Method for extracting lutein from marigold

Abstract

The invention relates to a method for extracting lutein from marigold, which comprises the following steps: S1, putting aspergillus niger into sterile water to obtain an aspergillus niger spore suspension; s2, grinding the dried marigold petals into powder, and sieving to obtain marigold petal powder; s3, soaking the marigold petal powder with sterile water; s4, adding the aspergillus niger spore suspension into the soaked marigold petal powder to obtain a marigold product of which the xanthophyll is to be extracted; and step S5, carrying out xanthophyll extraction on the marigold product from which xanthophyll is to be extracted, so as to obtain food-grade or drug-grade xanthophyll. According to the method for extracting the xanthophyll from the marigold, the yield of the xanthophyll in the marigold can be increased within a short time, and the method is simple to operate, low in cost and free of pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of lutein extraction, and in particular to a method for extracting lutein from marigold. Background Art

[0002] Lutein is a dihydroxy derivative of α-carotene and a well-known carotenoid with a wide range of applications in healthcare. A large amount of epidemiological evidence shows that lutein has a protective effect on vision, prevents cataracts, arteriosclerosis, and enhances immunity. It plays an important role in preventing cancer and delaying cancer development. The lutein market is divided into pharmaceutical, dietary supplement, food, animal and fish feed industries, and lutein is very popular among the public.

[0003] Currently, marigold petals are the main source of lutein, and studies have shown that the lutein content in processed marigold granules and dry powder is between 20-30 g / kg granules.

[0004] In view of the above benefits of lutein, different researchers have studied and obtained some methods for extracting and isolating lutein from marigold. The most commonly used of these methods is solvent extraction. However, most of the organic solvents used for lutein extraction come from petrochemical sources, which may cause harm to health and pollute the environment. These solvents may also cause irreversible degradation of the product. In addition, the yield of lutein extracted by these methods is not high.

[0005] In order to solve the above problems, it is necessary to develop an environmentally friendly method for extracting lutein and improve the extraction and purification efficiency of lutein.

[0006] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of the present application. Summary of the invention

[0007] The object of the present invention is to provide a method for extracting lutein from marigold to solve at least one of the above-mentioned background technology problems.

[0008] To solve the above problems, the technical solution of the present invention is achieved as follows: A method for extracting lutein from marigold comprises the following steps: Step S1: Place Aspergillus niger in sterile water and shake well to obtain a spore concentration of 1×10 6 / mL-1×10 7 / mL suspension of Aspergillus niger spores; Step S2, grinding the dried marigold petals into powder, and sieving to obtain marigold petal powder with a particle size of 212 μm-300 μm; Step S3, soaking the marigold petal powder with sterile water, wherein the marigold petal powder and the sterile water are mixed in a ratio of (g) to volume (mL) of 5:4, and soaking for 15-30 minutes to obtain soaked marigold petal powder; Step S4, adding the Aspergillus niger spore suspension to the soaked marigold petal powder, wherein the amount (mL) of the Aspergillus niger spore suspension is 0.5%-1% of the mass (g) of the soaked marigold petal powder; controlling the temperature at 26° C.-32° C., and placing for 4-7 days to obtain a marigold product for extracting lutein; Step S5, extracting lutein from the marigold product from which lutein is to be extracted to obtain food or pharmaceutical grade lutein.

[0009] In some embodiments, in step S1, sterile distilled water is used to suspend Aspergillus niger spores, and the pH value and concentration of the Aspergillus niger spore suspension are adjusted so that the pH value of the Aspergillus niger spore suspension is 4.0-6.0 and the Aspergillus niger spore content is 1×10 6 / mL-1×10 7 Pieces / mL.

[0010] In some embodiments, in step S2, the marigold petals are dried in a hot air furnace at a temperature of 35° C. for 24 hours in a dark environment to obtain dried marigold petals.

[0011] In some embodiments, in step S2, the dried marigold petals are placed in a high-speed grinder for crushing, and the marigold petal powder is obtained after passing through a 50-70 mesh sieve.

[0012] In some embodiments, in step S5, the marigold product from which lutein is to be extracted is placed in an oven for drying at a temperature of 35° C. for 5 h.

[0013] In some embodiments, step S5 further includes: soaking the sieved marigold product from which lutein is to be extracted with ethanol in a ratio of 1:10 in terms of mass (g) to volume (mL).

[0014] In some embodiments, step S5 further comprises: performing ultrasonic extraction 3-4 times on the marigold product to be extracted with lutein after soaking in ethanol with an ultrasonic processor, each extraction time being 30 minutes, and then concentrating the extract with a rotary evaporator, and obtaining food or pharmaceutical grade lutein after spin drying.

[0015] In some embodiments, the ethanol purity is 95%, and the soaking time is greater than 12 h.

[0016] In some embodiments, step S5 includes: mixing and evenly mixing the sieved marigold product from which lutein is to be extracted and an aqueous surfactant solution at a ratio of mass (g) to volume (mL) of 1:19, wherein the hydrophile-lipophile balance (HLB) value of the aqueous surfactant solution is 5-7.

[0017] In some embodiments, the method further comprises: using an ultrasonic processor to ultrasonically extract the mixture of the marigold product from which lutein is to be extracted and the surfactant aqueous solution for 3-4 times, each extraction time being 30 minutes, and then concentrating the extract with a rotary evaporator, spinning dry and weighing to obtain food or pharmaceutical grade lutein.

[0018] The beneficial effects of the technical solution of the present invention are: Compared with the prior art, the method for extracting lutein from marigold of the present invention can increase the yield of lutein in marigold in a short time, and has simple operation, low cost and no pollution.

[0019] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art description are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. The drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention, and together with the specification, are used to explain the principles of the present invention.

[0021] Figure 1 is a scanning electron microscope image of crushed marigold petals particles according to an exemplary embodiment; Figure 2 , Figure 3 is a high performance liquid chromatogram of lutein detection according to an exemplary embodiment; Figure 4 is a graph showing lutein content in marigold at various temperatures according to an exemplary embodiment. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the embodiments of the present invention clearer and more understandable, and to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0023] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those described herein. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0024] The marigold involved in the embodiment is obtained by drying (such as baking or sun-drying) marigold flowers, and can be directly purchased on the market.

[0025] The present invention provides a method for extracting lutein from marigold, comprising the following steps: Step S1: put Aspergillus niger into sterile water and shake well to obtain a spore of Aspergillus niger with a spore content of 1×10 6 / mL-1×10 7 Spore suspension of 100 / mL; Specifically, Aspergillus niger is placed in a centrifuge tube filled with sterile water, and shaken on a laboratory shaker to obtain a spore suspension; the pH value and concentration of the spore suspension are adjusted to obtain a pH value of 4.0-6.0 and an Aspergillus niger spore content of 1×10 6 / mL-1×10 7 / mL suspension of Aspergillus niger spores; It should be noted that there is no special regulation for the ratio of Aspergillus niger to sterile water. Sterile water or Aspergillus niger can be gradually added according to the concentration of the spore suspension to ensure that the Aspergillus niger spore content is 1×10 6 / mL-1×10 7 In some embodiments, sterile water can also be added to the centrifuge tube containing Aspergillus niger, and then shaken to adjust the concentration of the spore suspension to obtain an Aspergillus niger spore content of 1×106 / mL-1×10 7 / mL of Aspergillus niger spore suspension.

[0026] In some embodiments, sterile distilled water can be used to suspend Aspergillus niger spores, and the pH value and concentration of the Aspergillus niger spore suspension can be adjusted so that the Aspergillus niger spore content is 1×10 6 / mL-1×10 7 Pieces / mL.

[0027] Step S2, grinding the dried marigold petals into powder, wherein the particle size of the powder is 212 μm-300 μm; Specifically, the marigold petals are dried in a hot air furnace at a temperature of 35° C. for 24 hours in a dark place to obtain dried marigold petals; Grinding dried marigold petals into powder, wherein the particle size of the powder is 212 μm-300 μm; sterilizing the marigold petal powder and storing it away from light; In some embodiments, dried marigold petals are placed in a high-speed grinder for grinding, and the marigold petals are passed through a 50-70 mesh sieve to obtain marigold petal powder.

[0028] In some embodiments, the marigold petal powder is packaged in aluminum foil and then stored at a temperature controlled at 4°C-10°C.

[0029] Step S3, soaking the marigold petal powder with sterile water, wherein the marigold petal powder and the sterile water are mixed in a ratio of (g) to volume (ml) of 5:4, and soaking for 15-30 minutes to obtain the soaked marigold petal powder; Specifically, marigold petal powder and sterile water are mixed in a ratio of mass (g) to volume (mL) of 5:4 (e.g., 5 g marigold petal powder, 4 mL sterile water) in a sterile container to soak the marigold petal powder for 15-30 minutes to obtain soaked marigold petal powder; In the experiment, 5 g of marigold petal powder and 4 mL of sterile water were placed in a sterile Erlenmeyer flask for soaking.

[0030] Step S4, adding Aspergillus niger spore suspension to the soaked marigold petal powder, wherein the amount of Aspergillus niger spore suspension is 0.5%-1% of the mass of the soaked marigold petal powder; controlling the temperature at 26° C.-32° C., and placing for 4-7 days to obtain a marigold product for extracting lutein; Step S5, extracting lutein from the marigold product from which lutein is to be extracted to obtain food or pharmaceutical grade lutein.

[0031] Specifically, the marigold product from which lutein is to be extracted is placed in an oven for drying at a temperature of 35° C. for 5 h.

[0032] Reference Figure 1 As shown, Figure 1 The present invention is a photograph of marigold product granules to be extracted lutein treated by the present invention and marigold petal granules not treated by the present invention observed under an electron microscope. The magnifications of A1 and B1 are the same, and the magnifications of A2 and B2 are the same. In the figure, the magnifications of A1 and B1 are 500 times, and the magnifications of A2 and B2 are 3000 times. Figure 1 It can be seen that the surface of the untreated marigold petal crushed particles is well-organized and smooth; while the surface of the marigold product crushed particles that have been treated to extract lutein is deformed, there is biological molecule leaching, and there are many pores on the surface of the biomass. This is because the extracellular enzymes produced by Aspergillus niger transform and modify the chemical composition of marigold petals, increase lycopene, β-carotene and other lutein precursors, thereby promoting the increase of lutein production in marigold; on the other hand, when Aspergillus niger spore suspension is added to the marigold petal powder, new active metabolites are produced after fermentation, which promotes the production of lutein.

[0033] from Figure 1 As shown in B1 and B2, the surface of the crushed particles of the marigold product from which lutein is to be extracted is not oxidized and has many pores. This is because the fermentation of Aspergillus niger promotes the release of phenolic acid in the hemicellulose of marigold petals, enhances the antioxidant properties of the marigold product from which lutein is to be extracted, and protects lutein from oxidation and loss of activity. The antioxidant capacity of lutein is improved, the activity is maintained, and subsequent extraction is facilitated.

[0034] The sieved marigold product to be extracted with lutein was soaked in ethanol at a ratio of mass (g) to volume (mL) of 1:10, wherein the purity of the ethanol was 95% and the soaking time was greater than 12 h; The marigold product to be extracted with lutein after being soaked in ethanol is subjected to ultrasonic extraction for 3-4 times by using an ultrasonic processor, and the extraction time for each time is 30 minutes (min). The extract is then concentrated by a rotary evaporator, and the extract is weighed after being spin-dried to obtain food or pharmaceutical grade lutein.

[0035] In other embodiments, step S5 may be: mixing and evenly mixing the sieved marigold product from which lutein is to be extracted and the surfactant aqueous solution in a ratio of mass (g) to volume (mL) of 1:19, wherein the hydrophile-lipophile balance (HLB) value of the surfactant aqueous solution is 5-7.

[0036] The mixture of marigold product to be extracted with surfactant aqueous solution is ultrasonically extracted 3-4 times with an ultrasonic processor, each extraction time is 30 minutes, and then the extract is concentrated with a rotary evaporator, and the extract is weighed after spin drying to obtain food or pharmaceutical grade lutein.

[0037] Reference Figure 2 , Figure 3 As shown, Figure 2 This is a high performance liquid chromatography test chart of lutein content in marigold treated by the method of the present invention; Figure 3 This is a high performance liquid chromatography detection diagram of the lutein content in marigold that has not been treated by the method of the present invention; from the comparison in the figures, it can be seen that the lutein content extracted from marigold treated by the method of the present invention is significantly increased.

[0038] Reference Figure 4 As shown, Figure 4 The figure is a diagram of the lutein content in marigold under various controlled temperature conditions after adding Aspergillus niger spore suspension to marigold petal powder in an embodiment of the present invention. Through experiments, it can be seen from the figure that the temperature can be controlled between 26°C and 32°C, with 30°C being the optimal.

[0039] The following description will be made by taking 5 g of dried marigold powder particles as an example.

[0040] A method for extracting lutein from marigold comprises the following steps: a) Place Aspergillus niger in a centrifuge tube filled with sterile water and shake on a shaker to obtain 1×10 6 / mL-1×10 7 The spore suspension of 1000 / mL is ready for use; b) placing the dried marigold petals in a high-speed grinder to grind and sieve to obtain marigold petal powder particles with a size of 212 μm-300 μm, and sterilizing the marigold petal powder particles; c) Take 5 g of marigold petal powder and 4 mL of sterile water and place them in a 50 mL sterile Erlenmeyer flask and soak for 30 minutes; d) adding Aspergillus niger spore suspension to the soaked marigold petal powder (the mass of Aspergillus niger spore suspension is 0.5% of the mass of the soaked marigold petal powder), controlling the temperature at 28° C. for 5 days to obtain a marigold product with luxuriant mycelium for later use.

[0041] Comparative Example 1 In Comparative Example 1, the following steps are included: a) crushing, sieving and sterilizing marigold petals in a high-speed grinder to obtain marigold petal powder particles; b) Take 5 g of marigold petal powder and 4 mL of sterile water and place them in a 50 mL sterile Erlenmeyer flask and soak for 30 minutes; c) placing the soaked marigold petal powder in an environment at room temperature of 28° C. for 5 days to obtain soaked marigold petal powder for later use.

[0042] Effect Experiment Example 1 The marigold product with luxuriant mycelium obtained in Example 1 and the marigold petal powder soaked in Comparative Example 1 were subjected to alcohol extraction to extract lutein, and the lutein yield in the extract was measured. The lutein amount obtained in Example 1 was 5 times higher than that in Comparative Example 1. The alcohol extraction method is specifically as follows: The product in Example 1 and the product in Comparative Example 1 were placed in an oven for drying at 35°C for 5 h; after drying, they were crushed by a high-speed pulverizer and passed through a 60-mesh sieve; after drying, 5 g of each was taken, 50 mL of 95% ethanol was added, soaked overnight, and then ultrasonically extracted three times with an ultrasonic processor, each for 30 minutes, and then the extract was concentrated with a rotary evaporator (40°C), and weighed after spin drying, and then fixed to volume with methanol, so that the final marigold extract concentration was 10 mg / mL. The yield of lutein in the marigold extract was determined by high performance liquid chromatography, and the final result showed that the amount of lutein obtained in Example 1 was 5 times higher than that in Comparative Example 1.

[0043] The difference between Example 2 and Example 1 is that the soaking time in step c) is 25 minutes; the mass of the Aspergillus niger spore suspension in step d) is 0.75% of the mass of the soaked marigold petal powder, the temperature is controlled at 30° C., and the soaking time is 4 days.

[0044] Comparative Example 2 The difference between Comparative Example 2 and Comparative Example 1 is that the soaking time in step b) is 25 minutes, and the temperature in step c) is 30° C. and the time is 4 days.

[0045] Effect Experiment Example 2 Lutein was extracted using the method in Experimental Example 1. The final results showed that the amount of lutein obtained in Example 2 was 14 times higher than that in Comparative Example 2.

[0046] The difference between Example 3 and Example 1 is that the soaking time in step c) is 15 minutes; the mass of the Aspergillus niger spore suspension in step d) is 1% of the mass of the soaked marigold petal powder, the temperature is controlled at 28° C., and the soaking time is 7 days.

[0047] Comparative Example 3 The difference between Comparative Example 3 and Comparative Example 1 is that the soaking time in step b) is 15 minutes, and the temperature in step c) is 28° C. and the time is 7 days.

[0048] Effect Experiment Example 3 Lutein was extracted using the method in Experimental Example 1. The final results showed that the amount of lutein obtained in Example 3 was 19 times higher than that in Comparative Example 3.

[0049] The method for extracting lutein from marigold of the present invention can increase the yield of lutein in marigold in a short time, and has simple operation, low cost and no pollution.

[0050] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary techniques in the art that are not disclosed in this disclosure. The specification and examples are to be considered as exemplary only, and the true scope and spirit of the present invention are indicated by the following claims.

[0051] It is understandable that the above content is a further detailed description of the invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the invention is limited to these descriptions. For ordinary technicians in the technical field to which the invention belongs, without departing from the concept of the invention, they can also make several substitutions or modifications to the described embodiments, and these substitutions or modifications should be deemed to belong to the scope of protection of this patent. In the description of this specification, the description of reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention.

[0052] In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradicting each other. Although the embodiments and advantages of the invention have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the scope defined by the appended claims.

[0053] Furthermore, the scope of the invention is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described in the specification. One of ordinary skill in the art will readily appreciate that the above disclosures, processes, machines, manufactures, compositions of matter, means, methods, or steps currently existing or to be developed that perform substantially the same functions as the corresponding embodiments described herein or obtain substantially the same results as the embodiments described herein may be utilized. Therefore, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, means, methods, or steps within their scope.

Claims

1. A method for extracting lutein from marigold, characterized in that: The steps include: Step S1: Place Aspergillus niger in sterile water and shake well to obtain a spore concentration of 1×10 6 / mL-1×10 7 / mL suspension of Aspergillus niger spores; Step S2, grinding the dried marigold petals into powder, and sieving to obtain marigold petal powder with a particle size of 212 μm-300 μm; Step S3, soaking the marigold petal powder with sterile water, wherein the marigold petal powder and the sterile water are mixed in a ratio of (g) to volume (mL) of 5:4, and soaking for 15-30 minutes to obtain soaked marigold petal powder; Step S4, adding the Aspergillus niger spore suspension to the soaked marigold petal powder, wherein the amount (mL) of the Aspergillus niger spore suspension is 0.5%-1% of the mass (g) of the soaked marigold petal powder; controlling the temperature at 26° C.-32° C., and placing for 4-7 days to obtain a marigold product for extracting lutein; Step S5, extracting lutein from the marigold product from which lutein is to be extracted to obtain food or pharmaceutical grade lutein.

2. The method for extracting lutein from marigold according to claim 1, characterized in that: In step S1, sterile distilled water is used to suspend Aspergillus niger spores, and the pH value and concentration of the Aspergillus niger spore suspension are adjusted so that the pH value of the Aspergillus niger spore suspension is 4.0-6.0 and the Aspergillus niger spore content is 1×10 6 / mL-1×10 7 Pieces / mL.

3. The method for extracting lutein from marigold according to claim 1, characterized in that: In step S2, the marigold petals are dried in a hot air furnace at a temperature of 35° C. for 24 hours in a dark environment to obtain dried marigold petals.

4. The method for extracting lutein from marigold according to claim 3, characterized in that: In step S2, the dried marigold petals are put into a high-speed grinder for grinding, and the marigold petal powder is obtained after passing through a 50-70 mesh sieve.

5. The method for extracting lutein from marigold according to claim 1, characterized in that: In step S5, the marigold product from which lutein is to be extracted is placed in an oven for drying at a temperature of 35° C. for 5 h.

6. The method for extracting lutein from marigold according to claim 5, characterized in that: Step S5 also includes: soaking the marigold product from which lutein is to be extracted with ethanol at a ratio of 1:10 in terms of mass (g) to volume (mL).

7. The method for extracting lutein from marigold according to claim 6, characterized in that: The step S5 also includes: using an ultrasonic processor to perform ultrasonic extraction 3-4 times on the marigold product to be extracted with lutein after being soaked in ethanol, each extraction time is 30 minutes, and then concentrating the extract with a rotary evaporator, and obtaining food or pharmaceutical grade lutein after spin drying.

8. The method for extracting lutein from marigold according to claim 6, characterized in that: The purity of the ethanol is 95%, and the soaking time is greater than 12 hours.

9. The method for extracting lutein from marigold according to claim 5, characterized in that: Step S5 comprises: mixing and evenly mixing the sieved marigold product from which lutein is to be extracted and the surfactant aqueous solution at a ratio of mass (g) to volume (mL) of 1:19, wherein the hydrophile-lipophile balance (HLB) of the surfactant aqueous solution is 5-7.

10. The method for extracting lutein from marigold according to claim 9, characterized in that: Also includes: The mixture of marigold product to be extracted with surfactant aqueous solution is ultrasonically extracted 3-4 times with an ultrasonic processor, each extraction time is 30 minutes, and then the extract is concentrated with a rotary evaporator, and the extract is weighed after spin drying to obtain food or pharmaceutical grade lutein.

Images