CN113632922A - Lysate, preparation method and application - Google Patents
Lysate, preparation method and application Download PDFInfo
- Publication number
- CN113632922A CN113632922A CN202110936150.3A CN202110936150A CN113632922A CN 113632922 A CN113632922 A CN 113632922A CN 202110936150 A CN202110936150 A CN 202110936150A CN 113632922 A CN113632922 A CN 113632922A
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- CN
- China
- Prior art keywords
- lysate
- lactobacillus
- oat
- fermented oat
- bifidobacterium
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000005303 weighing Methods 0.000 description 1
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Abstract
The invention provides a lysate, a preparation method and application, wherein the preparation method comprises the following steps: inoculating probiotics into fermented oat liquid for culture, centrifuging the fermented oat liquid after culture is finished, and taking supernatant, wherein the supernatant is lysate. The invention takes the fermented oat liquid as the culture medium to replace the traditional culture medium with chemical components. The probiotics are strains which are safe and beneficial to human bodies, and the fermentation substrate is food-grade raw materials, so that the safety of the raw materials is high, and the prepared lysate is high and is widely applied; the preparation process is simple, and large-scale production can be realized; the lysate components are of various kinds. The product comprises lactic acid, acetic acid, beta-glucan, polysaccharide, polyphenol compounds, amino acid, superoxide dismutase, glutathione peroxidase, small molecule active peptide and the like.
Description
Technical Field
The invention relates to the technical lysate field, in particular to a lysate, a preparation method and application thereof.
Background
At present, under the conditions of pressure, environmental pollution and the like of human bodies, skins are easy to be in an oxidative stress state, and the demands of people, particularly women, on cosmetics which are natural in source and have the functions of oxidation resistance and aging resistance are continuously increased. In recent years, more and more probiotic fermentation lysates (such as a yeast lysate of saccharomyces bifidus) have been added to cosmetics. How to produce safe and functional probiotic lysate raw materials becomes a constantly optimized technical problem.
Disclosure of Invention
In view of the above, the invention provides a lysate, a preparation method and an application thereof, wherein the lysate is obtained by taking fermented oat liquid as a culture substrate and inoculating probiotics for culture, and has safe components, contains various antioxidant substances and outstanding antioxidant function.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of preparing a lysate comprising: inoculating probiotics into fermented oat liquid for culture, centrifuging the fermented oat liquid after culture is finished, and taking supernatant, wherein the supernatant is lysate.
Further, the probiotic is lactobacillus and/or bifidobacterium;
the lactobacillus comprises one or more of lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus casei, lactobacillus paracasei, lactobacillus gasseri, lactobacillus reuteri, lactobacillus curvatus and lactobacillus crispatus; the Bifidobacterium comprises one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis and Bifidobacterium adolescentis.
Further, the fermented oat liquid comprises enzymolysis oat powder and water;
or the fermented oat liquid comprises enzymolysis oat powder, yeast extract powder and water;
wherein the content of the enzymolysis oat powder in the fermented oat liquid is 6-15% (w/v), and the content of the yeast extract powder is 0-5% (w/v).
Further, the inoculation amount of the probiotics is 1% -3%.
Further, the culture time is 18-24 h, and the culture temperature is 30-37 ℃.
Further, after the completion of the culture, the fermented oat liquid is sheared, homogenized, centrifuged, and the supernatant is collected and stored after sterilization to obtain the lysate.
Further, the rotating speed of the shearing is 2500-; the homogenization is carried out for 2-4 cycles at 100-400bar and for 5-12 cycles at 600-1500 bar.
In a second aspect, the present invention provides a lysate prepared by the above-described preparation method.
Further, the lysate comprises:
antioxidant enzyme, antioxidant substance, antibacterial substance and other active substances;
wherein the antioxidant enzyme comprises at least one of superoxide dismutase and glutathione peroxidase;
the antioxidant substance comprises at least one of polysaccharide, beta-glucan and polyphenol compound;
the bacteriostatic substance comprises at least one of lactic acid, acetic acid and small molecular polypeptide;
the other active substance comprises at least one of amino acid and lipoteichoic acid.
In a third aspect, the present invention provides the use of a lysate as described above in a cosmetic, food, pharmaceutical product.
The technical scheme of the invention has the following beneficial effects:
the present invention provides a method for preparing a lysate, comprising: inoculating probiotics into fermented oat liquid for culture, centrifuging the fermented oat liquid after culture is finished, and taking supernatant, wherein the supernatant is lysate. The invention has at least the following advantages:
(1) the invention takes the fermented oat liquid as the culture medium to replace the traditional culture medium with chemical components. The probiotics are strains which are safe and beneficial to human bodies, and the fermentation substrate is food-grade raw materials, so that the safety of the raw materials is high, and the prepared lysate is high and is widely applied;
(2) the preparation process comprises probiotic fermentation, high-speed shearing, high-pressure homogenization, high-speed centrifugation and inactivation. The fermentation process is simple, and large-scale production can be realized;
(3) different from the method for obtaining lysate after crushing thalli in the prior art, the method for obtaining the lysate by taking the supernatant has the following advantages: A. the functional components are more abundant. The lysate not only contains the probiotic lysate and metabolites, but also contains the effective active ingredients of the oat, the probiotic lysate is rich in antioxidant enzyme, small molecular active peptide, lipoteichoic acid and the like, the metabolites of the probiotic comprise fatty acid such as lactic acid, acetic acid and the like and small molecular active substances, and the oat contains rich antioxidant and anti-aging substances such as beta-glucan, polysaccharide, polyphenol compounds, amino acid and the like. In contrast to the traditionally used yeast lysate, only the intracellular active ingredient of the probiotic bacteria is contained. B. The application range is wider. The lysate is the collected fermentation supernatant, has no obvious flocculent precipitate, and can be applied to the watery skin care products such as toner and the like. Unlike traditional probiotic lysates, most are flocculent precipitates. C. The stability in shelf life is better. Because the lysate of the invention is supernatant, the thalli and fragments are greatly removed, and the possibility of bacterial contamination and further metabolism is not easy to occur in the shelf life.
(4) The lysate has outstanding efficacy: can regulate skin microecological balance, effectively remove free radicals, improve skin antioxidase activity, inhibit skin pathogenic bacteria, reduce skin wrinkle, and delay aging.
Drawings
FIG. 1 is a flow diagram of a method of preparing a probiotic fermented oat lysate;
FIG. 2 is the free radical scavenging capacity of probiotic fermented oat lysate;
FIG. 3 is a graph of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity of probiotic fermented oat lysates;
FIG. 4 is the inhibitory effect of probiotic fermented oat lysate on pathogenic bacteria;
FIG. 5 is a cell viability curve of a Bifidobacterium longum lysate;
FIG. 6 is a cell viability curve of a Bifidobacterium infantis lysate;
FIG. 7 is a cell viability curve of a Lactobacillus paracasei lysate;
FIG. 8 shows the results of the cytomorphological examination of Bifidobacterium longum lysate by its action;
FIG. 9 shows the results of the cytomorphological examination of Bifidobacterium infantis lysate by its action;
FIG. 10 shows the results of the cytomorphological examination of the Lactobacillus paracasei lysate by the action thereof;
FIG. 11 is a histogram of lysate Reactive Oxygen Species (ROS) content.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention in conjunction with the following examples, but it will be understood that the description is intended to illustrate the features and advantages of the invention further, and not to limit the invention.
In a first aspect, the present invention provides a method of preparing a lysate comprising: inoculating probiotics into fermented oat liquid for culture, centrifuging the fermented oat liquid after culture is finished, and taking supernatant, wherein the supernatant is lysate.
Specifically, oats are rich in beta-glucan, amino acids, polysaccharides, and the like. The lysate of probiotics, especially lactobacillus and bifidobacterium is rich in antioxidant enzyme, organic acid, lipoteichoic acid, small molecular active peptide, polysaccharide and the like, has the effects of resisting ultraviolet injury, promoting the DNA repair of skin cells and providing skin resistance, can be used for various emulsified, water-based and hydroalcoholic systems of skin care, sun protection and after-sun care products, and helps to prevent the photoaging of epidermis and dermis. The invention takes the fermented oat liquid as the culture medium, is different from the existing culture medium, and has food-grade safety. According to the invention, probiotics are inoculated into fermented oat liquid for culture, after the culture is finished, the fermented oat liquid is centrifuged, and supernatant is taken, wherein the supernatant is lysate. The probiotics in the invention are beneficial strains safe to human body, the fermentation substrate has food-grade safety, so the obtained lysate has high safety, and simultaneously has products after the probiotics are fermented and small molecular substances in the fermented oat liquid, and the lysate has various components and outstanding efficacy.
According to some embodiments of the invention, the probiotic is lactobacillus and/or bifidobacterium; the lactobacillus comprises one or more of lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus casei, lactobacillus paracasei, lactobacillus gasseri, lactobacillus reuteri, lactobacillus curvatus and lactobacillus crispatus; the Bifidobacterium comprises one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis and Bifidobacterium adolescentis. In the invention, the probiotics are activated before being inoculated into the fermented oat liquid, and the activation method comprises the following steps: inoculating probiotic bacteria into seed culture medium at an inoculation amount of 1% -3%, culturing at 37 deg.C for 18-24 h, and activating twice, wherein lactobacillus is cultured in aerobic environment, and Bifidobacterium is cultured in anaerobic environment. Specifically, the seed culture medium comprises the following components: 1% of peptone, 0.8% of beef extract powder, 0.4% of yeast extract powder, 2% of glucose, 0.1% of sorbitan monooleate, 0.2% of dipotassium phosphate, 0.2% of ammonium citrate tribasic, 0.5% of sodium acetate trihydrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate tetrahydrate, 0.05% of L-cysteine hydrochloride and 95% of double distilled water.
According to some embodiments of the invention, the fermented oat liquid comprises enzymatically hydrolyzed oat flour and water; or the fermented oat liquid comprises enzymolysis oat powder, yeast extract powder and water; wherein the content of the enzymolysis oat powder in the fermented oat liquid is 6-15% (w/v), and the content of the yeast extract powder is 0-5% (w/v). The enzymolysis oat flour is obtained by soaking oat grains, homogenizing, carrying out enzymolysis on alpha-amylase, beta-amylase and pullulanase, carrying out enzyme inactivation and carrying out spray drying.
According to some embodiments of the invention, the probiotic is inoculated in an amount of 1% to 3%.
According to some embodiments of the invention, the incubation time is 18h-24h and the incubation temperature is 30-37 ℃. In the invention, the probiotics are firstly activated, the activated probiotics are inoculated into the fermented oat liquid in the inoculation amount of 1-3%, and the fermented oat liquid is cultured in a constant temperature box for 18-24 h at the temperature of 30-37 ℃.
According to some embodiments of the invention, the fermented oat solution is sheared, homogenized, centrifuged, and the supernatant is collected after the cultivation is completed, and the supernatant is sterilized and stored to obtain the lysate.
According to some embodiments of the invention, the shear rotation speed is 2500-; the homogenization is carried out for 2-4 cycles at 100-400bar and for 5-12 cycles at 600-1500bar, wherein 1 cycle refers to the time from the liquid entering the homogenizer to the discharge.
According to some embodiments of the invention, the preparation method specifically comprises the steps of:
(1) inoculating 1-3% of probiotics into a seed culture medium, culturing at 37 ℃ for 18-24 h, and activating twice, wherein lactobacillus is cultured in an aerobic environment, and bifidobacterium is cultured in an anaerobic environment;
(2) and eluting the activated probiotic bacteria liquid for three times by using normal saline, and transferring the activated probiotic bacteria liquid into the fermentation substrate fermented oat liquid by using the inoculation amount of 1-3% after heavy suspension. Culturing in a constant temperature box for 18-24 h at 30-37 deg.C;
(3) shearing the fermented oat liquid for 15-20min at 2500-3000rpm by a high-speed shearing machine;
(4) homogenizing the fermented oat liquid by a homogenizer, and homogenizing for 2-4 cycles at a first-stage pressure of 100-;
(5) centrifuging the fermented oat homogeneous solution at 8000-12000 rpm for 5-10 min to obtain light yellow clear supernatant; adjusting pH to 4.0-4.5 with 10% sodium hydroxide solution;
(6) sterilizing the supernatant at 90-100 deg.C for 10-15min, placing in brown bottle, and refrigerating for storage.
In a second aspect, the present invention provides a lysate prepared by the above-described preparation method.
According to further embodiments of the invention, the lysate comprises: antioxidant enzyme, antioxidant substance, antibacterial substance and other active substances; wherein the antioxidant enzyme comprises at least one of superoxide dismutase and glutathione peroxidase; the antioxidant substance comprises at least one of polysaccharide, beta-glucan and polyphenol compound; the bacteriostatic substance comprises at least one of lactic acid, acetic acid and small molecular polypeptide; the other active substance comprises at least one of amino acid and lipoteichoic acid. The lysate prepared by the preparation method provided by the invention has various component types and outstanding effect, can adjust the microecological balance of skin, effectively eliminate free radicals, improve the activity of antioxidant enzyme of the skin, inhibit skin pathogenic bacteria, reduce skin wrinkles and delay aging.
In a third aspect, the present invention provides the use of a lysate as described above in a cosmetic, food, pharmaceutical product. Wherein the food and health care product is not limited to fruit and vegetable juice, protein beverage, milk-containing beverage, fermented milk-containing beverage, and plant beverage; the cosmetic is not limited to cream, toner, eye cream, facial mask, essence cream, spray. The medicine is not limited to external ointment, external spray and oral liquid products.
The invention is further illustrated by the following specific examples.
Example 1 preparation of probiotic fermented oat lysate
The probiotic fermentation oat lysate protocol is shown in figure 1.
(1) Seed activation: inoculating lactobacillus paracasei seed liquid into MRS liquid culture medium (OXOID) according to the proportion of 1%, and aerobically standing and culturing for 24h at 37 ℃; this step was followed for activation twice. Centrifuging the activated bacteria liquid for 10min by a high-speed centrifuge at the rotating speed of 8000rpm to obtain high-activity bacteria; eluting with 0.9% physiological saline three times, adjusting the concentration to 1 x 109CFU/ml；
(2) Preparing a fermentation culture medium: weighing 8g of enzymolysis oat powder and 2g of food-grade yeast extract powder, supplementing 100mL of distilled water, stirring in a magnetic stirrer at 50 ℃ for 20min, homogenizing by a homogenizer, selecting a first-stage pressure of 200bar, homogenizing for 2 cycles, and sterilizing at 90 ℃ for 10 min;
(3) fermenting probiotics: inoculating lactobacillus paracasei into a fermentation culture medium by 1 percent of inoculation amount, and standing and culturing for 24 hours at 37 ℃;
(4) high-speed shearing: shearing the cultured fermented oat liquid for 20min by a high-speed shearing machine, wherein the shearing rate is 2500 rpm;
(5) high-pressure homogenization: homogenizing the fermented oat liquid by a homogenizer, and homogenizing for 4 cycles at a first-stage pressure of 200bar and for 10 cycles at 850 bar;
(6) centrifuging and inactivating: and centrifuging the fermented oat homogeneous solution for 5min at 8500rpm of a high-speed centrifuge to obtain a light yellow and clear supernatant. The pH was adjusted to 4.2 with 10% sodium hydroxide solution. The supernatant was sterilized and sterilized at 90 ℃ for 10 minutes. Placing in a brown bottle, and storing at 4 ℃;
(7) and (3) detecting microorganisms: the supernatant was tested for total viable and pathogenic bacteria. The total number of the live bacteria is less than 10CFU/mL, the pathogenic bacteria are not detected, and the requirements that the total number of the cosmetics in the cosmetic hygiene standard GB7916-87 is less than 1000CFU/mL are met.
Comparative examples 1-10 were obtained by searching and optimizing the process, and Lactobacillus paracasei was selected as the probiotic.
Comparative example 1
A probiotic fermented oat lysate was prepared as described in example 1, except that the oat concentration in step (2) was changed to 6%.
Comparative example 2
A probiotic fermented oat lysate was prepared as described in example 1, except that the oat concentration in step (2) was changed to 10%.
Comparative example 3
A probiotic fermented oat lysate was prepared as described in example 1, except that the oat concentration in step (2) was changed to 12%.
Comparative example 4
A probiotic fermented oat lysate was prepared as described in example 1, except that the oat concentration in step (2) was changed to 15%.
Comparative example 5
A probiotic fermented oat lysate was prepared as described in example 1, except that the yeast extract concentration in step (2) was changed to 0.
Comparative example 6
A probiotic fermented oat lysate was prepared as described in example 1, except that the yeast extract concentration in step (2) was changed to 5%.
Comparative example 7
A probiotic fermented oat lysate is prepared as in example 1, except that the homogenization step in step (5) is changed to a first pressure of 200bar homogenization for 4 cycles and 600bar homogenization for 10 cycles.
Comparative example 8
A probiotic fermented oat lysate is prepared as in example 1, except that the homogenization step in step (5) is changed to a first pressure of 200bar homogenization for 4 cycles and 1500bar homogenization for 10 cycles.
Comparative example 9
A probiotic fermented oat lysate was prepared as described in example 1, except that the centrifugation speed in step (6) was changed to 3000 rpm.
Comparative example 10
A probiotic fermented oat lysate was prepared as described in example 1, except that the centrifugation rate in step (6) was changed to 0.
The processes of example 1 and comparative examples 1 to 10 were compared by measuring the pH, number of bacteria and two antioxidant capacities after lactobacillus paracasei fermentation. SOD is superoxide dismutase, and T-AOC is total antioxidant capacity. The antioxidant index is determined by using a kit purchased by Nanjing manufacturer according to an operation instruction.
The results of comparing the fermentation conditions, the antioxidant ability, and the lysate morphology of example 1 with those of comparative examples 1 to 10 are shown in Table 1.
TABLE 1 comparison of probiotic fermented oat lysates prepared by different processes
Example 1 in comparison with comparative examples 1-4, it was found that the higher the number of bacteria of the probiotic bacteria, the stronger the antioxidant capacity as the concentration of oats was increased. When the oat concentration was 8%, the antioxidant capacity of the lysate had reached a higher level. By combining the cost performance of the process materials, the effectiveness of high-pressure homogenization and the oxidation resistance, the oat concentration of 8% is preferably selected.
Example 1 in comparison with comparative examples 5, 6, it was found that yeast extract could promote the proliferation of probiotics, but the lysate obtained in comparative example 6 was darker and, considering the wide range of applications, optimally 2% yeast extract was chosen.
Example 1 in comparison to comparative examples 7 and 8, it was found that the antioxidant capacity of the lysate increased with increasing homogenization pressure, but there was no significant difference. In view of energy consumption, machine maintenance and product cost performance during large-scale production, the homogenizing pressure is preferably 850 bar.
In example 1 and comparison with comparative examples 9 and 10, it can be seen that after homogenization, centrifugation is required to obtain a clarified solution, and macromolecular substances and disrupted cell products which are not easily absorbed and utilized are removed. The non-centrifuged and 3000rpm centrifuged group lysates contained flocculent precipitates, and optimally the centrifugation speed was 8500rpm, considering the universality and effectiveness of the application.
The conditions in example 1 are considered superior based on a comparison of example 1 with 10 comparative examples.
Example 2 antioxidant performance assay of probiotic fermented oat lysate
Four lysates were obtained by separately fermenting oat base with four different species (bifidobacterium longum, bifidobacterium infantis, lactobacillus paracasei and lactobacillus plantarum) according to the preparation method described in example 1. Wherein Bifidobacterium longum and Bifidobacterium infantis are cultured in anaerobic mode.
The four lysates were subjected to antioxidant performance measurements, including hydroxyl radical scavenging assay (. OH), DPPH radical scavenging assay, total antioxidant capacity assay (T-AOC), superoxide dismutase (SOD). The principle of the colorimetric method adopted in the DPPH free radical scavenging test is that the free radical scavenger provides an electron to be paired with a lone pair of electrons of DPPH free radicals, so that the purple of the free radical scavenger is changed into yellow, the absorbance at the wavelength of 517nm is reduced, and the change degree of the free radical scavenger and the free radical scavenging degree are in a linear relation, namely, the stronger the scavenging capability of the free radical scavenger is, the smaller the absorbance is. Other indexes were determined by using a kit purchased from Nanjing Kangji according to the instructions.
Table 2 determination of the antioxidant capacity of probiotic lysates
Note that: the substrate lysate refers to a culture medium prepared from oat and yeast powder, and the supernatant obtained by the same steps is fermented without adding probiotics. Product S is a commercially available lysate product.
The results are shown in table 2, fig. 2, and fig. 3. The data show that oat lysate fermented with four different species has a stronger antioxidant capacity compared to the positive control (commercial product S and 0.05% vitamin C). The lysate presented in the patent can effectively remove hydroxyl free radicals and DPPH free radicals, and the removal capacity is higher than 94%; the lysate has excellent total antioxidant capacity and superoxide dismutase activity, the total antioxidant capacity is higher than 17U/mL, and the superoxide dismutase activity is higher than 25U/mL; wherein the Bifidobacterium longum lysate has the highest superoxide dismutase capacity; the total antioxidant capacity of lactobacillus plantarum lysate is strongest.
Therefore, the probiotic lysate can efficiently remove free radicals, improve the activity of antioxidant enzyme and effectively relieve skin oxidation and aging.
Example 3 product analysis of probiotic fermented oat lysate
This example analyzes functional components such as antioxidant components and amino acid components in probiotic fermented oat lysate. The antioxidant component comprises polyphenol compounds and polysaccharide; the amino acid detection types are glutamic acid, aspartic acid, histidine, threonine, methionine, proline and glycine.
(1) Detection method of polyphenol compounds
The lysate is centrifuged through a membrane, the supernatant is extracted with 90% acetone and the proteins, saccharides and the like are precipitated, a standard curve is prepared with pyrogallic acid, the standard curve and hydroxyl groups in the sample are oxidized by a folin phenol reagent to make the standard curve blue, and the content (in terms of phenolic hydroxyl molar concentration) is determined by measuring the absorbance value with an ultraviolet spectrophotometer.
(2) Polysaccharide detection method
The lysate was ethanol precipitated to remove polysaccharides and the residue was washed several times with 80% ethanol. Dissolving the residue with water, hydrolyzing with phenol-sulfuric acid method (glucose curve synchronous hydrolysis, result is calculated by glucose), and measuring absorbance value with ultraviolet spectrophotometer to determine content.
(3) Amino acid detection method
Each amino acid was auto-derived by pre-column sampler, separated by amino acid analytical column, detected by uv detector (and/or fluorescence detector), qualitative by retention time, and quantitative by peak area external standard.
TABLE 3 analysis of lysate functional Components
And (3) detection results:
the selected fermentation strains of this example were bifidobacterium longum, bifidobacterium infantis, lactobacillus paracasei and lactobacillus plantarum, and lysates were prepared according to the method in example 1. Commercial product S was used as a control. The results are shown in Table 3. The lysate contains polyphenol compounds, and the polyphenol has strong antioxidant activity; the polysaccharide comprises oat polysaccharide and polysaccharide substances generated by metabolism of probiotics. Research has shown that polysaccharides have antioxidant effects. The four lysates contained higher levels of polyphenols and polysaccharides than product S. Wherein, the lactobacillus plantarum lysate has the highest content of polyphenols and polysaccharides.
Four lysates had a variety of amino acids, all in higher amounts than product S. The amino acid has excellent antioxidant function, and can repair skin barrier and maintain skin health. Glutamic acid and histidine help to prevent UV damage, aspartic acid can build up skin DNA; threonine promotes the synthesis of elastin and collagen; methionine can remove free radical and delay senility; proline helps to reduce wrinkles in the skin canthus; glycine is beneficial for skin healing.
Example 4 bacteriostatic ability analysis of probiotic fermented oat lysate
This example analyzes the bacteriostatic components of the lysate and simultaneously determines the inhibitory ability of the lysate against pathogenic bacteria.
(1) Method for detecting bacteriostatic component
The lysate is converted into esters after derivatization, extracted by isooctane, and separated by a gas chromatographic column, and is qualitative by retention time and quantitative by an external standard method. The detection indexes are lactic acid, acetic acid and formic acid.
(2) Determination of the inhibitory Capacity of lysates on pathogenic bacteria
Preparing a pathogenic bacterium liquid: inoculating a seed solution into a nutrient broth culture medium by taking staphylococcus aureus ATCC6538 and escherichia coli ATCC8739 as indicator bacteria, and performing shake culture at 37 ℃ for 24 hours; the activated bacteria liquid is transferred to a fresh nutrient broth culture medium by 3 percent and cultured for 6 hours by shaking. Adjusting the bacterial liquid concentration of the pathogenic bacteria to OD 0.1 for later use.
Lysate bacteriostatic ability assay: transferring the culture solution with OD of 0.1 into nutrient broth (sample group) containing 10% lysate and nutrient broth (blank group) alone, aerobically culturing at 37 deg.C for 16h, and measuring OD of each group600Value and number of pathogens. The rate of suppression of pathogenic bacteria by the lysate was calculated.
The formula is 1- (number of bacteria in sample group/number of bacteria in blank control group) × 100%.
TABLE 4 lysate analysis of bacteriostatic composition
TABLE 5 inhibitory Properties of lysates on pathogenic bacteria
The analysis of the bacteriostatic components is shown in Table 4. The lysates differ in lactic acid, acetic acid, and formic acid content. The bacteriostatic content of the four lysates was higher than that of the matrix lysate and product S. Among them, bifidobacterium longum and lactobacillus plantarum lysate have the highest lactic acid content, and bifidobacterium infantis lysate has the most abundant acetic acid content.
The results of the determination of the inhibitory ability of the lysate against pathogenic bacteria are shown in Table 5 and FIG. 4. The data show that the four probiotic lysates provided by the embodiment have inhibitory activity on staphylococcus aureus and escherichia coli, and the inhibitory rate is as high as 71.96% -79.53%; the inhibition was higher than that of matrix lysate and product S.
Therefore, the lysate in the embodiment has rich bacteriostatic components, thereby facilitating the storage of products and preventing the pollution of mixed bacteria; on the other hand, the skin moisturizing agent can inhibit skin pathogenic bacteria and moisturize skin, and strengthen skin surface.
Example 5 probiotic fermented oat lysate in skin keratinocyte cytotoxicity assay
Bifidobacterium longum, Bifidobacterium infantis and Lactobacillus paracasei fermented oat lysate are selected for detecting skin keratinocyte cytotoxicity.
1. Cytotoxicity detection method
(1) Inoculating keratinocyte into 96-well plate at inoculation density of 1.00E +04 cells/well, culturing in incubator (37 deg.C, 5% CO)295% RH) overnight. The experiment was set up with a blank control group, a positive control group and a sample group. In the sample set, 8 concentration gradients (0.0078%, 0.0156%, 0.0313%, 0.0625%, 0.125%, 0.25%, 0.5%, and 1%) were set for each sample, and 3 replicates were set for each concentration gradient.
(2) And (3) administration is carried out when the cell plating rate in the 96-well plate reaches 40-60%. Adding 200 mu L of cell culture solution into each hole of the blank control group; adding 200 mu L of culture solution containing 10% DMSO into each well of the positive control group; add 200. mu.L of culture medium containing lysate of the corresponding concentration to each well of the sample set; null wells were seeded without cells and only 200 μ L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO)295% RH).
(3) After 24h of cell incubation culture, the supernatant was discarded, MTT working solution (0.5mg/mL, now ready for use) was added, incubation was carried out at 37 ℃ in the dark for 4h, after the incubation was completed, the supernatant was discarded, 150. mu.L of DMSO was added to each well, and the OD was read at 490 nm.
(4) Calculating the relative activity of the cells: calculated according to the following formula:
relative cell viability (%) × (sample well OD-zero well OD)/(blank control well OD-zero well OD) × 100%.
2. Results of cytotoxicity assay
Each sample was set to 8 administration concentrations (0.0078%, 0.0156%, 0.0313%, 0.0625%, 0.125%, 0.25%, 0.5%, and 1%) and cytotoxicity test experiments were performed on keratinocytes, the MTT test results are shown in table 6, and the cell viability trend is shown in fig. 5 to 7.
TABLE 6 probiotic lysate MTT test results (cell viability)
The results showed that the cell viability was higher than 90% in all three groups of samples at sample concentrations equal to or lower than 0.5%. At a sample concentration of 1%, cell viability was about 82%. Based on the MTT assay results, the following 5 concentrations (0.0625%, 0.125%, 0.25%, 0.5%, and 1%) were selected for morphological examination for all three samples.
Example 6 probiotic fermentation of oat lysate on skin keratinocyte morphological examination
Bifidobacterium longum, Bifidobacterium infantis and Lactobacillus paracasei fermented oat lysate are selected for carrying out morphological detection on skin keratinocytes.
1. Morphological detection method
(1) Cell inoculation: and (3) setting a sample group and a solvent control group, wherein each group is provided with two multiple holes. The cells were seeded at the corresponding seeding density in 24-well plates, incubators (37 ℃ C., 5% CO)295% RH) overnight.
(2) Preparing liquid: according to the MTT detection result, determining the morphological observation concentration of the detection sample, and preparing the working solution of the test object with different concentrations (0.0625%, 0.125%, 0.25%, 0.5% and 1%).
(3) Administration: when the cell plating rate of the 24-well plate reached 40%, the administration was carried out, the lysate was added to the sample group at different concentrations, the keratinocyte culture solution was added to the solvent control group, and the incubator (37 ℃, 5% CO)295% RH) for 24 h.
(4) And (3) cell observation: after incubation, cell morphology was observed under a microscope and photographed.
2. Results of morphological examination
The results of the morphological examination are shown in FIGS. 8-10.
When the concentration of the bifidobacterium longum lysate is 0.5%, the relative activity value of the cells is 90.14%, and the cell state is not obviously different from that of a solvent control group under a microscope. Therefore, based on the MTT results and morphological results, the samples showed no significant cytotoxicity to keratinocytes at a concentration range of 0.5%.
When the concentration of the bifidobacterium infantis lysate is 0.5%, the relative activity value of the cells is 91.77%, and the cell state is not obviously different from that of a solvent control group under a microscope. Therefore, based on the MTT results and morphological results, the samples showed no significant cytotoxicity to keratinocytes at a concentration range of 0.5%.
At a concentration of 0.5% of the Lactobacillus paracasei lysate, the relative viability value of the cells was 90.13%, and the cell status was not significantly different from that of the solvent control group under a mirror microscope. Therefore, based on the MTT results and morphological results, the samples showed no significant cytotoxicity to keratinocytes at a concentration range of 0.5%.
Example 7 evaluation of efficacy of probiotic fermented oat lysate on skin keratinocytes
Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus paracasei fermented oat lysate were selected for evaluation of skin keratinocyte efficacy.
1. Efficacy evaluation method
And the active oxygen content (ROS) is selected as a detection index for the efficacy evaluation. The method comprises the following steps:
(1) cell inoculation: the keratinocytes were inoculated in 6 well plates at a cell inoculum size of 2.8E + 05/well, incubators (37 ℃ C., 5% CO)295% RH) overnight.
(2) Preparing liquid: the test article working solutions were prepared in accordance with the experimental groups (table 7).
TABLE 7 Experimental groups
(3) Administration: according to the experimental grouping design of table 7, when the plating rate of the cells in the 6-well plate reaches 40% -60%, the grouped drug delivery is carried out, the drug delivery amount of each hole is 2mL, and each group is provided with 3 multiple holes. Incubator (37 ℃, 5% CO)295% RH) for 24 h.
(4) UVB irradiation: the groups requiring UVB irradiation were subjected to UVB irradiation (irradiation dose 300 mJ/cm) in experimental groups (Table 7)2)。
(5) ROS content detection
a) After irradiation, each well of cells was washed directly with PBS 3 times;
b) flow detection: add 1mL of DCFH-DA probe in 10. mu.M active oxygen kit per well, incubate (37 ℃ C., 5% CO)295% RH) for 30 min; discarding culture solution containing DCFH-DA, washing with PBS for 3 times, after pancreatin (0.25%) digests cells, washing cells with PBS for 1 time, adding 0.5mL fresh PBS, and performing flow detection;
c) and (4) analyzing results: the MFI (mean fluorescence intensity) of each group was pooled and subjected to statistical analysis.
Results statistical analysis was plotted using GraphPad Prism Program software, and t-test statistical analysis was performed on the sample and control groups, with P <0.05 indicating significant difference and P <0.01 indicating significant difference.
2. Detection result of content of Reactive Oxygen Species (ROS)
Based on the test method, the ROS content was measured, the ROS content measurement results are shown in table 8, and the change in ROS content among the groups is shown in fig. 11:
TABLE 8 summary of Reactive Oxygen Species (ROS) content measurements
Note: summarizing MFI (mean fluorescence intensity) values of the samples, and when the statistical analysis is carried out by a t-test method, the significance of a negative control group is represented by # compared with a blank control group, the p value is less than 0.05 and is represented by #, and the p value is less than 0.01 and is represented by #; the significance of the sample group and the positive control group was expressed as x, p value <0.05 was expressed as x, and p value <0.01 was expressed as x, compared to the negative control group.
Compared with a blank control group, the active oxygen content of the negative control group is obviously increased, which indicates that the UVB stimulation condition of the experiment is effective. Compared with a negative control group, the active oxygen content of the positive control VE is obviously reduced under the administration concentration of 0.05 percent, which indicates that the experiment is effective. Compared with the negative control group, the active oxygen content of the three samples is extremely reduced at the administration concentration of 0.5%. Three samples (bifidobacterium longum lysate, bifidobacterium infantis lysate, lactobacillus paracasei lysate) were shown to have a significant inhibitory effect on the reactive oxygen species produced by UVB stimulation.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method of producing a lysate, comprising: inoculating probiotics into fermented oat liquid for culture, centrifuging the fermented oat liquid after culture is finished, and taking supernatant, wherein the supernatant is lysate.
2. The method of claim 1, wherein the probiotic bacteria are lactobacilli and/or bifidobacteria;
the lactobacillus comprises one or more of lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus casei, lactobacillus paracasei, lactobacillus gasseri, lactobacillus reuteri, lactobacillus curvatus and lactobacillus crispatus; the Bifidobacterium comprises one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis and Bifidobacterium adolescentis.
3. The preparation method according to claim 1, wherein the fermented oat liquid comprises enzymolyzed oat flour and water;
or the fermented oat liquid comprises enzymolysis oat powder, yeast extract powder and water;
wherein the content of the enzymolysis oat powder in the fermented oat liquid is 6-15% (w/v), and the content of the yeast extract powder is 0-5% (w/v).
4. The method according to claim 1, wherein the probiotic bacteria are inoculated in an amount of 1-3%.
5. The method according to claim 1, wherein the culturing time is 18 to 24 hours and the culturing temperature is 30 to 37 ℃.
6. The method according to claim 1, wherein the fermented oat solution is sheared and homogenized after completion of the cultivation, and then centrifuged to obtain a supernatant, and the supernatant is sterilized and stored to obtain the lysate.
7. The method as claimed in claim 6, wherein the shearing speed is 2500-3000rpm, and the shearing time is 15-20 min; the homogenization is carried out for 2-4 cycles at 100-400bar and for 5-12 cycles at 600-1500 bar.
8. A lysate produced by the production method according to any one of claims 1 to 7.
9. The lysate of claim 8, comprising therein:
antioxidant enzyme, antioxidant substance, antibacterial substance and other active substances;
wherein the antioxidant enzyme comprises at least one of superoxide dismutase and glutathione peroxidase;
the antioxidant substance comprises at least one of polysaccharide, beta-glucan and polyphenol compound;
the bacteriostatic substance comprises at least one of lactic acid, acetic acid and small molecular polypeptide;
the other active substance comprises at least one of amino acid and lipoteichoic acid.
10. Use of a lysate as claimed in claim 8 or 9, wherein the lysate is used in cosmetics, food, pharmaceuticals.
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