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CN1467290A - Lactob.plantarum ST-III strain and application in regulating blood fat - Google Patents

Lactob.plantarum ST-III strain and application in regulating blood fat Download PDF

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CN1467290A
CN1467290A CNA031163777A CN03116377A CN1467290A CN 1467290 A CN1467290 A CN 1467290A CN A031163777 A CNA031163777 A CN A031163777A CN 03116377 A CN03116377 A CN 03116377A CN 1467290 A CN1467290 A CN 1467290A
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cholesterol
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CN1207382C (en
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蒋能群
孙立国
郭本恒
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Bright Dairy and Food Co Ltd
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SHANGHAI GUANGMING DAIRY INDUSTRY Co Ltd
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    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

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Abstract

A Lactobacillus plantarum ST-IIICGMCC No.0847 and its application in regulating blood fat are disclosed.

Description

Plant lactobacillus ST-III bacterial strain and the application aspect regulating blood fat thereof
Technical field
The present invention relates to a kind of new bacterial strain of lactobacillus, and by cultivating the application of this bacterial strain aspect regulating blood fat.
Background technology
The enteron aisle of humans and animals, a large amount of profitable strains in perch such as traditional fermented food, is all bringing into play important physiological action at the growth and the aspects such as growth, immunity and antagonism of the digestion of nutritive substance and absorption, body.In recent years, Bacterium lacticum, bifidus bacillus and the faecalis etc. in the microecology man discovery beneficial flora have direct relation with the metabolism of cholesterol.In external a large amount of body and in vitro study all find and confirmed that some probiotic lactobacillus has the effect of the serum cholesterol content that reduces human body or animal.And abundant this type of microorganism resource of not found by the western sciences man is as yet arranged in China's traditional food, remain further to be developed.
Summary of the invention
The inventor is at above-mentioned problem, having studied with China's traditional food pickles is the target flora with the micropopulation of pickling in the clover, use the hypercholesterolemia substratum, therefrom screen probiotic lactobacillus, and further study its application aspect regulating blood fat with reducing cholesterol effect.
Thereby the purpose of this invention is to provide a kind of plant lactobacillus ST-III;
Another object of the present invention is to disclose the application of above-mentioned plant lactobacillus ST-III aspect regulating blood fat.
Plant lactobacillus of the present invention (Lactobacillus plantarum) ST-III preparation process is as follows: kimchi juice or clover juice are refrigerated as sample, measure its pH value, and be inoculated in MRS flat board (Merck company buys) and carry out total enumeration; Take above-mentioned sample then, according to PBS buffered soln immersion mixing and the gradient dilution of said determination result with pH6.8;
Draw suitable extent of dilution (every milliliter contains 100 left and right sides bacteriums) 100 microlitres and coat the LBS flat board, cultivate;
The picking colonies typical is transferred to the MRS flat board and is further purified;
The desirable single bacterium colony of picking is transferred and is increased bacterium in MRS liquid, obtains lactic bacterium strains and stored refrigerated;
Above-mentioned fresh lactic bacterium strains is had the decreasing cholesterol function with the external model preliminary screening probiotic bacterium (USMAN and A.HOSONO.Bile tolerance, taurocholate deconjugation, andbinding of cholesterol by lactobacillus gasseri strains.J.Dairy Sci.199982:243-248.), carry out strain identification, wherein can obtain plant lactobacillus ST-III.
Plant lactobacillus ST-III of the present invention on December 6th, 2002 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north, Institute of Microorganism, Academia Sinica) preservation, and receive preservation registration number CGMCC No.0847.
The Microbiological Characteristics of this plant lactobacillus ST-III (CGMCC No.0847) is shown in table 1.
The Microbiological Characteristics of table 1 plant lactobacillus ST-III
Test subject result Test subject result
The shaft-like formation gemma-catalase of Gram-positive cellular morphology-oxidizing ferment-growth+amphimicrobian growth+O/F test fermentation produces lactic acid+carbohydrate aerogenesis in air Carbohydrate product acid (continuing) trehalose+wood sugar-fructose+ribose+gossypose+lactose+melezitose+salicin+
Glucose-gluconic acid sodium salt+carbohydrate product sour grapes sugar+gluconic acid sodium salt+cellobiose+arabinose+sucrose+ Rhamnose-aesculin+mannose+sweet mellow wine+maltose+sorbierite+melibiose+galactolipin+
Aforesaid operations all carries out under aseptic condition.
Plant lactobacillus ST-III of the present invention is except that the beneficial effect with general Bacterium lacticum, this bacterial strain is applied to the regulating blood fat aspect, as the milk-product of development functionality,, also play the effect of the auxiliary people of reduction serum cholesterol concentration as the human diet supplement.
By being described in more detail the present invention by following examples.Following examples only are illustrative, and the present invention is not limited by these embodiment.
Plant lactobacillus ST-III of the present invention on December 6th, 2002 in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, and receive preserving number CGMCCNO.0847.
Embodiment
Embodiment 1 sample collecting
Sample is:
Kimchi juice: Caulis et Folium Brassicae capitatae pickles, dish leaf and the juice homogenate that obtains of homogeneous together;
Clover juice: gather the clover of pickling of Sky-High Hill, Suzhou farmers', get 5 grams, add the homogenate that 10 ml sterile water homogeneous obtain;
Embodiment 2 collection and milk-acid bacteria screening
With the above-mentioned sample aseptic rib collar bottle of packing into, screw 4 ℃ of refrigerations.Measure the pH of kimchi juice and clover juice in 24 hours, and be inoculated in its total enumeration of the dull and stereotyped mensuration of MRS, handle according to the following steps then.
Embodiment 3LBS plate isolation
Get kimchi juice and clover juice, according to the result who is measured among the embodiment 2, aseptic condition is down with PBS buffered soln (pH6.8) gradient dilution, draw suitable extent of dilution solution (every milliliter contains 100 left and right sides bacteriums) 100 microlitres and coat the LBS flat board, flat board is inverted into the anaerobism incubator, 37 ℃ of anaerobism were cultivated 36-72 hour, observed the colony growth situation, dull and stereotyped 4 ℃ of preservations.
Embodiment 4 bacterial strain microscopies
Aseptic technique, transfering loop picking embodiment 3 gained colonies typical cultures are applied to slide glass, film-making and gramstaining, oily sem observation thalli morphology, the picking gram positive bacterium is as the purpose bacterial strain.
The dull and stereotyped purifying of embodiment 5 bacterial strains
Under the aseptic condition, 4 preliminary purpose bacterium colonies of determining of transfering loop picking embodiment, line separates on the MRS flat board, is inverted anaerobism for 37 ℃ and cultivates 24-36 hour, and picking list bacterium colony carries out microscopy, and is purified until the bacterial strain of determining gained.
The bacterium process that increases of embodiment 6 bacterial strains
Desirable single bacterium colony of picking embodiment 5 gained, be transferred to 1 milliliter of MRS liquid, 37 ℃ leave standstill cultivation about 24 hours, inoculum size by 1% (volume ratio) is transferred to new MRS liquid with nutrient solution again, 37 ℃ leave standstill cultivation 24 hours, repeat amplification protcol 2 times in liquid MRS again, 4 ℃ of preservations of gained nutrient solution.
Embodiment 7 has the probiotic bacterium of decreasing cholesterol function with the external model preliminary screening
The hypercholesterolemia nutrient solution MRSO-CHOL that it is the cholesterol source with aseptic egg yellow liquor that 15 milliliters of aseptic tool plug centrifuge tubes add 10 milliliters inserts the fresh bacterial strain that embodiment 6 obtains by the inoculum size of 1% (volume ratio), cultivates 24 hours for 37 ℃.Nutrient solution 12, centrifugal 10 minutes of 000 * g, measure the method for cholesterol according to o-phthalaldehyde(OPA) and measure content of cholesterol in the supernatant, with nonvaccinated MRSO-CHOL in contrast, calculate the reduced rate (S.E.Glliland of each experimental strain cholesterol, C.R.Nelson, C.Maxwell.Applied and EnviromentalMicrobiology, 1985,49:377～381.), therefrom select and have the bacterial strain of reducing cholesterol effect preferably, the results are shown in Table 2.
The reduced rate calculation formula of cholesterol is: D = C - A C × 100 %
Wherein A is each experimental strain fermented supernatant fluid OD 550nm[the absorbance value that wavelength 550nm (nanometer) locates];
C is the OD of blank 550nm
D is the cholesterol reduced rate.
Table 2 decreasing cholesterol probiotic lactobacillus results of preliminary screening
Cholesterol reduced rate bacterial strain is counted the ratio source
(%) (strain) (%) pickled vegetable clover
More than 40 6 9.09 42
30-40???????????9??????????13.64?????5??????????2
20-30???????????13?????????19.70?????2??????????8
Below 20 38 57.28 12 16
Wherein, ST-III has the highest cholesterol reduced rate, reaches 47.68%, and corresponding cholesterol reduction amount is 244.84 mcg/ml.Also has the cholesterol reduced rate of 5 strain bacterial strains in addition more than 40%.
Embodiment 8ST-III strain identification
The Microbiological Characteristics of this ST-III such as above-mentioned table 1 show.
Embodiment 9 thallus suspension liquid preparation methods
Plant lactobacillus ST-III of the present invention (CGMCC No.0847) is inserted MRS liquid, and 32 ℃ leave standstill cultivation 16 hours, counting; According to count results with skimmed milk do suitable dilution, suspending obtains as bacterial concentration required in the table 3.The dead bacterium suspension preparation of ST-III method: suspend 10 8Cfu/ml (institute's mycetome number in every milliliter) ST-III skimmed milk placed 100 ℃ of heating in water bath 15 minutes, cooling, and refrigerator is preserved stand-by.
Application Example serum cholesterol-lowering functional experiment (experiment in the body, experimentation on animals)
Animal model probiotic lactobacillus reduce the application that obtains in the research of serum cholesterol functional experiment more and more widely (Zheng Jianxian. functional foodstuff (the 3rd volume). Beijing: China Light Industry Press, 1999.9.408-412.).High fat high cholesterol diet brings out the Tallow, beef metabolism disorder, blood lipid level changes: serum cholesterol (TC) and triglyceride level (TG) level and low density lipoprotein cholesterol (LDL-C) content raise, and high density lipoprotein cholesterol (HDL-C) content reduces.The common way of serum cholesterol-lowering functional experiment is, when feeding high fat high cholesterol diet, the laboratory animal laboratory sample of feeding is determined the impact effect of laboratory sample to animal lipid by the variation of measuring the animal serum lipid content.
The probiotic lactobacillus that the present invention's screening obtains is plant lactobacillus (Lactobacillus plantarum) ST-III (CGMCC No.0847), and this bacterial strain has the ability of higher reducing cholesterol concentration in experiment in vitro.
The present invention adopts the SD rat, adds weight ratio and is 1% cholesterol, 5% lard and 0.25% pig cholate in conventional feed and bring out SD rat hypercholesterolemia.Feed the simultaneously skimmed milk of suspension plant Bacterium lacticum detects lipids contents such as SD rat blood serum cholesterol at last, judges whether this bacterial strain has the effect that reduces serum cholesterol.
1, laboratory animal and Animal House condition
Male SD rat (8 age in week), 64.Conventional feed was fed 3 days, was divided into 8 groups at random, 8 every group, divided cage to feed.
The Animal House condition: 22 ± 2 ℃ of temperature, humidity 56 ± 5%, 1 week was changed sawdust 2-3 time.Control illumination in 12 hours, 12 hours dark.
Feed: conventional feed is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center;
High cholesterol diet: conventional feed interpolation weight ratio is 1.0% cholesterol, and 5% lard and 0.25% Iocholic acid salt are entrusted Chinese Academy of Sciences's Shanghai Experimental Animal Center processing;
All feeds advance the sterilization of Animal House previous irradiation.
2, the functional animal experiment method of probiotic lactobacillus ST-III
(1) the laboratory animal grouping and the arrangement of feeding
64 SD rats are divided into 8 groups at random, feed different feeds and thallus suspension liquid, and concrete grouping is as shown in table 3.
The grouping of table 3 laboratory animal and the plan experiment grouping of feeding aDaily feeding bIrritate 3 milliliters// days 1. blank conventional feeds of stomach+water water 2. high fat contrast high lipid food+water water 3. high fat SKM cContrast high lipid food+water SKM4.L.bulgaricus dRight
High lipid food+aqueous suspension 2.5 * 10 8The SKM of cfu/ml L.bulgaricus is according to 5.L.acidophilus eRight
High lipid food+aqueous suspension 2.5 * 10 8The SKM of cfu/ml L.acidophilus is according to the high high lipid food+aqueous suspension 2.5 * 10 of 6.ST-III viable bacteria 8The SKM7.ST-III viable bacteria of cfu/ml viable bacteria ST-III hangs down high lipid food+aqueous suspension 2.5 * 10 6The dead bacterium high lipid food+aqueous suspension 2.5 * 10 of the SKM8.ST-III of cfu/ml viable bacteria ST-III 8The SKM of the dead bacterium ST-III of cfu/ml
Annotate: aEvery group of 8 SD rats
bThe experiment of feeding continues 21 days altogether
cSKM---10% skimmed milk of sterilizing
dL.bulgaricus---lactobacillus bulgaricus
eL.acidophilus---Lactobacterium acidophilum
(2) daily administration of laboratory animal and feeding
The SD rat is divided into 1-8 group at random by table 3, except that blank group 1 is fed the conventional feed, and the 2-8 group high lipid food of all feeding.According to scheme in the table 3, at 8 in morning every day is to 3 milliliters// days of SD rat oral gavages, record feed consumption every day, experiment beginning and finish the body weight of weighing rat.
(3) detection of rat blood serum collection and blood fat and serum total cholic acid concentration
After the experiment of feeding in 21 days finishes; fasting 12 hours; the ether deep anaesthesia is plucked eyeball and is got blood, the aseptic centrifuge tube of packing into; centrifugal (3000 * g; 10 minutes) collect serum, serve hospital inspection center, the extra large Huashan and measure total cholesterol (TC) in the serum, triglyceride level (TG); high density lipoprotein cholesterol (HDL-C) low density lipoprotein cholesterol (LDL-C) and and the content of total cholic acid (TBA).
(4) statistical study
Data one-way analysis of variance on the same group.
Not on the same group between t check, p＜0.05 difference has statistical significance, p＜0.01 and p＜0.001 difference has the statistical significance of height.
3, interpretation of result
(1) measurement result of Serum TC and TG concentration
After the experiment of feeding in 21 days finished, rat blood serum was collected in fasting 12 hours, send Shanghai Huashan Hospital to carry out the serum lipid analysis, measures TC, TG, HDL-C, LDL-C and TBA content.Concrete outcome sees Table 4 and table 5.
Table 4 Serum TC and TG assay
The experiment grouping aTC (mmol/L) TG (mmol/L)
1???????????1.4±0.29???????????0.68±0.20
2???????????2.64±0.36??????????0.99±0.10
3???????????2.56±0.23??????????0.97±0.19
4???????????2.36±0.29??????????0.90±0.32
5???????????2.27±0.21 *????????0.98±0.22
6???????????2.01±0.30 **???????0.80±0.25
7???????????2.26±0.23 *????????0.93±0.35
8???????????2.39±0.24??????????0.86±0.08
Annotate: aThe experiment grouping sees Table 3
Group 4-8 carries out the t check with group 3 respectively: *P＜0.05 difference has statistical significance, *P＜0.01 difference has the statistical significance of height.
Blank group 1 in the comparison sheet 4 and the TC of high fat control group 2 and TG content, as can be seen, the TC of group 2 and TG content are respectively than high 88.57% and 45.59% of group 1, and difference has the statistical significance (p＜0.001) of height and statistical significance (p＜0.05) is arranged.In this experiment, the high lipid food of feeding can bring out high TC of rat and TG level thus, meets the report and the requirement of experiment of document.
Group 3 is compared with group 2, and TC and TG content have descended 3.03% and 2.02% respectively, difference not statistically significant (p＞0.05), and this experiment confirm SKM does not reduce effect to TC content.Compare with group 3, among the group 4-8 of the thalline of feeding, the TG content of group 6 is minimum, has descended 17.52% than group 3, but difference not statistically significant (p＞0.05), and other TG content of 4 groups is compared difference with group 3 does not have statistical significance (p＞0.05) yet.May feed thalline to the not tangible influence of the reduction of rat TG content.
With regard to TC, the dead bacterium of the group 4 of the SKM suspension L.bulgaricus that feeds and ST-III group 8 is compared with the high fat control group 3 of the SKM that feeds, has descended 7.81% and 6.64% respectively, difference not statistically significant (p＞0.05); And compare with group 3, the rat TC content of the group 7 that the group 5 of SKM suspension L.acidophilus of feeding and ST-III viable bacteria are low has descended 11.33% and 11.72% respectively, difference has statistical significance (p＜0.05), the high group 8 rat TC content of the ST-III viable bacteria of feeding have descended 21.48%, and difference has the statistical significance (p＜0.01) of height.Group 6 has reduced by 11.06% TC content than group more than 7.As seen ST-III of the present invention has the effect that reduces TC.
(2) influence of ST-III thalline of feeding to rat HDL-C and LDL-C
As table 5 is experimental rat HDL-C and LDL-C measurement result.
Table 5 rat HDL-c and LDL-c Determination on content
The experiment grouping aHDL-C (mmol/L) LDL-C (mmol/L) AI b
1????????1.02±0.21?????????????0.32±0.14???????????0.32
2????????0.75±0.10?????????????1.54±0.29???????????2.05
3????????0.79±0.10?????????????1.51±0.22???????????1.93
4????????0.90±0.12?????????????1.15±0.32 *?????????1.30 **
5????????1.05±0.25 *???????????1.04±0.16 ***???????1.06 ***
6????????0.91±0.07 *???????????0.94±0.24 **????????1.03 ***
7????????0.94±0.12 *???????????1.13±0.22 **????????1.21 **
8????????1.09±0.20 **??????????1.10±0.28 **????????1.07 ***
Annotate: group 4-8 carries out the t check with group 3 respectively: *P＜0.05 difference has statistical significance, *P＜0.01, * *P＜0.001 difference has the statistical significance of height
aThe experiment grouping sees Table 3; bAI-atherosclerosis risk factor, AI=LDL-C/HDL-C
As can be seen from Table 5, the HDL-C content of high fat control group 2 is than the decline 26.47% of blank group 1, difference has statistical significance (p＜0.05), LDL-C content has risen 381.25%, difference has the statistical significance (p＜0.001) of height, atherosclerosis factors A I has risen 540.62%, and difference has the statistical significance (p＜0.001) of height.As can be seen, high lipid food has brought out the lipoprotein metabolism disorder of rat, and HDL-c content descends and LDL-c content raises, and AI risk factor value raises very obvious.
The feed rat blood serum HDL-C of SKM and LDL-C content of group 3 raises 5.33% and reduced by 1.95% than high fat control group 2 respectively, difference not statistically significant (p＞0.05), and AI factor difference is not statistically significant also.Illustrate that SKM does not have regulating effect to lipoprotein metabolism.
Organize 4 rat blood serum HDL-C content and LDL-C content respectively than high fat SKM control group 3 risings 13.92% and reduction by 23.84%, but difference not statistically significant (p＞0.05).The lipoprotein metabolism not influence of L.bulgaricus to rat is described.
It is higher by 32.91% than group 3 to organize 5 rat blood serum HDL-c content, and difference has statistical significance (p＜0.05), and LDL-C content is lower by 31.12% than group 2, and difference has the statistical significance (p＜0.01) of height; And the AI factor has reduced by 45.08% (p＜0.001), and difference also has the statistical significance of height.Illustrate that L.acidophilus has the effect of rising rat HDL-C and reduction LDL-C content.
10 of the SKM that feeds suspension 8The rat blood serum HDL-C content of the high group 6 of cfu/ml concentration ST-III viable bacteria has raise 15.19% than group 3, and difference has statistical significance (p＜0.05), and LDL-C content has reduced by 37.75% respectively, and difference has the statistical significance (p＜0.01) of height; The AI factor has descended 46.63% simultaneously, and difference also has the statistical significance (p＜0.001) of height.
10 of the SKM that feeds suspension 6The rat blood serum HDL-C content of the low group 7 of cfu/ml concentration ST-III viable bacteria has raise 18.98% than group 3, and difference has statistical significance (p＜0.05), and LDL-C content has reduced by 37.75%, and difference has the statistical significance (p＜0.01) of height; The AI factor has descended 46.63% simultaneously, and difference has the statistical significance (p＜0.001) of height.
10 of the SKM that feeds suspension 8The rat blood serum HDL-C content of the dead bacterium group 8 of cfu/ml concentration ST-III has raise 37.98% than group 3, and difference has the statistical significance (p＜0.01) of height, and LDL-C content has descended 27.15%, and difference has the statistical significance (p＜0.01) of height; The AI factor has descended 44.56% simultaneously, and difference has the statistical significance (p＜0.001) of height.
Group 6, the experimental result explanation of group 7 and group 8, ST-III viable bacteria and dead bacterium have regulating effect to the rat blood serum lipoprotein metabolism, and the HDL-C content that can raise reduces LDL-C content.Find that relatively biomass raises, effect is obvious.

Claims (2)

1, a kind of plant lactobacillus (Lactobacillus plantarum) ST-III CGMCC NO.0847.
2, the application of the described plant lactobacillus of claim 1 aspect regulating blood fat.
CNB031163777A 2003-04-11 2003-04-11 Lactob.plantarum ST-III strain and application in regulating blood fat Expired - Lifetime CN1207382C (en)

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AU2003252423A AU2003252423A1 (en) 2003-04-11 2003-06-23 Lactobacillus plantarum strain st-iii and its use in adjusting blood fat
PCT/CN2003/000485 WO2004090118A1 (en) 2003-04-11 2003-06-23 Lactobacillus plantarum strain st-iii and its use in adjusting blood fat

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WO2006034611A1 (en) * 2004-09-29 2006-04-06 Bright Dairy & Food Co., Ltd. Use of lactobacillus plantarum st-iii in losing weight
CN100337653C (en) * 2004-12-21 2007-09-19 上海交大昂立股份有限公司 Combination of containing Lactobobacillus plantarum
CN102342327A (en) * 2011-10-21 2012-02-08 上海光明奶酪黄油有限公司 Cheddar cheese containing probiotics and preparation method thereof
CN102453767A (en) * 2011-12-23 2012-05-16 光明乳业股份有限公司 Method and kit for rapidly and quantitatively detecting lactobacillus plantarum ST-III
CN102533790A (en) * 2011-12-23 2012-07-04 光明乳业股份有限公司 PCR detection method for detecting lactobacillus plantarum ST-III and primer and kit thereof
CN102732470A (en) * 2012-07-20 2012-10-17 光明乳业股份有限公司 Purely natural and low-cost lactobacillus (Lb.) plantarum ST-III culture method and products thereof, and application of products
CN103284028A (en) * 2013-05-13 2013-09-11 陈正军 Pickle starter culture and preparation method of pickle starter culture and application method in pickle starter culture in vegetable pickling
CN103598594A (en) * 2013-10-28 2014-02-26 天津科技大学 Application of lactobacillus plantarum in reducing blood fat and assisting fat-reducing
CN104430851A (en) * 2013-12-25 2015-03-25 广东燕塘乳业股份有限公司 Fermented milk capable of reducing cholesterol and preparation method of fermented milk
CN105820989A (en) * 2016-06-15 2016-08-03 苏州健世星生物科技有限公司 High-density culture method of direct-feeding-type leavening agent probiotic lactic bacteria
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CN109385389A (en) * 2017-08-08 2019-02-26 光明乳业股份有限公司 A kind of lactobacillus plantarum ST-III and preparation method thereof lacking ldhA gene
CN107897368A (en) * 2017-11-15 2018-04-13 光明乳业股份有限公司 A kind of probiotics fermention breast and preparation method thereof
CN108517308A (en) * 2018-05-23 2018-09-11 光明乳业股份有限公司 A kind of microecological microbial agent and its preparation method and application
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CN111004754B (en) * 2019-12-30 2022-05-06 光明乳业股份有限公司 Anti-oxidation lactobacillus preparation and preparation method thereof

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