质以及营养价值起到很好的改善作用。发酵的小米粉提取物有抗氧化、抗菌活性。事实上,从发酵小米中提取具有天然抗氧化活性、抗菌活性、以及抗酶解活性的肽是可行的。
Millets are a major food source in arid and semi-arid parts of the world. Millets are goodsources of energy. They provide protein, fatty acids, minerals, vitamins, dietary fibre andpolyphenols. Typical millet protein contains high quantity of essential amino acidsespecially the sulphur containing amino acids (methionine and cysteine). Processing milletcausingenhance the viability or functionality of probiotics with significant health benefits.Traditionally fermented foods and beverages obtained from millet or millet mixed withother cereals (corn and sorghum) include koko (millet porridge), fura, mangishi, jandh, uji,burukutu, kunu-zaki, ogi and bushera while dambu, masvusvu and roti are representativesof unfermented millet based products. Solid-state bioprocessing of foxtail millet byLactobacillus paracasei Fn032is a biotechnological strategy to produce fermented foxtailmillet meal with more beneficial components.
The effect of L. paracasei Fn032fermentation and heat-moisture treatment (HMT)on the physicochemical properties of foxtail millet (Setaria italica) flour was investigated.The results obtained showed a significant (P <0.05) increase in proximate chemicalcomposition in general after fermentation and HMT. Differential scanning calorimetryanalysis showed high decomposition temperature (Td) trend of180.59and189.82oC afterHMT. However, there was significant (P <0.05resulted in variation of slow digestible starch and resistant starch count from6.83to18.42%and7.61to22.68%respectively, after fermentation and HTM. Following this observation,it was ascertained that in X-ray diffraction; pasting viscosity and fluorescencespectrophotometry show greater HMT influenced on the flour components. Findings fromthe scanning electron microscopy analysis showed microstructure differences of the flourssamples.
Evaluation of antioxidant, antimicrobial properties and nutritional values of waterextracts from fermented foxtail millet flour and its bran with and without protease by L.paracasei Fn032was done. Fermented foxtail millet flour with added protease extractshowed higher scavenging ability on DPPH radicals as well as reducing power than fermented foxtail millet flour and fermented foxtail millet bran extracts. Both extracts,fermented foxtail millet flour and fermented foxtail millet flour with protease, showedsignificant (P <0.05) effectiveness inhibition abilities on microbial growth whencompared with fermented foxtail millet bran extracts. Amino acid profile revealed thatfermented foxtail millet flour with protease, with relatively strongest antioxidant,antimicrobial activity, also had the highest total hydrophobic amino acids content (51.39%)and hydrophobic index (8.47Kj/moL amino acid residue). Moreover, fermented foxtailmillet flour with protease revealed the highest protein content, predicted protein efficiencyratio, and protein digestibility. Molecular weight of the whole extracts varied from180–5000Da.
Purification by RP-HPLC and amino acid sequencing by LC-MS of peptidesderived from foxtail millet (Setaria italica) meal fermented by L. paracasei Fn032werecarried out. The purified foxtail millet peptide fractions (FFMp) of Tyrosine/Leucine-rich(FFMp4=756.84, FFMp6=678.74and FFMp10=678.87Da) showed significant (P <0.05)scavenging activities for DPPH, and superoxide anion radicals. FFMp peptides(synthesized) have shown fairly inhibition of the Escherichia coli ATCC8099growth.Furthermore, FFMp4and FFMp6peptides showed resistance to trypsin proteolysis whileFFMp10appeared to have partial hydrolysis.
The effect of chemically synthesized short peptides (6residues), previouslyidentified from a fermented foxtail millet meal fraction (FFMp10) on the cell surfacehydrophobicity (CSH), biomass growth, DNA binding ability of Escherichia coli ATCC8099was verified. The proteolytic responses of these peptides were also tested using thehigh performance liquid chromatography. Changes made in the FFMp10sequence were:Mp1, Mp2, Mp3and Mp4peptides sequences by Arg and Lys amino acids substitutions.The results revealed that all the peptides resisted to pepsin treatment for1h. However,Mp2, Mp3and Mp4showed no significant HPLC elution profile changes after the trypsintreatment. The values of CSH were significantly higher (P <0.05) with FFMp10, Mp1andMp22h treatment on bacteria, whereas, lowered with Mp3and Mp4treatment. Overallpeptides treatment lowered the rate of bacterial biomass growth when compared to control.Slight E. coli ATCC8099DNA bindings were observed in lane2,3and lane3respectivelyfor Mp1and Mp2incubation.
Fermentation and heat moisture treatment methods present a possible way ofchanging or improving the physicochemical properties and add nutritional value to foxtailmillet meal. Fermented foxtail millet flour extracts were relatively effective in theantioxidant, antimicrobial properties assayed. Indeed, it is feasible to derive naturalantioxidants peptides along with antimicrobial activity and resistance to enzyme fromfermented foxtail millet meal.
本文研究了副干酪乳杆菌Fn032发酵和湿热处理对小米粉理化性质的影响。结果表明,发酵和湿热处理方法能显著增强小米粉中功能性化学成分的含量(P<0.05)差示扫描量热分析显示湿热处理,小米粉出现了较高的分解温度(Td),温度范围在180.59oC到189.82oC之间,而热焓量(△H)显著下降。发酵和湿热处理处理后,慢消化淀粉和抗性淀粉含量分别为6.83-18.42%,以及7.61-22.68%。另外,x射线衍射对糊化特性的分析及荧光分光光度计结果显示湿热处理对小米粉的组成有很大的影响。扫描电镜分析结果显示了小米粉样品微观结构的差异性。
对副干酪乳杆菌Fn032发酵以及蛋白酶处理的小米粉及其米糠水提物的抗氧化、抗菌性能及营养价值评价结果表明。发酵小米粉中添加蛋白酶后的水解提取物与发酵小米面粉和发酵米糠提取物相比有较高的DPPH自由基清除能力。小米发酵物中添加或不添加蛋白酶,与发酵米糠提取物相比,表现较强的抑菌能力。氨基酸图谱分析表明发酵小米中添加蛋白酶组,具有较强的抗氧化、抗菌活性,其总疏水性氨基酸含量达到最高(51.39%),其疏水性指数为(8.47日/moL氨基酸残基)。此外,蛋白酶水解的小米发酵物组蛋白质含量达到最高、有效提高蛋白比和蛋白质消化率。提取物的分子量分布范围为180-5000Da。反相液相色谱纯化副干酪乳杆菌Fn032发酵小米肽,并通过液质联用测定分离产物的氨基酸序列。富含酪氨酸／亮氨酸的小米肽(FFMp)(FFMp4=756.84,FFMp6=678.74和FFMp10=678.87Da)有较强的(P<0.05)DPPH和超氧阴离子自由基的清除能力。合成的FFMp多肽能够显著抑制大肠杆菌ATCC8099的生长。此外,FFMp4和FFMp6显示了抗胰蛋白酶水解能力,而FFMp10有部分被水解。化学合成短肽(含有6个氨基酸残基),在大肠杆菌ATCC8099细胞表面疏水性,微生物增长,与DNA的结合水平等方面,与小米发酵肽(FFMp10)不同。通过高效液相色谱法也对多肽的蛋白水解能力进行了分析。FFMp10的氨基酸序列是在Mp1、Mp2、Mp3、Mp4多肽序列的基础上用Arg和Lys氨基酸替换。结果表明所有多肽对胃蛋白酶水解1h均有抗性。而Mp2、Mp3和Mp4高效液相色谱洗脱分析表明其对胰蛋白酶水解具有抗性。与Mp3和Mp4相比,Mp10、Mp1和Mp2处理大肠杆菌2小时,表现出较高的抑菌活性。与正常对照组相比,所有肽处理均能降低细菌增长。对Mp1和Mp2于大肠杆菌共培养分析的电泳图分析表明,肽能够与大肠杆菌ATCC8099的DNA结合。
发酵及湿热处理方法对小米理化性质以及营养价值起到很好的改善作用。发酵的小米粉提取物有抗氧化、抗菌活性。事实上,从发酵小米中提取具有天然抗氧化活性、抗菌活性、以及抗酶解活性的肽是可行的。
Millets are a major food source in arid and semi-arid parts of the world. Millets are goodsources of energy. They provide protein, fatty acids, minerals, vitamins, dietary fibre andpolyphenols. Typical millet protein contains high quantity of essential amino acidsespecially the sulphur containing amino acids (methionine and cysteine). Processing milletcausingenhance the viability or functionality of probiotics with significant health benefits.Traditionally fermented foods and beverages obtained from millet or millet mixed withother cereals (corn and sorghum) include koko (millet porridge), fura, mangishi, jandh, uji,burukutu, kunu-zaki, ogi and bushera while dambu, masvusvu and roti are representativesof unfermented millet based products. Solid-state bioprocessing of foxtail millet byLactobacillus paracasei Fn032is a biotechnological strategy to produce fermented foxtailmillet meal with more beneficial components.
The effect of L. paracasei Fn032fermentation and heat-moisture treatment (HMT)on the physicochemical properties of foxtail millet (Setaria italica) flour was investigated.The results obtained showed a significant (P <0.05) increase in proximate chemicalcomposition in general after fermentation and HMT. Differential scanning calorimetryanalysis showed high decomposition temperature (Td) trend of180.59and189.82oC afterHMT. However, there was significant (P <0.05resulted in variation of slow digestible starch and resistant starch count from6.83to18.42%and7.61to22.68%respectively, after fermentation and HTM. Following this observation,it was ascertained that in X-ray diffraction; pasting viscosity and fluorescencespectrophotometry show greater HMT influenced on the flour components. Findings fromthe scanning electron microscopy analysis showed microstructure differences of the flourssamples.
Evaluation of antioxidant, antimicrobial properties and nutritional values of waterextracts from fermented foxtail millet flour and its bran with and without protease by L.paracasei Fn032was done. Fermented foxtail millet flour with added protease extractshowed higher scavenging ability on DPPH radicals as well as reducing power than fermented foxtail millet flour and fermented foxtail millet bran extracts. Both extracts,fermented foxtail millet flour and fermented foxtail millet flour with protease, showedsignificant (P <0.05) effectiveness inhibition abilities on microbial growth whencompared with fermented foxtail millet bran extracts. Amino acid profile revealed thatfermented foxtail millet flour with protease, with relatively strongest antioxidant,antimicrobial activity, also had the highest total hydrophobic amino acids content (51.39%)and hydrophobic index (8.47Kj/moL amino acid residue). Moreover, fermented foxtailmillet flour with protease revealed the highest protein content, predicted protein efficiencyratio, and protein digestibility. Molecular weight of the whole extracts varied from180–5000Da.
Purification by RP-HPLC and amino acid sequencing by LC-MS of peptidesderived from foxtail millet (Setaria italica) meal fermented by L. paracasei Fn032werecarried out. The purified foxtail millet peptide fractions (FFMp) of Tyrosine/Leucine-rich(FFMp4=756.84, FFMp6=678.74and FFMp10=678.87Da) showed significant (P <0.05)scavenging activities for DPPH, and superoxide anion radicals. FFMp peptides(synthesized) have shown fairly inhibition of the Escherichia coli ATCC8099growth.Furthermore, FFMp4and FFMp6peptides showed resistance to trypsin proteolysis whileFFMp10appeared to have partial hydrolysis.
The effect of chemically synthesized short peptides (6residues), previouslyidentified from a fermented foxtail millet meal fraction (FFMp10) on the cell surfacehydrophobicity (CSH), biomass growth, DNA binding ability of Escherichia coli ATCC8099was verified. The proteolytic responses of these peptides were also tested using thehigh performance liquid chromatography. Changes made in the FFMp10sequence were:Mp1, Mp2, Mp3and Mp4peptides sequences by Arg and Lys amino acids substitutions.The results revealed that all the peptides resisted to pepsin treatment for1h. However,Mp2, Mp3and Mp4showed no significant HPLC elution profile changes after the trypsintreatment. The values of CSH were significantly higher (P <0.05) with FFMp10, Mp1andMp22h treatment on bacteria, whereas, lowered with Mp3and Mp4treatment. Overallpeptides treatment lowered the rate of bacterial biomass growth when compared to control.Slight E. coli ATCC8099DNA bindings were observed in lane2,3and lane3respectivelyfor Mp1and Mp2incubation.
Fermentation and heat moisture treatment methods present a possible way ofchanging or improving the physicochemical properties and add nutritional value to foxtailmillet meal. Fermented foxtail millet flour extracts were relatively effective in theantioxidant, antimicrobial properties assayed. Indeed, it is feasible to derive naturalantioxidants peptides along with antimicrobial activity and resistance to enzyme fromfermented foxtail millet meal.
引文
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