WO2023108790A1 - 一种黑玉米花青素的提取工艺 - Google Patents

一种黑玉米花青素的提取工艺 Download PDF

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WO2023108790A1
WO2023108790A1 PCT/CN2021/141103 CN2021141103W WO2023108790A1 WO 2023108790 A1 WO2023108790 A1 WO 2023108790A1 CN 2021141103 W CN2021141103 W CN 2021141103W WO 2023108790 A1 WO2023108790 A1 WO 2023108790A1
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corn
extraction
ultrasonic
black corn
black
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PCT/CN2021/141103
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English (en)
French (fr)
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范庆君
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海南拉风农业科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins

Definitions

  • the application relates to the technical field of extraction of natural product active substances, in particular to an extraction process of anthocyanins from black corn.
  • Black corn (Black corn, Zeamays L.) is also known as black glutinous rice and purple corn.
  • Black corn is a variety of corn varieties. Compared with the original corn varieties, black corn has higher sugar content, more black pigment and is deposited in the cuticle of the grain, so it has a beautiful black appearance and high nutritional value. It can be used as food or medicine , under the new trend of the same source of medicine and food, and the combination of medicine and food, the black corn industry is developing rapidly, and because it is easy to plant and has a high yield in one season, it is produced all over the country. Such a huge production volume provides a solid foundation for the commercialization of black corn A large number of sources.
  • the black corn anthocyanins extracted from black corn have the effects of non-toxicity, high safety, anti-oxidation, improvement of blood circulation, inhibition of inflammation and allergy, and anti-cancer effects. It is a pure natural anti-aging nutritional supplement. Studies have shown that It is the most effective antioxidant found by humans today, its antioxidant performance is 50 times higher than that of vitamin E, and 200 times higher than that of vitamin C.
  • Anthocyanins in black corn are polyhydroxy compounds, easily soluble in water, slightly soluble in organic solutions such as ethanol and methanol; however, the total extraction rate of anthocyanins from black corn is currently low, and a part of anthocyanins and still Stay in black corn, the utilization rate is low, patent CN108531356A " black corn vinegar and the method for extracting black corn anthocyanin ", the anthocyanin content of extraction is not good; Patent CN106473127A " a kind of black corn anthocyanin extraction method " adopts Ultrasound-assisted enzymatic hydrolysis extracts anthocyanins from black corn, but if the enzymatic hydrolysis temperature is too high, the anthocyanins will be thermally decomposed or denatured, resulting in a low content of the extracted anthocyanins.
  • the application proposes a process for extracting black corn anthocyanins to solve the above problems.
  • Raw material pretreatment select the selected mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 50-70 mesh sieve to obtain corn powder, and set aside;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5000-6000 rpm for 10-15 minutes, and measure the absorbance of the obtained supernatant.
  • the enzyme activity of cellulase in the S2 step is 800-1000U/g
  • the enzyme activity of citrate synthase is 600-800U/g
  • the enzyme activity of ⁇ -amylase is 1200-1500U/g .
  • the enzymatic hydrolysis time in the step S3 is 15-20 hours.
  • the constant temperature concentration temperature in the step S3 is 20-30° C.
  • the concentration time is 15-30 minutes.
  • the ultrasonic extraction conditions in the step S3 are power of 400-600W, temperature of 40-60°C, and ultrasonic frequency of 28-30kHz.
  • the ultrasound is extracted once at intervals of 2-4 minutes, and each ultrasound is 5-8 minutes.
  • the mass volume ratio g/mL of the corn powder and the enzyme liquid is 1 ⁇ 5:8.
  • the content of anthocyanins extracted by the black corn anthocyanin extraction process of the present application is relatively high, and the cell wall of black corn grains is broken through ultrasonic-assisted enzymatic extraction, releasing more anthocyanins, which greatly improves the extraction of anthocyanins Efficiency, among them, the use of biologically active enzyme liquid, scientifically proportioned, cellulase degrades the cellulose component in the cell wall, which is conducive to cell crushing, and ⁇ -amylase can hydrolyze the amylose and sugar in black corn into glucose and maltose , maltotriose, and citrate synthase can regulate and cooperate with hydrolysis of some proteins and lipids, fully extract anthocyanins from black corn in an all-round way, and then adjust the parameters of the ultrasonic process to accelerate anthocyanins production under appropriate parameter conditions. Molecular diffusion and activity rate to achieve effective extraction of black corn anthocyanins.
  • a kind of extraction technology of black corn anthocyanin comprise the following steps:
  • Raw material pretreatment select the selected mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 50-70 mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:3 to 8:0.6 to 1.5 to obtain an enzyme liquid for later use.
  • the enzyme activity of the cellulase is 800U/g
  • the enzyme activity of citrate synthase is 600U/g
  • the enzyme activity of ⁇ -amylase is 1200U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass volume ratio of corn powder and enzyme solution g/mL is 1-5:8, shake and shake at 18-28°C for 15 hours for enzymatic hydrolysis , and then ultrasonically extracted 2 to 4 times.
  • the ultrasonic extraction conditions are power 400 ⁇ 600W, temperature 40 ⁇ 60°C, ultrasonic frequency 28 ⁇ 30kHz, ultrasonic extraction once every 2 ⁇ 4min, each ultrasonic 5 ⁇ 8min, combined collection
  • the filtrate is concentrated at a constant temperature in a rotary evaporator, the temperature of the constant temperature concentration is 20-30°C, and the concentration time is 15-30min to obtain a concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5000-6000 rpm for 10-15 minutes, and measure the absorbance of the obtained supernatant.
  • a kind of extraction technology of black corn anthocyanin comprise the following steps:
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 70-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid hydrolyze cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:8:1.5 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 1000 U/ g
  • the enzyme activity of citrate synthase is 800U/g
  • the enzyme activity of ⁇ -amylase is 1500U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 5:8, shake and shake at 28°C for 20 hours, and then perform ultrasonication Extraction 4 times, ultrasonic extraction conditions are power 600W, temperature 60°C, ultrasonic frequency 30kHz, ultrasonic interval 4min extraction once, each ultrasonic 8min, combined and collected filtrates were concentrated in a rotary evaporator at constant temperature, the temperature of constant temperature concentration was 30°C , concentration time 30min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 6000 rpm for 15 minutes, and measure the absorbance of the obtained supernatant.
  • a kind of extraction technology of black corn anthocyanin comprise the following steps:
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 22°C for 18 hours, and then use ultrasound Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • the ultrasonic extraction conditions in the S3 step are power 500W, temperature 70°C, and ultrasonic frequency 40kHz; specifically the following steps:
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 22°C for 18 hours, and then use ultrasound Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 70°C, ultrasonic frequency 40kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Example 3 The difference between this example and Example 3 is that the extraction process of black corn anthocyanins, the temperature of the constant temperature concentration is 40°C, and the concentration time is 10min, which specifically includes the following steps:
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • the enzyme activity of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 22°C for 18 hours, and then use ultrasound Extraction 3 times, ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at constant temperature, the temperature of constant temperature concentration was 40°C , concentration time 10min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Example 3 The difference between this comparative example and Example 3 is that the enzyme solution is prepared by mixing cellulase, citrate synthase, and ⁇ -amylase at a mass ratio of 1:2:4.
  • the extraction process of black corn anthocyanins comprises the following steps:
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • the enzyme liquid is prepared by mixing cellulase, citrate synthase, and ⁇ -amylase at a mass ratio of 1:2:4, and is used for future use.
  • the enzyme activity of the cellulase is 900U/g, lemon
  • the enzyme activity of acid synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 22°C for 18 hours, and then use ultrasound Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • Enzyme liquid cellulase with an enzyme activity of 900U/g
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 22°C for 18 hours, and then use ultrasound Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Example 3 The difference between this comparative example and Example 3 is that in the S3 step, the corn powder and the enzyme solution are shaken and shaken at 10°C for enzymolysis;
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate at 10°C for 18 hours, and then perform ultrasonication Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Example 3 The difference between this comparative example and Example 3 is that in the step S3, the corn powder and the enzyme solution are shaken and shaken at 50° C. for enzymolysis.
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 3:8, oscillate and shake at 50°C for 18 hours, and then perform ultrasonication Extraction 3 times, ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • Example 3 The difference between this comparative example and Example 3 is that the mass volume ratio g/mL of corn powder and enzyme solution is 1:1.
  • Raw material pretreatment select mature black corn, peel off the corn kernels, dry, pulverize with a high-speed pulverizer, pass through a 60-mesh sieve to obtain corn powder, and set aside;
  • enzyme liquid mix cellulase, citrate synthase, and ⁇ -amylase with a mass ratio of 1:5:1 to obtain an enzyme liquid for use.
  • the enzyme activity of the cellulase is 900 U/g
  • the enzyme activity of citrate synthase is 700U/g
  • that of ⁇ -amylase is 1300U/g;
  • Ultrasound-assisted extraction mix the corn powder obtained in S1 with the enzyme solution of S2, the mass-volume ratio of corn powder and enzyme solution g/mL is 1:1, oscillate and shake at 22°C for 18 hours, and then perform ultrasonication Extraction 3 times, the ultrasonic extraction conditions are power 500W, temperature 50°C, ultrasonic frequency 29kHz, ultrasonic interval 3min extraction, each ultrasonic 6min, combined and collected filtrates were concentrated in a rotary evaporator at a constant temperature, and the temperature of constant temperature concentration was 25°C , concentration time 23min, obtain concentrated crude filtrate;
  • Centrifugation Put the crude filtrate into a high-speed refrigerated centrifuge, centrifuge at a speed of 5500 rpm for 13 minutes, and measure the absorbance of the obtained supernatant.
  • A--absorbance value, A (A max -A 700 ) pH1.0- (A max -A 700 ) pH4.5 ;
  • the content of the anthocyanins extracted in this application is relatively high, and the extraction content is relatively high.
  • the embodiment group is 7.58-7.89mg/g.
  • the ratio of the enzyme solution plays a certain role in promoting its enzymatic hydrolysis and extraction.
  • anthocyanin is a water-soluble substance, and the enzyme solution can enzymatically hydrolyze the cell wall of anthocyanin at an appropriate ratio to achieve a better extraction effect.
  • the enzyme solution prepared by the present application can fully extract the anthocyanins in black corn in all directions;
  • the enzymatic hydrolysis temperature is too low, so that the diffusion of the cell membrane pathway is small, and the anthocyanins It cannot be released and extracted;
  • Comparative Example 4 the temperature is higher than 28°C, and reaching 50°C will cause anthocyanins to be thermally decomposed or denatured;
  • Comparative Example 5 the ratio between corn powder and enzyme solution directly affects the extraction effect , If the ratio is too high, the extraction will be insufficient and the extraction rate will be low.

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Abstract

提供一种黑玉米花青素的提取工艺，包括以下步骤：S1原料预处理、S2制备酶液、S3超声辅助提取、S4离心等步骤提取得到黑玉米花青素成分，通过超声波辅助酶解提取，将黑玉米籽粒细胞壁破碎，释放更多的花青素，极大提高了花青素的提取效率，制备的酶液能够全方位将黑玉米中的花青素充分提取，再调控超声工艺参数，在适当的参数条件下，加快花青素分子扩散和活动速率，达到黑玉米花青素的有效提取。

Description

一种黑玉米花青素的提取工艺 技术领域
本申请涉及天然产物活性物质提取技术领域，特别涉及一种黑玉米花青素的提取工艺。
背景技术
黑玉米(Black corn，Zeamays L.)又称为黑糯米、紫玉米，是被子植物门单子叶植物纲，为禾本目玉蜀黍属植物。黑玉米是玉米品种中的变种，较原玉米品种含糖量高，黑色色素多且沉积于籽粒角质层中因而有漂亮的黑色外观，且营养价值极高，即可作为粮食食用也可药用，在现如今药食同源，医食并用的新潮流下，黑玉米产业迅速发展，又因其易于种植且一季多产而产地遍布全国，如此巨大的生产量，对于黑玉米商品化提供了大量产源。而从黑玉米中提取出的黑玉米花青素具有无毒性、安全性高、抗氧化、改善血液循环、抑制炎症与过敏以及抗癌的功效，纯天然的抗衰老的营养补充剂，研究证明是当今人类发现最有效的抗氧化剂，它的抗氧化性能比维生素E高出五十倍，比维生素C高出二百倍。黑玉米中的花青素是多羟基化合物，易溶于水，微溶于乙醇、甲醇等有机溶液；而目前从黑玉米提取花青素中总提取率较低，且一部分花青素与仍留在黑玉米中，利用率低，专利CN108531356A“黑玉米醋及提取黑玉米花青素的方法”，提取的花青素含量欠佳；专利CN106473127A“一种黑玉米花青素提取方法”采用超声辅助酶解法提取黑玉米中的花青素，但酶解温度过高，都会使得花青素的受热分解或变性，导致提取出的花青素含量过低。
发明内容
鉴于此，本申请提出一种黑玉米花青素的提取工艺，解决上述问题。
本申请的技术方案是这样实现的：一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过50～70目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:3～8:0.6～1.5的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合酶解，在18～28℃下振荡摇匀，再经超声提取2～4次，合并收集滤液于旋转蒸发仪中恒温浓缩，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5000～6000rpm离心10～15min，得到上清液测定其吸光度。
进一步的，所述S2步骤中所述纤维素酶的酶活力为800～1000U/g，柠檬酸合酶的酶活力为600～800U/g，β-淀粉酶的酶活力为1200～1500U/g。
进一步的，所述S3步骤中酶解时间15～20h。
进一步的，所述S3步骤中恒温浓缩的温度为20～30℃，浓缩时间15～30min。
进一步的，所述S3步骤中超声提取条件为功率400～600W，温度为40～60℃，超声频率为28～30kHz。
进一步的，所述S3步骤中超声间隔2～4min提取一次，每次超声5～8min。
进一步的，所述玉米粉末和酶液的质量体积比g/mL为1～5:8。
与现有技术相比，本申请的有益效果是：
本申请的黑玉米花青素提取工艺提取的花青素含量较高，通过超声波辅助酶解提取，将黑玉米籽粒细胞壁破碎，释放更多的花青素，极大提高了花青素的提取效率，其中，采用生物活性酶液，经科学配比，纤维素酶降解细胞壁中的纤维素成分，利于细胞破碎，β-淀粉酶能够将黑玉米中直链淀粉和糖类水解为葡萄糖、麦芽糖、麦芽三糖，柠檬酸合酶能够调控配合水解的部分蛋白质和脂质，全方位将黑玉米中的花青素充分提取，再调控超声工艺参数，在适当的参数条件下，加快花青素分子扩散和活动速率，达到黑玉米花青素的有效提取。
具体实施方式
为了更好理解本申请技术内容，下面提供具体实施例，对本申请做进一步的说明。
本申请实施例所用的实验方法如无特殊说明，均为常规方法。
本申请实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过50～70目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:3～8:0.6～1.5的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为800U/g，柠檬酸合酶的酶活力为600U/g，β-淀粉酶的酶活力为1200U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为1～5:8，在18～28℃下振荡摇匀酶解15h，再经超声提取2～4次，超声提取条件为功率400～600W，温度为40～60℃，超声频率为28～30kHz，超声间隔2～4min提取一次，每次超声5～8min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为20～30℃，浓缩时间15～30min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5000～6000rpm离心10～15min，得到上清液测定其吸光度。
实施例2
一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过70目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:8:1.5的纤维素酶、柠檬酸合酶、β-淀粉酶混合酶解，得到酶液，备用，所述纤维素酶的酶活力为1000U/g，柠檬酸合酶的酶活力为800U/g，β-淀粉酶的酶活力为1500U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为5:8，在28℃下振荡摇匀酶解20h，再经超声提取4次，超声提取条件为功率600W，温度为60℃，超声频率为30kHz，超声间隔4min提取一次，每次超声8min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为30℃，浓缩时间30min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速6000rpm离心15min，得到上清液测定其吸光度。
实施例3
一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
实施例4
本实施例与实施例3的区别在于，黑玉米花青素的提取工艺，所述S3步骤中超声提取条件为功率500W，温度为70℃，超声频率为40kHz；具体为以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为70℃，超声频率为40kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
实施例5
本实施例与实施例3的区别在于，黑玉米花青素的提取工艺，所述恒温浓缩的温度为40℃，浓缩时间10min，具体包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为40℃，浓缩时间10min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
对比例1
本对比例与实施例3的区别在于，酶液为质量比1:2:4的纤维素酶、柠檬酸合酶、β-淀粉酶混合制得。
具体为黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：酶液为质量比1:2:4的纤维素酶、柠檬酸合酶、β-淀粉酶混合制得，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
对比例2
本对比例与实施例3的区别在于，酶液为纤维素酶；
具体为一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、酶液：酶活力为900U/g的纤维素酶的；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
对比例3
本对比例与实施例3的区别在于，所述S3步骤将玉米粉末和酶液在10℃下振荡摇匀酶解；
具体为一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在10℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
对比例4
本对比例与实施例3的区别在于，所述S3步骤将玉米粉末和酶液在50℃下振荡摇匀酶解。
具体为一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为3:8，在50℃下振荡摇匀酶解18h，再经超声提取3 次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
对比例5
本对比例与实施例3的区别在于，玉米粉末和酶液的质量体积比g/mL为1:1。
具体为一种黑玉米花青素的提取工艺：包括以下步骤：
S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，使用高速粉碎机粉碎，过60目筛，得到玉米粉末，备用；
S2、制备酶液：将质量比为1:5:1的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用，所述纤维素酶的酶活力为900U/g，柠檬酸合酶的酶活力为700U/g，β-淀粉酶的酶活力为1300U/g；
S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合，玉米粉末和酶液的质量体积比g/mL为1:1，在22℃下振荡摇匀酶解18h，再经超声提取3次，超声提取条件为功率500W，温度为50℃，超声频率为29kHz，超声间隔3min提取一次，每次超声6min，合并收集滤液于旋转蒸发仪中恒温浓缩，恒温浓缩的温度为25℃，浓缩时间23min，得到浓缩粗滤液；
S4、离心：将粗滤液放入高速冷冻离心机内，以转速5500rpm离心13min，得到上清液测定其吸光度。
一、花青素得率测定
将实施例1～5和对比例1～5取上清液稀释6倍后通过紫外分光光度计在最大吸收波长和700nm测定其吸光度，利用公式进行花青素得率计算，公式如下：
Figure PCTCN2021141103-appb-000001
式中：
A--吸光值，A＝(A max-A 700) pH1.0-(A max-A 700) pH4.5；
MW--矢车菊素-3-葡萄糖苷分子量，449.2g/mol；
DF--稀释倍数；
V--提取液总体积，mL；
Ε--矢车菊素-3-葡萄糖苷的摩尔消光系数；
L--光程，1cm；
M--样品质量，g。
计算结果各平均值如下表：
  mg/g
实施例1 7.58
实施例2 7.78
实施例3 7.89
实施例4 7.60
实施例5 7.62
对比例1 6.27
对比例2 6.03
对比例3 6.18
对比例4 6.20
对比例5 6.38
本申请所提取的花青素含量较高，提取含量较高，实施例组在7.58～7.89mg/g，与对比例1比较，酶液的比例对其酶解和提取起到一定的促进作用，花青素为水溶性物质，酶液在适当的比值下能够将花青素酶解细胞壁达到较好的提取效果，比值过低或过高会导致酶液失活或者达到饱和无法在进行酶解；与对比例2比较，本申请制备的酶液能够全方位将黑玉米中的花青素充分提取；与对比例3比较，酶解温度过低，使得细胞膜通路扩散较小，花青素无法被释放和提取；与对比例4比较，温度高于28℃，达到50℃后会导致花青素受热分解或变性；与对比例5比较，玉米粉末和酶液的比例关系直接影响提取效果，比例过高会导致提取不充分，提取率较低。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (7)

  1. 一种黑玉米花青素的提取工艺，其特征在于：包括以下步骤：
    S1、原料预处理：选取经挑选成熟的黑玉米，剥落玉米籽粒，干燥，粉碎，过50～70目筛，得到玉米粉末，备用；
    S2、制备酶液：将质量比为1:3～8:0.6～1.5的纤维素酶、柠檬酸合酶、β-淀粉酶混合，得到酶液，备用；
    S3、超声辅助提取：将S1得到的玉米粉末和S2的酶液混合酶解，在18～28℃下振荡摇匀，再经超声提取2～4次，合并收集滤液于旋转蒸发仪中恒温浓缩，得到浓缩粗滤液；
    S4、离心：将粗滤液放入高速冷冻离心机内，以转速5000～6000rpm离心10～15min，得到上清液测定其吸光度。
  2. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述S2步骤中所述纤维素酶的酶活力为800～1000U/g，柠檬酸合酶的酶活力为600～800U/g，β-淀粉酶的酶活力为1200～1500U/g。
  3. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述S3步骤中酶解时间15～20h。
  4. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述S3步骤中恒温浓缩的温度为20～30℃，浓缩时间15～30min
  5. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述S3步骤中超声提取条件为功率400～600W，温度为40～60℃，超声频率为28～30kHz。
  6. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述S3步骤中超声间隔2～4min提取一次，每次超声5～8min。
  7. 如权利要求1所述的一种黑玉米花青素的提取工艺，其特征在于：所述玉米粉末和酶液的质量体积比g/mL为1～5:8。
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