CN106244509A - Lactobacillus rhamnosus culture medium and cultural method - Google Patents
Lactobacillus rhamnosus culture medium and cultural method Download PDFInfo
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- CN106244509A CN106244509A CN201610888771.8A CN201610888771A CN106244509A CN 106244509 A CN106244509 A CN 106244509A CN 201610888771 A CN201610888771 A CN 201610888771A CN 106244509 A CN106244509 A CN 106244509A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The open a kind of lactobacillus rhamnosus culture medium of the present invention, there is basal medium and Optimal Medium, basal medium includes component: yeast protein peptone, glucose, yeast extract, sodium acetate, magnesium sulfate, Tween 80, oligomeric isomaltose, potassium dihydrogen phosphate, surplus are sterilized water, pH value 6.20～6.80；Optimal Medium includes component: yeast protein peptone, glucose, yeast extract, sodium acetate, magnesium sulfate, Tween 80, oligomeric isomaltose, potassium dihydrogen phosphate, surplus are sterilized water, pH value 6.20～6.80.This culture medium is suitable to the breeding of lactobacillus rhamnosus and cultivates, and the method utilizing this culture medium culturing lactobacillus rhamnosus, can not only cultivate lactobacillus rhamnosus by Effective multiplication, also help and realize large-scale industrial production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of lactobacillus rhamnosus culture medium and cultural method.
Background technology
Lactobacillus rhamnosus is subordinated to Lactobacillus, for Gram-positive facultative anaerobe, plasmid-free；Breast can not be utilized
Sugar, but can metabolism monosaccharide；Can grow very well under anaerobic conditions, have CO2In the presence of also can grow.
Lactobacillus rhamnosus is one of Body normal flora, and intestinal Stickiness is high, and colonization ability is strong, is that human research is the widest
One of general probiotic bacteria, have balance and improve gastrointestinal function, strengthen human autoimmune's ability, promote bacillus bifidus and addicted to
Lactobacillus lactis grows and acts on, prevents and help treating diarrhoea, preventing respiratory tract infection, discharge toxin, pre-anti-caries, prevented
Quick grade acts on.
Research to lactobacillus rhamnosus at present has focused largely on its functional characteristic, in terms of this bacterial strain enrichment culture
Research few.Be applied to now lactobacillus rhamnosus cultivate culture medium most be MRS culture medium, be mainly used for utilize
Lactobacillus rhamnosus produces lactic acid.And the present invention is the purpose for reaching lactobacillus rhamnosus raised growth, finds and be suitable to Fructus rhamni (Rhamnus davurica Pall.)
Premised on the culture medium of sugar lactobacillus propagation, thus realize the large-scale industrial production of lactobacillus rhamnosus lyophilized powder.
Summary of the invention
The present invention provides a kind of lactobacillus rhamnosus culture medium and cultural method, is suitable to the breeding training of lactobacillus rhamnosus
Support, and the method utilizing this culture medium culturing lactobacillus rhamnosus, lactobacillus rhamnosus can not only be cultivated by Effective multiplication, also
It is advantageously implemented large-scale industrial production, so that the production rule of the lactobacillus rhamnosus lyophilized powder as food additive
Mould industrialization.
The technical solution used in the present invention is:
Lactobacillus rhamnosus culture medium, has basal medium and Optimal Medium,
Described basal medium includes following component: yeast protein peptone 6.0～10.0g/L, glucose 19.0～25.0g/
L, yeast extract 2.0～4.0g/L, sodium acetate 4.5～6.0g/L, magnesium sulfate 0.4～0.6g/L, Tween 80 0.5～
0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～
6.80；
Described Optimal Medium includes following component: yeast protein peptone 11.0～15.0g/L, glucose 25.0～30.0g/
L, yeast extract 6.0～8.0g/L, sodium acetate 2.0～3.0g/L, magnesium sulfate 0.10～0.15g/L, Tween 80 0.5～
0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～
6.80。
Described lactobacillus rhamnosus culture medium, described basal medium preparation method is as follows, weighs according to formula proportion
Each component, mixing, heating for dissolving, if the pH value of basal medium is less than 6.20, then adjust by 1mol/L food stage NaOH solution
Medium's PH Value is to sterilizing 30min at 6.20～6.80,115 DEG C；
The preparation method of described Optimal Medium is as follows, weighs each component according to formula proportion, mixes, heating for dissolving, as
Really the pH value of culture medium is less than 6.20, then adjust Medium's PH Value to 6.20～6.80,115 DEG C by 1mol/L food stage NaOH solution
Lower sterilizing 30min.
A kind of cultural method utilizing described lactobacillus rhamnosus culture medium, it is characterised in that comprise the steps:
1) fermentation strain is prepared；
1.1) streak culture: after the dilution of lactobacillus rhamnosus mycopowder sterilized water, sectional streak in MC culture medium, line
After end, plate is inverted, 37 DEG C of incubator constant temperature culture 48～72 hours；
1.2) one-level purification is cultivated: in the single colony inoculation on picking plate to the test tube equipped with MRS fluid medium, examination
The seal of tube, in 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtains one-level bacteria suspension；
1.3) two grades of purification are cultivated: by 3～5% inoculum concentration, by cultured one-level bacterial suspension inoculation to equipped with MRS liquid
In the triangular flask of culture medium, culture medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, in 37 DEG C of incubator constant temperature
Quiescent culture 17～24 hours, obtain two grades of bacteria suspensions；
1.4) bacterium is clay standby with preservation: two grades of purification are cultivated the two grades of bacteria suspensions obtained and is centrifuged, 12000rpm from
The heart obtains bacterium mud after 10 minutes, be dissolved in the composition mixed solution of MRS fluid medium and 50% glycerite by bacterium mud,
Bacterium mud mixed liquor, is then divided in bacterium mud mixed liquor in sterilized cryopreservation tube, and sealed membrane seals, frozen in-40～-80 DEG C
In cryogenic refrigerator, obtain lactobacillus rhamnosus bacterium mud cryopreservation tube, standby；
2) strain fermentation；
2.1) frozen bacterium mud recovery: take the lactobacillus rhamnosus bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37
Carry out strain recovery in DEG C water-bath, all melt to frozen liquid in pipe；
2.2) strain one-level is cultivated: by bacterium mud the mixed liquor 1～2ml direct inoculation recover extremely equipped with basal medium
In triangular flask, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.3) strain two grades cultivation: according to 2～6% inoculum concentration, one-level is cultivated the bacterial suspension inoculation terminated to equipped with base
In the triangular flask of basal culture medium, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.4) strain third stage culture: according to 2～6% inoculum concentration, two grades are cultivated the bacterial suspension inoculation terminated to equipped with base
In the fermentation tank of basal culture medium, opening fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is 0,37 DEG C of constant temperature culture 8～15
Hour；
2.5) strain level Four fermentation: according to 2～6% inoculum concentration, the bacterial suspension inoculation terminated by third stage culture is to equipped with excellent
Changing in the fermentation tank of culture medium, open fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is 0,37 DEG C of constant temperature culture, is sending out
After ferment starts 3 hours, the permanent pH maintaining bacterium solution is 5.50～6.00, and cultivation cycle 8～15 hours, until monitoring bacteria suspension
OD600Value stops increasing, and stops fermentation immediately, and bacteria suspension does cooling process.
Described cultural method, step 1.4) bacterium clay standby with preserve described in MRS fluid medium and 50% glycerol
The volume ratio of solution is 1:1～1.5；The mixed solution of described MRS fluid medium and 50% glycerite composition with centrifugal before
The volume ratio of two grades of bacteria suspensions is 1:10.
Described cultural method, step 2.3) basal medium dress liquid amasss and accounts for the 40% of triangular flask volume；Step 2.4)
Basal medium dress liquid amasss and accounts for the 60% of fermenter volume；Step 2.5) Optimal Medium dress liquid amasss and accounts for fermenter volume
60%.
Described cultural method, step 2.5) by stream add food stage NaOH solution maintain the permanent pH of bacterium solution be 5.50～
6.00。
Compared with prior art, the present invention has an advantage highlighted below:
1, the component raw material used for the culture medium of lactobacillus rhamnosus in the present invention is food stage, it is adaptable to advise greatly
Mould industrialized production is as the lactobacillus rhamnosus lyophilized powder of food additive.
2, compared with being applied to now the lactobacillus rhamnosus widest MRS culture medium of cultivation, the present invention is directed to rhamnose breast
Bacillus purification is cultivated and strain fermentation has been respectively adopted the different basal medium being more suitable for thalline different growth phases and excellent
Change culture medium, add somatomedin oligomeric isomaltose in the medium, be not only suitable for lactobacillus rhamnosus thalli growth, numerous
Growing speed fast, number of viable is big, and reduces the cost of culture medium.
3, in the present invention, the strain for fermentation uses the preserving type that centrifugal bacterium mud is frozen, compared with the most generally should
Inclined-plane and the most frozen mode of bacteria suspension, it is thus achieved that the yield of bacteria suspension and viable count higher.
4, adding food stage NaOH solution by stream, to control pH in sweat constant, is more beneficial for being enriched with rhamnose breast bar
Bacterium thalline.
5, classification amplification culture can promote thalline quantity quickly to increase, and can obtain substantial amounts of thalline, viable bacteria in the short time
Number can reach 109More than CFU/ml, and strain is stable, makes a variation, it is few to degenerate.
Accompanying drawing explanation
Fig. 1 is the viable count comparison diagram using 4 kinds of variety classes peptone zymocyte suspensions in embodiment 4.
Fig. 2 is the viable count comparison diagram using 6 kinds of variety classes somatomedin zymocyte suspensions in embodiment 5.
Fig. 3 is the consumption of the oligomeric isomaltose impact on viable count in embodiment 5
Fig. 4 is lactobacillus rhamnosus culture process flow chart.
Detailed description of the invention
A kind of lactobacillus rhamnosus culture medium
Basal medium formulation and preparation method: yeast protein peptone 6.0～10.0g/L, glucose 19.0～25.0g/L,
Yeast extract 2.0～4.0g/L, sodium acetate 4.5～6.0g/L, magnesium sulfate 0.4～0.6g/L, Tween 80 0.5～0.75g/
L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～6.80；
According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20, then adjust by 1mol/L food stage NaOH solution
Medium's PH Value is to sterilizing 30min at 6.20～6.80,115 DEG C；
Optimization culture based formulas and preparation method: yeast protein peptone 11.0～15.0g/L, glucose 25.0～30.0g/L,
Yeast extract 6.0～8.0g/L, sodium acetate 2.0～3.0g/L, magnesium sulfate 0.10～0.15g/L, Tween 80 0.5～
0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～
6.80；According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20, then by 1mol/L food stage NaOH
Solution adjusts Medium's PH Value to sterilizing 30min at 6.20～6.80,115 DEG C；
Nutrient media components raw material is food stage.
Utilize the method for above-mentioned culture medium culturing lactobacillus rhamnosus as shown in Figure 4:
1, the preparation of fermentation strain
1) streak culture: after the dilution of lactobacillus rhamnosus mycopowder sterilized water, four rides in MC culture medium, line knot
Shu Hou, plate inversion, 37 DEG C of incubator constant temperature culture 48～72 hours；
2) one-level purification is cultivated: single colony inoculation that on picking plate, molten calcium circle is bigger is to equipped with 5ml MRS liquid culture
In the test tube of base, test tube seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
3) two grades of purification are cultivated: by 3～5% inoculum concentration, trained to equipped with MRS liquid by cultured one-level bacterial suspension inoculation
Supporting in the triangular flask of base, culture medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, and 37 DEG C of incubator constant temperature stand
Cultivate 17～24 hours；
4) bacterium is clay standby with preservation: two grades of purification being cultivated the bacteria suspension obtained and is centrifuged, 12000rpm is centrifuged 10 points
Obtain bacterium mud after clock, bacterium mud is dissolved in by MRS fluid medium and 50% glycerite (MRS fluid medium and 50% sweet
The volume ratio of oil solution is 1:1～1.5) in the mixed solution that forms, mixed solution with centrifugal before the volume ratio of bacteria suspension be 1:
10, bacterium mud mixed liquor is divided in sterilized cryopreservation tube, sealed membrane seals, frozen in-40～-80 DEG C of cryogenic refrigerators,
Standby；
Note: 1) 4) operation all must aseptic operating platform and aseptic in perform；
2, strain fermentation
5) frozen bacterium mud recovery: take the lactobacillus rhamnosus bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37 DEG C
Strain recovery is carried out in water-bath, 30～45s, all melt to frozen liquid in pipe；
6) strain one-level is cultivated: by bacterium mud the mixed liquor 1～2ml direct inoculation recover extremely equipped with 10ml basal medium
50ml triangular flask in, triangular flask seal, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
7) strain two grades cultivation: according to 2～6% inoculum concentration, one-level is cultivated the bacterial suspension inoculation terminated to equipped with basis
In the triangular flask of culture medium, basal medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, 37 DEG C of incubator perseverances
Temperature quiescent culture 17～24 hours；
8) strain third stage culture: according to 2～6% inoculum concentration, two grades are cultivated the bacterial suspension inoculation terminated to equipped with basis
In the fermentation tank of culture medium, basal medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, and rotating speed is
100rpm, ventilation is 0,37 DEG C of constant temperature culture 8～15 hours；
9) strain level Four fermentation: according to 2～6% inoculum concentration, the bacterial suspension inoculation terminated by third stage culture is to equipped with optimization
In the fermentation tank of culture medium, Optimal Medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, and rotating speed is
100rpm, ventilation is 0,37 DEG C of constant temperature culture, after fermentation starts 3 hours, adds food stage NaOH solution by stream and maintains
The permanent pH of bacterium solution is 5.50～6.00, cultivation cycle 8～15 hours, until monitoring bacteria suspension OD600Value stops increasing, immediately
Stop fermentation, and bacteria suspension is done cooling process.
Using above-mentioned culture medium and cultural method to cultivate lactobacillus rhamnosus, the viable count of bacteria suspension reaches 109CFU/ml
Above.
Note: 5) 9) operation be both needed to carry out in 100,000 grades of cleaning shops.
Embodiment 1 lactobacillus rhamnosus culture medium and cultural method
One, lactobacillus rhamnosus culture medium, including basal medium and Optimal Medium.
The formula of basal medium and preparation: yeast protein peptone 8.0g/L, glucose 25.0g/L, yeast extract 3.0g/
L, sodium acetate 6.0g/L, magnesium sulfate 0.5g/L, Tween 80 0.5g/L, oligomeric isomaltose 1.0g/L, potassium dihydrogen phosphate 3.0g/
L, surplus are sterilized water, pH value 6.20；Weigh each component, mixing, heating for dissolving according to formula proportion, use 1mol/L food stage
NaOH solution adjust Medium's PH Value to 6.20, sterilizing 30min at 115 DEG C.
The formula of Optimal Medium and preparation: yeast protein peptone 14.0g/L, glucose 30.0g/L, yeast extract
6.0g/L, sodium acetate 3.0g/L, magnesium sulfate 0.10g/L, Tween 80 0.5g/L, oligomeric isomaltose 2.0g/L, potassium dihydrogen phosphate
3.0g/L, surplus are sterilized water, pH value 6.80；Weigh each component, mixing, heating for dissolving according to formula proportion, eat with 1mol/L
Grade NaOH solution adjust Medium's PH Value to 6.80, sterilizing 30min at 115 DEG C.
Two, above-mentioned lactobacillus rhamnosus culture medium culturing lactobacillus rhamnosus is utilized.
Comprise the steps:
1, the preparation of fermentation strain
1) streak culture: after the dilution of lactobacillus rhamnosus mycopowder sterilized water, four rides in MC culture medium, line knot
Shu Hou, plate is inverted, 37 DEG C of incubator constant temperature culture 60 hours；
2) one-level purification is cultivated: single colony inoculation that on picking plate, molten calcium circle is bigger is to equipped with 5ml MRS liquid culture
In the test tube of base, test tube seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
3) two grades of purification are cultivated: by 3% inoculum concentration, by cultured one-level bacterial suspension inoculation to equipped with 100ml MRS liquid
In the 250ml triangular flask of body culture medium, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
4) bacterium is clay standby with preservation: two grades of purification being cultivated the 100ml bacteria suspension obtained and is centrifuged, 12000rpm is centrifuged
Obtaining bacterium mud after 10 minutes, bacterium mud is dissolved in 10ml mixed solution, and (mixed liquor is by 5ml MRS fluid medium and 5ml50%
Glycerite mix homogeneously is formulated) in, bacterium mud mixed liquor is divided in sterilized 1ml cryopreservation tube, sealed membrane seals,
Frozen in-80 DEG C of cryogenic refrigerators, standby；
Note: 1) 4) operation all must aseptic operating platform and aseptic in perform；
2, strain fermentation
5) frozen bacterium mud recovery: take the lactobacillus rhamnosus bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37 DEG C
Strain recovery is carried out in water-bath, 30～45s, all melt to frozen liquid in pipe；
6) strain one-level is cultivated: by the 1ml bacterium mud mixed liquor direct inoculation recover extremely equipped with 10ml basal medium
In 50ml triangular flask, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
7) strain two grades cultivation: according to 2% inoculum concentration, cultivates the bacterial suspension inoculation terminated to equipped with 100ml base by one-level
In the 250ml triangular flask of basal culture medium, inoculating 4 bottles by this, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
8) strain third stage culture: according to 2% inoculum concentration, cultivates the bacterial suspension inoculation terminated to equipped with 18L basis by two grades
In the 30L fermentation tank of culture medium, opening fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is that 0,37 DEG C of constant temperature culture 12 are little
Time；
9) strain level Four fermentation: according to 5% inoculum concentration, the bacterial suspension inoculation terminated by third stage culture optimizes to equipped with 300L
In the 500L fermentation tank of culture medium, opening fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is 0,37 DEG C of constant temperature culture,
After fermentation starts 3 hours, adding food stage NaOH solution by stream and maintain constant pH to be 5.60 ± 0.1, cultivation to 11 is little constantly,
Monitor bacterium solution OD600Value stops increasing, and stops fermentation immediately, and bacterium solution is done cooling process；
10), after fermentation ends, take bacteria suspension and use colony counting method detection viable count to be 7.9 × 109CFU/ml。
Note: 5) 9) operation be both needed to carry out in 100,000 grades of cleaning shops.
Embodiment 2 is for the fungi preservation way choice experiment of fermentation
After the cultivation of lactobacillus rhamnosus scribed line, one-level, two grades of purification are cultivated, bacteria suspension respectively with inclined-plane, bacteria suspension and
Use the preserving type of centrifugal bacterium mud in embodiment 1 to carry out frozen, adopt with the strain that these three preserving type is frozen the most respectively
Ferment with identical condition of culture, after fermentation ends, measure viable count and the yield of bacteria suspension respectively.
The assay method of viable count: use colony counting method；
The assay method of yield:
Table 1 uses the strain of different preserving type to carry out the impact fermented on yield and viable count
| Preserving type | Yield (%) | Viable count (CFU/ml) |
| Inclined-plane preserves | 1.33 | 1.09×109 |
| Bacteria suspension preserves | 1.22 | 1.14×109 |
| Centrifugal bacterium mud preserves | 1.44 | 3.20×109 |
Shown by the data in table 1: use the strain that in embodiment 1, centrifugal bacterium mud mode preserves to ferment, viable count
It is above inclined-plane and bacteria suspension preserving type with yield.
The single factor test screening experiment of embodiment 3 nutrient media components
Be directed to be applied at present culture medium that lactobacillus rhamnosus cultivates most be MRS culture medium, be mainly used for
Utilizing lactobacillus rhamnosus to produce lactic acid, we are the purpose reaching lactobacillus rhamnosus raised growth, find and are suitable to rhamnose
The culture medium of lactobacillus propagation, is applied to the large-scale industrial production lactobacillus rhamnosus lyophilized powder as food additive,
MRS culture medium is carried out improved experimental.
Owing to beef powder, dipotassium hydrogen phosphate, Triammonium citrate and manganese sulfate do not meet the relevant state that food additive uses
Family's standard and regulation, therefore cannot be used for fermenting and producing food stage lactobacillus rhamnosus.For the beef in MRS culture medium prescription
Powder, Triammonium citrate, dipotassium hydrogen phosphate and manganese sulfate component, carry out experiment of single factor, investigates each component to cultivating rhamnose
The impact of the viable count of lactobacillus, according to each group culture medium prescription preparation culture medium in table 2, and under identical condition of culture
Cultivate lactobacillus rhamnosus, the viable count of detection bacteria suspension.
Table 2 experiment of single factor culture medium prescription (unit: g/L)
The viable count result of table 3 experiment of single factor bacteria suspension
The result of table 3 shows, the viable count of A and C group and MRS group are without significant difference, thus draw beef powder and citric acid
The viable count of cultivation lactobacillus rhamnosus is affected without obvious by three ammoniums as the component of culture medium；The viable count of D group and MRS group
Variant, thus show that the viable count cultivating lactobacillus rhamnosus is had an impact by manganese sulfate as the component of culture medium, but shadow
Sound is not highly significant；The viable count of B group and MRS group have the difference of highly significant, thus show that dipotassium hydrogen phosphate is as cultivation
The viable count cultivating lactobacillus rhamnosus is had by the component of base obviously to be affected, and therefore replaces with food grade monopotassium phosphate
For dipotassium hydrogen phosphate.
The optimization experiment of peptone kind in embodiment 4 culture medium
Peptone in culture medium selects fish peptone, Os Bovis seu Bubali peptone, casein peptone and yeast protein peptone these 4 kinds respectively
Peptone, uses identical condition of culture to ferment, measures the viable count of bacteria suspension after fermentation ends respectively.Shown by Fig. 1,
Using yeast protein peptone to ferment, the viable count recording bacteria suspension is the highest.
Somatomedin kind and the optimization experiment of consumption in embodiment 5 culture medium prescription
For reaching the purpose of the viable count of lactobacillus rhamnosus enrichment culture, raising bacteria suspension, add rush in the medium
Somatomedin, selects oligomeric isomaltose, oligomeric galactose, stachyose, vitamin B2, vitamin B5, vitamin B12As rush
Somatomedin, uses identical condition of culture to ferment, measures the viable count of bacteria suspension after fermentation ends respectively.Shown by Fig. 2
Showing, using oligomeric isomaltose to carry out fermenting as somatomedin, the viable count recording bacteria suspension is the highest, and stachyose is the most secondary
It, but the price of stachyose is more than ten times of oligomeric isomaltose price, considering cost factor, the most oligomeric different wheat
Bud sugar is as somatomedin.
Selecting oligomeric isomaltose as somatomedin, the consumption of oligomeric isomaltose uses gradient concentration to add, training
Support other components of base identical with addition, under same culture conditions, investigate the impact on viable count.As seen from Figure 3, low
When the addition of IMO is between 1.0-3.0g/L, the bacteria suspension viable count obtained is all 7.0 × 109CFU/ml with
On, but when addition is 2.5g/L, viable count reaches the highest, begins to afterwards have a declining tendency, therefore from cost and
Impact on viable count considers, and the addition selecting oligomeric isomaltose is 1.0-2.5g/L.
Whether embodiment 6 level Four sweat controls the constant pH impact experiment on fermentation
In level Four sweat, the most identical at other condition of culture, add food stage NaOH solution by stream respectively and tie up
Hold constant pH and not stream add any soda acid under natural pH, investigate the OD to bacteria suspension600, viable count and the impact of yield, such as table
4。
In table 4 level Four sweat, natural pH and control constant pH are to OD600, viable count and the impact of yield
| PH control mode | OD600 | Viable count (CFU/ml) | Yield (%) |
| Natural pH | 7.232 | 3.4×109 | 1.22 |
| Control constant pH | 11.584 | 7.9×109 | 1.85 |
Shown by the data in table 4, add food stage NaOH solution by stream and maintain constant pH, the bacteria suspension that fermentation obtains
OD600, viable count and yield the highest, this is because in lactobacillus rhamnosus sweat produce lactic acid, cause nature
PH is on a declining curve, and the phase is enriched with adverse influence to thalline after fermentation, adds food stage NaOH solution by stream and maintains
Constant pH is more beneficial for the enrichment of thalline.
Claims (6)
1. lactobacillus rhamnosus culture medium, it is characterised in that there is basal medium and Optimal Medium,
Described basal medium includes following component: yeast protein peptone 6.0～10.0g/L, glucose 19.0～25.0g/L, ferment
Female extractum 2.0～4.0g/L, sodium acetate 4.5～6.0g/L, magnesium sulfate 0.4～0.6g/L, Tween 80 0.5～0.75g/L,
Oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～6.80；
Described Optimal Medium includes following component: yeast protein peptone 11.0～15.0g/L, glucose 25.0～30.0g/L, ferment
Female extractum 6.0～8.0g/L, sodium acetate 2.0～3.0g/L, magnesium sulfate 0.10～0.15g/L, Tween 80 0.5～0.75g/
L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, surplus are sterilized water, pH value 6.20～6.80.
2. lactobacillus rhamnosus culture medium as claimed in claim 1, it is characterised in that described basal medium preparation method is such as
Under, weigh each component, mixing, heating for dissolving according to formula proportion, if the pH value of basal medium is less than 6.20, then use
1mol/L food stage NaOH solution adjusts Medium's PH Value to sterilizing 30min at 6.20～6.80,115 DEG C；
The preparation method of described Optimal Medium is as follows, weighs each component, mixing, heating for dissolving according to formula proportion, if trained
The pH value supporting base is less than 6.20, then adjust Medium's PH Value to go out at 6.20～6.80,115 DEG C by 1mol/L food stage NaOH solution
Bacterium 30min.
3. one kind utilize as described in claim 1-2 the cultural method of lactobacillus rhamnosus culture medium, it is characterised in that include as
Lower step:
1) fermentation strain is prepared；
1.1) streak culture: after the dilution of lactobacillus rhamnosus mycopowder sterilized water, sectional streak in MC culture medium, line terminates
After, plate is inverted, 37 DEG C of incubator constant temperature culture 48～72 hours；
1.2) one-level purification is cultivated: in the single colony inoculation on picking plate to the test tube equipped with MRS fluid medium, test tube is close
Envelope, in 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtains one-level bacteria suspension；
1.3) two grades of purification are cultivated: by 3～5% inoculum concentration, by cultured one-level bacterial suspension inoculation to equipped with MRS liquid culture
In the triangular flask of base, culture medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, and stands in 37 DEG C of incubator constant temperature
Cultivate 17～24 hours, obtain two grades of bacteria suspensions；
1.4) bacterium is clay standby with preservation: two grades of purification being cultivated the two grades of bacteria suspensions obtained and is centrifuged, 12000rpm is centrifuged 10
Obtain bacterium mud after minute, bacterium mud is dissolved in the composition mixed solution of MRS fluid medium and 50% glycerite, obtain bacterium mud
Mixed liquor, is then divided in bacterium mud mixed liquor in sterilized cryopreservation tube, and sealed membrane seals, frozen in-40～-80 DEG C of low temperature
In refrigerator, obtain lactobacillus rhamnosus bacterium mud cryopreservation tube, standby；
2) strain fermentation；
2.1) frozen bacterium mud recovery: take the lactobacillus rhamnosus bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37 DEG C of water
Carry out strain recovery in bath, all melt to frozen liquid in pipe；
2.2) strain one-level is cultivated: by bacterium mud the mixed liquor 1～2ml direct inoculation recover extremely equipped with the triangle of basal medium
In Ping, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.3) strain two grades cultivation: according to 2～6% inoculum concentration, one-level is cultivated the bacterial suspension inoculation terminated and cultivates to equipped with basis
In the triangular flask of base, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.4) strain third stage culture: according to 2～6% inoculum concentration, two grades are cultivated the bacterial suspension inoculation terminated and cultivates to equipped with basis
In the fermentation tank of base, opening fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is 0,37 DEG C of constant temperature culture 8～15 hours；
2.5) strain level Four fermentation: according to 2～6% inoculum concentration, the bacterial suspension inoculation terminated by third stage culture is to equipped with optimization culture
In the fermentation tank of base, opening fermentation tank stirring paddle, rotating speed is 100rpm, and ventilation is 0,37 DEG C of constant temperature culture, in fermentation beginning 3
After hour, the permanent pH maintaining bacterium solution is 5.50～6.00, and cultivation cycle 8～15 hours, until monitoring bacteria suspension OD600Value is stopped
Only increase, stop fermentation immediately, and bacteria suspension is done cooling process.
Cultural method the most as claimed in claim 3, it is characterised in that step 1.4) bacterium clay standby with preserve described in MRS
The volume ratio of fluid medium and 50% glycerite is 1:1～1.5；Described MRS fluid medium and 50% glycerite composition
The volume ratio of mixed solution and centrifugal the first two grade of bacteria suspension be 1:10.
5. cultural method as claimed in claim 3, it is characterised in that step 2.3) basal medium dress liquid is long-pending accounts for triangular flask
The 40% of volume；Step 2.4) basal medium dress liquid amasss and accounts for the 60% of fermenter volume；Step 2.5) Optimal Medium dress liquid
Volume accounts for the 60% of fermenter volume.
6. cultural method as claimed in claim 3, it is characterised in that step 2.5) add food stage NaOH solution by stream and tie up
The permanent pH holding bacterium solution is 5.50～6.00.
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