CN111012901B - Artificial saliva containing ovomucin and lysozyme and preparation method and application thereof - Google Patents
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Abstract
The invention discloses artificial saliva containing ovomucin and lysozyme, a preparation method and application thereof, and belongs to the field of biology. The invention creatively compounds natural ovomucin and lysozyme to simulate the composition of the mucin and the lysozyme in the oral cavity, combines ultrasonic treatment and137the Cs irradiation treatment homogenized the solution for degassing and sterilization. The artificial saliva prepared by the method has chemical composition and physical property parameters similar to those of natural saliva, effectively relieves the xerostomia problem of specific people, has an antibacterial rate of more than 97.8 percent on typical harmful bacteria in the oral cavity, and has performance stability of more than 9 months.
Description
Technical Field
The invention relates to the field of biology, in particular to artificial saliva which is prepared from ovomucin and lysozyme and has functions of relieving xerostomia and inhibiting bacteria, a preparation method and application thereof.
Background
Saliva is a liquid secreted in the oral cavity of a human body and has the effects of moisturizing, lubricating, inhibiting bacteria and the like, and is one of the most important components in the oral cavity. The components of the compound are mainly water (more than 99 percent), and the organic matters are mainly salivary amylase, mucin, lysozyme, mucopolysaccharide and inorganic ions such as potassium, sodium, calcium and the like. However, there are various causes that the salivary secretion amount inside the oral cavity is reduced, thereby causing some discomfort, wherein xerostomia and canker sore are two typical symptoms.
Xerostomia refers to the sensation inside the oral cavity caused by the change of the saliva components inside the oral cavity or the reduction of the secretion amount, and the cause of xerostomia is many, but the phenomenon is not an independent disease. Due to the lubricating and protective effects of saliva, people with reduced salivary secretion often have burning sensation, eating difficulty, bleeding of oral mucosa and other symptoms in the oral cavity.
The great cause of the oral ulcer is the unbalance of oral flora, because the saliva contains lysozyme, when the saliva secretion is in a normal level, the propagation of harmful bacteria in the oral cavity can be effectively inhibited, and conversely, when the xerostomia happens, the unbalance of the harmful bacteria and beneficial bacteria in the oral cavity can be caused by insufficient saliva secretion, and the immunity of the organism is reduced, so the oral ulcer is finally caused.
Disclosure of Invention
Based on the background, the invention provides a preparation method of artificial saliva containing ovomucin and lysozyme, which has stable properties and has the effects of relieving xerostomia and bacteriostasis.
It is an object of the first aspect of the invention to provide an artificial saliva comprising ovomucin and lysozyme.
In order to solve the problems, the invention adopts the technical scheme that:
an artificial saliva containing ovomucin and lysozyme is prepared from deionized water as main raw material, and raw materials including ovomucin, lysozyme, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and sweetener. The raw materials and the mass fraction thereof are as follows: 0.1 to 0.4 percent of ovomucin, 0.05 to 0.2 percent of lysozyme, 0.03 to 0.08 percent of sodium chloride, 0.05 to 0.1 percent of potassium chloride, 0.02 to 0.05 percent of calcium chloride, 0.008 to 0.01 percent of magnesium chloride, 0 to 0.5 percent of sweetening agent and the balance of deionized water.
Further, the sweetener is one or more of xylitol, stevioside and glycyrrhizin; the ovomucin and lysozyme are extracted from natural eggs or are pure products purchased, and the ovomucin and lysozyme are preferably extracted from natural eggs.
The second aspect of the invention aims at providing a method for preparing artificial saliva containing ovomucin and lysozyme, which comprises the following steps:
step (1), preparing the following raw material components in percentage by mass: 0.1 to 0.4 percent of ovomucin, 0.05 to 0.2 percent of lysozyme, 0.03 to 0.08 percent of sodium chloride, 0.05 to 0.1 percent of potassium chloride, 0.02 to 0.05 percent of calcium chloride, 0.008 to 0.01 percent of magnesium chloride, 0 to 0.5 percent of sweetening agent and the balance of deionized water;
step (2), dissolving ovomucin in the deionized water at 25 ℃ according to the mass fraction to obtain a solution I for later use;
step (3), dissolving lysozyme in partial deionized water at 25 ℃ according to the mass fraction to obtain a solution II for later use;
dissolving sodium chloride, potassium chloride, calcium chloride and magnesium chloride in part of deionized water at 25 ℃ according to the mass fraction to obtain a solution (c) for later use;
step (5), dissolving the sweetening agent into the residual deionized water at 25 ℃ according to the mass fraction to obtain solution (IV) for later use;
step (6), stirring the obtained solution (III) at the stirring speed of 200r/min for 30min at the temperature of 25 ℃ until the solution is uniformly mixed to obtain solution (V) for later use;
step (7), ultrasonically treating the solution for 10min to 20min at the power of 200W to 500W until no small bubbles are generated in the solution, thus obtaining solution;
step (8), use the solution137And (5) sterilizing the Cs by irradiation, wherein the dosage is 10-50kGy, and the time is 1-10 min, and obtaining the product.
The preparation method is adopted to prepare the colorless and transparent viscous liquid which has the viscosity similar to that of natural saliva and has the effects of relieving xerostomia and resisting and inhibiting bacteria, and the viscous liquid can be further prepared into oral spray or mouthwash, and is a novel biomedical preparation.
In a third aspect, the invention provides the use of an artificial saliva comprising ovomucin and lysozyme for alleviating xerostomia and inhibiting bacteria.
Mechanism of action
Mechanism of alleviating xerostomia: ovomucin is an important protein in egg white, has negative charges, can show higher viscosity in a solution and can maintain a thicker state, so the ovomucin can simulate mucin in human saliva to play a role in moisturizing. And the ovomucin and mucin in saliva of an organism belong to homologous protein, so that the tolerance of the organism is higher, and the rejection phenomenon is not easy to generate. The lysozyme is a protein existing in egg white and saliva at the same time, has high stability and good heat resistance, has positive charges, so that electrostatic attraction exists between the lysozyme with the positive charges and the ovomucin with the negative charges, the lysozyme can play a role similar to bridging between the ovomucin molecules, and finally ultrasonic treatment is assisted, the molecular motion is accelerated, and the bridging effect is further enhanced. The interaction enables the two to form a complex body more quickly, so that a membrane structure with larger surface area and more stable property is formed in the oral cavity, and a better moisturizing effect can be achieved after the membrane structure is attached to the oral mucosa.
The mechanism of bacteriostasis and antibiosis is as follows: (1) the antibacterial and bacteriostatic mechanism of the ovomucin is as follows: the ovomucin is a sulfated egg white glycoprotein and is composed of alpha-ovomucin and beta-ovomucin, wherein the beta-ovomucin contains sialic acid. Sialic acid is a general term for derivatives in which amino acids or hydroxyl hydrogen is substituted in neuraminic acid, is also present in human salivary mucin, can enable saliva to generate smooth feeling, and more importantly, sialic acid can prevent pathogen invasion through a competitive mechanism of a binding site, has anti-infection capacity, and has an inhibiting effect on common viruses and bacteria. In addition, sialic acid can increase the viscosity of mucin, shield sugar chains on the mucin, so that the sugar chains are not easy to hydrolyze by glycosidase, and the protein is protected from being hydrolyzed by protease, so that the ovomucin or mucin is more stable. (2) The antibacterial and bacteriostatic mechanism of lysozyme is as follows: bacterial cell walls contain peptidoglycans, particularly abundant in the cell wall of gram-positive bacteria. Since lysozyme has a positive charge and most bacteria have a negative charge, lysozyme attaches to the bacterial surface by electrostatic attraction and binds peptidoglycan between its two domains. Then lysozyme twists the fourth sugar on the glucose hexaose into a half-chair conformation, the twisted conformation has higher energy, and the glycosidic bond is easy to break, thereby achieving the purposes of cracking the cell wall of bacteria, resisting bacteria and inhibiting bacteria.
In addition, the ovomucin and the lysozyme can mutually react in the solution due to the electrostatic attraction to form a complex, and the complex has the bacteriostatic function of two proteins, so that the synergistic effect is achieved in the aspect of antibiosis and bacteriostasis.
The invention has the following beneficial effects:
the artificial saliva prepared by the preparation method disclosed by the invention has the effects of relieving xerostomia and inhibiting bacteria, and is comfortable and fresh in taste; no irritant components such as alcohol and the like, and high human body adaptability; no preservative is used, and the consumer acceptance is high; can remarkably relieve xerostomia and canker sore symptoms caused by various factors; the texture is thick, the retention time in the oral cavity is long, and the stability is good.
The artificial saliva provided by the invention is placed at room temperature for more than 9 months, the bacteriostasis rate is not significantly reduced, and various physical and chemical indexes are not significantly changed.
According to the artificial saliva provided by the invention, both ovomucin and lysozyme with antibacterial effects are added, and a synergistic interaction effect exists between the ovomucin and the lysozyme, so that the antibacterial and antibacterial performance of the product is greatly improved compared with that of the existing similar products.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.
Example 1:
an artificial saliva containing ovomucin and lysozyme mainly comprises deionized water, and is weighed by the following raw materials in parts by weight: ovomucin 0.2 g; 0.15g of lysozyme; 0.04g of sodium chloride; 0.05g of potassium chloride; 0.05g of calcium chloride; 0.008g of magnesium chloride; 99.502g of deionized water.
The preparation method comprises the following steps:
dissolving 0.2g of ovomucin in partial deionized water at 25 ℃ to obtain a solution I for later use;
0.15g of lysozyme is dissolved in part of deionized water at 25 ℃ to obtain a solution II for later use.
0.04g of sodium chloride, 0.05g of potassium chloride, 0.05g of calcium chloride and 0.008g of magnesium chloride are dissolved in partial deionized water at 25 ℃ to obtain a solution (c) for later use.
Transferring the solution (i) to a volumetric flask, and using the residual deionized water to fix the volume to 100 mL.
And transferring the solution with constant volume to a beaker, and stirring at the stirring speed of 200r/min for 30min until the solution is uniformly mixed to obtain a solution IV for later use.
And (4) performing ultrasonic treatment on the solution (IV) at the power of 300W for 20min until no small bubbles are generated in the solution, thus obtaining a solution (V) for later use.
Mixing the solution with137And (5) sterilizing by irradiation of Cs at the dose of 20kGy for 6min to obtain an artificial saliva product, and measuring the pH value to be 6.7.
Example 2:
an artificial saliva containing ovomucin and lysozyme comprises deionized water as main ingredient, and the following raw materials by weight: ovomucin 0.35 g; 0.15g of lysozyme; 0.04g of sodium chloride; 0.05g of potassium chloride; 0.02g of calcium chloride; 0.01g of magnesium chloride; 99.38g of deionized water.
The preparation method comprises the following steps:
dissolving 0.35g of ovomucin in deionized water at 25 ℃ to obtain a solution I for later use;
0.15g of lysozyme is dissolved in deionized water at 25 ℃ to obtain a solution II for later use.
0.04g of sodium chloride, 0.05g of potassium chloride, 0.02g of calcium chloride and 0.01g of magnesium chloride are dissolved in deionized water at 25 ℃ to obtain a solution (c) for later use.
Transferring the solution to a volumetric flask, and fixing the volume to 100mL by using deionized water.
And transferring the solution with constant volume to a beaker, and stirring at the stirring speed of 200r/min for 30min until the solution is uniformly mixed to obtain a solution IV for later use.
And (4) carrying out ultrasonic treatment on the solution (IV) for 15min under the power of 200W until no small bubbles are generated in the solution, thus obtaining a solution (V) for standby.
Mixing the solution with137And (5) sterilizing by irradiation of Cs at the dose of 18kGy for 7min to obtain an artificial saliva product, and measuring the pH value to be 7.0.
Example 3:
a sweet artificial saliva containing ovomucin and lysozyme comprises deionized water as main ingredient, and is prepared from the following raw materials by weight: ovomucin 0.15 g; 0.15g of lysozyme; 0.07g of sodium chloride; 0.065g of potassium chloride; 0.05g of calcium chloride; 0.01g of magnesium chloride; 0.5g of xylitol; 99.005g of deionized water.
The preparation method comprises the following steps:
dissolving 0.15g of ovomucin in deionized water at 25 ℃ to obtain a solution I for later use;
0.15g of lysozyme is dissolved in deionized water at 25 ℃ to obtain a solution II for later use.
0.07g of sodium chloride, 0.065g of potassium chloride, 0.05g of calcium chloride and 0.01g of magnesium chloride are dissolved in deionized water at 25 ℃ to obtain a solution (c) for later use.
0.5g of xylitol is dissolved in deionized water at 25 ℃ to obtain a solution (r) for later use.
Transferring the solution to a volumetric flask, and adding deionized water to a constant volume of 100 mL.
Transferring the solution with constant volume into a beaker, stirring at a stirring speed of 200r/min for 30min until the solution is uniformly mixed to obtain a solution for later use.
Ultrasonically treating the solution for 10min under the power of 450W until no small bubbles are generated in the solution, and obtaining solution (c) for later use.
The solution is prepared by137And (5) sterilizing by irradiation of Cs at the dose of 47kGy for 1.5min to obtain an artificial saliva product, and measuring the pH value to be 6.4.
Example 4:
a sweet artificial saliva containing ovomucin and lysozyme comprises deionized water as main ingredient, and is prepared from the following raw materials by weight: ovomucin 0.4 g; 0.17g of lysozyme; 0.046g of sodium chloride; 0.1g of potassium chloride; 0.036g of calcium chloride; 0.008g of magnesium chloride; stevioside 0.35 g; 98.89g of deionized water.
The preparation method comprises the following steps:
0.4g of ovomucin is dissolved in deionized water at 25 ℃ to obtain solution I for later use.
0.17g of lysozyme is dissolved in deionized water at 25 ℃ to obtain a solution II for later use.
0.046g of sodium chloride, 0.1g of potassium chloride, 0.036g of calcium chloride and 0.008g of magnesium chloride are dissolved in deionized water at 25 ℃ to obtain a solution (III) for later use.
0.35g of stevioside is dissolved in deionized water at 25 ℃ to obtain a solution (r) for later use.
Transferring the solution to a volumetric flask, and adding deionized water to a constant volume of 100 mL.
Transferring the solution with constant volume into a beaker, stirring at a stirring speed of 200r/min for 30min until the solution is uniformly mixed to obtain a solution for later use.
Ultrasonically treating the solution for 17min under the power of 300W until no small bubbles are generated in the solution, and obtaining solution (c) for later use.
The solution is prepared by137And (5) sterilizing by irradiation of Cs at the dose of 30kGy for 4min to obtain an artificial saliva product, and measuring the pH value to be 6.9.
The inhibitory effects of the artificial saliva obtained in examples 1 to 4 on anaerobic streptococci, Actinomyces naeslundii and Staphylococcus aureus were measured within 10min by the method of QBT-2738-2012, and the results are shown in Table 1 below, using the artificial saliva without ovomucin as control example 1 and the artificial saliva without lysozyme as control example 2, respectively.
Table 1: the saliva obtained in the examples shows the inhibitory effect on anaerobic streptococcus, actinomyces naeslundii and staphylococcus aureus.
The bacteriostatic stability of the artificial saliva prepared in examples 1 to 4 after 9 months of storage was determined by the method of QBT-2738-2012 using Staphylococcus aureus as a test strain, and the results are shown in Table 2 using the artificial saliva without ovomucin as control example 1 and the artificial saliva without lysozyme as control example 2, respectively.
Table 2 bacteriostatic stability effect of the saliva prepared in example during storage.
| Example 1 | Example 2 | Example 3 | Example 4 | Comparative example 1 | Comparative example 2 | |
| Bacteriostatic stability | 99.55％ | 99.01％ | 98.89％ | 99.21％ | 70.19％ | 63.82％ |
The bacteriostasis stability is the bacteriostasis rate of the artificial saliva after storage/the bacteriostasis rate of the fresh artificial saliva multiplied by 100 percent
The embodiments described above are intended to describe the present invention in detail, but it is only a part of the embodiments of the present invention, rather than all embodiments, and one can also obtain other embodiments without inventive step according to the embodiments, and these embodiments all belong to the protection scope of the present invention.
Claims (6)
1. An artificial saliva containing ovomucin and lysozyme, characterized in that: the composite material consists of the following raw materials in percentage by mass: 0.1-0.4% of ovomucin, 0.05-0.2% of lysozyme, 0.03-0.08% of sodium chloride, 0.05-0.1% of potassium chloride, 0.02-0.05% of calcium chloride, 0.008-0.01% of magnesium chloride, 0-0.5% of sweetening agent and the balance of deionized water, wherein the artificial saliva is high-stability artificial saliva with functions of relieving xerostomia and inhibiting bacteria.
2. An artificial saliva containing ovomucin and lysozyme according to claim 1, wherein: the sweetener is one or more of xylitol, stevioside and glycyrrhizin.
3. A method for preparing an artificial saliva containing ovomucin and lysozyme according to claim 1 or 2, comprising the steps of:
step (1), preparing the following raw material components in percentage by mass: 0.1 to 0.4 percent of ovomucin, 0.05 to 0.2 percent of lysozyme, 0.03 to 0.08 percent of sodium chloride, 0.05 to 0.1 percent of potassium chloride, 0.02 to 0.05 percent of calcium chloride, 0.008 to 0.01 percent of magnesium chloride, 0 to 0.5 percent of sweetening agent and the balance of deionized water;
step (2), dissolving ovomucin in the deionized water at 25 ℃ according to the mass fraction to obtain a solution I for later use;
step (3), dissolving lysozyme in partial deionized water at 25 ℃ according to the mass fraction to obtain a solution II for later use;
dissolving sodium chloride, potassium chloride, calcium chloride and magnesium chloride in part of deionized water at 25 ℃ according to the mass fraction to obtain a solution (c) for later use;
step (5), dissolving the sweetening agent into the residual deionized water at 25 ℃ according to the mass fraction to obtain solution (IV) for later use;
step (6), mixing the obtained solution (III), (IV) and stirring to obtain solution (V);
step (7), carrying out ultrasonic treatment on the solution obtained in the step (6) to obtain a solution;
and (8) irradiating the solution obtained in the step (7) to obtain the artificial saliva product.
4. A method of producing artificial saliva containing ovomucin and lysozyme according to claim 3, wherein: the stirring treatment in the step (6) is specifically as follows: stirring at 25 deg.C and 200r/min for 30min until the solution is uniformly mixed to obtain solution.
5. A method of producing artificial saliva containing ovomucin and lysozyme according to claim 3, wherein: the ultrasonic treatment in the step (7) is specifically as follows: ultrasonic treatment is carried out for 10min to 20min at the power of 200W to 500W until no small bubbles are generated in the solution.
6. A method of producing artificial saliva containing ovomucin and lysozyme according to claim 3, wherein: the irradiation treatment in the step (8) is specifically as follows: by using137And (5) sterilizing the Cs by irradiation, wherein the dosage is 10-50kGy and the time is 1-10 min.
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| CN111012901A (en) | 2020-04-17 |
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