摘要
南瓜戊糖是存在于南瓜中的一类珍贵单糖,南瓜中六碳糖和多糖含量较高,利用生物技术可将其转化为低能量的戊糖,戊糖在人体内代谢不需要胰岛素参与,开发适于糖尿病、肥胖人群功能食品和药品,对南瓜采后增值具有重要意义。在从1635株芽孢杆菌中筛选出了转化葡萄糖为戊糖优良菌株J503和B106的研究基础上,本研究以六碳糖为对象,利用枯草芽孢杆菌J503和B106进行生物转化,采用HPLC, E-Nose和GC-MS等技术检测戊糖含量和类型。通过蛋白质组学方法,采用液相-质谱分析关键蛋白,做蛋白质同源分析。推断六碳糖转化为戊糖的关键酶蛋白及体内代谢途径,为南瓜功能开发提供科学依据。
主要结果和结论如下：
(1)通过高效液相色谱(HPLC)方法定量检测戊糖(木糖和阿拉伯糖),确定葡萄糖和甘露糖能转化生成戊糖(木糖和阿拉伯糖)。选择菌株J503转化葡萄糖为戊糖生成量进行发酵条件筛选,应用中心响应面法优化出枯草芽孢杆菌J503发酵葡萄糖生成戊糖的发酵条件：发酵温度37℃,发酵时间36h,底物葡萄糖浓度3.36%,pH7。在此工艺条件下,菌株J503发酵葡萄糖,得到戊糖为2.26±0.03mg/mL,非常接近方程预测值2.28mg/mL。
(2)经固相微萃取技术(SPME)富集、气相色谱-嗅辨-质谱(GC-O-MS)分析,木糖及J503和B106发酵六碳糖样品和亮氨酸(L-leu)反应形成主要挥发性成分为丙酮、2-甲基呋喃、3-甲基正丁醛、3-甲基丁酸、5-甲基己酮、6-甲基-2-庚酮、4-甲基戊酸、2-异戊基-6-甲基吡嗪和丁羟甲苯9种物质。阿拉伯糖及发酵样品和甘氨酸(Gly)反应形成主要挥发性成分为丙酮醛、糠醛、苯甲醛、1,5-二氮杂二环-5-酮、壬醛、2,4-叔丁基苯酚、邻苯二甲酸异丁辛酯和邻苯二甲酸二丁酯8种物质。根据主要香气成分含量得出,J503和B106发酵葡萄糖比甘露糖生成戊糖含量高,六组样品生成戊糖大小排序为：(J503+葡萄糖)>(J503+甘露糖)>(B106+葡萄糖)>(B106+甘露糖)>(J503+B106+葡萄糖)>(J503+B106+甘露糖)。
(3)戊糖比六碳糖更易发生美拉德反应,木糖比阿拉伯糖较易发生美拉德反应,葡萄糖比甘露糖较易发生美拉德反应,氨基酸显色反应强弱顺序为Leu> Gly> Cys> Asp。处理组色差反应产物的颜色均比对照颜色浅,综合色差深浅结果为J503+B106混合发酵葡萄糖和甘露糖>J503发酵葡萄糖和甘露糖>B106发酵葡萄糖和甘露糖。
(4)电子鼻能区分开不同戊糖和四种氨基酸(半胱氨酸、甘氨酸、亮氨酸和天冬氨酸)发生美拉德反应产生的挥发性成分,同时也能区分两株枯草芽孢杆菌(J503和B106)不同组合发酵葡萄糖,甘露糖和南瓜汁,所有样品两个主成分的累计贡献率在78%-85%之间,均能有效反应样品有效感应值。PCA降维区分效果和顶空固相微萃取气质分析挥发性成分结果相似。
(5)J503菌株发酵葡萄糖提取蛋白质种类和含量最高,多数蛋白分子量主要分布在43KD以下,分子量在26KD的蛋白质含量最高,使用液相-质谱方法鉴定出菌株J503发酵葡萄糖转化为戊糖过程490种蛋白质。经数据库(NCBI/BLAST)匹配推断7种可能参与菌株J503转化六碳糖为五碳糖的蛋白。关键蛋白①戊烯加双氧酶,关键蛋白②X1yB,关键蛋白③内源木聚糖酶endo-xylanase,关键蛋白④糖差向异构酶,关键蛋白⑤阿拉伯半乳聚糖内源半乳糖苷酶,关键蛋白⑥木聚糖酶xynC,关键蛋白⑦PenP。推测出菌株J503转化六碳糖为戊糖的代谢机理图。
Pumpkin pentose is a kind of precious monosaccharide in Pumpkin, which has not been developed and utilized widely for its low content. However, there are a great number of six-carbon sugars and polysaccharides in Pumpkin, which could be transformed to pentoses by biotechnology. Pumpkin pentose can be metabolized in human body without insulin and it can be used to explore functional food and medicines for diabetes and obese patients, which will greatly increase the additional post-harvest value of pumpkin. Strain J503and B106were screened from1635Bacillus which can transform glucose to pentose. In this study, hexose was used for biotransformation by Bacillus subtilis B106and J503, which was deteced by HPLC-MS, E-Nose and GC-MS for content and type of pentose. By proteomics method, key protein analysis by LC-MS, protein homologous analysis, key enzyme proteins of pentose and metabolic were deduced, which provided a scientific basis for the functional development of the functional pumpkin products.
The main results and conclusions were made as follows:
(1Quantitative of pentose (xylose and arabinose) was detected by HPLC, glucose and mannose were detected could be converted to pentose (xylose and arabinose). Strain J503was chosen to convert glucose to generate pentose. Response surface method was used to optimise the fermentation condition: Fermentation temperature for37℃， fermentation time for36h, the substrate glucose concentration for3.4%, pH value for7. Under this condition,2.26±0.03mg/mL pentose had been fermentated by strain J503, which was very close to the predicted value equation2.28mg/mL.
(2) By solid phase micro extraction (SPME) enrichment, gas chromatography-olfactometry resolution-mass spectrometry (GC-O-MS) analysis, characterized volatile products of Maillard reaction of xylose and four amino acids (cysteine, glycine, leucine, and days aspartic acid) were furfural, naphthalene, butylated hydroxytoluene,(2-methyl-furan-),(1-Propanone,1-(2-furanyl)-),(Ethanone,1-(2-furanyl)-),(phenyl methoxy oxime) and (2-Isoamyl-6-methylpyrazine). The main nine volatile components from the reaction for leucine with xylose and fermentation products by Strain J503and B106were acetone,2-methyl furan,3-methyl n-butyl aldehyde,3-methyl butyric acid,5-methyl hexanone,6-methyl-2-4-methyl heptyl methadone, pentanoic acid,2-isoamyl-6-methyl pyrazine and butylated hydroxytoluene.The main eight volatile components from the reaction for glycine with arabinose and fermentation products by Strain J503and B106wereacetone aldehyde, furfural, benzaldehyde,1,5-dinitrogen miscellaneous-5-ketone, nonyl aldehyde and2,4-tertiary butyl phenol, phthalic acid different butyl octyl ester and butyl phthalate. According to the main content of aroma components, the pentose contents of fermentation by glucose were higher than those fermentation by mannose using J503and B106. The generated pentose order of all samples is (J503+glucose)>(J503+mannose)>(B106+glucose)>(B106+mannose)>(J503+B106+glucose)>(B106J503++mannose)
(3) Pentoses were more likely to have Maillard reaction than hexose, xylose were more likely to have Maillard reaction than arabinose, glucose were more likely to have Maillard reaction than mannose. The order of response intensity of color reaction of amino acid was Leu> Gly> Cys> Asp. The color of treated-group was lighter than control, the order of color difference was glucose and mannose fermentation by J503+B106> glucose and mannose fermentation by J503> glucose and mannose fermentation by B106.
(4) Electronic nose could distinguish volatile components of Maillard reaction by different pentose and four amino acids (cysteine, glycine, leucine, and aspartic acid) and glucose, mannose, and pumpkin juice fermentation by Bacillus subtilis (J5O3and B106).The cumulative contribution of all the samples between the two main components was78%-85%, which could effectively responded effectively induction value of the sample. The results determined by dimensionality reduction of PCA were similar with headspace solid phase micro-extraction analysis of volatile components.
(5) Glucose germentation by J503had the most protein species and content, the molecular weight of majority of the protein was below43KD, protein of26KD was;at the most content. Liquid-mass spectrometry (LC-MS) method was used to identify490kinds of pentose proteins which could participated in glucose conversion by J503. The database (NCBI/BLAST) matched seven proteins which may be involved in the transformation:①1,2-dihydroxy-3-keto-5-methylthiopentenedioxygenase [Bacillus amyloliquefaciens subsp. plantarum CAU B946],②XlyB [Bacillus amyloliquefaciens FZB42],③endo-xylanase [Bacillus subtilis subsp. inaquosorum KCTC13429],④sugar-phosphate epimerase/isomerase [Bacillus subtilis subsp. subtilis str.168],⑤arabinogalactan endo-1,4-beta-galactosidase [Bacillus amyloliquefaciens subsp. plantarum CAU B946],⑥glucuronoxylanase xynC [Bacillus amyloliquefaciens] and⑦PenP [Bacillus amyloliquefaciens FZB42]. The mechanism was deduced which converted six-carbon sugar pentose metabolism by strain J503.
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