WO2022032803A1 - 燕窝提取方法、提取物及其产品 - Google Patents

燕窝提取方法、提取物及其产品 Download PDF

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WO2022032803A1
WO2022032803A1 PCT/CN2020/116289 CN2020116289W WO2022032803A1 WO 2022032803 A1 WO2022032803 A1 WO 2022032803A1 CN 2020116289 W CN2020116289 W CN 2020116289W WO 2022032803 A1 WO2022032803 A1 WO 2022032803A1
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nest
bird
extract
powder
hours
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PCT/CN2020/116289
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English (en)
French (fr)
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余的军
石培松
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广西自贸区燕握生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a method for extracting from bird's nest and various extracts obtained by said method.
  • More than half of the ingredients in bird's nest are protein, and a considerable proportion is water-soluble protein, including protein that promotes the growth of human tissue, and contains 18 kinds of amino acids that are essential and conditionally necessary for the human body (including 8 kinds of essential amino acids).
  • Bird's nest acid in bird's nest also known as "N-acetylneuraminic acid (NeuAc)" of sialic acid, is an acetyl derivative of aminoglycosylneuraminic acid. Sialic acid has been found to have various beneficial effects on the nervous system, immune system, and the like.
  • bird's nest also contains epidermal growth factor (EGF), which has strong mitogenic, proliferation, differentiation, and regeneration effects on a variety of tissue cells, and promotes proliferation and differentiation of hematopoietic stem cells at different developmental stages.
  • EGF epidermal growth factor
  • Stimulating factor (CSF) a glycoprotein that can be aggregated to improve bone strength and dermal thickness and various glycoproteins that can inhibit or induce killing of cancer cells and restore stem cell viability after aggregation.
  • CSF a glycoprotein that can be aggregated to improve bone strength and dermal thickness and various glycoproteins that can inhibit or induce killing of cancer cells and restore stem cell viability after aggregation.
  • the content of these proteins, sialic acid and other components has become an important indicator to reflect the nutritional value of bird's nest products.
  • the common bird's nest extraction products or ready-to-eat bird's nest products on the market are also processed by steaming and stewing at medium and low temperature, or the enzymatic hydrolysis method, or even the direct hydrolysis method.
  • enzymatic hydrolysis and hydrolysis methods may not only destroy amino acids with poor stability and sialic acids in the form of glycoproteins, but also introduce additional chemical substances, and these methods will lose some nutrients, resulting in unsatisfactory extraction efficiency. .
  • the main purpose of the present invention is to provide a bird's nest extraction method and a bird's nest extract obtained by the method, wherein the method can retain the nutrients in the bird's nest to a greater extent, and decompose the large molecular weight protein into a suitable molecular weight. And more uniform low molecular weight protein, so as to provide more valuable bird's nest extract.
  • one aspect of the present invention provides a method for extracting bird's nest, the method comprising the following steps:
  • the bird's nest slurry is added to water at a weight ratio of 1 to 5 times, and the first extract is obtained by continuously stirring at 200 to 1000 rpm for 3 to 8 hours at 70 to 120 ° C;
  • step D firstly, the mixture is stirred at a stirring speed of 100-300 rpm for 15-60 minutes, and then allowed to stand for 3-4 hours.
  • step E firstly, the mixture is stirred at a stirring speed of 100-300 rpm for 15-60 minutes, and then allowed to stand for 2-3 hours.
  • step C the stirring is continued at a stirring speed of 300-800 rpm for 4-6 hours.
  • the above-mentioned step B includes adding the cleaned bird's nest powder into water 15-40 times its weight, mixing and soaking for 4-12 hours, preferably 6-10 hours.
  • the clean bird's nest powder is mixed with water and then subjected to ultrasonic crushing for 15-30 minutes before the soaking. More preferably, the soaking is performed at a temperature of 40°C to 50°C.
  • step A obtaining clean bird's nest powder.
  • the clean bird's nest powder can be obtained by any suitable method. Generally speaking, the following steps can be included:
  • the method may further comprise step G: further processing the liquid bird's nest extract to obtain a concentrated or solid bird's nest extract.
  • the solid bird's nest extract can be obtained by drying.
  • the drying method is not particularly limited, and any suitable method can be used. Examples include, but are not limited to, spray drying, vacuum drying, freeze drying, and the like.
  • the concentrated bird's nest extract can be obtained by methods such as distillation under reduced pressure, but not limited thereto.
  • the solid bird's nest extract is a powder
  • the method of the present invention further comprises step H: combining the first lower layer liquid and the second lower layer liquid as fertilizer.
  • the method of the present invention further comprises step 1: putting the hair and impurities removed in the step A into the swiftlet house, to attract the swiftlet to build the nest.
  • a bird's nest extract obtained according to the above-mentioned method for extracting bird's nest.
  • a bird's nest extract wherein the protein with a molecular weight of less than 1000 Da in the bird's nest extract accounts for more than 45 wt% of the total protein in the extract, and the content of 8 essential amino acids accounts for the total amino acid content more than 45wt%.
  • the protein with a molecular weight below 3000 Da in the bird's nest extract accounts for more than 70 wt % of the total protein in the extract, and can even reach 75 wt %.
  • the content of sialic acid in the bird's nest extract accounts for more than 10wt% of the total solid weight of the extract, particularly preferably more than 12wt%t, or even more than 13wt%.
  • a bird's nest product comprising the above-mentioned bird's nest extract.
  • the bird's nest product can be one of food, health products and cosmetics.
  • the food may be a drink.
  • the bird's nest extraction method of the present invention adopts stirring for a certain time at a certain temperature and stirring speed, combined with further standing, to promote the breaking of part of the amide bonds in the macromolecular protein, and form a small and concentrated extract with a small molecular weight that is beneficial to the human body. .
  • the method is mild and does not add additional chemical substances (such as acid, enzymes, etc.), and better retains the nutrients in the bird's nest.
  • the extract obtained according to this method is analyzed by HPLC (High Performance Liquid Chromatography), and the content of small molecular peptides with molecular weight within 1000 Da accounts for more than 45wt%; the amount of small molecular peptides and polypeptides with molecular weight within 3000 Da can reach about 75wt% or more.
  • HPLC High Performance Liquid Chromatography
  • the extract contains 18 kinds of amino acids, and according to a preferred embodiment, the weight ratio of 8 kinds of amino acids necessary for the human body accounts for 48% by weight of all 18 kinds of amino acids; the sialic acid content is based on the total amount of the extract (dry weight), Up to 13.8wt%, so as to achieve targeted and effective nutrient retention and application maximization of bird's nest; meanwhile, the method of the present invention can obtain excellent extraction rate, and in a preferred embodiment, the bird's nest extract (dry weight) accounts for the input bird's nest The weight ratio can reach more than 90wt%.
  • FIG. 1 is a flow chart of a method for extracting bird's nest according to an embodiment of the present invention
  • Example 2 is a histogram of molecular weight distribution of protein peptides in the bird's nest extract obtained in Example 3;
  • Fig. 3 is an SDS-PAGE (sodium dodecyl sulfonate-polyacrylamide gel electrophoresis) photograph of the molecular weight distribution of the bird's nest extract obtained in Example 3, Comparative Example 1 and Comparative Example 2, respectively.
  • first ⁇ second involved in this article is only to distinguish similar objects, and does not represent a specific ordering of objects. It is understood that “first ⁇ second” can be mutually change to a specific order or sequence. It should be understood that the “first ⁇ second” distinctions are interchangeable under appropriate circumstances, so that the methods of the invention described herein can be performed in sequences other than those illustrated or described herein.
  • proteins they can be divided into macromolecular proteins with a molecular weight greater than 10,000 Da and proteins with a molecular weight of less than 10,000 Da according to their molecular weight.
  • Proteins with smaller molecular weights can be subdivided into proteins with molecular weights between 3000Da and 10000Da, which can also be called long peptides; proteins with molecular weights between 1000Da and 3000Da are often called polypeptides; and proteins with molecular weights less than 1000Da are often called peptides.
  • proteins with molecular weights less than 1000Da are often called peptides.
  • the proportion of polypeptides with a molecular weight of less than 3000 Da in the extract obtained by the method of the present invention can reach about 75 wt%, and the proportion of small molecular peptides (or small peptides) less than 1000 Da can reach 45 wt%, therefore, the term "protein peptides" herein It refers to the general term for polypeptides and small molecular peptides in bird's nest extract.
  • FIG. 1 the bird's nest extraction method of the present invention and its advantages will be described according to an embodiment of the present invention.
  • the obtained bird's nest material such as bird's nest, must first remove the feathers and sundries in the bird's nest raw material.
  • it is also possible to directly use the processed bird's nest materials such as clean bird's nest or bird's nest.
  • step A is firstly performed to pulverize the bird's nest material to remove impurities.
  • the bird's nest or the swallow's strips from which feathers and sundries are roughly removed can be crushed into bird's nest powder by hand or a grinding mill at room temperature. Since the unremoved feathers are lighter than the bird's nest powder after being pulverized, and the inorganic impurities such as dust and small stones are heavier, the hair and foreign matter in the bird's nest powder are removed by suction, sieving and centrifugal separation successively to obtain a small and clean clean. bird's nest powder. Specifically, for example, a 40-80 mesh vibrating sieve can be used to obtain bird's nest powder with uniform particle size, and the particles with larger particle size can be returned to step A for re-grinding.
  • the bird's nest or strip can also be sprayed with clean water on the place where there are hairs and foreign objects, and after softening, remove the hair and impurities with tweezers, and then grind to obtain clean bird's nest powder.
  • the softened swallow cup should be dried before grinding.
  • a heating pulverizer temperature 30°C-80°C to pulverize to obtain dry bird's nest powder.
  • the material In the process of grinding bird's nest into powder, whether by hand or by machine, the material should be prevented from turning into a gel.
  • the methods of removing feathers and sundries in the raw materials of bird's nest include any one of pure manual, manual plus semi-automatic machinery and fully automatic cleaning robot.
  • the present invention is not particularly limited to this.
  • carry out step B carry out pretreatment to bird's nest powder, namely, soak the bird's nest powder of cleaning with water to make the particle of bird's nest powder expand.
  • the clean bird's nest powder and water are mixed in a certain proportion and soaked until the bird's nest powder expands to obtain bird's nest slurry.
  • the bird's nest pulp can be obtained after 15-30 minutes of ultrasonic crushing (for example, 40KHz frequency and 250W power), and then soaking for 4-12 hours, preferably 6-10 hours.
  • the mixing ratio (weight ratio) of bird's nest powder to water is 1:15 to 1:40.
  • the soaking step can also be carried out at a certain temperature, for example, at 40°C to 50°C, so that the best expansion of the bird's nest powder can be obtained.
  • the water used in any step of the present invention can be one of purified water, deionized water, or distilled water (eg, double distilled water).
  • step C add water to the bird's nest slurry obtained in the previous step at a weight ratio of 1:1 to 1:5, and at a temperature of 70 to 120° C., at a temperature of 200 to 1000 rpm, preferably 300 to 800 rpm.
  • the stirring is continued for 3 to 8 hours at the rotational speed, preferably for 4 to 6 hours.
  • This step can promote the loosening of the nutrients in the bird's nest, and break at least part of the amide bonds of the peptide chains in the proteins with large molecular weights, thereby reducing the molecular weight of the proteins.
  • the first extraction solution GY1 is obtained.
  • step D put the first extraction solution GY1 into a mixing and stirring vessel, heat at 30-40° C. and stir at 100-300 rpm for 15-60 minutes, for example, 20-40 minutes, and then let stand. Stirring for a certain period of time helps to release impurities and further loosens the peptide bonds in the protein into smaller molecular weight peptides.
  • the standing time can be determined according to the actual situation. For example, it can be allowed to stand for 3 to 4 hours, so that most impurities are precipitated, and there is sufficient time for the protein to complete the peptide bond cleavage reaction to generate polypeptides with smaller molecular weights, and these cleavage polypeptides are dissolved in water. Then the lower sediment containing secondary metabolites and gray matter was removed, and the supernatant was taken out as the second extract GY2.
  • step E the remainder in the mixing and stirring vessel, that is, the middle layer, is also stirred at 30-40° C. at a rotational speed of 100-300 rpm for 15-60 minutes, for example, 20-40 minutes, and then allowed to stand.
  • the standing time can be 2-3 hours, so that the impurities can be fully precipitated.
  • the lower sediment was likewise drained, and the supernatant (generally the entire remaining liquid in this step) was extracted as the third extract GY3.
  • step F the obtained second extraction solution GY2 and third extraction solution GY3 are combined to form a fourth extraction solution GY4, that is, a liquid bird's nest extract.
  • the liquid bird's nest extract obtained in step F can be further subjected to step G to obtain a concentrated or solid extract.
  • the fourth extract GY4 can be put into a spray dryer with an automatic feeding system, and spray-dried to obtain a powdery solid.
  • Concentrated/solid bird's nest extract can also be obtained by methods such as lyophilization, vacuum distillation, vacuum drying, etc.
  • the form of the bird's nest extract is not particularly limited in the present invention, and further processing methods can be selected according to needs.
  • the effective utilization rate (ie extraction rate) of the bird's nest components in the bird's nest extract obtained by the method of the present invention is over 90%.
  • Most of the proteins in the extract are present as polypeptides with lower molecular weights.
  • the molecular weights of proteins and polypeptides have a narrow normal distribution, wherein the content of polypeptides with lower molecular weights ( ⁇ 1000Da) accounts for more than 45wt%, and the content of polypeptides with molecular weights less than 3000Da can reach about 75wt%.
  • the narrow normal distribution of this small molecular weight peptide allows for better utilization.
  • the extract obtained by the method of the present invention contains 18 kinds of amino acids and the content ratio of 8 kinds of amino acids necessary for human body is above 45wt%, even up to 48wt%; the content of sialic acid in the extract (dry weight) is above 10wt%, which can be Up to 13.8 wt%. Nutrient and value components have significant advantages over other extraction methods.
  • Bird's nest contains a high proportion of protein, but due to its tertiary supercoiled structure, the protein is not easily absorbed due to its large molecular weight (which can be 10000Da to 300000Da, or even higher). Even for polypeptides with a molecular weight of about 3000 Da, the absorption efficiency of humans is not high (about 40%). Small molecular peptides with molecular weights between polypeptides and amino acids (molecular weights below 1500Da) can be directly absorbed through the intestinal epidermis, enter the blood and be transported to the required parts of the human body, and still maintain their nutrients and ingredients. Excellent biological activity and physiological function. Most of the extracts of the present invention are short peptides, and the proportion is much higher than that of bird's nest products obtained by conventional methods, which is more conducive to the absorption and utilization of the human body.
  • the method of the present invention may further comprise steps H and I for recycling the accumulated waste in the method.
  • Step H involves using the lower sediments (secondary metabolites and ash matter) obtained in Steps D and E above, to be used as fertilizers as biodegradables.
  • Step 1 includes inedible swiftlet feathers and impurities, which are reused in swiftlet houses to attract more swallows to build nests with their taste. Achieving "zero waste” and “zero emissions” in waste management to reduce waste disposal that utilizes energy and produces methane and carbon dioxide through product gases, effectively protecting the earth's environment.
  • the bird's nest extract of the present invention can be further formed into products, such as food, health products, beauty cosmetics and the like.
  • the food or health care product of the present invention contains any form of the bird's nest extract mentioned above.
  • the product may also contain other components such as additives, adjuvants, excipients, and the like.
  • the additives can be such as pigments, pigments, flavoring agents, fragrances, sweeteners and the like.
  • the product may also contain further active ingredients, such as other nutrients, which may be complementary or synergistic with the nutrients in the bird's nest.
  • SDS-PAGE In order to more intuitively compare the distribution of protein molecular weights in the extract obtained by the method of the present invention and the method of the prior art, SDS-PAGE (sodium dodecyl sulfonate-polyacrylamide gel was used) Electrophoresis) method to determine the distribution of protein molecular weight in the extract sample.
  • FD-201 electrophoresis apparatus purchased from Shanghai Medical Analytical Instrument Factory
  • small vertical electrophoresis accessory mold and TEMED purchased from BIO-RAD
  • peptide molecular mass standard Mr range 348–16949 purchasedd from In Pharmacia LKB company
  • Sephadex G-75 gel the chromatography column size is 1.0 ⁇ 15cm.
  • sialic acid content High performance liquid chromatograph (Waters2695 and Waters2489 UV detector) was used to test the sialic acid content of the extract samples of the embodiment extracts and the comparative examples and commercially available canned rock sugar bird's nests.
  • the sialic acid reference substance (purity ⁇ 99%) was purchased from Sigma Company, the chromatographic column was Waters Atlantis T 3 (4.6 mm ⁇ 150 mm, 5 ⁇ m), and the mobile phase was acetonitrile-water (40:60).
  • bird's nest raw materials (Swiftlet Garden, Desaru Bio Valley, Johor, Malaysia) 800g. Remove the feathers and sundries from the raw materials of bird's nest, and grind the bird's nest or strips into bird's nest powder at room temperature with a grinding mill. 680g of fine and clean bird's nest powder was obtained by removing the hair and foreign matter on the bird's nest successively by suction, filtration and centrifugation. In order to avoid the material becoming gel due to shear heat, the temperature of the grinding and crusher cavity was set at 30°C.
  • GY0-1 was taken out, pure water was added at a weight ratio of 1:4, stirring was continued at a rotating speed of 500 rpm, and heated at 120° C. for 4 hours to promote the loosening of nutrients in the bird’s nest to obtain a total of 71400 g of extract GY1-1.
  • the extract GY1-1 was put into a container with a stirrer, heated at 30° C. and stirred at 300 rpm for 30 minutes. After standing for 3 hours, the lower sediment was drained to obtain a total of 13138 g of the supernatant GY2-1.
  • the extract was passed through a spray dryer and spray-dried to obtain 626 g of powdered bird's nest extract.
  • the dry extract obtained in this example has an extraction rate of the bird's nest component (the final extract accounts for the weight of the input bird's nest) of 92% relative to the weight of the bird's nest material.
  • Get bird's nest raw material (Malaysia Desaru biological valley Swiftlet Garden) 800g, obtain and process according to the method of embodiment 1 to obtain bird's nest powder 710g.
  • a total of 22010 g of bird's nest slurry GY0-2 was obtained, waiting for effective extraction.
  • GY0-2 Take out GY0-2, add it into distilled water at a weight ratio of 1:3, continue stirring at 300 rpm, and heat at 80°C for 5 hours to loosen the nutrients in the bird's nest, and extract a total of 88040g of extract GY1-2.
  • the extract GY1-2 was placed in a stirred vessel, heated at 40°C and stirred at 200 rpm for 30 minutes, left standing for 4 hours to remove the lower sediment, and a total of 17520 g of supernatant GY2-2 was obtained.
  • small molecule peptides with molecular weight ⁇ 1000Da accounted for about 48.9wt%
  • protein peptides with molecular weight ⁇ 3000Da accounted for 77.7wt%.
  • the extract GY4-2 was put into a spray dryer with an automatic feeding system, and spray-dried to obtain 642 g of powdered bird's nest extract.
  • the dry extract obtained in this example has an extraction rate of the obtained bird's nest component (the final extract accounts for the weight ratio of the input bird's nest) to 90.4% relative to the weight of the bird's nest material.
  • Get bird's nest raw material (Malaysia Desaru biological valley Swiftlet Garden) 800g, obtain bird's nest powder 701g according to the method of embodiment 1. Put the crushed bird's nest powder into the clean water for 10 hours at a weight ratio of 1:40, and then pass through the auxiliary ultrasonic vibrator (ultrasonic frequency 40KHz, ultrasonic power 250W), and after ultrasonic crushing for 20 minutes, it is better to heat to 40 °C to allow the bird's nest powder particles to expand to the best. A total of 28741 g of bird's nest slurry GY0-3 with the maximum expansion effect of bird's nest powder particles was obtained, waiting for effective extraction.
  • auxiliary ultrasonic vibrator ultrasonic frequency 40KHz, ultrasonic power 250W
  • the extract GY1-3 was placed in a stirring vessel, heated at 40°C and stirred at 100 rpm for 30 minutes, left standing for 3 hours to remove the lower sediment, and a total of 8567 g of supernatant GY2-3 was obtained.
  • the residue in the container was stirred at 40° C. for 30 minutes at a rotation speed of 100 rpm, and left to stand for 3 hours to remove the lower sediment, and a total of 30,417 g of the supernatant GY3-3 was obtained.
  • the weight ratio and the number-average molecular weight are as shown in the following table 3 and Fig. 2 under each molecular weight range that comprises protein peptide in the above-mentioned method for measuring the extraction solution GY4-3:
  • the extract GY4-3 was put into a spray dryer with an automatic feeding system, and spray-dried to obtain a total of 638 g of powdered bird's nest extract.
  • the dry extract obtained in this example has an extraction rate of the bird's nest component (the final extract accounts for the weight of the input bird's nest) of 91% relative to the weight of the bird's nest material.
  • This comparative example uses the enzymatic hydrolysis method to extract bird's nest.
  • the content of small molecular peptides with molecular weight less than 1000Da in the obtained bird's nest extract is 31wt%, and the content of small molecular peptides and polypeptides below 3000Da is 47wt%.
  • the sialic acid content detected by spectrophotometry on the sialic acid sample was 9.51%.
  • the ratio of essential amino acids (EAA) in the extract to the total amino acids was 39.27wt% detected by ninhydrin colorimetry.
  • the bird's nest was extracted by the traditional method of boiling, steaming and stewing.
  • the content ratio (area under the curve) of small molecular peptides with molecular weight less than 1000Da in the bird's nest obtained was 14%, and the content of small molecular peptides and polypeptides below 3000Da (area under the curve) was 14%. ) is 23%.
  • the sialic acid content detected by spectrophotometry on the sialic acid sample was 9%.
  • the content of essential amino acids (EAA) in the extract was 39.29% of the total amino acids detected by ninhydrin colorimetry.
  • the molecular weight of protein peptides in the bird's nest extract obtained by the extraction method of the present invention is not only small ( ⁇ 3000Da), but also uniformly distributed and has obvious normal distribution characteristics.
  • FIG. 3 it corresponds to Table 4 showing the protein peptides in the extracts obtained by the method of the present invention (Example 3), the enzymatic hydrolysis method (Comparative Example 1) and the boiling and stewing method (Comparative Example 2).
  • SDS-PAGE photograph of molecular weight distribution Photo 1 in FIG. 3 is a gel electrophoresis photo of the protein peptides in the extract obtained by the extraction method of the present invention. The molecular weight distribution of protein peptides is narrow and a high proportion is distributed in the region of less than 3000 Da.
  • Photo 2 is the molecular weight distribution of proteins in the extract obtained by the enzymatic hydrolysis method of Comparative Example 1.
  • Photo 3 shows the molecular weight distribution of proteins in the extract obtained by the boiling and stewing method of Comparative Example 2. Since the number of small molecular peptides and polypeptides obtained is small, most of them are distributed in the area with molecular weight greater than 10000 Da, which cannot be displayed in the picture.
  • the comparison results of the following table 5 show: compared with the extract of the bird's nest enzymolysis extraction method and the traditional hydrolysis steamed and stewed bird's nest extraction method, in the bird's nest extract obtained by the extraction method of the present invention, it contains 18 kinds of amino acids, and its 8 kinds of human body essential
  • the weight ratio of amino acids (EAA) is about 48wt%, which is significantly higher than that of essential amino acids in the extracts obtained in Comparative Example 1 and Comparative Example 2 (39.27wt% and 39.29wt%, respectively). This shows that the method of the present invention better retains the protein in the bird's nest.
  • the invention removes impurities by the methods of sucking wool, sieving and centrifuging after crushing the swallow cup, avoiding water washing and retaining more soluble protein.
  • Table 5 Examples The content of amino acids in the extracts of Examples 1 to 3, Comparative Example 1 and Comparative Example 2
  • Example 1 Example 2
  • Example 3 Comparative Example 1 Comparative Example 2
  • Sialic acid (wt%) 13.82 13.81 13.85 9.51 9.08 1.04
  • Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Extraction rate (wt%) 92 90.4 91 68 14.2

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Abstract

燕窝提取方法、提取物及其产品。该方法包括：B浸泡洁净的燕窝粉以获得燕窝浆；C将所述燕窝浆以其1～5倍的重量比加入水，在70℃～120℃下以200～1000rpm持续搅拌3～8小时获得第一提取液；D将所述第一提取液在30℃～40℃降低的温度下搅拌均匀并静置，待杂质沉淀后排除包含沉淀物的第一下层液体，并取出上清液作为第二提取液；E将步骤D中的剩余液体继续在30℃～40℃下搅拌均匀并静置，待进一步的杂质沉淀后排除包含沉淀物的第二下层液体，以获得第三提取液；F合并所述第二提取液和所述第三提取液得到液态的燕窝提取物。该方法温和、无额外化学品，更好保留了燕窝中的营养成分；所得提取物中低分子量肽占45wt％以上，更利于吸收，同时提取率高，可达90wt％以上。

Description

燕窝提取方法、提取物及其产品 技术领域
本发明涉及从燕窝提取物的方法和由所述方法获得的各种提取物。
背景技术
我国久有食用燕窝以作滋补的传统。关于燕窝的记载，始见于1537年明代嘉靖年间陈懋仁所撰写的《泉南杂志》。燕窝的功效最早记载于清代名医张璐的《本经逢原》。
燕窝中一半以上的成分为蛋白质，而且相当比例是水溶性蛋白质，其中包括促进人体组织生长的蛋白质、含有人体必需的和条件必需的18种氨基酸(其中含8种人体必需氨基酸)。燕窝中的燕窝酸，也被称为唾液酸(Sialic acid)的“N-乙酰基神经氨酸(NeuAc)”，是氨基糖神经氨酸的乙酰基衍生物。已发现唾液酸具有针对神经***、免疫***等的多种有益功效。此外，燕窝中还含有对多种组织细胞有强烈的促***和增殖分化、再生作用的表皮生长因子(EGF)，对不同发育阶段的造血干细胞起促增殖、分化的作用以及抗流感病毒的集落刺激因子(CSF)，可以聚合提成改善骨强度和真皮厚度的糖蛋白以及可以聚合提成后抑制或诱导杀死癌细胞的和恢复干细胞活力的各种糖蛋白。这些蛋白质、唾液酸等成分的含量成为体现燕窝产品的营养价值的一个重要指标。
人们传统上食用燕窝的方式是：清洗、浸泡、冲洗、加水蒸炖。相当一部分燕窝的水溶性蛋白质已经在2到3次的清洗、浸泡、冲洗和新水蒸炖的过程中流失了。由于燕窝中的蛋白质多为大分子量的蛋白质，传统食用方式很难把这些蛋白质变成人体易直接吸收的小分子蛋白质。
目前，市场上常见的燕窝提取产品或即食燕窝产品，要么也同样采用中低温蒸炖的加工方式，要么采用的酶解法，甚至直接水解法。其中，酶解法和水解法不但 有可能破坏稳定性较差的氨基酸和以糖蛋白形式存在的唾液酸，还会引入额外的化学物质，并且这些方法均会损失部分营养物质，导致提取效率不够理想。
因此，仍需要改进燕窝的加工方式以更好地发挥燕窝的价值。
发明内容
有鉴于此，本发明的主要目的在于提供一种燕窝提取方法以及由该方法获得的燕窝提取物，其中该方法能够更大限度地保留燕窝中的营养物质，并使大分子量蛋白质分解为分子量适当且较为均匀的低分子量蛋白质，从而提供更有价值的燕窝提取物。
为此，本发明一方面提供一种燕窝提取的方法，所述方法包括以下步骤：
B、浸泡洁净的燕窝粉以获得燕窝浆；
C、将所述燕窝浆以其1～5倍的重量比加入水，在70℃～120℃下以200～1000rpm持续搅拌3～8小时获得第一提取液；
D、将所述第一提取液在30℃～40℃降低的温度下搅拌均匀并静置，待杂质沉淀后排除包含沉淀物的第一下层液体，并取出上清液作为第二提取液；
E、将剩余液体继续在30℃～40℃下搅拌均匀并静置，待进一步的杂质沉淀后排除包含沉淀物的第二下层液体，以获得第三提取液；
F、合并所述第二提取液和所述第三提取液得到液态的燕窝提取物。
根据一种实施方式，步骤D中先在100～300rpm的搅拌速度下搅拌15～60分钟，再静置3～4小时。
根据一种实施方式，步骤E中先在100～300rpm的搅拌速度下搅拌15～60分钟，再静置2～3小时。
根据较优的实施方式，步骤C中以300～800rpm的搅拌速度持续搅拌4～6小时。
根据一种实施方式，上述步骤B包括将所述洁净的燕窝粉加入其重量15～40倍的水中混合并浸泡4～12小时，优选6～10小时。较优地，所述洁净的燕窝粉与水混合后先进行15～30分钟的超声破碎再进行所述浸泡。更优地，所述浸泡在40℃～50℃的温度下进行。
本发明的方法中，还包括步骤A：获得洁净的燕窝粉。本发明中，洁净的燕窝粉可以通过任何适宜方法获得。通常来说，可以包括以下步骤：
a)手工或机器把燕盏或燕条粉碎成燕窝粉，然后经过吸毛、过筛和离心分离方式去除毛和异物得到洁净的燕窝粉；或者
b)把燕盏或燕条用水软化后手工清除毛和杂质，然后手工或机器进行粉碎得到洁净的燕窝粉。
根据本发明的一种实施方式，所述方法可进一步包括步骤G：将所述液态燕窝提取物进一步处理以获得浓缩的或固态的燕窝提取物。
根据本发明，固态燕窝提取物可通过干燥获得。干燥的方法没有特别限制，任何适宜的方法均可使用。可列举的例如有喷雾干燥、真空干燥、冷冻干燥等，但不限于此。浓缩的燕窝提取物可通过诸如减压蒸馏等方法获得，但不限于此。
较优地，所述固态燕窝提取物是粉状物
本发明的方法进一步包括步骤H：将所述第一下层液体和所述第二下层液体合并后作为肥料。
本发明的方法进一步包括步骤I：将步骤A中除去的毛和杂质放入燕屋，以吸引金丝燕前来建窝。
根据本发明的第二方面，提供一种根据上述燕窝提取方法获得的燕窝提取物。
根据本发明的第三方面，提供一种燕窝提取物，所述燕窝提取物中分子量在1000Da以下的蛋白质占提取物中蛋白质总量的45wt％以上，且8种 人体必需氨基酸含量占总氨基酸含量的45wt％以上。
根据优选的方案，所述燕窝提取物中分子量在3000Da以下的蛋白质占提取物中蛋白质总量的70wt％以上，甚至可达到75wt％。
更优选所述燕窝提取物中唾液酸的含量占提取物固体总重量的10wt％以上，特别优选达到12wt％t以上，甚至达到13wt％以上。
根据本发明的第四方面，提供一种燕窝产品，所述燕窝产品包括上述燕窝提取物。
根据本发明，所述燕窝产品可以是食品、保健品和化妆品中的一种。
所述食品可以是饮品。
本发明的燕窝提取方法采用在一定的温度、搅拌速度下搅拌一定的时间，并结合进一步的静置，促使大分子蛋白质中部分酰胺键断裂，形成利于人体吸的分子量较小且集中的提取物。所述方法温和且不额外添加化学物质(如酸、酶等)，更好地保留了燕窝中的营养成分。根据该方法获得的提取物经HPLC(高效液相凝胶色普法)分析，其中分子量在1000Da以内的小分子肽含量占45wt％以上；分子量在3000Da以内的小分子肽和多肽的量可达约75wt％以上。并且该提取物中含18种氨基酸，而且根据较优的实施例，人体必需8种氨基酸占全部18种氨基酸的重量比达48wt％；唾液酸含量以提取物(干重)的总量计，高达13.8wt％，从而有针对性而且有效地实现燕窝的营养保留和应用最大化；同时，本发明的方法可获得优异的提取率，较佳实施方式中燕窝提取物(干重)占投入燕窝重量比可达90wt％以上。
附图说明
图1为根据本发明一种实施方式进行燕窝提取的方法流程图；
图2为实施例3中获得的燕窝提取物中蛋白肽的分子量分布柱状图；
图3为实施例3、对比例1和对比例2中分别获得的燕窝提取物分子量 分布的SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)照片。
具体实施方式
下面将结合本发明的具体实施方式及附图，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明的一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者产品不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者是还包括为实施方法或者产品所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的方法或者产品中还存在另外的相关要素。
需要说明的是，本文所涉及的术语“第一\第二”仅仅是区别类似的对象，不代表针对对象的特定排序，可以理解地，“第一\第二”在允许的情况下可以互换特定的顺序或先后次序。应该理解“第一\第二”区分的对象在适当情况下可以互换，以使这里描述的本发明的方法能够以除了在这里图示或描述的那些以外的顺序实施。
通常，对于蛋白质，按其分子量可划分为分子量大于10000Da的大分子蛋白质，以及分子量小于10000Da的蛋白质。分子量较小的蛋白质又可细分为分子量在3000Da～10000Da的蛋白质，又可称为长肽；分子量在 1000Da～3000Da之间的蛋白质，又经常称为多肽；而分子量小于1000Da的蛋白质，常称为小分子肽或寡肽。由于本发明方法获得的提取物中分子量小于3000Da的多肽占比可达约75wt％，而小于1000Da的小分子肽(或小肽)占比可达45wt％，因此，本文所称“蛋白肽”特指燕窝提取物中的多肽和小分子肽的总称。
以下，参考图1，根据本发明一种实施方式来说明本发明的燕窝提取方法及其优点。
获得的燕窝材料如燕盏，首先要去除燕窝原材料中的羽毛和杂物。当然也可以直接使用处理好的干净的燕盏或燕条等燕窝材料。
如图1所示，首先进行步骤A，对燕窝材料进行粉碎除杂。具体地，可将大致除去羽毛和杂物的燕盏或燕条通过手工或研磨粉碎机在常温下先把燕盏或燕条粉碎成燕窝粉。由于未除去的羽毛粉碎后较燕窝粉碎物轻，而灰尘、小石子等无机杂质又较重，因此先后经过吸毛、过筛和离心分离方式去除燕窝粉中毛和异物后得到细小且干净的洁净的燕窝粉。具体地，可例如使用40～80目的振动筛获得粒径均匀的燕窝粉，粒径较大的颗粒可重新返回步骤A进行再次粉碎。
可替代地，也可以把燕盏或燕条针对存在毛和异物处喷洒洁净水，软化后用镊子清除毛和杂质后，再进行研磨得到洁净的燕窝粉。在该方法中，研磨前应先把软化的燕盏干燥。或者使用加热粉碎机(温度30℃-80℃)粉碎，以获得干燥的燕窝粉。
将燕窝研磨成粉末状的过程中，无论是人工研磨还是机器研磨，均应当避免材料变成凝胶。
去除燕窝原材料中的羽毛和杂物的方法包括纯人工，人工加半自动机械和全自动清洗机械手中的任何一种。本发明对此并无特别限制。
接着，进行步骤B，对燕窝粉进行预处理，即，把洁净的燕窝粉用水 浸泡使燕窝粉的颗粒膨胀。具体地，将洁净的燕窝粉和水以一定的比例混合后浸泡至燕窝粉膨胀得到燕窝浆。优选可经过超声破碎(可使用例如40KHz频率、250W功率)15～30分钟后，浸泡4～12小时，优选6到10小时，获得燕窝浆。燕窝粉与水的混合比例(重量比)为1:15到1:40。该浸泡的步骤还可以在一定的温度下进行，例如在40℃～50℃下，这样可获得燕窝粉的最佳膨胀。
本发明中任何步骤中所用的水可以是纯净水、去离子水或蒸馏水(如双蒸水)中的一种。
在步骤C中，将上一步获得的燕窝浆，按其与水的重量比为1:1到1:5加入水，在70～120℃的温度下，以200～1000rpm，优选300～800rpm的转速持续搅拌3到8小时，优选持续搅拌4到6小时。该步骤可促使燕窝中的营养物质解松，并使至少部分大分子量的蛋白质中肽链的部分酰胺键断裂，使蛋白质的分子量减小。该步骤获得第一提取液GY1。
接着进行步骤D，将第一提取液GY1放入混合搅拌容器，加热温度30～40℃并以100～300rpm的转速搅拌15～60分钟，例如20～40分钟后，静置。进行一定时间的搅拌有助于杂质释放出来并且有助于蛋白质中肽键的进一步松脱断裂成为分子量较小的多肽。静置的时间可根据实际情况确定。例如可静置3～4小时，以使大部分杂质沉淀，并能够有充分的时间使蛋白质完成肽键断裂的反应以生成分子量较小的多肽，并让这些断裂的多肽溶于水中。然后排走包含次级代谢产物及灰质的下层沉淀物，并取出上清物作为第二提取液GY2。
在步骤E中，将混合搅拌容器中的剩余物，即，中间层，同样在30～40℃下以100～300rpm的转速搅拌15～60分钟，例如20～40分钟后，再静置。静置时间可为2-3小时，使杂质能充分沉淀。同样排走下层沉淀物，然后提取上清物(在该步骤中通常为全部剩余的液体)作为第三提取液GY3。
在步骤F中，将所得到的第二提取液GY2和第三提取液GY3合并形成第四提取液GY4，即液态燕窝提取物。
仍参考图1，在步骤F中得到的液体燕窝提取物可进一步经过步骤G，获得浓缩的或固态的提取物。例如，可把第四提取液GY4放入具有自动送料***的喷雾干燥机，喷雾干燥，获得粉状固体。也可以通过诸如冻干、减压蒸馏、真空干燥等方法获得浓缩/固体燕窝提取物。本发明对燕窝提取物的形式没有特别限制，可根据需要选择进一步的处理的方式。
本发明方法获得的燕窝提取物中燕窝成分有效利用率(即提取率)达90％以上。提取物中的蛋白质多数以具有较低分子量的多肽存在。蛋白质和多肽的分子量呈窄正态分布，其中分子量较低(＜1000Da)的多肽含量占比45wt％以上，分子量小于3000Da的多肽含量可达到约75wt％。这种小分子量的多肽的窄正态分布，使得利用功效更佳。根据本发明方法获得的提取物中含18种氨基酸而且人体必需8种氨基酸含量比例在45wt％以上，甚至可达48wt％；唾液酸在提取物(干重)中的含量在10wt％以上，可高达13.8wt％。营养及价值成分相比其他提取方法具有显著的优势。
燕窝中含有高比例的蛋白质，但蛋白质由于其三级超螺旋结构，分子量大(可以是10000Da～300000Da，甚至更高)不易吸收。即便是分子量在3000Da左右的多肽，人的吸收效率也不高(约40％)。分子量介于多肽和氨基酸之间的小分子短肽(分子量约1500Da以下)则可以透过肠道表皮直接被吸收，并进入血液被输送到人体所需部位，仍能保持其具有的营养成分以及优良的生物活性及生理功能。本发明提取物不仅多数是短肽，比例远远高于常规方法获得的燕窝产品，更利于人体的吸收利用。
本发明的方法还可进一步包括步骤H和I，对方法中的积累废物进行回收利用。步骤H包括将以上步骤D和E中获得的下层沉淀物(次级代谢产 物及灰质)，将被作为生物降解物用作肥料。步骤I包括不可食用的毛燕羽毛和杂质，重复使用在燕屋，以味道吸引更多燕子过来建窝。在废物管理中实现“零废物”和“零排放”以减少利用能源和通过产品气体产生甲烷和二氧化碳的废物处理，有效保护地球环境。
本发明的燕窝提取物可进一步形成产品，如食品、保健品、美容化妆品等。本发明的食品或保健品中包含上述任何一种形式的燕窝提取物。所述产品还可包含其他组分，如添加剂、佐剂、赋形剂等。其中添加剂可以是诸如色素、颜料、调味剂、香料、甜味剂等。所述产品还可包含进一步的活性成分，例如其他营养成分，可以是能够与燕窝中的营养物质相互补充或相互协同的其他营养成分。
以下通过具体实施例进一步说明本发明的优点。
实施例
分析方法：
1、蛋白质分子量及分子量分布的测定：以下实施例和对比例获得的提取物样品采取高效液相凝胶色谱法(Agilent 1300 series，HPLC)进行分子量及其分布分析。具体分析条件如下：色谱柱采用TSKgelG2000SWXL凝胶色谱柱，分子量分级范围100～20000，二级管阵列检测器，检测波长为220nm，柱温为30℃；流动相为乙/水/三氟乙酸＝45/55/0.1(v/v/v)，流速为0.5mL/分钟，进样量5-20μl。结果示于表1～4。
2、SDS-PAGE：为了更直观的比较本发明的方法和现有技术的方法获得的提取物中蛋白质分子量的分布状况，采用SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)法测定提取液样品中的蛋白质分子量的分布状态。
分析仪器及材料：FD-201电泳仪(购自于上海医用分析仪器厂)、小型垂直式电泳附件模具及TEMED(购自于BIO-RAD)、肽分子质量标准 Mr范围348–16949(购自于Pharmacia LKB公司)、Sephadex G-75凝胶，层析柱规格为1.0×15cm。
具体分析方法：在电泳仪的内槽装阴极缓冲液，外槽用阳极缓冲液，恒压电泳，在40V下通电1小时，当样品进入分离胶时，电压升至60V，进行2小时。电泳完毕后胶置于染色液﹝25g/L考马斯亮蓝，R250的乙醇(V)：冰乙酸(V)：水(V)为9:2:9﹞中振荡染色1.5～2小时，转至脱色液(400ml/L乙醇，40ml/L的冰乙酸)中扩散脱色，直到背景清晰。拍照得到较直观的提取液样品的分子量分布凝胶电泳图片，见图3。
3、提取物中总氨基酸种类及含量的测定：根据GB/T5009.124-2003《食品中氨基酸的测定》，使用氨基酸自动分析仪(日立L-8900氨基酸自动分析仪)，把提取物检测样品经盐酸水解为游离氨基酸，经氨基酸自动分析仪的离子交换柱分离后，与茚三酮溶剂(日本和光纯药工业公司)产生颜色反应，以测定本发明实施例燕窝提取物及对比例样品的氨基酸的种类及含量。色谱柱规格为4.6×60mm，填料为3μm磺酸型阳离子树脂分离柱。
具体分析方法：1)样品前处理：取以下实施例1、2、3的提取液GY4-1，GY4-2，GY4-3和对比例1和对比例2的提取液各约0.5g(精确到0.0001g)样品于水解管中，加6mol/L盐酸15mL，加入新蒸馏苯酚3滴后将水解管放入冷冻剂中冷冻5分钟，再抽真空(接近0Pa)后充入高纯氮气，再抽真空充氮气，重复3次后在充氮状况下封口，将已封口的水解管放在110±1℃的恒温干燥箱(上海博迅BSC-150)内水解22小时后取出冷却。打开水解管将水解液过滤后用离子水冲洗多次后将水解液全部转移到50mL容量瓶并用去离子水定容。吸取滤液2mL于50mL烧杯中，水浴蒸干，残留物用1-2mL水溶解再干燥并反复进行两次。最后蒸干后用10mL的pH2.2的缓冲液溶解，混匀压滤后供测定 用。2)定量分析：准确吸取0.200mL的18种不同氨基酸的每种标准氨基酸溶液(来自Fluka AAS18)，用pH2.2的缓冲液稀释到5mL，此标准稀释液浓度为2.0nmol/20μL，为上机测定用的氨基酸标准。用氨基酸自动分析仪以外标法测定每一种提取物样品的氨基酸成分及对应的含量。结果见表5。
4、唾液酸含量的测定：采用高效液相色谱仪(Waters2695及Waters2489紫外检测器)对实施例提取物及对比例的提取物样品以及市售的罐装冰糖燕窝的进行唾液酸含量测试。唾液酸对照品(纯度≥99％)购自于Sigma公司，色谱柱为Waters Atlantis T 3(4.6mm×150mm,5μm)，流动相为乙腈-水(40:60)。称取各种提取液样品各100mg于锥形瓶中，准确加入1.0％磷酸溶液60mL，并加入茚三酮指示剂2mL于100℃沸水浴中水解20分钟，冷却到室温，补足减失重量，摇匀后用0.45μm滤膜过滤。检测流速为1.0mL/min,检测波长为192nm，进样体积20μL。检测结果见表6。
实施例1
取燕窝原材料(马来西亚柔佛州迪沙鲁生物谷Swiftlet Garden)800g。去除燕窝原材料中的羽毛和杂物，用研磨粉碎机在常温下把燕盏或燕条粉碎成燕窝粉。先后经过吸毛，过滤和离心分离方式去除燕窝上的毛和异物后得到细小及干净的燕窝粉680g。为避免材料因剪切热而变成凝胶，把研磨破碎机腔体温度设定在30℃。
以1:20的重量比将粉碎的燕窝粉放入纯净水中，通过超声波震动器(超声频率40KHz，超声功率250W)，超声破碎20分钟后，并加热到50℃浸泡6小时，让燕窝粉颗粒膨胀至最佳，得到窝浆体GY0-1共14280g，以待有效提取。
将GY0-1取出，按重量比1:4加入纯净水，以500rpm的转速持续搅 拌，并加热120℃，4小时促使燕窝中的营养物质解松，获得提取物GY1-1共71400g。
将提取物GY1-1放入带有搅拌器的容器，加热温度30℃并以300rpm的转速搅拌30分钟后，静止3小时排走下层沉淀物后取得上清液GY2-1共13138g。
将容器中的剩余物，加热到40℃并以100rpm的转速搅拌30分钟后，静止2小时排走下层沉淀物后取得上清液GY3-1共52550g。
将所述得到上清液GY2-1和上清液GY3-1混合成为提取液GY4-1共65688g。
按上述分析方法获得该提取液包含蛋白肽的各蛋白质的分子量范围下所占重量比例及数均分子量如下表1所示
表1：实施例1燕窝提取液中包含蛋白肽的
蛋白质分子量大小、对应分子量的蛋白质重量百分比及数均分子量
分子量范围(Da) 重量百分比(wt％，λ＝220nm) 数均分子量
＜180 1.43  
180～500 16.97 380
501～1000 27.61 641
1001～2000 19.23 1313
2001～3000 8.77 2416
3001～5000 6.96 3709
5001～8000 4.89 5616
8001～10000 4.28 8197
＞10000 9.86 18031
由表1可见，分子量＜1000Da的小分子肽占比约46wt％，分子量＜3000Da的蛋白肽(包含小分子肽和多肽，下同)所占比例达到74wt％。
把提取液通过喷雾干燥机，喷雾干燥获得粉状燕窝提取物626g。本实施例获得的干燥的提取物相对燕窝材料重量，获得的燕窝成分的提取率(最终提取物占投入燕窝的重量比)达92％。
实施例2
取燕窝原材料(马来西亚柔佛州迪沙鲁生物谷Swiftlet Garden)800g，按照实施例1的方法获处理得到燕窝粉710g。以1:30的重量比将粉碎的燕窝粉放入蒸馏水中，通过超声波震动器(超声频率40KHz，超声功率250W)，超声破碎20分钟后，在40℃浸泡8小时，让燕窝粉颗粒膨胀至最佳。得到燕窝浆体GY0-2共22010g，以待有效提取。
将GY0-2取出，按重量比1:3加入蒸馏水中，以300rpm的转速持续搅拌，并加热80℃，5小时促使燕窝中的营养物质解松，提取获得提取物GY1-2共88040g。
将提取物GY1-2放入带有搅拌的容器中，加热温度40℃并以200rpm的转速搅拌30分钟后，静置4小时排走下层沉淀物后取得上清液GY2-2共17520g。
将容器中的剩余物在30℃以100rpm的转速搅拌30分钟后，静置2小时排走下层沉淀物取得上清物GY3-2共62116g。
将所得上清液GY2-2和上清液GY3-2混合成为提取液GY4-2共79636g。
测定该提取液GY4-2中包含蛋白肽的各分子量范围下所占重量比例及数均分子量如下表2所示:
表2：实施例2燕窝提取液中包含蛋白肽的
分子量大小、对应分子量的蛋白质重量百分比及数均分子量
分子量范围(Da) 重量百分比(wt％，λ＝220nm) 数均分子量
＜180 1.58  
180～500 18.12 389
501～1000 29.22 703
1001～2000 19.63 1314
2001～3000 9.11 2411
3001～5000 6.12 4021
5001～8000 4.29 5333
8001～10000 3.76 8461
＞10000 8.17 16976
其中，分子量＜1000Da的小分子肽占比约48.9wt％，分子量＜3000Da的蛋白肽所占比例达到77.7wt％。
把提取液GY4-2放入具有自动送料***的喷雾干燥机，喷雾干燥，获得粉状燕窝提取物642g。本实施例获得的干燥的提取物相对燕窝材料重量，获得的燕窝成分的提取率(最终提取物占投入燕窝的重量比)达90.4％。
实施例3
取燕窝原材料(马来西亚柔佛州迪沙鲁生物谷Swiftlet Garden)800g，按照实施例1的方法获得燕窝粉701g。以1:40的重量比将粉碎的燕窝粉放入所述的洁净水浸泡10小时，通过辅助超声波震动器(超声频率40KHz，超声功率250W)，超声破碎20分钟后，更优的加热到40℃让燕窝粉颗粒膨胀至最佳。得到燕窝粉颗粒最大膨胀效果的燕窝浆体GY0-3共28741g，以待有效提取。
将GY0-3取出，按重量比1:2加入蒸馏水，以200rpm的转速持续搅拌，并加热75℃，6小时促使燕窝中的营养物质解松，提取获得提取物GY1-3共42840g。
将提取物GY1-3放入带有搅拌的容器中，加热温度40℃并以100rpm的转速搅拌30分钟后，静置3小时排走下层沉淀物后取得上清液GY2-3共8567g。
将容器中的剩余物在40℃以100rpm的转速搅拌30分钟后，静置3小时排走下层沉淀物后取得上清液GY3-3共30417g。
将所述得到上清物GY2-3和上清物GY3-3混合成为提取液GY4-3共38984g。
按上述方法测定提取液GY4-3中包含蛋白肽的各分子量范围下所占重 量比例及数均分子量如下表3以及图2所示:
表3：实施例3燕窝提取液中包含蛋白肽的
分子量大小、对应分子量的蛋白质重量百分比及数均分子量
分子量范围(Da) 重量百分比(wt％，λ＝220nm) 数均分子量
＜180 1.31  
180～500 17.51 372
501～1000 28.96 613
1001～2000 19.65 1176
2001～3000 10.21 2721
3001～5000 5.36 3879
5001～8000 4.32 5733
8001～10000 4.01 8417
＞10000 8.67 18189
其中，分子量＜1000Da的小分子肽占比约48.9wt％，分子量＜3000Da的蛋白肽所占比例达到77.7wt％。从图2更能够直观看到本发明方法获得的提取液中蛋白肽的分子量呈窄正态分布。
把提取液GY4-3放入具有自动送料***的喷雾干燥机，喷雾干燥，获得粉状燕窝提取物共638g。本实施例获得的干燥的提取物相对燕窝材料重量，获得的燕窝成分的提取率(最终提取物占投入燕窝的重量比)达91％。
对比例1
本对比例使用酶解法对燕窝进行提取。
取燕窝原材料(马来西亚柔佛州迪沙鲁生物谷Swiftlet Garden)粉碎、除杂、清洗干净并过40/80目过滤筛后得到10克燕窝粉，以1:60重量比加入水在室温浸泡4小时后置于密闭容器，以90℃水浴中加热30分钟，后冷却至50℃。以3000rpm搅拌10分钟。向匀浆的燕窝加入稀盐酸，调节pH至7，然后加入13mg木瓜蛋白酶(20万U/g酶活力，购自南宁庞博生物工程有限公司)和13mg碱性蛋白酶(20万U/g酶活力，购自南宁庞博生物工程有限公司)，于50℃恒温摇床反应3小时。将酶解产物加热至95℃，30分 钟灭酶。然后，用200目滤布过滤除杂，得澄清酶解液。最后，经喷雾干燥得到粉状燕窝提取物6.8克。提取率为68％。
经高效液相凝胶色谱法(HPLC)检测，所得的燕窝提取物中分子量小于1000Da的小分子肽含量比例为31wt％，在3000Da以下的小分子肽和多肽的含量为47wt％。通过分光度法对唾液酸样品检测到的唾液酸含量为9.51％。采取茚三酮显色法检测到提取物中的人体必需氨基酸(EAA)占总氨基酸的含量比例为39.27wt％。
对比例2
本对比例采用传统水煮蒸炖制备方法对燕窝进行提取
取燕窝原材料(马来西亚柔佛州迪沙鲁生物谷Swiftlet Garden)，粉碎、除杂、清洗干净并过40/80目过滤筛后得到10克燕窝粉，与水按重量比为1:10混合，于室温浸泡4小时。将浸泡过的燕窝置于密闭容器，以90℃高温炖煮6小时，冷却后用孔径为200目的滤膜过滤浓缩。经喷雾干燥后得到粉状提取物1.42克，提取率仅为14.2％。经高效液相凝胶色谱法(HPLC)检测，所得的燕窝中分子量小于1000Da的小分子肽含量比例(曲线下面积)为14％，在3000Da以下的小分子肽和多肽的含量(曲线下面积)为23％。通过分光度法对唾液酸样品检测到的唾液酸含量为9％。采取茚三酮显色法检测到提取物中的人体必需氨基酸(EAA)占总氨基酸的含量比例为39.29％。
测试结果：
1、采用上述高效液相凝胶色谱法(HPLC)分别检测对比例1中用酶解提取法获得的提取液，对比例2中用水煮蒸炖法获得的提取液以及实施例3的提取液中蛋白质和多肽的分子量分布结果对比示于下表4。
表4：实施例3、对比例1和对比例2的提取液中蛋白质/多肽的分子量分布
Figure PCTCN2020116289-appb-000001
Figure PCTCN2020116289-appb-000002
由表4可以看到相对于其他燕窝提取方法，本发明提取方法获得的燕窝提取物中蛋白肽分子量不仅小(<3000Da)，而且分布均一集中，具有较明显的正态分布特征。
进一步参见图3，其对应于表4示出了用本发明的方法(实施例3)、酶解法(对比例1)和水煮蒸炖法(对比例2)获得的提取液中蛋白肽的分子量分布的SDS-PAGE照片。图3中照片1为本发明提取方法获得的提取液中提取物蛋白肽的凝胶电泳照片，蛋白肽的分子量分布窄而且高比例的分布在小于3000Da的区域。照片2为对比例1酶解法获得的提取液中的蛋白质分子量分布，尽管小分子肽和多肽具有一定比例，但各分子量的分布较散，而且3000-10000Da这区间的比例显著高于实施例3，达到了44％。照片3则为对比例2水煮蒸炖法获得的提取液中蛋白质的分子量分布，由于得到的小分子肽和多肽数量较少，而多数都分布在分子量大于10000Da的区域，图片中无法显示。可见，酶解法虽然可以获得分子量较小的肽，但是分子量的分布不集中，仍有较大比例分子量较大而且分布比较分散的蛋白质，这会影响产品功效的专一性和稳定性。而传统的水煮蒸炖法则难以得到分子量较小的肽，提取物中的蛋白质大部分仍具有很高的分子量而影响营养价值的吸收。
2、采用上述高效液相色谱法(HPLC)原理的氨基酸自动分析仪来测定实施例1～3和对比例1～2的提取液样品的氨基酸成分及含量对比如表5所示。
下表5的对比结果显示：相比于燕窝酶解提取法和传统水解蒸炖燕窝提取法的提取物，本发明提取方法获得的燕窝提取物中，其含有18种氨基酸，其8种人体必需氨基酸(EAA)的重量含比达48wt％左右，明显高于对比例1和对比例2获得的提取液中的人体必需氨基酸的重量比(分别为39.27wt％和39.29wt％)。这说明本发明的方法更好地保留了燕窝中的蛋白质。本发明对燕盏粉碎后通过吸毛、过筛和离心的方法除去杂质，避免了水洗，保留了更多的可溶性蛋白质。
表5：实施例实施例1～3、对比例1和对比例2的提取液中氨基酸的含量
Figure PCTCN2020116289-appb-000003
Figure PCTCN2020116289-appb-000004
3、按照上述检测方法，采取分光光度法对实施例1～3中的燕窝提取液及对比例1和2以及市售冰糖燕窝样品中的唾液酸进行检测，每个样品检测6次取平均值，结果对比如下表6所示。
表6：实施例1～3、对比例1、对比例2的提取液和市售产品中唾液酸的含量
  实施例1 实施例2 实施例3 对比例1 对比例2 市售产品
唾液酸(wt％) 13.82 13.81 13.85 9.51 9.08 1.04
上表结果显示：本发明提取方法获得的燕窝提取液中唾液酸平均含量达13.82wt％，远远高于酶解法、水煮蒸炖法获得的燕窝提取液中的9.51wt％、9.08wt％以及市售的冰糖燕窝的1.04wt％。
4、根据本发明中实施例1、2、3的提取率和对比例1和2的提取率对比，提取率(％)＝提取物干重/燕窝投入重量，结果如表7所示。
表7：实施例1～3、对比例1、对比例2的提取率对比
  实施例1 实施例2 实施例3 对比例1 对比例2
提取率(wt％) 92 90.4 91 68 14.2
以上结果显示：本发明提取方法获得的燕窝提取物含量比例达90wt％以上，提取效率远高于酶解法和水煮蒸炖法获得的提取率(68％wt％和14.2wt％)。
以上所述仅为本发明的优选实施方式，并非因此限制本发明的专利范 围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。

Claims (19)

  1. 一种燕窝提取的方法，所述方法包括以下步骤：
    B、浸泡洁净的燕窝粉以获得燕窝浆；
    C、将所述燕窝浆以其1～5倍的重量比加入水，在70℃～120℃下以200～1000rpm持续搅拌3～8小时获得第一提取液；
    D、将所述第一提取液在30℃～40℃降低的温度下搅拌均匀并静置，待杂质沉淀后排除包含沉淀物的第一下层液体，并取出上清液作为第二提取液；
    E、将步骤D中的剩余液体继续在30℃～40℃下搅拌均匀并静置，待进一步的杂质沉淀后排除包含沉淀物的第二下层液体，以获得第三提取液；和
    F、合并所述第二提取液和所述第三提取液得到液态的燕窝提取物。
  2. 根据权利要求1所述的方法，其中，步骤D中在100～300rpm的搅拌速度下搅拌15～60分钟，再静置3～4小时。
  3. 根据权利要求1所述的方法，其中，步骤E中在100～300rpm的搅拌速度下搅拌15～60分钟，再静置2～3小时。
  4. 根据权利要求1所述的方法，其中，步骤C中以300～800rpm的搅拌速度持续搅拌4～6小时。
  5. 根据权利要求1所述的方法，其中，步骤B包括将所述洁净的燕窝粉加入其重量15～40倍的水混合并浸泡4～12小时。
  6. 根据权利要求5所述的方法，其中，步骤B包括将所述洁净的燕窝粉加入其重量15～40倍的水混合并浸泡6～10小时。
  7. 根据权利要求5所述的方法，其中，所述洁净的燕窝粉与水混合后先进行15～30分钟的超声破碎再进行所述浸泡。
  8. 根据权利要求7所述的方法，其中，所述浸泡在40℃～50℃的温度下进行。
  9. 根据权利要求1所述的方法，其中，所述方法还包括以下步骤：
    A、获得所述洁净的燕窝粉，该步骤包括：
    a)手工或机器把燕盏或燕条粉碎成燕窝粉，然后经过吸毛、过筛和离心分离方式去除毛和异物得到洁净的燕窝粉；或者
    b)把燕盏或燕条用水软化后手工清除毛和杂质，然后手工或机器进行粉碎得到洁净的燕窝粉。
  10. 根据权利要求1所述的方法，其中，所述方法进一步包括以下步骤：
    G、将所述液态燕窝提取物进一步处理以获得浓缩的或固态的燕窝提取物。
  11. 根据权利要求1所述的方法，其中，所述方法进一步包括以下步骤：
    H、将所述第一下层液体和所述第二下层液体合并后作为肥料。
  12. 根据权利要求7所述的方法，其中，所述方法进一步包括以下步骤：
    I、将步骤A中除去的毛和杂质放入燕屋，以吸引金丝燕前来建窝。
  13. 一种根据权利要求1～10中任一项所述的燕窝提取的方法获得的燕窝提取物。
  14. 一种燕窝提取物，其中所述燕窝提取物中分子量在1000Da以下的蛋白质占提取物中蛋白质总量的45wt％以上，且8种人体必需氨基酸含量占总氨基酸含量的45wt％以上。
  15. 根据权利要求14所述的燕窝提取物，其中所述燕窝提取物中分子量在3000Da以下的蛋白质占提取物中蛋白质总量的70wt％以上。
  16. 根据权利要求14所述的燕窝提取物，其中所述燕窝提取物中唾液酸的含量占提取物固体总重量的10wt％以上。
  17. 一种燕窝产品，所述燕窝产品包括权利要求14～16中任一项所述的燕窝提取物。
  18. 根据权利要求17所述的燕窝产品，其中所述燕窝产品是食品、保健品和化妆品中的一种。
  19. 根据权利要求18所述的燕窝产品，其中所述食品是饮品。
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