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CN103695515B - The method of preparing a protein chestnut - Google Patents

The method of preparing a protein chestnut Download PDF

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CN103695515B
CN103695515B CN 201310589684 CN201310589684A CN103695515B CN 103695515 B CN103695515 B CN 103695515B CN 201310589684 CN201310589684 CN 201310589684 CN 201310589684 A CN201310589684 A CN 201310589684A CN 103695515 B CN103695515 B CN 103695515B
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CN 201310589684
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CN103695515A (en )
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王丰俊
吕佼
王建中
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北京林业大学
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Abstract

本发明公开了制备板栗蛋白的方法,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;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;利用第一α-淀粉酶将第一酶解产物进行第二酶解处理;将第二酶解处理产物进行碱提处理;将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-淀粉酶将该蛋白质碱提液进行第三酶解处理;利用等电点沉淀法将第三酶解产物进行酸沉;将酸沉产物依次进行静置和第二离心,以便收集获得沉淀;以及将该沉淀进行水洗至中性,以便获得所述板栗蛋白质。 The present invention discloses a method of preparing chestnut protein, the method comprising the steps of: an aqueous solution of chestnut flour ultrasonic crushing treatment; Chestnut aqueous cellulase enzymes after ultrasonic disintegration of the powder in a first process enzymatic treatment; using a first α- amylase second product of the first enzymatic hydrolysis process; the product of the second enzymatic treatment process for alkaline extraction; the first alkaline extraction product was centrifuged and the supernatant was collected to obtain an alkali extract protein ; using the second alkaline α- amylase protein extract was subjected to the third enzymatic treatment; isoelectric precipitation using a third acid hydrolysates sink; the product acid precipitation and the second centrifugal sequentially allowed to stand, the precipitate so obtained was collected; and the precipitate was washed with water to neutral, in order to obtain a chestnut protein. 利用该方法能够有效提高板栗蛋白提取率,并且由于提取条件温和,所以不会破坏蛋白的活性,制得的板栗蛋白活性好。 Using this method can effectively improve protein extraction rate chestnut, and due to the mild extraction conditions, it will not destroy the activity of the protein, the obtained chestnut good protein activity.

Description

制备板栗蛋白质的方法 The method of preparing a protein chestnut

技术领域 FIELD

[0001] 本发明涉及制备板栗蛋白质的方法。 [0001] The present invention relates to a process for preparing chestnut proteins.

背景技术 Background technique

[0002] 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。 [0002] chestnut containing 5% ~ 11% protein content than rice, and lysine, isoleucine, methionine, cysteine, threonine, valine, phenylalanine, tyrosine amino acid content than the standard FA0 / WH0, and is the first limiting amino acid lysine rice, wheat, corn, and soy, and threonine rice, wheat second limiting amino acid, tryptophan and a second limiting amino acids are 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口ο Thus, the direct consumption of edible chestnut or chestnut protein can complement cereals and legumes limiting amino acid deficiency, can make dietary nutrients more complete mouth ο

[0003] 然而,现阶段提取制备板栗蛋白质的方法仍有待改进。 [0003] However, the method of preparing chestnut extract protein at this stage could be improved.

发明内容 SUMMARY

[0004] 本发明是基于发明人的下列发现而完成的: [0004] The present invention is based on the discovery by the inventors completed the following:

[0005] 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。 [0005] Extraction method abroad chestnut proteins are mainly two, one is the traditional alkaline method, a disadvantage of this method is the extraction rate. 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 subcritical water extraction method, the disadvantage of this method is likely to cause the process to extract the protein denatured proteins.

[0006] 本发明旨在至少解决现有技术中存在的技术问题之一。 [0006] The present invention aims to solve the technical problems of the prior art at least one present. 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 provide a high extraction rate, a good activity of the protein preparation method chestnut protein.

[0007] 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。 [0007] In accordance with one aspect of the present invention, the present invention provides a process for preparing chestnut protein. 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 present invention, the method comprising the steps of: chestnut powder subjected to ultrasonic crushing treatment aqueous solution to obtain an aqueous solution of chestnut flour after ultrasonic disintegration treatment; Chestnut aqueous cellulase enzymes after ultrasonic disintegration of the powder treatment for the first a hydrolysis process, in order to obtain a first hydrolyzate; using a first α - amylase digestion product of the first enzymatic process for a second, in order to obtain a second hydrolyzate; the second enzyme alkali treated product stripping treatment, alkaline extraction to obtain the product; the first alkaline extraction product was centrifuged and the supernatant was collected to obtain an alkali extract protein; using a second α - amylase to the alkaline extraction of the protein third liquid enzymatic process, in order to obtain a third hydrolysates; isoelectric precipitation method using the third hydrolysates acid precipitation, acid precipitation to obtain a product; the product acid precipitation was allowed to stand in sequence and a second centrifugation to collect the precipitate obtained; and the precipitate was washed with water to neutral, in order to obtain a 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, using the present invention method can effectively extract protein prepared chestnut, chestnut and 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 present method using ultrasonic waves and cellulase hydrolysis chestnut promote dissolution of protein and enzymatic damage using α- amylase protein (also sometimes herein "protein" abbreviated as "protein") and the envelope of the starch, thus effectively improving the extraction rate chestnut protein, protein extraction rate of up to 66%, relative to the conventional method is increased by 20%, and chestnut protein obtained in the protein content may be up to 88%, high purity, quality well, and due to the mild extraction conditions can be well maintained active chestnut protein.

[0008] 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: [0008] Further, the method for preparing a protein chestnut above embodiment of the present invention may also have the following additional technical features:

[0009] 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。 [0009] Incidentally, the raw material suitable for 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 feedstock, will produce on the quality and yield of the final product certain effect, but high efficiency than a prior art protein prepared chestnut, good quality.

[0010] 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。 [0010] According to an embodiment of the present invention, the ultrasonic power at 400w, ultrasonic cell disruption process 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 loosening of cellular structure, to strengthen the effect of a subsequent cellulase. 根据本发明的实施例,所述板栗粉水溶液的料液比为lg:15mlο换句话说,所述板栗粉水溶液是通过将板栗粉和水按照lg:15ml的比例混合配制获得的。 According to an embodiment of the present invention, the liquid feed solution Chestnut Powder ratio of lg: 15mlο words, the chestnut powder is obtained by aqueous solution and water chestnut powder according lg: 15ml mixing ratio formulated obtained. 由此,有利于后续蛋白的溶出和提取。 This is advantageous in 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。 Further, it is noted that the chestnut powder can be obtained by the following preparation methods: use non-fresh mildew infestation chestnut as raw material, peeled, sliced, after lyophilization pulverizing mill, made of 60 mesh chestnut flour.

[0011] 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。 [0011] According to an embodiment of the present invention is carried out at 40-60 ° C preferably 50 ° C, pH 4-6 preferably pH4.5 5-15min condition of the first enzymatic process, 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。 Accordingly, chestnut protein can effectively promote dissolution, and can be maintained 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 other embodiments of the present invention, when the first enzymatic process, the ratio of the cellulase solution with the Chestnut Powder after ultrasonic disintegration 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 destroys the cell walls to promote dissolution of protein, it is conducive to the subsequent protein extraction.

[0012] 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。 [0012] According to an embodiment of the present invention is carried out at 30-50 ° C preferably 40 ° C, pH 5.5-6.5 preferably pH6.1 condition of the second enzymatic process 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。 Thus, effective starch hydrolysis chestnut, further starch can be effectively released envelope protein to facilitate protein eluted. 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 other embodiments of the present invention, when the second enzymatic process, the first α - amylase 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。 Accordingly, it is possible to effectively improve the efficiency of enzymatic hydrolysis of chestnut starch, starch removal of the envelope protein, and capable of maintaining the activity of the protein.

[0013] 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。 [0013] According to some preferred examples of the present invention, at 40 degrees Celsius, according to the proportion 120U / g using the first α - amylase for the second enzymatic treatment 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。 Thereby, starch hydrolysis high efficiency, low envelope protein, and capable of maintaining the protein structure and activity.

[0014] 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。 [0014] According to an embodiment of the present invention, the alkaline extraction treatment comprising: a second enzymatic treatment the product was adjusted to pH 10.0 is preferably 8.0 to 10.0, and the at 30-50 ° C preferably 40 ° C said alkaline extraction 25_35min, preferably 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。 Accordingly, it is possible to improve the dissolution rate of the protein, a protein enriched in the alkaline extraction 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pH10.0,并于40摄氏度下进行所述碱提30min。 According to some examples of the present invention, the second product was subjected to enzymatic treatment the pH was adjusted to pH 10.0, and the alkaline extraction 30min at 40 degrees Celsius. 由此,能够使蛋白高效溶出。 This enables efficient elution of the protein.

[0015] 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。 [0015] According to an embodiment of the present invention, the residue obtained after a first centrifugation and washed with deionized water, sonicated and then centrifuged, the supernatant was collected and then incorporated into the protein extract base, in order to improve protein chestnut the extraction rate.

[0016] 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。 [0016] Incidentally, the inventors have found that alkaline protein extract starch content is still high, therefore, the method of the present invention is added to the starch hydrolysis wherein the protein in the alkaline extraction solution, to separate the protein. 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 present invention is carried out at 30-50 ° C preferably 40 ° C, pH 5.5-6.5 preferably pH6.1 third condition of the 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 envelope protein of the starch, is to facilitate the subsequent 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 present invention, when the third enzymolysis, the second α - amylase ratio of base to the protein extract was 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, high efficiency of dissolved starch chestnut, chestnut envelope protein good starch removal, and does not destroy the activity of the protein.

[0017] 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。 [0017] According to an embodiment of the present invention, the acid precipitation is carried out at the isoelectric point at pH4.2 conditions. 由此,能够使板栗蛋白质有效沉淀。 This enables effective to precipitate proteins chestnut.

[0018] 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。 [0018] According to an embodiment of the present invention, the method of preparing chestnut protein further comprises the protein chestnut vacuum freeze-drying. 由此,能够有效获得板栗蛋白粗提取物。 Accordingly, it is possible to efficiently obtain a crude protein extract of 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 present invention, by a vacuum freeze dryer for 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。 Accordingly, it is possible to achieve an effective vacuum freeze-dried to obtain a crude protein extract of chestnut.

[0019] 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。 [0019] According to some specific examples of the present invention, a process for preparing a protein of the present invention chestnut may further comprise the steps of: chestnut flour with water at 1:15 (g / mL) ratio of aqueous Chestnut Powder obtained was dissolved, and then in ultrasonic crushing treatment under ultrasonic power 400w lOmin. 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。 Next, in proportion 100U / g of cellulase added to the aqueous solution after ultrasonic disintegration Chestnut Powder treated, adjusted to pH 4.5, placed in 50 ° C water bath with stirring hydrolysis 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, a low-temperature α - amylase, enzyme added amount 120U / g, and further stirred at 40 ° C for hydrolysis 30min, then with Imol / mLNaOH adjusted to pH 10.0, 40 ° into C water bath extraction 30min, then centrifuged and the supernatant, pH of the supernatant was adjusted to 6.1 after press 120U / g added in the amount of enzyme added low temperature α - amylase, and further stirred at 40 ° C for after hydrolysis 30min, carried out under isoelectric point conditions of pH4.2 acid precipitation, the precipitate was centrifuged, washed with water to neutral precipitated protein is obtained after chestnut, then vacuum lyophilization of.

[0020] 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。 [0020] Incidentally, with respect to conventional methods, the method of preparing a protein of the present invention chestnut important consideration in the structure of the protective protein Chestnut Chestnut protein while increasing the extraction rate is not destroyed by α- amylase, cellulase and ultrasonic cell disrupter role in promoting protein eluted chestnut, chestnut residual starch is reduced, and due to the mild extraction conditions, it is possible to effectively maintain the activity of the protein, thereby facilitating the subsequent biological and functional activity of the protein and the industrial production.

[0021] 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。 [0021] Additional aspects and advantages of the invention will be set forth in part in the description which follows, from the following description in part be apparent from, or learned by practice of the present invention.

附图说明 BRIEF DESCRIPTION

[0022] 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: [0022] The foregoing and / or other aspects and advantages of the invention will be described with reference to embodiments in conjunction with the embodiments become apparent and more readily appreciated below, wherein:

[0023] 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。 [0023] FIG. 1 shows a schematic flowchart of a method according to an embodiment of preparing a protein chestnut embodiment of the present invention.

具体实施方式 detailed description

[0024] 下面将结合实施例对本发明的方案进行解释。 [0024] below with reference to embodiments 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 by way of example 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。 Technical literature or the art in accordance with the conditions described in the specific technique or conditions are not specified in the examples or 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 not specified by the manufacturer, are the conventional products available through the city.

[0025] —般方法: [0025] - general procedure:

[0026] 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: [0026] Referring to FIG 1, according to an embodiment of the present invention, a process for preparing chestnut protein of the invention generally comprises the steps of:

[0027] 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。 [0027] First, an aqueous solution of chestnut flour subjected to ultrasonic crushing treatment so as to obtain an aqueous solution of chestnut flour after sonication treatment.

[0028] 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。 [0028] Next, using an aqueous solution of cellulase chestnut powder after ultrasonic disintegration of the processing performed first enzymatic process, so as to obtain a first hydrolyzate.

[0029] 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。 [0029] Again, using the first α - amylase digestion product of the first enzymatic process for a second, in order to obtain a second hydrolyzate.

[0030] 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。 [0030] Next, the second enzymatic process product stripping treatment with alkali, alkaline extraction in order to obtain the product.

[0031] 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。 [0031] Next, the first alkaline extraction product was centrifuged and the supernatant was collected to obtain an alkali extract protein.

[0032] 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。 [0032] Then, the second α - amylase protein to the third base extract enzymatic process, in order to obtain a third hydrolyzate.

[0033] 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。 [0033] Next, isoelectric precipitation the third hydrolysates acid precipitation, acid precipitation to obtain a product.

[0034] 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。 [0034] Next, the acid precipitation and the product was allowed to stand sequentially second centrifugation to collect a precipitate is obtained.

[0035] 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。 [0035] Then, the precipitate was washed with water to neutral, in order to obtain a chestnut protein.

[0036] 实施例1 [0036] Example 1

[0037] 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: [0037] According to the general procedure previously described with reference to FIG. 1, chestnut protein prepared according to the following steps:

[0038] 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。 [0038] Selection no mildew infestation fresh chestnut as raw material, peeled, sliced, pulverizing mill after lyophilization, 60 mesh 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, the chestnut powder and deionized water in proportion 1:15 (g / mL) was dissolved to obtain an aqueous solution of chestnut flour and placed sonicator (JAV-1V type sonifier cell disrupter, ultrasonic Jining Aoboor Electric Co.) , in the ultrasonic power 400w, the processing 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 100U / g ratio of cellulase added to the aqueous Chestnut Powder treated after ultrasonic disintegration (Sigma-Aldrich, Cat. No. 22178), and then adjusted to pH 4.5, transferred to 50 ° C constant temperature water bath (W201B thermostat water bath, Shanghai Shenshun Sheng Technology Co., Ltd.) was stirred 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。 Next, adjust pH = 6.1, was added α - amylase (Sigma Aldrich, Catalog No. A1031), an enzyme additive amount of 120U / g, incubated at 40 ° C for hydrolysis was stirred 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 adjusted to pH 10.0 and placed in a 40 ° C water bath (W201B type constant temperature water bath, Shanghai Shen Shunsheng Technology Co., Ltd.) leaching after 30min, the supernatant was centrifuged, and then transferred the supernatant to pH 6.1, and the amount of enzyme added according to 120U / g added α - amylase (Sigma Aldrich, Cat Α1031), and at 40 ° C for 30min after the enzymatic incubation continued stirring, the isoelectric point pH4 .2 performed under conditions of acid precipitation, then the precipitate by centrifugation, washed with water to give the precipitate to neutral proteins chestnut, which is then freeze-dried in vacuo (Thermo Fisher LL1500 lyophilizer U.S. Thermo Fisher Scientific Co.) saved.

[0039] 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。 [0039] calculated, chestnut protein extraction rate was 66%, and because the mild extraction conditions, and the obtained chestnut very good protein activity. 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%ο Further, (KDY-9830 Kjeldahl apparatus, Beijing-Run Electromechanical Technology Co., Ltd.) by the Kjeldahl method chestnut crude protein content measured is 88.43% ο protein

[0040] 对比例1 [0040] Comparative Example 1

[0041] 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。 [0041] Selection no mildew infestation fresh chestnut as raw material, peeled, sliced, pulverizing mill after lyophilization, 60 mesh 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 prepared following the steps shown in the flow:

[0042] 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 HC1 调pH 到4.2)— [0042] Degreasing water chestnut powder material according to a ratio of 1:15 (g / mL) was added distilled water adjusted to pH 9.0 - 140min leaching in a water bath at 45 ° C - centrifugation (4000r / min, lOmin) - a supernatant acid precipitation (with lmol / L HC1 adjusted to pH 4.2) -

收集沉淀。 The precipitate was collected.

[0043] 所述沉淀即为板栗蛋白,经计算,板栗蛋白提取率为39.69%。 [0043] The precipitated protein is the chestnut, calculated, chestnut protein extraction rate was 39.69%.

[0044] 对比例2 [0044] Comparative Example 2

[0045] 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。 [0045] The use non-fresh mildew infestation chestnut as raw material, peeled, sliced, pulverizing mill after lyophilization, 60 mesh 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, subcritical water extraction, chestnut extract protein prepared according to the procedure shown in the following scheme:

[0046] 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,lOmin)—取上清液一酸沉(用lmol/L HC1调pH到4.2)—收集沉淀。 9.0 [0046] Degreasing water chestnut powder material according to a ratio of 1:15 (g / mL) was added distilled water adjusted pH - (extraction temperature at 180 ° C, the extraction time and extraction pressure is 4.0MPa conditions 25min) cooling a subcritical water extraction a centrifugation (4000r / min, lOmin) - a acid precipitation and the supernatant (with lmol / L HC1 adjusted to pH 4.2) - the precipitate was collected.

[0047] 所述沉淀即为板栗蛋白,经计算,板栗蛋白提取率为45.28%。 [0047] The precipitated protein is the chestnut, calculated, chestnut protein extraction rate was 45.28%.

[0048] 对比实施例1、对比例1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,相对于传统碱水法提高了26%,相对于亚临界水提取法提高了21%。 [0048] Comparative Example 1, Comparative Example 1 and Comparative Example 2 shows that protein extraction rate Chestnut method of preparing a protein of the present invention chestnut, with respect to the conventional alkaline method to improve by 26%, relative to the increase subcritical water extraction 21%. 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。 With respect to the conventional method showed, chestnut protein extraction rate of the process for preparing chestnut protein of the invention is significantly improved. 此外,由实施例1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,蛋白含量可尚达88%。 Further, seen from Example 1, process for preparing high protein chestnut chestnut protein of the present invention obtained the purity, activity, and protein content can still 88%.

[0049] 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。 [0049] In the description of the present specification, reference to the term "one embodiment," "some embodiments", "an example", "a specific example", or "some examples" means that a description of the exemplary embodiment or embodiments described a particular feature, structure, material, or characteristic is included in at least one embodiment of the present invention, embodiments or examples. 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, a schematic representation of the above terms 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 embodiments or examples may be at any one or more in a proper manner.

[0050] 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。 [0050] While there has been illustrated and described embodiments of the present invention, those of ordinary skill in the art can be appreciated: that various changes may be made to these embodiments without departing from the principles and spirit of the invention, modifications, substitutions and modifications, the scope of the present invention is defined by the claims and their equivalents.

Claims (17)

1.一种制备板栗蛋白质的方法,其特征在于,包括以下步骤: 将板栗粉水溶液进行超声波破碎处理,于400W超声功率下,进行所述超声波破碎处理6-14min,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,于40-60摄氏度,pH 4-6条件下进行所述第一酶解处理5-15min,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,于30-50摄氏度,pH5.5-6.5条件下进行所述第二酶解处理25-35min,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,于30-50摄氏度,pH5.5-6.5条件下进行所述第三酶解处理25-35min, 1. A method for preparing a protein chestnut, characterized by comprising the steps of: an aqueous solution of chestnut flour ultrasonic crushing treatment, ultrasonic power at 400W, the ultrasonic crushing treatment for 6-14min, after ultrasonic disintegration treatment to obtain the chestnut powder in water; aqueous cellulase enzymes chestnut powder after ultrasonic disintegration process the first enzymatic process performed at 40-60 ° C, carried out at pH 4-6 5-15min condition of the first enzymatic treatment, so as to obtain a first hydrolyzate; using a first α- amylase enzyme product of the first enzymatic process for a second, 30-50 degrees C, for the second enzyme under conditions pH5.5-6.5 processing 25-35min, to obtain a second hydrolyzate; the second enzymatic treatment product is alkaline extraction treatment, alkaline extraction in order to obtain the product; the first alkaline extraction product was centrifuged and the supernatant was collected, a base to obtain a protein extract; α- amylase with a second protein to the third base extract enzymolysis at 30-50 degrees Celsius for 25 enzymatic treatment under the third condition pH5.5-6.5 -35min, 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。 So as to obtain a third hydrolysates; isoelectric precipitation method using the third hydrolysates acid precipitation, acid precipitation to obtain a product; the product acid precipitation and the second centrifugal sequentially allowed to stand to collect obtain the precipitate; and the precipitate was washed with water to neutral, in order to obtain a chestnut protein.
2.根据权利要求1所述的方法,其特征在于,于400W超声功率下,进行所述超声波破碎处理lOmin。 2. The method according to claim 1, characterized in that the ultrasonic power at 400W, the ultrasonic crushing treatment for lOmin.
3.根据权利要求1所述的方法,其特征在于,于50摄氏度,pH 4.5条件下进行所述第一酶解处理lOmin。 3. The method according to claim 1, characterized in that, within 50 ° C, under conditions of pH 4.5 the first enzymatic treatment lOmin.
4.根据权利要求1所述的方法,其特征在于,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。 4. The method according to claim 1, wherein, when the first enzymatic process, the ratio of the cellulase solution with the Chestnut Powder after ultrasonic disintegration treatment is 50-150U / g.
5.根据权利要求4所述的方法,其特征在于,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100U/g。 5. The method as claimed in claim 4, wherein, when the first enzymatic process, the ratio of the cellulase solution with the Chestnut Powder after ultrasonic disintegration treatment is 100U / g.
6.根据权利要求1所述的方法,其特征在于,于40摄氏度,pH 6.1条件下进行所述第二酶解处理30min。 6. The method according to claim 1, characterized in that, carried out at 40 ° C, pH 6.1 condition of the second enzymatic treatment 30min.
7.根据权利要求1所述的方法,其特征在于,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。 7. The method according to claim 1, wherein, when the second enzymatic process, the first α - amylase ratio of the first hydrolyzate was 80_160U / g.
8.根据权利要求7所述的方法,其特征在于,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120U/g。 8. The method according to claim 7, wherein, when the second enzymatic process, the first α - amylase ratio of the first hydrolyzate was 120U / g.
9.根据权利要求1所述的方法,其特征在于,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,并于30-50摄氏度下进行所述碱提25-35min。 9. The method according to claim 1, wherein said alkaline extraction treatment comprising: a second enzymatic treatment the product was pH adjusted to pH 8.0-10.0, and 30-50 degrees at the said alkali extraction 25-35min.
10.根据权利要求9所述的方法,其特征在于,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10.0,并于40摄氏度下进行所述碱提30mino 10. The method according to claim 9, wherein said alkaline extraction treatment comprising: a second enzymatic treatment the product was adjusted to pH 10.0, and the alkaline extraction at 40 ° C 30mino
11.根据权利要求1所述的方法,其特征在于,于40摄氏度,pH 6.1条件下进行所述第三酶解处理30min。 11. The method according to claim 1, characterized in that, carried out at 40 ° C, pH 6.1 condition of the third enzymatic treatment 30min.
12.根据权利要求1所述的方法,其特征在于,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。 12. The method according to claim 1, wherein, when said third enzymatic treatment, the second α - amylase ratio of base to the protein extract was 80_160U / g.
13.根据权利要求12所述的方法,其特征在于,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120U/g。 13. The method according to claim 12, wherein, when said third enzymatic treatment, the second α - amylase ratio of base to the protein extract was 120U / g.
14.根据权利要求1所述的方法,其特征在于,所述酸沉是在等电点pH 4.2条件下进行的。 14. The method according to claim 1, wherein the acid precipitation is 4.2 and the like under conditions of pH of the isoelectric point.
15.根据权利要求1所述的方法,其特征在于,进一步包括将所述板栗蛋白进行真空冷冻干燥。 15. The method according to claim 1, characterized in that, further comprising the freeze-dried in vacuo chestnut protein.
16.根据权利要求15所述的方法,其特征在于,利用真空冷冻干燥机进行所述真空冷冻干燥。 16. The method according to claim 15, characterized by a vacuum freeze dryer for the vacuum freeze-drying.
17.根据权利要求1所述的方法,其特征在于,所述板栗粉水溶液的料液比为lg:15mlο 17. The method according to claim 1, wherein the liquid feed solution Chestnut Powder ratio of lg: 15mlο
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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