JP5985795B2 - Method for producing sericin peptide - Google Patents

Description

  The present invention relates to a sericin peptide (a silk protein derived sericin peptide), and more particularly to a method for producing a sericin peptide that can be suitably applied to cosmetics, medical products and the like.

The silkworm cocoons are mainly made of fibroin and sericin, and the silk fibers obtained from this silkworm have a double structure in which the surface of fibroin, an oriented fiber protein, is coated with sericin, a gelatinous protein. The ingredient is composed of about 25% sericin for about 75% fibroin.
By the way, since the excellent luster and texture of silk in silk fibers are exhibited by fibroin, sericin in the process of making silk thread (silk fiber) or silk fabric is made of alkali, acid, surfactant, proteolytic enzyme, ionic water, It is extracted and discarded (refined) with hot water and the like, and only fibroin is a component fiber of the final product of silk knitted fabric.
However, discarded sericin contains 30% or more of serine and is formed as a sericin polypeptide that is rich in amino acids such as aspartic acid, glutamic acid, glycine, alanine, and tyrosine. Employees of silk fabric factories have reviewed the sericin itself that has been extracted and discarded as a functional material because of its clean hand skin even during winter water work. The use in new fields such as cosmetics and food has been widely studied.

  By the way, as a method for separating and recovering sericin from silkworm / silk fabric, etc., a method of treating silkworm / silk fabric with high-temperature and high-pressure water and freezing, thawing and extracting the resulting aqueous solution of sericin (Patent Document 1), etc. Although proposed and put into practice, sericin obtained by this method has an average molecular weight of 100,000 or more.

However, high molecular weight sericin having an average molecular weight of 50,000 or more has poor solubility in water, and the aqueous solution becomes a gel at 0 to 40 ° C.
In other words, such a high molecular weight sericin is very troublesome and poor in appearance because sericin does not completely dissolve, becomes cloudy and has a high viscosity, and forms a colloidal gel over time or aggregates and precipitates. There was a characteristic that would end up.

  Such a high molecular weight sericin that is poorly soluble and gels at room temperature is difficult to use in applications and fields that require high uniformity and transparency. Even if it is used as a raw material for cosmetics such as hair care, the effect of sericin on the skin and hair can hardly be expected.

  On the other hand, as a method for obtaining sericin having a low molecular weight (sericin peptide), for example, a method of hydrolyzing silk sericin with ionic water obtained by an ionic water generator, or a silk protein raw material derived from rabbit or tengu Is dissolved in a neutral salt aqueous solution, and a peptide bond between the specific amino acid residues of the silk protein is cleaved using a peptide bond cleaving substance such as a proteolytic enzyme, whereby the silk having an average molecular weight of 10,000 to less than 200,000 A method for producing an aqueous protein solution (Patent Document 2) is disclosed.

  Furthermore, although it relates to a method for producing a fibroin peptide instead of sericin, a technique for degrading silk fibroin with a neutral salt and hydrolyzing it with a proteolytic enzyme is also disclosed (Patent Document). 3).

  However, the conventional method of eluting high molecular weight sericin or fibroin peptide from silk thread into an aqueous solution containing a high concentration of neutral salt requires a desalting step such as dialysis after hydrolysis with a peptide bond cleaving substance, which is a large-scale process. New problems have been pointed out, such as requiring equipment and increasing manufacturing costs.

  JP-A-11-131318   JP 2004-300142 A   Japanese Patent Application Laid-Open No. 07-0667686

  Under such circumstances, it is an object of the present invention to provide a method for efficiently producing low molecular weight sericin (sericin peptide) that can be suitably applied to cosmetics, medical products and the like. is there.

  In order to solve this problem, the present invention has been practiced in Japan for a long time, and has focused on the fermentation technology when producing origami, for example, miso and soy sauce. The added raw material sericin is subjected to primary fermentation, then subjected to secondary fermentation, and further, a process of aging this secondary fermentation sericin is performed to decompose sericin, and this is filtered and extracted.

  Specifically, minerals and purified water are added to the raw material sericin and mixed, and 10 wt% to 30 wt% of Aspergillus oryzae and saccharides are added to the raw material sericin mixture and held at a predetermined temperature for primary fermentation. Next, the primary fermentation sericin is subjected to secondary fermentation by adding lactic acid bacteria or acetic acid bacteria belonging to the genus Streptococcus or Lactobacillus singly or 10% to 20% by weight of a mixture thereof and a saccharide, and maintaining the temperature at a predetermined temperature. Further, a procedure is adopted in which the secondary fermented sericin is aged at 35 ° C. to 40 ° C. and then filtered and extracted.

  In this case, it is preferable to use a previously dissociated (ionized) mineral as a mineral added to the raw material sericin. Specifically, a raw material obtained by mixing starch and / or cereals, seeds and eggshells at a predetermined ratio is used. By crushing, then adding purified water and koji mold and fermenting and aging to dissociate calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese and other minerals contained in the raw material, followed by filtration The obtained fermentation ripening liquid is preferably used.

  As Aspergillus spp. To be added to the raw material sericin mixture, Aspergillus oryzae, Aspergillus soe, Aspergillus niger, Aspergillus awamori, Aspergillus revens alone or a mixture thereof can be used.

  On the other hand, maturation of secondary fermentation sericin is preferably performed while flowing in a far-infrared irradiation and / or electron supply atmosphere.

  By adopting this procedure, it is possible to prepare a sericin peptide having a molecular weight of about 2800 and having a reduced molecular weight.

  According to the method for producing a sericin peptide according to the present invention, a halfway sericin such as an aqueous silk protein solution having an average molecular weight of 10,000 or more and less than 200,000, which has been conventionally proposed, is further costly to use. The sericin mixture used as a raw material does not need to be treated, and the peptide bond between specific amino acid residues constituting sericin is appropriately cleaved by fermentation action by Aspergillus, lactic acid bacteria, acetic acid bacteria, etc., and a divalent mineral at each cut end Is further appropriately combined, and this state is aged in an organic acid produced by fermentation and then filtered and extracted, so that a sericin peptide having a much lower molecular weight than that of the prior art can be produced efficiently and inexpensively. .

  In addition, the sericin peptide obtained by the method of the present invention has a molecular weight of about 2800 and is appropriately bound with a divalent mineral. Therefore, the sericin peptide does not form a gel at room temperature and can retain water solubility. Conditioning effect that gives firmness and stiffness to hair and setting effect can be greatly improved, and keratin tissue such as hair is cross-linked with low molecular weight sericin with divalent ions such as calcium, The hair quality can be improved without leaching even when shampulins are used, and the desired desired effect can be sufficiently expected with a small amount.

It is procedure explanatory drawing which shows embodiment in the manufacturing method of the sericin peptide which concerns on this invention. It is a mass spectrometry result (M / Z 1-3000) of the sericin peptide based on this invention obtained by the procedure shown in FIG. 1, Comprising: It is an ion peak characteristic (molecular weight distribution) figure of molecular weight 2800 vicinity. It is a mass spectrometry result (M / Z 1-20000) of the sericin peptide based on this invention obtained by the procedure shown in FIG. 1, Comprising: It is an ion peak characteristic (molecular weight distribution) figure of molecular weight 20,000 or less.

Next, an embodiment of the method for producing a sericin peptide according to the present invention is illustrated and described in detail below.
In FIG. 1, the raw material sericin 10 used in the method for producing a sericin peptide according to the present invention includes, for example, known means (alkali, acid, surfactant, proteolysis in the process of making silk thread or silk fabric). High molecular weight sericin (sericin polypeptide) extracted by enzyme, ionized water, hot water, etc., or sericin having a low molecular weight to some extent, such as conventionally proposed silk protein aqueous solutions, can be used.

  The raw material sericin 10 itself is made up of “raw yarn” forming the cocoon itself, “raw yarn (which is an aggregate of cocoon yarns) formed by pulling a plurality of cocoon yarns together. A)), sterilized in advance of litter that is commonly referred to as spider silk such as “waste candy”, “cutting candy”, “raw skin candy”, “fuzz” Is used.

In particular, “fluff” is the first thread spited out to form a scaffold for anchoring the heel when the heel makes the heel, and contains about 50% by weight of sericin. Therefore, it is possible to extract about twice the amount of sericin from the same amount of yarn compared to a normal cocoon yarn having a sericin content of 20-30% by weight. Low price and cheap. Therefore, if such fluff is used as a raw material, the production cost of the sericin peptide according to the present invention can be further reduced.
In addition, “cut cocoon” is a cocoon after opening the cocoon to take out the cocoon, and this cut cocoon is inexpensive because the silk thread is cut and cannot be used as raw material, and Since there is relatively little contamination and other contaminants found in other spider yarns, it can be used very suitably as a raw material, and furthermore, the subspider yarns can be used alone or a mixture thereof. Can do.

Next, a container 12 is prepared, and raw water sericin 10, mineral 14, and purified water 16 obtained by removing chlorine or the like from Tapwater such as tap water in advance are put into the container 12, The raw material sericin mixture (dispersion) 18 is prepared.
In this case, the mixing ratio of the raw material sericin 10 and the purified water 16 is preferably about 8 to 9 with respect to the raw material sericin 1-2, and the amount of mineral 14 added is the raw material sericin 10 and the purified water 16. It is preferable to set it in the range of 10 wt% to 20 wt% with respect to the total amount.

  On the other hand, the mineral 14 added to the raw material sericin 10 is a previously dissociated (ionized) mineral. As the ionized mineral, for example, starch and / or cereals, seeds and eggshells are mixed at a predetermined ratio. The raw material is pulverized, and then purified water and koji mold are added and fermented and matured to dissociate minerals such as calcium, magnesium, sodium, potassium, iron, copper, zinc, and manganese contained in the pulverized raw material, followed by filtration. It is preferable to use the fermentation ripening liquid obtained.

Next, 10 wt% to 30 wt% of Aspergillus gonococcus 20 with respect to the raw material sericin mixture (dispersion) 18 and approximately the same amount of saccharide 22 as that of Aspergillus 20 are added and heated at 35 ° C. By maintaining the temperature at 40 ° C., primary fermentation of the raw sericin mixture 18 is performed.
In addition, in this primary fermentation, good fermentation may not be obtained depending on the condition of the koji mold, so that the koji mold 20 and the carbohydrates 22 are added as needed while constantly observing the holding temperature and fermentation state. Fermentation is ensured by adjusting.

Further, as the Aspergillus gonococcus 20 added to the raw material sericin 18, for example, Aspergillus oryzae, Aspergillus soe, Aspergillus niger, Aspergillus awamori, Aspergillus revens can be used alone or a mixture thereof.
In addition, if the amount of koji mold 20 added to the raw material sericin mixture 18 is less than 10% by weight, sufficient fermentation becomes difficult, and if it exceeds 30% by weight, the amount is too high and the economy is lowered. It is preferable to perform the fermentation while paying sufficient attention to the points.

  Further, in the fermentation of the raw material sericin mixture 18, it is preferable to promote the fermentation of koji mold by appropriately stirring with a known means such as a stirrer and supplying oxygen, and the raw material sericin has its oxygen bond by the fermentation action in this fermentation step. It is cut into a viscous primary fermentation sericin mixture 24.

  The primary fermented sericin mixture 24 thus obtained is newly supplemented with about 5% by weight of the saccharide 22 of the primary fermented sericin mixture 24, and is also a lactic acid bacterium belonging to the genus Streptococcus or Lactobacillus, and belonging to the genus Acetobacter. The acetic acid bacteria are subjected to secondary fermentation by adding 10 wt% to 20 wt% of these acetic acid bacteria alone or by holding them again at 35 ° C to 40 ° C for about 10 days.

If the amount of Streptococcus or Lactobacillus lactic acid bacteria, Acetobacter acetic acid bacteria, or a mixture 26 added to the primary fermentation sericin mixture 24 is less than 10% by weight, secondary fermentation is not sufficiently performed. If it exceeds wt%, the amount will be too great and the economy will be reduced.
In addition, during this secondary fermentation, carbohydrate 22 and lactic acid bacteria, acetic acid bacteria, or acetic acid bacteria or lactic acid bacteria are observed while observing the fermentation state and holding temperature with lactic acid bacteria belonging to the genus Streptococcus or Lactobacillus, acetic acid bacteria belonging to the genus Acetobacter, or a mixture thereof. It is important to ensure the secondary fermentation by adjusting the amount of the mixture 22 introduced.

And the obtained secondary fermentation sericin mixture 28 is kept at 35 ° C. to 40 ° C. as it is by using a conventionally known heater or the like, and is aged for a predetermined period, for example, about one week.
In the ripening of the secondary fermentation sericin mixture 28, preferably, the secondary fermentation sericin mixture 28 is allowed to flow slowly with stirring under an infrared magnetic field and / or electron (-e) supply atmosphere under far-infrared irradiation. The liquefaction of 28 is promoted and sericin is dissociated (ionized) in an organic acid solution such as lactic acid or acetic acid produced by fermentation of lactic acid bacteria, acetic acid bacteria or a mixture 26 thereof.
By these fermentation and ripening actions, the sericin mixture cleaves peptide bonds between specific amino acid residues constituting sericin as appropriate, and divalent minerals such as calcium are appropriately bound to these ends. As a result, it is suitably retained as a fermented and aged sericin peptide aqueous solution having a low molecular weight (in which sericin is dispersed) that does not form a gel at room temperature.

  The fermented and aged sericin peptide aqueous solution 30 is allowed to stand for 2 to 3 weeks, sterilized again by means of heating or ultraviolet irradiation, and then filtered and extracted to obtain a sericin peptide 32 having a transparent or yellowish color. It was.

  Next, mass spectrometry of the sericin peptide 32 according to the method of the present invention thus obtained was performed, and the results shown in FIGS. 2 and 3 were obtained.

The mass analysis procedure and analysis conditions are as follows.
Equipment used: AXIMA-Performance (manufactured by Shimadzu Corporation)
Laser ionization time-of-flight mass spectrometer (MALDI-TOFMS)
Extraction voltage: 20 kv
Flight mode; Linear
Detection ion; positive ion matrix; sinapinic acid (SA)
10 mg / ml in 0.1% TFA, 50% MeCNsolution
Sample pretreatment: Sericin peptide 32 was centrifuged and 9 μl of the supernatant was collected, and 1% TFA was added to prepare a 0.1% TFA solution. This sample solution is purified by ZipTipC18 (the operation follows the protocol attached to ZipTip), the purified sample is directly applied to the MALDI plate, air-dried, then the matrix solution is overlaid, air-dried, and then applied to the mass spectrometer. The plate was mounted and mass spectrometry was performed.

According to this mass measurement, although some peaks were observed, a distribution having a peak top around 2800 (see FIGS. 2 and 3) was obtained, and it was confirmed that the molecular weight was about 2800. .
Thus, a low molecular weight sericin peptide having a molecular weight of about 2800 can be obtained because the peptide bond between specific amino acid residues constituting sericin by fermentation including aging by Neisseria gonorrhoeae, lactic acid bacteria, acetic acid bacteria, etc. This is because, in this case, a divalent mineral such as calcium is appropriately bonded to these ends, and this state is maintained in a balanced manner.

  For comparison, the raw material sericin 10 was also measured in the same manner using the above-described mass spectrometer, and the molecular weight could not be measured because it exceeded the measuring range of the device. From these descriptions, it is easily guessed that the average molecular weight of the raw material sericin 10 is 100,000 or more.

When the sericin peptide 32 according to the present invention thus obtained is blended in a cosmetic composition, for example, a cosmetic for hair, the molecular weight is about 2800 and it is appropriately combined with a divalent mineral, so that it is gelled even at room temperature. In spite of its low molecular weight, the conditioning effect that gives the hair firmness and stiffness, and the setting effect can be greatly improved.
In addition, sericin peptide 32 obtained by the method of the present invention is different from the conventional one in which gel-like sericin or low molecular weight keratin is used for keratin tissue such as hair and does not enter the tissue or causes precipitation. Sericin, which has been reduced in molecular weight by divalent ions such as calcium, is cross-linked to the keratinous tissue, so that it can improve hair quality without leaching even with shampulence, and hair loss caused by coloring, permanents, etc. The desired effect could be sufficiently obtained with a small amount, such as a suitable repair of the above.

  In addition, although the compounding quantity of the sericin peptide 32 mix | blended with cosmetics composition is not specifically limited, Usually, it mix | blends within the range of 5.0-10.0 weight% as sericin density | concentration in cosmetics compositions. It is preferable to do this.

  In addition, as cosmetic compositions, various components generally blended, for example, alcohols such as ethanol, glycerin and sorbitol, sorbitan fatty acid esters such as sorbitan monolaurate, sorbitan monoisostearate and sorbitan tristearate, glycerol Glycerin fatty acid esters such as monostearate and glycerol monooleate, polyoxyethylene hydrogenated castor oil, polyether-modified silicone surfactants, surfactants such as dimethicone polyols, plasticizers, antioxidants, UV absorbers, Moisturizers, fragrances, dyes, pigments, pigments, preservatives, vitamins, hormones, deodorants, pH adjusters, fixing agents, and the like can be appropriately blended, and the use of this cosmetic composition includes hair gels, Hair shampoo, hair treatment, Various hair care cosmetics such as alins, styling lotion, hair mist, styling mousse, hair spray, skin care cosmetics such as lotion, milky lotion, moisture cream, moisturizing lotion, makeup cosmetics such as mascara and foundation. Needless to say, the present invention is not particularly limited.

10. Raw material sericin 12. Container
14. Mineral,
16. Purified water,
18..Sericin raw material mixture (dispersion),
20. Aspergillus oryzae mixture 22. Sugars such as sugar,
24 .. Primary fermentation sericin mixture,
26 .. Lactic acid bacteria, acetic acid bacteria or a mixture thereof,
28 .. Secondary fermentation sericin mixture,
30. Fermentation-ripened sericin peptide aqueous solution,
32..Sericin peptide,

Claims (5)

  Mineral and purified water are added to and mixed with raw material sericin, and 10% to 30% by weight of Aspergillus oryzae and saccharides are added to this raw material sericin mixture and maintained at a predetermined temperature for primary fermentation, and then this primary fermentation. A secondary fermentation is carried out by adding a saccharide of Streptococcus or Lactobacillus lactic acid bacteria or acetic acid bacteria to sericin alone or a mixture thereof and adding a saccharide to 10% by weight to 20% by weight and maintaining a predetermined temperature. A method for producing a sericin peptide, which comprises aging sericin at a temperature of 35 ° C. to 40 ° C. for a predetermined period and then extracting the sericin by filtration.   The method for producing a sericin peptide according to claim 1, wherein a mineral ionized (dissociated) in advance is used as the mineral. Pre-ionized (dissociated) minerals are contained in the raw material by pulverizing the raw material in which starch and / or cereals, seeds and eggshell are mixed in a predetermined ratio, and then fermenting and aging with purified water and koji mold. The method for producing a sericin peptide according to claim 2 , which is a fermentation ripening liquid obtained by dissociating minerals such as calcium, magnesium, sodium, potassium, iron, copper, zinc, and manganese, and further filtering. The sericin according to any one of claims 1 to 3, which comprises using Aspergillus oryzae, Aspergillus soe, Aspergillus niger, Aspergillus awamori, Aspergillus revens alone or a mixture thereof as an Aspergillus genus fungus. A method for producing a peptide. The method for producing a sericin peptide according to any one of claims 1 to 4 , wherein the secondary fermentation sericin is ripened while flowing in a far-infrared irradiation and / or an electron supply atmosphere.

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