CN116590169B

CN116590169B - Bifidobacterium longum and application thereof

Info

Publication number: CN116590169B
Application number: CN202211636604.6A
Authority: CN
Inventors: 何俊杰; 张继勇; 黄桂州; 陆丽杰; 李燕琴; 郑华生; 张鹏; 谢培镇; 陈杰烽; 陈彦烁; 杜克斯; 程建华
Original assignee: Guangzhou Chao Hui Biological Technology Co ltd; Guangzhou Zengcheng Chaohui Biotechnology Co ltd; Dongguan Huagong Chuangwei Biotechnology Co ltd
Current assignee: Guangzhou Chao Hui Biological Technology Co ltd; Guangzhou Zengcheng Chaohui Biotechnology Co ltd; Dongguan Huagong Chuangwei Biotechnology Co ltd
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2024-01-30
Anticipated expiration: 2042-12-15
Also published as: CN116590169A

Abstract

The invention belongs to the technical field of microorganisms, and discloses bifidobacterium longum and application thereof. The invention screens a bifidobacterium longum which is bifidobacterium longum Bifidobacterium longum HGCW-18 and has a preservation number of CCTCC NO: m20221015. The bifidobacterium longum HGCW-18 has high biomass, more secreted active peptide and simple preparation process, and is suitable for industrial production. The bifidobacterium longum fermentation product filtrate prepared by the bifidobacterium longum HGCW-18 has the effects of improving the smoothness, the glossiness and the elasticity of the skin, tightening pores, repairing skin barriers and improving the integral state of the skin, and is suitable for the fields of cosmetics and foods.

Description

Bifidobacterium longum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum and application thereof.

Background

Bifidobacteria are an important flora colonizing the intestinal tract of humans and animals. The bifidobacterium longum is used as an important probiotics of bifidobacterium, is widely planted in intestinal tracts of human bodies, has a plurality of potential probiotics functions on human bodies, can synthesize and secrete various polypeptide substances and enzymes such as antibacterial peptides, protease, lipase, amylase and the like, and promotes the decomposition of nutrient substances so as to be beneficial to the absorption and utilization of the human bodies. Lactic acid produced after fermentation of bifidobacterium longum can improve utilization of iron, phosphorus and calcium and absorption of vitamin D; can synthesize multiple vitamins such as vitamin B, vitamin C, vitamin K, folic acid, etc.; can also synthesize gamma-aminobutyric acid, conjugated linoleic acid, exopolysaccharide and other substances, directly provide the nutrient substances for hosts, and can also remove free radicals in human bodies so as to delay the aging of organisms. Bifidobacterium longum can increase the activity and content of superoxide dismutase (superoxide dismutase, SOD) in human blood, thereby accelerating the elimination of free radicals in the body and slowing down aging, and thus has wide application.

However, in the application process, the bifidobacterium longum has the problems of slow growth, low activity, easiness in bacterial contamination, insufficient biomass, less monomer metabolites and the like, so that the bifidobacterium longum is difficult to meet the requirements of industrialization and improvement of the efficacy performance of products.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide bifidobacterium longum and application thereof. The strain has high biomass and high secretion of active peptide, and can be used in cosmetic and food fields.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention screens a Bifidobacterium longum which is Bifidobacterium longum sp.HGCW-18 with a preservation number of CCTCC NO: m20221015.

The bifidobacterium longum HGCW-18 has high biomass, more secreted active peptide and simple preparation process, and is suitable for industrial production. The bifidobacterium longum fermentation product filtrate prepared by the bifidobacterium longum HGCW-18 has the effects of improving the smoothness, the glossiness and the elasticity of the skin, tightening pores, repairing skin barriers and improving the integral state of the skin, and is suitable for the fields of cosmetics and foods.

As a preferred embodiment of the bifidobacterium longum of the present invention, the 16SrDNA base sequence of the bifidobacterium longum is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a bifidobacterium longum fermentation product, prepared by fermentation of said bifidobacterium longum.

In a third aspect, the invention provides a method for preparing a bifidobacterium longum fermentation product, comprising the steps of:

inoculating the bifidobacterium longum into a fermentation culture medium, and performing anaerobic culture at 35-39 ℃ for 36-60 h to obtain the bifidobacterium longum. Preferably, the fermentation culture is MRS medium.

In a fourth aspect, the invention provides a bifidobacterium longum fermentation product filtrate, which is prepared by fermentation of the bifidobacterium longum.

In a fifth aspect, the present invention provides a method for preparing a bifidobacterium longum fermentation product filtrate, comprising the steps of:

taking the bifidobacterium longum fermentation product; centrifuging, collecting supernatant, and filtering the supernatant.

As a preferred embodiment of the method for preparing the bifidobacterium longum fermentation product filtrate, the rotational speed of the centrifugal treatment is 4000 rpm-7000 rpm, and the time is 15 min-30 min; the filtration treatment is to pass the supernatant through a 0.45um and/or 0.20um water-based filter membrane.

In a sixth aspect, the invention provides the use of said bifidobacterium longum in cosmetics or food.

As a preferred embodiment of the application according to the present invention, the cosmetic comprises at least one of a fermentation product, a fermentation product filtrate, a fermentation product lysate, and a fermentation product extract of bifidobacterium longum.

As a preferred embodiment of the application of the present invention, the fermentation product filtrate of bifidobacterium longum is added to the cosmetic in an amount of 1 to 20% by mass.

As a preferred embodiment of the application of the invention, the bifidobacterium longum can be mixed with streptococcus thermophilus and lactobacillus bulgaricus to be inoculated into raw milk, and after heat preservation and fermentation, the yoghurt is prepared.

Compared with the prior art, the invention has the beneficial effects that:

the bifidobacterium longum HGCW-18 (with the preservation number of CCTCC NO: M20221015) has high biomass, more secreted active peptide and simple preparation process, and is suitable for industrial production. The bifidobacterium longum fermentation product filtrate prepared by the bifidobacterium longum HGCW-18 has the effects of improving the smoothness, the glossiness and the elasticity of the skin, tightening pores, repairing skin barriers and improving the integral state of the skin, and is suitable for the fields of cosmetics and foods.

Drawings

FIG. 1 is a colony morphology of isolated bifidobacterium longum HGCW-18;

FIG. 2 is a gram stain of bifidobacterium longum HGCW-18 under a microscope;

FIG. 3 is an agarose gel electrophoresis of the 16S rDNA PCR product of bifidobacterium longum HGCW-18.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

Example 1: isolation and characterization of bifidobacterium longum HGCW-18

1. Isolation and purification and morphological identification

Samples of cheese products were collected from team Cheng Jianhua in Chuxiong City, yunnan, 6 months in 2018, and the microorganisms in the cheese products were isolated with TPY solid medium. Culturing the separated microorganism in a workstation at 37 ℃ for 48 hours, picking dominant bacterial colonies on a flat plate, streaking and separating to obtain pure bacterial colonies, carrying out morphological observation through gram staining, inoculating the pure bacterial colonies into a TPY solid culture medium, carrying out anaerobic culture at 37 ℃ for 48 hours, and preserving at 4 ℃ after the culture is finished. A single strain HGCW-18 was obtained.

After the strain HGCW-18 is purified, single colony is formed in TPY solid culture medium, its form is shown in figure 1, and its form is almost identical, most of colony form is round, white and its surface roughness is drier. Can grow in the environment of 30-45 ℃, is negative in contact with enzyme, motility, nitrate reduction, indole and H2S production and gelatin liquefaction tests,

the results of gram staining are shown in FIG. 2, and the morphology of purple rod-shaped cells was observed under a microscope, and one of the lactic acid bacteria was determined.

2. Thin layer chromatography detection and identification

The strain HGCW-18 is identified by detecting the production of organic acids during glycolysis of different sugar sources by thin layer chromatography using the characteristic that lactic acid and acetic acid are mainly produced by glycolysis of lactobacillus.

Culturing the strain HGCW-18 separated in the above steps in culture medium containing different sugar sources at 37deg.C for 24 hr, centrifuging at 5000rpm for 20min, collecting supernatant, and detecting by thin layer chromatography with mixed solution of methylation and bromophenol blue dissolved in 70% alcohol as indicator.

The results are shown in Table 1.

TABLE 1 thin layer chromatography assay for strain HGCW-18

Detecting items	Detection result
		Arabinose (Arabic sugar)	+
Ribose	+
		Lactose and lactose	+
Cellobiose	+
		Melezitose	+
Cellobiose	+
		Sorbitol	+
Xylose	+
		Mannose	－
Fructose	+
		Sucrose	+
Maltose	+
		Trehalose	－
Melibiose	+
		Mannitol (mannitol)	+
Chrysanthemic phenol	－
		Salicin	－

3. 16S rDNA identification

The bacterial 16S rDNA sequence is highly conserved among different species, with the 16S rDNA sequence being different among different species. Therefore, by PCR amplification and sequencing of the 16S rDNA sequence, the bacterial species can be determined by comparison with the known sequences in GenBank, and they can be classified into specific genera or species. Identification of 16S rDNA (16S rRNA is a subunit of ribosomal RNA, 16S rDNA is a gene encoding 16S rRNA) refers to the identification of bacterial species by sequencing the bacterial 16S rDNA sequence.

(1) Extracting and purifying the DNA of the bacterial strain HGCW-18 by using a bacterial genome DNA extraction kit, and amplifying the extracted genome DNA as a 16S rDNA-PCR template.

The TSINGKE plant DNA extraction kit (universal) was used, and the specific steps were as follows:

1) Placing the spin column in a collecting tube, adding 250 μl of buffer, centrifuging at 12000rpm for 1min, and activating silica gel film;

2) The dried tissue (not more than 20 mg) was sampled and sufficiently ground by adding liquid nitrogen. Grinding, placing into a 1.5mL centrifuge tube, adding 400 mu L of buffer gP1, vortex oscillating for 1min, and carrying out water bath at 65 ℃ for 20min, wherein the materials can be taken out, inverted and uniformly mixed for full cleavage;

3) 150 μl of buffer gP2 is added, vortexed and oscillated for 1min, and ice-bath for 5min;

4) Centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;

5) Adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all the liquid into a rotary column, centrifuging at 12000rpm for 30s, and discarding waste liquid;

6) Adding 500 mu L buffer power (absolute ethanol is added before use) into the rotary column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

7) Adding 500 mu L of washing buffer (absolute ethanol is added before use) into the rotary column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

8) Repeating the operation step 7);

9) Putting the rotary column back into a collecting pipe, centrifuging at 12000rpm for 2min, uncovering and airing for 1min;

10 Taking out the spin column, placing the spin column into a clean centrifuge tube, adding 80 mu LTE buffer (preheated TE buffer at 65 ℃) at the center of the adsorption film, placing the spin column at 20-25 ℃ for 2min, and centrifuging at 12,000rpm for 2min.

(2) PCR amplification

1) Universal primer for bacterial strain identification

Primer information is shown in Table 2.

Table 2 general primers

2) The extracted DNA sample was diluted in a proper amount and amplified as a PCR template, and the components of the amplification system are shown in Table 3.

TABLE 3 PCR amplification System

1×TSE101 gold medal mix	45μL
		F(10P)	2μL
R(10P)	2μL
		DNA template	1μL

The above amplification system was amplified according to the amplification procedure shown in Table 4.

TABLE 4 PCR amplification procedure

3) Electrophoresis detection

The amplified PCR products were subjected to agarose gel electrophoresis (2. Mu.L of sample+6. Mu.L of bromophenol blue) at 300V for 12 minutes to obtain an identification gel. The electrophoretogram is shown in fig. 3.

(3) Sequencing

The PCR product was subjected to first generation sequencing (27F/1492R as sequencing primer) and the sequencing result was shown in SEQ ID NO. 1.

The base sequence of SEQ ID NO.1 is:

CGGGAACGCATTCACCGCGACGTTGCTGATTCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCGCGTCGCCGCGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCGGCATAATCCGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAGCCGTATCTCTACGACCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACACGGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCCGATAAAAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCACGGTGGGCCGTTACCCCGCCGTCAAGCTGATAGGACGCGACCCCATCCCATACCGCGAAAGCTTTCCCAGAAGACCATGCGATCAACTGGAGCATCCGGCATTACCACCCGTTTCCAGGAGCTATTCCGGTGTATGGGGCAGGTCGGTCACGCATTACT。

the sequencing results were aligned for homology to the GenBank gene library by the BLAST program. The 16S rDNA homology analysis results show that the homology of the strain with known bifidobacterium longum in GenBank database is up to 100%.

The isolated lactobacillus is identified as Bifidobacterium longum sp.hgcw-18, which was deposited at the China center for type culture collection (CCTCC NO) at 7.4 of 2022 by combining morphological identification, detection and identification by thin layer chromatography and 16S rDNA homology analysis: m20221015, which is classified and named as Bifidobacterium longum HGCW-18, and the preservation unit address is Jiuqiu No. 299 university of Wuhan, hubei province, china.

Example 2: preparation of Bifidobacterium longum HGCW-18 fermentation product filtrate

Taking the bifidobacterium longum HGCW-18 separated in the example 1, selecting a loop, inoculating the loop into a sterilized 100mLMRS culture medium, and performing anaerobic culture for 48 hours at 37 ℃ to obtain a bifidobacterium longum fermentation product.

Centrifuging the fermentation product of Bifidobacterium longum at 5000rpm for 20min, collecting supernatant, and sequentially passing the supernatant through 0.45um and 0.20um water-based filter membrane to obtain filtrate of fermentation product of Bifidobacterium longum.

Comparative example 1: preparation of Bifidobacterium longum ATCC55814 fermentation product filtrate

The difference from example 2 is that the bifidobacterium longum strain was changed to a commercially available bifidobacterium longum strain (ATCC 55814, available from beijing deposited biotechnology ltd) to prepare a bifidobacterium longum fermentation product filtrate.

Comparative example 2: preparation of Bifidobacterium longum ATCC55816 fermentation product filtrate

The difference from example 2 is that the bifidobacterium longum strain was changed to a commercially available bifidobacterium longum strain (ATCC 55816, available from beijing deposited biotechnology ltd) to prepare a bifidobacterium longum fermentation product filtrate.

Comparative example 3: preparation of Bifidobacterium longum CICC6068 fermentation product filtrate

The difference from example 2 is that the bifidobacterium longum strain was changed to a commercially available bifidobacterium longum (cic c6068, available from the Shanghai collection biotechnology center) to prepare a bifidobacterium longum fermentation product filtrate.

Test example 1: biomass testing

10mL of the fermentation products of bifidobacterium longum prepared in example 2 and comparative examples 1 to 3 were respectively taken, centrifuged at 5000rpm for 20min, the supernatant and the precipitate were separated, the precipitate was washed with deionized water for 2 times, the precipitate was transferred to a weighing bottle, and dried in an oven at 105 ℃ to constant weight, the net weight of the cells was calculated, and the dry weight concentration (g/L) of bifidobacterium longum was calculated from the volume of the sample solution.

The dry weight concentration of bifidobacterium longum in the fermentation product of example 2 was calculated to be 3.7g/L, the dry weight concentration of bifidobacterium longum in the fermentation product of comparative example 1 was calculated to be 1.5g/L, the dry weight concentration of bifidobacterium longum in the fermentation product of comparative example 2 was calculated to be 1.8g/L, and the dry weight concentration of bifidobacterium longum in the fermentation product of comparative example 3 was calculated to be 2.0g/L.

The test results show that the dry weight concentration of bifidobacterium longum of example 2 is higher than that of the prior art, indicating that bifidobacterium longum of example 2 has a higher biomass.

Test example 2: active peptide secretion assay

Drawing a standard curve: 10 volumetric flasks of 10mL were used to prepare Gly-Gly-Tyr-Arg tetrapeptide standard solutions of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8mg/mL in 5% TCA. Taking 6.0mL of standard solution respectively, adding 4.0mL of biuret reagent, uniformly mixing on a vortex mixer, standing for 10min, centrifuging at 2000rpm for 10min, taking supernatant, measuring an OD value (taking 0.0mg/mL as a blank control) at 540nm, taking the concentration of peptide as an abscissa X (mg/mL), taking the OD value as an ordinate Y, and drawing a standard curve.

2.5mL of the bifidobacterium longum fermentation product prepared in example 2 was taken, 2.5mL of 10% (W/V) aqueous trichloroacetic acid (TCA) solution was added, the mixture was mixed uniformly on a vortex mixer, the mixture was allowed to stand for 10min, and then centrifuged at 4000rpm for 15min, the supernatant was transferred to a 50mL volumetric flask, and the supernatant was subjected to shaking with 5% TCA to scale. 6.0mL of the solution is taken to be placed in another test tube, 4.0mL of biuret reagent (sample solution: biuret reagent=3:2, V/V) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, centrifuged at 2000rpm for 10min, the supernatant is taken to measure the OD value at 540nm, and the polypeptide concentration C (mg/mL) of the bifidobacterium longum fermentation product is obtained by comparing a standard curve.

And (3) repeating the operation by taking 2.5mL of the bifidobacterium longum fermentation products prepared in the comparative examples 1-3, and comparing the standard curve to obtain the polypeptide concentration C (mg/mL) of the bifidobacterium longum fermentation products in the comparative examples 1-3.

The concentration of the polypeptide in the fermentation product of Bifidobacterium longum of example 2 was 0.48mg/ml, and the concentrations of the polypeptide in the fermentation products of Bifidobacterium longum of comparative examples 1 to 3 were 0.05mg/ml, 0.08mg/ml, and 0.15mg/ml.

The test results show that the active peptide secretion of the bifidobacterium longum of the example 2 is far higher than that of the bifidobacterium longum in the prior art.

Test example 3: skin smoothness Stm test

The smoothness of the skin was tested using the bifidobacterium longum fermentation product filtrate of example 2 and the bifidobacterium longum fermentation product filtrate of comparative examples 1 to 3.

Test sample: bifidobacterium longum fermentation product filtrate (10% aqueous solution) of example 2; the bifidobacterium longum fermentation product filtrate (10% aqueous solution) of comparative examples 1 to 3.

The instrument used for the test: the digital optical three-dimensional image analyzer evaskun (EOTECH, france) is a rapid optical imaging system that photographs the local surface morphology of the skin.

Subject selection: screening 120 healthy men or women (30 volunteers per sample, excluding the person receiving dermatological treatment, or the conditions of affected result judgment and other possible effects on the test result such as the passing of the test site within one month, the cosmetology, the wound, abrasion, temperature rise, pimple and the like) of 18-35 years old.

The testing steps are as follows: the facial region of the subject is cleaned. The test subject takes 2-3mL of sample to be smeared on the test area, and the sample is used for 2 times a day, and each time in the morning and evening. The skin smoothness analysis was performed on the pictures taken by evaskun before, 2 and 4 weeks after the sample was used, and the test results were averaged.

Mean change rate from initial value= (mean after use-mean before use)/mean before use x 100%.

The skin smoothness Stm obtained by the test picture analysis is used as a parameter for evaluating the skin smoothness, and the smaller the skin smoothness Stm value is, the smoother the skin is. In addition, a sample is considered to have an effect of repairing the skin barrier function if the difference in skin smoothness Stm at any point of access after use of the sample is significantly reduced from the pre-use contrast.

The results of the skin smoothness Stm test before, after 2 weeks and after 4 weeks of use of the samples are shown in Table 5. The smaller the skin smoothness Stm value, the smoother the skin.

Table 5 skin smoothness Stm descriptive statistics (n=30)

The test results show that the bifidobacterium longum fermentation product filtrate of example 2 has a certain improvement in skin smoothness after 4 weeks of use, and has a better effect of repairing skin barrier function, compared with the bifidobacterium longum fermentation product filtrate of comparative examples 1 to 3.

Test example 4: skin gloss test

Test sample: same as in test example 3.

The instrument used for the test: skin gloss tester glossmeter (GL 200, courage and Khazaka, germany).

Subject selection: same as in test example 3.

The testing steps are as follows: the facial region of the subject is cleaned. The test subject takes 2-3mL of sample to be smeared on the test area, and the sample is used for 2 times a day, and each time in the morning and evening. The pictures taken by the Glossymeter were subjected to skin gloss analysis before, 2 weeks after and 4 weeks after the sample was used, and the test results were averaged.

The results of the skin gloss test before, after 2 weeks and after 4 weeks of use of the samples are shown in Table 6. The greater the skin gloss value, the better the skin gloss.

Table 6 skin gloss descriptive statistics (n=30)

The test results showed that the subjects showed a significant improvement in skin gloss after 4 weeks of use of the bifidobacterium longum fermentation product filtrate of example 2, as compared to the bifidobacterium longum fermentation product filtrate of comparative examples 1 to 3.

Test example 5: pore tightening test

Test sample: same as in test example 3.

The instrument used for the test: face image analyzer VISIA-CR (Canfield, USA).

Subject selection: same as in test example 3.

The testing steps are as follows: the facial region of the subject is cleaned. The test subject takes 2-3mL of sample to be smeared on the test area, and the sample is used for 2 times a day, and each time in the morning and evening. Before using the sample, after using the sample for 2 weeks and 4 weeks, collecting face front pictures of the volunteers, and carrying out image analysis on the cross polarized light pictures to obtain quantitative index pore area ratio, and evaluating shrinkage condition of the face pores.

Mean change rate from initial value = (mean after use-mean before use)/mean before use x 100%

The smaller the pore area in the skin, the smaller the pores in the skin, indicating that the better the pore tightening effect of the sample.

The results of the pore area ratio test before, after 2 weeks and after 4 weeks of sample application are shown in Table 7.

Table 7 pore area ratio descriptive statistics (n=30)

The test results show that the pore area ratio is significantly reduced after the subject uses the bifidobacterium longum fermentation product filtrate of example 2 for 4 weeks, compared with the bifidobacterium longum fermentation product filtrate of comparative examples 1 to 3, indicating that the bifidobacterium longum fermentation product filtrate of example 2 has good pore tightening efficacy.

Test example 6: TEWL test for percutaneous moisture loss

Test sample: same as in test example 3.

The instrument used for the test: skin moisture loss tester Aqua Flux (AF 200, BIOX, uk).

Subject selection: same as in test example 3.

The testing steps are as follows: the facial region of the subject is cleaned. The subjects took 2-3mL samples to smear on the test area, 2 times a day, each in the morning and evening. The skin of the face skin was tested for its transcutaneous water loss TEWL value before, after 2 weeks and 4 weeks of sample use.

The smaller the transcutaneous water loss TEWL value, the smaller the amount of transcutaneous water loss per unit time, the better the skin barrier.

The results of the test for the percutaneous water loss TEWL values before, after 2 weeks and after 4 weeks of the use of the samples are shown in table 8.

Table 8 descriptive statistics of the TEWL values for transdermal moisture loss (n=30)

Test results show that the bifidobacterium longum fermentation product filtrate of the embodiment 2 can effectively reduce the percutaneous water loss of skin and is beneficial to the repair of skin barriers compared with the bifidobacterium longum fermentation product filtrate of the comparative examples 1-3.

Test example 7: questionnaire survey

After using the bifidobacterium longum fermentation product filtrate of the present invention, the subjects self-assessed the improvement of the skin of the face after the use of the sample and the suitability of the sample in the form of an answer questionnaire.

Test samples and subjects were as in test example 3.

The results of feedback from the efficacy evaluation of 30 subjects on the samples were collected before, after 2 weeks and after 4 weeks of use of the samples and are shown in tables 9 to 12.

Effective number = improvement under the investigation index the sum of the numbers of people who compare consent and very consent is chosen. Crowd available rate = available number/answer population x 100%.

Table 9 effect evaluation volunteer self-evaluation results after use of the sample of example 2 (n=30)

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Table 10 effect evaluation volunteer self-evaluation results after use of the sample of comparative example 1 (n=30)

Table 11 effect evaluation volunteer self-evaluation results after use of the sample of comparative example 2 (n=30)

Table 12 effect evaluation volunteer self-evaluation results after use of the sample of comparative example 3 (n=30)

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The questionnaire results show that, compared with the bifidobacterium longum fermentation product filtrate of comparative examples 1 to 3, the subject has the advantages that after using the bifidobacterium longum fermentation product filtrate of example 2 for 2 to 4 weeks, the dry state of the facial skin is relieved, the moist smooth feeling is improved, the oil-out condition of the facial skin is improved, the facial wrinkles are reduced or become shallow, the roughness of the facial skin is improved, the glossiness of the facial skin is improved, the elasticity of the facial skin is improved, the pores of the facial skin are smaller, the skin is finer, the barrier capacity and the tolerance of the facial skin are better, and the overall state of the facial skin is better.

Example 3: compound strain compound preparation of yoghourt

The preparation method comprises the following specific steps:

(1) Preparation of raw milk

The mass percentages are as follows: mixing 87% of commercially available milk, 6% of white granulated sugar, 2% of fructo-oligosaccharide, 1% of low-ester pectin and 4% of modified starch uniformly, and sterilizing at 105 ℃ for 20min to obtain raw milk;

(2) Inoculating Bifidobacterium longum HGCW-18, streptococcus thermophilus CICC 20386 (from Beijing deposited biosciences Co., ltd.) and Bacillus bulgaricus CICC 20353 (from Beijing deposited biosciences Co., ltd.) of example 1 to raw milk of step (1) at 40℃in a mass ratio of 1:1:1 (total addition amount of three bacteria is 1×10) ⁷ CFU/mL), fermenting at 40 ℃, and stopping fermenting when the pH of the milk is reduced to 4.8, thus obtaining the yoghourt.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, 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 equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A Bifidobacterium longum, characterized in that the Bifidobacterium longum is Bifidobacterium longum (bifidobacteria sp.) HGCW-18 with a preservation number of CCTCC NO: m20221015.

2. A bifidobacterium longum fermentation product, characterized in that it is produced by fermentation of bifidobacterium longum according to claim 1.

3. A method for preparing a bifidobacterium longum fermentation product, which is characterized by comprising the following steps:

inoculating the bifidobacterium longum in the fermentation medium, and performing anaerobic culture for 36-60 h at the temperature of 35-39 ℃ to obtain the bifidobacterium longum.

4. A bifidobacterium longum fermentation product filtrate, characterized in that it is obtained by fermentation of bifidobacterium longum according to claim 1.

5. A method for preparing a bifidobacterium longum fermentation product filtrate, which is characterized by comprising the following steps:

taking the bifidobacterium longum fermentation product of claim 3; centrifuging, collecting supernatant, and filtering the supernatant.

6. The method for preparing a fermentation product filtrate of bifidobacterium longum according to claim 5, wherein the rotational speed of the centrifugal treatment is 4000rpm to 7000rpm for 15min to 30min; the filtration treatment is to pass the supernatant through a 0.45um and/or 0.20um water-based filter membrane.

7. Use of bifidobacterium longum as claimed in claim 1 in cosmetics or food.

8. The use according to claim 7, wherein the cosmetic comprises at least one of a fermentation product made by the bifidobacterium longum, a filtrate of the fermentation product of the bifidobacterium longum, and a lysate of the fermentation product of the bifidobacterium longum.

9. The use according to claim 8, characterized in that the fermentation product filtrate of bifidobacterium longum is added in an amount of 1-20% by mass in the cosmetic.