CN114292773B - Bifidobacterium adolescentis and screening and culturing methods and application thereof - Google Patents
Bifidobacterium adolescentis and screening and culturing methods and application thereof Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 76
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a bifidobacterium adolescentis and screening and culturing method and application thereof, wherein the bifidobacterium adolescentis is preserved in the Guangdong province microorganism strain preservation center, and the name is Bifidobacterium adolescentis JK-BIF-BIF-001-02-B1, and the strain preservation number is: GDMCC No:62063. the lysate of Bifidobacterium adolescentis can improve the activity and quantity of human fibroblasts, and has good anti-aging effect. The invention also provides a fermentation culture method of the bifidobacterium adolescentis, which greatly improves the yield of thalli by adopting a fermentation culture medium with a specific nitrogen source and a growth factor and optimizing the industrial fermentation process conditions, so that the yield of the thalli at the industrial level of the bifidobacterium adolescentis reaches more than 3%.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to bifidobacterium adolescentis and a screening and culturing method and application thereof.
Background
The skin is the first line of defense of the human body and is responsible for resisting external invasion and preventing water loss; meanwhile, the display screen is also a display screen for the physiological state and pathological state of the human body. When the organism ages, the skin can be dry and rough, the skin is loose, the wrinkles are increased, the wrinkles are deepened, the elasticity is lacked, the skin is chromatophoresis and the like; the underlying mechanism is injury and aging of skin fibroblasts. Thereby resulting in reduced collagen content and elastin denaturation.
The epidermis layer, dermis layer and tissue of the skin appendages all change with age, but the most characteristic changes are also changes in the composition of the dermis layer. The intrinsic cause of the changes in the composition of the dermis is fibroblast aging. Fibroblasts are responsible for synthesizing matrices such as collagen fibers, reticular fibers, elastic fibers, aminopolysaccharides and proteoglycans, which are structural components in dermis, wherein the content of collagen fibers is the most abundant. In the skin aging process, the collagen content is gradually reduced, and the thickness of dermis is thinned; elastic fiber is denatured and reduced, and loses elasticity and tension.
Bifidobacterium adolescentis is a strictly anaerobic probiotic and mainly originates from living parts such as human, animal intestinal tracts, bovine rumen and the like. The anti-aging effect of the lysate on skin has been reported in many documents, for example, patent document CN112708581A describes bifidobacterium adolescentis DH162, fermentation products and applications thereof, and patent document CN113521111A describes the application of bifidobacterium adolescentis in aging delaying and mildew preventing.
However, when bifidobacterium adolescentis is produced industrially, the requirements on growth conditions are severe, the concentration and the yield of the thallus obtained by the current fermentation culture are low (the yield of the thallus at the current industrial level of bifidobacterium adolescentis is about 2.5%), and how to improve the yield of the thallus is a study on the way to improve the yield of the thallus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide bifidobacterium adolescentis and a screening and culturing method and application thereof. The lysate of the bifidobacterium adolescentis provided by the invention can improve the activity and the quantity of human fibroblasts, and has good anti-aging effect. The invention also adopts the fermentation culture medium with specific nitrogen source and growth factor, optimizes the industrial fermentation process conditions, and greatly improves the yield of thalli, so that the yield of thalli at the industrial level of bifidobacterium adolescentis reaches more than 3%.
The invention aims at realizing the following technical scheme:
in a first aspect, the present invention provides a bifidobacterium adolescentis deposited with the cantonese province microorganism strain deposit under the name Bifidobacterium adolescentis JK-BIF-001-02-B1, strain deposit No.: GDMCC No:62063.
in a second aspect, the invention provides a screening method of bifidobacterium adolescentis, comprising the following steps:
A. diluting the human intestinal contents, and then adding the diluted human intestinal contents into a screening culture medium for culturing under anaerobic conditions;
B. selecting single colonies growing out for gram staining, and selecting strains with gram positive and bifidobacterium morphological characteristics;
C. inoculating the strain selected in the step B into an MRS liquid culture medium for culture, culturing the strain which grows spirally at the bottom of the culture medium under aerobic and anaerobic conditions, and selecting the strain which grows only under anaerobic conditions;
D. c, carrying out catalase test on the strain selected in the step C, and selecting the strain with negative result;
E. d, detecting lactic acid and acetic acid of metabolites of the strain selected in the step D, and selecting a strain with mass concentration of acetic acid being greater than that of lactic acid;
F. and E, identifying the strain selected in the step E by 16S rRNA to obtain the bifidobacterium adolescentis strain.
Preferably, in step a, the screening medium comprises the following components: 10g/L of soybean peptone, 10g/L of glucose, 5g/L of yeast extract powder, 0.4g/L of dipotassium hydrogen phosphate, 0.5g/L of diammonium hydrogen citrate, 0.02g/L of magnesium sulfate, 15g/L of agar, 1g/L of L-cysteine hydrochloride, 30-200 mu g/mL of neomycin sulfate and 50-100 mu g/mL of mupirocin lithium salt.
Preferably, in step F, the primer sequences used for the identification are shown in SEQ ID NO.1 and SEQ ID NO. 2.
In a third aspect, the invention provides a fermentation medium for bifidobacterium adolescentis, comprising the following components: 12-18g/L of nitrogen source, 15-25g/L of glucose, 1-2g/L of dipotassium hydrogen phosphate, 1-2g/L of monopotassium phosphate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 0.1-2g/L of growth factor, and manganese sulfate (MnSO 4 ·H 2 O)0.15-0.25g/L；
The nitrogen source is at least one selected from soybean peptone, tryptone, wheat peptone, yeast peptone, cottonseed peptone, corn steep liquor and potato steep liquor;
the growth factor is at least one selected from vitamins, oligosaccharides and amino acids. Specifically, the vitamins include, but are not limited to VA, VB, VC, VE, the oligosaccharides include, but are not limited to fructo-oligosaccharides, xylo-oligosaccharides, malto-oligosaccharides, and the amino acids include, but are not limited to, amino acids such as, but not limited to, arginine, gamma-aminobutyric acid.
Preferably, the nitrogen source is selected from at least one of soy peptone and tryptone;
the growth factor is at least one selected from gamma-aminobutyric acid, tyrosine, arginine, fructo-oligosaccharide, VE and VC.
Preferably, the growth factor is gamma-aminobutyric acid, and the addition amount of the gamma-aminobutyric acid is 1-2g/L.
More preferably, the fermentation medium comprises the following components: 15g/L soybean peptone, 20g/L glucose, 1.5g/L dipotassium hydrogen phosphate, 1.5g/L potassium dihydrogen phosphate, 0.49g/L magnesium sulfate heptahydrate and gamma-ammonia1g/L of hydroxybutyric acid, manganese sulfate (MnSO 4 ·H 2 O)0.21g/L。
The fermentation medium of the invention can be used for fermentation culture of the screened bifidobacterium adolescentis GDMCC No.62063 and fermentation culture of other known bifidobacterium adolescentis.
In a fourth aspect, the present invention provides a fermentation culture method of bifidobacterium adolescentis, comprising the steps of:
s1, inoculating bifidobacterium adolescentis into a fermentation culture medium for culturing under anaerobic conditions, and not stirring and introducing nitrogen after inoculation until the bifidobacterium adolescentis enters a logarithmic growth phase;
s2, stirring and introducing nitrogen until fermentation is finished after the logarithmic growth phase is started, and carrying out pH regulation and carbon source feeding in the later section of the logarithmic growth phase;
s3, after entering a stabilization period, introducing a certain amount of air in the early stage of the stabilization period;
after fermentation is finished, the yield of the obtained thalli is more than 2.8 percent;
in the step S2, the ventilation amount of the nitrogen is 0-0.1vvm;
in the step S3, the ventilation amount of the air is 0-0.2vvm.
In the invention, the thallus yield refers to the mass percentage of wet thallus obtained by centrifugation per 100g of fermentation liquor.
Bacterial activity refers to the number of viable bacteria and the activity of the bacterial cells.
The amount of cells is positively correlated with two factors, namely the number of cells (including dead cells and living cells) and the volume of cells.
Therefore, there is a large difference between the fermentation culture process targeting the number of viable bacteria and the fermentation culture process targeting the yield of the cells.
The fermentation culture method of the invention not only can be used for fermentation culture of the screened bifidobacterium adolescentis GDMCC No.62063, but also can be used for fermentation culture of other known bifidobacterium adolescentis, and can improve the yield of thalli.
According to the invention, static culture is adopted before fermentation enters the logarithmic growth phase, and stirring or stirring and nitrogen introducing treatment are carried out at the beginning of entering the logarithmic growth phase, so that the yield of the bifidobacterium adolescentis industrial level thallus is obviously improved.
Preferably, in step S1, the fermentation medium comprises the following components: 12-18g/L of nitrogen source, 15-25g/L of glucose, 1-2g/L of dipotassium hydrogen phosphate, 1-2g/L of monopotassium phosphate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 0.1-2g/L of growth factor, and manganese sulfate (MnSO 4 ·H 2 O)0.15-0.25g/L；
The nitrogen source is at least one selected from soybean peptone, tryptone, wheat peptone, yeast peptone, cottonseed peptone, corn steep liquor and potato steep liquor;
the growth factor is at least one selected from vitamins, oligosaccharides and amino acids.
Preferably, the nitrogen source is selected from at least one of soy peptone and tryptone;
the growth factor is at least one selected from gamma-aminobutyric acid, tyrosine, arginine, fructo-oligosaccharide, VE and VC.
Preferably, the growth factor is gamma-aminobutyric acid, and the addition amount of the gamma-aminobutyric acid is 1-2g/L.
More preferably, the fermentation medium comprises the following components: 15g/L soybean peptone, 20g/L glucose, 1.5g/L dipotassium hydrogen phosphate, 1.5g/L potassium dihydrogen phosphate, 0.49g/L magnesium sulfate heptahydrate, 1g/L gamma-aminobutyric acid, manganese sulfate (MnSO 4 ·H 2 O)0.21g/L。
Preferably, in step S1, anaerobic conditions are achieved by introducing nitrogen into the fermenter at a volume of 1-2 times before inoculation.
Preferably, in step S2, the stirring speed is 50-100rpm;
the pH is regulated and controlled to 5.0-5.5; the fed-batch carbon source is at least one of glucose, lactose, sucrose and galactose, and the feeding speed is 0.5 per mill-1 per mill of the total volume of the fed-batch fermentation liquor per minute;
the period of the fermentation is 22-28h.
Preferably, in step S3, air is introduced between 19 and 24 hours of fermentation, and the ventilation amount of the air is 0.05 to 0.15vvm. According to the invention, by using an oxygen stress method in the later fermentation stage, namely, a certain amount of air is introduced in the stationary phase, the oxygen in the air can stress the metabolism of the strain, and the free radical (the free radical causes aging) scavenging capacity of the cell lysate is further enhanced.
Preferably, the yield of the obtained cells is 3% or more after the fermentation is completed.
In a fifth aspect, the invention provides the use of a bifidobacterium adolescentis according to the preceding description or obtained according to the preceding fermentation culture method, for the preparation of an anti-ageing product.
Preferably, the preparation of anti-aging products is carried out on lysate obtained after crushing bifidobacterium adolescentis; the lysate is added in an effective amount of 0.05-5%.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a bifidobacterium adolescentis strain derived from the intestinal tract of young people, and a lysate of the bifidobacterium adolescentis strain can effectively increase the characteristics of skin fibroblasts and can be stably passaged;
(2) The invention provides a culture medium adopting a specific nitrogen source and a growth factor, which can obviously improve the thallus yield of bifidobacterium adolescentis;
(3) The invention further adopts static culture before fermentation enters the logarithmic growth phase, and carries out low-speed stirring and a small amount of nitrogen treatment after entering the logarithmic growth phase, so that the yield of bifidobacterium adolescentis thalli can be improved; in addition, a certain amount of air is introduced into the fermentation later stage, so that the free radical scavenging capability of the cell lysate can be improved.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 shows the form of bifidobacterium adolescentis obtained in the present invention;
FIG. 2 is a flow chart of a process for screening and obtaining bifidobacterium adolescentis strains according to the invention;
FIG. 3 is a graph showing the effect of 0.5% lysates of different strains obtained according to the invention on cell viability;
FIG. 4 shows the effect of 0.5% lysate of different strains obtained according to the present invention on fibroblast proliferation;
FIG. 5 shows the yield of cells obtained using different nitrogen source media according to the present invention;
FIG. 6 shows the yield of cells obtained using different growth factors of the invention;
FIG. 7 shows the effect of the use of different amounts of gamma-aminobutyric acid on the yield of bacterial sludge.
FIG. 8 shows the effect of using different stirring speeds and aeration rates on fermentation of bifidobacterium adolescentis according to the present invention;
FIG. 9 shows the effect of static culture and static+aeration culture on fermentation of Bifidobacterium adolescentis according to the present invention;
FIG. 10 is a graph of the fermentation process of bifidobacterium adolescentis according to the invention;
FIG. 11 shows the concentration results of lysates obtained with different aeration rates for 50% inhibition of free radicals according to the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1
The embodiment provides a method for acquiring bifidobacteria, most of bacteria in the rear section (large intestine) of human intestinal tracts are strict anaerobes, and the method is suitable for growth and propagation of the bifidobacteria; therefore, in this example, the object strain was isolated by screening using the intestinal contents of young people as a sample. The process flow is shown in fig. 2, and the specific steps are as follows:
1. samples taken from the later sections of the young (20-30 years old) intestinal tracts are taken out from a refrigerator at-80 ℃ and thawed, and diluted sequentially with sterilized normal saline at a gradient of 10 times, wherein each sample is prepared by taking 50-100 mu L of diluted liquid with three gradients of 4, 5 and 6 respectively, and placing the diluted liquid into a disposable sterile plate with the diameter of 9cm, and 3 gradients are parallel.
2. The sterilized screening culture medium (formula: 10g of soybean peptone, 10g of glucose, 5g of yeast extract powder, 0.4g of dipotassium hydrogen phosphate, 0.5g of diammonium hydrogen citrate, 0.02 of magnesium sulfate, 15g of agar, 1g of L-cysteine hydrochloride, 1000mL of distilled water, adjusting the pH to 6.8-7.0,121 ℃ for sterilization for 20min, adding 30-200 mu g/mL of neomycin sulfate and 50-100 mu g/mL of mupirocin lithium salt) when the temperature is reduced to 45-50 ℃, rapidly pouring plates with different gradient diluents into each plate, adding about 20mL of the plates, covering the plates, stacking together, and shaking the sample liquid and the culture medium clockwise and anticlockwise; the dish was then opened and the mixed media allowed to solidify.
In the screening culture medium, the added neomycin sulfate is an aminoglycoside antibiotic, and the neomycin sulfate with a certain concentration has good antibacterial effect on enterobacteriaceae bacteria such as staphylococcus, corynebacterium, escherichia coli, klebsiella, proteus and the like, and has no inhibition effect on lactobacillus and bifidobacterium. The added mupirocin lithium salt with a certain concentration can effectively inhibit most lactic acid bacteria, but not inhibit the growth of bifidobacteria.
3. And pouring about 10mL of screening culture medium at 50 ℃ into the plate, covering the plate cover after the culture medium is solidified, drying the water vapor on the wall of the plate, stacking the plates together, inversely placing the plates into a 10# self-sealing bag, placing an anaerobic strip capable of consuming oxygen (the culture needs strict anaerobic conditions, and the oxygen in the strain culture environment can be exhausted through the anaerobic strip), sealing the self-sealing bag, and then placing the self-sealing bag in an incubator at 37 ℃ for static culture for 2-4 days.
4. When the monoclonal colonies were grown large enough, the culture was stopped.
5. According to the colony and thallus characteristics of bifidobacteria: after 48-72h culture, the single colony forms a plurality of milky or off-white opaque colonies with the diameter of 0.5-1 mm; the thallus is in a shape of arc, bending and spatula, has different sizes at two ends and a width of about 0.5 μm and a length of 1-6 μm, and has bending and bifurcation phenomena to form a special V or Y shape of bifidobacterium.
Single colonies of white or milky opaque character on the plates were picked, gram stained and examined under a microscope. Selecting strains with gram positive and bifidobacterium morphological characteristics under a microscope, inoculating the strains into an MRS liquid culture medium for culture, selecting strains which spirally grow at the bottom of a test tube, coating a flat plate (100 mu l of bacterial liquid per plate), culturing under aerobic and anaerobic conditions, and selecting strains which only grow under anaerobic conditions.
6. And (3) carrying out catalase detection on the strain selected in the step (5), and selecting the strain with negative catalase.
7. And (3) detecting lactic acid and acetic acid from metabolites of the strain selected in the step (6), and selecting the strain with acetic acid concentration larger than that of lactic acid (mass concentration ratio).
8. The strain selected in the step 7 is identified by 16S rRNA, and the primer is S4-F ataatgcggccg cacgggcggtgtgtrc (SEQ ID NO. 1); S4-R taatagcggccg cagcmgccgcggtaatwc (SEQ ID NO. 2). A total of 5 bifidobacteria including 2 bifidobacteria adolescentis, 2 bifidobacteria longum and 1 bifidobacteria animalis were obtained, which were designated as JK-BB-001, JK-BB-002, JK-BB-003, JK-BB-004 and JK-BB-005 in this order.
Example 2
The 5 bifidobacteria obtained in example 1 were disrupted using a high-pressure cell disruptor to obtain bacterial lysates by: preparing bacterial cells into bacterial liquid with concentration of 10-15% by using distilled water, circularly breaking walls for three times under the pressure of 850-1000bar, and controlling the temperature at 50 ℃ or below during breaking; and centrifuging the bacterial liquid after wall breaking, and taking the supernatant to obtain lysate of each bacterial body.
A model is built by human fibroblasts, and the anti-aging efficacy characteristics of lysates of different strains are compared by using the model.
96-well plates were seeded with 0.8-1 ten thousand human fibroblasts (purchased from North Naloba No. BNCCC340988) and human primary fibroblasts were cultured using a method in which human primary fibroblasts were replaced with primary liquid every 2-3 days and passaged every 7 days. Selecting seventh generation (P7) fibroblasts in logarithmic growth phase, inoculating into a 96-well plate according to 1.2 ten thousand per hole, continuously incubating for 24 hours, discarding the culture solution, adding 150 mu L of serum-free culture medium, adding 0.5% concentration of lysate of different sources prepared by 50 mu L of serum-free culture medium, continuously culturing for 48 hours, discarding the supernatant, and evaluating the cell viability by using neutral red staining. Neutral red staining method: 150. Mu.L of neutral red staining solution (final concentration: 33. Mu.g/mL) was added to each well for staining for 3 hours, the staining solution was discarded, 100. Mu.L of eluent was added to each well, and the OD540 absorbance was measured using an ELISA reader. At the end of neutral staining, photographs were observed under an inverted microscope. The quantitative analysis results of the cell viability are shown in FIG. 3, and the cell state is photographed as shown in FIG. 4. The analysis of the results shows that the 5 lysates at a concentration of 0.5% promote proliferation of human primary fibroblasts relative to the CONTROL group (CONTROL group), but the strain with the most remarkable proliferation promoting effect is JK-BB-002 (fig. 3 and 4 show the results of adding each bifidobacterium lysate at a concentration of 0.5%, wherein the 0# CONTROL in fig. 4 is bifidobacterium longum with a preservation number of CGMCC 13013, which shows a better anti-aging effect in earlier studies). The results in FIG. 4 also show that the concentration of fibroblasts in JK-BB-002 is highest, the growth vigor is denser, and the cell morphology is normal; the concentration of cells in JK-BB-005 was lower than that in JK-BB-002, and the morphology of the cells was somewhat abnormal, indicating that the cells were not in a healthy state.
The thus-screened JK-BB-002 is bifidobacterium adolescentis which is deposited in the microorganism strain collection of Guangdong province at 11/15 of 2021, and the biological material name is Bifidobacterium adolescentis JK-BIF-BIF-001-02-B1, the classification name is Bifidobacterium adolescentis, and the deposit number is: GDMCC No:62063. the morphology of this strain is shown in FIG. 1. The 16S rRNA sequence is shown as SEQ ID NO.3, and the homology analysis with the existing bifidobacterium adolescentis proves that the homology with the Bifidobacterium adolescentis ATCC15703 strain is the highest and is 100%. Further analysis of the strain of the present invention and ATCC15703 strain showed that the sources of the two strains were completely different and that the whole gene sequences of the two strains were greatly different.
SEQ ID NO.3：
GCTGATCCGCGATTACTAGCGACTCCGCCTTCATGGAGTCGGGTTGCAGACTCCAATCCGAACTGAGACCGGTTTTAAGGGATCCGCTCCCCCTCACGAGGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCCCGTGAGTTCCCACCACGACGTGCTGGCAACACAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAGGCCCCATCTCTGGGGCTGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGAGACCGTGGAATGGTCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAAACCGCCTACGAGCCCTTTACGCCCATAATT
Example 3
In the embodiment, bifidobacterium adolescentis JK-BB-002 is fermented and cultured, and the formula of the culture medium is optimized to improve the yield of thalli. The method comprises the following specific steps:
1. a comparison experiment is carried out on a nitrogen source influencing the yield of the thalli, and the adopted culture medium formula is as follows: 15g/L of nitrogen source, 20g/L of glucose, 1.5g/L of dipotassium hydrogen phosphate, 1.5g/L of potassium dihydrogen phosphate, 0.49g/L of magnesium sulfate heptahydrate, 0.38g/L of isomaltooligosaccharide and manganese sulfate (MnSO 4 ·H 2 O) 0.21g/L. The JK-BB-002 screened in example 2 was inoculated onto a medium of different nitrogen sources (tryptone, soybean peptone, corn steep liquor, yeast peptone, wheat peptone, cottonseed peptone, potato steep liquor) and then horizontally fermented in a triangular flask under the following conditions: the cells were cultured under stringent anaerobic conditions (the triangular flask was placed in a closed container having an anaerobic bag) at 37℃for 48 hours, and the obtained cell yields were shown in FIG. 5. The results showed that the cell yields of soybean peptone and tryptone were highest, and soybean peptone was a non-animal source nitrogen source more suitable for the cosmetic industry, so soybean peptone was optimally selected as the nitrogen source.
2. A comparison experiment is carried out on growth factors influencing proliferation, and the adopted culture medium formula comprises the following components:15g/L soybean peptone, 20g/L glucose, 1.5g/L dipotassium hydrogen phosphate, 1.5g/L potassium dihydrogen phosphate, 0.49g/L magnesium sulfate heptahydrate, 0.38g/L growth factor, manganese sulfate (MnSO 4 ·H 2 O) 0.21g/L. The JK-BB-002 screened in example 2 was inoculated onto a medium of different growth factors (vitamin (VA, VB, VC, VE), oligosaccharides (fructo-oligosaccharides, xylo-oligosaccharides), amino acids (amino acids, arginine,), gamma-aminobutyric acid) and then horizontally fermented in a flask under the following conditions: the cells were cultured under stringent anaerobic conditions (the triangular flask was placed in a closed container with an anaerobic bag) at 37℃for 48 hours, and the cell yields were as shown in FIG. 6 (the control group was a medium without addition of growth factors). The results showed that the enhancement effect of gamma-aminobutyric acid was best, and the obtained cell yield was highest, so that gamma-aminobutyric acid was optimally selected as a growth factor.
3. The comparison experiment is carried out on different addition amounts of the growth factor gamma-aminobutyric acid influencing proliferation, the same culture medium formula and method in the step 2 are adopted, the influence of different addition amounts of gamma-aminobutyric acid (0.1 g/L, 0.5g/L, 1g/L, 1.5g/L and 2 g/L) on the yield of the cultured and obtained frontal fungus body is discussed, and the result shows that the yield of the fungus body can reach more than 2.7% when the addition amount of gamma-aminobutyric acid is 1-2g/L.
Example 4
In the embodiment, bifidobacterium adolescentis JK-BB-002 is fermented and cultured, and culture conditions are optimized to improve the yield of thalli. The method comprises the following specific steps:
1. the JK-BB-002 selected in example 2 was inoculated onto a medium of the following formulation: 15g/L soybean peptone, 20g/L glucose, 1.5g/L dipotassium hydrogen phosphate, 1.5g/L potassium dihydrogen phosphate, 0.49g/L magnesium sulfate heptahydrate, 1g/L gamma-aminobutyric acid, manganese sulfate (MnSO 4 ·H 2 O)0.21g/L。
2. The following three groups of different process conditions are adopted to respectively carry out fermentation culture in a 5L fermentation tank:
2.1 static culture process: after inoculation, the fermentation tank is not stirred until the fermentation tank enters the logarithmic growth phase, nitrogen is not introduced, stirring is started after the fermentation tank enters the logarithmic growth phase, the rotating speeds are respectively 50rpm, 100rpm and 200rpm, and the rotating speeds are kept until the fermentation is finished; after 5-10L of nitrogen was introduced before inoculation, the off-gas valve was closed throughout the fermentation stage. (the cells deposited on the bottom of the pot cannot be stirred up when stirring is < 50rpm, and high shear force is disadvantageous for the cells due to high rotation speed)
2.2, stirring and culturing: setting two gradients of stirring rotation speed after inoculation, namely 50rpm and 200rpm respectively, and keeping the rotation speed until fermentation is finished; after 5-10L of nitrogen was introduced before inoculation, the off-gas valve was closed throughout the fermentation stage.
2.3 aeration culture process: nitrogen is not introduced before inoculation, in order to ensure positive pressure in the fermentation tank, nitrogen is introduced after inoculation, the aeration rate is set to be 0.1VVM and 0.4VVM respectively, and the nitrogen is introduced until the fermentation is finished (stirring is not started in the whole fermentation stage).
2.4 static+aeration culture Process: introducing 5-10L of nitrogen before inoculation, after inoculation, not stirring the fermentation tank until the fermentation tank enters the logarithmic phase, not introducing nitrogen, stirring the fermentation tank after entering the logarithmic phase, and introducing nitrogen until the fermentation is finished; the rotational speed + aeration rate used was 100rpm +0.1vvm, 100rpm +0.4vvm, respectively.
When the four groups of the culture are cultured under different process conditions, the same pH regulation and control (pH is regulated to 5.2) and carbon source feeding process (glucose is used as a carbon source, and the feeding amount per minute is 1 per mill of the total volume of the fermentation liquor) are adopted in the later stage of the logarithmic phase. The results show that stirring and aeration have a relatively pronounced effect on the initiation and proliferation of bifidobacteria. As shown in FIG. 8, the curves at 50rpm and 200rpm are the results obtained using the different stirring speed process of 2.2, and the curves at 0.1vvm and 0.4vvm are the results obtained using the different aeration rate process of 2.3. As shown in FIG. 9, 50rpm, 100rpm, 200rpm are the results obtained using the different stirring speed process of 2.1, and 100rpm+0.1vvm, 100rpm+0.4vvm are the results obtained using the different aeration rate process of 2.4. As is clear from the results of FIGS. 8 and 9, the cell yields were highest in the case of static culture at 50-100rpm and static culture at 100rpm+0.1vvm+aeration, and were 3.21-3.34%. The results of fig. 9 also show that: when the stirring rotation speed is 50rpm and 100rpm, the fermentation process and the thallus yield have no obvious difference. 200rpm can lead to slow fermentation process, and the thallus yield is obviously reduced. At a rotational speed of 100rpm, the aeration rate was substantially unchanged at 0.1vvm, and when the aeration rate was increased to 0.4vvm, both the fermentation process and the cell yield were slightly decreased.
Example 5
The JK-BB-002 selected in example 2 was cultured by the static culture process of example 4 in the same manner as in step 1 and step 2.1 of example 4. The fermentation process curve of the strain thus obtained is shown in FIG. 10. Selecting A, B, C time points in a fermentation period for comparison in the later stage of fermentation, wherein the point A is at the last stage of the logarithmic phase (18-21 h), the activity of the thalli is highest, the thalli proliferation capability is strong, and compared with the point B, C, the thalli yield is low; the yield of the thalli at the point B (21-24 h) and the yield of the thalli at the point C (24-26 h) are close, and the wall breaking efficiency of the thalli at the point C is higher under the same wall breaking condition, so that the yield of the thalli reaches 3.3% by selecting the point C as the fermentation end point.
This example further discusses the effect of a small amount of air introduced between points B and C (1-2 h after point B, i.e., any time point between 22-26h of fermentation) on the free radical scavenging capacity of the bacterial strain lysate obtained after cultivation, in the fermentation cycle of FIG. 10. At this time, the bacterial concentration reaches the maximum value, and the yield of the bacterial cells is not affected by the introduction of oxygen. The concentration results of 50% inhibition of free radicals with different aeration rates (0 VVM, 0.03VVM, 0.1VVM, 0.3VVM, 1 VVM) for the same aeration time (5 min) are shown in FIG. 11. When the ventilation is 0.1VVM, the lysate of the obtained strain has the strongest scavenging ability on free radicals.
There are many ways in which the invention may be practiced, and what has been described above is merely a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications may be made without departing from the principles of the invention, and such modifications are intended to be within the scope of the invention.
Sequence listing
<110> Shanghai JiaKai biotechnology Co., ltd
<120> A bifidobacterium adolescentis and screening and culturing method and application thereof
<130> PO5211370
<160> 3
<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial Sequence)
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ataatgcggc cgcacgggcg gtgtgtrc 28
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<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
taatagcggc cgcagcmgcc gcggtaatwc 30
<210> 3
<211> 806
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gctgatccgc gattactagc gactccgcct tcatggagtc gggttgcaga ctccaatccg 60
aactgagacc ggttttaagg gatccgctcc ccctcacgag gtcgcatccc gttgtaccgg 120
ccattgtagc atgcgtgaag ccctggacgt aaggggcatg atgatctgac gtcatcccca 180
ccttcctccg agttgacccc ggcggtcccc cgtgagttcc caccacgacg tgctggcaac 240
acagggcgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 300
gacgaccatg caccacctgt gaacccgccc cgaagggagg ccccatctct ggggctgtcg 360
ggaacatgtc aagcccaggt aaggttcttc gcgttgcatc gaattaatcc gcatgctccg 420
ccgcttgtgc gggcccccgt caatttcttt gagttttagc cttgcggccg tactccccag 480
gcgggatgct taacgcgttg gctccgacac ggagaccgtg gaatggtccc cacatccagc 540
atccaccgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc 600
gctcctcagc gtcagtgacg gcccagagac ctgccttcgc cattggtgtt cttcccgata 660
tctacacatt ccaccgttac accgggaatt ccagtctccc ctaccgcact caagcccgcc 720
cgtacccggc gcggatccac cgttaagcga tggactttca caccggacgc gacgaaaccg 780
cctacgagcc ctttacgccc ataatt 806
Claims (9)
1. A bifidobacterium adolescentis characterized in that the bifidobacterium adolescentis is deposited in the microorganism strain deposit center of Guangdong province under the name ofBifidobacterium adolescentisJK-BIF-BIF-001-02-B1, strain accession number is: GDMCC No:62063.
2. use of a fermentation medium in the fermentation culture of bifidobacterium adolescentis according to claim 1, characterized in that it comprises the following components: 12-18g/L of nitrogen source, 15-25g/L of glucose, 1-2g/L of dipotassium hydrogen phosphate, 1-2g/L of monopotassium phosphate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 0.1-2g/L of growth factor and 0.15-0.25 g/L of manganese sulfate;
the nitrogen source is at least one selected from soybean peptone, tryptone, wheat peptone, yeast peptone, cottonseed peptone, corn steep liquor and potato steep liquor;
the growth factor is selected from at least one of gamma-aminobutyric acid, tyrosine, arginine, isomaltooligosaccharide, fructooligosaccharide, VE and VC.
3. The use according to claim 2, wherein in the fermentation medium the nitrogen source is selected from at least one of soy peptone, tryptone;
the growth factor is at least one selected from gamma-aminobutyric acid, tyrosine, arginine, fructo-oligosaccharide, VE and VC.
4. A fermentation culture method of bifidobacterium adolescentis as claimed in claim 1, comprising the steps of:
s1, inoculating bifidobacterium adolescentis into a fermentation culture medium for culturing under anaerobic conditions, and not stirring and introducing nitrogen after inoculation until the bifidobacterium adolescentis enters a logarithmic growth phase;
s2, stirring and introducing nitrogen until fermentation is finished after the logarithmic growth phase is started, and carrying out pH regulation and carbon source feeding in the later section of the logarithmic growth phase;
s3, after entering a stable period, introducing air;
after fermentation is finished, the yield of the obtained thalli is more than 2.8 percent;
in the step S2, the ventilation amount of the nitrogen is 0-0.1vvm;
in the step S3, the ventilation amount of the air is 0-0.2vvm.
5. The fermentation culture method of bifidobacterium adolescentis according to claim 4, wherein in step S1, the fermentation medium comprises the following components: 12-18g/L of nitrogen source, 15-25g/L of glucose, 1-2g/L of dipotassium hydrogen phosphate, 1-2g/L of monopotassium phosphate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 0.1-2g/L of growth factor and 0.15-0.25 g/L of manganese sulfate;
the nitrogen source is at least one selected from soybean peptone, tryptone, wheat peptone, yeast peptone, cottonseed peptone, corn steep liquor and potato steep liquor;
the growth factor is at least one selected from gamma-aminobutyric acid, tyrosine, arginine, isomaltooligosaccharide, fructooligosaccharide, VE and VC.
6. The fermentation culture method of bifidobacterium adolescentis according to claim 4, characterized in that in step S2, the rotational speed of the stirring is 50-100rpm;
the pH is regulated and controlled to 5.0-5.5; the fed-batch carbon source is at least one of glucose, lactose, sucrose and galactose, and the feeding speed is 0.5 per mill-1 per mill of the total volume of the fed-batch fermentation liquor per minute;
the period of the fermentation is 22-28h.
7. The fermentation culture method of bifidobacterium adolescentis according to claim 4, wherein in step S3, air is introduced between 19 and 24 hours of fermentation, and the ventilation amount of the air is 0.05 to 0.15vvm.
8. Use of bifidobacterium adolescentis according to claim 1 or obtainable by a fermentation culture process according to any of claims 4-7 in the manufacture of an anti-ageing product.
9. The use according to claim 8, characterized in that it comprises the preparation of anti-aging products from the lysate obtained after disruption of bifidobacterium adolescentis; the lysate is added in an effective amount of 0.05-5%.
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| WO2010027117A1 (en) * | 2008-09-04 | 2010-03-11 | Marine Bioprocess Co., Ltd. | Preparation method of gaba by fermentation of seaweed |
| CN111748512A (en) * | 2020-07-15 | 2020-10-09 | 江南大学 | Nitrogen source suitable for efficiently proliferating bifidobacterium adolescentis and application thereof |
| CN113234597A (en) * | 2021-05-28 | 2021-08-10 | 江南大学 | Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof |
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| WO2010027117A1 (en) * | 2008-09-04 | 2010-03-11 | Marine Bioprocess Co., Ltd. | Preparation method of gaba by fermentation of seaweed |
| CN111748512A (en) * | 2020-07-15 | 2020-10-09 | 江南大学 | Nitrogen source suitable for efficiently proliferating bifidobacterium adolescentis and application thereof |
| CN113234597A (en) * | 2021-05-28 | 2021-08-10 | 江南大学 | Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof |
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