High-internal-phase emulsion gel of microcapsule compounded coffee essential oil and preparation method of high-internal-phase emulsion gel

Abstract

The invention provides high-internal-phase emulsion gel with microcapsules compounded with coffee essential oil and a preparation method of the high-internal-phase emulsion gel, and belongs to the field of plant essential oil processing. The high internal phase emulsion gel comprises a water phase and an oil phase, and the oil phase accounts for 72-90% of the total volume of the system; the water phase is a microcapsule and is prepared by mixing a core material, a wall material and an emulsifier in water and then performing ultrasonic treatment; the oil phase is coffee essential oil and is prepared by dissolving a coffee raw material in an organic solvent, extracting, filtering and carrying out rotary evaporation. According to the invention, a novel high internal phase emulsion delivery system is developed by combining microcapsules and a high internal phase emulsion technology, so that the efficacy, the bioavailability and the stability of the coffee essential oil are improved, and the preparation method is a key and a premise for further application of the coffee essential oil.

Description

Technical Field

[0001] The invention relates to the field of plant essential oil processing, in particular to a high internal phase emulsion gel of microcapsule composite coffee essential oil and a preparation method thereof. Background Art

[0002] Coffee is a popular beverage in the world and is commonly grown in tropical and subtropical regions. Currently, coffee is mainly grown in Yunnan, Hainan and other regions in my country. It is a unique agricultural and food resource. Coffee essential oil is an essential oil extracted from coffee, accounting for about 10% of the weight of coffee. It is a natural cosmetic raw material with multiple functional activities. For example, it has antioxidant, antibacterial and anti-aging capabilities, and can also help improve skin condition. Studies have shown that it is beneficial to improve enlarged pores, reduce acne, and improve skin inflammation.

[0003] Because coffee essential oil has unique functional characteristics, it is loved by consumers as a cosmetic raw material and has a broad market prospect. However, there are still some difficulties in utilizing coffee essential oil. For example, as an oily substance, coffee essential oil is not only easily restricted in its scope of application, but also prone to decomposition and deterioration during processing and storage, and has a high transportation cost. In addition, the bioavailability of coffee essential oil remains to be improved. The above many factors limit the application and promotion of coffee essential oil. Therefore, it is of great significance to find a method to improve the bioavailability and stability of coffee essential oil and to improve its scope of application.

[0004] In recent years, high internal phase emulsions have been widely used in the cosmetics field. High internal phase emulsions refer to emulsions in which the volume of the oil phase accounts for more than 74%. Compared with traditional emulsions, they have unique physical properties. They not only have a large interface area, but also have adjustable flow properties. These physical properties give high internal phase emulsions higher loading capacity, stability and controllability. However, the performance improvement of high internal phase emulsions alone is limited, and how to further improve the various properties of high internal phase emulsions has become the focus of research. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a microcapsule composite coffee essential oil high internal phase emulsion gel and a preparation method thereof. The present invention develops a novel high internal phase emulsion delivery system by combining microcapsule and high internal phase emulsion technology, thereby improving the efficacy, bioavailability and stability of coffee essential oil, which is the key and prerequisite for the further application of coffee essential oil.

[0006] The technical solution of the present invention is as follows:

[0007] A microcapsule composite coffee essential oil high internal phase emulsion gel comprises a water phase and an oil phase, wherein the water phase is microcapsules and the oil phase is coffee essential oil; the oil phase accounts for 72-90% of the total volume of the system.

[0008] Wherein, the preparation of the microcapsules comprises the following steps:

[0009] S1, firstly dissolving the wall material and emulsifier in water and performing the first ultrasonic treatment;

[0010] S2, then adding the core material for a second ultrasonic treatment;

[0011] The mass ratio of the core material to the wall material is 1:8-1:15.

[0012] Preferably, the wall material includes any one of hydroxypropyl-β-cyclodextrin and β-cyclodextrin; the concentration of the wall material in water is 2-6%; the emulsifier includes any one of whey protein and zein; the concentration of the emulsifier in water is 2-6%; the core material includes any one of caffeic acid, caffeine, and chlorogenic acid.

[0013] Preferably, the power of the first ultrasonic treatment is 300-500 W, the time is 30-60 min, and the ultrasonic temperature is 40-60° C.; the power of the second ultrasonic treatment is 300-500 W, the time is 30-60 min, and the ultrasonic temperature is 40-60° C.

[0014] Wherein, the preparation of coffee essential oil comprises the following steps:

[0015] M1. Grind the coffee raw material and extract it using an organic solvent extraction method; after the extraction is completed, filter and obtain a filtrate;

[0016] M2. The filtrate is filtered again after being decontaminated with activated carbon, and the organic solvent is removed by rotary evaporation of the filtrate, followed by centrifugation to obtain coffee essential oil.

[0017] Preferably, the coffee raw material in step M1 is ground to a mesh size of 40-100 mesh, the organic solvent is ethanol or ethyl acetate, and the solid-liquid ratio of the coffee raw material to the organic solvent is 1:5-1:20 g / ml; the extraction conditions are extraction temperature of 50-80° C. and extraction time of 60-150 min.

[0018] Preferably, the mass of the activated carbon in step M2 is 1-5% g / ml of the coffee raw material; the rotary evaporation conditions are a rotary evaporation temperature of 40-60°C and a rotary evaporation speed of 40-70rpm; and the centrifugation conditions are a centrifugal speed of 4000-10000rpm and a centrifugal time of 30-60min.

[0019] Furthermore, the water phase and the oil phase are mixed and then fully homogenized and emulsified to obtain the high internal phase emulsion gel; the homogenization and emulsification rate is 10000-20000 rpm / min, the temperature is 50-70°C, and the time is 4-12 min.

[0020] The beneficial technical effects of the present invention are:

[0021] 1. The present invention is the first to use microcapsule technology combined with high internal phase emulsion technology for the stabilization study of coffee essential oil. Caffeic acid and coffee essential oil are combined as active ingredients in the form of microcapsules and high internal phase oil phase using high internal phase emulsion, which play a synergistic role with each other and improve the sample loading, bioavailability and stability of coffee essential oil.

[0022] 2. The main components of the high internal phase emulsion are the water phase and the oil phase. The present invention improves the various properties of the high internal phase emulsion by modifying the water phase. The present invention uses microcapsule technology to modify the water phase. Not only can the core material of the microcapsule further increase the various functional capabilities of the coffee essential oil, but the wall material of the microcapsule can also further enhance the stability of the high internal phase emulsion, and together play a synergistic role.

[0023] 3. The present invention uses hydroxypropyl-β-cyclodextrin as the wall material of microcapsules for the aqueous phase preparation of high internal phase emulsions, thereby improving the loading capacity, stability and bioavailability of coffee essential oil in high internal phase emulsions.

[0024] 4. The composite high internal phase emulsion gel prepared by the present invention further expands the application scope of coffee essential oil and reduces transportation costs.

[0025] 5. The preparation process is simple and fast. There are no complicated preparation conditions such as high temperature and high pressure in the production process. The energy consumption cost of the preparation process is low, the safety is good, and it is green and environmentally friendly, which is suitable for large-scale industrial production.

[0026] 6. The composite coffee essential oil high internal phase emulsion gel prepared by the present invention is in a white gel emulsion state, can be taken orally or applied externally, is easy to use and store, and has no toxic effect on the body. The combination of the two methods can play a role in whitening and regulating the skin. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] This embodiment provides a method for preparing a high internal phase emulsion gel by combining caffeic acid microcapsules with coffee essential oil, and the specific steps are as follows:

[0030] (1) Add 2 g of hydroxypropyl-β-cyclodextrin and 2 g of whey protein into 100 mL of water, and perform ultrasonic treatment for 30 min at an ultrasonic power of 300 W and an ultrasonic temperature of 40° C., and wait for complete dissolution to obtain a mixed solution.

[0031] (2) 0.25 g of caffeic acid was added as a core material to the mixed solution obtained in step (1), and then ultrasonic treatment was performed at an ultrasonic power of 300 W and an ultrasonic temperature of 40° C. for 30 min, and the solution was completely dissolved to obtain a microcapsule solution.

[0032] (3) 100 g of coffee was added to 500 mL of ethyl acetate and extracted at 50° C. for 60 min. After the extraction was completed, the filtrate was filtered to obtain a filtrate. 1 g of activated carbon was added to the filtrate to remove impurities. After the impurities were removed, the filtrate was filtered again. The organic solvent was removed by rotary evaporation at 40° C. and 40 rpm. After the rotary evaporation was completed, the upper layer was centrifuged at 4000 rpm for 60 min to obtain coffee essential oil.

[0033] (4) 2.5 mL of microcapsule solution was used as the aqueous phase and high-speed sheared at 50° C. and 10,000 rpm / min for 3 min, followed by slow addition of 7 mL of coffee essential oil, followed by homogenization under the same conditions and then standing at room temperature to obtain a high internal phase emulsion gel of caffeic acid microcapsules composited with coffee essential oil.

[0034] Embodiment 2:

[0035] This embodiment provides a method for preparing a high internal phase emulsion gel by combining caffeic acid microcapsules with coffee essential oil, and the specific steps are as follows:

[0036] (1) 1 g of hydroxypropyl-β-cyclodextrin and 1 g of whey protein were added to 25 mL of water, and ultrasonically treated for 40 min at an ultrasonic power of 400 W and an ultrasonic temperature of 50° C., and then completely dissolved to obtain a mixed solution.

[0037] (2) 0.1 g of caffeic acid was added as a core material to the mixed solution obtained in step (1), and then ultrasonic treatment was performed at an ultrasonic power of 400 W and an ultrasonic temperature of 50° C. for 40 min, and the solution was completely dissolved to obtain a microcapsule solution.

[0038] (3) 100 g of coffee was added to 1000 mL of ethyl acetate and extracted at 70° C. for 100 min. After the extraction was completed, the filtrate was filtered to obtain a filtrate. 2 g of activated carbon was added to the filtrate to remove impurities. After the impurities were removed, the filtrate was filtered again. The organic solvent was removed by rotary evaporation at 50° C. and 50 rpm. After the rotary evaporation was completed, the upper layer was centrifuged at 6000 rpm for 40 min to obtain coffee essential oil.

[0039] (4) 1 mL of microcapsule solution was used as the aqueous phase, and high-speed shearing was performed at 60° C. and 15,000 rpm / min for 4 min, followed by slow addition of 4 mL of coffee essential oil, followed by homogenization under the same conditions, and then allowed to stand at room temperature to obtain a high internal phase emulsion gel of caffeic acid microcapsules composited with coffee essential oil.

[0040] Embodiment 3:

[0041] This embodiment provides a method for preparing a high internal phase emulsion gel by combining caffeic acid microcapsules with coffee essential oil, and the specific steps are as follows:

[0042] (1) Add 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein into 100 mL of water, and perform ultrasonic treatment for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60° C., and wait for complete dissolution to obtain a mixed solution.

[0043] (2) 0.4 g of caffeic acid was added as a core material to the mixed solution obtained in step (1), and then ultrasonic treatment was performed at an ultrasonic power of 500 W and an ultrasonic temperature of 60° C. for 60 min, and the solution was completely dissolved to obtain a microcapsule solution.

[0044] (3) 100 g of coffee was added to 2000 mL of ethyl acetate and extracted at 80° C. for 150 min. After the extraction was completed, the filtrate was filtered to obtain a filtrate. 5 g of activated carbon was added to the filtrate to remove impurities. After the impurities were removed, the filtrate was filtered again. The organic solvent was removed by rotary evaporation at 60° C. and 70 rpm. After the rotary evaporation was completed, the upper layer was centrifuged at 10,000 rpm for 30 min to obtain coffee essential oil.

[0045] (4) 5 mL of microcapsule solution was used as the aqueous phase, and high-speed shearing was performed at 70° C. and 20,000 rpm / min for 6 min, followed by slow addition of 25 mL of coffee essential oil, followed by homogenization under the same conditions, and then allowed to stand at room temperature to obtain a high internal phase emulsion gel of caffeic acid microcapsules composited with coffee essential oil.

[0046] Test Example 1: Material Performance Evaluation of High Internal Phase Emulsion

[0047] Sample 1, high internal phase emulsion water phase not modified by microencapsulation technology:

[0048] Add 10 g of whey protein to 100 mL of water, and perform ultrasonic treatment for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60° C., wait for complete dissolution, and obtain an aqueous phase of a high internal phase emulsion for use; high-speed shear 5 mL of the aqueous phase at 70° C. and 20,000 rpm / min for 6 min, then slowly add 25 mL of coffee essential oil, and then homogenize under the same conditions, and then leave at room temperature to obtain sample 1.

[0049] Sample 2: Modification of high internal phase emulsion water phase using microencapsulation technology:

[0050] 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein were added to 100 mL of water, and ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a wall material solution. 0.4 g of caffeic acid was added to the wall material solution as a core material, and then ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a microcapsule solution. 5 mL of the microcapsule solution was used as the aqueous phase, and high-speed shearing was performed at 70°C and 20,000 rpm / min for 6 min, and then 25 mL of coffee essential oil prepared in Example 1 was slowly added, followed by homogenization under the same conditions, and then placed at room temperature to obtain sample 2.

[0051] The particle size distribution of the two samples was measured by a laser particle size analyzer, and the particle size distribution of the two samples was compared. The test data is shown in Table 1.

[0052] Table 1 Particle size, Zeta potential and PDI analysis

[0053] sample Particle size (nm) Zeta potential (mv) PDI Sample 1 314.53±10.64 28.75±1.64 0.38±0.03 Sample 2 271.78±12.89 33.35±2.29 0.19±0.02

[0054] The performance of high internal phase emulsion was evaluated by particle size, Zeta potential and PDI. The results showed that after the water phase was modified by microencapsulation technology, the particle size of the emulsion decreased, the Zeta potential increased, and the PDI value decreased, indicating that the water phase modified by microencapsulation technology and the high internal phase emulsion technology itself played a synergistic role and improved the strength of the high internal phase emulsion material itself.

[0055] Test Example 2: Evaluation of the bio-drug loading of high internal phase emulsions

[0056] Sample 1, high internal phase emulsion water phase not modified by microencapsulation technology:

[0057] Add 10 g of whey protein to 100 mL of water, and perform ultrasonic treatment for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60° C., wait for complete dissolution, and obtain a high internal phase emulsion aqueous phase for use; high-speed shear 5 mL of the aqueous phase at 70° C. and 20,000 rpm / min for 6 min, then slowly add 25 mL of coffee essential oil, and then homogenize under the same conditions, and then leave at room temperature to obtain sample 1.

[0058] Sample 2: Modification of high internal phase emulsion water phase using microencapsulation technology:

[0059] 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein were added to 100 mL of water, and ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a wall material solution. 0.4 g of caffeic acid was added to the wall material solution as a core material, and then ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a microcapsule solution. 5 mL of the microcapsule solution was used as the aqueous phase, and high-speed shearing was performed at 70°C and 20,000 rpm / min for 6 min, and then 25 mL of coffee essential oil prepared in Example 1 was slowly added, followed by homogenization under the same conditions, and then placed at room temperature to obtain sample 2.

[0060] The above samples were used to calculate the bio-drug loading of the high internal phase emulsion according to the following formula:

[0061] Bio-drug loading = m1 / m0×100%

[0062] Wherein m1 represents the amount of coffee essential oil embedded in the emulsion (g), and m0 represents the total weight of the emulsion (g).

[0063] After measurement, it was found that the bio-drug loading of sample 1 was 84.93%, and the bio-drug loading of sample 2 was 92.37%. The results showed that the bio-drug loading of the emulsion was improved after the aqueous phase was modified by microencapsulation technology, indicating that the microcapsules formed by hydroxypropyl-β-cyclodextrin had a synergistic effect on the emulsion itself, thereby increasing the bio-drug loading of the high internal phase emulsion.

[0064] Test Example 3: Evaluation of thermal stability of high internal phase emulsion

[0065] Sample 1, coffee essential oil:

[0066] Sample 2, high internal phase emulsion water phase not modified by microencapsulation technology:

[0067] Add 10 g of whey protein to 100 mL of water, and perform ultrasonic treatment for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, wait for complete dissolution, and obtain a high internal phase emulsion aqueous phase for use; high-speed shear 5 mL of the aqueous phase at 70°C and 20,000 rpm / min for 6 min, then slowly add 25 mL of coffee essential oil, and then homogenize under the same conditions, and then leave at room temperature to obtain sample 2.

[0068] Sample 3, using microencapsulation technology to modify the aqueous phase of high internal phase emulsion:

[0069] 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein were added to 100 mL of water, and ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a wall material solution. 0.4 g of caffeic acid was added to the wall material solution as a core material, and then ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, and the solution was completely dissolved to obtain a microcapsule solution. 5 mL of the microcapsule solution was used as the aqueous phase, and high-speed sheared at 70°C and 20,000 rpm / min for 6 min, and then 25 mL of coffee essential oil prepared in Example 1 was slowly added, followed by homogenization under the same conditions, and then placed at room temperature to obtain Sample 3.

[0070] The above samples were heated in an oil bath at 180°C for 2 h. The acid value was used as an evaluation index of thermal stability to compare the thermal stabilities of the three samples. The results are shown in Table 2.

[0071] Table 2 Thermal stability evaluation

[0072]

[0073] The thermal stability of the high internal phase emulsion was evaluated by the acid value before and after heating. The results showed that the thermal stability of coffee essential oil was poor and the acid value increased significantly after heating. In addition, the acid value of the high internal phase emulsion after the aqueous phase was modified by microencapsulation technology increased the least, indicating that the microcapsules formed by adding hydroxypropyl-β-cyclodextrin had a synergistic effect with the high internal phase emulsion, thereby improving the thermal stability of the high internal phase emulsion.

[0074] Test Example 4: Comparison of Antioxidant Effects of High Internal Phase Emulsions

[0075] Sample 1, the aqueous phase was modified by microencapsulation technology, but caffeic acid was not added when preparing the microcapsules:

[0076] 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein were added to 100 mL of water, and ultrasonically treated for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60° C., and the mixture was completely dissolved to obtain a wall material solution. 5 mL of the wall material solution was used as the aqueous phase, and high-speed shearing was performed at 70° C. and 20,000 rpm / min for 6 min. Subsequently, 25 mL of coffee essential oil prepared in Example 1 was slowly added, and the mixture was homogenized under the same conditions, and then placed at room temperature to obtain Sample 1.

[0077] Sample 2, the aqueous phase was modified by microencapsulation technology, and caffeic acid was added when preparing microcapsules:

[0078] Add 5 g of hydroxypropyl-β-cyclodextrin and 5 g of whey protein to 100 mL of water, and ultrasonically treat them for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, wait for complete dissolution, and obtain a wall material solution. Add 0.4 g of caffeic acid as a core material to the wall material solution, and then ultrasonically treat it for 60 min at an ultrasonic power of 500 W and an ultrasonic temperature of 60°C, wait for the solution to be completely dissolved, and obtain a microcapsule solution. Use 5 mL of the microcapsule solution as the aqueous phase, and high-speed shear it at 70°C and 20,000 rpm / min for 6 min. Then slowly add 25 mL of coffee essential oil prepared in Example 1, and then homogenize under the same conditions. Then, place it at room temperature to obtain Sample 2.

[0079] The same volume of the above samples was measured and their antioxidant capacity was evaluated using DPPH free radicals. The results showed that different samples had different scavenging abilities for the same concentration of DPPH free radicals at the same concentration. The scavenging ability of sample 1 was 62.16%, while that of sample 2 was 85.68%. This indicates that caffeic acid as the core material of the microcapsule has a synergistic effect with the oil phase in the high internal phase emulsion, thereby increasing the antioxidant capacity of the high internal phase emulsion.

[0080] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A high internal phase emulsion gel of microcapsule composite coffee essential oil, characterized in that: It comprises a water phase and an oil phase, wherein the water phase is microcapsules and the oil phase is coffee essential oil; the oil phase accounts for 72-90% of the total volume of the system.

2. The high internal phase emulsion gel according to claim 1, characterized in that The preparation of the microcapsules comprises the following steps: S1, firstly dissolving the wall material and emulsifier in water and performing the first ultrasonic treatment; S2, then adding the core material for a second ultrasonic treatment; The mass ratio of the core material to the wall material is 1:8-1:

15.

3. The high internal phase emulsion gel according to claim 2, characterized in that: The wall material includes any one of hydroxypropyl-β-cyclodextrin and β-cyclodextrin; the concentration of the wall material in water is 2-6%.

4. The high internal phase emulsion gel according to claim 2, characterized in that: The emulsifier includes any one of whey protein and zein; the concentration of the emulsifier in water is 2-6%.

5. The high internal phase emulsion gel according to claim 2, characterized in that: The core material includes any one of caffeic acid, caffeine and chlorogenic acid.

6. The high internal phase emulsion gel according to claim 2, characterized in that: The power of the first ultrasonic treatment is 300-500W, the time is 30-60min, and the ultrasonic temperature is 40-60°C; the power of the second ultrasonic treatment is 300-500W, the time is 30-60min, and the ultrasonic temperature is 40-60°C.

7. The high internal phase emulsion gel according to claim 1, characterized in that: The coffee essential oil preparation comprises the following steps: M1. Grind the coffee raw material and extract it using an organic solvent extraction method; after the extraction is completed, filter and obtain a filtrate; M2. The filtrate is filtered again after being decontaminated with activated carbon, and the organic solvent is removed by rotary evaporation of the filtrate, followed by centrifugation to obtain coffee essential oil.

8. The high internal phase emulsion gel according to claim 7, characterized in that: The coffee raw material in step M1 is ground to a mesh size of 40-100 mesh, the organic solvent is ethanol or ethyl acetate, and the solid-liquid ratio of the coffee raw material to the organic solvent is 1:5-1:20 g / ml; the extraction conditions are an extraction temperature of 50-80° C. and an extraction time of 60-150 min.

9. The high internal phase emulsion gel according to claim 7, characterized in that: The mass of the activated carbon in step M2 is 1-5% of the coffee raw material; the rotary evaporation conditions are a rotary evaporation temperature of 40-60°C and a rotary evaporation speed of 40-70rpm; the centrifugal conditions are a centrifugal speed of 4000-10000rpm and a centrifugal time of 30-60min.

10. The high internal phase emulsion gel according to claim 1, characterized in that: The water phase and the oil phase are mixed and then fully homogenized and emulsified to obtain the high internal phase emulsion gel; the homogenization and emulsification rate is 10000-20000 rpm / min, the temperature is 50-70°C, and the time is 4-12 minutes.

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