|Veröffentlichungsdatum||2. Apr. 2014|
|Eingetragen||20. Nov. 2013|
|Prioritätsdatum||20. Nov. 2013|
|Auch veröffentlicht unter||CN103695515B|
|Veröffentlichungsnummer||201310589684.9, CN 103695515 A, CN 103695515A, CN 201310589684, CN-A-103695515, CN103695515 A, CN103695515A, CN201310589684, CN201310589684.9|
|Erfinder||王丰俊, 吕佼, 王建中|
|Zitat exportieren||BiBTeX, EndNote, RefMan|
|Patentzitate (1), Nichtpatentzitate (3), Klassifizierungen (1), Juristische Ereignisse (3)|
|Externe Links: SIPO, Espacenet|
制备板栗蛋白质的方法 Chestnut protein preparation methods
 本发明涉及制备板栗蛋白质的方法。  The present invention relates to a method for preparing chestnut protein.
 板栗中含蛋白质5%~11%，含量高于稻米，并且赖氨酸、异亮氨酸、蛋氨酸、半胱氨酸、苏氨酸，缬氨酸、苯丙氨酸、酪氨酸等氨基酸的含量超过FA0/WH0的标准，而赖氨酸是水稻，小麦，玉米，和大豆类的第一限制性氨基酸，苏氨酸是水稻，小麦的第二限制性氨基酸，色氨酸和蛋氨酸分别是玉米和豆类的第二限制性氨基酸。  chestnut containing 5% to 11% protein content is higher than rice, and lysine, isoleucine, methionine, cysteine, threonine, valine, phenylalanine, tyrosine amino acid content than FA0 / WH0 standards, and lysine is the first limiting amino acid in rice, wheat, corn, and soy, and threonine are rice, wheat second limiting amino acid, tryptophan and are the second limiting amino acid methionine corn and beans. 由此可见，食用板栗或者直接食用板栗蛋白质可以补充禾谷类和豆类中限制氨基酸的不足，可使膳食的营养成分更加完善ο Thus, the direct consumption of edible chestnut chestnut or protein supplement cereals and legumes limiting amino acid deficiency, dietary nutrients can be more perfect ο
 然而，现阶段提取制备板栗蛋白质的方法仍有待改进。  However, the method of preparation stage chestnut extract proteins could be improved.
 本发明是基于发明人的下列发现而完成的:  The present invention is based on the following findings of the inventors accomplished:
 国内外对板栗蛋白的提取方法主要有两种，一种是传统的碱水法，这种方法的缺点是提取率低。  chestnut protein extraction methods at home and abroad are mainly two types, one is the traditional alkaline method, the disadvantage of this method is to extract low. 另一种是亚临界水萃取法，这种方法的缺点是提取蛋白的过程容易造成蛋白的变性。 Another is subcritical water extraction method, the disadvantage of this method is the process of extracting the protein is likely to cause protein denaturation.
 本发明旨在至少解决现有技术中存在的技术问题之一。  The present invention aims to solve the technical problems of the prior art, at least the presence of one. 为此，本发明的一个目的在于提出一种提取率高、蛋白活性好的制备板栗蛋白的方法。 To this end, an object of the present invention is to propose a high extraction rate, good protein activity chestnut protein preparation methods.
 根据本发明的一个方面，本发明提出了一种制备板栗蛋白的方法。  In accordance with one aspect of the present invention, the present invention provides a method for preparing a protein of chestnut. 根据本发明的实施例，该方法包括以下步骤:将板栗粉水溶液进行超声波破碎处理，以便获得经过超声波破碎处理的板栗粉水溶液；利用纤维素酶对所述经过超声波破碎处理的板栗粉水溶液进行第一酶解处理，以便获得第一酶解产物；利用第一α -淀粉酶将所述第一酶解产物进行第二酶解处理，以便获得第二酶解产物；将所述第二酶解处理产物进行碱提处理，以便获得碱提产物；将所述碱提产物进行第一离心，并收集上清液，以便获得蛋白质碱提液；利用第二α -淀粉酶将所述蛋白质碱提液进行第三酶解处理，以便获得第三酶解产物；利用等电点沉淀法将所述第三酶解产物进行酸沉，以便获得酸沉产物；将所述酸沉产物依次进行静置和第二离心，以便收集获得沉淀；以及将所述沉淀进行水洗至中性，以便获得所述板栗蛋白质。 According to an embodiment of the invention, the method comprising the steps of: an aqueous solution of chestnut flour ultrasonic disintegration treatment, so as to obtain an aqueous solution of chestnut flour after sonication treatment; use of cellulase on the chestnut powder solution through an ultrasonic crushing treatment for the first an enzymatic treatment, in order to obtain a first hydrolyzate; using the first α - amylase the first hydrolyzate second enzymatic treatment, in order to obtain a second hydrolyzate; the second enzyme processed products were alkaline extraction process in order to obtain alkali extraction product; the product was first centrifuged alkaline extraction and the supernatant was collected, in order to obtain a protein extract base; by the second α - amylase mention the protein base Third liquid enzymatic treatment, in order to obtain a third hydrolysates; isoelectric precipitation method utilizing the third hydrolysates acid precipitation, acid precipitation in order to obtain a product; the product of the acid precipitation in turn stand and a second centrifugation, in order to obtain a precipitate was collected; and the precipitate was washed with water until neutral, so as to obtain the chestnut protein. 发明人惊奇地发现，利用本发明的方法能够有效提取制备板栗蛋白，并且板栗蛋白提取率高，活性好。 The inventors have surprisingly found that the use of the method of the present invention can effectively prepare chestnut extract protein, and chestnut protein extraction rate, good activity. 具体地，本发明的方法利用超声波和纤维素酶进行酶解作用促进板栗蛋白的溶出，且利用α-淀粉酶酶解破坏蛋白质(在本文中有时也将“蛋白质”简称为“蛋白”)与淀粉的包络作用，从而有效提高了板栗蛋白的提取率，蛋白提取率可高达66%，相对于传统方法提高了20%，并且获得的板栗蛋白中蛋白含量可高达88%，纯度高，质量好，并且由于提取条件温和，能够很好地保持板栗蛋白的活性。 In particular, the method of the present invention is the use of ultrasonic and cellulase enzymatic hydrolysis promoting dissolution chestnut protein, and the use of α- amylase hydrolysis disrupt protein (also sometimes herein "protein" referred to as "protein") and the envelope of the starch, which effectively increases the protein extraction rate of chestnut, protein extraction rate can be as high as 66%, compared to traditional methods increased by 20%, and get chestnut protein up to 88% protein content, high purity, quality good, and due to the mild extraction conditions, can well retain activity chestnut protein.
 另外，根据本发明上述实施例的制备板栗蛋白质的方法还可以具有如下附加的技术特征: 需要说明的是，适用于本发明的原料包括鲜板栗、干燥过的板栗、板栗粉。  Further, the method for preparing the protein of the present invention, the above-described chestnut embodiment may also have the following additional technical features:  It should be noted that the starting material useful in the present invention include fresh chestnut, dried chestnut, chestnut powder. 本发明的方法中所采用的主要萃取剂为去离子水。 The main method of extraction agent used in the present invention is deionized water. 原料的品种、品质以及蛋白的含量，对终产品的品质与得率将产生一定的影响，但均比采用现有技术制备板栗蛋白质的效率高，品质好。 Variety, quality and protein content of the raw materials, the quality and yield of the final product will have some impact, but high art than chestnut protein preparation efficiency, good quality.
 根据本发明的实施例，于400w超声功率下，进行超声波细胞破碎处理6_14min，优选lOmin。  According to an embodiment of the invention, at 400w ultrasonic power, ultrasonic cell disruption treatment 6_14min, preferably lOmin. 由此，能够使细胞结构松动，加强后续纤维素酶的作用效果。 Thereby, the cell structure is loose, the effect of strengthening the role of the follow-up of cellulases. 根据本发明的实施例，所述板栗粉水溶液的料液比为Ig:15ml。 According to embodiments of the present invention, the chestnut flour aqueous liquid ratio of Ig: 15ml. 换句话说，所述板栗粉水溶液是通过将板栗粉和水按照Ig:15ml的比例混合配制获得的。 In other words, the water chestnut flour is produced by chestnut flour and water in accordance with the Ig: 15ml mixing ratio of the preparation obtained. 由此，有利于后续蛋白的溶出和提取。 Thereby, beneficial dissolution and subsequent protein extraction. 此外，需要说明的是，前述的板栗粉可以通过以下方法制备获得:选用无霉变虫蛀的新鲜板栗为原料，经过去皮、切片、冷冻干燥后经粉碎机粉碎，过60目筛制成板栗粉。 In addition, you need to note is that the aforementioned chestnut powder can be prepared by obtaining: no mildew infestation selection of fresh chestnut as raw material, peeled, sliced, freeze-dried after crushing mill, is made through a 60 mesh sieve chestnut flour.
 根据本发明的实施例，于40-60摄氏度优选50摄氏度，PH4-6优选pH4.5条件下进行所述第一酶解处理5-15min，优选lOmin。  According to an embodiment of the invention, carried out at 40-60 degrees Celsius preferably 50 degrees Celsius, PH4-6 preferably pH4.5 conditions the first enzymatic treatment 5-15min, preferably lOmin. 由此，能够有效促进板栗蛋白溶出，且能够保持蛋白活性。 Thereby, it is possible to effectively promote chestnut protein eluted, and can maintain protein activity. 根据本发明的另一些实施例，进行所述第一酶解处理时，所述纤维素酶与所述经过超声波破碎处理的板栗粉水溶液的比例为50-150U/g，优选100U/g。 According to further embodiments of the present invention, when performing the first enzymatic treatment, the proportion of the cellulase solution with the chestnut powder after ultrasonic disruption treatment is 50-150U / g, preferably 100U / g. 由此，能够有效破坏细胞壁促进蛋白溶出，有利于后续的蛋白提取。 This effectively destroy the cell wall protein to promote dissolution, in favor of the subsequent protein extraction.
 根据本发明的实施例，于30-50摄氏度优选40摄氏度，pH5.5-6.5优选pH6.1条件下进行所述第二酶解处理25-35min，优选30min。  According to an embodiment of the invention, carried out at 30-50 degrees Celsius preferably 40 degrees Celsius, pH5.5-6.5 preferably pH6.1 conditions of the second enzymatic treatment 25-35min, preferably 30min. 由此，能够使板栗淀粉有效酶解，进而能够有效解除淀粉对蛋白质的包络，从而有利于蛋白溶出。 This enables effective chestnut starch hydrolysis, thus can effectively relieve starch to protein envelope, thus contributing to protein dissolution. 根据本发明的另一些实施例，进行所述第二酶解处理时，所述第一α -淀粉酶与所述第一酶解产物的比例为80-160U/g，优选120U/g。 According to further embodiments of the present invention, when performing the second enzymatic treatment, the first α - amylase and the ratio of the first hydrolyzate was 80-160U / g, preferably 120U / g. 由此，能够有效提高板栗淀粉的酶解效率，去除淀粉对蛋白质的包络，且能够保持蛋白质的活性。 Thereby, it is possible to effectively improve the efficiency of chestnut starch hydrolysis, removal of starch envelope protein, and can maintain activity of the protein.
 根据本发明的一些优选示例，于40摄氏度下，按照120U/g的比例利用第一α -淀粉酶进行所述第二酶解处理30min。  According to some preferred examples of the present invention, at 40 degrees Celsius, according to the ratio of 120U / g using a first α - amylase digestion of the second processing 30min. 由此，淀粉酶解效率高，对蛋白质的包络少，且能够保持蛋白质的结构和活性。 As a result, high-starch hydrolysis efficiency, less envelope proteins, and to maintain the structure and activity of proteins.
 根据本发明的实施例，所述碱提处理包括:将所述第二酶解处理产物进行pH调节至pH为8.0-10.0优选10.0,并于30-50摄氏度优选40摄氏度下进行所述碱提25_35min，优选30min。  According to an embodiment of the invention, the alkaline extraction treatment comprising: the product of the second enzymatic treatment the pH was adjusted to a pH of 8.0 to 10.0 preferably 10.0, and the at 30-50 degrees Celsius preferably 40 degrees Celsius said base mention 25_35min, preferred 30min. 由此，能够提高蛋白的溶出率，获得富含蛋白质的碱提产物。 Thereby, it is possible to improve the dissolution rate of the protein, protein-rich alkaline extraction to obtain the product. 根据本发明的一些具体示例，将所述第二酶解处理产物进行PH调节至ρΗΙΟ.0，并于40摄氏度下进行所述碱提30min。 According to some specific examples of the present invention, the second product was subjected to enzymatic treatment PH was adjusted to ρΗΙΟ.0, and carried out at 40 ° C the alkali extraction 30min. 由此，能够使蛋白高效溶出。 This enables efficient elution of the protein.
 根据本发明的实施例，可以将经过第一离心后获得的滤渣加去离子水洗涤、超声破碎后再离心，然后收集上清液合并入所述蛋白质碱提液，以便提高板栗蛋白的提取率。  According to an embodiment of the invention, the filter residue can be obtained after the first centrifugation was washed with deionized water, sonicated and then centrifuged, the supernatant was collected and then incorporated into the base of the protein extract, in order to improve protein chestnut The extraction rate.
 需要说明的是，发明人发现，蛋白质碱提液中淀粉含量仍然较高，因而，本发明的方法在蛋白质碱提液中加入淀粉酶水解其中的淀粉，以便蛋白的分离。  Note that, the inventors have found that alkaline protein extract starch content is still high, and therefore, the process of this invention is added amylase hydrolyze the starch in the protein extract in a base, in order to separate proteins. 根据本发明的实施例，于30-50摄氏度优选40摄氏度，pH5.5-6.5优选pH6.1条件下进行所述第三酶解处理25-35min，优选30min。 According to an embodiment of the invention, carried out at 30-50 ° C preferably 40 ° C, pH5.5-6.5 preferably pH6.1 condition said third enzymatic treatment 25-35min, preferably 30min. 由此，能够有效打破淀粉对蛋白的包络作用，方便后续酸沉的进行。 This can effectively break the starch envelope protein to facilitate the subsequent conduct acid precipitation. 根据本发明的实施例，进行所述第三酶解处理时，所述第二α-淀粉酶与所述蛋白质碱提液的比例为80-160U/g，优选120U/g。 According to an embodiment of the invention, when said third enzymatic treatment, the proportion of the second α- amylase and alkaline extract of the protein of 80-160U / g, preferably 120U / g. 由此，板栗淀粉溶解效率高，对包络板栗蛋白质的淀粉去除效果好，且不会破坏蛋白质的活性。 Thus, the high chestnut starch dissolved efficiency, good for the envelope proteins chestnut starch removal, and will not destroy the activity of the protein.
 根据本发明的实施例，所述酸沉是在等电点pH4.2条件下进行的。  According to an embodiment of the invention, the acid precipitation at the isoelectric point is carried out under conditions of pH4.2. 由此，能够使板 Thereby, the plate
栗蛋白质有效沉淀。 Li protein effective precipitation.  根据本发明的实施例，该制备板栗蛋白质的方法进一步包括将所述板栗蛋白进行真空冷冻干燥。  According to an embodiment of the invention, the protein preparation chestnut method further comprises the protein was subjected to vacuum freeze-dried chestnut. 由此，能够有效获得板栗蛋白粗提取物。 This makes it possible to efficiently obtain a crude extract protein chestnut. 具体地，根据本发明的一些实施例，利用真空冷冻干燥机进行所述真空冷冻干燥。 Specifically, according to some embodiments of the invention, by a vacuum freeze-drying machine of the vacuum freeze-drying. 由此，能够有效实现真空冷冻干燥，获得板栗蛋白粗提取物。 Thus, the effective realization of vacuum freeze-dried to obtain a crude extract protein chestnut.
 根据本发明的一些具体示例，本发明的制备板栗蛋白质的方法还可以包括以下步骤:将板栗粉与水按1:15( g/mL)比例溶解获得板栗粉水溶液，然后将其于超声波功率400W下进行超声波破碎处理lOmin。  According to some specific examples of the present invention, a method for preparing chestnut protein present invention may further comprise the steps of: chestnut flour and water by 1:15 (g / mL) ratio of aqueous chestnut powder obtained was dissolved, and then in ultrasonic disintegration treatment lOmin ultrasonic power 400W. 接着,按100U/g的比例向经过超声波破碎处理的板栗粉水溶液中加入纤维素酶，调PH到4.5，放入50°C恒温水浴搅拌酶解lOmin。 Then, according to the proportion of 100U / g of the aqueous solution after sonication treatment chestnut flour added cellulase, adjust PH to 4.5, into the 50 ° C water bath with stirring enzymatic lOmin. 然后调pH=6.1，加入低温α-淀粉酶，酶添加量为120U/g，并于40°C下继续保温搅拌酶解30min，接着，用Imol/mLNaOH调pH到10.0后，放入40°C水浴锅中浸提30min，然后，离心取上清液，调上清液的PH至6.1后，按120U/g的酶添加量加入低温α -淀粉酶，并于40°C下继续保温搅拌酶解30min后，在等电点pH4.2条件下进行酸沉，离心取沉淀，水洗沉淀至中性后即得板栗蛋白质，然后将其真空冷冻干燥保存。 Then adjust pH = 6.1, was added cold α- amylase enzyme dosage 120U / g, and at 40 ° C under stirring and incubation was continued hydrolysis 30min, followed by Imol / mLNaOH pH adjusted to 10.0, into the 40 ° C water bath extraction 30min, then centrifuged and the supernatant, the supernatant was adjusted to 6.1 after PH, according to 120U / g of enzyme dosage adding low α - amylase, and continue incubation at 40 ° C under stirring After digestion 30min, at the isoelectric point of pH4.2 acid precipitation conditions, precipitation centrifugation, washed to neutrality after precipitation was chestnut protein, and then vacuum freeze drying preservation.
 需要说明的是，相对于传统方法，本发明的制备板栗蛋白质的方法着重考虑在提高板栗蛋白提取率的同时保护板栗蛋白的结构不被破坏，通过α-淀粉酶、纤维素酶和超声波细胞破碎仪的作用，促进板栗蛋白的溶出，减少了板栗淀粉的残留，并且由于提取条件温和，能够有效保持蛋白质的活性，从而有利于生物活性和功能性蛋白质的后续研究及工业化生产。  It should be noted that, with respect to the traditional methods, the method for preparing a protein chestnut important consideration of the present invention is to improve the structure of protein extraction rate chestnut while protecting chestnut protein is not destroyed by α- amylase, cellulase and ultrasonic cell disrupter role in promoting the dissolution chestnut protein, reducing the residual chestnut starch, and because mild extraction conditions, can effectively maintain the activity of the protein, which is conducive to biological activity and subsequent functional protein and industrial production.
 本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。  Additional aspects and advantages of the invention will be given in part in the description which follows, from the following description in part will become apparent, or learned by practice of the invention.
附图说明 Brief Description
 本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中:  The foregoing and / or additional aspects and advantages of the invention will be described in embodiments in conjunction with the following drawings for become apparent from and be readily understood, in which:
 图1显示了根据本发明一个实施例的制备板栗蛋白质的方法的流程示意图。  Figure 1 shows a schematic diagram of a process for preparing a protein chestnut one embodiment of the invention a method. 具体实施方式 DETAILED DESCRIPTION
 下面将结合实施例对本发明的方案进行解释。  below with reference to examples of the present invention will be explained. 本领域技术人员将会理解，下面通过参考附图描述的实施例是示例性的，仅用于说明本发明，而不应视为限定本发明的范围。 Those skilled in the art will appreciate that the following examples described with reference to the accompanying drawings are exemplary only to illustrate the invention and should not be construed as limiting the scope of the present invention. 实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。 Embodiments does not indicate the specific technical or conditions, or conditions in accordance with the technical literature in the art or described in accordance with product instructions. 所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。 The reagents or equipment manufacturers were not indicated, are conventional commercially available products can be obtained.
 一般方法:  general approach:
 参照图1，根据本发明的实施例，本发明的制备板栗蛋白质的方法一般包括以下步骤:  Referring to FIG. 1, according to an embodiment of the invention, a method for preparing chestnut proteins of the invention generally comprises the steps of:
 首先，将板栗粉水溶液进行超声波破碎处理，以便获得经过超声波破碎处理的板栗粉水溶液。  First, an aqueous solution of the chestnut powder subjected to ultrasonic crushing treatment so as to obtain an aqueous solution of chestnut flour after sonication treatment.
 其次，利用纤维素酶对所述经过超声波破碎处理的板栗粉水溶液进行第一酶解处理，以便获得第一酶解产物。  Secondly, the use of cellulase on the chestnut flour solution after sonication treatment were first enzymatic treatment, in order to obtain the first hydrolyzate.
 再次，利用第一α -淀粉酶将所述第一酶解产物进行第二酶解处理，以便获得第二酶解产物。  Once again, using the first α - amylase the first hydrolyzate second enzymatic treatment, in order to obtain a second hydrolyzate.
 接着，将所述第二酶解处理产物进行碱提处理，以便获得碱提产物。  Next, the second product was subjected to enzymatic treatment alkali extraction treatment to obtain alkali extraction product.
 接下来，将所述碱提产物进行第一离心，并收集上清液，以便获得蛋白质碱提液。  Next, the alkali extraction products were first centrifuged and the supernatant was collected, in order to obtain a protein extract base.
 然后，利用第二α -淀粉酶将所述蛋白质碱提液进行第三酶解处理，以便获得第三酶解产物。  Then, using the second α - amylase protein alkaline extract the third enzymatic treatment, in order to obtain a third hydrolysates.
 接着，利用等电点沉淀法将所述第三酶解产物进行酸沉，以便获得酸沉产物。  Subsequently, isoelectric precipitation method the third hydrolysates acid precipitation, acid precipitation in order to obtain the product.
 接下来，将所述酸沉产物依次进行静置和第二离心，以便收集获得沉淀。  Next, the product of the acid precipitation in turn standing and second centrifugation to obtain a precipitate was collected.  然后，将所述沉淀进行水洗至中性，以便获得所述板栗蛋白质。  Then, the precipitate was washed with water until neutral, so as to obtain the chestnut protein.
 实施例1  Example 1
 根据前面所述的一般方法，参照图1，按照以下步骤制备板栗蛋白质:  According to the general method described above with reference to FIG. 1, follow these steps to prepare chestnut protein:
 选用无霉变虫蛀的新鲜板栗为原料，经过去皮、切片、冷冻干燥后经粉碎机粉碎，过60目筛制成板栗粉。  used without mildew infestation fresh chestnut as raw material, peeled, sliced, freeze-dried after crushing mill, through a 60 mesh sieve made of chestnut flour. 然后，将板栗粉与去离子水按照1:15 (g/mL)的比例溶解获得板栗粉水溶液并置于超声破碎仪(JAV-1V型超声波细胞粉碎仪，济宁市奥波超声波电器有限公司)中，在400w超声功率下，处理lOmin。 Then, with deionized water chestnut powder in accordance with 1:15 (g / mL) to obtain the proportion of dissolved water chestnut flour and placed sonicator (JAV-1V ultrasonic cell smash, Jining City Aoboor ultrasound Electric Co., Ltd.) , under 400w ultrasonic power, handling lOmin. 然后按照100U/g的比例向经过超声波破碎处理的板栗粉水溶液中加入纤维素酶(西格玛奥德里奇公司，货号22178)，再调pH至4.5，转入50°C恒温水浴锅(W201B型恒温水浴锅，上海申顺生物科技有限公司)搅拌酶解lOmin。 Then follow the 100U / g ratio of flour to water chestnut after sonication treatment added cellulase (Sigma-Aldrich, Item 22178), and then adjusted to pH 4.5, into the 50 ° C temperature water bath (W201B type thermostat water bath, Shanghai Shenshun Sheng Technology Co., Ltd.) for enzymatic lOmin. 接着，调pH=6.1，加入α-淀粉酶(西格玛奥德里奇公司，货号A1031)，酶添加量为120U/g，于40°C下保温搅拌酶解30min。 Then, adjust pH = 6.1, adding α- amylase (Sigma-Aldrich, Item A1031), the enzyme dosage 120U / g, and incubated at 40 ° C with stirring enzymatic 30min. 然后，用Imol/mL NaOH调pH到10.0,并放入40°C水浴锅(W201B型恒温水浴锅，上海申顺生物科技有限公司)中浸提30min后，离心取上清液，再调上清液pH至6.1，然后按照120U/g的酶添加量加入α -淀粉酶(西格玛奥德里奇公司，货号Α1031)，并于40°C下继续保温搅拌酶解30min后，在等电点pH4.2条件下进行酸沉，然后离心取沉淀，水洗沉淀至中性即得板栗蛋白质，再将其真空冷冻干燥(Thermo Fisher LL1500冻干机，美国赛默飞世尔科技有限公司)保存。 Then, Imol / mL NaOH pH adjusted to 10.0, and placed in 40 ° C water bath (W201B constant temperature water bath, Shanghai 申顺生 Technology Co., Ltd.) in 30min after extraction, centrifugation and the supernatant, and then transferred on Serum pH to 6.1, and then follow the 120U / g of enzyme dosage join α - amylase (Sigma-Aldrich, Item Α1031), and at 40 ° C under stirring digestion after 30min incubation continued at the isoelectric point pH4 .2 conducted under conditions of acid precipitation, and sedimentation centrifugation, washed to neutrality that was precipitated protein chestnut, and then vacuum freeze-drying (Thermo Fisher LL1500 lyophilizer, USA Thermo Fisher Scientific Co.) saved.
 经计算，板栗蛋白质提取率为66%，且因为提取条件温和，制得的板栗蛋白活性非常好。  After calculation, chestnut protein extraction rate of 66%, and because the extraction under mild conditions, chestnut protein activity prepared very well. 另外，通过凯氏定氮法(KDY-9830凯氏定氮仪，北京市通润源机电技术有限责任公司)测得板栗粗蛋白的蛋白含量为88.43%。 In addition, by Kjeldahl method (KDY-9830 Kjeldahl apparatus, Beijing Tongrun source Electrical Technology Co., Ltd.) measured protein chestnut crude protein content of 88.43%.
 对比例I  Comparative Example I
 选用无霉变虫蛀的新鲜板栗为原料，经过去皮、切片、冷冻干燥后经粉碎机粉碎，过60目筛制成板栗粉。  used without mildew infestation fresh chestnut as raw material, peeled, sliced, freeze-dried after crushing mill, through a 60 mesh sieve made of chestnut flour. 然后，利用传统碱水法，按照以下流程所示的步骤提取制备板栗蛋白质: Then, using conventional alkaline method to extract protein chestnut preparation process by following the steps shown below:
 脱脂板栗粉末一按料水比1:15 (g/mL)加入蒸馏水调pH到9.0 —在45°C水浴下浸提140min —离心(4000r/min, lOmin)—取上清液一酸沉(用lmol/L HCl 调pH 至Ij 4.2)—  chestnut powder skimmed by a water ratio 1:15 (g / mL) of distilled water was added to adjust the pH to 9.0 - at 45 ° C water bath extraction 140min - centrifugation (4000r / min, lOmin) - a supernatant acid precipitation (with lmol / L HCl pH was adjusted to Ij 4.2) -
收集沉淀。 The precipitate was collected.
 所述沉淀即为板栗蛋白，经计算，板栗蛋白提取率为39.69%。  The precipitated protein is the chestnut, the calculated rate of 39.69% chestnut protein extraction.
 对比例2  Comparative Example 2
 选用无霉变虫蛀的新鲜板栗为原料，经过去皮、切片、冷冻干燥后经粉碎机粉碎，过60目筛制成板栗粉。  used without mildew infestation fresh chestnut as raw material, peeled, sliced, freeze-dried after crushing mill, through a 60 mesh sieve made of chestnut flour. 然后，利用亚临界水提取法，按照以下流程所示的步骤提取制备板栗蛋白质: Then, using subcritical water extraction, protein extract was prepared according to the procedure shown below chestnut process:
 脱脂板栗粉末一按料水比1:15 (g/mL)加入蒸馏水调pH到9.0 —亚临界水萃取(提取温度180°C、提取时间25min和提取压力4.0MPa条件下)一冷却一离心(4000r/min，IOmin)—取上清液一酸沉(用lmol/L HCl调pH到4.2)—收集沉淀。  chestnut powder skimmed by a water ratio 1:15 (g / mL) of distilled water was added to adjust the pH to 9.0 - subcritical water extraction (extraction temperature of 180 ° C, extraction time 25min and extraction pressure 4.0MPa conditions) a cooling a centrifugal (4000r / min, IOmin) - supernatant an acid precipitation (with lmol / L HCl pH adjusted to 4.2) - The precipitate was collected.
 所述沉淀即为板栗蛋白，经计算，板栗蛋白提取率为45.28%。  The precipitated protein is the chestnut, the calculated rate of 45.28% chestnut protein extraction.
 对比实施例1、对比例I和对比例2可知，本发明的制备板栗蛋白质的方法的板栗蛋白质提取率，相对于传统喊水法提闻了26%,相对于亚临界水提取法提闻了21%。  Comparative Example 1, Example I and Comparative Example 2 shows that the rate of protein extraction chestnut chestnut protein preparation method of the present invention, compared with the traditional shout Water Act mention the smell by 26% relative to the subcritical water extraction 21% mention the smell. 表明相对于传统方法，本发明的制备板栗蛋白质的方法的板栗蛋白质提取率显著提高。 Showed that compared with the traditional method, a protein extract was prepared chestnut chestnut protein according to the present invention significantly improved. 此外，由实施例I可知，本发明的制备板栗蛋白质的方法获得的板栗蛋白纯度高，活性好，蛋白含量可闻达88%。 In addition, I can see from Example, high protein preparation method of the present invention chestnut obtained chestnut protein purity, activity, and protein content can be heard up to 88%.
 在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。  In the description of the present specification, reference to the term "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc., means that in connection with the description of the embodiments or examples described that a particular feature, structure, material, or characteristic included in the present invention, at least one embodiment or example. 在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。 In the present specification, the term of the above schematic representation is not necessarily referring to the same embodiment or example. 而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。 Furthermore, the particular features, structures, materials, or characteristics described may be in any one or more embodiments or examples combined in a suitable manner.
 尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。  While there have been shown and described embodiments of the invention, one of ordinary skill in the art will appreciate that: made without departing from the principles and spirit of the present invention may be made in these embodiments, various changes, modifications, substitutions and modifications, the scope of the invention being indicated by the claims and their equivalents.
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|2. Apr. 2014||C06||Publication|
|30. Apr. 2014||C10||Entry into substantive examination|
|30. März 2016||C14||Grant of patent or utility model|