Preparation method of blueberry anthocyanin compound

Abstract

The invention discloses a preparation method of a blueberry anthocyanin compound, which is characterized in that blueberry pomace powder is used as a raw material, blueberry pomace anthocyanin is extracted by adopting ultrahigh pressure microjet assisted enzymolysis, pea starch (C-type starch) is used as an anthocyanin protective agent, and the compound is formed by interaction of the blueberry anthocyanin and the pea starch by adopting a damp-heat method. The blueberry anthocyanin-pea starch compound with high binding rate is prepared. The blueberry anthocyanin compound prepared by the invention is not easily degraded by digestive enzymes in gastrointestinal fluid in the simulated digestion process, and when digestion is finished, the retention rate of blueberry anthocyanin in the compound is 47.28% and is obviously higher than that of a blueberry anthocyanin monomer (35.97%), so that the bioavailability of the blueberry anthocyanin is improved.

Description

Technical Field

[0001] The invention relates to a method for preparing a blueberry anthocyanin complex and belongs to the field of food processing. Background Art

[0002] Blueberry is one of the popular health foods in recent years. Blueberry anthocyanidins have multiple physiological functions such as preventing brain nerve aging, lowering blood sugar, lowering blood pressure, lowering blood lipids, anti-oxidation, anti-cancer and enhancing body immunity. However, anthocyanidins are easily degraded to varying degrees due to their structure and external environmental conditions, resulting in the instability of blueberry anthocyanidins and their low bioavailability in the human body. Secondly, the absorption of anthocyanidins in the human body is limited. Animal and human studies have confirmed that anthocyanidins have extremely low absorption rates, and most anthocyanidins disappear rapidly in the gastrointestinal tract after oral administration for several hours. Therefore, the development of blueberry anthocyanidin complexes, improving the stability of anthocyanidins, and then improving the bioavailability of anthocyanidins are of great practical significance for the efficient utilization of such anthocyanidin substances.

[0003] The interaction between blueberry anthocyanins and starch is a new way to improve the stability of anthocyanins. Starch is divided into amylose and amylopectin according to its chemical structure. The special structure of starch provides the possibility for its interaction with other small molecules. Studies have shown that in aqueous solution or acidic conditions, anthocyanins react with the hemiacetal hydroxyl groups in the starch glycosyl to form anthocyanin-starch complexes. Pea starch is an excellent anthocyanin protective agent that can reduce the degradation rate of anthocyanins and improve the stability of anthocyanins. Pea starch is a C-type starch. Due to its repeatability, biodegradability and low cost, it can encapsulate a wide range of bioactive ingredients and nutrients and is used in the fields of nanocomposites. The structure of blueberry anthocyanins is mainly composed of C6-C3-C6 as the basic C skeleton. The phenolic hydroxyl structure of its side chain, especially the ortho-hydroxyl group in catechol or pyrogallol, can react with the hydroxyl group in the pea starch molecule to form a non-inclusive complex through hydrogen bonds, which provides a new idea for improving the stability and bioavailability of blueberry anthocyanins.

[0004] At present, a large amount of pomace produced during the processing of blueberries has not been fully utilized. Blueberry pomace contains a large amount of anthocyanins, which is an important by-product in blueberry processing. It is still used as fertilizer or animal feed, resulting in a huge waste of resources. Therefore, the use of reasonable methods to extract anthocyanins from blueberry pomace and improve the comprehensive utilization rate of blueberry pomace resources can greatly improve its application and economic value. Dynamic ultra-high pressure microfluidization technology is an emerging functional ingredient extraction technology and a non-thermal extraction method. This technology promotes the rupture of plant cell walls and the efficient dissolution of bioactive ingredients through instantaneous strong shearing, high-speed collision, instantaneous pressure release and other effects. This method has the advantages of high yield, short time consumption, and basically no destruction of the structure of active ingredients. The present invention uses ultra-high pressure microfluidization assisted enzyme method to extract anthocyanins from blueberry pomace, which greatly improves the extraction rate and purity of anthocyanins.

[0005] After checking the literature and patents, there are few reports on the research of extracting anthocyanins from blueberry pomace using ultra-high pressure microfluidics, and no reports on the preparation method of blueberry anthocyanins and pea starch complex were found. Summary of the invention

[0006] Technical issues

[0007] The present invention aims to provide a method for preparing a blueberry anthocyanidin complex, by extracting anthocyanidins from blueberry pomace and utilizing the strong interaction between blueberry anthocyanidins and pea starch molecules to form a natural complex, thereby preparing a blueberry anthocyanidin-pea starch complex, thereby improving the stability of blueberry anthocyanidins, reducing the degradation rate of blueberry anthocyanidins, and releasing them into the gastrointestinal tract to be better absorbed by the human body.

[0008] Technical Solution

[0009] The technical scheme of the present invention is summarized as follows: using blueberry pomace powder as raw material, adopting ultrahigh pressure microfluidization assisted enzymatic hydrolysis extraction method to effectively extract anthocyanins from blueberry pomace, and then using pea starch (C-type starch) as an anthocyanin protective agent, using a wet heat method to make blueberry anthocyanins and pea starch interact to form a complex, and preparing a blueberry anthocyanin-pea starch complex with a high embedding rate. The specific steps include:

[0010] (1) Extraction of anthocyanins from blueberry pomace: According to a known method, the blueberry pomace powder is subjected to conventional ultrafine grinding and then sieved through a 200-400 mesh sieve. Accurately weigh the blueberry pomace powder and dissolve it in distilled water. After repeated treatment with an ultrahigh pressure microfluidizer at 100-160 MPa for 3 times, pectinase is added, and the amount of pectinase added is 0.1%-0.8% of the mass of the blueberry pomace powder. Perform enzymatic hydrolysis at 50°C for 2-5 hours, centrifuge the enzymatic hydrolyzate at 6000 r / min for 20 minutes, collect the supernatant, and concentrate it under reduced pressure at 55°C to 100 mL. After concentration, freeze-dry to obtain a blueberry pomace anthocyanin extract sample.

[0011] (2) Preparation of blueberry anthocyanidin-pea starch complex: The blueberry anthocyanidin-pea starch complex was prepared by wet heat method. Accurately weigh pea starch and dissolve it in distilled water to prepare a pea starch solution of a certain concentration, gelatinize it at 70°C for 10 to 30 minutes, and cool it to room temperature after gelatinization. The blueberry anthocyanidin extract obtained in step (1) was added to the prepared blueberry anthocyanidin solution at a mass ratio of 1:5 to 4:5 with pea starch, and the pH was adjusted to 3.0 with 0.1M hydrochloric acid buffer. The mixture was stirred at 100 to 200 rpm at 50°C for 20 minutes, and the mixture was taken out and placed in a 4°C refrigerator away from light for 1 to 2 hours. After centrifugation at 4500r / min for 15 minutes, the precipitate was taken, the supernatant was discarded, and vacuum freeze-dried to obtain the blueberry anthocyanidin-pea starch complex.

[0012] In step (1), for 1L of distilled water, the mass of blueberry pomace powder added is 100 to 250 g.

[0013] In step (2), the purity of the blueberry anthocyanin extract is higher than 70%. The particle size distribution value of the pea starch is d(0.9) of 39.50-41.0 μm, d(0.5) of 24.86-25.17 μm, and d(0.1) of 17.20-17.26 μm. For the pea starch solution, the added mass of pea starch is 50-100 g for 1 L of distilled water.

[0014] Beneficial Effects

[0015] Compared with the prior art, the preparation method of the blueberry anthocyanin complex of the present invention has the following advantages:

[0016] 1. The present invention uses an ultra-high pressure microfluidization-assisted enzymatic hydrolysis extraction method to extract anthocyanins unique to blueberry pomace, thereby greatly improving the extraction rate of anthocyanins from blueberry pomace and significantly increasing the purity, thus providing a theoretical basis for deep processing of blueberries.

[0017] 2. The present invention uses pea starch to prepare a blueberry anthocyanin complex. The obtained blueberry anthocyanin-pea starch complex has significantly higher stability than the blueberry anthocyanin monomer in an in vitro simulated digestion process, effectively improves the stability of blueberry anthocyanins, and is not easily degraded by digestive enzymes in gastrointestinal fluid, which can greatly improve its application potential in disease prevention.

[0018] 3. The present invention utilizes blueberry pomace, an important by-product in blueberry juice processing, as a raw material, with a low production cost. At the same time, it avoids the waste of resources caused by direct discharge of blueberry pomace, realizes the value-added utilization of blueberry processing by-products, and can greatly improve its application and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of different mass ratios on the binding rate of blueberry anthocyanin complexes

[0020] Figure 2 Effects of different pH values ​​on the binding rate of blueberry anthocyanin complex

[0021] Figure 3 Retention rate of blueberry anthocyanins during in vitro simulated digestion DETAILED DESCRIPTION

[0022] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0023] The raw materials used in the present invention are as follows: blueberry pomace powder comes from Shandong Hongfuhui Technology Co., Ltd.; pectinase is purchased from Shanghai Yuanye Biotechnology Co., Ltd. According to a known method, the blueberry pomace powder is subjected to conventional ultrafine grinding, passed through a 200-400 mesh sieve, and stored at -18°C for later use. The purity of the blueberry pomace anthocyanin extract is higher than 70%. Pea starch is extracted by this laboratory according to a known method, and the particle size distribution of starch is determined by a laser diffraction particle size analyzer. The particle size distribution value of pea starch is d(0.9) of 39.50-41.0 μm, d(0.5) of 24.86-25.17 μm, and d(0.1) of 17.20-17.26 μm.

[0024] Embodiment 1:

[0025] Extraction of anthocyanins from blueberry pomace: 100 g of blueberry pomace powder was accurately weighed and dissolved in 1 L of distilled water. After being treated three times by an ultra-high pressure microfluidizer at 120 MPa, pectinase was added, and the amount of pectinase added was 0.8% of the mass of the blueberry pomace powder. The enzymatic hydrolysis was performed at 50°C for 2 h, and the enzymatic hydrolyzate was centrifuged at 6000 r / min for 20 min. The supernatant was collected and concentrated to 100 mL at 55°C under reduced pressure. After concentration, it was freeze-dried to obtain a blueberry pomace anthocyanin extract sample.

[0026] Preparation of blueberry anthocyanidin-pea starch complex: The blueberry anthocyanidin-pea starch complex was prepared by wet heat method. 55 g of pea starch was accurately weighed and dissolved in 1 L of distilled water to prepare a pea starch solution of a certain concentration. The solution was gelatinized at 70°C for 10 min. After gelatinization, the solution was cooled to room temperature. The blueberry pomace anthocyanidin extract obtained in step (1) was added to the prepared blueberry pomace anthocyanidin solution in a mass ratio of 1:5 to pea starch. The solution was adjusted to pH 3.0 with 0.1 M hydrochloric acid buffer and stirred at 200 rpm for 20 min at 50°C. The mixture was taken out and placed in a 4°C refrigerator away from light for 1 h. After centrifugation at 4500 r / min for 15 min, the precipitate was taken, the supernatant was discarded, and the mixture was freeze-dried in vacuum to obtain a blueberry anthocyanidin-pea starch complex with a binding rate of 72.19%.

[0027] Embodiment 2:

[0028] Extraction of anthocyanins from blueberry pomace: 210 g of blueberry pomace powder was accurately weighed and dissolved in 1 L of distilled water. After being treated three times by an ultra-high pressure microfluidizer at 160 MPa, pectinase was added, and the amount of pectinase added was 0.5% of the mass of the blueberry pomace powder. The enzymatic hydrolysis was performed at 50°C for 5 h, the enzymatic hydrolyzate was centrifuged at 6000 r / min for 20 min, the supernatant was collected, and the supernatant was concentrated to 100 mL at 55°C under reduced pressure. After concentration, it was freeze-dried to obtain a blueberry pomace anthocyanin extract sample.

[0029] Preparation of blueberry anthocyanidin-pea starch complex: accurately weigh 100g of pea starch and dissolve it in 1L of distilled water to prepare a pea starch solution of a certain concentration, gelatinize at 70°C for 30min, and cool to room temperature after gelatinization. The blueberry anthocyanidin extract obtained in step (1) is added to the prepared blueberry anthocyanidin solution at a mass ratio of 3:5 to pea starch, and adjusted to pH 3.0 with 0.1M hydrochloric acid buffer, and stirred at 150rpm for 20min at 50°C. The mixed solution is taken out and placed in a 4°C refrigerator away from light for 1.5h, and centrifuged at 4500r / min for 15min, and the precipitate is taken, the supernatant is discarded, and vacuum freeze-dried to obtain a blueberry anthocyanidin-pea starch complex with a binding rate of 80.06%.

[0030] Embodiment 3:

[0031] Extraction of anthocyanins from blueberry pomace: accurately weigh 250 g of blueberry pomace powder and dissolve it in 1 L of distilled water. Repeat the treatment three times with an ultra-high pressure microfluidizer at 130 MPa, then add pectinase in an amount of 0.1% of the mass of the blueberry pomace powder. Perform enzymolysis at 50°C for 3 h, centrifuge the enzymolysis solution at 6000 r / min for 20 min, collect the supernatant, concentrate it under reduced pressure at 55°C to 100 mL, concentrate it, and freeze-dry it to obtain a blueberry pomace anthocyanin extract sample.

[0032] Preparation of blueberry anthocyanidin-pea starch complex: accurately weigh 80g of pea starch and dissolve it in 1L of distilled water to prepare a pea starch solution of a certain concentration, gelatinize at 70°C for 10-30min, and cool to room temperature after gelatinization. The blueberry anthocyanidin extract obtained in step (1) is added to the prepared blueberry anthocyanidin solution at a mass ratio of 4:5 to pea starch, and adjusted to pH 3.0 with 0.1M hydrochloric acid buffer, and stirred at 120rpm for 20min at 50°C, and the mixed solution is taken out and placed in a 4°C refrigerator away from light for 1-2h, and centrifuged at 4500r / min for 15min, and the precipitate is taken, the supernatant is discarded, and vacuum freeze-dried to obtain a blueberry anthocyanidin-pea starch complex with a binding rate of 78.12%.

[0033] Experimental Example 4:

[0034] The blueberry anthocyanin complex and anthocyanin monomer obtained in Example 1 were subjected to in vitro simulated digestion to determine their gastrointestinal digestion characteristics. In vitro digestion includes three main stages: simulated oral cavity, simulated stomach, and simulated small intestine. After 5 minutes of simulated oral digestion, 1 hour and 2 hours of stomach digestion, and 1 hour and 2 hours of intestinal digestion, a total of 5 time points, 10 mL of aliquots were taken out and the enzymes were inactivated in a boiling water bath, cooled to room temperature, centrifuged, and the supernatant was taken. The anthocyanin content was determined by the known method pH differential method, and the binding rate and retention rate were calculated. The specific process is as follows:

[0035] (1) Simulate oral digestion process: The reagents used to adjust pH are 6 mol / L HCl and 0.9 mol / L NaHCO3. Take several test tubes, mix 10 mL of supernatant sample with 0.25 mL of artificial saliva (16.25 mg α-amylase dissolved in 12.5 mL 1 mmol / LCaCl2, and adjust the pH to 7.0) and place them in a constant temperature shaker at 37°C and shake at a constant temperature of 120 r / min for 5 minutes. After 5 minutes, take out a test tube and bathe it in boiling water for 5 minutes to stop the reaction. Cool to room temperature, centrifuge at 4500 r / min for 10 minutes, and the supernatant obtained is the sample of the oral digestion group. The remaining test tubes are adjusted for pH and then subjected to gastric digestion.

[0036] (2) Simulated gastric digestion process: The remaining test tubes after oral digestion were adjusted to pH 3.0, and 0.25 mL of simulated gastric fluid (0.4 g pepsin dissolved in 10 mL 0.1 mmol / L HCl) was added to each test tube. The test tubes were placed in a constant temperature shaker at 37°C and shaken at a constant temperature of 120 r / min for 120 min. One test tube was taken out every 60 min, and the pH was adjusted to 7.0 with NaHCO3 to stop the reaction. The digestive fluid was centrifuged at 4500 r / min for 10 min, and the supernatant obtained was the gastric digestion group sample. The remaining test tubes were adjusted to pH and then subjected to intestinal digestion.

[0037] (3) Simulated intestinal digestion process: The pH of the remaining test tubes after gastric digestion was adjusted to 7.5, and 2.5 mL of simulated intestinal fluid (1 g pancreatic enzyme and 6 g bile salt dissolved in 50 mL 1 mol / L NaHCO3) was added to each test tube. The test tubes were placed in a constant temperature shaker at 37°C and shaken at 120 r / min for 120 min. A test tube was taken out every 60 min, and the digestive fluid was centrifuged at 4500 r / min for 10 min. The supernatant obtained was the intestinal digestion group sample.

[0038] Anthocyanin content, binding rate and retention rate were calculated using the following formulas:

[0039] Anthocyanin concentration was calculated as follows (the result was calculated as cyanidin-3-glucoside):

[0040]

[0041] Where: A=(A 520nm -A 700nm )pH 1.0 -(A 520nm -A 700nm )pH 4.5 ; MW is the relative molecular mass of cyanidin-3-glucoside, 449.2 g / mol, DF is the dilution factor, V is the volume of the measurement solution system (mL), ε is the molar extinction coefficient of cyanidin-3-glucoside, 26900 L / (mol·cm), and L is the optical path length, 1 cm.

[0042] The binding rate is calculated as follows:

[0043]

[0044] The retention rate is calculated as follows:

[0045]

[0046] Figure 3The simulated in vitro digestion results of Example 4. As the in vitro digestion time increases, the retention rates of the complex and anthocyanin monomers show an overall downward trend. When the oral digestion stage is completed (gastric digestion begins), due to the short oral digestion stage, the difference in retention rates between the two is not significant. Anthocyanins can exist stably under acidic conditions, are unstable under alkaline conditions, and are easily degraded into small molecular phenolic substances under the neutral conditions of intestinal fluid. Therefore, the retention rate of anthocyanins in the intestinal digestion stage decreases significantly. At the end of digestion, the anthocyanin retention rate in the blueberry anthocyanin-pea starch complex was 47.28%, which was significantly higher than that of the blueberry anthocyanin monomer (35.97%), thereby improving the bioavailability of blueberry anthocyanins.

[0047] Experimental example:

[0048] 1. Effect of different mass ratios of blueberry anthocyanins to pea starch on binding rate

[0049] Figure 2 The effect of different mass ratios of blueberry anthocyanins to pea starch on the binding rate. As the mass ratio of blueberry anthocyanins increases, the binding rate first increases and then decreases. When the mass ratio is 6:10, the binding rate reaches the maximum, which is 80.06%. When the proportion of blueberry anthocyanins continues to increase, the binding rate decreases instead. Therefore, the mass ratio of blueberry anthocyanins to pea starch is 6:10 as the best.

[0050] 2. Effect of different pH on binding rate

[0051] Figure 3 The effect of different pH values ​​on the binding rate. As the pH value increases, the binding rate decreases. The maximum binding rate is 79.53% at a pH of 3. This is because the hemiacetal hydroxyl group of starch is protonated in an acidic environment, which increases the local positive charge on the central carbon and promotes its electrophilicity, which is beneficial to the nucleophilic reaction of the hydroxyl group in anthocyanin, and gradually increases the binding rate; but when the pH is too high, the protonation ability will be weakened, which is not conducive to the occurrence of nucleophilic reactions, resulting in a decrease in the binding rate. In addition, blueberry anthocyanins are sensitive to pH. As the pH increases, the acidic conditions of the solution gradually weaken, and the degradation rate of anthocyanins gradually increases, which also leads to a decrease in the binding rate. Therefore, pH 3 is the best choice.

Claims

1. A method for preparing a blueberry anthocyanin complex, characterized in that: The steps include: (1) Extraction of anthocyanins from blueberry pomace: The blueberry pomace powder was subjected to conventional ultrafine grinding and passed through a 200-400 mesh sieve. The blueberry pomace powder was accurately weighed and dissolved in distilled water. The powder was subjected to ultrahigh pressure microfluidizer homogenization at 100-160 MPa for 3 times. Then, 0.1%-0.8% pectinase by weight of the blueberry pomace powder was added and enzymolysis was performed at 50° C. for 2-5 h. The enzymolysis solution was centrifuged at 6000 r / min for 20 min. The supernatant was collected, concentrated under reduced pressure at 55° C., and then freeze-dried to obtain a blueberry pomace anthocyanin extract sample. (2) Preparation of blueberry anthocyanidin-pea starch complex: The blueberry anthocyanidin-pea starch complex was prepared by wet heat method. Accurately weigh pea starch and dissolve it in distilled water to prepare a pea starch solution of a certain concentration. Gelatinize at 70°C for 10 to 30 minutes. After gelatinization, cool to room temperature. The blueberry pomace anthocyanidin extract obtained in step (1) was added to the prepared blueberry pomace anthocyanidin solution at a mass ratio of 1:5 to 4:5 with pea starch, and adjusted to pH 3.0 with 0.1M hydrochloric acid buffer. The mixture was stirred at 50°C at 100 to 200 rpm for 20 minutes. The mixture was taken out and placed in a 4°C refrigerator away from light for 1 to 2 hours, then centrifuged at 4500 r / min for 15 minutes, the precipitate was taken, the supernatant was discarded, and freeze-dried to obtain a blueberry anthocyanidin-pea starch complex powder sample.

2. The method for preparing the blueberry anthocyanin complex according to claim 1, characterized in that: In step (1), for 1L of distilled water, the mass of blueberry pomace powder added is 100 to 250 g.

3. The method for preparing the blueberry anthocyanin complex according to claim 1, characterized in that: In step (2), the purity of the blueberry pomace anthocyanin extract is higher than 70%. The particle size distribution value of the pea starch is d(0.9) of 39.50-41.0 μm, d(0.5) of 24.86-25.17 μm, and d(0.1) of 17.20-17.26 μm.

4. The method for preparing the blueberry anthocyanin complex according to claim 1, characterized in that: In step (2), for 1L of distilled water, the added mass of pea starch is 50-100g.

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