Preparation method and application of blueberry anthocyanin extract

Abstract

The invention discloses a preparation method and application of a blueberry anthocyanin extract, and belongs to the technical field of natural product extraction. The preparation method comprises the following steps: mixing blueberry pomace powder and water according to a solid-liquid ratio of 1: (10-50), exhausting, performing ultrasonic-assisted extraction, adding hemicellulase accounting for 0.6-1.4% of the mass of the pomace powder, extracting at 40-60 DEG C for 1-4 hours, and finally centrifuging to obtain a blueberry anthocyanin extracting solution. According to the method, the blueberry pomace is cooperatively treated by ultrasonic waves and hemicellulase, so that the defects of low efficiency and easy degradation of anthocyanin in a traditional extraction method are overcome, and the method has the outstanding advantages of high extraction rate, good activity maintenance and green and environment-friendly process. The obtained extract shows excellent DPPH and ABTS < + > free radical scavenging ability, and can be widely applied to functional food, health care products and cosmetics as a natural antioxidant.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a method for preparing and applying blueberry anthocyanin extract. Background Technology

[0002] blueberry( Vaccinium Blueberries (Spp.) are hailed as the "Queen of Fruits," and their health benefits are primarily attributed to their rich anthocyanin content. Blueberry anthocyanins are a class of water-soluble flavonoids formed by glycosidic bonds between aglycones such as cyanidin, delphinidin, and morning glory pigments and glycosyl groups such as glucose and galactose. Studies have shown that blueberry anthocyanins possess remarkable antioxidant, anti-inflammatory, vision-protective, and cardiovascular disease-preventing properties, demonstrating broad application prospects in functional foods, health products, and cosmetics.

[0003] Currently, traditional methods for extracting anthocyanins from blueberries mainly include solvent extraction and hot reflux extraction. These methods generally suffer from low extraction efficiency, long extraction times, and high solvent consumption. More importantly, anthocyanins are sensitive to conditions such as light, heat, and pH. The prolonged high-temperature heating in traditional hot extraction processes can easily lead to the degradation and inactivation of anthocyanins, thereby reducing the yield and bioactivity of the final product.

[0004] To overcome the drawbacks of traditional methods, some emerging extraction technologies have been developed, such as ultrasound-assisted extraction and enzymatic extraction. Ultrasound-assisted extraction utilizes cavitation and mechanical effects to effectively break down plant cell walls, accelerating the dissolution of internal components and shortening extraction time. Enzymatic extraction uses enzymes such as cellulase and pectinase to specifically degrade structural polysaccharides in plant cell walls, releasing the target product gently and efficiently. However, ultrasound extraction alone may not thoroughly destroy certain components in the cell wall, while enzymatic extraction alone often requires a long processing time. Although some studies have attempted to combine ultrasound and enzymatic methods, optimizing the selection of enzyme types and synergistic process parameters to achieve the highest extraction efficiency and maximize the preservation of anthocyanin activity remains a pressing issue for blueberry processing byproducts (such as pomace).

[0005] Furthermore, blueberry juice processing generates a large amount of pomace, mainly composed of the peel, seeds, and some pulp. Notably, over 80% of the anthocyanins in blueberries are concentrated in the peel. Currently, aside from a small amount used as animal feed or discarded, this pomace is not utilized for high-value purposes, resulting in significant resource waste. Therefore, developing a specific process for efficiently, greenly, and cost-effectively extracting highly active anthocyanins from blueberry pomace not only has significant scientific research value but also holds considerable promise for industrial applications and economic benefits.

[0006] In summary, there is an urgent need for an optimized method in the existing technology that can effectively overcome the defects of low efficiency and easy loss of activity in the extraction of anthocyanins, taking into account the characteristics of blueberry pomace. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for preparing blueberry anthocyanin extract and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing blueberry anthocyanin extract, comprising the following steps: a) Mix blueberry pomace powder with distilled water at a ratio of 1:(10-50) to obtain a mixture; exhaust the air at 50°C for 3 minutes. b) The mixture was subjected to ultrasonic-assisted extraction at a power of 220W for 10 minutes. c) Add hemicellulase to the above-mentioned ultrasonicated solution, wherein the amount of hemicellulase added is 0.6%-1.4% of the mass of the blueberry pomace powder; d) Extract the system from step c) at 40-60℃ for 1-4 hours; e) Centrifuge and collect the supernatant to obtain blueberry anthocyanin extract.

[0009] Based on the above scheme, the material-liquid ratio is 1:(30~35).

[0010] Based on the above scheme, the material-to-liquid ratio is 1:35.

[0011] Based on the above scheme, the amount of hemicellulase added is 1.0% of the mass of blueberry pomace powder.

[0012] Based on the above scheme, the extraction temperature in step d) is 55-56℃.

[0013] Based on the above scheme, the extraction temperature in step d) is 56℃.

[0014] Based on the above scheme, the extraction time for step d) is 2 hours.

[0015] A blueberry anthocyanin extract was prepared by the method described above.

[0016] The application of a blueberry anthocyanin extract as described above in the preparation of antioxidant functional foods, health products, or cosmetics.

[0017] An antioxidant composition comprising the above-mentioned blueberry anthocyanin extract and a food- or cosmetically acceptable carrier.

[0018] Advantages of the technical solution of this invention Compared with the prior art, the preparation method of blueberry anthocyanin extract of the present invention has the following significant advantages and beneficial effects: 1. High extraction efficiency and significantly improved yield. This invention creatively combines ultrasonic physical disruption of cell walls with hemicellulase biodegradation, producing a powerful synergistic effect on the cell wall structure of blueberry pomace. The cavitation effect of ultrasound loosens the cell wall structure, providing a larger contact area for enzyme action; while hemicellulase can precisely and efficiently degrade hemicellulose, a key component of the cell wall, completely breaking down the mass transfer barrier. This synergistic process overcomes the limitations of single technologies, enabling rapid and complete dissolution of anthocyanins. Experimental results show that the final extraction rate of this process can reach 6.64 mg / g, significantly higher than that of the traditional hot reflux method and pure enzymatic method.

[0019] 2. It preserves the bioactivity of anthocyanins to the greatest extent possible. The process conditions of this invention are mild, achieving optimal extraction results at a relatively low extraction temperature (56°C) and a short extraction time (2 hours), effectively avoiding the thermal degradation and inactivation of anthocyanins caused by prolonged high-temperature heating. Simultaneously, the transient effect of ultrasound may, to some extent, inhibit the activity of endogenous oxidases in the raw materials, further reducing oxidative losses. The final extract exhibits good DPPH and ABTS results. + It exhibited strong antioxidant activity similar to that of vitamin C in free radical scavenging experiments, proving that its biological activity was well preserved.

[0020] 3. The process is green and environmentally friendly, and the cost is low. This invention uses blueberry pomace, a byproduct of blueberry juice processing, as the main raw material, achieving high-value utilization of agricultural waste, conforming to the concept of circular economy, and with extremely low raw material costs. Simultaneously, the entire extraction process uses water as a solvent, completely avoiding the use of traditional organic solvents (such as methanol and acetone), fundamentally eliminating the safety risks of solvent residues and environmental pollution, making the process clean and green.

[0021] 4. Broad application prospects and high product value The blueberry anthocyanin extract prepared by this invention is not only highly pure, but also has excellent antioxidant capacity as proven by experiments. It can be used as a highly efficient natural antioxidant and functional raw material, and can be widely applied in functional foods, health products, cosmetics and pharmaceuticals, greatly enhancing the added value of the blueberry processing industry chain. Attached Figure Description

[0022] Figure 1 The effect of solid-liquid ratio on the extraction rate of blueberry anthocyanins; Figure 2The effect of extraction temperature on the extraction rate of blueberry anthocyanins; Figure 3 The effect of extraction time on the extraction rate of blueberry anthocyanins; Figure 4 The effect of enzyme dosage on the extraction rate of blueberry anthocyanins; Figure 5 Scavenging rate of DPPH· by different concentrations of extract and vitamin C; Figure 6 Scavenging rate of ·OH by different concentrations of extract and vitamin C; Figure 7 Different concentrations of extract and vitamin C on ABTS + The clearance rate.

[0023] In the above figures, different letters indicate that different samples have significant differences in free radical scavenging rates, P<0.05. Detailed Implementation

[0024] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0026] The blueberry pomace powder used in the following examples was provided by Shandong Hongfuhui Technology Co., Ltd., and the hemicellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 6000 U / g. L(+)-Ascorbic acid (V C Xilong Scientific Co., Ltd.

[0027] Example 1 A method for preparing blueberry anthocyanin extract, comprising the following steps: Blueberry pomace powder was weighed and added to distilled water at a ratio of 1:30. The mixture was then placed in a 50℃ water bath for 3 minutes to remove air. Ultrasonic extraction was performed for 10 minutes at a power of 200W. Hemicellulase was added at a concentration of 1.0% (m / m). Extraction was continued at 50℃ for 2 hours. After extraction, the mixture was centrifuged at 10000 rpm for 5 minutes to obtain blueberry anthocyanin extract. The extraction rate of blueberry anthocyanins was determined to be 5.23 mg / g. Example 2 A method for preparing blueberry anthocyanin extract, comprising the following steps: Weigh blueberry pomace powder and add it to distilled water at a ratio of 1:40. In a 50℃ water bath, remove air for 3 minutes. Perform ultrasonic-assisted extraction for 10 minutes at a power of 200W. Add hemicellulase at a concentration of 1.0% (m / m). Extract at 55℃ for 2 hours. After extraction, centrifuge at 10000 r / min for 5 minutes to obtain blueberry anthocyanin extract. The extraction rate of blueberry anthocyanins was determined to be 4.76 mg / g.

[0028] Example 3 A method for preparing blueberry anthocyanin extract, comprising the following steps: Weigh blueberry pomace powder and add it to distilled water at a ratio of 1:35. In a 50℃ water bath, remove air for 3 minutes. Perform ultrasonic-assisted extraction for 10 minutes at a power of 200W. Add hemicellulase at a concentration of 1.0% (m / m). Extract at 56℃ for 2 hours. After extraction, centrifuge at 10000 r / min for 5 minutes to obtain blueberry anthocyanin extract. The extraction rate of blueberry anthocyanins was determined to be 6.64 mg / g.

[0029] In the above embodiments, the anthocyanin content of blueberries was determined according to the method of Zhang Lixia et al., using the known pH differential method to measure the total anthocyanin content in the blueberry extract. The extracted anthocyanin supernatant was diluted to a certain factor, and 1 mL of each sample was taken and diluted to 10 mL with a pH=1.0 buffer solution (0.2 mol / L KCl and 0.2 mol / L HCl in a volume ratio of 25:67) and a pH=4.5 buffer solution (1 mol / L NaAc, 1 mol / L HCl, and H2O in a volume ratio of 100:60:90), respectively. The solutions were then allowed to stand in the dark for 2 h, and the absorbance was measured at 520 nm and 700 nm, respectively. The anthocyanin content was calculated using the following formula (results expressed as cyanidin-3-glucoside):

[0030] In the formula: A = (A 520 nm -A 700 nm pH 1.0 - (A 520 nm -A 700 nm 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 test solution (mL); ε is the molar extinction coefficient of cyanidin-3-glucoside, 26900 L / (mol·cm); L is the optical path length, 1 cm.

[0031] The formula for calculating the anthocyanin extraction rate of blueberry pomace powder is: W=CV / m Where W is the anthocyanin extraction rate (mg / g); C is the anthocyanin content in the solution (mg / mL); V is the total volume of the anthocyanin extract (mL); and m is the accurate mass (g) of the weighed blueberry pomace powder.

[0032] References: Zhang Lixia, Zhou Jianzhong, Liu Hongjin, et al. Optimization of process for preparing clarified blackberry juice by double-enzyme hydrolysis [J]. Transactions of the Chinese Society of Agricultural Engineering, 2010, 26(10):372-376. I. Effect of extraction conditions on the extraction rate of anthocyanins from blueberry pomace 1. Effect of solid-liquid ratio on blueberry anthocyanin extraction rate Under the conditions of a fixed enzyme dosage of 0.8%, extraction temperature of 45℃, extraction time of 2 h, ultrasonic power of 200 W, and ultrasonication for 10 min, the effects of different material-to-liquid ratios (1:10, 1:20, 1:30, 1:40, 1:50) on the anthocyanin extraction rate from blueberry pomace powder were investigated. The results are as follows Figure 1 As shown, the extraction rate of blueberry anthocyanins gradually increases with the increase of the liquid-to-solid ratio. When the liquid-to-solid ratio reaches 1:30, further increasing the amount of solvent does not significantly improve the extraction rate; therefore, the optimal liquid-to-solid ratio is 1:30.

[0033] 2. Effect of extraction temperature on extraction efficiency The effects of different extraction temperatures (40℃, 45℃, 50℃, 55℃, and 60℃) on the anthocyanin extraction rate from blueberry pomace powder were investigated under the following conditions: enzyme dosage of 0.8%, material-to-liquid ratio of 1:10 (g / ml), extraction time of 2 h, ultrasonic power of 200 W, and ultrasonication for 10 min.

[0034] The results are as follows Figure 2 As shown, the extraction rate of blueberry anthocyanins gradually increases and then decreases with increasing extraction temperature. This is mainly because higher temperatures intensify the molecular motion and collision frequency of blueberry anthocyanins, promoting plant cell rupture and facilitating the exudation and diffusion of anthocyanins into the solvent. However, the extraction rate decreases when the temperature reaches 60℃, because anthocyanins are heat-sensitive substances, and high temperatures can damage their molecular structure, leading to the decomposition of some anthocyanins. Therefore, the extraction temperature of 55℃ yields the best results.

[0035] 3. The effect of extraction time on extraction results The effects of different extraction times (1h, 2h, 3h, 4h) on the anthocyanin extraction rate in blueberry pomace powder were investigated under the conditions of fixed enzyme dosage of 0.8%, material-to-liquid ratio of 1:10 (g / ml), extraction temperature of 45℃, ultrasonic power of 200W, and ultrasonication for 10min.

[0036] The results are as follows Figure 3 As shown, the extraction rate of blueberry anthocyanins initially increased and then decreased with prolonged extraction time. At 2 hours, the extraction rate was 2.26%. Further extending the extraction time resulted in a decrease in the extraction rate. Therefore, an extraction time of 2 hours yielded the best extraction effect.

[0037] 4. Effect of enzyme dosage on extraction efficiency The effects of different enzyme dosages (0.6%, 0.8%, 1.0%, 1.2%, and 1.4%) on the anthocyanin extraction rate from blueberry pomace powder were investigated under the following conditions: a fixed material-to-liquid ratio of 1:10 (g / mL), an extraction temperature of 45℃, an extraction time of 2 h, an ultrasonic power of 200 W, and ultrasonication for 10 min.

[0038] The results are as follows Figure 4 As shown, the extraction rate of blueberry anthocyanins gradually increases with the increase of enzyme dosage. When the enzyme dosage is greater than 1.0%, the difference in anthocyanin extraction rate is not significant, indicating that the enzyme has reached saturation. Therefore, the optimal enzyme dosage is 1.0%.

[0039] II. Optimization of Blueberry Anthocyanin Extraction Process To further improve the extraction rate of blueberry anthocyanins, based on the results of single-factor experiments, a Box-Behnken experimental design was adopted, fixing the enzyme dosage at 0.8%, ultrasonic power at 200W, and ultrasonic treatment for 10 min. The material-to-liquid ratio, extraction temperature, and extraction time were selected as independent variables, with the blueberry pomace powder extraction rate as the response value. Response surface methodology was used to optimize the conditions for ultrasonic-assisted enzymatic extraction of blueberry anthocyanins. The results are shown in Tables 1 and 2. The optimal process conditions were found to be: when the ultrasonic power was 200W and the ultrasonic treatment time was 10 min, the optimal extraction conditions for blueberry anthocyanins were a material-to-liquid ratio of 1:35, an extraction temperature of 56℃, an extraction time of 2 h, and an enzyme dosage of 1.0%. Under these conditions, the blueberry anthocyanin extraction rate was 6.64 mg / g.

[0040] Table 1 Response Surface Design and Results

[0041] Table 2. Analysis of Variance of Regression Model

[0042] Note: P<0.05 indicates a significant difference; P>0.05 indicates a non-significant difference; P<0.01 indicates a highly significant difference.

[0043] III. Determination of Antioxidant Indicators of Blueberry Anthocyanins DPPH scavenging rate determination: The method of Peng Cheng and Amoussa et al. was followed with slight modifications. Blueberry anthocyanin extract was diluted to a certain factor, and the above sample solution was prepared into new sample solutions of specific concentrations using distilled water. The determination system contained 2 mL of sample solution and 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, denoted as A1. The blank system used distilled water instead of the sample solution, denoted as A0. The control group did not contain a colorimetric reagent and was denoted as A2. The DPPH free radical scavenging rate was calculated using the following formula:

[0044] • OH scavenging rate determination: The method of Li Ruiguang and Amoussa et al. was followed with slight modifications. Blueberry anthocyanin extract was diluted to a certain factor, and new sample solutions of specific concentrations were prepared using distilled water. The determination system contained 3 mL of sample solutions of different concentrations, 0.5 mL of 0.9 mmol / L FeSO4, 0.5 mmol / L 0.9 mmol / L salicylic acid-ethanol solution, and 0.5 mL of 0.88 mmol / L H2O2 solution, denoted as A1. In the blank system, distilled water was used instead of the sample solution, denoted as A0. The control group did not contain a colorimetric reagent and was denoted as A2. • OH free radical scavenging rate was calculated using the following formula:

[0045] ABTS + Scavenging rate determination: The method of Yu and Amoussa et al. was followed with slight modifications. Before the determination, an equal volume of 7.4 mol / L ABTS stock solution and 2.6 mol / L K₂S₂O₈ were mixed to prepare the ABTS working solution. This solution was stored at 4 ℃ in the dark for 12–16 h. Immediately before use, the ABTS working solution was diluted with a buffer solution at pH 7.4 to achieve an absorbance of 0.7 ± 0.02 at 734 nm. Blueberry juice was diluted to a certain factor, and new sample solutions of specific concentrations were prepared using distilled water. The determination system contained 2 mL of ABTS working solution and 2 mL of sample solution, denoted as A1. The blank system used distilled water instead of the sample and was denoted as A0. The control group did not contain ABTS working solution and was denoted as A2. ABTS + The clearance rate is calculated using the following formula:

[0046] References: Peng Cheng, Xue Haijun, Chang Xiaoxiao, et al. Quality and antioxidant capacity of juice from different varieties of wampee [J]. Journal of South China Normal University (Natural Science Edition), 2020, 52(1): 70-76. Li Ruiguang, Liu Linwei, Zheng Haiyan, et al. Study on the in vitro antioxidant properties of flavonoid extract from reed [J]. Northwest Agricultural Journal, 2009, 18(04):310-314. Yu C, Liu X, Pei J, et al. Grafting of laccase-catalysed oxidation ofbutyl paraben and p-coumaric acid onto chitosan to improve its antioxidant and antibacterial activities[J]. Reactive and Functional Polymers, 2020, 149:104511. Amoussa AMO, Zhang L, Lagnika C, et al. Effects of preheating and drying methods on pyridoxine, phenolic compounds, ginkgolic acids, and antioxidant capacity of Ginkgo biloba nuts,[J]. Journal of Food Science, 2021, 86:1-12. Blueberry anthocyanin extract was prepared under optimal extraction conditions, and the anthocyanin concentration was determined. The extract was diluted at different ratios to obtain concentrations of 25 mg / L, 20 mg / L, and 15 mg / L, designated as Sample 1, Sample 2, and Sample 3, respectively. Vitamin C was used as a control group, and its antioxidant activity was measured. Vitamin C, as a common antioxidant, has a certain scavenging effect on all three types of free radicals and can be used as a reference to compare the antioxidant capacity of the blueberry pomace powder extract and the samples. The results are as follows: Figures 5-7 As shown.

[0047] Depend on Figures 5-7 It can be seen that anthocyanin extract has effects on DPPH· and ABTS. + The scavenging rates of the two free radicals were relatively high, reaching 99.54% and 99.64% respectively, which were similar to the scavenging effect of vitamin C. This indicates that the blueberry pomace powder extract has a strong ability to scavenge these two free radicals and has a strong antioxidant capacity. However, the scavenging rate of ·OH was significantly lower than that of the other two free radicals and also lower than that of vitamin C. This indicates that its ability to scavenge ·OH is relatively weak, and its antioxidant effect has a certain degree of free radical selectivity.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a blueberry anthocyanin extract, characterized in that, The steps are as follows: a) Mix blueberry pomace powder with distilled water at a ratio of 1:(10~50) to obtain a mixture; exhaust the air at 50℃ for 3 minutes; b) The mixture was subjected to ultrasonic-assisted extraction at a power of 220W for 10 minutes. c) Add hemicellulase to the above-mentioned ultrasonicated solution, wherein the amount of hemicellulase added is 0.6%-1.4% of the mass of the blueberry pomace powder; d) Extract the system from step c) at 40-60℃ for 1-4 hours; e) Centrifuge and collect the supernatant to obtain blueberry anthocyanin extract.

2. The method for preparing blueberry anthocyanin extract according to claim 1, characterized in that, The material-to-liquid ratio is 1:(30~35).

3. The method for preparing blueberry anthocyanin extract according to claim 2, characterized in that, The material-to-liquid ratio is 1:

35.

4. The method for preparing blueberry anthocyanin extract according to claim 1, characterized in that, The amount of hemicellulase added is 1.0% of the mass of the blueberry pomace powder.

5. The method for preparing blueberry anthocyanin extract according to claim 1, characterized in that, The extraction temperature in step d) is 55-56℃.

6. The method for preparing blueberry anthocyanin extract according to claim 5, characterized in that, The extraction temperature in step d) is 56℃.

7. The method for preparing blueberry anthocyanin extract according to claim 1, characterized in that, The extraction time for step d) is 2 hours.

8. A blueberry anthocyanin extract, characterized in that, Prepared by the method according to any one of claims 1 to 7.

9. The use of the blueberry anthocyanin extract as described in claim 8 in the preparation of antioxidant functional foods, health products, or cosmetics.

10. An antioxidant composition, characterized in that, It comprises the blueberry anthocyanin extract as described in claim 8 and a food or cosmetically acceptable carrier.

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