【作者】 李积华；

【导师】 郑为完；

【作者基本信息】 南昌大学 ， 食品科学， 2007， 博士

【摘要】 绿豆汤在我国甚受欢迎，是我国人民消暑、解渴的主要传统饮品之一，然而目前市场上还没有令人满意的相关产品。本文以酶法水解为主要手段，开发两种绿豆饮料新产品——全绿豆速溶固体饮料和全绿豆仿乳固体饮料，并从实用与理论相结合的角度出发，就产品开发过程中的关键问题进行研究，主要研究结果如下：采用粗纤维测定法、中性洗涤纤维测定法、酸性洗涤纤维测定法以及改良的Southgate法分别测定了绿豆中的纤维含量，测定结果分别为5.812％，10.56％，6.921％和12.62％，测定结果从大到小依次为：改良的Southgate法＞中性洗涤纤维测定法＞酸性洗涤纤维测定法＞粗纤维测定法。首次分析了改良的Southgate法分析绿豆纤维素的具体条件，并分析了绿豆膳食纤维组成及其在绿豆皮和子叶中的分布情况：（1）绿豆含膳食纤维12.62％，其中水溶性非消化性多糖、水不溶性非纤维素多糖、纤维素及木质素含量分别为0.8473％、6.272％、4.361％和1.143％；（2）绿豆皮含膳食纤维64.56％，其中水溶性非消化性多糖、水不溶性非纤维素多糖、纤维素和木质素含量分别为9.063％、9.183％、32.84％和13.47％；（3）绿豆中豆皮的干物质含量为8.42％，但它含有绿豆43.07％的膳食纤维，而且绿豆水溶性非消化性多糖和木质素90％以上都来自豆皮。建立了绿豆在20℃～40℃时的吸水动力学模型：y=f（x，T）=（0.0697×T²-4.8181×T+134）ln（x）-0.5069×T²+36.631×T-851.65式中，y为吸水百分率；x为浸泡时间，单位为min；T为浸泡温度，单位为℃。对20℃、25℃、30^C、35℃、和40℃下模型吸水进程进行拟合所得的总体回归系数r²均大于0.99。吸水实验研究结果同时表明，无论采用室温浸泡还是高温浸泡，从吸水率角度考虑，热烫工序没有必要。首次研究了绿豆浸泡过程中Mg、Fe、Mn、Ca、Zn和Cu等微量元素的溶出情况。室温浸泡对Mg、Zn和Mn的含量没有明显影响，对Fe、Cu和Ca的含量影响相对明显但这种影响基本与浸泡时间及过程中绿豆状态（皮裂开与否）无关。热烫处理对浸泡过程中Fe和Ca的流失影响不显著，但对Mg、Cu、Zn及Mn的流失有加速作用，尤其加速Mg的损失，因此，从微量元素损失角度考虑，热烫工序不可取。水煮处理并没有很好地使绿豆中的微量元素溶于水中，即使煮沸45min至烂，绿豆中的Mn、Zn和Fe仍然很难进入水相，其提取率分别只有8.01％、18.0％、23.3％，其它三种微量元素除Mg之外，也有60％以上残留在渣中，因此，传统的“绿豆汁”工艺或只喝绿豆汤而弃其渣的习惯对绿豆中的微量元素利用率不高。确定了α-耐高温淀粉酶的最佳水解工艺条件，即：加酶量0.5％，酶解温度98℃，酶解时间3小时，pH6.2及底物浓度1∶8（绿豆∶水），淀粉提取率为96.34％。从氮收率和产物风味两个指标出发，考察了Alcalase 2.4L FG酶、复合蛋白酶、木瓜蛋白酶、风味蛋白酶对绿豆分离蛋白的水解特性。从水解度方面考虑，Alcalase 2.4L FG酶具有最高的氮收率，其次是木瓜蛋白酶、复合蛋白酶，风味蛋白酶的氮收率最低；从风味方面考虑，Alcalase 2.4L FG酶和木瓜蛋白酶水解产物均有苦味，而复合蛋白酶和风味蛋白酶水解产物无苦味；风味蛋白酶水解产物虽无苦味，却有明显的鲜味，不适合清爽型饮料开发。通过SAS软件进行响应面分析，建立了Alcalase 2.4L FG酶解绿豆蛋白工艺中酶浓度、水解温度、水解时间，底物浓度和pH值对水解度的数学模型（见表5.3）。Alcalase 2.4L FG酶水解绿豆蛋白工艺参数：酶浓度为0.21 mL·10 g^-1底物，反应温度54.7℃，反应时间1.52 hr，底物浓度11.28 g·100 mL^-1水，反应pH值8.8，在实验条件下，该酶催化水解绿豆蛋白反应的动力学参数如下：K_m=0.2057mol·L^-1，V_max=0.2001 mol·hr^-1·L^-1。以提取绿豆渣水溶性纤维素量为指标，考察了Viscozyme L、Celluclast 1.5L以及两种酶的互配混合酶的水解效果。在开始7小时水解时间内，Celluclast 1.5L和互配混合酶的作用效果比Viscozyme L更好，但在后面的水解时间里，Viscozyme L的水解效果明显更佳，因此，选择Viscozyme L酶为本实验用酶。建立了Viscozyme L酶解绿豆渣提取水溶性纤维素的数学模型，如下：RE=44.482727+0.3741665x₁-3.1308335x₂-0.56000x₅-0.537727x₁^*x₁-0.471250x₂^*x₁-1.967727x₂^*x₂-0.261477x₃^*x₃+0.59125x₄^*x₃+0.30875x₅^*x₂式中，RE为纤维素提取百分率，x₁、x₂、x₃、x₄、x₅分别是水解时间、水解温度、酶用量、水解pH和水与底物质量比等因素的标准化参数。优化了工艺参数：酶解时间25.4小时、温度41.5℃、加酶量[E]／[S]0.0223mL·g^-1豆渣、pH4.64、水体积／绿豆渣干重13.1倍，该条件下，绿豆渣纤维素提取率为49.39％。采用原子吸收法分析了绿豆渣中的微量元素含量和绿豆渣在Viscozyme L酶水解过程中各微量元素溶出情况：绿豆渣中的各微量元素含量比绿豆中的高得多，通过Viscozyme L酶解，提取出了绿豆渣中63.05％的Mg、31.82％的Fe、33.00％的Cu、55.55％的Ca、59.36％的Zn和61.04％Mn，从微量元素角度说明了在绿豆饮料开发过程中水解绿豆纤维素的必要性。通过GC／MS分析了绿豆汤及绿豆分步酶解过程中的香气成分。经NISTO2数据库检索，确定绿豆汤十二种香气成分，分别是1-乙酯基-z，z-10，12-十六二烯、苯并噻唑、1，3，5，7，9-五乙基-环五硅氧烷、1，1，3-三甲基-1-硅烷基-环丁烷、2-溴代乙醇、1-氟十六烷、1，3-二丙氧基-十八烷烃、2，4，6-三甲基葵烷、4，6-二-特-丁基-m-甲酚、3-乙基甲基胺-丙腈、9-十四碳烯酸、2，2，4，4，5，5，7，7-八甲基-3，6-二氧-2，4，5，7-四硅辛烷。研究结果表明，绿豆汤香气清淡的原因来自两方面：一是本身所含香气成分种类少，二是各香气组分含量低。对分步酶解产物香气成分的GC／MS分析结果表明，酶解对绿豆香气成分改变不多。绿豆经水煮、淀粉酶水解、淀粉酶水解+蛋白酶水解、淀粉酶水解+蛋白酶水解+纤维素酶水解等四种处理方式对绿豆总黄酮的提取率（溶出率）依次是0.1745％、0.2157％、0.2424％和0.3056％；相对水煮，三步酶解处理方式的黄酮溶出量依次增加了23.6％、38.9％和75.1％。通过对这四种处理方式所得产物中总黄酮的HPLC／MS谱图分析，结果表明：在绿豆汤中得到8个HPLC峰，分子量依次为323.9、684.5、341.2、263.8、164.7、448.1、432.1和432.1，其中第2、5、6、7、8分子量的峰在其它三步酶解产物黄酮中均出现。绿豆极其分步酶解过程中的微量元素变化分析结果表明：（1）通过酶解，绿豆中Se、Cu、Zn、Mn、Fe的总提取率依次为97.79％、68.84％、51.84％、63.97％和30.40％，而水煮对各微量元素的提取率依次只有19.26％、36.22％、17.58％、7.85％和22.99％；（2）绿豆酶解产物中Se主要以有机态形式存在，其中蛋白酶解产物中Se有机态含量最高，其次是淀粉酶解产物，酶解产物中Se的有机态分布系数为60％左右，远大于绿豆汤中Se的有机态分布系数（3.64％）；（3）绿豆酶解产物中Cu主要无机态形式存在，与绿豆汤中的存在形式相同；（4）绿豆酶解产物中Zn主要以有机态形分布系数为26.17％，绿豆汤中Zn的有机态分布系数只有6.64％，其中蛋白酶解产物中Zn有机态分布系数最高，其次是纤维素酶解产物；（5）绿豆汤中Mn元素几乎完全以无机态形式存在，而绿豆酶解产物中Mn的有机态形系数为14.76；（6）绿豆汤和绿豆酶解产物中Fe元素均以无机态形式存在。对绿豆及其分步酶解产物的氨基酸组成进行了分析，结果表明：（1）绿豆中17种氨基酸总含量为18.41％，其中含量在1.0％以上氨基酸的有天门冬氨酸、谷氨酸、精氨酸、缬氨酸、亮氨酸、苯丙氨酸、赖氨酸；蛋氨酸、半胱氨酸和组氨酸含量最低，不到0.1％；（2）总体上，氨基酸分布在蛋白酶解产物中的含量最高，其次是淀粉酶解产物，在残渣中的含量很低，仅占总氨基酸的2.1％，说明通过酶解工艺，氨基酸得到充分利用；（3）工艺加工给氨基酸带来了一定损失，损失比较严重的几种氨基酸依次是赖氨酸（50.97％）、丝氨酸（28.79％）、苯丙氨酸（20.11％）、甘氨酸（13.58％）和半胱氨酸（10.32％），其它氨基酸损失不显著，在10％以内；但总氨基酸的损失只有7.92％。采用HPLC对绿豆蛋白酶解产物分子量分布进行了分析：85％的蛋白酶解产物出峰时间在32.9min，分子量在301～612范围，产物苦味不重。采用正交试验设计优化了绿豆固体饮料调味配方，即：基料粉用量10％、蔗糖用量3％、多孔淀粉用量0.05％。采用正交试验设计优化了绿豆固体饮料的喷雾干燥条件，即：进风温度170℃，进料速度22.0mL·min^-1，气流压力0.07Mpa，出风温度为95℃。产品呈亮黄绿色，清爽可口，甜香宜人，绿豆利用率达到92.1％。探讨了Alcalase 2.4LFG酶水解米渣蛋白的工艺条件（深度水解）为：温度T=60℃，pH=9，酶浓度（[E]／[S]）=0.033 mL·g^-1渣，底物浓度=0.0833 g·mL^-1，水解时间=1 h，该实验条件下，Alcalase 2.4L FG是一种典型的蛋白构象比较稳定的米氏酶，该酶催化水解米渣蛋白反应的动力学参数如下：K_m=0.6598 mol·L^-1，V_max=0.00351 mol·min^-1·L^-1。探讨了Alcalase 2.4LFG有限水解米渣蛋白的工艺条件（低度水解）：温度T=60℃，pH=9.0，酶浓度为0.003 mL·g^-1渣，底物浓度10 g渣／120mL水，水解时间约为30min。在实验条件下，该酶催化反应的底物临界浓度为22.25g渣／120mL。在一定水解条件(0.003 mL·g^-1渣、pH值9.0、温度为60℃、反应时间不大于25min)下，Alcalase 2.4LFG酶催化有限水解米渣蛋白反应符合如下动力学模型：x=1／（37.76846）ln[1+（-0.0008[S]₀+0.0178）×37.76846×t]式中，x为水解度（％），[S]₀为底物初始浓度(g·mL^-1)，t为水解时间（min）。通过有限水解，得到了高乳化性的米渣蛋白肽。研究了新型绿豆仿乳固体饮料制备工艺，建立了以“自制的酶法有限水解米渣蛋白肽（DH=4.59）+阿拉伯胶+单甘酯+蔗糖酯+大豆磷脂”为复合乳化剂的新乳化体系，其中米渣蛋白肽完全替代了价格昂贵的酪朊酸钠。优化了各工艺参数，并通过中试生产制备了新型全绿豆仿乳固体饮料（如图10．10），油脂包埋率达到95％以上。对所制备的全绿豆仿乳固体饮料新产品的理化指标、有害重金属元素含量、微生物指标进行了测定分析，结果表明，各项指标均符合相关要求。采用分形理论对SEM电子束轰击粉末油脂微胶囊表面产生应力裂纹进行了分析：（1）应力裂纹具有良好自相似性，可以采用分形维数进行定量分析；（2）在实验设计条件下，粉末油脂微胶囊经SEM电子束轰产生应力裂纹盒子维数与相应产品的油脂包埋率存在非常显著的二次关系，其中相关系数R²=0.9969。这对微胶囊粉末油脂的质量评价和制备机理研究具有重要意义。更多还原

【Abstract】 Mungbean soup is one of the most primary traditional drinks to remove summer-heat and to quench the thirst and is greatly popular in our country. But still, there is no good cognate product in the market. In this text, two new kinds of mungbean drinks were developed with enzymatic treatments: one is the whole mungbean solid beverage and the other is the whole mungbean juice-like milk solid beverage. And also, some significant problems in the process were discussed. The main results were as follows.Dietary fiber in mungbean was analyzed by four methods, such as crude fiber method, neutral detergent fiber method （NDF）, acid detergent fiber method （ADF） and improved Southgate method. The results showed significant differences among these methods. The sequence of total fiber content from high to low was as follows: improved Southgate method> neutral detergent fiber method> acid detergent fiber method> crude fiber method. The improved Southgate method determining conditions were firstly studied with the mungbean as the substrate. The soluble non-digestive polysaccharide, insoluble non-digestive polysaccharide, cellulose, and lignin in mungbean and its spermoderm were determined by the improved Southgate method. The results were 0.8473%, 6.272%, 4.361%, 1.143% respectively and 9.063%, 9.183%, 32.84%, 13.47% respectively.The mungbean water absorbing kinetic model in 20℃to 40℃range was established as follow:y=f（x, T） = （0.0697×T² - 4.8181×T + 134） ln（x） -0.5069×T² + 36.631×T - 851.65 where y is the water absorbing percentage, x is soaking time in minutes, T is soaking temperature in centigrade. Regression coefficients in fitting experiments were above 0.99. Influence of blanching on water absorbing percentage was also studied and proved no significant value to improve the percentage.Dissolution properties of Mg, Fe, Mn, Ca, Zn, and Cu elements from mungbean in the course of soaking were studied. In room temperature, parts of Fe, Cu and Ca released in water when soaking, but the amounts got no significant influences of soaking time and seed states, whether its coat broken or not. While little Mg, Zn and Mn released in water in the course of soaking. Blanching accelerated dissolution of Mg, Cu, Zn and Mn, especially of Mg. Cooking treatment didn’t release great amount of trace elements in water, except Mg. Only 8.01% Mn, 18.0% Zn and 23.3 %Fe were in water after 45 min boiling treatment.Optimization of mungbean starch hydrolysis conditions withα- amylase was studied by orthogonal design experiment with the following results, pH was 6.2, the hydrolytic time was 3h, temperature was 98℃, the substrate concentration was 1:8（mungbean to water）, respectively. Starch extraction rate was 96.34%.The hydrolysis properties of mungbean protein with Alcalase 2.4L FG, protamex, caroid and flavourzyme were studied on the basis of nitrogen yield and product flavor. Alcalase 2.4L FG proved the first choice when nitrogen yield was considered, then were caroid, protamex and flavourzyme respectively. To product flavor aspect, Alcalase 2.4L FG and caroid got bitter-tastes while the others didn’t. Protamex was relatively better than flavourzyme as a non-bitter-taste protease in developing bean beverage. Because the latter brought other uncomfortable taste. In short, Alcalase 2.4L FG was suited for getting highnitrogen yield and protamex was suited for improving flavor in mungbean protein hydrolysis.A mungbean protein hydrolysis kinetic model of Alcalase 2.4L FG was established with Second Order Regression Rotation Designing in SAS analysis System. The optimum hydrolysis conditions were got by Ridge of Maximum Response analysis with the following results, pH was 8.8, the hydrolytic time was 1.52h, temperature was 54.7℃, the substrate concentration was 11.28 g·100 ml^-1, the enzyme concentration was 0.21 ml·10 g^-1. the kinetic parameters of Km and Vmax were 0. 2057mol·L^-1 and 0. 2001 mol·hr^-1·L^-1.The hydrolysis characters of fiber in mungbean residues were studied with Viscozyme L, Celluclast 1.5L and compound of the two on the primary goal to improve fiber solubility. Viscozyme L behaved the best one in total. The kinetic model of the process of extraction soluble fibre from mungbean residue with Viscozyme L was established with Second Order Regression Rotation Designing in SAS analysis system and the result was as follow.RE= 44.482727 + 0.3741665x₁ - 3.1308335x₂ - 0.56000x₅ - 0.537727x₁*x₁ - 0.471250x₂*x₁ - 1.967727x₂*x₂ - 0.261477x₃*x₃ + 0.59125x₄*x₃ + 0.30875x₅*x₂ where RE was the soluble fiber extraction rate, x1, x2, x3, x4, and x5 stood for the normalized parameters of reaction time, temperature, enzyme concentration, pH and substrate concentration, respectively. The optimum hydrolysis conditions were as follows, pH was 4.6, the hydrolytic time was 25.4h, temperature was 41.5℃, the substrate concentration was 1:13.1 （residue to water）, the enzyme concentration was 0.0223 ml·g^-1 and the soluble fibre extraction rate was 49.39% in the repeated experiment. Also, the shift on the trace elements in the residue during the course of the process was analyzed. The extraction rates of trace elements were as follows, Mg 63.05%, Fe 31.82%, Cu 33.00%, Ca 55.55%, Zn 59.36%, Mn 61.04%.Twelve aroma compounds in mungbean were determined by GC/MS analysis and NIST02 index library. They were z, z-10, 12-Hexadecadien-1-ol acetate, benzothiazole, 1, 3, 5, 7, 9-Pentae-thylcyclopentasiloxane, 1, 1, 3-Trimethyl-1-silacyclo-butane, 2-bromo-ethanol, 1-flouro-dodecane, 1, 3-propanediylbis- octadecane, 2, 4, 6-trimethyl-decane, 4, 6-di-tert-butyl-m-cresol, 3-ethylmethy- lamin-propane-nittrile, myfistoleic acid and 2, 2, 4, 4, 5, 5, 7, 7-octamethyl-3, 6-dioxa-2, 4, 5, 7- tetra-silaoctane. It depended on both the lack of the quantity and content of aroma compounds which made mungbean aroma taste light. Aroma analysis of the enzymatic hydrolysates showed little change in aroma with enzymatic treatment.The extraction rates and HPLC/MS spectrum of total flavonoids were analyzed in mungbean with different treatments, such as cooking, amylase hydrolysis, amylase and protease hydrolysis, amylase and protease and cellulose hydrolysis. Total flavonoids extraction rates of the four treatments were 0.1745%, 0.2157%, 0.2425% and 0.3056%. To cooking treatment, the other enzymatic ones improved extraction rates over 23.6%, 38.9%, 75.1%. Eight peaks were got in the HPLC spectrum of flavonoids in mungbean soup and their molecular masses were analyzed to be 323.9, 684.5, 341.2, 263.8, 164.7, 448.1, 432.1 and 432.1, respectively. Among these, peak2, peak5, peak6, peak7 and peak8 appeared in other samples with enzymatic treatments.Trace elements speciation distributions in mungbean during enzymatic hydrolization were investigated with AFS-230E atom fluorescence photometer. The results showed: （1） Se, Cu, Zn, Mn and Fe elements total extraction rates by enzymatic hydrolization were 97.79%, 68.84%, 51.84%, 63.97% and 30.40%, respectively. （2） Se was mostly in organic form in the hydrolysates. Organic speciation was primarily in protease hydrolysate, and then in amylase hydrolysate. The distribution coefficient of Se in organic form was about 60 % in total enzymatic product and 3.64 % in mungbean soup. （3） Cu was in inorganic form in both mungbean hydrolysate and soup. （4） The distribution coefficient of Zn in organic form was about 26.17 % in total enzymatic product and 6.64 % in mungbean soup. Zn organic speciation was primarily in protease hydrolysate, and then in cellulase hydrolysate. （5） Mn was mainly in inorganic form in mungbean soup while there was 14.76% in total hydrolysates. （6） Fe was in inorganic form in both mungbean hydrolysate and soup.Amino acid compositions in mungbean and hydrolysis products were investigated. （1） Total amino acids content in mungbean was 18.14%. Aminouccinic acid, glutamic acid, arginine, aminoisovaleric acid, leucine, phenylalanine and lysine had contents above 1.0%. Methionine, halfcystine and histidine got lowest contents which were under 0.1%. （2） In total, most amino acids were. in protease hydrolysate, then were in amylase hydrolysate. Only 2.1% total amino acids was in the hydrolysis residue, which showed high utilization rate of amino acids by enzymatic hydrolysis process. （3） The process load to some loose in lysine（50.97）, serine（28.79）, phenylalanine（20.11%）, glycocine（13.58） and halfcystine（10.32%）. But total loose rate was only 7.92%.Exclusion chromatography analysis showed that 85% mungbean protein hydrolysate molecular weight rage was in 301 to 612, wthich indicated that mungbean protein was in oligopeptide form in product.Flavouring formula was optimized by orthogonal experiment design with results as follows, 10% mungbean hydrolysate, 3% sugar and 0.05% poIyporate starch. Spray drying process was also optimized by orthogonal experiment design with results as follows, inlet air temperature 170℃, outlet air temperature 95℃, feeding rate 22.0mL·min^-1, air pressure 0.07MPa. The new product of whole mungbean beverage was relaxed and palatable, and with comfortable sweetness and aroma. The mungbean utilization rate in the whole process was 92.1%.The hydrolysis characters of rice residue protein were studied with Alcalase 2.4L FG in high hydrolysis degree and with the results as follows, pH was 9.0, the hydrolytic time was 1h, temperature was 60℃, the substrate concentration was 0.0833 g·ml^-1, the enzyme concentration was 0.5 ml·g^-1（the enzyme was diluted to 10% concentration）, the kinetic parameters of Km and Vmax were 0.6598mol·L^-1 and 0.00351mol·hr^-1·L^-1.A rice residue-protein hydrolysis kinetic model of Alcalase 2.4L FG was established with Second Order Regression Rotation Designing in SAS analysis system. The optimum hydrolysis conditions were got by Ridge of Maximum Response analysis with the following results, pH was 8.8, the hydrolytic time was 1.52h, temperature was 60℃, the substrate concentration was 11.28 g·100 ml^-1, the enzyme concentration was 0.21 ml·10 g^-1. the kinetic parameters of Km and Vmax were 0.2057mol·L^-1 and 0.2001 mol·hr^-1·L^-1.The hydrolysis characters of fiber in mungbean residues were studied with Viscozyme L, Celluclast 1.5L and compound of the two on the primary goal to improve fiber solubility. Viscozyme L behaved the best one in total. The kinetic model of the process of extraction soluble fibre from mungbean residue with Viscozyme L was established with Second Order Regression Rotation Designing in SAS analysis system and the result was as follow.The characters of rice residue protein limited hydrolysis were studied with Alcalase 2.4L FG in light hydrolysis degree and with the results as follows, pH was 9.0, the hydrolytic time was 30min, temperature was 60℃, the substrate concentration was 10 g·120ml^-1water, the enzyme concentration was 0.03 ml·120ml^-1 water（the enzyme was diluted to 10% concentration）. The critical concentration of the substrate was 22.25 g·120ml^-1water. A limited hydrolysis model of rice residue protein was established as follow.where x stood for hydrolysis degree, [S]₀ was substrate concentration, t was the reaction time.The rice protein peptid with good emulsibility was got with limited hydrolysis process.The process of new whole mungbean juice-like milk solid beverage was discussed and a pragmatic process flow was got in fig. 10. 10. In this process, a new emulsification system was established with rice residue peptide （DH=4.59） as the protein emulsifier. A good quality product was got in semi-works production with a microencapsulation efficiencie over 95%.The fractral analysis on cracks in microcapsule of powder oil occurred during SEM electron bean scanning was carried out. （1） The cracks could be regarded as having self-similarity, and could be studied according to fractal theory. （2） The fractal dimensions of the cracks showed second order relationship with the microencapsulation efficiencies of the products and the coefficient correlation was 0.9969. It indicated a new load to study on the quality evaluation and process mechanism of powder oil.更多还原