Citrus peel oil sac separating agent and use method thereof
By using a separator of high concentration pectinase solution and high osmotic regulation buffer, the white cortex of citrus peel was removed, and the problem of difficulty in separating oil cells in the prior art was solved, and the integrity of oil cells structure and efficient development of essential oils were achieved.
Patent Information
- Application Number
- CN202510043575.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to effectively isolate and study citrus peel oil cells, resulting in damage to the oil cell structure and research errors, hindering the efficient development of citrus essential oils and improving flavor quality.
Using high-concentration pectinase solution and high-osmotic regulation buffer as separation agents, the white skin of the peel was removed by enzymatic removal to obtain clean peel oil cells.
It achieves rapid and efficient separation of citrus peel oil cells, maintains the integrity of the oil cell structure, reduces the loss of volatile essential oils, and supports oil cell research and essential oil development.
Smart Images
Figure CN120025964A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of citrus processing, and in particular to a separating agent for citrus peel oil cells and a use method thereof. Background Art
[0002] Citrus peel is mainly divided into two layers: the outer peel and the inner peel. The outer peel is also called the oil cell layer, and the inner peel is also called the white peel. The peel oil cells mainly originate from the epidermal cells of the peel in the oil cell layer, and develop and mature in the oil cell layer and the white peel. During the development to maturity stage, the oil cells can continuously secrete and accumulate volatile oil substances inside. After industrial refining, essential oils can be obtained. Its components are mainly volatile fractions composed of monoterpenoids and a small amount of sesquiterpenoids, which account for 94%-98% of the essential oils in total; it also contains a small amount of sterols, carotenoids, waxes, coumarins and polymethoxyflavonoids, accounting for 2%-6% of the essential oils. These volatile substances not only repel pathogens and pests, but also give the fruit a special smell, attracting animals to eat and thus promoting seed dissemination and reproduction. At the same time, the essential oil extracted from the peel has been widely used in the fields of cleaning and antibacterial, food processing and medical treatment.
[0003] At present, the isolation research of secretory structures is mainly focused on plant trichomes, such as Artemisia annua glandular hairs that produce a large amount of artemisinin, tobacco glands that secrete nicotine, and cannabis glands that can accumulate cannabinoids. In recent years, the isolation methods of specialized glandular hairs on the outside of plants have been continuously derived and improved, and this type of separable secretory structure has been widely used in the synthesis of metabolites and molecular biology research.
[0004] However, as an embedded secretory structure, the oil cells in citrus peel are closely connected to the surrounding white cortex tissue and cannot be directly used to separate other exocrine structures. Limited by the current separation technology, the oil cell layer of citrus peel is often equated with the oil cell structure to conduct research on the synthesis and regulation of volatile substances. This sampling process will inevitably destroy the integrity of the oil cell structure, and it is impossible to achieve more detailed research at the oil cell tissue level, which brings errors to the experimental results.
[0005] The current separation technology system based on the embedded secretory structure of plant fruits is still immature, which hinders the identification of characteristic metabolites inside citrus oil cells and basic research progress such as the developmental regulation mechanism. Therefore, it is urgent to develop a method for rapid and efficient separation of peel oil cells, and then better combine it with metabolomics, transcriptomics and other methods to provide important technical support for the improvement of citrus flavor quality and the efficient development of citrus essential oils. Summary of the invention
[0006] In order to solve the defects and deficiencies in the above-mentioned prior art, the present invention provides a citrus peel oil cell separation agent and a method of using the same, wherein the separation agent can remove the white skin layer of the peel to obtain clean peel oil cells. The separation agent and the method of using the same are simple, fast, low-cost, can effectively reduce the loss of volatile essential oil substances in the oil cells, and maintain the integrity of the oil cell structure, laying a foundation for the relevant theoretical basic research of oil cells and the development of essential oils.
[0007] In order to achieve the purpose of the present invention, the present invention uses a high concentration pectinase solution and a high osmotic adjustment buffer as the main components of the separation agent, and enzymatically removes the white cortex tissue of the peel to obtain clean peel oil cells. The specific technical scheme is:
[0008] A citrus peel oil cell separation agent, the separation agent is a mixed solution of pectinase solution and buffer solution, the buffer solution contains sorbitol, KOH and CaCl 2 .
[0009] Furthermore, the concentration of the sorbitol solution in the buffer is 0.5-0.93 mol / L, the concentration of the KOH solution is 4-6 mmol / L, and the concentration of the CaCl 2 The concentration of the solution is 0.5-1.5mmol / L, and the pH value of the buffer is 5.5-6.0.
[0010] Furthermore, the concentration of the buffer solution is 0.1-0.93 mol / L, the pectinase solution and the buffer solution are mixed in a volume ratio of 1:(1-3), and the concentration of the pectinase in the separation agent after mixing is 800-2000 U / ml.
[0011] Furthermore, the concentration of the buffer solution is 0.5-0.93 mol / L, the pectinase solution and the buffer solution are mixed in a volume ratio of 1:(1-2), and the concentration of the pectinase in the separation agent after mixing is 800-2000 U / ml.
[0012] Furthermore, the pectinase is p-4716 pectinase.
[0013] Another object of the present invention is to provide a method for separating citrus peel oil cells using the separation agent, the method comprising the following steps:
[0014] (1) Using citrus fruits as materials, removing a large piece of peel including a white peel layer, and thinning the white peel layer of the peel;
[0015] (2) applying the prepared separating agent evenly to the thinned white peel layer of the citrus and allowing it to stand for a period of time; the standing time is determined according to the citrus variety and the thickness of the white peel layer of the peel;
[0016] (3) When the white skin layer on the surface of the oil vesicles can be gently scraped off, directly rinse with running water to remove the enzymatically degraded white skin layer in the pericarp; if there are still white skin filaments remaining between the oil vesicles after cleaning, they can be directly picked out with tweezers.
[0017] Further, in step (1), without damaging the oil vesicles, try to scrape off the white skin layer as much as possible to shorten the time for enzymatically degrading the white skin layer.
[0018] Further, the citrus white skin layer coated with the separating agent is left standing for 3 - 5 hours under the culture conditions of 25 - 30 °C.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] (1) The separating agent and its using method of the present invention can separate complete and clean citrus pericarp oil vesicles, providing a reference idea for the separation and collection of embedded secretory structures in plant fruits.
[0021] (2) Through the proportion coordination of the buffer solution and the pectinase solution, on the one hand, the present invention can reduce the usage amount of the pectinase solution, saving the usage cost of the pectinase solution. On the other hand, the buffer solution also has a penetration effect, accelerating the enzymatic hydrolysis effect of the pectinase solution on the white filaments between citrus oil vesicles. The white skin layer and the white skin filaments between the oil vesicles can be effectively removed within a short time, maintaining the integrity of the citrus oil vesicle structure, reducing the loss of volatile essential oils inside the oil vesicles, and being conducive to subsequent oil vesicle research and industrial development and utilization.
[0022] (3) The separating agent and its using method of the present invention are applicable to the separation of oil vesicles of most citrus varieties, providing a valuable material basis for exploring the differences in the accumulation of citrus volatile substances inside the oil vesicles of different citrus germplasms, and also having important guiding significance for citrus essential oil development and output value improvement. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Referring to the accompanying drawings, the disclosure of the present invention will become more understandable. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention.
[0024] Figure 1 It is a photo of the ponkan materials selected for the present invention and the pericarp with the white skin layer thinned.
[0025] Figure 2 It is a photo of the white skin layer of the pericarp after being coated with the separating agent of the present invention.
[0026] Figure 3 It is a photo of the oil vesicles and the front side of the pericarp after enzymatic hydrolysis with the separating agent of the present invention.
[0027] Figure 4 It is a photo and an enlarged view of the ponkan pericarp oil vesicles separated by the separating agent of the present invention.
[0028] Figure 5 The photograph and enlarged view of the oil cells in the peel of Majiayou obtained by separation using the separation agent of the present invention are shown.
[0029] Figure 6 The photograph and enlarged view of the oil cells in the peel of Gannan Early Navel Orange obtained by separation using the separation agent of the present invention are shown.
[0030] Figure 7 This is a photo of the oil cells in the peel of tangerine peel separated by a separation agent mixed with pectinase solution and buffer in a ratio of 1:1 and 1:2.
[0031] Figure 8 This is a photo of the oil cells in the peel of tangerine peel separated by a separation agent mixed with pectinase solution and buffer in a ratio of 1:3.
[0032] Fig. 9 This is a diagram showing the separation effects of different types of separators on the peel of tangerine fruit.
[0033] Fig.10 This is a photo of enzymatic hydrolysis of tangerine fruit peel after being soaked in a separator solution for 2 hours. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of this application.
[0035] The separating agent and the method of use provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0036] Example 1
[0037] A citrus peel oil cell separation agent is prepared, wherein the separation agent is a mixed solution of pectinase solution and buffer solution, wherein the buffer solution contains sorbitol, KOH and CaCl 2 ; The concentration of sorbitol solution in the buffer is 0.93 mol / L, the concentration of KOH solution is 5 mmol / L, and the concentration of CaCl 2 The concentration of the solution is 1 mmol / L, the pH value of the buffer solution is 5.5; the pectinase solution and the buffer solution are mixed in a volume ratio of 1:1, and the concentration of the pectinase in the separation agent after mixing is 2000 U / ml. The pectinase solution is purchased from Sigma, specifically p-4716 pectinase.
[0038] Fresh tangerine fruits were picked at the National Citrus Breeding Center in Wuhan, Hubei Province. The tangerine fruits were in the development stage and immature. The method for separating the oil cells from the peel of tangerine fruits using the separation agent provided in this embodiment includes the following three steps:
[0039] (1) Take the tangerine fruit, first peel a large piece of peel containing the white skin, and remove as much of the white skin tissue as possible without destroying the oil cells. By removing the excess white skin, the enzymatic hydrolysis time can be shortened and the loss of volatile substances can be reduced.
[0040] (2) The prepared separating agent in this example is evenly applied to the white skin layer of the fruit peel in step (1), and the fruit peel is placed at 30° C. with the white skin layer facing upward for 3 hours. The white skin layer of the fruit peel is removed by the enzymatic hydrolysis of the separating agent.
[0041] (3) Wash the white cortex tissue of step (2) with running water. The bottom of the oil cell is clearly visible. The white cortex fibers left around are directly pulled out with tweezers to obtain a complete and clean oil cell.
[0042] Figure 1 The material for tangerine peeling used in this embodiment of the present invention and the photo of the peel with the white skin layer thinned.
[0043] Figure 2 This is a photo of the white skin layer of the fruit peel after being coated with the separating agent of the present invention.
[0044] Figure 3 The photos are of the oil cells of the peel and the front side of the peel after enzymatic hydrolysis using the separation agent of the present invention.
[0045] Figure 4 The photograph and enlarged view of the oil cells of the tangerine peel separated by the separating agent of the present invention are shown.
[0046] Example 2
[0047] The method is basically the same as Example 1, except that the pomelo fruits were picked at the National Citrus Breeding Center in Wuhan, Hubei as the objects for separation of peel oil cells.
[0048] Example 3
[0049] The method is basically the same as Example 1, except that Gannan Early Navel Orange fruits were picked from Ganzhou Citrus Research Institute as the objects for separation of peel oil cells.
[0050] Figure 5 The photograph and enlarged view of the oil cells in the peel of Majiayou obtained by separation using the separation agent of the present invention are shown. Figure 6 The photograph and enlarged view of the oil cells in the peel of Gannan Early Navel Orange obtained by separation using the separation agent of the present invention are shown.
[0051] According to the observation under stereo microscope in Examples 1-3, it can be seen that the contours around the oil cells are clear and the white skin layer is rarely attached. The structure of the oil cells in the peel of Examples 1 and 2 is complete, and the bottom of the oil cells of Gannan Early Navel Orange in Example 3 is slightly damaged, mainly because the enzymatic hydrolysis time is long. The enzymatic hydrolysis time of the peel can be appropriately shortened according to the thickness of the white skin layer.
[0052] Comparative Example 1
[0053] The components and method for preparing the separation agent are basically the same as those in Example 1, except that the buffer concentrations are respectively prepared to 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, 0.7 mol / L and 0.93 mol / L, and the pectinase solution and the buffer are mixed in a volume ratio of 1:2.
[0054] Figure 7 This is a photo of the oil cells in the peel of tangerine peel separated by a separation agent mixed with pectinase solution and buffer in a ratio of 1:1 and 1:2.
[0055] Comparative Example 2
[0056] The components and method for preparing the separation agent are basically the same as those in Example 1, except that the concentrations of the buffer solution are respectively prepared to be 0.5 mol / L, 0.7 mol / L and 0.93 mol / L, and the pectinase solution and the buffer solution are mixed in a volume ratio of 1:3.
[0057] Figure 8 This is a photo of the oil cells in the peel of tangerine peel separated by a separation agent mixed with pectinase solution and buffer in a ratio of 1:3.
[0058] By comparison, it can be seen that when the ratio of pectinase solution to buffer is 1:(1-2), the higher the buffer concentration, the faster the penetration of pectinase solution, and the better the enzymatic removal effect of the white cortex and the white cortex fibers between oil cells. When the ratio of pectinase solution to buffer is 1:3, the enzymatic efficiency of pectinase on the white cortex begins to decline significantly, which is specifically manifested in that when the buffer concentration is lower than 0.5 mol / L, the white cortex remains; when the buffer concentration is 0.5-0.93 mol / L, the white cortex on the surface can be cleaned, but the white cortex fibers between oil cells are difficult to remove and the degree of attachment is tight. Therefore, controlling the buffer concentration to 0.5-0.93 mol / L and the ratio of pectinase solution to buffer to 1:(1-2) can enable the separation agent to achieve a better separation effect.
[0059] Comparative Example 3
[0060] The separation agent ratio and use method of this comparative example are basically the same as those of Example 1, except that the p-4716 pectinase solution is replaced by PASE pectinase.
[0061] Comparative Example 4
[0062] The ratio of the separating agent and the method of use of this comparative example are basically the same as those of Example 1, except that the p-4716 pectinase solution is replaced by RS-10 cellulase solution.
[0063] Fig. 9 The following is a diagram showing the effect of different types of separation agents on the peel of the tangerine fruit. As can be seen from the figure, the separation agent containing p-4716 pectinase can effectively remove the white cortex material of the peel, and the oil cell structure is fully displayed; the separation agent containing PASE pectinase can also effectively remove the white cortex material of the peel, but some white cortex material will remain between the oil cells, and the separation effect is not as good as the separation agent containing p-4716 pectinase; the separation agent containing RS-10 cellulase cannot effectively enzymatically hydrolyze the white cortex and cannot achieve the purpose of separating the oil cells.
[0064] Comparative Example 5
[0065] The peel of the tangerine was directly placed into the separating agent solution of Example 1, and the enzymatic hydrolysis process of the peel was observed. Fig.10 This is a photo of the enzymatic hydrolysis of the peel of the tangerine fruit after being immersed in the separation agent solution for 2 hours. Fig.10 It can be seen that when the tangerine peel is treated by immersion, the peel tissue can be directly softened by enzymatic hydrolysis, the outer skin turns brown, and the oil cells embedded in the white skin are also enzymatically hydrolyzed, making it impossible to achieve the purpose of separating the oil cells.
[0066] Comparative Example 6
[0067] The separating agent solution of Example 1 was applied to the yellow peel layer of the tangerine peel to observe the separating effect of the separating agent on the oil cells in the tangerine peel.
[0068] After the yellow peel of the tangerine peel was applied with the separator solution for 10 hours, the yellow peel was softened and the oil cells could be peeled out from the peel, but there were the following problems: 1. The oil cells themselves developed from the upper epidermal cells, and part of the yellow peel belonged to the top part of the oil cells. Destroying the yellow peel was equivalent to destroying the oil cell structure; 2. The enzymatic hydrolysis time was too long, resulting in the loss of the main volatile components in the oil cells, and it was impossible to conduct accurate and detailed research on the oil cells separated in this way.
[0069] Comparative Example 7
[0070] The separation agent method and use method of this comparative example are basically the same as those of Example 1, except that the concentration of the pectinase solution after mixing in this comparative example is 250U / ml. When the separation agent prepared with the low concentration pectinase solution of this comparative example is used to treat the peel of the tangerine, the separation agent only enzymatically decomposes the white peel layer without contacting the yellow peel layer, and cannot effectively separate the oil cells in the peel.
[0071] The above contents are further detailed descriptions of the present invention in combination with specific / preferred implementations, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.
Claims
1. A citrus peel oil cell separation agent, characterized in that: The separating agent is a mixed solution of pectinase solution and buffer solution, and the buffer solution contains sorbitol, KOH and CaCl2.
2. The separating agent for citrus peel oil cells according to claim 1, characterized in that: The concentration of the sorbitol solution in the buffer solution is 0.5-0.93 mol / L, the concentration of the KOH solution is 4-6 mmol / L, the concentration of the CaCl2 solution is 0.5-1.5 mmol / L, and the pH value of the buffer solution is 5.5-6.
0.
3. A citrus peel oil cell separation agent according to any one of claims 1-2, characterized in that: The concentration of the buffer solution is 0.1-0.93 mol / L, and the pectinase solution and the buffer solution are mixed in a volume ratio of 1:(1-3). After mixing, the concentration of the pectinase in the separation agent is 800-2000 U / ml.
4. A citrus peel oil cell separation agent according to any one of claims 1 to 3, characterized in that: The concentration of the buffer solution is 0.5-0.93 mol / L, and the pectinase solution and the buffer solution are mixed in a volume ratio of 1:(1-2). After mixing, the concentration of the pectinase in the separation agent is 800-2000 U / ml.
5. A citrus peel oil cell separation agent according to any one of claims 1 to 4, characterized in that: The pectinase is p-4716 pectinase.
6. A method for using a citrus peel oil cell separation agent as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Using citrus fruits as materials, removing a large piece of peel including a white peel layer, and thinning the white peel layer of the peel; (2) applying the prepared separating agent evenly to the thinned white peel layer of the citrus and allowing it to stand for a period of time; the standing time is determined according to the citrus variety and the thickness of the white peel layer of the peel; (3) When the white skin layer on the surface of the oil cells can be gently scraped off, use running water to directly rinse and remove the enzymatically hydrolyzed white skin layer in the peel; if there are still white skin fibers remaining between the oil cells after cleaning, use tweezers to directly remove them to obtain complete oil cells.
7. The method for using the separating agent for citrus peel oil cells according to claim 6, characterized in that: Without destroying the oil cells, try to remove the white cortex as much as possible to shorten the time used for enzymatic hydrolysis of the white cortex.
8. The method for using the citrus peel oil cell separation agent according to claim 6, characterized in that: The citrus white peel layer coated with the separator is allowed to stand at 25 to 30° C. for 3 to 5 hours.