CN103897998A - Lactobacillus salivarius, applications thereof, functional food compositions and preparation method of the functional food composition - Google Patents
Lactobacillus salivarius, applications thereof, functional food compositions and preparation method of the functional food composition Download PDFInfo
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Abstract
The invention provides lactobacillus salivarius. The lactobacillus salivarius is characterized in that the accession number of lactobacillus salivarius is CGMCC No.6288. The invention also provides a functional food composition and a preparation method thereof. The preparation method of the functional food composition includes inactivating living bacteria of the lactobacillus salivarius to obtain a bacterial substance. The invention also provides a functional food composition comprising the living bacteria of the lactobacillus salivarius. The invention also provides applications of the lactobacillus salivarius in preparation of the anti-ageing functional food compositions. The living bacteria of the lactobacillus salivarius and the inactivated bacterial substance both have anti-aging functions.
Description
Technical field
The present invention relates to lactobacillus salivarius (Lactobacillus salivarius) and application and functional food composition and preparation method thereof, particularly, relate to a kind of lactobacillus salivarius, contain functional food composition of this lactobacillus salivarius and preparation method thereof, and application in the functional food that delays senility in preparation of this lactobacillus salivarius.
Background technology
Aging is that the degeneration that body tissue, organ dysfunction occur with age growth changes (Turnheim, 2003), is the general performance of the various biochemical reactions of body.Economic fast development, the progress of science and technology, makes developed country and some developing countries step into aging society.The problem of an aging population has become global social concern.By the end of the year 2009, China 60 years old and above elderly population reach 1.6714 hundred million, account for 12.5% of total population, expect the year two thousand twenty and will reach 2.43 hundred million.With advancing age and the aging of body, the disease relevant to aging, as cardiovascular, nervous system disorders, tumour, diabetes etc., the life-span and the quality of life that are threatening the mankind.Therefore, increasing research is absorbed in to find and is had the material of anti-aging effects and study its anti-ageing mechanism.Current multiple natural product has been proved has anti-aging effects.But the Material Source with anti-aging effects of thinking is at present comparatively single, and separation and purification cost is high, the medicine with anti-aging effects has again certain side effect, thereby has limited the widespread adoption of these materials.Probiotic bacterium security is higher, and separation and purification cost is low, if can specify the antisenility function of probiotic bacterium, and determines its mechanism of resisting senility, and the prospect using probiotic bacterium as anti-aging product will be very wide.
Lactobacillus salivarius is the Bacterium lacticum extensively existing in human body and animal body.In the oral cavity of human body, breast milk, woman vagina, can detect lactobacillus salivarius (the few equality of Lee, 1991; Olivares et al., 2006).It is the ancestral home bacterium of human body, is one of normal microflora being present in human body; Except human body, from the enteron aisles such as chicken, pig, rabbit, be also separated to lactobacillus salivarius (Chen Guiyin, 2006; Tang Xiaoli, 2007; Luo Rui, 2011).And lactobacillus salivarius do not belong to common pathogenic bacterium, seldom there is the diseases induced report of lactobacillus salivarius, the lactobacillus salivarius strains that therefore a lot of research all obtains screening carry out functional study, use to the probiotic bacterium as human and animal.
Summary of the invention
The object of this invention is to provide a kind of lactobacillus salivarius with deferring senility.
To achieve these goals, on the one hand, the invention provides a kind of lactobacillus salivarius (Lactobacillus salivarius), it is characterized in that, the deposit number of described lactobacillus salivarius is CGMCC No.6288.
Second aspect, the invention provides a kind of method of preparing functional food composition, and described method comprises: by the viable bacteria body deactivation of lactobacillus salivarius as above, obtain thalline material.
The third aspect, the invention provides a kind of functional food composition of being prepared by method as above.
Fourth aspect, the invention provides a kind of functional food composition, it is characterized in that, the viable bacteria body that described functional food composition contains lactobacillus salivarius as above.
The 5th aspect, the invention provides the application in the functional food composition that lactobacillus salivarius as above delays senility in preparation.
Thalline material after viable bacteria body and the deactivation of lactobacillus salivarius provided by the invention all has the effect delaying senility.
Other features and advantages of the present invention are described in detail the embodiment part subsequently.
Biological preservation
Lactobacillus salivarius of the present invention (Lactobacillus salivarius), be deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 25th, 2012, Institute of Microorganism, Academia Sinica, postcode: 100101) (depositary institution be abbreviated as CGMCC), deposit number is CGMCC No.6288.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
On the one hand, the invention provides a kind of lactobacillus salivarius (Lactobacillus salivarius), the deposit number of this lactobacillus salivarius is CGMCC No.6288.
Lactobacillus salivarius of the present invention separates the ight soil from Bama County of Guangxi long lived elder.
Lactobacillus salivarius provided by the invention is through cultivating the viable bacteria body that can produce a large amount of lactobacillus salivarius, and the method for described cultivation does not have special requirement, as long as making described lactobacillus salivarius propagation, and for example can be by 10
7the inoculation of magnitude is in Bacterium lacticum substratum, and under anaerobism or aerobic condition, cultivates after 8-72 hour at the temperature of 15-38 DEG C, obtains nutrient solution.Described Bacterium lacticum substratum can be the substratum that known various applicable Bacterium lacticum is cultivated, can be for example milk and/or " milk-acid bacteria---Basic of Biology and application " (Yang Jiebin, light industry press, 1996 publish) described in milk-acid bacteria (MRS) substratum.
The present invention can further separate the viable bacteria body of the lactobacillus salivarius in above-mentioned nutrient solution, the method of described separation has no particular limits, as long as can be from nutrient solution enrichment thalline, for example can realize by method centrifugal and/or that filter, described condition centrifugal and described filtration can be known condition, and the present invention does not repeat them here.
Second aspect, the invention provides a kind of method of preparing functional food composition, and described method comprises: by the viable bacteria body deactivation of lactobacillus salivarius as above, obtain thalline material.
The present inventor is surprisingly discovery under study for action, and the thalline material obtaining after deactivation 0.5-1.5h at 65-85 DEG C, has the effect delaying senility more significantly.Therefore, the condition optimization of deactivation comprises: temperature is 65-85 DEG C, and the time is 0.5-1.5h.
The inventive method also comprises adds thalline material in food to.
According to the present invention, although thalline material is added in food, can realize object of the present invention, play the effect delaying senility, but under preferable case, taking the gross weight of functional food composition as benchmark, the addition of thalline material is 10-70 % by weight, more preferably 30-50 % by weight.Under above-mentioned preferable case, the effect delaying senility of functional food composition is more remarkable.
In the present invention, food can be the food of any type, such as juice product, milk-product, bean product etc.Food also can be according to the difference of edible object and different.In described functional food composition, can also contain conventional additive, such as spices, mineral substance, VITAMIN, stablizer, thickening material, sanitas etc.
The third aspect, the invention provides a kind of functional food composition of being prepared by method as above.
Fourth aspect, the invention provides a kind of functional food composition, the viable bacteria body that this functional food composition contains lactobacillus salivarius as above.
According to the present invention, although the viable bacteria body that functional food composition contains lactobacillus salivarius as above can be realized object of the present invention, play the effect delaying senility.But under preferable case, taking the gross weight of functional food composition as benchmark, the content of the viable bacteria body of lactobacillus salivarius is 10
5-10
10cFU/g, more preferably 10
7-10
9cFU/g.Under above-mentioned preferable case, the effect delaying senility of functional food composition is more remarkable.
The viable bacteria body of the lactobacillus salivarius that the functional food composition that meets above-mentioned requirements can comprise the nutrient solution of lactobacillus salivarius (for example through this lactobacillus salivarius ferment the cultured milk prod making), separate etc.
In the present invention, functional food composition also contains food, and food as previously mentioned, does not repeat them here.
The 5th aspect, the invention provides the application in the functional food composition that lactobacillus salivarius as above delays senility in preparation.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, also can carry out arbitrary combination between various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Embodiment
The present invention is further illustrated for following embodiment, but therefore do not limit the present invention.
(1) adopt nematode as study subject
Nematode is growth, apoptosis, old and feeble area research model animals the most clearly, its life cycle is short, and environment sensitive to external world, and because the homology of old and feeble signal is high, the research of unicellular lower eukaryote has direct reference (Greer and Brunet, 2008) to Mammals and human senility.Therefore, adopt in the following Examples and Comparative Examples nematode as study subject.
In following embodiment and comparative example:
Nematode is the strain of Caenorhabditis elegans worm, and wild-type Caenorhabditis elegans N2, purchased from U.S.'s nematode genetics center (Caenorhabditis Genetics Center(CGC)).
Bacterial classification: nematode standard food intestinal bacteria (E.coli) OP50, purchased from U.S.'s nematode genetics center; Lactobacillus salivarius A is that (this bacterial strain was deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center to lactobacillus salivarius of the present invention on June 25th, 2012, Institute of Microorganism, Academia Sinica, postcode: 100101) (depositary institution be abbreviated as CGMCC), deposit number is CGMCC No.6288).
Nematode elementary operation method is carried out with reference to Wormbook standard eelworm culture method.
1M potassium phosphate buffer (pH 6.0): 108.3g KH
2pO
4, 46.8g K
2hPO
43H
2o, distilled water is settled to 1L, and 121 DEG C of sterilizing 15min are for subsequent use.
NGM substratum: 3.0g NaCl, 20g agar powder, 2.5g bacto peptone, adds distilled water 970mL, and 121 DEG C of sterilizing 15min are placed in 60 DEG C of water-baths, add following composition under aseptic condition: 1mL 1M MgSO
4, 1mL 1M CaCl
2, 25mL 1M potassium phosphate buffer, the ethanolic soln of 3.2mL 5mg/mL cholesterol; After mixing, pour plate.After flat board solidifies, be placed in room temperature 2 days, volatilization moisture and inspection are dull and stereotyped to be placed in 4 DEG C of watertight chests and to save backup without living contaminants.
MNGM substratum: add FUdR, Pyocianil to the final concentration of degerming to be after filtration respectively 50 μ M, 0.5mM under aseptic condition before NGM substratum pour plate, mix pour plate, solidify and be placed on 2 days volatilization moisture of room temperature, flat board can be coated with bacterium and use or be placed in preservation in 4 DEG C of watertight chests afterwards, in 1 month, finishes using.
M9 damping fluid: 3g KH
2pO
4, 15.12g Na
2hPO
412H
2o, 5g NaCl, adding distil water is settled to 1L, and 121 DEG C of sterilizing 15min are cooled to the aseptic 1mL of adding 1M MgSO after room temperature
4, mix for subsequent use.
Nematode lysate: 1mL 8M NaOH, 0.6mL 9% chlorine bleach liquor, the aseptic ddH of 3.4mL
2o, mixes rear use, matching while using.
LB substratum: Tryptones 10g, yeast soaks powder 5g, NaCl 10g(solid medium adds 15g agar), add 950mL distilled water, regulate pH to 7.0,121 DEG C of sterilizing 15min are for subsequent use.
MRS substratum: be purchased from Beijing overpass company.
Embodiment 1.1
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) intestinal bacteria OP50 is seeded to LB liquid nutrient medium with 1% inoculum size, in 37 DEG C of 220rpm shaking culture 8h of constant-temperature table to logarithm latter stage, obtains intestinal bacteria OP50 nutrient solution; Lactobacillus salivarius A is seeded to MRS liquid nutrient medium with 1% inoculum size, cultivates 12h to logarithm latter stage in filling 37 DEG C, nitrogen anaerobism bottle, obtain lactobacillus salivarius A nutrient solution.
By the nutrient solution of above-mentioned intestinal bacteria OP50 and lactobacillus salivarius A, collect thalline with the centrifugal 15min of 4000 × g respectively, by M9 damping fluid repeated centrifugation washed twice, 4 DEG C of centrifugal 15min of 16000 × g, remove whole supernatant liquors afterwards, weigh thalline weight in wet base.Thalline is resuspended with a certain amount of M9, and making thalline final concentration is 400mg/mL, is sub-packed in 1.5mL centrifuge tube, as concentrated thalline.
Respectively by 75 DEG C of hot deactivation 1h of above-mentioned two kinds of concentrated thalline, after cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed to get to mixture by the weight ratio of 3:2 (being that lactobacillus salivarius is 40 % by weight), mixture is suspended in M9 damping fluid according to the ratio of 400mg/mL, coat the mNGM planar surface of diameter 60mm, making every dull and stereotyped biomass is 10mg, totally 6 flat boards, 4 DEG C of sealings save backup.
(2) in diameter 90mm NGM media surface, be evenly coated with 200 μ L intestinal bacteria OP50 nutrient solutions, cultivate 10h for 37 DEG C, to go down to posterity with dull and stereotyped as nematode, 4 DEG C of sealings save backup.
Under aseptic condition, there is the NGM flat board of nematode to cut the fritter substratum of about 1cm × 1cm from growth with scalpel, facing down is buckled in nematode and goes down to posterity with dull and stereotyped upper, is placed in 25 DEG C of cultivations, and 60mm plate goes down to posterity once for general 7 days, keeps nematode activity.
Get after going down to posterity adult mostly the flat board in the spawning time for nematode synchronization:
In flat board, add 2mL M9 damping fluid, with aseptic straw piping and druming, sweep away the polypide on flat board as far as possible, polypide is transferred to 1.5mL centrifuge tube, the centrifugal 1min of 800 × g, abandons supernatant and collects polypide, adds 1mL M9 recentrifuge washing polypide, exhausts supernatant as far as possible.
Add the nematode lysate that 1mL is fresh, vortex oscillation 10s at once, the centrifugal 1min of 800 × g, pipettor siphons away supernatant.
Add at once 1mL M9 washing polypide, the centrifugal 1min of 800 × g, double, remove residual lysate.The M9 suspension liquid that finally in pipe, residue approximately 100 μ L contain worm's ovum.
Use aseptic straw that worm's ovum is dripped in the NGM flat board of coating OP50, cultivate 48h for 25 DEG C, obtain the nematode of synchronization 48h.
(3) by the nematode of synchronization 48h, be placed in stereomicroscope observation, using platinum shovel picking gonotreme is on the mNGM flat board that obtains in step (1) of transparent semi-moon shaped L4 phase telianthus worm, 10 of every dull and stereotyped pickings, and flat board is placed in 25 DEG C of cultivations.Observe nematode survival state every day, as polypide touches and do not have movable reaction to think polypide death platinum shovel.Get rid of hatching and unnatural death nematode in missing, body.All end is tested in nematode death.Calculate nematode mean lifetime in table 1.
Embodiment 1.2
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) intestinal bacteria OP50 is seeded to LB liquid nutrient medium with 1% inoculum size, in 37 DEG C of 220rpm shaking culture 8h of constant-temperature table to logarithm latter stage, obtains intestinal bacteria OP50 nutrient solution; Lactobacillus salivarius A is seeded to MRS liquid nutrient medium with 1% inoculum size, cultivates 12h to logarithm latter stage in filling 37 DEG C, nitrogen anaerobism bottle, obtain lactobacillus salivarius A nutrient solution.
By the nutrient solution of above-mentioned intestinal bacteria OP50 and lactobacillus salivarius A, collect thalline with the centrifugal 15min of 4000 × g respectively, by M9 damping fluid repeated centrifugation washed twice, 4 DEG C of centrifugal 15min of 16000 × g, remove whole supernatant liquors afterwards, weigh thalline weight in wet base.Thalline is resuspended with a certain amount of M9, and making thalline final concentration is 400mg/mL, is sub-packed in 1.5mL centrifuge tube, as concentrated thalline.
Respectively by 65 DEG C of hot deactivation 1.5h of above-mentioned two kinds of concentrated thalline, after cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed to get to mixture by the weight ratio of 7:3 (being that lactobacillus salivarius is 30 % by weight), mixture is suspended in M9 damping fluid according to the ratio of 400mg/mL, coat the mNGM planar surface of diameter 60mm, making every dull and stereotyped biomass is 10mg, totally 6 flat boards, 4 DEG C of sealings save backup.
(2) in diameter 90mm NGM media surface, be evenly coated with 200 μ L intestinal bacteria OP50 nutrient solutions, cultivate 10h for 37 DEG C, to go down to posterity with dull and stereotyped as nematode, 4 DEG C of sealings save backup.
Under aseptic condition, there is the NGM flat board of nematode to cut the fritter substratum of about 1cm × 1cm from growth with scalpel, facing down is buckled in nematode and goes down to posterity with dull and stereotyped upper, is placed in 25 DEG C of cultivations, and 60mm plate goes down to posterity once for general 7 days, keeps nematode activity.
Get after going down to posterity adult mostly the flat board in the spawning time for nematode synchronization:
In flat board, add 2mL M9 damping fluid, with aseptic straw piping and druming, sweep away the polypide on flat board as far as possible, polypide is transferred to 1.5mL centrifuge tube, the centrifugal 1min of 800 × g, abandons supernatant and collects polypide, adds 1mL M9 recentrifuge washing polypide, exhausts supernatant as far as possible.
Add the nematode lysate that 1mL is fresh, vortex oscillation 10s at once, the centrifugal 1min of 800 × g, pipettor siphons away supernatant.
Add at once 1mL M9 washing polypide, the centrifugal 1min of 800 × g, double, remove residual lysate.The M9 suspension liquid that finally in pipe, residue approximately 100 μ L contain worm's ovum.
Use aseptic straw that worm's ovum is dripped in the NGM flat board of coating OP50, cultivate 48h for 25 DEG C, obtain the nematode of synchronization 48h.
(3) by the nematode of synchronization 48h, be placed in stereomicroscope observation, using platinum shovel picking gonotreme is on the mNGM flat board that obtains in step (1) of transparent semi-moon shaped L4 phase telianthus worm, 10 of every dull and stereotyped pickings, and flat board is placed in 25 DEG C of cultivations.Observe nematode survival state every day, as polypide touches and do not have movable reaction to think polypide death platinum shovel.Get rid of hatching and unnatural death nematode in missing, body.All end is tested in nematode death.Calculate nematode mean lifetime in table 1.
Embodiment 1.3
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) intestinal bacteria OP50 is seeded to LB liquid nutrient medium with 1% inoculum size, in 37 DEG C of 220rpm shaking culture 8h of constant-temperature table to logarithm latter stage, obtains intestinal bacteria OP50 nutrient solution; Lactobacillus salivarius A is seeded to MRS liquid nutrient medium with 1% inoculum size, cultivates 12h to logarithm latter stage in filling 37 DEG C, nitrogen anaerobism bottle, obtain lactobacillus salivarius A nutrient solution.
By the nutrient solution of above-mentioned intestinal bacteria OP50 and lactobacillus salivarius A, collect thalline with the centrifugal 15min of 4000 × g respectively, by M9 damping fluid repeated centrifugation washed twice, 4 DEG C of centrifugal 15min of 16000 × g, remove whole supernatant liquors afterwards, weigh thalline weight in wet base.Thalline is resuspended with a certain amount of M9, and making thalline final concentration is 400mg/mL, is sub-packed in 1.5mL centrifuge tube, as concentrated thalline.
Respectively by 85 DEG C of hot deactivation 0.5h of above-mentioned two kinds of concentrated thalline, after cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed to get to mixture by the weight ratio of 1:1 (being that lactobacillus salivarius is 50 % by weight), mixture is suspended in M9 damping fluid according to the ratio of 400mg/mL, coat the mNGM planar surface of diameter 60mm, making every dull and stereotyped biomass is 10mg, totally 6 flat boards, 4 DEG C of sealings save backup.
(2) in diameter 90mm NGM media surface, be evenly coated with 200 μ L intestinal bacteria OP50 nutrient solutions, cultivate 10h for 37 DEG C, to go down to posterity with dull and stereotyped as nematode, 4 DEG C of sealings save backup.
Under aseptic condition, there is the NGM flat board of nematode to cut the fritter substratum of about 1cm × 1cm from growth with scalpel, facing down is buckled in nematode and goes down to posterity with dull and stereotyped upper, is placed in 25 DEG C of cultivations, and 60mm plate goes down to posterity once for general 7 days, keeps nematode activity.
Get after going down to posterity adult mostly the flat board in the spawning time for nematode synchronization:
In flat board, add 2mL M9 damping fluid, with aseptic straw piping and druming, sweep away the polypide on flat board as far as possible, polypide is transferred to 1.5mL centrifuge tube, the centrifugal 1min of 800 × g, abandons supernatant and collects polypide, adds 1mL M9 recentrifuge washing polypide, exhausts supernatant as far as possible.
Add the nematode lysate that 1mL is fresh, vortex oscillation 10s at once, the centrifugal 1min of 800 × g, pipettor siphons away supernatant.
Add at once 1mL M9 washing polypide, the centrifugal 1min of 800 × g, double, remove residual lysate.The M9 suspension liquid that finally in pipe, residue approximately 100 μ L contain worm's ovum.
Use aseptic straw that worm's ovum is dripped in the NGM flat board of coating OP50, cultivate 48h for 25 DEG C, obtain the nematode of synchronization 48h.
(3) by the nematode of synchronization 48h, be placed in stereomicroscope observation, using platinum shovel picking gonotreme is on the mNGM flat board that obtains in step (1) of transparent semi-moon shaped L4 phase telianthus worm, 10 of every dull and stereotyped pickings, and flat board is placed in 25 DEG C of cultivations.Observe nematode survival state every day, as polypide touches and do not have movable reaction to think polypide death platinum shovel.Get rid of hatching and unnatural death nematode in missing, body.All end is tested in nematode death.Calculate nematode mean lifetime in table 1.
Embodiment 1.4
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Carry out nematode longevity test according to the method for embodiment 1.1, different, in step (1), after deactivation is cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed by the weight ratio of 3:7 (being that lactobacillus salivarius is 70 % by weight).Calculate nematode mean lifetime in table 1.
Embodiment 1.5
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Carry out nematode longevity test according to the method for embodiment 1.1, different, in step (1), after deactivation is cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed by the weight ratio of 9:1 (being that lactobacillus salivarius is 10 % by weight).Calculate nematode mean lifetime in table 1.
Embodiment 1.6
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Carry out nematode longevity test according to the method for embodiment 1.1, different, deactivation temperature is 90 DEG C, and inactivation time is 0.3h.Calculate nematode mean lifetime in table 1.
Embodiment 1.7
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Carry out nematode longevity test according to the method for embodiment 1.1, different, deactivation temperature is 60 DEG C, and inactivation time is 2.0h.Calculate nematode mean lifetime in table 1.
Comparative example 1.1
Carry out nematode longevity test according to the method for embodiment 1.1, different, with the alternative lactobacillus salivarius A of the disclosed bacterial strain of CN 101538545A.Calculate nematode mean lifetime in table 1.
Comparative example 1.2
Carry out nematode longevity test according to the method for embodiment 1.1, different, after deactivation is cooling, intestinal bacteria OP50 is suspended in M9 damping fluid according to the ratio of 400mg/mL, coat mNGM planar surface,, mNGM planar surface is only coated with the cooled intestinal bacteria OP50 of deactivation.Calculate nematode mean lifetime in table 1.
Table 1
| Nematode mean lifetime (my god) | |
| Embodiment 1.1 | 24.1±0.4 |
| Embodiment 1.2 | 23.1±0.6 |
| Embodiment 1.3 | 24.3±0.5 |
| Embodiment 1.4 | 18.9±0.4 |
| Embodiment 1.5 | 18.7±0.9 |
| Embodiment 1.6 | 19.1±0.2 |
| Embodiment 1.7 | 20.0±0.3 |
| Comparative example 1.1 | 17.8±0.2 |
| Comparative example 1.2 | 17.2±1.0 |
Embodiment 1.1 is compared and can be found out with comparative example 1.1 and comparative example 1.2 respectively, and lactobacillus salivarius of the present invention has the effect delaying senility, after deactivation for the nematode of feeding, life-span that can significant prolongation nematode.
Embodiment 1.1 is compared and can be found out with embodiment 1.4 and embodiment 1.5 respectively, after deactivation is cooling, intestinal bacteria OP50 and lactobacillus salivarius A are mixed, taking the gross weight of mixture as benchmark, the addition of lactobacillus salivarius A is 30-50 % by weight, more the life-span of significant prolongation nematode; Embodiment 1.1 is compared and can be found out with embodiment 1.6 and embodiment 1.7 respectively, at 65-85 DEG C of deactivation 0.5-1.5h, be more conducive to extend the life-span of nematode.
(2) adopt mouse as study subject
In following embodiment and comparative example:
Male Kunming strain mice: purchased from Beijing company of dimension tonneau China, body weight 20 ± 2g.
Lactobacillus salivarius A is that (this bacterial strain was deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center to lactobacillus salivarius of the present invention on June 25th, 2012, Institute of Microorganism, Academia Sinica, postcode: 100101) (depositary institution be abbreviated as CGMCC), deposit number is CGMCC No.6288).
MRS substratum: be purchased from Beijing overpass company.
Coomassie brilliant blue G250: be purchased from the biological company limited of Shanghai, Shanghai space.
Iipofuscin content, brain MAO-B activity, brain SOD activity and brain Na
+/ K
+the mensuration of-atpase activity is referring to " anti-aging effects of Water-soluable Polysaccharide from Rhizoma Dioscoreae Opposite on Mice " (Zhan Tong, Tao Jing, Wang Shuru, pharmacy progress, the 6th phase of 23 volumes in 1999):
Iipofuscin assay: get liver, make 5% homogenate with chloroform-methanol mixed solution, 40 DEG C of warm water bath 5 minutes, with 3000rpm centrifugal 10 minutes, to get supernatant liquor and survey fluorescence, emission wavelength is 435nm, excitation wavelength is 365nm, put sensitivity × 1, slit 10nm, regulating instrument fluorescence intensity with the Quinine Sulphate Di HC of 0.1 μ g/mL is 50.
Brain MAO-B(B MAO-B B) determination of activity: get cerebral tissue, add phosphoric acid buffer (the 0.1mol/L K of ice-cold 0.2mol/LpH7.4
2hPO
480.2ml and 0.1mol/L KH
2pO
419.8ml mix), ultrasonic homogenate 2 times, in 4 DEG C with 3000rpm centrifugal 10 minutes, precipitation is suspended in PBS liquid (pH7.4), and water-bath immediately vibration mixes, with 3000rpm centrifugal 10 minutes, cyclohexane layer is surveyed to light absorption value in 242nm place, crude enzyme liquid protein concentration is measured with Coomassie brilliant blue G250, makes standard protein with bSA, and enzyme activity unit is defined as 37 DEG C of enzyme amounts that produce the optical density(OD) change of 0.01/3h.
Brain SOD(superoxide-dismutase) determination of activity: get cerebral tissue, tissue sample is measured the SOD ultramicron Fast Measurement reagent that adopts diease occurrence teaching and research group of Nanjing Railway College of Medicine, enzymic activity represents with U/mg enzyme amount, calculation formula: [(control tube optical density(OD)-mensuration pipe optical density(OD))/control tube optical density(OD)]/50/100 × diluted sample degree/sample protein concentration.Sample protein concentration is measured with Coomassie brilliant blue G250, and calf serum is made standard protein.
Brain Na
+/ K
+-atpase activity is measured: get cerebral tissue 0.1g, add rapidly 1ml extracting solution (to include sucrose 250mmol/L, Histidine 30mmol/L, EDTA 5mmol/L, Desoxycortone 10 % by weight.Ice bath homogenate, three layers of filtered through gauze, obtain crude enzyme liquid, and protein concentration is measured with Coomassie brilliant blue G250.Enzymatic reaction is divided into two groups: A group, adds the medium A TP liquid (MgCl of 0.4mL
26mmol/L, NaCl 100mmol/L, KCl 20mmol/L, Tris 30mmol/L, ATP 3mmol/L).50 μ L dielectric fluid (MgCl
26mmol/L, NaCl 100mmol/L, KCl 20mmol/L, Tris 30mmol/L) and crude enzyme liquid 50 μ L; B group, adds Quabain and suppresses Na
+/ K
+-ATP enzyme, replaces 50 μ L dielectric fluids with the 5mmol/L Quabain of 50 μ L, and all the other are identical.In 37 DEG C of water-baths, react respectively 20 minutes, add 30% ice-cold trichoroacetic acid(TCA) 0.3mL, move in ice bath, with 1500rpm centrifugal 5 minutes, get supernatant liquor 0.5ml, survey its content of inorganic phosphorus.Step is: get 0.5mL and measure liquid, add the 2mL developer (FeSO of 1 volume 10% ammonium molybdate+9 volume freshly prepared 9.15%
47H
2o) and 2mL distilled water mix, 25 DEG C of reactions 8 minutes, survey light absorption value in 652nm place, make typical curve with standard phosphorus solution, try to achieve content of inorganic phosphorus, finally Na
+/ K
+the content of inorganic phosphorus that-ATP enzyme decomposes is the amount of A group-B group.Enzymic activity is expressed as μ mol pi/hrmg albumen, and protein concentration is measured with Coomassie brilliant blue G250, and calf serum is made standard protein.
Embodiment 2.1
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) lactobacillus salivarius A is inoculated in to the MRS substratum of 10L with aseptic inoculation ring, and under anaerobic, at the temperature of 37 DEG C, cultivates after 12 hours, obtain nutrient solution.By the nutrient solution obtaining supernatant discarded after centrifugal 10 minutes under the speed of 4000 × g, obtain the viable bacteria body of lactobacillus salivarius A.
(2) choose immediately 15 Male Kunming strain mice, every day, retrobulbar injection D-semi-lactosi 0.12mg/g, and fed containing 1 × 10
9the tap water of the viable bacteria body of the lactobacillus salivarius A of CFU/g, continuous one month.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.2
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) lactobacillus salivarius A is inoculated in to the MRS substratum of 10L with aseptic inoculation ring, and under anaerobic, at the temperature of 37 DEG C, cultivates after 12 hours, obtain nutrient solution.By the nutrient solution obtaining supernatant discarded after centrifugal 10 minutes under the speed of 4000 × g, obtain the viable bacteria body of lactobacillus salivarius A.
(2) choose immediately 15 Male Kunming strain mice, every day, retrobulbar injection D-semi-lactosi 0.12mg/g, and fed containing 1 × 10
8the tap water of the viable bacteria body of the lactobacillus salivarius A of CFU/g, continuous one month.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.3
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
(1) lactobacillus salivarius A is inoculated in to the MRS substratum of 10L with aseptic inoculation ring, and under anaerobic, at the temperature of 37 DEG C, cultivates after 12 hours, obtain nutrient solution.By the nutrient solution obtaining supernatant discarded after centrifugal 10 minutes under the speed of 4000 × g, obtain the viable bacteria body of lactobacillus salivarius A.
(2) choose immediately 15 Male Kunming strain mice, every day, retrobulbar injection D-semi-lactosi 0.12mg/g, and fed containing 1 × 10
7the tap water of the viable bacteria body of the lactobacillus salivarius A of CFU/g, continuous one month.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.4
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.1 is tested, different, and in tap water, the content of the viable bacteria body of lactobacillus salivarius A is 1 × 10
10cFU/g.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.5
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.1 is tested, different, and in tap water, the content of the viable bacteria body of lactobacillus salivarius A is 1 × 10
5cFU/g.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.6
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.1 is tested, different, step (1) is obtained to viable bacteria body hot deactivation 1h at 75 DEG C of lactobacillus salivarius A, obtain thalline material, this thalline material is joined in tap water and do not add viable bacteria body, in tap water, the content of thalline material is 40 % by weight.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.7
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.1 is tested, different, step (1) is obtained to viable bacteria body hot deactivation 1.5h at 65 DEG C of lactobacillus salivarius A, obtain thalline material, this thalline material is joined in tap water and do not add viable bacteria body, in tap water, the content of thalline material is 50 % by weight.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.8
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.1 is tested, different, step (1) is obtained to viable bacteria body hot deactivation 0.5h at 85 DEG C of lactobacillus salivarius A, obtain thalline material, this thalline material is joined in tap water and do not add viable bacteria body, in tap water, the content of thalline material is 30 % by weight.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.9
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.6 is tested, different, and in tap water, the content of thalline material is 65 % by weight.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.10
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.6 is tested, different, and in tap water, the content of thalline material is 15 % by weight.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.11
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.6 is tested, different, and deactivation temperature is 90 DEG C, and inactivation time is 0.3h.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Embodiment 2.12
The present embodiment is for illustrating effect that lactobacillus salivarius of the present invention delays senility.
Method according to embodiment 2.6 is tested, different, and deactivation temperature is 60 DEG C, and inactivation time is 2.0h.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Comparative example 2.1
Method according to embodiment 2.1 is tested, different, with the alternative lactobacillus salivarius A of the disclosed bacterial strain of CN 101538545A.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Comparative example 2.2
Method according to embodiment 2.6 is tested, different, with the alternative lactobacillus salivarius A of the disclosed bacterial strain of CN 101538545A.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Comparative example 2.3
Method according to embodiment 2.1 is tested, different, does not add bacterial classification in tap water.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Comparative example 2.4
Method according to embodiment 2.1 is tested, different, and not injection of d-galactose does not add bacterial classification in tap water.Survey Iipofuscin content, brain MAO-B activity, brain SOD activity and the brain Na of Male Kunming strain mice
+/ K
+-atpase activity, the results are shown in Table 2.
Table 2
The free radical producing in metabolic process can act on the polyunsaturated fatty acid in biological film phospholipid, produce lipid peroxide, as mda (MDA), and then becoming macromolecular complex-lipofuscin (age pigment) with protein cross with phosphatidylethanolamine, this is the one mark of organism aging process.
In human brain, MAO-B activity increased with age growth after 45 years old, Fowier finds that the MAO-B activity in human brain 19 regions was proportionate with the age, in cerebral tissue, MAO-B can cause changes of Catecholamine Content disorder in brain with the variation at age, impels physiological activity imbalance, thereby causes old and feeble generation.
SOD is oxygen radical removing enzyme in the important body of a class, but with age growth, the SOD activity in body tissue lowers gradually.
Na
+/ K
+-ATP enzyme claims again sodium [potassium, is a kind of very important film enzyme extensively existing in organism, and this enzyme plays an important role in ionic equilibrium inside and outside maintaining physiological activity, body temperature and eubolism and cytolemma.When biological decay, there will be multiple biochemical variation, wherein the variation of function of brain cell is more obvious, and this may reduce relevant with brain cell energy.There is report, its Na in some tissues of old rats
+/ K
+the younger mouse of-atpase activity decreases.
Comparative example 2.3 and comparative example 2.4 are compared and can be found out, injection of d-galactose after eyeball of mouse, can accelerate the aging of mouse.Embodiment 2.1 to embodiment 2.12 is compared and can be found out with comparative example 2.3, and lactobacillus salivarius of the present invention has the effect delaying senility; Embodiment 2.1 is compared with comparative example 2.1, embodiment 2.6 is compared and can be found out with comparative example 2.2, lactobacillus salivarius of the present invention has than the disclosed bacterial strain of CN 101538545A the effect delaying senility more significantly.
Embodiment 2.1 is compared and can be found out with embodiment 2.4 and embodiment 2.5 respectively, and in tap water, the content of the viable bacteria body of lactobacillus salivarius is 10
7-10
9cFU/g, is more conducive to delay senility; Embodiment 2.6 is compared and can be found out with embodiment 2.9 and embodiment 2.10 respectively, and the content of the thalline material obtaining after the viable bacteria body deactivation of lactobacillus salivarius in tap water is 30-50 % by weight, is more conducive to delay senility; Embodiment 2.6 is compared and can be found out with embodiment 2.11 and embodiment 2.12 respectively, at 65-85 DEG C of deactivation 0.5-1.5h, be more conducive to delay senility.
Thalline material after viable bacteria body and the deactivation of lactobacillus salivarius provided by the invention all has the effect delaying senility.
Claims (10)
1. a lactobacillus salivarius (Lactobacillus salivarius), is characterized in that, the deposit number of described lactobacillus salivarius is CGMCC No.6288.
2. prepare a method for functional food composition, described method comprises: by the viable bacteria body deactivation of lactobacillus salivarius claimed in claim 1, obtain thalline material.
3. method according to claim 2, wherein, the condition of described deactivation comprises: temperature is 65-85 DEG C, the time is 0.5-1.5h.
4. according to the method in claim 2 or 3, wherein, described method also comprises, described thalline material is added in food.
5. method according to claim 4, wherein, taking the gross weight of described functional food composition as benchmark, the addition of described thalline material is 10-70 % by weight, is preferably 30-50 % by weight.
6. a functional food composition of being prepared by the method described in any one in claim 2-5.
7. a functional food composition, is characterized in that, the viable bacteria body that described functional food composition contains lactobacillus salivarius claimed in claim 1.
8. functional food composition according to claim 7, wherein, taking the gross weight of described functional food composition as benchmark, the content of the viable bacteria body of described lactobacillus salivarius is 10
5-10
10cFU/g, is preferably 10
7-10
9cFU/g.
9. according to the functional food composition described in claim 7 or 8, wherein, described functional food composition also contains food.
10. the application in the functional food composition that lactobacillus salivarius claimed in claim 1 delays senility in preparation.
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| CN110923172A (en) * | 2019-12-25 | 2020-03-27 | 汉臣氏（沈阳）儿童制品有限公司 | Lactobacillus salivarius with constipation relieving and cholesterol reducing effects and application thereof |
| CN111297914A (en) * | 2020-02-24 | 2020-06-19 | 北京农学院 | Application of lactobacillus fermentation clear liquid in antioxidation or preparation of product for antioxidation and antioxidation product |
| CN114558037A (en) * | 2022-02-24 | 2022-05-31 | 同济大学 | Application of AKK and LS in preparation of anti-aging product for improving cognition level |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105395575A (en) * | 2015-11-19 | 2016-03-16 | 徐州一统食品工业有限公司 | Chewable tablet containing inactivated lactobacillus salivarius and preparation method thereof |
| CN110923172A (en) * | 2019-12-25 | 2020-03-27 | 汉臣氏（沈阳）儿童制品有限公司 | Lactobacillus salivarius with constipation relieving and cholesterol reducing effects and application thereof |
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| CN114558037A (en) * | 2022-02-24 | 2022-05-31 | 同济大学 | Application of AKK and LS in preparation of anti-aging product for improving cognition level |
| CN114558037B (en) * | 2022-02-24 | 2023-08-15 | 同济大学 | Application of AKK and LS in preparation of anti-aging products for improving cognition level |
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|---|---|
| CN103897998B (en) | 2016-06-29 |
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