CN116459167A - Preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel - Google Patents
Preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel Download PDFInfo
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- CN116459167A CN116459167A CN202310460600.5A CN202310460600A CN116459167A CN 116459167 A CN116459167 A CN 116459167A CN 202310460600 A CN202310460600 A CN 202310460600A CN 116459167 A CN116459167 A CN 116459167A
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- fermentation product
- poria cocos
- hydrogel
- peach gum
- lysate
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Abstract
The invention discloses a preparation method of lactobacillus/peach gum and poria cocos fermentation product lysate hydrogel, which comprises the following steps: (1) preparing culture solution containing peach gum and poria cocos; (2) activating strains; (3) fermenting and culturing lactobacillus to obtain a fermentation product; (4) homogenizing by high-pressure micro-jet to obtain lysate of fermentation product; (5) preparation of fermentation product lysate hydrogel. The invention creatively utilizes a large amount of active ingredients such as plant polysaccharide, protein, amino acid and the like in the lactic acid bacteria fermentation product lysate, peach gum and poria cocos, and then adds the novel fermentation product xanthan gum, and the three-dimensional network hydrogel is formed by physical cross-linking and winding of a large amount of hydroxyl groups, carboxyl groups and amino groups in molecules in a hydrogen bond and other modes, so that the purposes of increasing the stability of protein, polypeptide active molecules and suspension systems are achieved, the low-temperature storage is not needed, no sediment is generated after long-term storage, and the stability is higher.
Description
Technical Field
The invention relates to the technical field of lysate of probiotics fermentation products, in particular to a preparation method of lysate hydrogel of lactobacillus peach gum and poria cocos fermentation products.
Background
The lactobacillus fermentation product lysate contains thalli and all active ingredients synthesized in the fermentation process, contains peptidoglycan, teichoic acid, protein, phospholipid, sterol, fatty acid, various enzymes, peptide, amino acid, nucleotide, extracellular polysaccharide and the like, not only provides rich nutrition for skin, but also has good anti-aging effect, and is widely applied to the field of cosmetic skin care products.
Peach gum is a gelatinous substance secreted by peach trees for faster wound healing, mainly contains glucuronic acid and galactose, also contains plant collagen, fat, carbohydrate and the like, is also a biological sugar collagen material, is a medical and aesthetic raw material similar to xanthan gum or Arabic gum in similar purposes, can reduce wrinkles and tender skin, and has certain effects of maintaining beauty and protecting skin. Poria is a fungus of Poria genus of Polyporaceae of Basidiomycetes, is rich in various effective components including pachymaran, triterpenes, lauric acid, ergosterol, adenine and choline, and has skin caring effects of whitening skin, resolving macula and eliminating wart. Based on the two-way fermentation technology, the synergistic effect of medicinal and edible plant peach gum and poria cocos on a lactobacillus liquid fermentation system is researched, so that more products with effects and environmental friendliness are developed, and the method has a wide market prospect. The term "bi-directional fermentation" refers to the use of Chinese medicinal materials or residues with certain active ingredients as drug-property matrix instead of conventional nutrient matrix, and adding preferred strains for microbial transformation. The bidirectional property of the probiotic bacteria is reflected in that the drug-resistant matrix provides nutrition required by the probiotics, and meanwhile, the probiotic bacteria are influenced by enzymes in the probiotics to change the tissues and components of the probiotic bacteria, so that a new taste function is generated, and the probiotic bacteria has the characteristics of higher automation degree, high substance transfer, good inheritance, strong artificial controllability, easier enrichment of active ingredients and easier industrial production, and fermentation liquor can be directly used as a raw material of cosmetics.
However, in the prior art, as raw materials in the catalogue of the cosmetic record, the related products of lactobacillus are mostly fermentation filtrate and lysate of fermentation products, the former is centrifuged to filter out lactobacillus cells, the latter is obtained after breaking the wall of the lactobacillus, but more active ingredients are remained, but more water-soluble suspension exists, layering and sedimentation can occur in the placing process, and the products need to be uniformly shaken before use, so that the problems of inconvenient use and uneven content in the formula, and finally the product appearance and the use experience are affected. In addition, lactic acid bacteria are anaerobic bacteria, certain anaerobic conditions are needed in production, meanwhile, the nutrition requirement is very strict, in order to increase the number of living bacteria or prolong the survival time of the living bacteria, nutrient solution such as beef broth and the like are sometimes needed to be added into a common MRS culture medium, so that the production cost is greatly increased, and therefore, the preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel is provided to solve the problems.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above-mentioned problems with the prior art lactic acid bacteria fermentation product lysate.
Therefore, the invention aims to provide a preparation method of lactobacillus peach gum and poria cocos fermentation product lysate hydrogel, which creatively utilizes a large amount of active ingredients such as plant polysaccharide, protein, amino acid and the like in the lactobacillus fermentation product lysate, peach gum and poria cocos, then adds in novel fermentation products such as xanthan gum and guar gum, and forms a three-dimensional network hydrogel through physical cross-linking and winding of a large amount of hydroxyl groups, carboxyl groups and amino groups in molecules in a manner of hydrogen bonding and the like, thereby achieving the purpose of increasing the stability of protein and polypeptide active molecules and suspension systems, and achieving the advantages of no need of low-temperature storage, no sediment after long-term storage and higher stability.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel comprises the following steps:
step S1, preparing 5% peach gum solution: 50g of peach gum is taken, washed and decontaminated, and then added with 950g of NaCO with the concentration of 0.1mol/L 3 And NaHCO 3 Soaking the solution at 80-90deg.C, hydrolyzing for 4 hr, pulverizing, filtering under reduced pressure, adjusting pH to 6.0-7.0 with dilute hydrochloric acid solution, and sterilizing to obtain 5% peach gum solution;
step S2, preparing 10% of poria cocos extracting solution: reflux-extracting Poria sclerotium powder 100g with 70% ethanol 500g in 95deg.C water bath for 3 hr, cooling to room temperature, vacuum filtering, extracting for 2 times, mixing filtrates, concentrating the filtrate under reduced pressure to recover ethanol, dissolving in water, filtering, adding water to 1000g, and sterilizing to obtain Poria extractive solution;
step S3, preparing a culture solution: respectively weighing 5% peach gum solution and 10% Poria cocos extract according to the weight ratio of 5%, adding into MRS liquid culture medium, and sterilizing to obtain culture solution;
step S4, preparing lysate of lactobacillus fermentation products:
pure seed activation: taking lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 20-30 hours at the temperature of 30-42 ℃ to obtain a seed culture medium;
fermentation culture: inoculating the activated seed culture medium into a culture solution containing peach gum and poria cocos according to a proportion of 1% -5%, and culturing at 30-42 ℃ for 20-30 hours to obtain a lactobacillus fermentation product;
the preparation of lactobacillus fermentation product lysate comprises taking lactobacillus fermentation product, homogenizing 1 time with a micro-jet high pressure homogenizer 18000-20000psi, homogenizing 2 times under the condition of homogenizing pressure 23000-25000psi, and controlling the temperature at 0-10deg.C to obtain lactobacillus fermentation product lysate.
Step S5, preparing a lysate hydrogel of the lactobacillus fermentation product:
adding functional auxiliary agent into the lysate of lactobacillus fermentation product, stirring uniformly, weighing xanthan gum accounting for 0.3% of the total amount and guar gum accounting for 0.3% of the total amount, mixing uniformly, spreading on the liquid surface, and stirring at room temperature to dissolve completely to obtain yellowish transparent hydrogel.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the concentration of the peach gum solution in the step S1 is 2% -8%, and the addition amount of the peach gum in the culture solution is 0.1% -0.5%.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the concentration of the poria cocos extracting solution in the step S2 is 5% -15%, and the adding amount of the poria cocos extracting solution in the culture solution is 0.3% -0.7%.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the MRS medium described in the step S3 includes: vitamin C, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, cysteine, bifidobacterium, yeast extract, beef extract, soybean peptone and beef peptone.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the addition amount of the seed culture medium in the step S4 is 1% -5% of the culture solution.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the micro-jet high-pressure homogenization times in the step S4 are 2-5 times, wherein the homogenization pressure gradient is increased, the homogenization is firstly carried out for 1 time by 18000-20000psi, then the homogenization pressure is increased to 23000-25000psi for 2 times, and the temperature is controlled to be 0-10 ℃.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the hydrogel material in step S5 includes one or more of xanthan gum, guar gum, carbomer, and sodium carboxymethyl cellulose.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the functional auxiliary agent in the step S5 comprises a preservative and a flavoring agent.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the preservative comprises one or more of sodium benzoate, potassium sorbate, sodium dehydroacetate, phenoxyethanol and ethyl hexyl glycerol.
As a preferable scheme of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, the preparation method comprises the following steps: the flavoring agent comprises one or more of peppermint essence, lavender essence, jasmine essence and rose essence.
The invention has the beneficial effects that:
1. the invention adopts a bidirectional liquid fermentation technology, combines the advantages of traditional Chinese medicine and modern biological fermentation technology, and develops the cosmetic raw material with Chinese characteristics.
2. According to the invention, natural peach gum and poria cocos rich in active ingredients such as polysaccharide, plant collagen and fatty carbohydrate are selected as medicinal and edible traditional Chinese medicines to be added into a traditional MRS culture medium, so that the natural peach gum and poria cocos can be used as nutritional ingredients of probiotics, namely lactobacillus, on one hand, the growth of living bacteria is promoted (protein nutrient solution such as beef broth is not required to be added into the MRS culture medium), and on the other hand, the active ingredients of peach gum and poria cocos are further metabolized and decomposed by utilizing lactobacillus, so that a synergistic effect is achieved.
3. The invention creatively utilizes a large amount of active ingredients such as plant polysaccharide, protein, amino acid and the like in the lactic acid bacteria fermentation product lysate, peach gum and poria cocos, and then adds the novel fermentation product xanthan gum, and the three-dimensional network hydrogel is formed by physical cross-linking and winding of a large amount of hydroxyl groups, carboxyl groups and amino groups in molecules in a hydrogen bond and other modes, so that the purposes of increasing the stability of protein, polypeptide active molecules and suspension systems are achieved, the low-temperature storage is not needed, no sediment is generated after long-term storage, and the stability is higher.
4. The hydrogel prepared by the method has the advantages of reversibility, good water solubility, convenience in direct use of the formula, simple production process and large-scale production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic diagram showing the effect of different amounts of peach gum added in the preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel on lactic acid bacteria growth;
FIG. 2 is a schematic diagram showing the effect of different amounts of Poria cocos extracts added to MRST culture solution containing 0.25% peach gum on lactobacillus growth;
FIG. 3 is a schematic diagram showing the detection result of flat live bacteria of lactic acid bacteria fermentation products before and after adding traditional Chinese medicines into MRS culture solution of the preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel;
FIG. 4 is a schematic diagram of microscopic photographs before and after homogenization of lactic acid bacteria fermentation products of the preparation method of lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel of the present invention;
FIG. 5 is a schematic diagram showing the detection results of live bacteria on a flat plate before and after homogenization of a lactic acid bacteria fermentation product by the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel;
FIG. 6 is a photograph of a lactic acid bacterium fermentation product lysate and a hydrogel thereof of the preparation method of the lactic acid bacterium peach gum and poria cocos fermentation product lysate hydrogel of the present invention;
FIG. 7 is a graph showing the clearance of DPPH free radicals from different samples of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel;
FIG. 8 is a graph showing the removal rate of superoxide anion radical by different samples of the preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Further, in describing the embodiments of the present invention in detail, the cross-sectional view of the device structure is not partially enlarged to a general scale for convenience of description, and the schematic is only an example, which should not limit the scope of protection of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in actual fabrication.
Example 1
And (3) examining the influence of the addition amount of peach gum in the MRS culture solution on the growth of lactobacillus:
(1) Preparation of 5% peach gum solution: taking 50g of peach gum, cleaning, removing impurities, adding 950g of NaCO3 and NaHCO3 solution (weight ratio of 1:1, pH 10-11) with the concentration of 0.1mol/L, soaking at 80-90 ℃ for 4 hours, hydrolyzing, crushing, filtering under reduced pressure, regulating the pH value to 6.0-7.0 by using a dilute hydrochloric acid solution, and sterilizing to obtain a 5% peach gum solution;
(2) Preparing a culture solution: respectively taking 0,0.5,1.5,5 g and 10g of 5% peach gum solution, adding an MRS liquid culture medium to 100g, and sterilizing to obtain an MRST culture solution;
(3) Pure seed activation: taking lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 24 hours at the temperature of 30-42 ℃ to obtain a seed culture medium;
(4) Fermentation culture: 1g of activated seed culture medium is taken and inoculated into the different 100g of MRST culture solutions, and the culture is carried out for 24 hours at the temperature of 30-42 ℃ to obtain lactobacillus fermentation products with different peach gum addition amounts.
As a result, the increase of the addition amount of the 5% peach gum solution is found, the amount of lactic acid bacteria deposited on the bottom is observed to be obviously increased by naked eyes, OD600 values (see figure 1) of different culture times are further measured on a spectrophotometer, after 24 hours of culture, the OD600 values of the MRST culture mediums added with different amounts of the peach gum solution are obviously increased compared with those of the MRST culture mediums alone, and when the addition amount of the 5% peach gum solution in the MRS culture mediums is more than 5% (accounting for 0.25% of the weight of the culture solution), the growth of the lactic acid bacteria can be obviously promoted, the increase of the peach gum dosage is continued, and the increase of the OD600 values is not obvious.
Further, anaerobic culture of viable bacteria is carried out on the sample cultured for 24 hours by adopting different dilutions on a culture medium, and the number of the viable bacteria is calculated, and is shown in Table 1:
table 15% peach gum solution different addition amounts promote the growth result of living bacteria
| 5% peach gum solution addition | OD600 | Number of viable bacteria |
| 0 | 1.024 | 1.0×10 9 |
| 0.5 | 1.329 | 1.2×10 9 |
| 1.5 | 1.504 | 1.6×10 9 |
| 5.0 | 1.895 | 2.8×10 9 |
| 10.0 | 1.923 | 3.1×10 9 |
As a result, the number of viable bacteria of the sample added with the peach gum solution after 24 hours of culture is obviously increased compared with that of the sample of the single RMS culture medium, which indicates that the addition of the peach gum solution with the mass concentration of 0.25% in the culture medium can obviously promote the growth of lactic acid bacteria and increase the number of viable bacteria.
Example 2
Examining the influence of the addition amount of the poria cocos extract in the MRST culture solution on the growth of lactic acid bacteria:
(1) Preparation of 10% Poria cocos extract: extracting Poria sclerotium powder 100g with 70% ethanol 500g under reflux in 95deg.C water bath for 3 hr, cooling to room temperature, vacuum filtering, extracting for 2 times, mixing filtrates, concentrating the filtrate under reduced pressure to recover ethanol, dissolving in water, filtering, adding water to 1000g, and sterilizing to obtain Poria extractive solution.
(2) Preparing a culture solution: 0,1.0,2.5,5, 10g of 10% Poria extract is respectively taken, MRST liquid culture medium is added to 100g, and the MRST culture solution is obtained after sterilization.
(3) Pure seed activation: taking the lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 24 hours at the temperature of 30-42 ℃ to obtain a seed culture medium.
(4) Fermentation culture: 1g of activated seed culture medium is taken and inoculated into the different 100g MRSTF culture solutions, and the culture is carried out for 24 hours at the temperature of 30-42 ℃ to obtain lactobacillus fermentation products with different peach gum addition amounts.
Taking each sample, directly measuring the OD600 value on a spectrophotometer, further carrying out anaerobic culture of living bacteria on a culture medium by adopting different dilutions of each sample, and calculating the number of the living bacteria, wherein the details are shown in fig. 2, 3 and table 2:
table 210% Poria cocos extract with different addition amounts for promoting the growth of living bacteria
| 10% of tuckahoe water extract | OD600 | Number of viable bacteria |
| 0 | 1.884 | 2.0×10 9 |
| 1.0 | 1.924 | 4.0×10 9 |
| 2.5 | 2.023 | 6.0×10 9 |
| 5.0 | 2.105 | 8.0×10 9 |
| 10.0 | 2.110 | 8.0×10 9 |
As a result, after 24 hours of culture, the OD600 value and the viable count of the samples added with different amounts of poria cocos extracts are obviously increased compared with those of the samples of the independent RMST culture medium, but the growing trend is that the samples are quick and slow, which shows that the addition of peach gum with the mass concentration of 0.25% and the poria cocos extract with the mass concentration of 0.5% into the culture medium can obviously promote the growth of lactic acid bacteria and increase the viable count.
Example 3
The influence of different bacteria breaking processes on the lysate of the fermentation product of the lactic acid bacteria peach gum and the poria cocos is examined:
(1) Pure seed activation: taking the lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 24 hours at the temperature of 30-42 ℃ to obtain a seed culture solution.
(2) Fermentation culture: adding 5% of 5% peach gum solution and 10% of Poria cocos extract into MRS liquid culture medium respectively, uniformly mixing, and sterilizing to obtain MRSTF culture solution; inoculating 1g of seed culture solution per 100g, culturing at 30-42 ℃ for 24H to obtain lactobacillus/peach gum and poria cocos fermentation products (MRSTF), respectively taking the fermentation products, homogenizing by using a high-pressure micro-jet homogenizer at different homogenizing pressures and different homogenizing times in an H10Z homogenizing cavity, and measuring OD600 values before and after homogenization, wherein the results are shown in Table 3:
TABLE 3 influence of different homogenization pressures and homogenization times on OD600 values
From the results of samples 1 to 5 in Table 3, it was found that the OD600 value decreased with the increase of the homogenization pressure under the same conditions of the number of homogenization, probably because lactic acid bacteria have relatively strong cell walls, and the cell walls can be effectively destroyed only when the homogenization pressure reaches over 20000Psi, but the homogenization pressure is high, the instrument loss is large, and in addition, the sedimentation is extremely easy in the homogenization process because of more thalli in the lactic acid bacteria fermentation broth, and the sediment is difficult to avoid blocking the homogenization cavity even if stirring is not stopped, and in order to facilitate the industrial mass production, samples 6 to 8 are homogenized for 1 time by adopting relatively low pressure and then homogenized for different times by high pressure respectively to optimize the process, and as a result, the OD600 of samples 7 and 8 are found to be basically equivalent.
Further selecting samples No. 7 before and after homogenization, and observing the viable bacteria by a gram-stained microscope, wherein the result is shown in fig. 4, and the microscopic photograph of the lactic acid bacteria before and after homogenization in fig. 4 shows that the purple lactic acid bacteria short-rod viable bacteria are obviously observed before homogenization, but only the lactic acid bacteria cell fragments are left after homogenization, so that the viable bacteria are difficult to find, and the breaking process of the sample No. 7 can fully prove that the requirements of the lactic acid bacteria breaking can be met.
Further diluting the homogenized samples No. 7 and No. 8 by 5 times to dilute the homogenized sample by 10 times 7 As a control, the number of viable bacteria was observed by plating 0.1. Mu.l each, and the results are shown in FIG. 5. As can be seen from FIG. 5, the number of viable bacteria before homogenization was 2.7X10 9 The number of viable bacteria after homogenization is only 50, basicallyIn order to meet the wall breaking requirement, in order to facilitate industrial production, a wall breaking process of a No. 7 sample is finally adopted, namely, 18000-20000Psi pressure is adopted for homogenizing for 1 time, and then the pressure is increased to 23000-25000Psi for homogenizing for 2 times.
Example 4
The preparation process of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel is examined:
(1) Pure seed activation: taking lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 24 hours at the temperature of 30-42 ℃ to obtain a seed culture solution;
(2) Fermentation culture: adding 5% peach gum solution and 10% Poria extract into MRS liquid culture medium respectively, mixing, sterilizing to obtain MRST culture solution, inoculating 1g seed culture solution per 100g, and culturing at 30-42deg.C for 24 hr to obtain lactobacillus/peach gum and Poria fermentation product (LRSTF-LF);
(3) Fermentation product lysate: respectively taking the lactobacillus/peach gum and poria cocos fermentation products, homogenizing the lactobacillus/peach gum and poria cocos fermentation products by a micro-jet high-pressure homogenizer for 1 time under 18000-20000Psi pressure, then homogenizing the lactobacillus/peach gum and poria cocos fermentation products for 2 times under 23000-25000Psi pressure, and controlling the temperature at 0-10 ℃ to obtain the lactobacillus/peach gum and poria cocos fermentation product lysate (MRSTF-LFL).
Taking 100g of each lysate, respectively adding 0.5% of ethyl hexyl glycerol and 0.5% of sodium benzoate, stirring to completely dissolve, respectively scattering 0.6g, 0.7 g and 0.8g of xanthan gum powder on the liquid surface, standing at room temperature for 1-2h, stirring at the rotating speed of 200 r/min to completely dissolve after swelling, and finally adding 0.05g of lavender essence, stirring to completely dissolve to obtain transparent light yellow hydrogel (MRSTF-LFL-HG).
The visual properties, pH, viscosity and stability were measured visually and compared with 4000 rpm for 15min, and the results are shown in Table 4 and FIG. 6.
TABLE 4 Effect of different xanthan gum usage on hydrogel stability
As can be seen from the data in table 4, the lysate without xanthan gum is obviously layered and precipitates after centrifugation, the viscosity is correspondingly increased along with the increase of the addition amount of xanthan gum, the centrifugal stability is correspondingly increased, the requirement can be met when the use amount of xanthan gum reaches more than 0.7%, the use amount of xanthan gum is continuously increased, the viscosity is too large, the subsequent formulation operation is inconvenient instead, and as can be seen from the lysate of lactic acid bacteria fermentation products and hydrogel photos thereof in fig. 6, the lysate without xanthan gum is light yellow suspension, stands, namely, layers, the bottom has obvious white precipitates, and the lysate with xanthan gum is sticky gel without layering and precipitation.
Example 5
Unlike example 4, carbomer was used as the hydrogel material, arginine was added as a pH adjustor in an amount of 0.7% by weight based on the total weight. Taking 200g of lactobacillus/peach gum and poria cocos fermentation product lysate prepared by a still method, adding 1% sodium benzoate, stirring to dissolve completely, scattering 1.4g of carbomer powder on the liquid surface, standing at room temperature for 1-2h, and swelling; stirring at 200 rpm to dissolve completely, adding 3g L-arginine, stirring while dissolving completely, adjusting pH to 6.0-7.5, and adding 0.05g jasmine essential oil, stirring to dissolve completely to obtain transparent pale yellow hydrogel.
Example 6
Unlike example 5, sodium carboxymethylcellulose was used as the hydrogel matrix, added in an amount of 2% of the total weight. Taking 200g of lactobacillus/peach gum and poria cocos fermentation product lysate prepared by a still method, adding 1% sodium benzoate, stirring to completely dissolve, scattering 4.0g of sodium carboxymethylcellulose powder on the liquid surface, standing at room temperature for 1-2h, and swelling; stirring at 200 rpm to dissolve completely, and adding 0.05g of rose essence, and stirring to dissolve completely to obtain transparent pale yellow hydrogel. As a result, it was found that the use of sodium carboxymethylcellulose as the hydrogel matrix in an amount of 2% was found to have a high tackiness and a slightly inferior use experience.
Example 7
Unlike example 6, guar gum was used as the hydrogel matrix, added in an amount of 1.0% of the total weight. Taking 200g of lactobacillus/peach gum and poria cocos fermentation product lysate prepared according to the method, adding 1% sodium benzoate, stirring to completely dissolve, adding 2.0g of guar gum powder, stirring at a rotating speed of 200 revolutions per minute to completely dissolve, and finally adding 0.05g of peppermint essential oil, stirring to completely dissolve to obtain transparent pale yellow hydrogel. As a result, guar gum is found to have good water solubility, and hydrogel can be prepared without swelling, so that time can be saved.
Example 8
In contrast to example 7, xanthan gum and guar gum were used as hydrogel matrices, both added in an amount of 0.3% of the total weight. Taking 200g of lactobacillus/peach gum and poria cocos fermentation product lysate prepared by a method, adding 1% sodium benzoate, stirring to completely dissolve, adding 0.6g of mixed powder of xanthan gum and guar gum which are uniformly mixed in advance, stirring at a rotating speed of 200 revolutions per minute to completely dissolve, and finally adding 0.05g of lavender, stirring to completely dissolve to obtain transparent light yellow hydrogel.
Example 9
As a comparative example, peach gum and tuckahoe were not added to MRS broth. Lactic acid bacteria fermentation product lysate (example 9-1, MRS-LFL) and lactic acid bacteria fermentation product lysate hydrogel (example 9-2, MRS-LFL-HG) were prepared according to the methods, respectively.
The respective indexes of the samples of examples 4-1,4-3, examples 5,6,7 and 8 and examples 9-1 and 9-2 were measured and compared, and the results are shown in Table 5:
TABLE 5 lactic acid bacteria/peach gum, poria fermentation product lysate and hydrogel Each performance comparison
As can be seen from Table 5, the lactic acid bacteria Lysate (LF), whether or not containing peach gum and Poria cocos (example 4-1 and example 9-1), is a suspension which is prone to delamination and sedimentation, and after being prepared into hydrogel, the physical stability is greatly improved, and the separation core is not delaminated after 15min at 4000 rpm and at room temperature. In addition, the common carbomer is adopted as the gel material (example 5), and the viscosity is maximum in the pH range of 6.0-7.5, so that the lactic acid content is reduced by adjusting the pH value with an alkaline substance, and other indexes including viscosity, solid content and nitrogen content are not obviously different.
Wherein, example 8 uses xanthan gum and guar gum as gel matrix, wherein, the xanthan gum is an anionic extracellular polysaccharide extracted from Mono-chrysosporium, and has double helix structure under low temperature and/or high ionic strength, and has stronger rigidity, thus having strong salt resistance, and the guar gum is a natural nonionic galactomannan extracted from endosperm of guar. The two are natural substances, a large number of hydrophilic groups exist in the molecule, for example, the xanthan gum has hydroxyl, acetyl, pyruvoyl and the like, the guar gum has a large number of hydroxyl groups, and strong hydrogen bonding exists among the molecules, so that the viscosity, stability and the like of an aqueous solution or a dispersion system can be obviously improved, and the two are mixed for use, so that the xanthan gum has obvious synergistic effect, the dispersion capability of the xanthan gum in water is greatly improved, and the working efficiency is improved.
Further, the samples of the above examples 4-1,8,9-1,9-2 were taken, and antioxidant effect was studied by taking the fermentation lysate CLR of the same product as the domestic lactobacillus in the current International medical and medical market as a positive control, and taking a blank MRS culture solution of 0.25% peach gum and 0.5% Poria as a negative control.
(1) DPPH radical scavenging Rate determination:
sequentially adding 1mL of DPPH solution and 0.5mL of samples to be tested with different concentrations into a test tube, uniformly mixing, standing in a dark place for 60min, transferring the solutions into a cuvette, measuring a light absorption value at a wavelength of 517nm by taking absolute ethyl alcohol as a reference (zeroing), and marking the light absorption value as A1; in addition, 1mL of absolute ethyl alcohol and 0.5mL of samples to be tested with different concentrations are sequentially added into a test tube, the samples are placed in the dark for 60min, the solution is transferred into a cuvette, the absorbance value is measured at the wavelength of 517nm by taking absolute ethyl alcohol as a reference (zeroing), and the absorbance value is recorded as A2; in addition, 1mL of DPPH solution and 0.5mL of absolute ethyl alcohol are added into a test tube, the mixture is placed in a dark place for 60min after uniform mixing, the solution is transferred into a cuvette, the absorbance value is measured at a wavelength of 517nm by taking absolute ethyl alcohol as a reference (zeroing), and the absorbance value is recorded as A0. Radical scavenging was calculated according to the following formula:
clearance (%) = [1- (A1-A2)/A0 ] ×100%
The test was repeated three times and the average value was taken as the final result, the result is shown in fig. 7;
the results of fig. 7 show that, in examples 4-1 and 9-1, the clearance of DPPH free radicals is 3.5 times and 4.0 times higher than that of the commercial CLR positive control, and in examples 8 and 9-2, the clearance of DPPH free radicals is slightly increased by 4.3 times and 4.6 times higher than that of the commercial CLR positive control, and after peach gum and poria cocos are added, the clearance of DPPH free radicals is increased, both the lactic acid bacteria fermented lysate and the hydrogel are fermented, which fully shows that the addition of peach gum and poria cocos is beneficial to the growth of lactic acid bacteria and the antioxidation effect is synergistically increased.
(2) Superoxide anion radical scavenging assay
Taking 4.5mL of Tris (hydroxymethyl) aminomethane (Tris) -HCl buffer solution in a test tube, preheating in a water bath at 25 ℃ for 20min, adding 0.5mL of sample solution with different concentrations and 0.5mL of 25mmol/L o-ring
After the phloroglucinol solution is uniformly mixed and reacts for 5min in a water bath at 25 ℃, 100 mu L of 8mol/L HCl is immediately added to terminate the reaction, then the absorbance of the reaction solution at 335nm is measured, and the scavenging rate of superoxide anion free radicals is calculated according to the following formula, wherein the result is shown in figure 8;
clearance (%) = [1- (Ai-Aj)/A0 ] ×100%
Note that: a0 is absorbance of the reaction solution without adding a sample (distilled water is used for replacing the sample); ai is absorbance of a reaction solution to which the sample and the pyrogallol are added; aj is absorbance of the reaction solution to which the sample was added and to which no pyrogallol was added (distilled water was used instead of pyrogallol).
The results show that the superoxide anion radical scavenging rate of the four products of the examples 4-1,8,9-1 and 9-2 is much higher than that of the CLR positive control and the blank culture solution sold in the market, the highest superoxide anion radical scavenging rate is improved by 9 times compared with the CLR positive control group, and after peach gum and poria cocos are added, the superoxide anion radical scavenging rate of the lactobacillus fermentation product lysate or hydrogel is obviously improved, so that the addition of the peach gum and the poria cocos is fully proved to be beneficial to the growth of lactobacillus and the antioxidation effect of the lactobacillus can be synergistically improved.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. The preparation method of the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel is characterized by comprising the following steps of:
step S1, preparing 5% peach gum solution: 50g of peach gum is taken, washed and decontaminated, and then added with 950g of NaCO with the concentration of 0.1mol/L 3 And NaHCO 3 Soaking the solution at 80-90deg.C, hydrolyzing for 4 hr, pulverizing, filtering under reduced pressure, adjusting pH to 6.0-7.0 with dilute hydrochloric acid solution, and sterilizing to obtain 5% peach gum solution;
step S2, preparing 10% of poria cocos extracting solution: reflux-extracting Poria sclerotium powder 100g with 70% ethanol 500g in 95deg.C water bath for 3 hr, cooling to room temperature, vacuum filtering, extracting for 2 times, mixing filtrates, concentrating the filtrate under reduced pressure to recover ethanol, dissolving in water, filtering, adding water to 1000g, and sterilizing to obtain Poria extractive solution;
step S3, preparing a culture solution: respectively weighing 5% peach gum solution and 10% Poria cocos extract according to the weight ratio of 5%, adding into MRS liquid culture medium, and sterilizing to obtain culture solution;
step S4, preparing lysate of lactobacillus fermentation products:
pure seed activation: taking lactobacillus glycerol bacteria frozen at the temperature of minus 80 ℃ and inoculating the lactobacillus glycerol bacteria on an MRS culture medium for activation, and culturing for 20-30 hours at the temperature of 30-42 ℃ to obtain a seed culture solution;
fermentation culture: inoculating the activated seed culture medium into a culture solution containing peach gum and poria cocos according to a proportion of 1% -5%, and culturing at 30-42 ℃ for 20-30 hours to obtain a lactobacillus fermentation product;
the preparation of lactobacillus fermentation product lysate comprises taking lactobacillus fermentation product, homogenizing 1 time with a micro-jet high pressure homogenizer 18000-20000psi, homogenizing 2 times under the condition of homogenizing pressure 23000-25000psi, and controlling the temperature at 0-10deg.C to obtain lactobacillus fermentation product lysate.
Step S5, preparing a lysate hydrogel of the lactobacillus fermentation product:
adding functional auxiliary agent into the lysate of lactobacillus fermentation product, stirring uniformly, weighing xanthan gum accounting for 0.3% of the total amount and guar gum accounting for 0.3% of the total amount, mixing uniformly, spreading on the liquid surface, and stirring at room temperature to dissolve completely to obtain yellowish transparent hydrogel.
2. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the concentration of the peach gum aqueous solution in the step S1 is 2% -8%, and the addition amount of the peach gum in the culture solution is 0.1% -0.5%.
3. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the concentration of the poria cocos extracting solution in the step S2 is 5% -15%, and the adding amount of the poria cocos extracting solution in the culture solution is 0.3% -0.7%.
4. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the MRS medium described in the step S3 includes: vitamin C, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, cysteine, bifidobacterium, yeast extract, beef extract, soybean peptone and beef peptone.
5. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the addition amount of the seed culture medium in the step S4 is 1% -5% of the culture solution.
6. The method for preparing the lactobacillus/peach gum, poria cocos fermentation product lysate hydrogel according to claim 1, which is characterized in that: the micro-jet high-pressure homogenization times in the step S4 are 2-5 times, wherein the homogenization pressure gradient is increased, the homogenization is firstly carried out for 1 time by 18000-20000psi, then the homogenization pressure is increased to 23000-25000psi for 2 times, and the temperature is controlled to be 0-10 ℃.
7. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the hydrogel material in step S5 includes one or more of xanthan gum, guar gum, carbomer, and sodium carboxymethyl cellulose.
8. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the functional auxiliary agent in the step S5 comprises a preservative and a flavoring agent.
9. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the preservative comprises one or more of sodium benzoate, potassium sorbate, sodium dehydroacetate, phenoxyethanol and ethyl hexyl glycerol.
10. The method for preparing the lactic acid bacteria peach gum and poria cocos fermentation product lysate hydrogel, which is characterized by comprising the following steps of: the flavoring agent comprises one or more of peppermint essence, lavender essence, jasmine essence and rose essence.
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