Preparation method of high-steady-state walnut oil based on stigmasterol and beta-sitosterol

Abstract

The invention discloses a preparation method of high-steady-state walnut oil based on stigmasterol and beta-sitosterol, and relates to the technical field of food processing. Comprising the following steps: S1, extracting walnut oil in walnut kernels by adopting an ultrasonic-assisted method; s2, adding a stigmasterol and beta-sitosterol mixture into the walnut oil, wherein the ratio of stigmasterol to beta-sitosterol in the mixture is 1: 2; s3, transferring the mixed solution to an ultrasonic cleaning instrument, setting the temperature to be 45 + / -1 DEG C, performing ultrasonic treatment for 27 minutes, pausing for 10 seconds every 5 minutes during the ultrasonic treatment, and slightly shaking the container to avoid local overheating; s4, after ultrasonic treatment of the mixed solution is completed, standing the sample for 5 minutes to balance the temperature, and then centrifuging to remove possible bubbles or undispersed particles; and S5, transferring the mixed solution to a brown glass bottle, purging with nitrogen for 1 minute, sealing, and keeping in a dark place at 4 DEG C. The invention provides the preparation method of the high-steady-state walnut oil, which can improve the oxidation stability of the walnut oil without changing the nutritional ingredients of the walnut oil.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for preparing highly stable walnut oil based on stigmasterol and beta-sitosterol. Background Art

[0002] Walnut oil, a new commodity vegetable oil, is favored by many consumers due to its rich content of polyunsaturated fatty acids. Its optimal ratio of polyunsaturated fatty acids, in particular, gives it a unique position in the edible oil market. However, this high concentration of polyunsaturated fatty acids also makes walnut oil more susceptible to oxidation from environmental factors, affecting its quality stability and shortening its shelf life, limiting its application.

[0003] Therefore, in order to solve the above technical problems, it is necessary to provide a new technical solution to solve this technical problem, especially a method for preparing highly stable walnut oil based on stigmasterol and β-sitosterol. Summary of the Invention

[0004] The purpose of the present invention is to solve the technical problems of easy oxidation and limited market application of existing walnut oil, and provide a method for preparing highly stable walnut oil based on stigmasterol and β-sitosterol.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The beneficial effects of the present invention are as follows: a method for preparing highly stable walnut oil based on stigmasterol and β-sitosterol comprises the following steps:

[0007] S1: extracting walnut oil from walnut kernels using ultrasound-assisted extraction; the ultrasound-assisted extraction comprises the following steps: mixing the walnut kernels with petroleum ether at a solid-liquid ratio of 1:15 (g / mL), setting the ultrasonic power to 225W, sonicating for 30 minutes, filtering and centrifuging, collecting the oil, and storing it at low temperature;

[0008] S2: adding a mixture of stigmasterol and β-sitosterol to walnut oil, wherein the ratio of stigmasterol to β-sitosterol in the mixture is 1:2; adding the mixture at 40° C. under magnetic stirring (300 rpm) for 5 minutes to completely dissolve the added mixture;

[0009] S3: Transfer the mixed solution to an ultrasonic cleaner (40 kHz, 300 W), set the temperature to 45 ± 1 °C, and sonicate for 27 min, pausing for 10 seconds every 5 min and gently shaking the container to avoid local overheating;

[0010] S4: After the sonication of the mixed solution was completed, the sample was allowed to stand for 5 minutes to equilibrate the temperature, and then centrifuged (3000 rpm, 5 minutes) to remove possible bubbles or undispersed particles;

[0011] S5: The mixed solution was transferred to a brown glass bottle, and nitrogen was purged for 1 minute to remove oxygen to obtain highly stable walnut oil. The highly stable walnut oil was sealed and stored at 4° C. in the dark.

[0012] Preferably, in step S2, stigmasterol and β-sitosterol are placed in dry brown glass bottles before being weighed, protected from light and moisture, and equilibrated at room temperature for 30 minutes to ensure stability.

[0013] Preferably, in step S2, the walnut oil is preheated in a 40° C. water bath for 10 minutes before adding stigmasterol and β-sitosterol to reduce the viscosity, thereby facilitating subsequent uniform mixing.

[0014] Beneficial effects:

[0015] By optimizing the ratio of endogenous antioxidant components and extraction process parameters, the present invention significantly improves the oxidative stability index (OSI) of walnut oil from 2 hours to more than 6.42 hours, increasing the oxidative stability index (OSI) by 221%, effectively extending the shelf life of the walnut oil and reducing the risk of oxidative deterioration. The invention completely relies on the endogenous antioxidant substances (β-sitosterol and stigmasterol) of the walnut oil itself to enhance the antioxidant performance, thereby retaining the natural properties and rich nutritional components of the walnut oil and meeting consumers' demand for natural and healthy edible oil.

[0016] The present invention selects the optimal ultrasound-assisted extraction method by comparing the extraction efficiency of walnut oil by different extraction methods, and simultaneously analyzes the influence of the oxidative stability of endogenous antioxidants in the walnut oil, discovering the dominant role of stigmasterol on the DPPH free radical scavenging activity and the dosage synergistic effect with beta-sitosterol. This overcomes the limitation of single antioxidant research and selects stigmasterol and beta-sitosterol, which have the strongest free radical scavenging ability, as additives without changing the natural nutritional components of the walnut oil. In addition, through research on different addition ratios, the optimal addition ratio of stigmasterol to beta-sitosterol is selected as 1:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the composition and content of endogenous antioxidant substances in walnut oil extracted using different methods of the present invention.

[0018] Figure 2 This is a graph showing the characteristic fatty acid content of walnut oil extracted using different extraction methods.

[0019] Figure 3 This is a graph showing the antioxidant capacity of walnut oil extracted using different methods.

[0020] Figure 4 This is a performance analysis diagram of significant correlation between the endogenous active substances and the antioxidant properties of the walnut oil of the present invention.

[0021] Figure 5 This is a significant correlation performance analysis diagram between the endogenous active substances in the walnut oil of the present invention and the single antioxidant property.

[0022] Figure 6 Graph showing the effect of the antioxidant ratio on the OSI of walnut oil.

[0023] Figure 7 This is a graph showing the effect of ultrasonic temperature on the OSI of walnut oil.

[0024] Figure 8 This is a graph showing the effect of ultrasound time on the OSI of walnut oil.

[0025] Figure 9 Graph showing the effect of the interaction of various factors on the OSI of walnut oil. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0029] Example 1

[0030] A method for preparing highly stable walnut oil based on stigmasterol and β-sitosterol comprises the following steps:

[0031] S1: extracting walnut oil from walnut kernels using ultrasound-assisted extraction; the ultrasound-assisted extraction comprises the following steps: mixing the walnut kernels with petroleum ether at a solid-liquid ratio of 1:15 (g / mL), setting the ultrasonic power to 225W, sonicating for 30 minutes, filtering and centrifuging, collecting the oil, and storing it at low temperature;

[0032] S2: Adding a mixture of stigmasterol and β-sitosterol to walnut oil, wherein the ratio of stigmasterol to β-sitosterol in the mixture is 1:2; adding the mixture under magnetic stirring at 40°C for 5 minutes (300 rpm) to completely dissolve the added mixture; before addition, stigmasterol and β-sitosterol are weighed and placed in dry brown glass bottles, protected from light and moisture, and equilibrated at room temperature for 30 minutes to ensure stability; before adding stigmasterol and β-sitosterol, the walnut oil is preheated in a 40°C water bath for 10 minutes to reduce viscosity and facilitate subsequent mixing;

[0033] S3: Transfer the mixed solution to an ultrasonic cleaner (40 kHz, 300 W), set the temperature to 45 ± 1 °C, and sonicate for 27 min, pausing for 10 seconds every 5 min and gently shaking the container to avoid local overheating;

[0034] S4: After the sonication of the mixed solution was completed, the sample was allowed to stand for 5 minutes to equilibrate the temperature, and then centrifuged (3000 rpm, 5 minutes) to remove possible bubbles or undispersed particles;

[0035] S5: The mixed solution was transferred to a brown glass bottle, and nitrogen was purged for 1 minute to remove oxygen to obtain highly stable walnut oil. The highly stable walnut oil was sealed and stored at 4° C. in the dark.

[0036] Comparative Example 1

[0037] Based on Example 1, walnut oil was extracted from walnut kernels by hot pressing. The extraction method is as follows: walnut kernels were placed in a screw press, pressed at a temperature of 120±2°C, and the oil was collected and stored at low temperature.

[0038] Comparative Example 2

[0039] Based on Example 1, walnut oil was extracted from walnut kernels by cold pressing. The extraction method was as follows: walnut kernels were squeezed with a screw press at room temperature, the pressing temperature was controlled at 40±2°C, and the oil was collected and stored at low temperature.

[0040] Comparative Example 3

[0041] Based on Example 1, walnut oil was extracted from walnut kernels using the Soxhlet extraction method. The extraction method was as follows: walnut kernels were mixed with petroleum ether at a solid-liquid ratio of 1:15 (g / mL), the extraction temperature was controlled at 60°C, the extraction time was 6 hours, the organic solvent was removed by rotary evaporation, and the oil was collected and stored at low temperature.

[0042] like Figures 1 to 3 As shown in the figure, ultrasound-assisted extraction is the best extraction method among the four different extraction methods. The reasons are as follows: Figure 1It was shown that walnut oil extracted by ultrasound-assisted extraction contained high levels of phytosterols and tocopherols, especially β-sitosterol and γ-tocopherol, which have a significant effect on improving oxidative stability. Figure 2 The results showed that walnut oil extracted by ultrasound-assisted extraction contained higher levels of unsaturated fatty acids such as linoleic acid and linolenic acid. The mild conditions of ultrasound-assisted extraction reduced the damage to unsaturated fatty acids caused by high temperatures, thus preserving the nutritional value of walnut oil. Figure 3 It was shown that the comprehensive antioxidant capacity (DPPH, ABTS and other free radical scavenging capacity) of the ultrasound-assisted extraction method was excellent, indicating that ultrasound-assisted extraction has advantages in balancing oxidative stability and nutrient retention.

[0043] like Figure 4 As shown, Figure 4 This study analyzed the significant correlation between the endogenous active substances in walnut oil and its antioxidant properties. PLS2 was used to analyze the correlation between oxidative stress indicators (DPPH free radicals, hydroxyl free radicals, iron ion reduction, and ABTS free radicals) and 11 bioactive components in walnut oil samples prepared by different methods. A PLS2 model was established using the 11 compounds as X variables and the oxidative stress indicators as Y variables. Figure 4 Figures A and B show the results of a PLS2 regression analysis. The labeled compounds in the figures are those with significant effects on the Y-variables (P ≤ 0.05) calculated using a kernel-knife uncertainty test. These compounds include oleic acid, linolenic acid, linoleic acid, stigmasterol, δ-tocopherol, γ-tocopherol, β-sitosterol, and campesterol. The cumulative explained variance of the PLS2 model is >80%. The predicted R-squared value is 0.9859892, and the actual R-squared value is 0.9630388, both exceeding 0.95, indicating good predictive power. The two large ellipses in the figure represent 50% and 100% of the explained variance of the PLS2 model, respectively. As can be seen in the figure, all X- and Y-variables fall between the two ellipses, indicating that the established PLS2 model has good explanatory power for these indicators.

[0044] Walnut oil samples prepared by four different methods are plotted along the horizontal axis. Ultrasonic-assisted extraction and cold-pressed walnut oil samples are located on the left side of the loading plot, Soxhlet-extracted walnut oil samples are located in the center, and hot-pressed oil samples are located on the right side. Hydroxyl radicals and iron reduction are located in the upper right of the loading plot, while ABTS and DPPH are located in the lower right, indicating that the four oxidative stress markers can be explained by Factor 2. Cold-pressed and ultrasonic-extracted oil samples show positive correlations with palmitic acid and stearic acid, but show significant negative correlations with oleic acid, linoleic acid, linolenic acid, sterols, tocopherols, and all oxidative stress markers. This may be due to the oxidation of unsaturated bonds in the oil during the cold-pressing and ultrasonic-extraction process. This result is consistent with the results of the antioxidant experiment, which showed that walnut oils prepared by cold-pressing and ultrasonic-assisted extraction had relatively weak scavenging abilities for DPPH radicals, hydroxyl radicals, iron ion reducing power, and ABTS radicals. The content of active ingredients in the cold-pressed and ultrasonic-extracted oils was also lower than that in the walnut oils prepared by the other two methods. For example, β-sitosterol, the most abundant walnut oil, is found in cold-pressed walnut oil at 875.51 ± 28.76 mg / kg, while ultrasound-assisted extraction yields 847.58 ± 21.63 mg / kg, a difference of 96.16 mg / kg from hot-pressed walnut oil. However, its antioxidant capacity is not the lowest, suggesting that unsaturated fatty acids and certain sterols may play a major role in the antioxidant process. The above correlation analysis preliminarily suggests a close correlation between sterols such as stigmasterol and β-sitosterol in walnut oil samples and free radical scavenging activity. To validate these results, we conducted a single-variable partial least squares regression analysis (PLS1).

[0045] like Figure 5 As shown, Figure 5This is a significant correlation performance analysis of the endogenous active substances in walnut oil and their single antioxidant properties. Regarding the DPPH free radical scavenging ability, the main correlates are stigmasterol (0.8224), linolenic acid (0.3903), and γ-tocopherol (0.3092). At the same time, differentiation was performed based on Soxhlet extraction of oils and fats to obtain differential coefficients. Combining the differential coefficients of various endogenous antioxidants, it was found that the coefficients of linolenic acid (0.53), γ-tocopherol (0.41), and δ-tocopherol (0.37) varied significantly, but their corresponding baseline values were relatively low. Other significant endogenous antioxidants, such as stigmasterol and β-sitosterol, had higher baseline levels and smaller differential coefficients (0.21 and 0.08, respectively). Therefore, it was concluded that in walnut oil with stigmasterol as the main agent for DPPH free radical scavenging, γ-tocopherol and linolenic acid also had significant scavenging efficacy against DPPH, and the differences between these samples were caused by the interaction between substances with low baseline levels. Similarly, the same conclusion was reached for other free radicals. Stigmasterol and β-sitosterol are the key endogenous antioxidants in walnut oil.

[0046] Therefore, in the present invention, stigmasterol and β-sitosterol are selected as the added components to improve the antioxidant capacity of walnut oil, which will not change the nutritional components of walnut oil while improving the antioxidant capacity of walnut oil.

[0047] like Figure 6-8 As shown, Figure 6 The effect of the antioxidant ratio of the present invention on the OSI of walnut oil. Different letters indicate significant differences when p < 0.05. As can be seen from the figure, the antioxidant ratio has a significant effect on the OSI of walnut oil. When the antioxidant ratio (β-sitosterol / stigmasterol) is 2:1, the OSI value of walnut oil is the highest.

[0048] Figure 7 The effect of ultrasonic temperature on the OSI of walnut oil during the present invention. Different letters indicate significant differences when p < 0.05. As can be seen from the figure, ultrasonic time has a significant effect on the OSI of walnut oil. Walnut oil has the highest OSI value when the ultrasonic temperature is 45°C. As the temperature increases, the OSI of walnut oil decreases, indicating that ultrasonic temperature affects the oxidative stability of walnut oil.

[0049] Figure 8 The effect of ultrasound time on the OSI of walnut oil was observed. When p < 0.05, different letters indicate significant differences. As can be seen from the figure, ultrasound time has a significant effect on the OSI of walnut oil. When the ultrasound time is 25 min, the OSI value of walnut oil is the highest.

[0050] Table 1 Antioxidant time of products obtained by different antioxidant ratios, ultrasonic temperature and ultrasonic time parameters

[0051]

[0052] Depend on Figure 6-8 As shown in Table 1, when the ratio of β-sitosterol to stigmasterol was 2:1, the ultrasonic temperature was 45°C, and the ultrasonic time was 25 minutes, the antioxidant time of the walnut oil product was the highest, which was 6.35 hours.

[0053] like Figure 9 As shown, Figure 9 The interaction of the various factors of the present invention on the OSI of walnut oil is shown in Figure 3. The more significant the effect on the OSI of walnut oil and the stronger the interaction, the steeper the curve; conversely, the smoother the curve. For the interactions between the antioxidant ratio (A) and ultrasonic temperature (B), the antioxidant ratio (A) and ultrasonic time (C), and the ultrasonic temperature (B) and ultrasonic time (C), the AB and BC response surface 3D plots showed steeper curves, indicating that the interaction between the antioxidant ratio, ultrasonic temperature, and ultrasonic time was more significant, and had a more significant impact on the OSI of walnut oil. The AC response surface 3D plot showed a flatter curve, indicating that the interaction between the antioxidant ratio and ultrasonic time had a smaller effect on the OSI of walnut oil, which is consistent with the results of the variance analysis.

[0054] Using response surface methodology, the optimal antioxidant addition conditions were determined: an antioxidant ratio (β-sitosterol:stigmasterol) of 2.182:1, an ultrasonic temperature of 45.289°C, and a duration of 27.096 minutes. Under these conditions, the OSI score for walnut oil was 6.422. Taking into account the actual experimental conditions, the optimal conditions were adjusted to an antioxidant ratio (β-sitosterol / stigmasterol) of 2:1, an ultrasonic temperature of 45°C, and a duration of 27 minutes. Three parallel experiments were conducted. The average OSI score was 6.424, with a relative error of no more than 1.5%. This small difference from the predicted value indicates that the mathematical model employed in this study has good predictive performance.

[0055] The invention analyzes the antioxidant factors in walnut oil, adds natural antioxidant substances existing in the walnut oil, and increases the antioxidant stability of the walnut oil.

[0056] The above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Ordinary technicians in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention. The scope of patent protection of the present invention should be defined by the claims.

Claims

1. A method for preparing highly stable walnut oil based on stigmasterol and β-sitosterol, characterized in that: The following steps are involved: S1: extracting walnut oil from walnut kernels using ultrasound-assisted extraction; the ultrasound-assisted extraction comprises the following steps: mixing the walnut kernels with petroleum ether at a solid-liquid ratio of 1:15 (g / mL), setting the ultrasonic power to 225W, sonicating for 30 minutes, filtering and centrifuging, collecting the oil, and storing it at low temperature; S2: adding a mixture of stigmasterol and β-sitosterol to walnut oil, wherein the ratio of stigmasterol to β-sitosterol in the mixture is 1:2; adding the mixture at 40° C. under magnetic stirring (300 rpm) for 5 minutes to completely dissolve the added mixture; S3: Transfer the mixed solution to an ultrasonic cleaner (40 kHz, 300 W), set the temperature to 45 ± 1 °C, and sonicate for 27 min, pausing for 10 seconds every 5 min and gently shaking the container to avoid local overheating; S4: After the sonication of the mixed solution was completed, the sample was allowed to stand for 5 minutes to equilibrate the temperature, and then centrifuged (3000 rpm, 5 minutes) to remove possible bubbles or undispersed particles; S5: The mixed solution was transferred to a brown glass bottle, and nitrogen was purged for 1 minute to remove oxygen to obtain highly stable walnut oil. The highly stable walnut oil was sealed and stored at 4° C. in the dark.

2. The method for preparing a highly stable walnut oil based on stigmasterol and β-sitosterol according to claim 1, wherein: In step S2, stigmasterol and β-sitosterol are placed in dry brown glass bottles before being weighed, protected from light and moisture, and equilibrated at room temperature for 30 minutes to ensure stability.

3. The method for preparing a highly stable walnut oil based on stigmasterol and β-sitosterol according to claim 1, wherein: In step S2, the walnut oil is preheated in a 40° C. water bath for 10 minutes before adding stigmasterol and β-sitosterol to reduce the viscosity, thereby facilitating subsequent uniform mixing.

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