WO2017012088A1 - 燕窝萃取物的萃取方法 - Google Patents

燕窝萃取物的萃取方法 Download PDF

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WO2017012088A1
WO2017012088A1 PCT/CN2015/084769 CN2015084769W WO2017012088A1 WO 2017012088 A1 WO2017012088 A1 WO 2017012088A1 CN 2015084769 W CN2015084769 W CN 2015084769W WO 2017012088 A1 WO2017012088 A1 WO 2017012088A1
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bird
nest
extract
extraction method
nest extract
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PCT/CN2015/084769
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English (en)
French (fr)
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陈天煌
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陈天煌
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum

Definitions

  • This invention relates to an extraction process, and more particularly to an extraction process suitable for extracting bird's nest extracts.
  • Bird's nest is one of the traditional and precious foods that China has been eating since ancient times. It refers to the saliva secreted by several swiftlets of the Swift and the swiftlet genus, and mixed with other substances (such as feathers). Built nests, not nests built by birds of the genus Yankee (such as Jiayan).
  • the protein purity and nutritional value of the swiftlet saliva are the highest. Produced in the southern coastal areas of China, Vietnam, Thailand, Malaysia, Indonesia and the Philippines. After the bird's nest is picked, it must be processed by steaming, soaking, removing impurities, picking, drying and other complex processing to make the finished bird's nest, including swallow, swallow, swallow, and swallow. Among them, the swallow is the whole high-quality bird's nest, which retains the original shape, so the price is particularly expensive.
  • bird's nest acid which is also known as sialic acid.
  • Gunnar Blix et al. isolated Sialic acid from submandibular gland mucin in 1952 and established the naming rules for sialic acid, but little is known about its physiological functions for a long time. Its chemical structure was determined exactly in 1960. With the rapid development of modern physics, chemistry and life sciences, the physiological activity of sialic acid has gradually been recognized.
  • Sialic acid is a generic term for a class of derivatives in which amino acids or hydroxy hydrogens are substituted in neuraminic acid, and usually also refers to N-acetylneuraminic acid, one of the most important members of such compounds.
  • Sialic acid is widely distributed in nature. The highest known sialic acid content in foods is found in bird's nest, which is about 12%. Sialic acid is also found in milk, eggs and cheese. It is mainly used as glycoprotein and ganglion. The component of the glucoside. Sialic acid substituents usually A sialic acid which is an acetyl group or a hydroxyacetyl group, but a methyl group, a sulfate group, or a phosphate group-substituted amino group has also been found.
  • sialyl monosaccharides there are about 50 species of sialyl monosaccharides found so far, all of which are derivatives of ceramides, each carrying a variety of different substituents, some of which are amine groups and some of which are hydroxyl groups. Hydroxyl groups).
  • N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and N-acetyl-9-O-acetylneuraminic acid (Neu5Ac) are most common in the sialic acid family. Among them, Neu5Gc is commonly found in animal cells, but only a small amount appears in tumor cells in humans, while normal cells do not have Neu5Gc.
  • sialic acid In addition to its structural changes, sialic acid also changes with the modification of its own molecules. In addition to this, the manner in which sialic acid and sialic acid are bonded to other sugars also produces a diversity of sialic acid bonding structures.
  • Neu5Ac binds to galactose in an ⁇ 2,3-linked or ⁇ 2,6-linked manner to form Neu5Ac- ⁇ 2,3-Gal and Neu5Ac- ⁇ 2,6-Gal. In human tissues and cells, only Neu5Ac is present, but there is no Neu5Gc.
  • influenza virus infection is related to sialic acid, so one of the anti-influenza drugs (oseltamivir and zanamivir) is a mimicking sialic acid analog that can interact with the virus.
  • the aminase is also a sialidase reaction, so that the virus cannot be released from the host cells normally, so it is no longer possible to infect other normal cells.
  • sialic acid is an important component of the structure and function of brain gangliosides and glycoproteins. Because sialic acid is a nutrient of nerve cell membrane and synapse, sialic acid can promote the differentiation, development and nerve regeneration of nerve cells, participate in synaptic transmission, maintain the normal function of cells, and participate in memory learning. Animal experiments have shown that supplementation with sialic acid can improve the learning behavior of animals, and supplementation with sialic acid can significantly increase the content of sialic acid in gangliosides and glycoproteins in the brain and cerebellum, which can improve the test results of experimental animals in maze test. . This result indicates that sialic acid is quite important for learning ability.
  • breastfed babies are able to take large amounts of sialic acid from breast milk, which may promote sialylation of gangliosides and glycoproteins in body fluids, tissues and glycosides, while further showing breastfeeding.
  • the development of the nervous system and intelligence of infants is superior to that of milk powder.
  • breast milk is an effective way to supplement sialic acid for infants because it contains about 0.3-1.5 mg of sialic acid per mL of breast milk.
  • bird's nest contains a glycoprotein that enhances the mitosis of mononuclear cells in human blood by concanavalin A (Con A) or phytohemagglutinin A (PHA).
  • Mitogenic stimulation factor It is like epidermic growth factor, which can stimulate cell division and proliferation and promote cell differentiation, so that damaged epithelial cells can be repaired quickly.
  • This glycoprotein and concanavalin A priming effect had the strongest effect in the first 10 hours.
  • its glycoprotein activity is very stable and does not disappear due to the action of trypsin or extraction with ether in an alkaline environment.
  • the current extraction methods adopt high temperature and high pressure extraction methods.
  • this extraction method will destroy the sialic acid contained in the bird's nest and the protein molecules such as epidermal growth factor and fibroblast growth factor attached to the glycoprotein and/or bird's nest attached to the sputum, resulting in saliva in the obtained extract.
  • the acid and protein content is not high, so the effect of taking such bird's nest extract is limited.
  • an object of the present invention is to provide a method for extracting bird's nest extract, and the extracted bird's nest extract still retains a considerable amount of sialic acid and protein.
  • a method for extracting a bird's nest extract comprises the steps of: providing an aqueous solution of a bird's nest pulverized material in a reaction tank for emulsification, heating to a first temperature, and vacuuming the reaction tank for a continuous period of For a time, to form a bird's nest emulsion, wherein the reaction tank has an emulsifier and an agitator surrounding the emulsifier, the first temperature is 30 to 60 degrees Celsius, and the first time is 30 minutes to 12 hours; while maintaining the emulsifier And in the case of operation of the agitator, after adjusting the bird's nest emulsion to the second temperature, the protease is added to the reaction tank and maintained at the second temperature for a second time, wherein the second temperature is 30 degrees Celsius to Celsius 40 degrees, and the second time is 1 to 12 hours; the protease reaction is terminated; the bird's nest emulsion is filtered to obtain bird'
  • the extraction method of the bird's nest extract further comprises the step of lyophilizing the bird's nest extract to make a bird's nest extract.
  • the vacuum pressure of the reaction tank is maintained at 10 to 200 Torr during the emulsification step.
  • the speed of the emulsifier is 1200 to 3600 revolutions per minute (rpm), and the speed of the agitator is 10 to 60 revolutions per minute (rpm).
  • the step of terminating the protease reaction is to heat the bird's nest emulsion to between 50 and 70 degrees Celsius for 30 to 120 minutes.
  • the step of filtering the bird's nest emulsion is carried out at a second pressure and the second pressure is between 400 and 720 Torr.
  • the step of concentrating the bird's nest filtrate has a pressure of 600 to 720 Torr and a concentration ratio of 2 to 4 times.
  • the step of sterilizing the bird's nest filtrate is an Ebba low temperature
  • the sterilization method is carried out.
  • the protease is at least one of papain, tryptone, or pineapple enzyme.
  • the bird's nest extract or bird's nest extract comprises from 1% to 13% by weight of sialic acid and from 4% to 15% by weight of protein, wherein the protein has a molecular weight distribution of from 100 Da to 300 kDa, and wherein less than 6.5 The protein of kDa accounts for 0.6% to 6.4% by weight of the total protein content.
  • the extraction method of the bird's nest extract of the present invention adopts an extraction step of vacuum decompression during emulsification, and maintains the emulsification temperature below 60 degrees Celsius, under the condition of maintaining considerable emulsification efficiency,
  • the active ingredients in the bird's nest are destroyed by the high temperature and high pressure process. Therefore, the bird's nest extract extracted by the extraction method of the present invention has a considerable amount of sialic acid and protein, so it has considerable utility, and can be made into shampoo, hair lotion, mask, makeup remover, eye cream, hand guard Creams, lotions, bath essences, beauty lotions, body creams, toothpastes, ointments, fondants, or soaps.
  • FIG. 1 is a schematic flow chart of an extraction method of a bird's nest extract according to a first preferred embodiment of the present invention.
  • Fig. 2 is a schematic view showing a reaction tank used in the extraction method of the embodiment shown in Fig. 1.
  • FIG. 1 is a schematic flow chart of a method for extracting bird's nest extract according to a first preferred embodiment of the present invention
  • FIG. 2 is a schematic view of a reaction tank used in the extraction method of the present embodiment.
  • the extraction method of the bird's nest extract of the present embodiment comprises the following steps.
  • Step S1 vacuum emulsification step: An aqueous solution of bird's nest pulverized material is supplied and placed in the reaction tank 1 for emulsification.
  • the aqueous solution of the bird's nest pulverized material is obtained by mechanically pulverizing the purchased scorpion, swallow cake or swallow silk by a pulverizer, and the pulverized body of the bird's nest is obtained in a particle size range of about 250 to 830 micrometers ( ⁇ m), and the bird's nest pulverized material is A suitable weight ratio is dissolved in water.
  • the water used is preferably sterile distilled water to reduce the risk of microbial contamination of the product.
  • the weight ratio of the bird's nest pulverized material to water is preferably 1:19 (bird's nest: water), so that an appropriate concentration of the aqueous solution is formed to prevent the emulsifier and the agitator from being effectively operated in the subsequent emulsification step.
  • the aqueous solution of the bird's nest pulverized material is heated to a first temperature and the reaction tank 1 is evacuated for a first time to form a bird's nest emulsion.
  • the first temperature is 30 to 60 degrees Celsius, and the first time is 30 minutes to 12 hours.
  • the reaction tank 1 has a central shaft 10, an emulsifier 11, an agitator 12, a first motor 14, and a second motor 15, and the emulsifier 11 is connected to a central shaft 10 disposed at the center of the reaction tank 1,
  • the agitator 12 is substantially The structure is disposed around the emulsifier 11 and has a plurality of first agitating blades 121 extending toward the central shaft 10.
  • the center shaft 10 is coupled to the first motor 14, and the agitator 12 is coupled to the second motor 15.
  • the first motor 14 and the second motor 15 are coaxially disposed, but are independently operable.
  • the first motor 14 rotates the emulsification blade 111 of the emulsifier 11 via the center shaft 10.
  • the second motor 15 drives the agitator 12 to rotate, so that the first agitating blades 121 extending toward the central shaft 10 rotate together to achieve the purpose of agitation.
  • a plurality of second agitating blades 13 may be further added to the central shaft 10, and the second agitating blades 13 may be an asymmetrical configuration with a narrower end at one end as shown in FIG. 2, thereby further increasing The agitating ability of the second agitating blade 13 enables the liquid in the reaction tank 1 to be mixed more quickly and uniformly. Because in this step S2, the agitator 12 is agitated around the emulsifier 11 while the emulsifier 11 is operating, and the rotational speeds of the two are quite different.
  • the rotational directions of the two can be reversed.
  • the other is clockwise rotation and the other is counterclockwise rotation.
  • the aqueous solution of the bird's nest pulverized material having a relatively high viscosity in the reaction tank 1 can be formed into a circulating vortex, and the liquid emulsified by the emulsifier 11 can be taken to another place, and the liquid which is not emulsified is brought to the emulsifier 11
  • Emulsification can improve the overall emulsification efficiency, so that the extracted product has a considerable amount of active ingredients (such as sialic acid and / or protein).
  • the vacuum pressure of the reaction tank 1 is preferably maintained at 10 to 200 Torr (t. equivalent to millimeters of mercury, mmHg), and the pressure actually used may be viscous according to the powder of the bird's nest powder. Depending on the degree, the higher the viscosity of the bird's nest powder solution, the lower the vacuum pressure of the reaction tank 1 in this step S2.
  • the rotation speed of the emulsifier 11 is preferably 1200 to 3600 revolutions per minute (rpm), and the S extraction method is performed, and the emulsification is not performed at a high temperature (30 degrees Celsius to 60 degrees Celsius). It can be used in the effective emulsification of bird's nest powder solution while avoiding the destruction of active ingredients in bird's nest (such as sialic acid and protein).
  • step S2 (enzymatic reaction step) is carried out: the bird's nest emulsion treated in step S2 in the reaction tank 1 is adjusted to the second temperature while the agitator 12 and the emulsifier 11 are continuously operated, and the protease is adjusted.
  • the reaction tank is added and maintained at the second temperature for a second time, wherein the second temperature is 30 degrees Celsius to 40 degrees Celsius, and the second time is 1 to 12 hours.
  • the preferred ratio of protease added in this step S2 is 0.1-0.3% by weight, and a suitable protease may be at least one of papain, tryptone, or pineapple enzyme, or any combination of the foregoing enzymes.
  • the pH is preferably maintained between 7 and 7.5 to maintain the activity of the protease.
  • step S3 terminatating the protease reaction step
  • the reaction of the protease is terminated, preferably the reaction of the protease is terminated in a heat deactivated manner
  • the bird's nest emulsion in the reaction tank 1 can be heated to At 50 to 70 degrees Celsius for 30 to 120 minutes, the protease is inactivated to terminate its reaction.
  • the pH in the reaction tank 1 is maintained between 7 and 7.5 to maintain the stability of the reaction environment and to avoid excessive destruction of the active ingredients in the bird's nest.
  • step S4 filtration step
  • the bird's nest emulsion treated in step S3 is filtered to obtain bird's nest filtrate. Since the bird's nest emulsion treated after step S3 still has considerable The viscosity is so that the step S4 is to perform filtration under reduced pressure.
  • the bird's nest emulsion after the step S3 is filtered through a column connected with an iron sieve, and the second pressure is preferably 400 to 720. Trust.
  • step S5 concentration sterilization step
  • the bird's nest filtrate is concentrated under a pressure of 600 to 720 Torr, and the final concentration ratio can be controlled to be 2 to 4 times.
  • the bird's nest filtrate is sterilized to obtain a bird's nest extract.
  • the sterilization treatment method in the step S5 is preferably sterilized by the Ebba low-temperature sterilization method, and maintained in a low-pressure environment so that it can be subjected to effective sterilization conditions while avoiding protein denaturation due to high temperature.
  • the bar type low temperature sterilization method used in the present embodiment is preferably maintained at a temperature of 65 degrees Celsius, a sterilization time of 30 minutes, and an ambient pressure of 10 to 200 Torr (less preferably 100 Torr).
  • the bird's nest extract obtained through the step S5 can be sampled and tested, and the sialic acid content, protein content and molecular weight distribution thereof are examined, and the number of bacteria is checked.
  • step S6 freeze-drying step
  • the bird's nest extract obtained by the step S5 is added to the excipient of 0 to 5% by weight, and lyophilized to prepare Bird's nest extract.
  • the purpose of adding the excipient is to avoid the generation of bubbles during the lyophilization process and to maintain the stability of the reaction process.
  • the excipient comprises at least one of starch, corn starch, maltodextrin, lactose, dextrin, or cellulose, and the bird's nest extract obtained in the present embodiment can be adapted to be made into various solid or semi-solid products. For example, capsules, emulsions, and the like.
  • the bird's nest extract and the bird's nest extract extracted by the extraction method of the present embodiment may each contain 1% to 13% by weight of sialic acid and 4% to 15%. % by weight of protein.
  • the above numerical range of 1% to 13% by weight of sialic acid means that any value between "1%” and "13%” is included, for example, 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, and any positive integer, rational number, and irrational number between the foregoing values, and encompasses the ranges covered by any combination of the foregoing various numerical values.
  • the bird's nest extract and the bird's nest extract obtained by the extraction may each contain at least one of an epidermal growth factor (EGF) or a fibroblast growth factor (FGF), and the protein
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • the molecular weight distribution is from 100 Da to 300 kDa.
  • proteins having a protein content of less than 6.5 kDa are present in an amount of from 0.6% to 6.4% by weight of the total protein.
  • the bird's nest extract obtained by the extraction step of the present embodiment may be in the form of powder and is in the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis map at 145.991 m/z, 172.000. m/z, 190.017 m/z, 211.987 m/z, 294.06 m/z, 335.104 m/z, 379.132 m/z, 496.354 m/z, and 1536.790 m/z have characteristic peaks.
  • MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the bird's nest extract and bird's nest extract obtained in this embodiment can be used to make shampoo, hair lotion, facial mask, make-up remover, eye cream, hand cream, lotion, bath essence, beauty liquid, body use. At least one of cream, toothpaste, ointment, fondant, or soap.
  • the extraction method of the bird's nest extract of the present invention takes the vacuum decompression extraction step during the emulsification, and maintains the emulsification temperature below 60 degrees Celsius. Under the condition of maintaining considerable emulsification efficiency, the active ingredients in the bird's nest are also avoided due to the high temperature and high pressure process. Therefore, the bird's nest extract extracted by the extraction method of the present invention has a considerable amount of sialic acid and protein, so It is very effective, and can be made into shampoo, hair lotion, facial mask, make-up remover, eye cream, hand cream, lotion, bath essence, beauty lotion, body cream, toothpaste, ointment, jelly, or Soap and other products.
  • protease which may be papain, tryptone, or pineapple enzyme
  • the bird's nest emulsion is heated to 50-70 degrees Celsius for 30-120 minutes to stop the enzyme reaction.
  • the liquid after the protease reaction is filtered under a pressure of 400 to 720 Torr to filter out undissolved impurities, and the filtrate is concentrated under a pressure of 600 to 720 Torr, followed by a low temperature sterilization method (temperature). Maintain a bird's nest extract at a temperature of 65 ° C, a sterilization time of 30 minutes, and an ambient pressure of 10 to 200 Torr.
  • bird's nest extract powder a powdery bird's nest extract
  • the bird's nest extract and the bird's nest extract powder obtained in the above experimental example 1 are tested by the Central Standards Bureau of the Ministry of Economics for the determination of crude protein in food (CNS No. 5035, class number N6116, also commonly known as "Kjeldahl method"
  • the determination of the crude protein content was carried out separately. Repeated tests were carried out on different samples prepared under the extraction conditions in Experimental Example 1. The results showed that 4 to 15 grams of crude protein per 100 grams of bird's nest extract and bird's nest extract powder were used. Quality (depending on the sample, each 100 grams of bird's nest extract and bird's nest extract powder may contain 4, 4.9, 11.57, 14.6, 15 grams of crude protein).
  • the bird's nest extract and the bird's nest extract powder obtained in the above Experimental Example 1 were subjected to high performance liquid chromatography (Hua Yongyou et al., Chinese Journal of Health Inspection, October 2010, Vol. 20, No. 10, pp. 2454-2456)
  • the sialic acid content of each sample was measured.
  • the results showed that the different samples prepared under the extraction conditions of Experimental Example 1 were repeatedly tested, and the percentage of sialic acid was 1% to 13% (depending on the sample, the bird's nest extract and the bird's nest extract powder may contain 1%). , 2.07%, 3.09%, 8.61%, 10.08%, 13% sialic acid).
  • the references listed above are all incorporated by reference in their entirety.
  • the bird's nest extract and the bird's nest extract powder obtained in the experimental example 1 contain a considerable amount of sialic acid, and therefore the person skilled in the art of the present invention has a general knowledge. According to the disclosure of the present specification, the present invention can be known.
  • the provided bird's nest extract composition and the food containing the bird's nest extract composition can have the functions of enhancing immunity, enhancing memory and preventing colds.
  • the bird's nest extract and the bird's nest extract powder obtained in the above Experimental Example 1 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the molecular weight distribution of the protein in the sample.
  • the sodium dodecyl sulfate polyacrylamide gel concentration was 12% and 15%, and 10% by weight of glycerol was added, and the colloid after the experiment was used as a Bio-rad gel DOC XR+ system image analysis device. Scanning and analysis were performed to calculate the protein content of different molecular weights in each sample. Repeated tests were carried out on different samples prepared under the extraction conditions of Experimental Example 1.
  • Table 1 Protein distribution of bird's nest extract and bird's nest extract powder
  • the bird's nest extract powder obtained in the above experimental example 1 was further analyzed for molecular weight distribution of each molecule in the sample by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and found to be 145.991 m/ in the analysis map.
  • MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the bird's nest extract obtained in the above experimental example 1 was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the molecular weight distribution of each molecule in the sample, and found that it was 171.966 m/z in the analysis map.
  • MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • the bird's nest extract and the bird's nest extract powder obtained in the above experimental example 1 were obtained by using commercial EGF and FGF kits (manufacturer name: Peprotech, and the box names were: Human EGF Mini ELISA Development Kit PEP-900-M05, Human FGF- The basic ELISA Development Kit 900-K0) was used to detect EGF and FGF in samples by sandwich enzyme-linked immunosorbent assay (Sandwich ELISA).
  • EGF standard was serially diluted twice from 1000 pg/mL (pg/mL) to 15.625 pg/mL (pg/mL), while the FGF standard was from 4000 pg/ml ( Pg/mL) was serially diluted twice to 62.5 pg/ml (pg/mL).
  • the capture antibody concentration was 100 ⁇ g/ml ( ⁇ g/mL)
  • the detection antibody concentration was 100 ⁇ g/ml ( ⁇ g/mL).
  • achromin-HRP conjugate (Avidin-HRP conjugate) and ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) color reaction
  • the absorbance of the read reaction at 450 nm/650 nm was measured every five minutes, and the absorbance of the standard controlling the highest concentration was not more than 1.2, and the absorbance of the standard of the lowest concentration was not more than 0.2.
  • the remaining experimental steps were performed in accordance with the instructions attached to the commercial EGF and FGF kits utilized.
  • each milliliter of bird's nest extract contained at least 0.04 ⁇ g of EGF, and each gram of bird's nest extract contained at least 0.0832 ⁇ g of EGF, and was tested with bird's nest food (ie, swallow) purchased from other sources.
  • the gram contains less than 31.26 pg of EGF.
  • Each milliliter of bird's nest extract contains at least 0.16 ⁇ g of FGF, and each gram of bird's nest extract contains 38.835 to 261.88 ng of FGF, and is tested with bird's nest food (ie, swallow) purchased from other sources.
  • the gram contains 228.67 to 300.172 pg of FGF.
  • the bird's nest extract and the bird's nest extract powder obtained in the above experimental example 1 do contain a considerable amount of EGF and FGF protein, so the bird's nest extract composition provided by the present invention and the bird's nest extract composition containing the same
  • the food may have the function of promoting epidermal growth, skin care, hair care or wound repair.
  • the bird's nest extract composition obtained in Experimental Example 1 was added with an appropriate amount of water, glycerin, aloe vera extract (Kidachi Aloe Ekisu) and isopropyl myristate, glycerol stearin at a weight percentage of 0.02% to 3%.
  • Glyceryl Stearate, cetyl alcohol, sorbitol, ceteth-15, sorbitan Stearate and other suitable emulsifiers To prepare a hand cream containing bird's nest extract.
  • the bird's nest extract composition obtained in the experimental example 1 was added with 65.7%-93.0% maltodextrin in a weight percentage of 2%-8.5%, and an appropriate amount (500-1000 mg) was packaged in a gelatin capsule to obtain a bird's nest extract composition.
  • the bird's nest extract composition obtained in the experimental example 1 is added with an appropriate amount of water, lauryl sulfate, lauryl polyoxyethylene sulfate, sodium cocoamphoacetate, coconut at a weight percentage of 0.02% to 3%.
  • the bird's nest extract composition obtained in the experimental example 1 is added with an appropriate amount of water and a surfactant (for example, sodium sulphate sodium sulphate (Sodium Lauryl Sulfate), sodium lauryl sulfate sodium salt (Sodium) in a weight percentage of 0.02% to 3%.
  • a surfactant for example, sodium sulphate sodium sulphate (Sodium Lauryl Sulfate), sodium lauryl sulfate sodium salt (Sodium) in a weight percentage of 0.02% to 3%.
  • Laureth Sulfate Laureth Sulfate
  • cocamide DEA Cocamide DEA
  • / or Glycol stearate emollient
  • milk Chemical agents such as olive oil, polysorbate, sodium chloride
  • humectants such as Tocopheryl Acetate E
  • preservatives such as hydroxymethyl
  • the bird's nest extract composition obtained in the experimental example 1 is added with an appropriate amount of arbutin, hyaluronic acid, elastin, seaweed extract, hydroxypropyl propyl ester, water, and coated at an appropriate base in a weight percentage of 0.02% to 3%.
  • a material for example, non-woven fabric is used to prepare a mask containing a bird's nest extract.
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, Cyclopentasiloxane, emollient (for example, Isohexadecane), perfume, surface at a weight percentage of 0.02% to 3%.
  • Active agents such as Hexylene Glycol, Sodium chloride
  • emulsifiers such as cyclohexasiloxane
  • preservatives such as benzyl alcohol, Phenoxyethanol
  • a fragrance such as sodium citrate
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, a humectant (for example, glycerin, Saccharide isomerate), and a silicon-based smoothing agent (for example, ring 2) in a weight percentage of 0.02% to 3%.
  • a humectant for example, glycerin, Saccharide isomerate
  • a silicon-based smoothing agent for example, ring 2
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, a humectant (for example, Dipropylene glycol, Butylene Glycol, glycerin), alcohol, in an amount of 0.02% to 3% by weight.
  • Emulsifiers such as Methyl gluceth-10, Polyglyceryl-2 diisostearate
  • surfactants eg PEG-60 hydrogenated castor oil (PEG-60 Hydrogenated) Castor Oil
  • Isostearic acid eg PEG-60 hydrogenated castor oil (PEG-60 Hydrogenated) Castor Oil
  • a emollient such as Triethylhexanoin
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, a humectant (for example, glycerin, propylene glycol), and a surfactant (for example, Polysorbate 20) in a weight percentage of 0.02% to 3%. ), PEG-150 distearate (Hydrogenated Castor Oil), emulsifier (such as glyceryl oleate), preservative (such as methyl isothiazolinone ( Methylisothiazolinone)), a suitable fragrance to make a lotion containing bird's nest extract.
  • a humectant for example, glycerin, propylene glycol
  • a surfactant for example, Polysorbate 20
  • PEG-150 distearate Hydrogenated Castor Oil
  • emulsifier such as glyceryl oleate
  • preservative such as methyl isothiazolinone ( Methylisothi
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, alcohol, a humectant (for example, glycerin, butylene glycol), an emulsifier (for example, hydrogenated lecithin) at a weight percentage of 0.02% to 3%. Hydrogenated lecithin), a emollient (such as acetyl glutamine (Acetyl Glutamine)), a suitable fragrance, and a preservative (such as Methylparaben) to produce a cosmetic liquid containing bird's nest extract.
  • a humectant for example, glycerin, butylene glycol
  • an emulsifier for example, hydrogenated lecithin
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, a humectant (for example, glycerin, propylene glycol, 1,2-pentanediol (Pentylene Glycol)) in an amount of 0.02% to 3% by weight, suitably Perfumes, emollients (such as Cyclopentasiloxane, Glyceryl Stearate), surfactants (such as PEG-60 Hydrogenated Castor Oil, antioxidants (such as citric acid) Sodium), an emulsifier (such as behenic acid), a preservative, and a suitable fragrance to prepare a body cream containing bird's nest extract.
  • a humectant for example, glycerin, propylene glycol, 1,2-pentanediol (Pentylene Glycol)
  • emollients such as Cyclopentasiloxane, Glyceryl Stearate
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, silicate, muscovite, fluoride, and a surfactant (for example, sodium dodecyl sulfate (SLS)) in a weight percentage of 0.02% to 3%.
  • a surfactant for example, sodium dodecyl sulfate (SLS)
  • SLS sodium dodecyl sulfate
  • a suitable flavor, glycerin, and sorbitol are used to prepare a toothpaste containing a bird's nest extract.
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, honey, rock sugar, and plants in a weight percentage of 0.02% to 3% (for example, Fritillaria, Camphor leaves, Radix Scutellariae, Campanulaceae, licorice, menthol, Almond) extract to make a cream containing bird's nest extract.
  • 0.02% to 3% for example, Fritillaria, Camphor leaves, Radix Scutellariae, Campanulaceae, licorice, menthol, Almond
  • the bird's nest extract composition obtained in Experimental Example 1 is added with an appropriate amount of water, oil (for example, palm oil), sodium stearate, appropriate flavors and dyes in an amount of 0.02% to 3% by weight to obtain a bird's nest extract. soap bar.
  • oil for example, palm oil
  • sodium stearate for example, sodium stearate
  • appropriate flavors and dyes in an amount of 0.02% to 3% by weight
  • the bird's nest extract composition obtained in the experimental example 1 is added with an appropriate amount of water, gelatin, glucose, sucrose, citric acid, and a suitable flavor and sweetener in a weight percentage of 0.02% to 3% to obtain a bird's nest extract.
  • Soft candy

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Abstract

一种燕窝萃取物的萃取方法，是将燕窝粉碎物水溶液在30-60℃下提取30分钟至12小时，同时搅拌溶液和抽取真空；随后将温度调整为30-40℃，加入蛋白酶水解1至12小时；终止蛋白酶反应；过滤，浓缩，灭菌，获得燕窝萃取液。所述方法有效保留了唾液酸和蛋白质。

Description

燕窝萃取物的萃取方法 技术领域
本发明是关于一种萃取方法，特别关于一种适用于萃取燕窝萃取物的萃取方法。
背景技术
燕窝是中国自古代以来开始被食用的传统名贵食品之一，是指雨燕目雨燕科部分雨燕和金丝燕属的几种金丝燕分泌出来的唾液，再混合其它物质(如：羽毛)所筑成的巢穴，而非雀形目燕科鸟类(如家燕)所筑巢穴。
其中以金丝燕唾液的蛋白质纯度和营养价值为最高。产自中国南部沿海一带、越南、泰国、马来西亚、印度尼西亚及菲律宾等地。燕窝被采摘之后，还要经过蒸细、浸泡、除杂、挑毛、烘干等复杂的加工才能制成燕窝成品，包括燕盏、燕条、燕饼、燕丝。其中燕盏是整只优质的燕窝经除杂保留原有形状而成，故价格特别昂贵。
而在燕窝的组成中，其含有一种名为燕窝酸的活性物质，其又称为唾液酸。贡纳尔·布利克斯等人于1952年从颌下腺粘蛋白中分离出唾液酸(Sialic acid)，并建立了唾液酸的命名规则，但在很长时期内人们对其生理功能了解甚少，其化学结构在1960年才被确切地测定出来。随着现代物理学、化学和生命科学技术的飞速发展，唾液酸的生理活性已逐渐被认识。唾液酸是神经氨酸中胺基酸或羟基氢被取代的一类衍生物的总称，通常也指代此类化合物中最重要的成员之一N-乙酰神经氨酸。唾液酸在自然界分布很广，目前已知的食物中唾液酸含量最高的是存在燕窝中，含量达12％左右，在牛奶、鸡蛋和奶酪中也存在唾液酸，其主要作为糖蛋白和神经节苷脂的组分。唾液酸取代基通常 为乙酰基或羟乙酰基，不过甲基、硫酸酯基、磷酸酯基取代氨基的唾液酸也已被发现。目前被发现的唾液酸单醣家族约有50种，都是神经胺酸的衍生物，每一种都携带着各种不同的取代基，有的是胺基(amine groups)、有的则是羟基(hydroxyl groups)。
而在唾液酸家族中又以N-乙酰神经氨酸(Neu5Ac)、N-乙醇酰神经氨酸(Neu5Gc)和N-乙酰-9-O-乙酰神经氨酸(Neu5Ac)最为常见。其中Neu5Gc普遍出现于动物细胞，但在人体中却只有少量出现于肿瘤细胞上，正常的细胞则无Neu5Gc存在。
唾液酸除了本身结构上的变化，也会随着本身分子修饰的不同而有所改变。除此之外，唾液酸与唾液酸之间或与其它的醣的键结方式，也产生唾液酸键结结构的多样性。像是Neu5Ac以α2，3-连结或是α2，6-连结的方式与半乳糖结合，形成Neu5Ac-α2，3-Gal和Neu5Ac-α2，6-Gal。而人类的组织和细胞中只有Neu5Ac，却没有Neu5Gc的存在。亦有文献显示，流感病毒的感染与唾液酸有关，因此其中一类的抗流感药物(克流感(oseltamivir)和瑞乐沙(zanamivir))，为模拟唾液酸的类似物，可与病毒的神经胺酸酶也就是唾液酸酶反应，使得病毒无法正常的从宿主细胞中释出，因此无法再感染其它正常细胞。
有文献显示，唾液酸是大脑神经节苷脂和糖蛋白的结构和功能上的重要组成部分。因唾液酸是神经细胞膜及突触的营养素，因此唾液酸可促进神经细胞的分化、发育和神经再生作用，参与突触传递、维持细胞的正常机能，参与记忆学习功能。动物实验研究表明，补充唾液酸可以提高动物的学习行为，而补充唾液酸可以明显增加大脑和小脑的神经节苷脂和糖蛋白中唾液酸的含量，可以提高实验动物在迷宫测试中的测试结果。此结果表明，唾液酸对于学习能力相当重要。亦有研究显示，大脑的生长和成熟与唾液酸所参与形成的酸性糖脂与蛋白含量的增加有关，而衰老和先天性发育迟缓综合征与其含量下降有关。通过动物实验发现，在没有唾液酸或唾液酸含量很低的情况下， 实验动物的学习及记忆能力均大大下降。
许多研究都证明，母乳喂养的婴儿能够从母乳中摄取大量的唾液酸，其有可能促进神经节苷脂和在体液、组织和配糖体中的糖蛋白的唾液酸化，同时进一步显示出母乳喂养对婴儿的神经系统和智力的发育要优于奶粉喂养。而在婴儿诞生后，母乳则是为婴儿补充唾液酸的有效途径，因为每mL母乳中含有约0.3-1.5mg的唾液酸。
更进一步的，有研究发现，燕窝含有一种醣蛋白，可增强刀豆蛋白A(Con A)或植物血凝素A(PHA)刺激人体血液中单核细胞的有丝分裂，因而称为「促进细胞分裂因子」(mitogenic stimulation factor)。其如同表皮生长因子(epidermic growth factor)能够刺激细胞分裂增殖和促进细胞分化，使得受损的上皮细胞得以快速修复。而此醣蛋白与刀豆蛋白A激发作用在最初的10小时保有最强的效果。除此之外，其醣蛋白的活性非常稳定，并不会因胰蛋白酶的作用或是在碱性环境下经由乙醚萃取而消失。
而在燕窝醣蛋白的寡糖研究方面，有文献显示燕窝醣蛋白上有着各式各样的含唾液酸的O-链接寡糖。而若以酶切下燕窝醣蛋白上N-链接寡糖进行分析，发现燕窝醣蛋白上唾液酸N-链接寡糖含有大量的L-岩藻糖(L-fucose)，而其含的唾液酸是多价的，并且在对抗疾病上可能扮演着重要的角色。另外也有研究指出，经胰酶水解的燕窝可以达到抗流感病毒的效果，可能和燕窝中所含的唾液酸有关。
为了萃取出燕窝中的营养成分，目前现有的萃取方法多采取高温高压的萃取方式。然而，此种萃取方式将破坏燕窝中所含的唾液酸与唾液酸所附随之醣蛋白及/或燕窝中的表皮生长因子、纤维母细胞生长因子等蛋白质分子，致使所得到的萃取物中唾液酸与蛋白质的含量不高，故而服用此种燕窝萃取物的效果有限。
因此，如何提供一种可萃取出具有相当含量的唾液酸及蛋白质的燕窝萃取物的萃取方法，使萃取所得的燕窝萃取物具有相当效用(例 如：提升免疫力、增强记忆力、预防感冒功能)，已成为重要课题之一。
发明内容
有鉴于上述课题，本发明的目的为提供一种燕窝萃取物的萃取方法，萃取出的燕窝萃取物仍保留有相当含量的唾液酸及蛋白质。
为达上述目的，依据本发明的一种燕窝萃取物的萃取方法，包含有以下步骤：提供燕窝粉碎物水溶液置于反应槽中进行乳化，加热至第一温度并将反应槽抽取真空并持续第一时间，以形成燕窝乳化液，其中反应槽具有乳化器及环绕乳化器的搅拌器，第一温度为摄氏30至60度，且第一时间为30分钟至12小时；在维持所述乳化器及所述搅拌器的运转的情况下，将燕窝乳化液调整至第二温度后，将蛋白酶加入反应槽，并维持在第二温度下持续第二时间，其中第二温度为摄氏30度至摄氏40度，且第二时间为1至12小时；终止蛋白酶的反应；过滤燕窝乳化液，获得燕窝滤液；浓缩燕窝滤液，并将浓缩后的燕窝滤液进行灭菌处理，以获得燕窝萃取液。
在一个实施例中，燕窝萃取物的萃取方法进一步包括将燕窝萃取液进行冻干的步骤，以制成燕窝萃取物。
在一个实施例中，在乳化步骤中，反应槽的真空压力维持在10～200托。
在一个实施例中，在乳化步骤中，乳化器的转速为每分钟1200～3600转(rpm)，且所述搅拌器的转速为每分钟10～60转(rpm)。
在一个实施例中，终止蛋白酶的反应的步骤是将燕窝乳化液加热至摄氏50～70度并持续30～120分钟。
在一个实施例中，过滤燕窝乳化液的步骤在第二压力下进行，且第二压力为400～720托。
在一个实施例中，浓缩燕窝滤液的步骤的压力为600～720托，且浓缩倍率为2～4倍。
在一个实施例中，将燕窝滤液进行灭菌处理的步骤是依巴式低温 杀菌法进行。
在一个实施例中，蛋白酶为木瓜酶、胰蛋白脢、或菠萝酶中的至少一种。
在一个实施例中，燕窝萃取液或燕窝萃取物包含有1％至13％重量百分比的唾液酸以及4％至15％重量百分比的蛋白质，其中蛋白质的分子量分布为100Da至300kDa，且其中小于6.5kDa的蛋白质占总蛋白质含量为0.6％～6.4％重量百分比。
承上所述，本发明的燕窝萃取物的萃取方法，因在进行乳化时采取真空减压的萃取步骤，并维持乳化温度在低于摄氏60度，在保有相当乳化效率的条件下，同时也避免了因高温高压工艺而破坏燕窝中的有效成分。因此，本发明的萃取方法所萃取出的燕窝萃取物，具有相当含量的唾液酸及蛋白质，故具有相当效用，而可制成洗发精、护发乳、面膜、卸妆液、眼霜、护手霜、化妆水、沐浴精、美容液、身体用乳霜、牙膏、枇杷膏、软糖、或肥皂等产品。
附图说明
图1为本发明第一优选实施例的燕窝萃取物的萃取方法流程示意图。
图2为图1所示的实施例的萃取方法中所使用的反应槽示意图。
具体实施方式
以下将参照相关附图，说明依本发明优选实施例的一种燕窝萃取物的萃取方法，其中相同的组件将以相同的参照符号加以说明。此外，以下各实施例及各实验例中所提及的数值范围，如有特别指示及说明者外，均包含各数值范围端点值及该端点值之间的任意数值，且亦涵盖任何正整数、有理数及无理数，以及包含前述各种数值的任意组合所涵盖的范围。
请参考图1及图2，图1为本发明第一优选实施例的一种燕窝萃取物的萃取方法流程示意图，图2为本实施例的萃取方法中所使用的反应槽示意图。本实施例的燕窝萃取物的萃取方法，包含有下述步骤。
步骤S1(真空乳化步骤)：提供燕窝粉碎物水溶液，并置于反应槽1中进行乳化。燕窝粉碎物水溶液的获得是将购得的燕盏、燕饼或燕丝以粉碎机机械粉碎后，获得燕窝粉碎物的粒径范围约在250～830微米(μm)，并将燕窝粉碎物以适当的重量比例溶于水中。所使用的水以无菌蒸馏水为佳，以降低产品的微生物污染风险。而燕窝粉碎物与水的重量比例，优选为1∶19(燕窝∶水)，使其形成适当浓度的水溶液，以免浓度过高导致后续乳化步骤中乳化器及搅拌器发生无法有效运作的情况。接着，将燕窝粉碎物水溶液加热至第一温度并将反应槽1抽取真空并持续第一时间，以形成燕窝乳化液。第一温度为摄氏30至60度，第一时间为30分钟至12小时。请同时参考图2，反应槽1具有中心轴10、乳化器11、搅拌器12、第一马达14、以及第二马达15，乳化器11为连接设置于反应槽1的中央的中心轴10，而搅拌器12大致上呈
Figure PCTCN2015084769-appb-000001
型结构，并环绕于乳化器11进行设置，且具有多个向中心轴10延伸的第一搅拌叶片121。中心轴10连接第一马达14，而搅拌器12则与第二马达15连接。第一马达14与第二马达15为同轴设置，但可独立运作。第一马达14经由中心轴10而带动乳化器11的乳化叶片111旋转。而第二马达15则带动搅拌器12旋转，使向中心轴10延伸的第一搅拌叶片121随同旋转以达成搅拌的目的。此外，在中心轴10上可进一步增设多个第二搅拌叶片13，而第二搅拌叶片13的可以是如图2中所示的一端较宽一端较窄的不对称构型，借此进一步增加第二搅拌叶片13的搅拌能力，使反应槽1中的液体能够混合的更快速以及均匀。由于本步骤S2中，搅拌器12是在乳化器11运作同时，绕着乳化器11进行搅拌，并且两者转速有相当的差异，而在其它实施方面中，两者的旋转方向可以相反，一者为顺时针旋转另一为逆时针旋转。借此，可使反应槽1中黏稠度相当高的燕窝粉碎物水溶液形成循 环涡流，使经过乳化器11乳化后的液体被带至他处，同时将未被乳化的液体带至乳化器11处进行乳化，故可提升整体乳化效率，使萃取后的产物具有相当量的有效成分(例如唾液酸及/或蛋白质)。
此外，在进行步骤S1的真空乳化步骤时，反应槽1的真空压力优选维持在10～200托(托，相当于毫米汞柱，mmHg)，而实际使用的压力则可依燕窝粉末溶液的黏稠度而定，即燕窝粉末溶液黏稠度越高，则反应槽1在此步骤S2中的真空压力则越低。而在本步骤S2的真空乳化步骤中，乳化器11的转速优选为每分钟1200～3600转(rpm)，且搅S萃取方式，并且不以高温(摄氏30度至60度)进行乳化，故可在燕窝粉末溶液进行有效的乳化情况下，同时避免破坏燕窝中的有效成分(例如唾液酸与蛋白质)。
接着，进行步骤S2(酶反应步骤)：将在反应槽1中经过步骤S2处理后的燕窝乳化液，在维持搅拌器12及乳化器11持续运转的情况下，调整至第二温度，将蛋白酶加入反应槽，并维持在第二温度下持续第二时间，其中第二温度为摄氏30度至摄氏40度，且第二时间为1至12小时。本步骤S2加入的蛋白酶，优选的比例是0.1-0.3％重量百分比，而适用的蛋白酶可为木瓜酶、胰蛋白脢、或菠萝酶所至少其中的一种，或前述酶的任意组合。而酶反应的环境，其pH值优选是维持在7至7.5之间，以保持蛋白酶的活性。
接着，进行步骤S3(终止蛋白酶反应步骤)，并在终止蛋白酶的反应时，优选是以加热失活(heat deactivate)的方式终止蛋白酶的反应，即可将反应槽1中的燕窝乳化液加热至摄氏50至70度并持续30至120分钟，使蛋白酶失去活性以终止其反应。而在终止蛋白酶反应步骤时，仍维持反应槽1中的pH值在7至7.5之间，以保持反应环境的稳定，避免过度破坏燕窝中的有效成分。
接着，进行步骤S4(过滤步骤)。为了要去除燕窝乳化液中不反应或未反应完全的杂质，则将经由步骤S3处理后的燕窝乳化液进行过滤，以获得燕窝滤液。由于经由步骤S3处理后的燕窝乳化液仍具有相当的 黏稠度，故本步骤S4中是采取减压方式进行过滤，即将步骤S3处理后的燕窝乳化液通过连接有铁筛的管柱，在第二压力下进行过滤，第二压力优选为400至720托。
接着，为了要减低燕窝滤液的体积以及降低滤液中的水分，以利后续长期保存燕窝萃取液，所以进行步骤S5(浓缩灭菌步骤)。本步骤S5中，是将燕窝滤液在600至720托的压力下进行浓缩，而最终的浓缩倍率可控制在2～4倍。浓缩之后，则燕窝滤液进行灭菌处理，以获得燕窝萃取液。而本步骤S5中的灭菌处理方式，优选为依巴式低温杀菌法进行灭菌，并维持低压环境，使其能进行有效的灭菌条件下，亦同时避免因高温所导致的蛋白质变性。
而本实施例中所采用的巴式低温杀菌法则以温度维持在摄氏65度，杀菌时间为30分钟，环境压力为10至200托(小于100托更佳)的条件为佳。
为了明了萃取效率，可将经由步骤S5所获得的燕窝萃取液采样送检，同时检验其唾液酸含量、蛋白质含量及分子量分布，以及检验其生菌数。
另外，为了长期保存的目的，可进一步进行步骤S6(冻干步骤)：将经由步骤S5所获得的燕窝萃取液，加入重量百分比为0至5％的赋型剂，进行冻干，以制成燕窝萃取物。加入赋型剂的目的则在于避免在冻干过程中产生气泡，维持反应过程的稳定。赋型剂包含有淀粉、玉米淀粉、麦芽糊精、乳糖、糊精、或纤维素中至少一种，而可使本实施例所得的燕窝萃取物适于制成各种固态或半固态的产品，例如胶囊、乳液等。
此外，经由本实施例的萃取方法所萃取出的燕窝萃取液及燕窝萃取物，经后续实验例的实验结果显示，其均可包含有1％至13％重量百分比的唾液酸以及4％至15％重量百分比的蛋白质。前述1％至13％重量百分比的唾液酸的数值范围是指「1％」及「13％」之间的任意数值均涵盖在内，例如1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、 11％、12％、13％以及前述各数值之间的任何正整数、有理数及无理数，并且包含前述各种数值的任意组合所涵盖的范围。前述4％至15％重量百分比的蛋白质的数值范围，亦同，是指「4％」及「15％」之间的任意数值均涵盖在内，且亦涵盖任何正整数、有理数及无理数，例如4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％，以及包含前述各种数值的任意组合所涵盖的范围。
而萃取所得的燕窝萃取液及燕窝萃取物，是均可包含有表皮生长因子(EGF，Epidermal Growth Factor)、或纤维母细胞生长因子(FGF，Fibroblast Growth Factor)至少其中的一种蛋白质，且蛋白质的分子量分布为100Da至300kDa。另外，小于6.5kDa的蛋白质占总蛋白质含量为0.6％～6.4％重量百分比。
进一步来说，经由本实施例的萃取步骤所得的燕窝萃取物，可呈粉状，且于基质辅助激光解吸电离飞行时间质谱分析法(MALDI-TOF MS)分析图谱中在145.991m/z、172.000m/z、190.017m/z、211.987m/z、294.06m/z、335.104m/z、379.132m/z、496.354m/z以及1563.790m/z具有特征峰。而当以燕窝萃取液状进行测试时，其于基质辅助激光解吸电离飞行时间质谱分析法(MALDI-TOF MS)分析图谱中在171.966m/z、189.971m/z、211.976m/z、233.971m/z、249.949m/z、379.067m/z、445.031m/z、655.995m/z具有特征峰。详细数据及实验方式如后所述。
而本实施例所制得的燕窝萃取液及燕窝萃取物，可用以制成洗发精、护发乳、面膜、卸妆液、眼霜、护手霜、化妆水、沐浴精、美容液、身体用乳霜、牙膏、枇杷膏、软糖、或肥皂中至少一种。
上述各项产品的添加成份，如后实验例六中所述。
综上所述，本发明的一种燕窝萃取物的萃取方法，发明的燕窝萃取物的萃取方法，因在进行乳化时采取真空减压的萃取步骤，并维持乳化温度在低于摄氏60度，在保有相当乳化效率的条件下，同时也避免了因高温高压工艺而破坏燕窝中的有效成分。因此，本发明的萃取方法所萃取出的燕窝萃取物，具有相当含量的唾液酸及蛋白质，故具 有相当效用，而可制成洗发精、护发乳、面膜、卸妆液、眼霜、护手霜、化妆水、沐浴精、美容液、身体用乳霜、牙膏、枇杷膏、软糖、或肥皂等产品。
接下来将以实验例具体说明本发明的前述优选实施例的燕窝萃取物的萃取方法的实际操作方式及效果。然需注意的是，以下的说明是用来详述本发明以使此熟习该项技术者能够据以实现，但并非用以限定本发明的范围。
实验例一：燕窝萃取组成物的萃取
首先，取天然燕窝磨碎后的燕窝粉末6公斤，加入114公升的无菌蒸馏水中并置于反应槽1中。接着进行真空减压萃取，即于摄氏30～50度环境中抽取真空(压力为10～200托(torr))维持30分钟至12小时，同时乳化器11的转速设定为每分钟1200～3600转，且搅拌器12的转速设定为每分钟10～60转。同时，待反应槽1中的溶液冷却至摄氏37度之后，加入228公克的蛋白酶(可为木瓜酶、胰蛋白脢、或菠萝酶)，维持在摄氏37度下作用1～12小时，之后将燕窝乳化液加热至摄氏50～70度并持续30～120分钟，以中止酶反应。
接着在400～720托的压力下过滤上述经过蛋白酶反应完毕后的液体，以滤除未溶解的杂质，并将滤液在600至720托的压力下进行浓缩后，以巴式低温杀菌法(温度维持在摄氏65度，杀菌时间为30分钟，环境压力为10至200托)进行杀菌，获得燕窝萃取液。
将前述燕窝萃取液混入0～10％重量百分比的糊精进行冻干处理，以获得粉状的燕窝萃取物(以下简称燕窝萃取粉)。
实验例二：燕窝萃取物中粗蛋白质含量的分析
将上述实验例一中获得的燕窝萃取液以及燕窝萃取粉，以经济部中央标准局食品中粗蛋白质的检验法(CNS总号5035，类号N6116，又俗称「凯氏法」(Kjeldahl method))分别进行粗蛋白质含量的测定。以实验例一中萃取条件下所制得的不同样本进行重复测验，结果显示每100公克的燕窝萃取液以及燕窝萃取粉中含有4至15公克的粗蛋白 质(依不同样本，每100公克的燕窝萃取液以及燕窝萃取粉中可含有4、4.9、11.57、14.6、15公克的粗蛋白质)。
实验例三：燕窝萃取物中唾液酸含量分析
将上述实验例一中获得的燕窝萃取液以及燕窝萃取粉，以高效液相色谱法(华永有等人，中国卫生检验杂志，2010年10月第20卷第10期，页2454-2456)检测各样本的唾液酸含量。结果显示，以实验例一的萃取条件下所制得的不同样本进行重复测验，其唾液酸的重量百分比为1％至13％(依不同样本，燕窝萃取液以及燕窝萃取粉中可含有1％、2.07％、3.09％、8.61％、10.08％、13％的唾液酸)。而上述所列的参考文献全体皆引用作为本说明书的公开内容。
经由上述实验结果可知，实验例一中获得的燕窝萃取液以及燕窝萃取粉含有相当含量的唾液酸，因此本发明所属技术领域具有通常知识者，依据本说明书的公开内容，当可得知本发明所提供的燕窝萃取组成物及含有此种燕窝萃取组成物的食品可具有提升免疫力、增强记忆力、预防感冒的功能。
实验例四：燕窝萃取物中蛋白质分子量分布分析
将上述实验例一中获得的燕窝萃取液以及燕窝萃取粉，以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)来分析样本中蛋白质的分子量分布。前述十二烷基硫酸钠聚丙烯酰胺凝胶浓度为12％及15％，且添加有10％重量百分比的甘油(glycerol)，并将实验后的胶体以Bio-rad gel DOC XR+系统影像分析装置扫描及分析计算各样本中的不同分子量的蛋白质含量。以实验例一的萃取条件下所制得的不同样本进行重复测验，结果显示，经实验例一所制得的燕窝萃取液以及燕窝萃取粉中，小于6.5kDa的蛋白质占总蛋白质含量约是0.6％至6.4％重量百分比，以其中样本进行前述分析的蛋白质分子量分布分析结果如下表1所示。
表1：燕窝萃取液以及燕窝萃取粉的蛋白质分布
  燕窝萃取粉 燕窝萃取液
分子量(kDa) 重量百分比(％) 重量百分比(％)
＞100 7.9-15.3 1.4-1.6
100-75 11.6-20.6 1.4-4.4
75-50 14.1-35.1 22.9-27.5
50-35 9.4-12.6 2.9-25.9
35-25 6.4-16.6 4.5-5.5
25-15 16.8-30 24.2-56
15-6.5 0.7-3.7 4.1-10.7
＜6.5 0.6-1.5 0.6-6.4
此外，将上述实验例一中获得的燕窝萃取粉，经由基质辅助激光解吸电离飞行时间质谱分析法(MALDI-TOF MS)进一步分析样本中各分子的分子量分布，发现在分析图谱中在145.991m/z、172.000m/z、190.017m/z、211.987m/z、294.065m/z、335.104m/z、379.132m/z、496.354m/z、1563.790m/z、5312.775m/z、6220.710m/z、5419.701m/z、7802.584m/z、5562.115m/z、24966.772m/z、以及5916.553m/z处有特征峰(peak)。
而将上述实验例一中获得的燕窝萃取液，经由基质辅助激光解吸电离飞行时间质谱分析法(MALDI-TOF MS)进一步分析样本中各分子的分子量分布，发现在分析图谱中在171.966m/z、189.971m/z、211.976m/z、233.971m/z、249.949m/z、379.067m/z、445.031m/z、655.995m/z、5308.455m/z、5417.040m/z、5553.870m/z、5865.525m/z、6219.320m/z、7593.220m/z、7824.713m/z、8147.532m/z、以及25756.060m/z处有特征峰(peak)。
上述基质辅助激光解吸电离飞行时间质谱分析法(MALDI-TOF MS)显示的「m/z」(质荷比)单位，即相当于分子量的道尔吞(Dalton，Da)单位。即上述基质辅助激光解吸电离飞行式时间质谱分析法于图谱中每1m/z相当于1Da的分子量。
上述结果显示，不论是燕窝萃取液以及燕窝萃取粉的样本中，均 含有寡肽(oligo-peptide)分子(其分子量小于1000Da)，特别有助于生物体(例如：人体)的经皮吸收或是经消化道吸收。因此本发明所属技术领域具有通常知识者，依据本说明书的公开内容，当可得知本发明所提供的燕窝萃取组成物及含有此种燕窝萃取组成物的食品，具有提升免疫力、增强记忆力、预防感冒的功能。
实验例五：燕窝萃取物中EGF与FGF检测
将上述实验例一中获得的燕窝萃取液以及燕窝萃取粉，是利用商用EGF与FGF试剂盒(厂商名：Peprotech，盒名分别为：Human EGF Mini ELISA Development Kit PEP-900-M05、Human FGF-basic ELISA Development Kit 900-K0)以三明治酶联免疫分析法(sandwich enzyme-linked immunosorbent assay，Sandwich ELISA)检测样本中的EGF及FGF。每个样本进行三次重复，而EGF标准品自1000皮克/毫升(pg/mL)连续两倍稀释至15.625皮克/毫升(pg/mL)，而FGF标准品则自4000皮克/毫升(pg/mL)连续两倍稀释至62.5皮克/毫升(pg/mL)。其中捕捉抗体(Capture Antibody)浓度为100微克/毫升(μg/mL)，而侦测抗体(Detection Antibody)浓度为100微克/毫升(μg/mL)。并以抗生物素蛋白-HRP缀合物(Avidin-HRP conjugate)与ABTS(2，2′-连氮双-(3-乙基苯并噻唑啉-6-磺酸))进行呈色反应，每五分钟测定一次在450nm/650nm读取反应的吸光值，并且控制最高浓度的标准品吸光值不可超过1.2，最低浓度的标准品吸光值不可超过0.2。其余实验步骤则依照所利用商用EGF与FGF试剂盒中所附的说明进行操作。
结果显示，每毫升的燕窝萃取液至少含有0.04μg的EGF，而每克的燕窝萃取粉至少含有0.0832μg的EGF，同时以其它来源购得的燕窝食品(即食燕)进行测试，其产品中每克含有的EGF小于31.26pg。而每毫升的燕窝萃取液中至少含有0.16μg的FGF，而每克的燕窝萃取粉含有38.835～261.88ng的FGF，同时以其它来源购得的燕窝食品(即食燕)进行测试，其产品中每克含有228.67～300.172pg的FGF。
由上述实验结果可知，经上述实验例一中获得的燕窝萃取液以及燕窝萃取粉确实含有相当含量的EGF及FGF蛋白质，故本发明所提供的燕窝萃取组成物及含有此种燕窝萃取组成物的食品可具有促进表皮生长、皮肤保养、护发或伤口修补的功能。
实验例六：燕窝萃取组成物所制得的各产品添加成份
A.护手霜
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、甘油、芦荟精华液(Kidachi Aloe Ekisu)与肉豆蔻酸异丙酯(isopropyl myristate)、甘油硬脂酸酯(Glyceryl Stearate)、鲸蜡醇(cetyl alcohol)、山梨糖醇(Sorbitol)、鲸蜡醇聚醚-15(Ceteth-15)、硬脂山梨坦(Sorbitan Stearate)及其它适当的乳化剂，以制得含有燕窝萃取物的护手霜。
B.燕萃胶囊
以实验例一所获得的燕窝萃取组成物以2％-8.5％的重量百分比添加65.7％-93.0％的麦芽糊精，并取适当量(500-1000毫克)包装在明胶胶囊中，以制得含有燕窝萃取物的燕萃胶囊。
C.洗发精
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、十二烷基硫酸盐、十二烷基聚氧乙烯硫酸盐、椰油基两性醋酸钠、椰油基单乙醇酰胺、聚二甲基硅氧烷(Dimethicone)、胶原蛋白、维生素、植物提取物(例如：芦荟精华液)、珠光剂、保湿剂、香精、色素、防腐剂、螯合剂(例如：EDTA)，以制得含有燕窝萃取物的洗发精。
D.护发乳
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、表面活性剂(例如硫酸月桂酸钠(Sodium Lauryl Sulfate)、月桂醇聚醚硫酸酯钠盐(Sodium Laureth Sulfate)、椰油酰胺DEA(Cocamide DEA)、和/或乙二醇硬脂酸酯(Glycol stearate))、柔润剂、乳 化剂(例如橄榄油、聚山梨醇酯(Polysorbate)、氯化钠(Sodium chloride))、保湿剂(例如醋酸盐维他命E(Tocopheryl Acetate E))、及其它适当的防腐剂(例如羟甲基甘氨酸钠)、及香料，以制得含有燕窝萃取物的护发乳。
E.面膜
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的熊果素、玻尿酸、弹力蛋白(Elastin)、海藻萃取物、羟苯丙酯、水，并涂附在适当基材(例如：不织布)上，以制得含有燕窝萃取物的面膜。
F.卸妆液
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、环戊硅氧烷(Cyclopentasiloxane)、柔润剂(例如异十六烷(Isohexadecane))、香料、表面活性剂(例如己二醇(Hexylene Glycol)、氯化钠(Sodium chloride))、乳化剂(例如环己硅氧烷(Cyclohexasiloxane))、防腐剂(例如苯甲醇、苯氧乙醇(Phenoxyethanol))、香料、及抗氧化剂(例如柠檬酸钠)，以制得含有燕窝萃取物的卸妆液。
G.眼霜
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、保湿剂(例如甘油、异构寡糖(Saccharide isomerate))，硅基顺滑剂(例如环二甲基硅酮)、植物油、抗氧化剂(例如维生素E)，以制得含有燕窝萃取物的眼霜。
H.化妆水
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、保湿剂(例如双丙甘醇(Dipropylene glycol、丁二醇(Butylene Glycol)、甘油)、酒精、乳化剂(例如甲基葡糖聚醚-10(Methyl gluceth-10)、聚甘油-2二异硬脂酸(Polyglyceryl-2diisostearate))、表面活性剂(例如PEG-60氢化篦麻油(PEG-60Hydrogenated Castor Oil)、 异硬脂酸(Isostearic Acid))、柔润剂(例如三异辛酸甘油酯(Triethylhexanoin))、适当防腐剂，以制得含有燕窝萃取物的化妆水。
I.沐浴精
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、保湿剂(例如甘油、丙烯乙二醇)、表面活性剂(例如聚山梨醇酯20(Polysorbate 20)、PEG-150二硬脂酸酯(PEG 150Distearate)、氢化蓖麻油(Hydrogenated Castor Oil))、乳化剂(例如甘油油酸酯(Glyceryl oleate))、防腐剂(例如甲基异噻唑啉酮(Methylisothiazolinone))、适当香料，以制得含有燕窝萃取物的化妆水。
J.美容液
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、酒精、保湿剂(例如甘油、丁二醇(Butylene Glycol))、乳化剂(例如氢化卵磷脂(Hydrogenated lecithin))、柔润剂(例如乙酰谷氨酰胺(Acetyl Glutamine))、适当香料、防腐剂(例如苯甲酸甲脂(Methylparaben))，以制得含有燕窝萃取物的美容液。
K.身体用乳霜，添加全成分
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、保湿剂(例如甘油、丙烯乙二醇、1，2-戊二醇(Pentylene Glycol))、适当香料、柔润剂(例如环戊硅氧烷(Cyclopentasiloxane)、甘油硬脂酸(Glyceryl Stearate))、表面活性剂(例如PEG-60氢化篦麻油(PEG-60Hydrogenated Castor Oil)、抗氧化剂(例如柠檬酸钠)、乳化剂(例如山嵛酸(Behenic acid))、防腐剂、适当香料，以制得含有燕窝萃取物的身体用乳霜。
L.牙膏
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、硅酸盐、白云母、氟化物、表面活性剂(例如十二烷基硫酸钠(SLS))、适当香料、甘油、山梨醣醇，以制得含有燕窝萃取物的牙膏。
M.枇杷膏
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、蜂蜜、冰糖、植物(例如川贝母、枇杷叶、北沙参、桔梗、甘草、薄荷脑、杏仁)萃取物，以制得含有燕窝萃取物的枇杷膏。
N.肥皂
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、油(例如棕榈油)、硬脂酸钠、适当香料和染料，以制得含有燕窝萃取物的肥皂。
O.软糖
以实验例一所获得的燕窝萃取组成物以0.02％-3％的重量百分比添加适量的水、明胶、葡萄糖、蔗糖、柠檬酸、及适当香料与甜味剂，以制得含有燕窝萃取物的软糖。
以上所述仅为举例性，而非为限制性者。任何未脱离本发明的精神与范畴，而对其进行的等效修改或变更，均应包含在所附权利要求中。

Claims (10)

  1. 一种燕窝萃取物的萃取方法，包含有：
    提供燕窝粉碎物水溶液置于反应槽中进行乳化，加热至第一温度并将所述反应槽抽取真空并持续第一时间，以形成燕窝乳化液，其中所述反应槽具有乳化器及环绕所述乳化器的搅拌器，所述第一温度为摄氏30至60度，且所述第一时间为30分钟至12小时；
    在维持所述乳化器及所述搅拌器的运转的情况下，将所述燕窝乳化液调整至第二温度后，将蛋白酶加入所述反应槽，并维持在所述第二温度下持续第二时间，其中所述第二温度为摄氏30度至摄氏40度，且所述第二时间为1至12小时；
    终止所述蛋白酶的反应；
    过滤所述燕窝乳化液，获得燕窝滤液；
    浓缩所述燕窝滤液，并将所述浓缩后的燕窝滤液进行灭菌处理，以获得燕窝萃取液。
  2. 如权利要求1所述的萃取方法，进一步包括将所述燕窝萃取液进行冻干的步骤，以制成燕窝萃取物。
  3. 如权利要求1所述的萃取方法，其中在所述乳化步骤中，所述反应槽的真空压力维持在10～200托。
  4. 如权利要求3所述的萃取方法，其中在所述乳化步骤中，所述乳化器的转速为每分钟1200～3600转，且所述搅拌器的转速为每分钟10～60转。
  5. 如权利要求1所述的萃取方法，其中终止所述蛋白酶的反应的步骤是将所述燕窝乳化液加热至摄氏50～70度并持续30～120分钟。
  6. 如权利要求1所述的萃取方法，其中过滤所述燕窝乳化液的步骤在第二压力下进行，且所述第二压力为400～720托。
  7. 如权利要求1所述的萃取方法，其中浓缩所述燕窝滤液的步骤的压力为600～720托，且浓缩倍率为2～4倍。
  8. 如权利要求1所述的萃取方法，其中将所述燕窝滤液进行灭菌处理的步骤是依巴式低温杀菌法进行。
  9. 如权利要求1所述的萃取方法，其中所述蛋白酶为木瓜酶、胰蛋白脢、或菠萝酶中的至少一种。
  10. 如权利要求1或2所述的萃取方法，其中所述燕窝萃取液或燕窝萃取物包含有；
    1％至13％重量百分比的唾液酸；以及
    4％至15％重量百分比的蛋白质，其中所述蛋白质的分子量分布为100Da至300kDa，且其中小于6.5kDa的蛋白质占总蛋白质含量为0.6％～6.4％重量百分比。
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CN110551788A (zh) * 2019-09-02 2019-12-10 佛山科学技术学院 一种富含抗氧化肽的燕窝小分子肽的制备方法
CN111034885A (zh) * 2019-12-31 2020-04-21 本草元素生物科技(厦门)有限公司 一种燕窝弹性蛋白肽植物饮品及其制备方法
CN111264849A (zh) * 2020-02-19 2020-06-12 福建省农业科学院农业工程技术研究所 绣球菌燕窝及其制作工艺
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