CN104178447B - A kind of method for improving lactic acid bacteria Bile salt resistance - Google Patents
A kind of method for improving lactic acid bacteria Bile salt resistance Download PDFInfo
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- CN104178447B CN104178447B CN201410445527.5A CN201410445527A CN104178447B CN 104178447 B CN104178447 B CN 104178447B CN 201410445527 A CN201410445527 A CN 201410445527A CN 104178447 B CN104178447 B CN 104178447B
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- lactic acid
- acid bacteria
- glutamine
- glutamine transaminage
- bile salt
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 241000894006 Bacteria Species 0.000 title claims abstract description 79
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 57
- 239000004310 lactic acid Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 57
- 239000003833 bile salt Substances 0.000 title claims abstract description 26
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 109
- 230000001580 bacterial effect Effects 0.000 claims abstract description 33
- 235000014897 Streptococcus lactis Nutrition 0.000 claims abstract description 26
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 23
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 23
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 23
- 239000010413 mother solution Substances 0.000 claims abstract description 15
- 239000002609 medium Substances 0.000 claims abstract description 14
- 241000186604 Lactobacillus reuteri Species 0.000 claims abstract description 8
- 229940001882 lactobacillus reuteri Drugs 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 241000194035 Lactococcus lactis Species 0.000 claims abstract 2
- 239000012530 fluid Substances 0.000 claims description 26
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 20
- 238000007792 addition Methods 0.000 claims description 13
- 229910021529 ammonia Inorganic materials 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000340 Transaminases Proteins 0.000 claims description 6
- 102000003929 Transaminases Human genes 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 150000001408 amides Chemical class 0.000 claims description 2
- 108010010779 glutamine-pyruvate aminotransferase Proteins 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims 1
- 229940099352 cholate Drugs 0.000 abstract description 42
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 abstract description 41
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 16
- 239000004472 Lysine Substances 0.000 abstract description 16
- 239000006041 probiotic Substances 0.000 abstract description 13
- 235000018291 probiotics Nutrition 0.000 abstract description 13
- 230000000529 probiotic effect Effects 0.000 abstract description 12
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- 235000013958 Lactobacillus casei Nutrition 0.000 abstract description 9
- 229940017800 lactobacillus casei Drugs 0.000 abstract description 9
- 210000002421 cell wall Anatomy 0.000 abstract description 8
- 230000000968 intestinal effect Effects 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 3
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- 235000018977 lysine Nutrition 0.000 description 15
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- 108010013639 Peptidoglycan Proteins 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
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- 230000012010 growth Effects 0.000 description 7
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- 102000004169 proteins and genes Human genes 0.000 description 7
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- 241000192125 Firmicutes Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- ZDXPYRJPNDTMRX-GSVOUGTGSA-N D-glutamine Chemical compound OC(=O)[C@H](N)CCC(N)=O ZDXPYRJPNDTMRX-GSVOUGTGSA-N 0.000 description 1
- LNLLNTMHVMIMOG-YUMQZZPRSA-N Gamma-glutamyl-Lysine Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CC[C@H](N)C(O)=O LNLLNTMHVMIMOG-YUMQZZPRSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of improve the method containing glutamine and the lactic acid bacteria Bile salt resistance of lysine in cell wall, methods described is lactic acid bacteria described in the culture medium culturing containing glutamine transaminage.The lactic acid bacteria being related in the present invention includes the lactic acid bacteria class bacterium clock containing glutamine and lysine in all cell wall such as 8148 bacterial strain of lactococcus lactis, Lactobacillus plantarum sp. strain, Lactobacillus reuteri and lactobacillus casei, preferably glutamine transaminage final concentration of 3～25U/mL in the medium of the invention, while the further raising lactic acid bacteria Bile salt resistance of the method that is repeatedly added to culture medium by glutamine transaminage mother solution.The method of the invention is simple, and cost is relatively low, it is easy to industrialized production, can significantly improve resistance of the existing commercially available lactic acid bacteria class probiotic bacteria to cholate by methods described, contributes to its function in the intestinal.
Description
Technical field
The present invention relates to the method for improving lactic acid bacteria Bile salt resistance, contains in more particularly to a kind of raising whole cell peptidoglycan
The method that the lactic acid bacteria of glutamine and lysine tolerates cholate.
Background technology
Probiotic bacteria refers to the living microorganisms for having certain promotion from host and to host health.And demonstrate,proved
It is bright be probiotic bacteria bacterial strain in, major part belongs to lactic acid bacteria.Numerous studies show, can be used as probiotic bacteria in human gi-tract
Lactic acid bacteria has antioxidation, reduces the important biomolecule functions such as cholesterol, enhancing immunity, antitumor.Probiotic bacteria can be in place
Have the survival volume of much degree in main body, and the fertility reached behind position decide its host is provided the dynamics of benefit or
Effect.This requires that the lactic acid bacteria in product can be reached intestinal and is colonized in intestinal with substantial amounts of survival bacterium by upper digestive tract
Mucosa, to play its benefit, thus lactobacillus cell should be with the toleration to cholate.
Cholate (Bile salt) is the sodium salt for being combined and being formed with glycine or taurine by the bile acid of hepatocytes secrete
Or potassium salt.The Main Function of cholate is to aid in digestion fat, so cholate has to phospholipid, fatty acid and memebrane protein in cell membrane
Certain destruction, so as to change cell permeability of the membrane, to cell damage, makes lactic acid bacteria class probiotics die.
Thus, cholate is had certain toleration be lactic acid bacteria can intestinal survival, the prerequisite that grows and play effect it
One, i.e.,：The bacterial strain of growth and metabolism can be only possible to survive in intestinal after normal physiological gallbladder salinity stress.Therefore, carry
High lactic acid bacteria is the key problem in technology of lactobacillus product development and exploitation to the toleration of cholate.
Existing method mainly includes the lactobacilli strain from nature separation screening bile tolerance, to existing commercialization bacterium
Strain is oriented selection-breeding, mutation or genetic manipulation, and utilizes encapsulation bacterial strain, to improve the ability of its bile tolerance.This
A little methods all be present in terms of application, and such as to have probiotic and bile tolerance bacterial strain difficulty concurrently larger for separation screening,
Follow-up safety evaluatio is also required to time and cost, and exists and cause not applicable asking as safety is not up to standard
Topic；For second method, as present consumer is to the favor of natural green food and the row to genetic modification food
Scold, if selection-breeding, mutation or genetic manipulation are oriented to the bacterial strain of practical application, there is Consumer acceptance relatively low
Risk；And the third method, various microencapsulation technologies have been developed at present both at home and abroad and is applied to probiotic bacteria, but each deposit
In some shortcomings, it is impossible to take into account industrialized production and keep contradiction intrinsic between bacterial activity.
The molecular weight that glutamine transaminage (transglutaminase, abbreviation TG) is made up of 331 amino is about
The monomeric protein at 38000 active center, its can catalytic proteins polypeptide occur intramolecular and intermolecular generation it is covalent
Crosslinking, so as to improve the 26S Proteasome Structure and Function of protein, to the property of protein such as：Foaminess, emulsibility, emulsion stability, heat
Stability, water-retaining property and gelling ability and other effects significantly, and then improve local flavor, mouthfeel, quality and outward appearance of food etc..
At present, not yet disclose and adopt glutamine transaminage to process lactic acid bacteria to improve the correlation text of its Bile salt resistance
Offer data.
The content of the invention
The present invention provides a kind of lactic acid bacteria containing glutamine and lysine in can improving whole cell peptidoglycan and tolerates gallbladder
The method of salt ability, methods described have simple to operate, low cost, the features such as practical.
A kind of method for containing the lactic acid bacteria Bile salt resistance of glutamine and lysine in raising whole cell peptidoglycan, it is described
Method is：With lactic acid bacteria described in the culture medium culturing containing glutamine transaminage；
Further, the glutamine transaminage point adds at least twice；
Further, the glutamine transaminage preferably point 2 to 5 additions；
The glutamine transaminage added in the time before preparing culture medium and starting to culture to terminate.
It is newborn that the lactic acid bacteria specifically includes 8148 bacterial strain of lactococcus lactis, Lactobacillus plantarum plant sp. strain, Luo Yishi
Contain the lactic acid bacteria class bacterium clock of glutamine and lysine in the whole cell peptidoglycan such as bacillus and lactobacillus casei；
After last time adds the glutamine transaminage, glutamine transaminage final concentration in the medium is made
For 3～25U/mL；
It is preferred that glutamine transaminage final concentration of 3～25U/mL in the medium；
Further preferably glutamine transaminage final concentration of 12U/mL in the medium；
The method for further improving lactic acid bacteria Bile salt resistance also includes being configured to the glutamine of 50～500U/mL with water
Transaminase's mother solution, with membrane filtration, glutamine transaminage mother solution is added in the lactic acid bacteria culturing medium；
The culture medium of above-mentioned lactic acid bacteria includes MRS fluid mediums, GM17 fluid mediums and MRS solid mediums.
Beneficial effects of the present invention
One：
Inventor contains the lactic acid bacteria class probiotic bacteria of glutamine and lysine in finding every whole cell peptidoglycan, can
Using the method for the present invention, its toleration to cholate is effectively improved.
It is former using the Bile salt resistance that lactococcus lactis and Lactobacillus plantarum etc. can be improved after glutamine transaminage process
Reason is as follows：
The simple structure of gram positive bacterial cell wall, is mainly made up of peptidoglycan layer, and Peptidoglycan is that abnormal shape is more
Sugared macromole, each peptidoglycan monomer contain three ingredients, i.e. dissacharide units, the small peptide linked up by four aminoacid
" tail " and peptide " bridge ".Small peptide " tail " and peptide " bridge " that different gram positive bacterias may have composition different.It is particularly
The composition of the small peptide of gram positive bacteria " tail " is L-Alanine → D-Gln → 1B → D-alanine, among these
The catalytic substrate glutamine and lysine of glutamine transaminage are provided with by chance.Therefore, think after inventor's research, adopt
After glutamine transaminage processes lactic acid bacteria, increase the cell wall degree of cross linking of lactic acid bacteria, the cell wall of encryption effectively can stop
Cholate molecule removes damaging cells film through cell wall, so as to impart using the anti-gallbladder of lactic acid bacteria after glutamine transaminage process
The good characteristic of salt.
Although the present invention can improve the Bile salt resistance of lactic acid bacteria, to acid stress DeGrain.Acid stress and gallbladder
Salt stress is widely different to the damage reason of cell.Acid stress can cause intracellular pH value to reduce, and suppress intracellular enzyme activity and transhipment system
The activity of system, causes glycolysiss rate reduction, so as to affect the generation of cellular energy.Meanwhile, intracellular low ph value can inducing cell
The energy of itself is consumed by intracellular excessive H+Pump out extracellular, will necessarily further increase the consumption of energy, so as to seriously press down
Cell growth processed.The Main Function of cholate is to aid in human consumption's fat, so cholate is to phospholipid, fatty acid and film in cell membrane
Albumen has certain destruction, so as to change cell permeability of the membrane, to cell damage.Result of study is also demonstrated that
Glutamine transaminage processes the acid stress to lactobacillus cell does not have an effect.
Which two：
Glutamine transaminage, glutamine transaminage are widely present in animal tissue, can rely ammonia by L- in catalytic proteins
Association reaction between acid and glutamine, thus can make between protein or polypeptide, covalent cross-linking, form covalent combination
The polymer of thing.People are catalyzed to form gamma-glutamyl lysine isopeptide bond containing glutamine transaminage edible always
Food such as fishery technology, Petaso, sausage, noodles, bean curd etc., therefore, it is prebiotic lactic acid bacteria class to be processed with glutamine transaminage
Bacteria strain is safe to human body.
The two subspecies are used by people to as Dairy fermentation agent lactococcus lactis all the time, with important economy
Value, meanwhile, it is also that type strain the most deep is studied in current lactic acid bacteria, therefore the experimental result obtained with which is very
The general character of lactic acid bacteria can be reflected in big degree.Lactobacillus plantarum is naturally occurring a kind of antibacterial in human body intestinal canal, takes plant
After thing lactobacilluss, there is specific medical care effect to healthy population and patient.At present, substantial amounts of lactobacillus plantarum strain
Commercial applications have been obtained as probiotic bacteria.
In sum, the present invention is perfectly safe in food security aspect, and several lactic acid bacterias studied have
The representativeness of this class lactic acid bacteria.
Which three：
The inventive method is simple, and cost is relatively low, it is easy to industrialized production, can significantly improve existing city by methods described
Resistance of the lactic acid bacteria class probiotic bacteria sold to cholate, contributes to its function in the intestinal.For example：According in the present embodiment 1
Method with 12U/mL glutamine transaminages process after lactococcus lactis grow 10h when to 0.04% Bile salt resistance ratio
It is untreated to improve 5.63 times；Lactobacillus plantarum plant subspecies after processing according to the method in embodiment 2 are when 10h is grown
7.59 times are improve than untreated to 0.04% Bile salt resistance.
Description of the drawings
Fig. 1：0.04% cholate coerces impact situation maps of the 2h to 8148 strain growth of lactococcus lactis；
Fig. 2：Glutamine transaminage feed postition coerces 2h tolerations to 8148 bacterial strain of lactococcus lactis, 0.04% cholate
Impact situation map；
Fig. 3：The process of variable concentrations glutamine transaminage is resistance to 8148 bacterial strain of lactococcus lactis, 0.04% cholate stress 2h
Situation map is affected by property；
Fig. 4：The process of variable concentrations glutamine transaminage is coerced to 0.04% cholate of Lactobacillus plantarum plant sp. strain
The impact situation map of 2h tolerations.
Specific embodiment
Embodiment 1
It is a kind of that 8148 bacterial strain of lactococcus lactis is processed by glutamine transaminage, improve the side of its tolerance cholate ability
Method.
In the present embodiment, M17 broth bouillons powder is bought from Qingdao Hai Bo.M17 broth bouillons are to use 42.3g M17
, in 1000ml distilled water, 121 DEG C of autoclavings 15 minutes are standby for broth bouillon powder heating for dissolving.
8148 bacterial strain of the lactococcus lactis is from Chinese industrial Microbiological Culture Collection administrative center, CICC numberings
20406。
The operating procedure of the present invention is followed successively by：
1st, GM17 fluid mediums are prepared：Concrete grammar is to add final concentration of 5g/L Fructus Vitis viniferaes in M17 broth bouillons
Sugar, obtains GM17 fluid mediums.
2nd, it is inoculated with：8148 bacterial strain of lactococcus lactis for having activated is inoculated in GM17 culture medium with 5% amount, at 30 DEG C
Quiescent culture 4h is used as seed liquor.
3rd, cultivate：Seed liquor is inoculated in by culture 8148 bacterium of lactococcus lactis in GM17 culture medium with 5% inoculum concentration again
Strain.
4th, glutamine transaminage solution is prepared：The glutamine transaminage mother solution of 200U/mL is configured to water, 0.45 is used
After μm membrane filtration, under conditions of placing 4 ± 2 DEG C, cold preservation is standby.
5th, glutamine transaminage is processed：Respectively at the glutamine transaminage that 2h, 4h, 6h addition equivalent step 4 is obtained
Mother solution, makes glutamine transaminage final concentration of 3～25U/mL in the medium.
6th, quiescent culture：Quiescent culture obtains 8148 bacterial strain of lactococcus lactis after glutamine transaminage is processed to 8h.
Wherein, it is as 8148 bacterial strain of lactococcus lactis is in the growth of GM17 culture medium that the present embodiment selects GM17 culture medium
Preferably, the method for the present embodiment is not limited to culture medium, and the GM17 culture medium of the present embodiment could alternatively be every can make lactic acid
The fluid medium of 8148 strain growth of Lactococcus.
The measure of stress resistance typically adopts survival rate method, this method to need multiple 10 times of dilutions, error to be difficult to avoid that.To subtract
Less due to diluting the error brought, the present invention determines 8148 bacterial strain of lactococcus lactis using based on the tolerance rate method of turbidimetry
Bile salt resistance.8148 bacterial strain of lactococcus lactis direct inoculation GM17 culture medium after 0.04% cholate stress 2h is cultivated.
Due to damage of the cholate to cell, viable count is caused to reduce, lag phase is obviously prolonged, logarithmic (log) phase growth rate is relatively slow,
But final Biomass is consistent with the matched group do not coerced.Cell is stronger to the toleration of cholate, then Biomass with do not coerced
Matched group closer to tolerance rate is higher, i.e., resistance is stronger, and vice versa.As shown in figure 1, cholate tolerance rate curve is presented
Go out early stage and be rapidly reduced to minimum, the characteristics of the later stage is gradually risen to 100%.The later stage of curve be enough to reflect cell Jing gallbladders
The energy for growth of living cells after salt stress, the i.e. toleration to cholate stress.Therefore, the later stage raised bench of cholate tolerance rate curve
The situation of section can be used to characterize the resistance size that cell coerces cholate.
The concrete assay method of cholate tolerance rate is：After strain culturing 8h, 5mL bacterium solutions are centrifuged, centrifugal condition is
4000r/min, 10min, abandon supernatant, by cell precipitation with brine once, be resuspended in containing 0.04% (w/v) cholate
Normal saline in, put water-bath 2h at 37 DEG C.It is centrifuged under 4000r/min, 10min, brine is once.With 5mL's
Culture medium re-suspended cell is precipitated, and is fully mixed as seed liquor, is inoculated in culture medium with 5% inoculum concentration, in 0h, 9h,
10h, 11h, 12h, 13h survey Biomass OD600.Cholate solution is replaced to carry out identical experimental implementation with normal saline simultaneously, with this
As control.Cholate stress-tolerance rate is calculated according to formula.
Cholate stress-tolerance rate (%)=ODStress/ODControl* 100%
Control experiment：First group of experimental group for being to be not added with glutamine transaminage, i.e., according to the step of embodiment 11,2,3,
After 6 operations, its cholate stress-tolerance rate is determined according to the method described above；Second group is disposably to add glutamine transaminage to end
The experimental group of concentration, i.e.,：Step 5 in embodiment 1 is replaced with and " disposably adds paddy in the medium at 0 moment of culture
Glutamine transaminase is to final concentration 3-25U/mL ", other are with embodiment 1；The 3rd group of experimental group for embodiment 1；
Wherein, in second group and the 3rd group, add the experimental result such as table 1 of glutamine transaminage to final concentration 9U/mL
Record.
Impact of the 1 glutamine transaminage feed postition of table to 8148 bacterial strain of lactococcus lactis, 0.04% Bile salt resistance
From accompanying drawing 2 and table 1, divide in 0 moment of culture disposably adds glutamine transaminage and incubation
The glutamine transaminage for adding final concentration of 9U/mL three times, can significantly improve 8148 bacterial strain of lactococcus lactis to cholate
Toleration.Also, the effect for being added in incubation in three times is substantially better than and disposably adds at 0 moment of culture.
Inventor also determines variable concentrations glutamine transaminage to 8148 bacterial strain of lactococcus lactis to 0.04% cholate
The impact of stress 2h tolerations, data are shown in accompanying drawing 3, as a result show that equivalent addition glutamine turns ammonia in three times in incubation
Enzyme, in the range of 0～12U/mL, with the increase of the glutamine transaminage concentration added, 8148 bacterial strain of lactococcus lactis
0.04% Bile salt resistance is also improved therewith, and the effect for especially adding 12U/mL is more significantly.But, when glutamine turns ammonia
When enzyme addition concentration is 15 and 20U/mL, effect is less than glutamine transaminage addition concentration on the contrary for 6U/mL, and paddy ammonia
Amide transaminase addition concentration is more worse than 15U/mL for the effect of 20U/mL.
Embodiment 2
It is a kind of that Lactobacillus plantarum plant sp. strain is processed by glutamine transaminage, improve its tolerance cholate ability
Method.
In the present embodiment, MRS broth bouillons powder is bought from Qingdao Hai Bo.The Lactobacillus plantarum plant sp. strain
From Chinese industrial Microbiological Culture Collection administrative center, CICC numberings 6073.
The operating procedure of the present invention is followed successively by：
1st, MRS fluid mediums are prepared：Concrete grammar is to weigh 52.4g MRS broth bouillon powder, heating for dissolving in
In 1000ml distilled water, 121 DEG C of autoclavings 15 minutes obtain MRS fluid mediums.
2nd, it is inoculated with：The Lactobacillus plantarum plant sp. strain for having activated is inoculated in into MRS fluid mediums with 5% amount
In, at 30 DEG C, quiescent culture 9.5h is used as seed liquor.
3rd, cultivate：It is inoculated in MRS fluid mediums with 5% inoculum concentration again, cultivates Lactobacillus plantarum plant subspecies bacterium
Strain.
4th, glutamine transaminage solution is prepared：The glutamine transaminage mother solution of 200U/mL is configured to water, is used
After 0.45um membrane filtrations, under conditions of placing 4 ± 2 DEG C, cold preservation is standby.
5th, glutamine transaminage is processed：Step is added when Lactobacillus plantarum plant sp. strain culture 2h, 4h, 6h
The rapid 4 glutamine transaminage mother solutions for obtaining, after last time addition glutamine transaminage, make glutamine transaminage in training
Final concentration of 3～25U/mL in foster base.
6th, quiescent culture：Quiescent culture obtains the Lactobacillus plantarum plant subspecies after glutamine transaminage is processed to 8h
Bacterial strain.
Wherein, the present embodiment selects MRS fluid mediums is trained in MRS liquid due to Lactobacillus plantarum plant sp. strain
Preferably, the method for the present embodiment is not limited to culture medium, and the MRS fluid mediums in the present embodiment could alternatively be for the growth of foster base
Every fluid medium that grow can Lactobacillus plantarum plant sp. strain.
Inventor is done using the Lactobacillus plantarum plant sp. strain after glutamine transaminage process to embodiment 2
The stress 2h experiments of 0.04% cholate, are as a result shown in accompanying drawing 4.
From accompanying drawing 4 as can be seen that equivalent addition glutamine transaminage 12U/mL is newborn to plant in three times in incubation
Bacillus plant subspecies improve the effect of anti-cholate ability preferably, but, when glutamine transaminage addition concentration is 15U/mL,
Effect adds concentration for 9U/mL's less than glutamine transaminage on the contrary.Therefore, this method improves cholate to lactobacilluss class probiotic bacteria
Resistance is equally effective.
Embodiment 3
It is a kind of that Lactobacillus plantarum plant sp. strain is processed by glutamine transaminage, improve its tolerance cholate ability
Method.
In the present embodiment, the purchase of MRS broth bouillons from Qingdao Hai Bo, the Lactobacillus plantarum plant sp. strain from
Chinese industrial Microbiological Culture Collection administrative center, CICC numberings 6073.
The operating procedure of the present invention is followed successively by：
1st, MRS fluid mediums are prepared：Concrete grammar is to weigh 52.4g MRS broth bouillon powder, heating for dissolving in
In 1000ml distilled water, 121 DEG C of autoclavings 15 minutes obtain MRS fluid mediums.
2nd, it is inoculated with：The Lactobacillus plantarum plant sp. strain for having activated is inoculated in into MRS fluid mediums with 5% amount
In, at 30 DEG C, quiescent culture 9.5h is used as seed liquor.
3rd, cultivate：It is inoculated in MRS fluid mediums with 5% inoculum concentration again, cultivates Lactobacillus plantarum plant subspecies bacterium
Strain.
4th, glutamine transaminage solution is prepared：The glutamine transaminage mother solution of 100U/mL is configured to water, filter membrane is used
Filter, it is standby.
5th, glutamine transaminage is processed：Start to culture to terminate front 1h's from culture Lactobacillus plantarum plant sp. strain
In random time, the glutamine transaminage mother solution of point two to five additions in fluid medium, last time addition paddy ammonia
After amide transaminase mother solution, the final concentration of 3～25U/mL of glutamine transaminage is made.
6th, quiescent culture：The Lactobacillus plantarum plant subspecies bacterium after glutamine transaminage is processed is obtained after quiescent culture
Strain.
Embodiment 4
Method that is a kind of that Lactobacillus reuteri is processed by glutamine transaminage, improving its tolerance cholate ability.
Wherein, the Lactobacillus reuteri is from Chinese industrial Microbiological Culture Collection administrative center, CICC numberings 6123.
The operating procedure of the present invention is followed successively by：
1st, it is inoculated with：The Lactobacillus reuteri for having activated is inoculated in the MRS broth bouillons of purchase, Luo Yishi is newborn for culture
Bacillus.
2nd, glutamine transaminage solution is prepared：The glutamine transaminage mother solution of 50～500U/mL is configured to water, is used
Membrane filtration, it is standby.
3rd, glutamine transaminage is processed：Random time before terminating from 0 moment of culture Lactobacillus reuteri to culture
Glutamine transaminage mother solution that is interior, being added in fluid medium at least one times, last time addition glutamine transaminage
After mother solution, the final concentration of 3～25U/mL of glutamine transaminage is made.
4th, quiescent culture：The Lactobacillus reuteri after glutamine transaminage is processed is obtained after quiescent culture.
After above-mentioned steps process, the Bile salt resistance of the bacterial strain is significantly improved.
Embodiment 5
Method that is a kind of that lactobacillus casei is processed by glutamine transaminage, improving its tolerance cholate ability.
Wherein, the lactobacillus casei is from Chinese industrial Microbiological Culture Collection administrative center, CICC numberings 6108.
The operating procedure of the present invention is followed successively by：
1st, MRS fluid mediums are prepared：52.4g MRS broth bouillon powder is weighed, heating for dissolving is distilled in 1000ml
In water, 121 DEG C of autoclavings 15 minutes obtain MRS fluid mediums.
2nd, glutamine transaminage is processed：The glutamine transaminage that point n times are added in MRS fluid mediums, finally
After once adding glutamine transaminage, the final concentration of 3～25U/mL of glutamine transaminage, wherein N is made to be whole more than 1
Number.
3rd, it is inoculated with：The lactobacillus casei for having activated is inoculated in MRS fluid mediums, lactobacillus casei is cultivated.
4th, quiescent culture：The lactobacillus casei after glutamine transaminage is processed is obtained after quiescent culture.
After above-mentioned steps process, the Bile salt resistance of the bacterial strain is significantly improved.
Inventor by with embodiment 1, embodiment 2, embodiment 3, the 5 same or analogous experiment of embodiment 4 and embodiment,
It was found that the method for the present invention is not suitable for all of lactic acid bacteria, part lactic acid bacteria is suitable only for, subsequent inventor is to suitable
Lactic acid bacteria class probiotic bacteria species for the present invention is studied, and is as a result found, 8148 bacterial strain of lactococcus lactis, plant breast bar
Bacterium plant subspecies etc. have general character suitable for the whole cell peptidoglycan of the lactic acid bacteria of the present invention.Result of study shows, plant breast bar
Contain 13.36 μ g glutamine and 19.05 μ g lysines, 8148 bacterial strain of lactococcus lactis in the every mg Peptidoglycans of bacterium plant sp. strain
Also glutamine and lysine are contained；The derivative strain MG1363 of document report lactococcus lactis 8148, its whole cell peptidoglycan
In also contain glutamine and lysine, it is adaptable to the present invention, in the same manner, the cell wall peptide of Lactobacillus reuteri and lactobacillus casei
Also glutamine and lysine are contained in polysaccharide simultaneously.Conversely, control is then lactococcus lactis NFL bacterial strains, Jing is determined, its cell
Glutamine, only lysine are not contained in wall Peptidoglycan, lysine content is 64.62 μ g/mg Peptidoglycans, turns ammonia with glutamine
After the culture medium of ferment treatment lactococcus lactis NFL bacterial strains, its cholate resistance is not changed in.
Measurement result shows the lactic acid bacteria suitable for the present invention, in its whole cell peptidoglycan must simultaneously containing glutamine and
Lysine.
Embodiment 6
It is a kind of that the lactic acid bacteria for containing glutamine and lysine in whole cell peptidoglycan is processed by glutamine transaminage, carry
The method of high its tolerance cholate ability.
The operating procedure of the present invention is followed successively by：
1. the selection of bacterial strain：First, the bacterial strain for meeting the present embodiment belongs to lactic acid bacteria, secondly, the cell wall of the bacterial strain
Peptidoglycan in contain glutamine and lysine simultaneously.
2. MRS solid mediums are prepared：Weigh 52.4g MRS broth bouillons powder and 15g agar powders, heating for dissolving in
In 1000ml distilled water, 121 DEG C of autoclavings 15 minutes obtain MRS fluid mediums.
3. glutamine transaminage is processed：It is disposable in the cool MRS fluid mediums to 45 DEG C to after sterilizing to add paddy ammonia
Amide transaminase, makes the final concentration of 3～25U/mL of glutamine transaminage.
4. it is inoculated with：The lactobacillus casei for having activated is inoculated in MRS solid mediums, after mixing, culture is quickly poured into
In ware, flat board is prepared into, cultivates lactic acid bacteria.
5. quiescent culture：The lactic acid bacteria after glutamine transaminage is processed is obtained after quiescent culture.
After above-mentioned steps process, the Bile salt resistance of lactic acid bacteria is significantly improved.
Embodiment described above is only that the preferred embodiment of the present invention is described, not the model to the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, technical side of the those of ordinary skill in the art to the present invention
Various modifications and improvement that case is made, all should fall in the protection domain of claims of the present invention determination.
Claims (7)
1. it is a kind of improve lactic acid bacteria Bile salt resistance method, the lactic acid bacteria selected from CICC numberings 20406 lactococcus lactis
The Lactobacillus reuteri of 8148 bacterial strains, the Lactobacillus plantarum sp. strain of CICC numberings 6073 and CICC numberings 6123, its feature
It is：Methods described includes：Add glutamine transaminage at least one times, make the final concentration of culture medium GLN transaminase
For 3～25U/mL, glutamine transaminage culture medium is obtained, and adopts lactic acid described in glutamine transaminage culture medium culturing
Bacterium.
2. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：The glutamine turns
2 to 5 additions of ammonia enzyme point.
3. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：The glutamine turns
Ammonia enzyme added in the time before preparing culture medium and starting to culture to terminate.
4. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：The glutamine turns
Ammonia enzyme final concentration of 12U/mL in the medium.
5. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：Also include with water and paddy
Glutamine transaminase is configured to the glutamine transaminage mother solution of 50～500U/mL, with membrane filtration, the paddy ammonia after filtration
Amide transaminase mother solution is added to glutamine transaminage in the lactic acid bacteria culturing medium.
6. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：The culture medium is liquid
Body culture medium or solid medium.
7. it is according to claim 1 improve lactic acid bacteria Bile salt resistance method, it is characterised in that：The culture medium is selected from
MRS fluid mediums, GM17 fluid mediums and MRS solid mediums.
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