A pecan oil microcapsule with high antioxidant activity using pecan cake peptide as wall material and its preparation method

Abstract

The present invention discloses a pecan oil microcapsule powder with high antioxidant activity using pecan cake meal peptide as the wall material and a preparation method thereof. The microcapsule powder is prepared by freeze-drying after microcapsules are formed by encapsulating pecan oil with pecan cake meal antioxidant peptide. The pecan cake meal antioxidant peptide is obtained by extracting from the fermented sample of pecan cake meal. The fermented sample of pecan cake meal is produced by fermenting Pleurotus eryngii in a solid fermentation medium prepared from pecan cake meal. The pecan oil microcapsule powder prepared by the present invention has significantly improved antioxidant activity compared with the microcapsule powder of conventional embedding wall materials; it improves the utilization value of pecan cake meal and further expands its application potential in the fields of food, health products, medicine, etc.; through the special treatment method of solid-state fermentation, an innovative method for preparing functional peptides is provided, which is of great significance for the development of new functional foods.

Description

Technical Field

[0001] The present invention relates to a high antioxidant activity pecan oil microcapsule powder with pecan cake meal peptide as the wall material and a preparation method thereof, belonging to the field of microcapsule preparation. Background Art

[0002] Carya illinoensis, also known as pecan, is a deciduous tree of the genus Carya in the family Juglandaceae. Its nuts are rich in crude fat and protein, and 7 out of 17 amino acids in the kernel protein are essential for the human body. The oil content of the kernel ranges from 51% to 69%, and the oil contained in the kernel is mainly composed of unsaturated fatty acids such as oleic acid and linoleic acid, with the total amount of unsaturated fatty acids exceeding 90%. It is a well-known dry fruit and oil tree species.

[0003] Pecan cake meal is the remaining fruit part after processing pecan oil. It is rich in protein and is generally used as feed or treated as waste. However, research shows that bioactive substances such as functional peptides, anthocyanins, and polyphenols in pecan cake meal also have good antioxidant and anti-tumor activities and can be used as functional raw materials for health products and functional foods. There is no report on using Pleurotus eryngii to prepare functional peptides by solid-state fermentation of pecan cake meal and using this functional peptide to encapsulate pecan oil to prepare microcapsule powder. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a high antioxidant activity pecan oil microcapsule powder with pecan cake meal peptide as the wall material and a preparation method thereof.

[0005] Technical Solution: To solve the above technical problem, the high antioxidant activity pecan oil microcapsule powder with pecan cake meal peptide as the wall material provided by the present invention is prepared by freeze-drying after forming microcapsules by encapsulating pecan oil with pecan cake meal antioxidant peptide; the pecan cake meal antioxidant peptide is obtained by extracting from the fermented sample of pecan cake meal; the fermented sample of pecan cake meal is produced by fermenting Pleurotus eryngii in a solid-state fermentation medium prepared from pecan cake meal.

[0006] Wherein, the fermentation time is 3 - 9 days.

[0007] Wherein, the fermentation temperature is 25 - 30°C.

[0008] The present invention also provides a method for preparing the microcapsule powder, which is characterized by including the following steps:

[0009] (1) Add the Pleurotus eryngii bacterial solution to the solid fermentation medium prepared from pecan cake meal. After mixing evenly, ferment to obtain the fermented pecan cake meal sample.

[0010] (2) Extract the crude protein solution from the fermented pecan cake meal sample described in step (1), freeze-dry it to obtain pecan cake meal antioxidant peptides, then add deionized water and pecan oil for emulsification, and freeze-dry to obtain microcapsule powder.

[0011] Among them, the preparation method of the solid fermentation medium described in step (1) includes the following steps: Mix pecan cake meal powder and distilled water evenly, and sterilize to obtain the solid fermentation medium prepared from pecan cake meal.

[0012] Among them, the mass-volume ratio of the pecan cake meal powder to distilled water is 1:1 - 5.

[0013] Among them, the sterilization temperature is 121 °C and the time is 15 - 20 minutes.

[0014] Among them, the mass-volume ratio of the pecan cake meal antioxidant peptides to deionized water in step (2) is 0.1 - 0.5:50 - 100 g / mL.

[0015] Among them, the mass-volume ratio of the pecan cake meal antioxidant peptides to pecan oil in step (2) is 0.1 - 0.5:5 - 10 g / mL.

[0016] Among them, when the extraction in step (2) includes the following steps: Take the fermented pecan cake meal sample described in step (1), adjust the pH to 9 - 12, stir at 25 - 45 °C for 1.0 - 3.0 hours, centrifuge, take the supernatant, adjust the pH to 4 - 4.5, let it stand for 1 - 2 hours, and take the precipitate; wash with ultrapure water and dissolve, adjust the pH value to 7.0 to obtain the crude protein solution.

[0017] The present invention prepares antioxidant peptides from fermented samples with different strains, different fermentation times, and different fermentation substrates, and selects the fermentation conditions with the strongest antioxidant activity for the antioxidant peptides as the fermentation strain being Pleurotus eryngii, the fermentation time being 9 days, and the fermentation substrate being pecan cake meal.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. High antioxidant activity pecan oil microcapsules are prepared. Compared with the pecan oil microcapsule powder prepared by conventional embedding wall materials (β-cyclodextrin), the antioxidant activity of the microcapsule powder prepared by the present invention is significantly higher than that of the control, and the microcapsule performance indicators such as its average particle size, Zeta potential, and encapsulation rate all reach or are better than those of the control; 2. The utilization value of pecan cake is improved, which has environmental protection significance, and further develops its application potential in the fields of food, health products, medicine, etc.; 3. An innovative method for preparing functional peptides is provided through a special treatment method of solid-state fermentation, which is of great significance for developing new functional foods. Description of the Drawings

[0019] Figure 1 is the DPPH free radical scavenging ability of antioxidant peptides prepared by different fermentation strains;

[0020] Figure 2 is the total antioxidant capacity of antioxidant peptides prepared by different fermentation strains;

[0021] Figure 3 is the total antioxidant capacity of antioxidant peptides prepared at different fermentation times;

[0022] Figure 4 is the DPPH free radical scavenging ability of antioxidant peptides prepared at different fermentation times;

[0023] Figure 5 is the morphological diagram of the microcapsule emulsion prepared from pecan cake peptides;

[0024] Figure 6 is the surface morphology structure diagram of the microcapsules prepared from pecan cake peptides. Detailed Embodiments

[0025] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0026] Pecan cake and pecan oil (pecan oil) were purchased from Xinyi Gejia Agricultural Development Company; soybean cake was purchased from Chenxi Organic Feed Business Department in Huainan City, Anhui Province; Bacillus subtilis (Bacillus subtilis) No.: CICC 10261, Cordyceps militaris (Cordyceps militaris) No.: CICC 14013, and Pleurotus eryngii (Pleurotus eryngii): No.: CICC 50125 were all purchased from China Center for Industrial Culture Collection. The BCA protein concentration assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0027] Example 1 Preparation of Fermentation Samples

[0028] Bacillus subtilis was cultured in mung bean sprout juice medium: 200 mL of concentrated mung bean sprout juice obtained by boiling mung bean sprouts was added with 10 g of sucrose (C 12 H 22 O 11 ), and sterilized at 121 °C for 15 min. Cordyceps militaris and Pleurotus eryngii were cultured in PDA liquid medium: 500 mL of potato extract, 10 g of glucose, 1.5 g of KH2PO4, and sterilized at 121 °C for 15 min. Solid fermentation medium prepared with pecan cake meal: The pecan cake meal was crushed, 100 g of the powder was taken, mixed evenly with 100 mL of distilled water, and sterilized at 121 °C for 15 min; Solid fermentation medium prepared with soybean cake meal: The soybean meal was crushed, 100 g of the powder was taken, mixed evenly with 100 mL of distilled water, and sterilized at 121 °C for 15 min.

[0029] In a laminar flow hood, 500 μL of the strains of Bacillus subtilis, Cordyceps militaris, and Pleurotus eryngii (hereinafter referred to as: Pleurotus eryngii) were respectively pipetted and inoculated into 5 mL of mung bean sprout juice or PDA liquid medium, and incubated in a constant temperature incubator at 30 °C for 5 d. After subculturing according to an inoculation amount of 10%, the fermented Bacillus subtilis, Cordyceps militaris, and Pleurotus eryngii bacterial solutions were respectively added to the solid fermentation media prepared with the above two kinds of pecan cake meal and soybean cake meal, mixed evenly, and fermented in a constant temperature incubator at 30 °C. They were taken out at 3 d and 9 d respectively, and the pecan cake meal fermentation samples and soybean cake meal fermentation samples fermented by Bacillus subtilis, Cordyceps militaris, and Pleurotus eryngii were obtained respectively.

[0030] Example 2 Selection of Fermentation Strains

[0031] 1. Preparation of Antioxidant Peptides

[0032] 10 g of the pecan cake meal fermentation samples prepared in Example 1 (the fermentation strains were Cordyceps militaris, Bacillus subtilis, and Pleurotus eryngii respectively; the fermentation days were 9 days) were respectively taken, added with 400 ml of ultrapure water, the pH was adjusted to 12 with 0.1 mol / L NaOH solution, and extracted in a water bath at 55 °C for 90 min; centrifuged at 4 °C and 10000 rpm for 20 min, and the supernatant was taken. Then the pH was adjusted to 4.5 with 0.1 mol / L HCl, extracted at room temperature for 1 h, centrifuged at 4 °C and 10000 rpm for 20 min, the precipitate was taken, and dissolved in an appropriate amount of 0.02 M PBS (PH = 7.4) buffer solution to obtain an antioxidant peptide aqueous solution. The antioxidant peptide aqueous solution was freeze-dried to obtain freeze-dried protein, which was the antioxidant peptide. Then its DPPH free radical scavenging ability and total antioxidant capacity were measured.

[0033] 2. Determination of DPPH Free Radical Scavenging Ability of Antioxidant Peptides

[0034] The DPPH radical scavenging ability of the prepared antioxidant peptides was measured using a DPPH radical scavenging ability kit. First, a 0.2 mM DPPH solution was prepared with absolute ethanol and stored in the dark at 4 °C. 2 mL of the DPPH solution and 2 mL of an 8.6 mg / mL antioxidant peptide aqueous solution were added to test tubes, and a blank group was set up for the control experiment. Then, 2 mL of the DPPH solution and 2 mL of the absolute ethanol solution were added successively, and the reaction was allowed to proceed in the dark at room temperature for 25 minutes. Subsequently, the absorbance was measured at 517 nm. To establish a standard curve, a series of Trolox concentration gradient standard solutions (0, 5, 10, 15, 20, 25 μg / mL) were prepared with 80% methanol. In addition, an 80% methanol solution without sample was used as a blank control. According to the measurement results, the DPPH radical scavenging ability value was expressed as the Trolox equivalent per gram of dry weight sample, with the unit of μg Trolox / mL. The standard curve was y = 0.019x - 0.0003, where x was the Trolox concentration and y was the scavenging rate, and R 2 = 0.9974. Based on the standard curve of the absorbance at 517 nm and the DPPH radical scavenging ability value and the measured absorbance, the DPPH radical scavenging ability of each microcapsule powder was obtained. The results are as Figure 1 shown. The antioxidant peptide prepared with the pecan cake meal peptide fermented by Pleurotus ostreatus as the wall material had the highest DPPH radical scavenging ability, indicating that Pleurotus ostreatus was the most suitable for preparing antioxidant peptides.

[0035] 3. Determination of the total antioxidant capacity of antioxidant peptides (FRAP method)

[0036] The total antioxidant capacity of the antioxidant peptide solution was determined using the FRAP method. In this experiment, a total antioxidant capacity assay kit was used. First, 2.5 mL of 10 mmol / L TPTZ solution, 25 mL of 0.3 mol / L sodium acetate buffer (pH = 3.6), and 2.5 mL of 20 mmol / L FeCl3 solution 2 were taken and mixed evenly. After incubating in a 35°C water bath for 30 min, the TPTZ working solution could be obtained, and this solution was prepared and used immediately. The TPTZ working solution and the antioxidant peptide solution (8.6 mg / mL) were allowed to react at 35°C for 25 min respectively, and the absorbance was measured at a wavelength of 593 nm. To establish a standard curve, FeSO4 solutions with concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM were prepared. Then, 0.1 mL of the antioxidant peptide solution was taken in a test tube, and 0.9 mL of the TPTZ working solution and 9 mL of the sodium acetate buffer were added successively. It was allowed to react at 35°C for 25 min. After the reaction was complete, the absorbance was measured at a wavelength of 593 nm. According to the measurement results, the FRAP result was expressed as the equivalent divalent iron antioxidant capacity (FEAC), with the unit of mmol / L FeSO4. According to the standard curve of the absorbance at 593 nm and the total antioxidant capacity and the measured absorbance of each antioxidant peptide, the total antioxidant capacity of each antioxidant peptide was obtained. The standard curve was y = 3.0322x + 0.0358, where y was the FeSO4 concentration and x was the absorbance value, and R 2 = 0.9984.

[0037] The results are as Figure 2 shown. The total antioxidant capacity of the antioxidant peptide from the pecan cake fermented by Pleurotus ostreatus is the best.

[0038] Example 3 Selection of Fermentation Time

[0039] 10 g of the pecan cake fermentation samples fermented for 3 days and 9 days prepared in Example 1 (the fermentation strain was Pleurotus ostreatus) were taken respectively, and the antioxidant peptides were prepared according to the method of Example 2, and their DPPH radical scavenging ability and total antioxidant capacity were measured. The results are as Figure 3 and Figure 4 shown. The total antioxidant capacity and DPPH radical scavenging ability of the antioxidant peptide from the pecan cake fermented by Pleurotus ostreatus for 9 days are both higher than those fermented for 3 days.

[0040] Example 4 Selection of Fermentation Medium

[0041] 10 g of the pecan cake fermentation sample and the soybean cake fermentation sample prepared in Example 1 (the fermentation strain was Pleurotus ostreatus; the fermentation time was 9 days) were taken respectively, and the antioxidant peptides were prepared according to the method of Example 2, and their DPPH radical scavenging ability and total antioxidant capacity were measured.

[0042] Table 1

[0043]

[0044] As shown in Table 1, the free radical scavenging ability and total antioxidant capacity of the antioxidant peptides prepared from the fermented samples of pecan meal are higher than those of the antioxidant peptides prepared from the fermented samples of soybean meal. Among them, A and B represent significant difference analysis, and A > B.

[0045] Example 5 Preparation of Microcapsule Powder and Determination of Performance Indexes

[0046] 1. Preparation of Microcapsule Powder

[0047] Respectively take 10 g of the fermented pecan meal sample prepared in Example 1 (the strain is Pleurotus ostreatus; the fermentation time is 9 days), add 400 ml of ultrapure water, adjust the pH to 12 with 0.1 mol / L NaOH solution, and extract at 55 °C in a water bath for 90 min; centrifuge at 4 °C and 10,000 rpm for 20 min, and take the supernatant. Then adjust the pH to 4.5 with 0.1 mol / L HCl, extract at room temperature for 1 h, centrifuge at 4 °C and 10,000 rpm for 20 min, take the precipitate, and dissolve it in an appropriate amount of 0.02 M PBS (PH = 7.4) buffer solution to obtain an aqueous solution of antioxidant peptides. Freeze-dry the aqueous solution of antioxidant peptides to obtain freeze-dried protein, which is the antioxidant peptide; weigh 0.5 g of freeze-dried protein, 100 mL of deionized water and 10 mL of pecan oil, and then circulate through a microfluidizer at 12,000 psi three times to obtain a microcapsule emulsion, which is made into microcapsule powder by freeze-drying.

[0048] 2. Measurement of the Average Particle Size and Zeta Potential of the Microcapsule Emulsion

[0049] Absorb 3 mL of the microcapsule emulsion into a special cuvette, and use a nanoparticle size and zeta potential analyzer to detect its average particle size and zeta potential. The measured zeta potential is -33.43 ± 0.02 A mV; the average particle size is 19.31 ± 0.71 A .

[0050] 3. Observation of the Morphology and Surface Topography Structure of the Microcapsule Emulsion

[0051] Morphology observation: Respectively absorb the microcapsule emulsion, evenly coat it on a glass slide, and observe its morphology under an optical microscope at a magnification of 40X. The results are as Figure 5 shown. There are many microcapsule particles in the microcapsule emulsion under the optical microscope.

[0052] Observation of the surface topography structure: Fix the glass slide carrying the microcapsule emulsion on a metal plate with double-sided tape, then perform sputtering treatment, and observe it under a scanning electron microscope. As Figure 6As shown, the microcapsules are in a complete form and exhibit a spherical structure, indicating the successful encapsulation by microencapsulation.

[0053] 4. Determination of the embedding rate of microcapsules

[0054] The ultrasonic oscillation method was used to break the wall and extract oil for determination. Weigh 2 g of microcapsule powder (M0) into a beaker, add 40 mL of petroleum ether, perform ultrasonic treatment for 15 min, immediately carry out vacuum filtration, then wash the filter residue four times with 40 mL of petroleum ether, transfer the combined filter residue to a weighed petri dish (the mass of the petri dish is denoted as M1), and then dry it to a constant weight at 105 °C (the mass is denoted as M2).

[0055] The total oil content of the microcapsules M3 = M0 - (M2 - M1)

[0056] Among them, M0 is the mass of the microcapsules; M1 is the mass of the petri dish; M2 is the mass of the petri dish and the microcapsule wall material.

[0057] Determination of the oil content on the surface of the microcapsules: Add 2 g of microcapsule powder to 30 mL of petroleum ether, extract for 2 min under sufficient shaking and then filter. Place the filtrate in a flask and evaporate the petroleum ether using a rotary evaporator, and calculate the weight difference before and after the flask as M4.

[0058] Embedding rate of microcapsules: The efficiency of microencapsulation can be expressed as the ratio of the core material encapsulated in the microcapsules to the total core material in the microcapsules, reflecting the degree to which the pecan oil is encapsulated by the carrier. Calculate according to the following formula:

[0059] Embedding rate (%) = (M3 - M4) × 100% / M3

[0060] Among them, M3 is denoted as the total oil content of the microcapsules; M4 is denoted as the oil content on the surface of the microcapsules.

[0061] The calculated embedding rate is 85.28 ± 1.02 A %.

[0062] Control group

[0063] Perform comparative tests on various indicators with the pecan oil microcapsule powder prepared with the conventional embedding wall material (β-cyclodextrin).

[0064] Table 2 Comparison of the performance of pecan oil microcapsules embedded with the conventional wall material (β-cyclodextrin)

[0065]

[0066]

[0067] As can be seen from Table 2, compared with the conventional embedding wall material, pecan meal peptides are more suitable as wall materials for preparing pecan oil microcapsules.

Claims

1. A method for preparing thin-shell pecan oil microcapsule powder with high antioxidant activity using thin-shell pecan cake antioxidant peptide as wall material, characterized in that: Comprising the following steps: (1)Add the Pleurotus eryngii ( Pleurotus eryngii ) bacterial liquid to the solid fermentation medium prepared from pecan meal. After mixing evenly, ferment for 3 - 9 days to obtain the fermented pecan meal sample; (2) Take the fermented sample of pecan meal obtained in step (1), adjust the pH to 9 - 12, stir at 25 - 45 °C for 1.0 - 3.0 hours, centrifuge, take the supernatant, adjust the pH to 4 - 4.5, let stand for 1 - 2 hours, and take the precipitate; wash with ultrapure water, dissolve, adjust the pH value to 7.0, after obtaining the crude protein solution, freeze-dry to obtain pecan meal antioxidant peptides, then add deionized water and pecan oil for emulsification, and freeze-dry to obtain microcapsule powder; the mass-volume ratio of the pecan meal antioxidant peptides to deionized water is 0.1 - 0.5:50 - 100 g / mL; the mass-volume ratio of the pecan meal antioxidant peptides to pecan oil is 0.1 - 0.5:5 - 10 g / mL.

2. The method according to claim 1, wherein The temperature of the fermentation in step (1) is 25 - 30 °C.

3. The method according to claim 1, wherein The preparation method of the solid-state fermentation medium described in step (1) comprises the following steps: mix pecan meal powder and distilled water evenly, and sterilize to obtain the solid-state fermentation medium prepared from pecan meal.

4. The method according to claim 3, wherein The mass-volume ratio of the pecan meal powder to distilled water is 1:1 - 5.

5. The method according to claim 3, characterized in that The sterilization temperature is 121 °C and the time is 15 - 20 minutes.

6. A highly antioxidant pecan oil microcapsule powder with pecan meal antioxidant peptides as the wall material prepared by the method according to any one of claims 1 - 5.

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