CN117210381B

CN117210381B - Lactobacillus paracasei and products for relieving visual fatigue and application thereof

Info

Publication number: CN117210381B
Application number: CN202311484864.0A
Authority: CN
Inventors: 秦琦; 林枫翔; 李秀娟; 侯莹
Original assignee: Hangzhou Gongyanlou Culture Development Co ltd; Harbin Meihua Biotechnology Co ltd
Current assignee: Hangzhou Gongyanlou Culture Development Co ltd; Harbin Meihua Biotechnology Co ltd
Priority date: 2023-11-09
Filing date: 2023-11-09
Publication date: 2024-02-09
Anticipated expiration: 2043-11-09
Also published as: CN117210381A

Abstract

The invention provides a cheese bacillus paracasei and a visual fatigue relieving product and application thereof; belongs to the field of microorganism. The cheese bacillus paracasei isLacticaseibacillus paracasei) Is cheese bacillus FG-14 with a preservation number of CGMCC No.24331 and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at the month of 01 and 17 of 2022. The lutein ester yield obtained by fermenting marigold with FG-14 is up to 31mg/g fermentation liquor, and can be used for preparing products for relieving asthenopia.

Description

Lactobacillus paracasei and products for relieving visual fatigue and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a cheese bacillus paracasei and a visual fatigue relieving product and application thereof.

Background

Lutein (Lutein) with molecular formula of C ₄₀ H ₅₆ O ₂ The relative molecular weight was 568.85. Orange yellow powder, slurry or liquid, insoluble in water, soluble in organic solvents such as hexane. It belongs to oxygen-containing carotenoid, its structure contains two secondary hydroxyl groups and a conjugated polyene structure and 10 methyl groups, and is a natural pigment with strong colouring ability and antioxidation ability, and because of its special nature, human beings can not synthesize lutein, and can only obtain it from rich food, for example, fruit, egg yolk, corn and olive oil, etc.. Lutein is widely found in dark green vegetables, most notably isolated from calendula. Lutein has proven to be effective in delaying the risk of age-related macular degeneration due to its ability to promote activation of target genes in human retinal pigment epithelial cells. The prior art shows that about 6-13mg lutein taken daily can reduce the risk of cataract development and macular degeneration, and clinical trials show that lutein can be taken at a dose of 18mg daily without producing any adverse reaction.

Lutein can reduce the risk of age-related macular degeneration (AMD) or cataracts. Macular Degeneration (AMD) is an age-related degenerative disease that ingests an appropriate amount of lutein, which can accumulate in the macular area of the human eye and absorb blue light, reducing damage to retinal cells, and many experiments have demonstrated that supplementation with lutein can help delay the onset and progression of ocular disease. Lutein has the potential of quenching singlet oxygen and scavenging active free radicals, so that the lutein can also be used as an active antioxidant, protect the health state of skin, reduce the damage of external radiation and ultraviolet rays and delay aging. Lutein has also been reported to play a critical role in the development of brain and cognitive functions. These functions are related to their neuroprotective effects, the ability to alter cell membrane fluidity, oxygen diffusion and ion exchange. Meanwhile, lutein has wide applicability as an edible pigment and bright color, can be used for foods, nutritional health products and pharmaceutical preparations, and especially has been used for years as a food additive. Lutein also has a certain resistance to cancer. The research shows that lutein can inhibit the incidence rate of colon cancer, esophagus cancer, cervical cancer, prostate cancer and the like. Therefore, research into drugs, health products or foods for alleviating and treating these diseases is important. In order to meet the application requirements, the lutein production process needs to be improved.

The lutein has poor light and heat stability and strong lutein ester stability; lutein is easily dissolved by gastric acid, is favorable for human body absorption, has lower acid resistance compared with lutein ester, and has lower consumption and loss of lutein ester in stomach.

The prior art CN110679934A discloses a lutein-containing liver-clearing and eyesight-improving ferment and a preparation method thereof, belongs to the technical field of ferment beverages, releases lutein ester in marigold flowers through enzymolysis wall breaking, and converts lutein into lutein easy to be absorbed through biological fermentation, and the lutein-containing liver-clearing and eyesight-improving ferment is directly prepared into a beverage. The fermentation process comprises fermenting for 2-4 days, enlarging bacterial activity, and post-ripening fermenting for 15 days; the fermentation method has long time.

The prior art CN114901254A discloses a chrysanthemum active microbial inoculum for improving age-related macular degeneration, and the preparation method comprises the steps of adding distilled water into medicinal chrysanthemum, heating for reflux or decoction, filtering, concentrating under reduced pressure, adding ethanol, standing, centrifuging, concentrating under reduced pressure, adding one or more of lactobacillus bacteria, and compounding or fermenting together to obtain the chrysanthemum active microbial inoculum. The preparation method has complicated steps and is not suitable for industrial production.

Therefore, the strain which has high yield of the fermented lutein ester and small application limit is excavated, and the strain has important significance for the application of lutein ester products.

Disclosure of Invention

In order to solve the problems, the invention develops a cheese bacillus paracasei and a visual fatigue relieving product and application thereof.

In one aspect, the invention provides a cheese bacillus paracaseiLacticaseibacillus paracasei）FG-14。

In particular, the cheese bacillus paracaseiLacticaseibacillus paracasei) FG-14 has a preservation number of CGMCC No.24331 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year 2022, month 01 and day 17.

The cell morphology and physicochemical experimental results of the cheese bacillus paracasei are shown in the following table:

the blank in the table indicates no result.

In another aspect, the invention provides a preparation of Lactobacillus paracasei FG-14.

Specifically, the preparation is prepared by the cheese bacillus paracasei FG-14, and the preparation comprises but is not limited to culture and/or freeze-dried powder; the culture includes, but is not limited to, fermentation broth and/or fermentation broth pellet.

Because of the characteristics of the Lactobacillus paracasei provided by the invention, lutein esters can be further contained in the preparation.

In yet another aspect, the invention provides a fermentation process for Lactobacillus paracasei FG-14.

Specifically, the fermentation method comprises inoculating the above Lactobacillus paracasei FG-14 to a culture medium.

Specifically, the culture medium comprises one or more of germinated oat, germinated black beans, germinated millet, germinated coriander, carrot, sugar, sodium glutamate, bamboo leaves and acanthopanax leaves.

Preferably, the culture medium comprises, by weight, 5-60 parts of bamboo leaves, 5-60 parts of acanthopanax leaves, 5-70 parts of carrots, 5-70 parts of germinated oats, 5-70 parts of germinated black beans, 5-60 parts of germinated millet, 5-60 parts of germinated coriander and 0.5-5 parts of sugar.

Preferably, the culture medium comprises, by weight, 15-40 parts of bamboo leaves, 15-35 parts of acanthopanax leaves, 15-55 parts of carrots, 20-50 parts of germinated oats, 15-50 parts of germinated black beans, 15-25 parts of germinated millet, 15-25 parts of germinated coriander and 0.5-1.5 parts of sugar.

Preferably, the germinated oat, germinated black beans, germinated millet and germinated coriander have a bud length of no more than 25% of the seed grains.

Specifically, the preparation method of the culture medium comprises the following steps: the oat, black beans, millet and lawn pennywort herb are germinated, and the bud length is not more than 25% of the seed grain.

Specifically, the germination treatment includes: pouring proper amount of warm water into oat, black beans, millet and lawn pennywort herb until the oat, black beans, millet and lawn pennywort herb are completely soaked, pouring water out after soaking for 4 hours, wrapping a container with a towel, placing the container in a shade place, sprinkling water every day to keep the container moist, and waiting for the oat, black beans, millet and lawn pennywort herb to sprout.

Specifically, the preparation method of the culture medium comprises the following steps: weighing folium Bambusae and folium Acanthopanacis Senticosi, adding water, boiling for 15-30min, filtering while hot, and removing residues to obtain folium Bambusae solution.

Specifically, the preparation method of the culture medium comprises the following steps: cleaning radix Dauci Sativae and cutting into pieces; weighing germinated oat, germinated black beans, germinated millet, germinated coriander and sugar, and treating with a tissue crusher for 5-15min; adding herba Lophatheri solution, homogenizing, and sterilizing at 105-121deg.C for 10-40min to obtain culture medium.

Preferably, the sugar includes, but is not limited to, glucose.

Specifically, the fermentation temperature may be 35-42 ℃.

Preferably, the fermentation temperature may be 36-37 ℃.

Specifically, the fermentation time is not less than 50 hours, and the fermentation mode can be closed anaerobic fermentation.

Preferably, the fermentation time is not less than 60 hours.

Specifically, the feeding of sodium glutamate solution is started from 8 to 12 hours after fermentation, and the concentration of the sodium glutamate solution is 0.5 to 6 percent w/v.

Preferably, the concentration of the sodium glutamate solution is 2-4% w/v.

Specifically, the sodium glutamate solution is sterilized at 105-130 ℃ for 10-40min.

Preferably, the sodium glutamate solution is sterilized at 105-121 ℃ for 15-25min.

Specifically, the stirring is carried out fully during the feeding.

In a further aspect, the invention provides the use of the above-described Lactobacillus paracasei FG-14 for the preparation of lutein esters.

In a further aspect, the invention provides the use of the above-mentioned Lactobacillus paracasei FG-14 or a preparation of the above-mentioned Lactobacillus paracasei FG-14 for the preparation of a food, a health product or a pharmaceutical product.

In particular, the use comprises adding Lactobacillus paracasei FG-14 or a preparation of Lactobacillus paracasei FG-14 to a food, a health product or a pharmaceutical product.

Alternatively, the food, health product or pharmaceutical product comprises lutein ester prepared by the cheese bacillus paracasei FG-14.

In a further aspect, a food, a health product or a pharmaceutical product comprising the above-described Lactobacillus paracasei FG-14 or a preparation of Lactobacillus paracasei FG-14.

The food can also comprise common auxiliary materials in the food field, including but not limited to: preservatives, sweeteners, acidity regulators, colorants, thickeners, anticaking agents, leavening agents, flavoring agents, emulsifiers, defoamers, and flavoring agents.

The health-care food is a health-care food, and the health-care food can also comprise common auxiliary materials of the health-care food, including but not limited to food pigments, sweeteners, emulsifying thickeners, vitamins, essence for food and other components.

Such other ingredients include, but are not limited to: beta-apo-8' -carotenal, beta-cyclodextrin, carnauba wax, benzoic acid and its sodium salt, glacial acetic acid, D-mannitol, DL-tartaric acid, DL-malic acid and DL-sodium malate, butyl Hydroxy Anisole (BHA), parabens and their sodium salts (sodium methyl parahydroxybenzoate, ethyl parahydroxybenzoate and its sodium salt), dibutyl hydroxy toluene (BHT), silica, titanium dioxide, beeswax, fumaric acid, glycerol, talc, sodium pyrophosphate, polyglyceryl fatty acid ester, polydextrose, polyvinyl alcohol, L-malic acid, L (+) -tartaric acid, sodium caseinate, phosphoric acid, monopotassium phosphate, dipotassium phosphate, disodium hydrogen phosphate, calcium hydrogen phosphate, tricalcium phosphate, sodium hexametaphosphate, calcium sulfate, rosemary extract, xylitol, citric acid, potassium citrate, sodium hydroxide, lactic acid, sodium lactate, sodium tripolyphosphate, sorbic acid and its potassium salts, calcium carbonate, sodium bicarbonate, hydrochloric acid, ethanol/alcohol, isomerized lactose solution, sodium acetate, ethyl acetate, stearic acid, calcium stearate, magnesium stearate, menthol, oligophospholipids, and caprylic acid.

Pharmaceutical excipients may also be included in the pharmaceutical product, including but not limited to: fillers, diluents, binders, wetting agents, disintegrants, lubricants, suspending agents, dispersing agents and preservatives.

In still another aspect, the present invention provides a method for preparing lutein esters.

Specifically, the preparation method comprises the fermentation method of the Lactobacillus paracasei FG-14.

As will be appreciated by those skilled in the art based on the teachings of the present invention, by the fermentation process described above, the Lactobacillus paracasei FG-14 of the present invention can produce a large amount of lutein esters to effect the preparation of lutein esters.

In addition, the preparation method may include a purification step of lutein ester and the like according to common general knowledge in the art.

The invention has the beneficial effects that:

the invention provides a cheese bacillus paracasei and a visual fatigue relieving product and application thereof; belongs to the field of microorganism. The cheese bacillus paracasei isLacticaseibacillus paracasei) Is cheese bacillus FG-14 with a preservation number of CGMCC No.24331 and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at the month of 01 and 17 of 2022. The lutein ester yield obtained by fermenting marigold with FG-14 is up to 31mg/g, and can be used for preparing products for relieving asthenopia.

Preservation description:

chinese academic name: cheese bacillus paracasei;

classification naming: cheese bacillus paracaseiLacticaseibacillus paracasei；

Strain number: FG-14;

preservation number: CGMCC No.24331;

preservation time: 2022, 01, 17;

preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);

preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Drawings

FIG. 1 shows the results of the temperature measurements of FG-14 and its metants and control three groups of fermentation broths in example 9.

FIG. 2 shows the results of pH measurements of FG-14 and its metants and control three groups of fermentation broths in example 9.

FIG. 3 is a reducing sugar standard curve in example 9.

FIG. 4 shows the reducing sugar content of FG-14 and its metants and control three fermentation broths in example 9.

FIG. 5 is a lutein ester standard curve in example 9.

FIG. 6 shows the lutein ester content of FG-14 and its metates and control three groups of fermentation broths in example 9.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 isolation of Lactobacillus paracasei FG-14

Collecting a sample in a healthy intestinal canal, screening 343 strains of lactic acid bacteria strains producing lutein esters from the sample by adopting a plate separation method, repeatedly performing primary screening and secondary screening to obtain a strain with high lutein esters yield, carrying out morphological and physiological biochemical identification on the strain, carrying out molecular genetic identification, and carrying out BLAST comparison with 16SrDNA submitted on GenBank, wherein the result shows that the strain belongs to LactobacillusLacticaseibacillus) Belongs to the field of technology. The phylogenetic tree constructed by MEGA6.0 software shows that the strain and the methodLacticaseibacillus paracaseiThe homology of the 16S rDNA sequence reaches 99 percent and is consistent with the physiological and biochemical test result, thus, the strain is determined to be the cheese bacillus paracasei(Lacticaseibacillus paracasei). The strain FG-14 is sent to Beijing microorganism research institute of China academy of sciences for identification, and the identification results are completely consistent. FG-14 cell morphology and physicochemical results are shown in Table 1:

TABLE 1 FG-14 cell morphology and physicochemical experimental results

The blank in the table indicates no result.

Detection and identification conclusion: under the laboratory condition, according to the cell morphology, physiological and biochemical characteristics, 16S rDNA gene sequence, dnaK gene sequence and other experimental data comprehensive analysis, refer to the "Bojie' S system bacteriology handbook" and International Journal of Systematic and Evolutionary Microbiology related research papers, the identification result of the strain FG-14 is:Lactibacillus paracaseilactobacillus paracasei (synonym:Lacticaseibacillus paracaseicheese bacillus paracasei).

The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24331 in 2022, 01 and 17.

Example 2 culture, fermentation and detection method of Lactobacillus paracasei FG-14

In the following examples, the preparation of plant substrates was referred to as follows:

(1) Respectively germinating herba Avenae Fatuae, semen Sojae Atricolor, semen Setariae and herba Coriandri under certain conditions, wherein the bud length is not more than 25% of seed;

(2) Weighing 5.0-35.0g of folium Bambusae respectively, 5.0-35.0g of radix Acanthopanacis Senticosi leaf, adding 100-1000mL of water, boiling, maintaining for 15-30min, filtering while hot, and removing residues to obtain folium Bambusae solution;

(3) Cleaning carrot, weighing 5.0-50.0g, and cutting; weighing 20.0-60.0g of germinated oat, 5.0-60.0g of germinated black beans, 5.0-60.0g of germinated millet, 5.0-60.0g of germinated lawn pennywort herb and 0.5-5.0g of glucose, and treating with a tissue crusher for 5-15min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105-121deg.C for 15-25min, and cooling to obtain plant culture medium (PM).

(4) Weighing 0.5-6.0g of sodium glutamate, dissolving in 100mL of purified water, and sterilizing at 105-121 ℃ for 15-25min for later use.

In the following examples, the culture methods of seed of Lactobacillus paracasei FG-14 strain were referred to as follows:

diluting the plant culture medium prepared in the step (3) by 2-5 times, inoculating FG-14 strain, and culturing in a 35-42 ℃ incubator for 12-24 hours to obtain mature seed of FG-14 strain.

In the following examples, the method for fermentation and control of Lactobacillus paracasei FG-14 is referred to as follows:

inoculating mature Lactobacillus paracasei FG-14 strain into the plant culture medium prepared in the above step (3), controlling temperature at 35-42deg.C, and performing closed anaerobic fermentation for 36-96 hr. The flow is started from 8 to 12 hours after fermentation, and the sodium glutamate solution prepared in the step (4) is added, the concentration of the solution is 0.5 to 6.0 percent w/v, and the flow acceleration is 10 to 100mL/h. The feeding was completed 12 hours before the fermentation was completed. The feeding can also be carried out in batches, the first feeding is started from 8 to 12 hours after fermentation, and the feeding is carried out once every 2 to 6 hours until the feeding is completed 12 hours before the fermentation is finished. In the feeding process, sufficient stirring is required.

In the following examples, the low temperature freeze drying process is referenced below:

after the fermentation is finished, the fermentation product with high lutein ester content is quickly placed in a freeze dryer for low-temperature freeze drying until the water content is less than or equal to 7.0 percent. Then pulverizing, and making into powder, tablet, capsule or adding into other foods.

In the following examples, the method for detecting the average lutein ester yield in the final fermentation broth is Berthelot colorimetric method.

Example 3 fermentation method 1 of Lactobacillus paracasei FG-14

(1) Pouring proper amount of warm water into oat, black beans, millet and lawn pennywort herb until the oat, black beans, millet and lawn pennywort herb are completely soaked, pouring water out after soaking for 4 hours, wrapping a container with a towel, placing the container in a shade place, sprinkling water every day to keep the container moist, waiting for the oat, black beans, millet and lawn pennywort herb to sprout, and controlling the sprout length not to exceed 25% of seed grains;

(2) Weighing 15.0g of bamboo leaves, 15.0g of acanthopanax leaves, adding 150mL of water, heating and boiling, maintaining for 15min, and filtering to remove residues while the bamboo leaves are hot to obtain a bamboo thorn leaf solution.

(3) Cleaning carrot, weighing 15.0g, and chopping; weighing 20.0g of germinated oat, 15.0g of germinated black beans, 15.0g of germinated millet, 15.0g of germinated coriander and 0.5g of glucose, and treating with a tissue crusher for 10min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105deg.C for 15min, and cooling to 35deg.C to obtain plant culture matrix.

(4) Inoculating mature Lactobacillus paracasei FG-14 strain to the plant culture medium obtained in step (3), controlling the fermentation temperature at 37 ℃, and feeding sodium glutamate solution at the concentration of 3.0% w/v (g/mL) at the beginning of fermentation to 8 hours, wherein the feeding speed is 10mL/h, and the total feeding amount is 150mL; sealing anaerobic fermentation for 72 hours to obtain fermentation liquor.

(5) And (3) after fermentation, rapidly placing the fermentation broth obtained in the step (4) into a freeze dryer for low-temperature freeze drying until the water content is less than or equal to 7.0%, and then crushing the fermentation broth to prepare powder, tabletting, capsules or adding the obtained fermentation broth into other various foods.

Example 4A fermentation method 2 of Lactobacillus paracasei

(1) Pouring proper amount of warm water into oat, black beans, millet and lawn pennywort herb until the oat, black beans, millet and lawn pennywort herb are completely soaked, pouring water after soaking for 4 hours, wrapping a container with a towel, placing the container in a shade place, sprinkling water every day to keep the container moist, waiting for the oat, black beans, millet and lawn pennywort herb to sprout, and controlling the sprout length to be not more than 25% of seed grains.

(2) Weighing 20.0g of bamboo leaves, 25.0g of acanthopanax leaves, adding 200mL of water, heating and boiling, maintaining for 15min, and filtering to remove residues while the bamboo leaves are hot to obtain a bamboo thorn leaf solution.

(3) Cleaning carrot, weighing 25.0g, and chopping; weighing 20.0g of germinated oat, 20.0g of germinated black beans, 15.0g of germinated millet, 10.0g of germinated coriander and 0.5g of glucose, and treating with a tissue crusher for 10min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105deg.C for 15min, and cooling to 35deg.C to obtain plant culture matrix.

(4) Inoculating mature Lactobacillus paracasei FG-14 strain to the plant culture medium obtained in step (3), and controlling fermentation temperature at 37deg.C; starting adding sodium glutamate solution at the time of fermentation until the 10 th hour, wherein the concentration of the solution is 2.5% w/v, adding 10.0mL every 2 hours, and finally adding 200mL; sealing anaerobic fermentation for 60 hours to obtain fermentation liquor.

Example 5A fermentation method 3 of Lactobacillus paracasei

(2) Weighing 25.0g of bamboo leaves, 25.0g of acanthopanax leaves, adding 250mL of water, heating and boiling, maintaining for 25min, and filtering to remove residues while the bamboo leaves are hot to obtain a bamboo thorn leaf solution.

(3) Cleaning carrot, weighing 35.0g, and chopping; weighing 30.0g of germinated oat, 35.0g of germinated black beans, 25.0g of germinated millet, 25.0g of germinated coriander and 1.5g of glucose, and treating with a tissue crusher for 10min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105deg.C for 20min, and cooling to 35deg.C to obtain plant culture matrix.

(4) Inoculating mature Lactobacillus paracasei FG-14 strain to the plant culture medium obtained in the step (3), controlling the fermentation temperature at 37 ℃, and feeding sodium glutamate solution at the concentration of 3.5% w/v (g/mL) at the beginning of fermentation to 8 hours, wherein the feeding speed is 20mL/h, and the total feeding amount is 250mL; sealing anaerobic fermentation for 72 hours to obtain fermentation liquor.

Example 6 fermentation method 4 of Lactobacillus paracasei

(2) Weighing 30.0g of bamboo leaves, 30.0g of acanthopanax leaves, adding 300mL of water, heating and boiling, maintaining for 25min, and filtering to remove residues while the bamboo leaves are hot to obtain a bamboo thorn leaf solution.

(3) The carrot is treated and washed, 55.0g is weighed and chopped. Weighing 50.0g of germinated oat, 40.0g of germinated black beans, 25.0g of germinated millet, 15.0g of lawn pennywort herb and 1.5g of glucose, and placing into a tissue crusher for treatment for 20min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105deg.C for 20min, and cooling to 35deg.C to obtain plant culture matrix.

(4) Inoculating mature Lactobacillus paracasei FG-14 strain to the plant culture medium obtained in step (3), controlling the fermentation temperature at 37 ℃, adding sodium glutamate solution at the beginning of fermentation until 10 hours, adding 15.0mL every 3 hours, and finally adding 250mL; and (4) performing closed anaerobic fermentation for 84 hours to obtain fermentation liquor.

Example 7A fermentation method of Lactobacillus paracasei 5

(2) Weighing 40.0g of bamboo leaves, 35.0g of acanthopanax leaves, adding 400mL of water, heating and boiling, maintaining for 25min, and filtering to remove residues while the bamboo leaves are hot to obtain a bamboo thorn leaf solution.

(3) Cleaning carrot, weighing 45.0g, and chopping; weighing 40.0g of germinated oat, 50.0g of germinated black beans, 25.0g of germinated millet, 20.0g of germinated coriander and 1.5g of glucose, and treating with a tissue crusher for 15min; adding herba Lophatheri solution, homogenizing in homogenizer, sterilizing at 105deg.C for 20min, and cooling to 35deg.C to obtain plant culture matrix.

(4) Inoculating mature Lactobacillus paracasei FG-14 strain into the plant culture medium obtained in step (3), fermenting at 37deg.C, and sealing for anaerobic fermentation for 96 hr. Starting the fermentation until 10 hours, adding 15.0mL every 3 hours until 400mL is added, wherein the concentration of the sodium glutamate solution is 3.5%w/v (g/mL); and (4) performing closed anaerobic fermentation for 84 hours to obtain fermentation liquor.

Example 8 determination of lutein ester content in fermentation broth

Detecting the average lutein ester yield in the fermentation broth by using a Berchelot colorimetric method; the specific operation is as follows: 0.1g of the sample was weighed, diluted with 90% aqueous ethanol to a concentration suitable for ultraviolet analysis, and the content was calculated from a standard curve.

The results of the lutein ester content detection in the fermentation broth obtained in the above examples are shown in table 2:

TABLE 2 lutein ester content in fermentation broths

The results show that the highest lutein ester content in the fermentation broth after fermentation by using the Lactobacillus paracasei FG-14 can reach 31.23mg/g.

Example 9 marigold fermentation test

Fermenting a substrate: adding 100g of marigold powder into 200mL of water; configuring 3 groups;

the fermentation method comprises the following steps: inoculating the 3 groups of fermentation substrates with Lactobacillus paracasei FG-14 or its metants respectively; the control group is added with distilled water with equivalent quantity, and the inoculation quantity is 5% w/w; fermenting at 36 deg.C for 95 hr to obtain flos Tagetis Erectae fermentation broth.

The preparation method of the metagen of FG-14 comprises the following steps: centrifuging the fermentation liquid of the Lactobacillus paracasei FG-14, and obtaining a supernatant as a metazoan.

(1) Temperature detection

Two thermometers are simultaneously inserted into different positions in the middle of the fermented marigold, and after the two thermometers are stabilized, the two thermometers are rapidly read, and the average value of the readings of the two thermometers is taken, so that the temperature is measured every 5 hours. Three sets of test results are shown in table 3 and fig. 1:

TABLE 3 fermentation broth temperature detection

The results show that as the fermentation time increases, the temperature of the fermentation liquor and the metazoan increases and then decreases, and the control group has no obvious change.

(2) PH detection

Taking 5g of each group of marigold fermentation liquid in a beaker, adding 10mL of water, stirring for 1min, standing for 3min, and measuring the pH. The test results are shown in Table 4 and FIG. 2.

TABLE 4 fermentation broth pH detection

The results showed that as the fermentation time increased, the pH decreased first and then increased, with no significant change in the control group.

(3) Reducing sugar determination

Preparing a DNS solution: accurately weighing 6.3g of 3, 5-dinitrosalicylic acid in a 500mL beaker, dissolving the 3, 5-dinitrosalicylic acid in a small amount of distilled water, adding 262mL of 2mol/L NaOH solution, adding the solution into 500mL of hot water solution containing 182g of potassium sodium tartrate, adding 5g of crystalline phenol and 5g of anhydrous sodium sulfate, and stirring to dissolve the crystalline phenol and the 5g of anhydrous sodium sulfate. After cooling, the mixture was transferred to a 1L volumetric flask, the volume was fixed, and the flask was stored in a brown flask and used after 7 days of storage.

The specific detection method comprises the following steps:

(1) and (3) standard curve preparation: 0-1mL (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mL) of glucose standard solution of 1mg/mL is respectively sucked into the test tubes, and distilled water is used for constant volume to 1mL. Adding DNS reagent, heating in boiling water bath for 5min, taking out, cooling with tap water, adding 18mL distilled water, and mixing; absorbance was measured at a wavelength of 540 nm; and drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate.

(2) Sample measurement: the sample was sucked, 1mL of DNS reagent was added, heated in a boiling water bath for 5min, taken out, cooled with tap water, and then 18mL of distilled water was added and mixed well. Absorbance was measured at a wavelength of 540 nm.

The results of the reducing sugar standard curve are shown in table 5 and fig. 3.

TABLE 5 gradient solution of reducing sugars

The reducing sugar content of the fermentation broth is shown in Table 6 and FIG. 4.

TABLE 6 reducing sugar content in fermentation broths

The results show that as the fermentation time increases, the reducing sugar content in the fermentation broth decreases and then increases, and the control group has no obvious change.

(4) Lutein ester content

(1) Drawing a standard curve: 0.3mg lutein ester standard (Sichuan Przeiow standard Co., ltd., product No. 127-40-2) is weighed and dissolved in 30mL 90% ethanol water solution, and the lutein ester solutions with concentration of 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mug/mL are prepared by gradient dilution, and are used as solutions to be tested, as shown in Table 7. Taking a 90% ethanol water solution as a blank solution control, scanning at 445nm by an ultraviolet-visible spectrophotometer, and determining the maximum absorption wavelength of lutein ester in a solvent; the standard curve is plotted as in fig. 5.

TABLE 7 lutein ester gradient solution

(2) Determination of lutein ester content in fermentation broth: weighing 0.1g of fermentation liquor, and diluting with 90% ethanol water solution to reach a concentration suitable for ultraviolet analysis; the content results are shown in Table 8 and FIG. 6 by standard curve calculation.

TABLE 8 lutein ester content in fermentation broth

The results show that the lutein ester content in the fermentation broth after fermentation by using FG-14 and the metagen is higher than that of the control group.

Test example 1

And (3) placing the marigold fermentation liquid obtained by fermenting the embodiment 9 in a freeze dryer for low-temperature freeze drying until the water content is less than or equal to 7.0%, and then crushing to prepare marigold powder.

Test article: according to the mass percentage, the preparation method comprises the steps of taking 6% of probiotics powder, 5% of fructo-oligosaccharide, 25% of isomaltooligosaccharide, 10% of inulin, 15% of marigold powder, 10% of medlar powder, 14% of mulberry powder and 15% of blueberry powder, and uniformly mixing.

Control: the marigold powder in the test sample is replaced by starch.

The probiotic powder, inulin, medlar powder, mulberry powder and blueberry powder are purchased from Tianjin Zhenif food industry Co., ltd, and the batch numbers of the products are as follows: inulin P1911255, medlar powder P2010473, mulberry powder H1919272, blueberry powder H1517231 and probiotic powder F6-22-10-12-1.

50 volunteers are randomly selected, and have different degrees of ocular discomfort; 25 persons are divided into a test group and a control group, wherein the control group comprises 25 persons, 13 men and 12 women; test group 25, men 12, women 13.

The two groups are respectively taken for 2 times daily for 30 days with 2g each time. Evaluation criteria are shown in Table 9; the results before and after administration of the two groups are shown in tables 10-11.

TABLE 9 method for determining visual fatigue symptom (semi-quantitative integration method)

Note that: "occasional" and "even feel" refer to 1-2 times/2 days; "sometimes" means 1-3 times/day; "frequent" means >3 times/day.

Table 10 control group administration of controls

Table 11 test group administration of test article

The above results indicate that the problem of ocular discomfort can be preferentially alleviated after administration of solid beverages containing Lactobacillus paracasei FG-14.

Claims

1. Cheese bacillus paracasei @Lacticaseibacillus paracasei) FG-14, wherein the preservation number is CGMCC No.24331, and the FG-14 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 2022, 01 and 17.

2. A preparation of lactobacillus paracasei FG-14, characterized in that it is prepared from the lactobacillus paracasei FG-14 of claim 1, said preparation being a culture or a lyophilized powder.

3. A fermentation method of lactobacillus paracasei FG-14, comprising inoculating the lactobacillus paracasei FG-14 of claim 1 to a culture medium.

4. A fermentation process according to claim 3, wherein the medium ingredients comprise germinated oats, germinated black beans, germinated millet, germinated coriander, carrots, sugar, sodium glutamate, bamboo leaves and acanthopanax leaves.

5. The fermentation method according to claim 4, wherein the medium comprises, by weight, 5-60 parts of bamboo leaves, 5-60 parts of acanthopanax leaves, 5-70 parts of carrots, 5-70 parts of germinated oats, 5-70 parts of germinated black beans, 5-60 parts of germinated millet, 5-60 parts of germinated coriander and 0.5-5 parts of sugar.

6. The fermentation method according to claim 5, wherein the medium comprises, by weight, 15-40 parts of bamboo leaves, 15-35 parts of acanthopanax leaves, 15-55 parts of carrots, 20-50 parts of germinated oats, 15-50 parts of germinated black beans, 15-25 parts of germinated millet, 15-25 parts of germinated coriander and 0.5-1.5 parts of sugar.

7. The fermentation process of any one of claims 4-6, wherein the sugar comprises glucose.

8. The fermentation process of claim 7, wherein the fermentation temperature is 35-42 ℃.

9. The fermentation process of claim 8, wherein the fermentation temperature is 36-37 ℃.

10. The fermentation method according to claim 9, wherein the fermentation time is not less than 50 hours, and the fermentation is closed anaerobic fermentation.

11. The fermentation process according to claim 10, wherein the feeding of the sodium glutamate solution is started at 8-12 hours of fermentation, said sodium glutamate solution having a concentration of 0.5-6% w/v.

12. The fermentation process of claim 11, wherein the sodium glutamate solution is at a concentration of 2% w/v to 4% w/v.

13. Use of lactobacillus paracasei FG-14 according to claim 1 for the preparation of lutein esters.

14. A process for the preparation of lutein esters, characterized in that said process comprises a fermentation process according to any one of claims 3-12.