CN1680544A - Acid and choline-resistant separated strain of lactobacillus with ability of reducing and assimilating cholesterol - Google Patents

Acid and choline-resistant separated strain of lactobacillus with ability of reducing and assimilating cholesterol Download PDF

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CN1680544A
CN1680544A CNA2005100652557A CN200510065255A CN1680544A CN 1680544 A CN1680544 A CN 1680544A CN A2005100652557 A CNA2005100652557 A CN A2005100652557A CN 200510065255 A CN200510065255 A CN 200510065255A CN 1680544 A CN1680544 A CN 1680544A
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lactobacillus
strain
cholesterol
test
acid
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CN1680544B (en
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刘玉茹
游金珠
谭静芬
廖啓成
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FOODSTUFF INDUSTRIAL AND DEVELOPMENT INST
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Abstract

The invention opened a new Lactobacillus which can resist the cholate, the gastric acid and assimilate the cholesterol. The strain or the progeny can be used to many kinds of the foods; and it can be used as the medicine for curing the gastrointestinal sickness and decreasing the cholesterol of the serum.

Description

Has the novel acidproof and bile tolerance lactobacillus separation strains that reduces with the assimilating cholesterol ability
The application submitted on October 30th, 2002, and denomination of invention is divided an application for the Chinese patent application 02146947.4 of " having the novel acidproof and bile tolerance lactobacillus separation strains that reduces with the assimilating cholesterol ability ".
Technical field
The present invention relates to have bile tolerance and stomach juice-resistant characteristic and have the novel lactobacillus separation strains of reduction serum cholesterol ability and its purposes simultaneously.
Background technology
" milk-acid bacteria (lactic acid bacteria) " is group energy fermentation carbohydrate and is the bacterium of primary product with lactic acid that its present well accepted form and physiological characteristic are: (1) gram-positive microorganism; (2) profile is coccus or bacillus; (3) catalase test is negative; (4) from it metabolic glucose can generate lactic acid more than 50%; (5) do not form endogenous spore; And (6) are many does not have a motility, can grow under little aerobic situation.
Well known general milk-acid bacteria comprised lactobacillus (Lactobacillus), streptococcus (Streptococcus), leuconos toc (Leuconostoc), Pediococcus 4 Pseudomonas (W.C. such as (Pediococcus) up to 1980 Frazier and D.C.Westhoff, 1978, Food Microbiology, 3rd ed.McGraw-Hill, Inc., New York, USA), and the generalized milk-acid bacteria also comprises genus bifidobacterium (Bifidobacterium) and these two Pseudomonas of Sporolactobacillus (Sporolactobacillus).And in recent years, microorganism can clearly independently have been become a taxonomical group according to dna homology and the analysis of rDNA sequence alignment on taxonomic hierarchies, and is endowed the position on the taxonomy.Known to the applicant, by in December, 1999, milk-acid bacteria family has been expanded to and has comprised 16 Pseudomonas and 223 species.
Via after the ingesting of the mankind or animal host, can improve microorganism is balanced each other in these mankind or animal host's the intestines single or mixed strains and be so-called probiotic bacterium (O ' sullivan etc., (1992), Trends in Food Sci.Technol., 3:309-314; Fuller, R., P.J.Heidt, V.Rush and D.van der Waaij. (eds.) (1995), Probiotics:prospects of use in opportunisticinfections.Old Herborn University Seminar Monograph No.8, pp.1), wherein most be that the probiotic bacterium that the people knew and used is Bacterium lacticum (Lactobacillus) and bifidus bacillus (Bifidobacterium).
From Eli Metchnikoff in 1908 propose " the edible yogurt that contains Bacterium lacticum; can replace the toxigenic bacterium body of normal presence in intestines; and not only good for healthly more can promote longevity " (EliMetchnikoff, 1908, The Prologation Of Life, Ed.P.Chalmers Mitchell, G.P.Putnam ' s Sons, The Knicherbocker Press, New York﹠amp; London) after the theory, research and clinical test results in recent years also shows " Bacterium lacticum and health have important association ".Therefore Bacterium lacticum is subjected to paying attention to widely.
Milk-acid bacteria can only not occupy critical role in the ecosystem of enteron aisle complexity, and the host is gone back benefiting on the unsoundness.The found effect of milk-acid bacteria comprises that the nutritional quality of promoting the food that the host ingested produces (J.Denter and B.Bisping with promoting the synthetic enzyme that reaches of VITAMIN, 1994, Int.J.Food Microbiol., 22:23-31), suppress in the growths of pathogenic bacterium in the enteron aisle and the stable enteron aisle the normal bacterium (Hose that balances each other, H. and Sozzi, T. (1991), J.Chem.Technol.And Biotech.51:540-544), producing antibody materials increases host's resistibility (H.Majamaa etc., (1995), Journal of Pediatric Gastro-enterology and Nutrition, 20:333-338) and reduce the risk of large bowel cancer and suppress tumour (E.J.Schiffrin etc., 1997, Am.J.Clin.Nutr., 66:515S-520S).Simultaneously, also studies show that, take the content that also can reduce serum cholesterol through the dairy products of lactobacillus ferment.
Cardiovascular disorder is listed in the people's of industrial country a underlying cause of death.In the U.S., because of the dead number of cardiovascular disorder more than cancer and other disease, it is owing to atherosclerosis and complication thereof that 3/4ths the cause of the death is wherein arranged, and high anteserum cholesterol is to cause one of cardiovascular disorder and atherosclerotic reason (Kannel etc., (1979), Ann.Intern.Med., 90:85-91; Pekkanen etc., (1990), New England J.Med., 322:1700-1707).In the Taiwan, the cerebrovascular and heart disease also rank ten big cause of the death firsts always in recent years, and the topmost cause of disease of the elderly and the cause of the death especially, and therefore, hypercholesterolemia is a cause of disease that can not be ignored.
Mann in 1974 and Spoerry find that the cultured milk of drinking behind lactobacillus ferment can reduce the human serum cholesterol concentration, and edible fresh milk does not then have influence (Am.J.Clin.Nutr., 27:464-469,1974).Therefore, people have carried out extensive studies to the processing and the effect of milk-acid bacteria reducing cholesterol in recent years.
Nineteen eighty-two, K.K.Grunewald is in J.of Food Science, report among the 47:2078-2079, after rat was lasted for 4 weeks by feeding with the food that contains 10% cultured milk that ferments through Lactobacterium acidophilum (Lactobacillus acidophilus), the serum cholesterol concentration of rat significantly descended.1989, people such as Danielson are in J.Anim.Sci., report among the 67:966-974, after the pig that comes feeding to be given the hypercholesterolemia diet with the acidophilic bacteria cultured milk (acidophilus yogurt) through Lactobacterium acidophilum LA16 strain isolated fermentation lasts 56 days, can reduce serum cholesterol and the low-density lipoprotein (LDL) of pig, to serum triglyceride and the then invalid usefulness of high-density lipoprotein (HDL) (HDL).
The paper of above-mentioned relevant Lactobacterium acidophilum LA16 strain isolated, be Dr.khem M.Shahani one of leader's the characteristic of the relevant Lactobacterium acidophilum that study group carried out and bioactive multinomial research.Especially; Dr.khem M.Shahani leader's study group once at the characteristic of the Lactobacterium acidophilum DDS-1 strain isolated of a human origin and biological activity and carry out considerable research; comprising the research that reduces serum cholesterol level; this DDS-1 strain isolated patented the power protection afterwards (referring to Nebraska Cultures; Inc. be disclosed on the internet internet data relevant for DDS-1TM Http:// www.nebraskacultures.com.benefits.html).
1997, cultured milk that people such as Akalin are more general [by thermophilus streptococcus (Streptococcus thermophilus) and lactobacillus delbruckii species (Lactobacillus delbrueckiisp.) fermentation] and the influence of acidophilic bacteria cultured milk [by thermophilus streptococcus and Lactobacterium acidophilum fermentation] for the mice serum cholesterol, and the ability of finding acidophilic bacteria cultured milk reduction serum cholesterol concentration obviously will be higher than general cultured milk (A.S.Akalin etc., (1997), J.Dairy Sci., 80:2721-2725).
People such as Smet were in British J.of Nutrition in 1998, report among the 79:185-194: pig is in the 3rd to 7 week of edible Lactobacterium acidophilum, output in the ight soil in the cholate is significantly increased, and in the 3rd to 7 week, the total serum cholesterol concentration also significantly decreases in the pig body.Therefore, the enzymic activity of the BSH that Bacterium lacticum had may be to cause one of mechanism of action that the serum cholesterol of test pig reduces.
The intravital cholesterol of people can be synthetic voluntarily by liver, also can be by absorbing in the animal food.The Excretion approach of cholesterol has two: (1) forms cholic acid because of the metabolism of liver, then combine and water soluble with glycine or taurine, form cholate such as GC (glycocholates) or taurocholate (taurocholates) etc. with potassium or sodium ion again, these salts can be excreted via ight soil; And (2) formation steroid hormone, the metabolism because of hormone excretes in urinating again, but this approach only accounts for small portion.
Cholate is the water-soluble end product material in the cholesterol metabolic process; and these cholate may enter in the enterohepatic circulation; because the cholate hydratase (BSH of bacterium [comprising: lactobacillus, enterococcus spp (Enterococcus), Peptostreptococcus (Peptostreptococcus), genus bifidobacterium, fusobacterium (Clostridium) and bacterioid (Bacteroid) etc.] in the intestines; E.C.3.5.1.24) enzyme effect can make cholic acid and glycine or taurine separate, and removes bonded cholate (deconjugated bile salts) and produce.These go the bonded cholate water insoluble, and easily with cholesterol in serum formation co-precipitation and be excreted.
Except that above-mentioned, according to report, the metabolic mechanism of cholesterol still has assimilation and co-precipitation.
1985, people such as S.E.Gilliland reported among the 49:377-381 at Appl.Environ.Microbiol, and Lactobacterium acidophilum has absorption and assimilation to cholesterol, and under the environment of 0.3% oxgall, the ability of Bacterium lacticum reducing cholesterol is preferable.Similarly, people such as Noh are in J.Dairy Sci. (1997), and 82:3107-3113 report, the cytolemma of Lactobacterium acidophilum can be in conjunction with cholesterol, and the cholesterol that is adsorbed can further assimilate and is metabolized to the cell desired substance.
On the other hand, F.A.M.Kalver and R.van der Meer are in Appl.Environ.Microbiol (1993), report among the 59:1120-1124, Bacterium lacticum and bifidus bacillus do not have assimilation for cholesterol, but be lower than at 6.0 o'clock in the pH value, because thalline increases (this phenomenon also may be relevant with the BSH activity that thalline is had) to the conjugation of cholate is active, and makes cholesterol and cholate form co-precipitation, and has reduced the cholesterol level in the substratum.
1997, M.M.Brashears and S.E.Gililland point out, under the situation of not controlling the pH value (being that normal ph is 4.5 to 5.5), Bacterium lacticum has good cholesterol co-precipitation, and if control pH value maintains about 6.0, the ability of this bacterium reducing cholesterol lowers (M.M.Brashears and S.E.Gililland (1997) significantly, Influences of pH during growth onremoval of cholesterol from MRS broth by Lactobacillus casei andLactobacillus acidophilus, Animal Science Research Report, pp.32-37; Http:// www.ansi.okstate.edu/research/1997rr/006.htm).
1998, Zhang Jiacheng and Luo Chengxiang tested with high fat breast, food oils, and think that " assimilation " is the main effect that milk-acid bacteria is minimized cholesterol.(Zhang Jiacheng and Luo Chengxiang (1998), " and milk-acid bacteria removes the research of effect to cholesterol in the food--the screening of lactic acid bacteria culturers (strain), " Food science (China), 19:20-22).
1999, Usman and Hosono reported among the 82:243-248 in J.Dairy Sci., and a kind of newfound lactobacilli Lactobacillus gasseri (Lactobacillus gasseri) can absorb cholesterol under the culture condition of no cholate.
Reducing the human serum cholesterol level with biological method is a kind of both economical and efficient manner.And by above documents and materials as can be known, milk-acid bacteria effectively reduces cholesterol effectiveness with external all may showing in vivo, and wherein possible mechanism of action has comprised going keying action, cholesterol and going the bonded cholate to produce the assimilation to cholesterol of coprecipitation and lactic-acid bacteria cells down in acid ph value of bile salt hydrolase.
But milk-acid bacteria is behind the human body that is ingested, can face human body stomach environmental stress and to the specificity of enteron aisle adsorptivity, therefore, milk-acid bacteria will be brought into play effect in enteron aisle, at first just must overcome gastral severe environment, and can settle down (colonization) at enteron aisle.Moreover, Lactobacterium acidophilum is the bacterial classification of a group nutritional requirement complexity, except stable in the cultured milk product, all the other commercially available various milk-acid bacterias are to process kenel (Powdered, granular and ingot shape) and make product with dry bacterium powder or other, thalline is all survived under room temperature or stored under refrigeration through for a long time and is difficult for, and causes the bacterium number to be difficult for maintaining the number of initial storage.Therefore, contained actual bacterium number in the product of commercially available non-cultured milk product, often not and the bacterium number that is indicated on the packing.So, in the production of milk-acid bacteria product, in sale and storage process, how to keep the bacterium number of milk-acid bacteria, be very important problem.And have bacterial strain screening acidproof, that bile tolerance reaches characteristics such as good reduction serum cholesterol ability, be an important goal of the good Bacterium lacticum product of exploitation.
From as can be known above-mentioned, if can be directly when strain screening screen, can save the expense of subsequent process at characteristics such as acidproof, the bile tolerance of bacterial strain and stability in storagies, more the bacterial strain that screens can be done widely and use.And separation source is also relevant with selecting of effective strain with region, and therefore, the human body source has become a main demand in bacterial strain screening source in recent years.
At present, in 171 specific food for health care that Japanese health ministry is checked and approved, have 36 (account for whole 21%) to be beneficial natural disposition bacterial classification, but the output value account for 82% of sum.In Europe, beneficial natural disposition bacterial classification in the output value of food products market up to 1,000,000,000 dollars.In the U.S., the sales value of sour buttermilk in 2000 promptly reaches 18.6 hundred million dollars.In Taiwan, in 2000, the market of benefit natural disposition bacterial classification has grown to 4,200,000,000 yuan, and the application form of product also increases year by year from fermented dairy prod, milk, ice-creams, candy, dietary supplements etc., and the object of application has more comprised adult, baby and various poultry, livestock.Can expect that thus there is a vast growing space in the market of beneficial natural disposition bacterial classification.
Up to now, numerous to the developmental research of milk-acid bacteria all over the world, wherein relevant for the acid resistance of lactobacterium strain and the disclosed patent of decreasing cholesterol ability with deliver document, mostly based on lactobacillus acidophilus strains, and mainly be to disclose cholate tolerance and acid resistance bacterial strain, or only emphasize reducing cholesterol ability and cholate tolerance part.The research of the cholate tolerance of at present relevant Bacterium lacticum is to emphasize that bacterial strain can contain growth under 0.3% GC (glycocholate), and acid resistance is then carried out the test of stomach juice-resistant state with the pH2 that comes across the gastric secretion initial stage.
Look into disclosed patent, Bacterium lacticum reduces the serum cholesterol ability about about 20%, and is delivering aspect the document, and the difference about 10～80% is arranged according to using method.
At US 4,839,281, mention that a kind of it can be grown in and contain in 0.15% cholate by the isolated Lactobacterium acidophilum GG of human feces (ATCC 53103) among the US 5,032,399, and its bacterium after cultivating 2 hours under the sour environment of pH1～2 to count extant number be 10 3CFU.
S.E.Gilliland and D.K.Walker are in J.Dairy Sci. (1990), report among the 73:905-911, be the Lactobacterium acidophilum ATCC 43121 (corresponding to CCRC 17064) in source and all can in containing 0.3% cholate, grow by pig, and have the serum cholesterol of reduction ability by the Lactobacterium acidophilum ATCC 4356 (corresponding to CCRC 10695) of human origin.
Again, people such as Danieloson are in J.Anim.Sci. (1989), that is reported among the 67:966-974 is studied and is become Nebraska Cultures by people such as pig isolated Lactobacterium acidophilum LA16 bacterial strain and Dr.khem M.Shahani, the Lactobacterium acidophilum DDS-1 strain isolated of the human origin of the patented product of Inc. (for the endogenous human bacterial strain) show and have the ability that reduces serum cholesterol (referring to Http:// www/nebraskacultures.com./benefits.html).
Usman and Hosono report among the 82:243-248 in J.Dairy Sci. (1999), and that the lactobacilli Lactobacillus gasseri that a strain is separated has is acidproof, bile tolerance and decreasing cholesterol ability.
Yoshio Saito and Jun Mizutani are at US 5,516,684 with US 5,707, two kinds of lactobacillus acidophilus strains are disclosed in 584, be Lactobacterium acidophilum FERM-P-14204 and Lactobacterium acidophilum FERM-P-14205, these bacterial strains can not represent going keying action, can not suppressing nutrient and absorb and can reduce cholesterol in blood and the liver of bile acide yet.
Lactobacterium strain with regard to the above-mentioned non-native source of carrying, if can separate by the native country and have local lactobacterium strain acidproof, that bile tolerance reaches characteristics such as good reduction serum cholesterol ability, should can guarantee its adaptability in compatriots' intestinal environment, local lactobacterium strain like this is as introduction or be added in the converted products, can guarantee that edible back effectively arrives in the intestines and the demand of settling down, and therefore improve the function of product.
Summary of the invention
So a first aspect of the present invention is to filter out to have lactobacillus separation strains (isolate) acidproof, that bile tolerance reaches multiple characteristics such as good reduction serum cholesterol ability from the human intestine's sample of native country.
The applicant originates as strains separation with native country, Taiwan healthy infants ight soil, with regard to stomach environment acid resistance, cholate tolerance and reducing cholesterol ability are screened isolated bacterial strain, and obtain the novel lactobacillus separation strains that 6 strains have these multiple characteristics, be Lactobacillus gasseri B21T1, B21T6, C21T1, X21B7 and B38T38, and acidophilus strain B 6T7, these strain isolateds have been preserved in " Biological resources are preserved and the research centre " (BCRC of Foodstuff Industrial and Development Inst. June 18 2002 Christian era, be called for short the production-goods center)] (No. 331,300 Xinzhu City food roads, Taiwan Province), preserving number is respectively CCRC 910195, CCRC 910196, CCRC910198, CCRC 910199, CCRC 910197 and CCRC 910194.These strain isolateds also have the regulation according to budapest treaty, be preserved in American type culture collection (ATCC June 21 2002 Christian era, P.O.Box 1549, Manassas, VA 20108, USA), preserving number is respectively PTA-4483, PTA-4484, PTA-4479, PTA-4480, PTA-4481 and PTA-4482.
Through with in the past strains lactobacillus acidophilus ATCC 43121, ATCC 4356 and DDS-1 relatively, novel lactobacillus separation strains of the present invention is proved has better reducing cholesterol ability.
A second aspect of the present invention provides a kind of go down to posterity composition of the mutant strain cultivating the offspring or derived by them and suitable vehicle of at least a foundation novel lactobacillus separation strains of the present invention or its that includes and is provided with preparing edible product such as beverage, cake, infant food, cultured milk, nutriment, animal-feed or the like.
A third aspect of the present invention provides the go down to posterity pharmaceutical composition of the mutant strain cultivating the offspring or derived by them of a kind of at least a foundation novel lactobacillus separation strains of the present invention that comprises useful living significant quantity or its, comes for being used for the treatment of and prevention of gastrointestinal and reduce serum cholesterol.
Description of drawings
The present invention is described in detail below in conjunction with drawings and Examples, thus the present invention in above-mentioned and other purpose and feature, can be by reference following description, the claims of enclosing and follow graphic and become more obvious, in the accompanying drawing:
Fig. 1 is for showing novel lactobacillus separation strains of the present invention and the comparison of lactobacterium strain on acid resistance in the past, wherein acid resistance is represented with Δ log (a-b), a is with the survivaling cell number of physiological saline (pH7) processing after 2 hours, and b is with the survivaling cell number of physiological saline (pH2) processing after 2 hours;
Fig. 2 is for showing novel lactobacillus separation strains of the present invention and the comparison of lactobacterium strain on the cholate tolerance in the past, wherein the cholate tolerance is represented with Δ log (c-d), c is with the survivaling cell number of MRS meat soup processing after 24 hours, and d is to be added with the survivaling cell number of MRS meat soup processing after 24 hours of 0.3% oxgall;
Fig. 3 is for showing novel lactobacillus separation strains of the present invention and lactobacterium strain in the past, and the growth fraction in the MRS meat soup that adds and do not add 0.3% oxgall;
Fig. 4 for the test that shows the present invention and carry out bacterial strain reducing cholesterol ability with reference to S.Razin etc., (1980), Biochimica Biophysica Acta, the phosphatidylcholine-cholesterol volume that 598:628-640 is prepared and the relation of cholesterol concentration.
Fig. 5 is the nucleotide sequence of the 16S rDNA that shows the isolated strain lactobacilli Lactobacillus gasseri B21T1 strain isolated of the present invention;
Fig. 6 show the present invention the nucleotide sequence of 16S rDNA of an isolated strain lactobacilli Lactobacillus gasseri B21T6 strain isolated;
Fig. 7 show the present invention the nucleotide sequence of 16S rDNA of an isolated strain lactobacilli Lactobacillus gasseri C21T1 strain isolated;
Fig. 8 show the present invention the nucleotide sequence of 16S rDNA of an isolated strain lactobacilli Lactobacillus gasseri X21B7 strain isolated;
Fig. 9 show the present invention the nucleotide sequence of 16S rDNA of an isolated strain lactobacilli Lactobacillus gasseri B38T38 strain isolated; And
Figure 10 show the present invention the 16S rDNA nucleotide sequence difference between an isolated strain acidophilus strain B 6T7 strain isolated and Lactobacterium acidophilum and the plant lactobacillus (Lactobacillus plantarum), wherein " to add the collimation mark person of showing " is the place, nucleotide difference address between this B6T7 strain isolated and the Lactobacterium acidophilum, and " with * sign person " is the place, nucleotide difference address between this B6T7 strain isolated and the plant lactobacillus.
Embodiment
For obtaining meeting the local Bacterium lacticum of native country habit with the intestinal environment of guaranteeing to adapt to the native, the applicant is with the ight soil that inhabits geographic 1 to 6 years old healthy infants of the Hsin-chu screening source as the expectation bacterial strain, use and a series ofly screen the doubtful bacterial strain of Bacterium lacticum (suspected strains), and from 43 samples, find the doubtful bacterial strain of 400 strains in 828 strain isolateds of gained with Rogosa agar based selective substratum.These doubtful bacterial strains are further tested whether have said characteristic in above-mentioned " summary of the invention " joint, just:
1. to the stability of acid,
2. to the stability of cholate, and
3. the ability that has reducing cholesterol,
And these doubtful bacterial strains are brought with open bacterial strain make comparisons with regard to above-mentioned characteristic, meet above-mentioned asking and the novel lactobacillus separation strains of tool good capacity and filter out 6 strains.
Resulting 6 strain strain isolateds further use the API identification systems, microorganism computer identification systems (Micro-IS System) wait with 16S rDNA sequential analysis and make identification of strains, and according to qualification result, this 6 strain strain isolated is classified called after Lactobacillus gasseri B21T1 respectively, B21T6, C21T1, X21B7 and B38T38, and acidophilus strain B 6T7, these strain isolateds are preserved in Foodstuff Industrial and Development Inst. (No. 331,300 Xinzhu City food roads, Taiwan Province) June 18 2002 Christian era, and preserving number is respectively CCRC 910195, CCRC 910196, CCRC 910198, CCRC 910199, CCRC 910197 and CCRC 910194.These bacterial strains also have the regulation according to budapest treaty, be preserved in American type culture collection (ATCC June 21 2002 Christian era, P.O.Box 1549, Manassas, VA 20108, USA), preserving number is respectively PTA-4483, PTA-4484, PTA-4479, PTA-4480, PTA-4481 and PTA-4482.
Based on above-mentioned advantageous feature, be suitable for as a kind of probiotic bacterium according to novel lactobacillus separation strains of the present invention.For example, these strain isolateds can be formulated in the edibility material of broad variety, and the edibility material comprises: fluid dairy products (milk, enriching milk), fermented dairy prod (Yogurt, yogurt, freezing yogurt, lactacidase fermenting beverage), milk powder, ice-creams, cheese, cheese, soymilk and beverages such as fermenting bean milk, fruit and vegetable juice, fruit juice and sports beverages, dessert, candy, infant food, dietary product, animal-feed, dietary supplement etc.The bacteria containing amount of every kind of product can be every g or every milliliter and has an appointment 10 6To 10 9Colony-forming unit (CFU).
Significantly, those skilled in the art can understand, novel lactobacillus separation strains of the present invention can be taken as the food added ingredients, by in the past method and when feedstock production, be added, or do not participate in fermentation and interpolation after fermenting process, to be formulated into the absorption that any suitable form supplies the mankind and non-human animal.Preferably, novel lactobacillus separation strains of the present invention can be formulated in the edibility material individually or with at least a other beneficial natural disposition biology jointly, described other beneficial natural disposition biology comprises: the lactobacillus species, as Lactobacterium acidophilum, lactobacillus delbruckii breast subspecies (Lactobacillus lactis), short lactobacillus (Lactobacillus brevis), lactobacterium casei (Lactobacillus casei), plant lactobacillus, lactobacillus salivarius (Lactobacillus salivarius), bifidobacterium (Lactobacillus bifidus), lactobacillus bulgaricus (Lactobacillusbulgaricus), bacillus caucasicus (Lactobacillus causasicus) and lactobacillus rhamnosus (Lactobacillus rhamnosus); The streptococcus species are as thermophilus streptococcus, streptococcus acidi lactici (Streptococcus lactis); Yeast is as Candida species Candida Kefyr, sieve rib yeast (Saccharomyces florentinus) not; Or the combination of these bacterial classifications.
Other has a kind of application form that novel lactobacillus separation strains of the present invention itself or the said products of containing this bacterial strain are made lyophilize powder or spray-dried powders, contains 10 approximately and make in each product 8To 10 9Above live lactobacillus sp thalline.This product is added with yeast powder, carbohydrate or other weighting agent, can make lozenge or capsule form again, for example contain the agent of digestion drug for controlling intestinal function or the instant thalline powder that brews food and supply directly to eat of Bacterium lacticum.
In addition, the present invention also expects above-mentioned novel lactobacillus separation strains used as the non-expectation of control enteric microorganism jointly individually or with other active constituent collect the medicine of giving birth in mammiferous digestive tube, to alleviate this non-uncomfortable stomach symptom of expecting that enteric microorganism is caused.Said composition can be formulated into solution, emulsion, powder, lozenge, capsule or supply to be used for other suitable form of oral administration.
Again, based on the reducing cholesterol ability that novel lactobacillus separation strains of the present invention had, the present invention expects that also these lactobacillus separation strains are used to prepare in order to the protective foods of reduction serum cholesterol and the use of OTC (over-the-counter) pharmaceuticals.
With regard to the above, it in the technological concept category of the present invention is that those have the bacterial strain with the bacterial characteristics of novel lactobacillus separation strains of the present invention that expection drops on, the offspring is cultivated in going down to posterity of novel lactobacillus separation strains of the present invention, or the mutant strain that is derived by novel lactobacillus separation strains of the present invention.
As used herein, term " mutant strain " means the tool genetic composition of a kind of institute has at least one nucleotide difference (for example, via due to replacement, insertion or the deletion of Nucleotide) with respect to reference strain or parental plant bacterial strain.Except that natural sudden change, mutant strain of the present invention can generate by several different methods.For example, these mutant strains can for example obtain by chemical mutation agent, transposon or radiation irradiation by the random mutagenesis of parental plant.In addition, mutant strain of the present invention can comprise the recombinant type nucleotide sequence.For instance, mutant strain can be the bacterial strain that has extra nucleotide sequence (for example being inserted into the intracellular sequence of parental plant through conversion, transduction or other modes).The poly-victory peptide that this extra nucleotide sequence can be encoded into normally or show conditionally.Other selects ground, and this extra nucleotide sequence can be encoded into one section nucleotide sequence that can change stechiology, for example antisense, ribozyme or other nucleotide sequence.In another example, the nucleic acid of insertion is inserted in the endogenous gene, and changes the function of (for example strengthening or destruction) this endogenous gene.For instance, the nucleic acid of insertion can be a kind of rejecting building that endogenous gene is deactivated (knockout construct); Or be a kind of artificial enhanser or promotor that can increase the Transcription of endogenous gene.
The present invention puts up with following embodiment and is described further, but will be appreciated that, these embodiment only as illustrating use, do not limit and should not be interpreted as enforcement of the present invention.
Embodiment 1The separation of lactobacillus separation strains and screening
Materials and methods:
One, substratum and diluent:
1. screening culture medium:
(A) show the tomato juice nutrient agar
Casein enzymatic hydrolysate (Sigrna, Louis, Mo, USA) 10g
Skimming milk 10g
Tomato juice 400ml
Agar 12g
Distilled water adds to 1L
(B) Rogosa nutrient agar
Rogosa agar (Merck, Darmstadt, Germany) 74.5g
Distilled water 1L
After the heated by microwave dissolving, to be cooled to 55 ℃, adjust the pH value to pH5.5 with acetic acid
(C) Bacterium lacticum is selected substratum (LBS)
Rogosa agar 8.4g
Tomato juice 40ml
Distilled water 60ml
After the heated by microwave dissolving, to be cooled to 55 ℃, adjust the pH value to pH5.5 with acetic acid
(D) Rogosa+X-glu nutrient agar
(MO USA) is added in the 0.2M Tris damping fluid (pH8.5) of 100ml for Sigma, Louis, and utilizes the sonic oscillation dissolving with 5-bromo-4-chloro-3-indyl 1-β-D-glycopyranoside (X-glu) of 80mg.The formed solution of 10ml is added in 55 ℃ of Rogosa agar of 190ml, promptly becomes and contain the Rogosa+X-glu agar that the X-glu ultimate density is 40 μ g/mL.
2. bacterium activation medium:
(A) Bacto Bacterium lacticum MRS meat soup (Difco Laboratories, Detroit, MI, USA)
(B) MRS agar: Bacto Bacterium lacticum MRS meat soup adds bacteria Agr (Agarbacteriological) (Scharlau Chemie S.A., Barcelona, Spain, European Union's manufacturing) (15g/L) again.
3. ribose usability substratum:
The composition of basic medium:
Bacto
Figure A20051006525500171
No. 3 10g of peptone
Bacto yeast extract 5g
(manufacturers of above-mentioned two kinds of components is Difco Laboratories, Detroit, and MI, USA)
Tween 80 1g
Ammonium hydrogen citrate 2g
Sodium acetate 5g
Sal epsom 0.1g
Manganous sulfate 0.05g
Dipotassium hydrogen phosphate 2g
Dichlorophenol sulfonphthalein 0.05%
Distilled water 1L
Utilize HCl that the pH value is adjusted to pH6.3
The basic medium branch for preparing is filled in the test tube, and every pipe contains 4.5ml, after 15 minutes, adds 10% ribose solution after 0.5ml sterilizes after filtration through 121 ℃ of sterilizations again.
4. diluent
5.1% peptone water
(BACTO TM?Peptone，Difco?Laboratories，Detroit，MI，USA)
Two, separate and screen the schedule of operation of bacterial strain:
1. get 1 to 6 years old child's ight soil stage casing, take out the sample of about one little section finger size with long bamboo let, and sample is added to one includes in the test tube of 9ml diluent (0.1% peptone water), and make inspection liquid once 10 times of dilutions, to leave standstill several minutes after the full and uniform stirring of the sample in the test tube pipe, so that contained microorganism is released in the diluent in the sample;
2. take out the above-mentioned dilution inspection liquid of 1ml, and will dilute and examine liquid and be incorporated in another test tube that includes the 9ml diluent, repeat serial dilution according to this until reaching 10 4-doubly dilution.
3. the serial dilution with regard to being done in the above-mentioned steps 2 takes out 0.2ml respectively through 10 2-, 10 3-, 10 4The inspection liquid of-times dilution, and the inspection liquid that will will take out individually places on the various screening culture medium (Rogosa agar, tomato juice agar, Bacterium lacticum are selected substratum and Rogosa+X-glu agar), after being smeared evenly with a L type glass rod, place an anaerobism incubator that is set under 37 ℃ the temperature (to include mixed gas: 5%H screening culture medium 2, 10%CO 2With 85%N 2) the interior cultivation 2 to 4 days;
4. select and on Rogosa+X-glu agar, present blueness and be the bacillus bacterium colony of Immobility (non-mobile) through microscopy, or outward appearance is translucent and also is the bacillus bacterium colony of Immobility through microscopy on other screening culture medium, choosing bacterium and the bacterium that will choose with bamboo let inserts one and contains in the test tube of pipe in a MRS meat soup and the fermentation, cultivation is after 1 to 2 day down in 37 ℃, and collection is grown but do not produced gas and be shown as the male bacterial strain through gramstaining.
5. with regard to the collected bacterial strain of above-mentioned steps 4, after twice of MRS meat soup activation, according to following described carry out growth temperature and the test of ribose usability:
(i) 1% inoculum is seeded in the MRS meat soup, and this substratum placed to leave standstill under 15 ℃ to cultivate last 14 days, if thalli growth is arranged, represent that then this bacterial strain can be in 15 ℃ of growths, and, represent that then this bacterial strain can't be in 15 ℃ of growths down if still do not have thalli growth in the 14th day;
(ii) 1% inoculum is seeded in the MRS meat soup, and this substratum placed to leave standstill under 45 ℃ to cultivate lasts 2 days,, represent that then this bacterial strain can be in 45 ℃ of growths, and if no thalli growth, represent that then this bacterial strain can't grow down in 45 ℃ if thalli growth is arranged;
(iii) 1% inoculum is seeded to a ribose usability substratum, and leaves standstill under 37 ℃ and cultivate a couple of days, if nutrient solution is by the purple stain Huang, the expression thalline can utilize the ribose growth and produce acid; If nutrient solution still be purple after 7 days, continue to leave standstill cultivation again, if the 14th day nutrient solution yellowing not yet represents that then this bacterial strain can't utilize the ribose growth and produce sour.
Three, result:
In this embodiment, the sieve bacterium culture medium of relevant Bacterium lacticum is mainly based on Rogosa agar, and what have then adds with tomato juice or X-glu again.Tomato juice contains and enriches vitamin b6 usp B group, and helps the growth of Bacterium lacticum, and the acetic acid in the Rogosa agar is except reducing pH value with antibacterial, and the acetate ion of high density also has the effect of inhibition microorganism growth simultaneously.
The outward appearance that Lactobacterium acidophilum is grown on the Rogosa agar is to present white colony, and is difficult for differentiating with other Bacterium lacticum.If in Rogosa agar, add X-glu, the outward appearance of Lactobacterium acidophilum bacterium colony promptly can become blueness, this is the β-D-glycosidic link that can interrupt X-glu because Lactobacterium acidophilum has β-D-glycosidase activity, and produce blue luminophor (chromogene), and then make the Lactobacterium acidophilum bacterium colony present blueness.But the microorganism that has this enzyme in cell so still must make further microscopy, gramstaining and carry out the test of Physiology and biochemistries such as above-mentioned carbohydrate and growth temperature, is chosen the doubtful bacterial strain with desirable properties not in minority again.
The applicant is from inhabit geographic 1 to the 6 years old healthy infants ight soil in Hsinchu City, Taiwan Province, 43 samples have been collected altogether, each sample is applied in each selective medium after dilution, after bacterial strain grows, from selective medium, choose the color bacterium colony different again and carry out microscopy,, carry out gramstaining again if the microscopy result is a bacillus with kenel, pick out gram-positive bacillus, and will be called strain isolated.These strain isolateds are placed the MRS meat soup that contains pipe in the fermentation, select the bacterial strain that can not produce gas again and carry out growth temperature and the test of ribose usability.If the bacterial strain of test can be 45 ℃ of down growths but can not be 15 ℃ of growths down, and can not utilize ribose then to preserve as the bacterial strain of the required carbon source of growth, be called doubtful bacterial strain in collected bacterial strain of this stage.
Referring to table 1, the applicant amounts in 43 collected samples and picks out 828 strain strain isolateds, and the doubtful bacterial strain of 400 strains is wherein arranged.About the strain number mode, act B6T7 is an example, and B is meant kinds of culture medium (LBS), and 6 are meant the sample source label, and T is meant bacterial strain color (appearance transparent), and 7 are meant the flowing water label of bacterium colony.
The sample that table 1. is collected *Label and strain isolated number thereof
The sample label The strain isolated number Doubtful bacterial strain The sample label The strain isolated number Doubtful bacterial strain
????1 ????40 ????0 ????23 ????3 ????0
????2 ????0 ????0 ????24 ????0 ????0
????3 ????0 ????0 ????25 ????100 ????100
????4 ????26 ????0 ????26 ????0 ????0
????5 ????45 ????0 ????27 ????0 ????0
????6 ** ????68 ????55 ????28 ????0 ????0
????7 ????0 ????0 ????29 ????0 ????0
????8 ????0 ????0 ????30 ????60 ????0
????9 ????0 ????0 ????31 ????0 ????0
????10 ????0 ????0 ????32 ????0 ????0
????11 ????0 ????0 ????33 ????0 ????0
????12 ????60 ????60 ????34 ????0 ????0
????13 ????0 ????0 ????35 ????0 ????0
????14 ????0 ????0 ????36 ????139 ????115
????15 ????0 ????0 ????37 ????0 ????0
????16 ????0 ????0 ????38 ** ????120 ????40
????17 ????0 ????0 ????39 ????0 ????0
????18 ????0 ????0 ????40 ????0 ????0
????19 ????0 ????0 ????42 ????60 ????0
????20 ????14 ????0 ????42 ????0 ????0
????21 ** ????30 ????30 ????43 ????60 ????0
????22 ????3 ????0 Sum ????823 ????400
*: sample source: 1 to 6 years old child's of Hsinchu City, Taiwan Province ight soil
*: the sample source numbering of strain isolated of the present invention
These doubtful bacterial strains then carry out the described acid resistance of following embodiment, cholate tolerance test and reducing cholesterol ability test, wherein 6 strains show the strain isolated with superperformance through test, be B21T1, B21T6, C21T1, X21B7, B38T38 and B6T7, being brought following bacterial strain in the past carries out the comparison of these character:
1. Lactobacterium acidophilum CCRC 17064 (, separate the source and be pig) corresponding to ATCC 43121, this bacterium is Bacterium lacticum (S.E.Gilliland etc., (1985), Appl.Environ.Microbiol., the 49:377-381 that tool reduces the serum cholesterol ability; S.E.Gilliland and D.K.Walker (1990), J.Dairy Sci., 73:905-911; F.A.M.Kalver and R.van der Meer (1993), Appl.Environ.Microbiol, 59:1120-1124; D.O.Noh etc., (1997), J.Dairy Sci., 82:3107-3113);
2. Lactobacterium acidophilum CCRC 10695 T(, separating the source) for human corresponding to ATCC 4356, this bacterium be from human body the Lactobacterium acidophilum type strain (D.K.Walker and S.E.Gilliland (1993), J.Dairy Sci., 76:956-961),
3. Lactobacterium acidophilum DDS-1 (separate source for human), this bacterium be present market product on sale (Nebraska Cultures, Inc.);
4. Lactobacterium acidophilum CCRC 14065[is corresponding to CSCO 2401, commercial can be available from Commonwealth Scientific Industrial Research Organization (CSIRO), Canberra, Austrilia]; And
5. the preservation thing Lactobacillus gasseri CCRC 14619 of a lactobacilli T(, separating the source) (Int.J.Syst.Bacteriol. (1980), 30,601 for human corresponding to ATCC 33323; E.Lauer and O.Kandler, Bakteriol.Parasitenkd.Infektionskr.Hyg.Abt.1Orig.Reihe C, 1980,1,75-78).
Embodiment 2Acid resisting test
One, experimental implementation program:
With reference to J.E.Holcombe etc., (1991), Cult.Dairy Prod.J., 26 (3): 4-5 and authorize disclosed method among people's such as Y.S.Yang the US 5,711,977, with the test of depositing viable count in neutral (pH7) and the imitative gastric acid environment (pH2).
After each test strain activates twice with MRS meat soup, get the centrifugal (3000rpm of its bacterium liquid, 10 minutes), after the removal supernatant liquor, add the normal saline solution solution (0.85%NaCl of 1ml, pH7), elder generation stirs thalline with bamboo let diffusing, and vibration is evenly got 0.5ml places pH7 and pH2 through the diffusing bacterium liquid that floats normal saline solution solution respectively then again, after in 37 ℃ constant incubator, leaving standstill 2 hours, survey it respectively and deposit viable count.The numerical value of Δ log bacterium number [=log (the bacterium number under pH7-under pH2 bacterium number)] is more little, is considered as the preferable bacterial strain of acid resistance.
Two, result:
Most microorganism does not all have tolerance in sour environment.Though milk-acid bacteria itself is an acid-producing bacteria, its growing environment can only reach pH3.2 to 4.5, and therefore, the environment (pH2.0 to 3.2) of extremely low pH also becomes the principal element that influences its survival under one's belt.Hydrochloric acid in gastric juice pH value can have the difference of pH1.5～4.5 with gastric content entry time and kind, on average last 2 hours, so adopt pH2 during test as representative, and because of acidic substance similar to hydrochloric acid, die with the normal saline solution (0.85%NaCl/0.01N HCl system) that through hydrochloric acid adjustment pH value is 2, after handling 2 hours under 37 ℃, test the bacterium number of survival, and make comparisons with the control group of pH7.
Test-results shows that through different pH values, 37 ℃/2h is the best (Fig. 1) with Lactobacterium acidophilum CCRC 17064 acid resistances after handling, and accepts bacterium number 1.9 log values of reduction of processing, and Lactobacterium acidophilum CCRC 10695 TWith Lactobacillus gasseri CCRC 14619 TAcid resistance relatively poor, the bacterium number reduces by 4.3 and 4.4 log values respectively.
The present invention is close with the acid resistance of CCRC 14065 in embodiment 1 resulting new strain isolated B21T1 (being classified as Lactobacillus gasseri after bacterial characteristics is identified, shown in following embodiment), and the bacterium number has reduced by 3.9 log values.The acid resistance of other in the present invention 5 strain isolateds (B21T6, C21T1, X21B7, B38T38 and B6T7) is close, and all reaches Δ log bacterium number less than 4 acid resistance screening pointer (referring to people's such as Y.S.Yang US 5,711,977).
Embodiment 3. the cholate tolerance test
One, experimental implementation program:
Each test strain with twice of MRS meat soup activation after, get respectively again that 1% inoculum is inoculated in 10ml MRS meat soup and 10mL contains in the MRS meat soup (MRSO) of 0.3% oxgall, in 37 ℃ cultivate 24 hours after, measure cell concentration (OD respectively with spectrophotometer 660).In addition, also count its remaining bacterium number, Δ log bacterium numerical value=log (the bacterium number of the bacterium number-MRSO meat soup of MRS meat soup) wherein, and Δ log bacterium numerical value is more little, the cholate tolerance of expression test strain is good more.
Two, result:
Milk-acid bacteria is after cholate is handled, and the survival rate between different strain has very big-difference, and therefore, the lactobacillus strain that screening has a cholate tolerance is being selected its importance is arranged on the probiotic bacterium (S.E.Gilliland etc., (1984), J.Dairy Sci., 67:3045-3051 for use; P.Marteau etc., (1997), J.Dairy Sci., 80:1031-1037).In previous research, oxgall is commonly used and comes bacterium in the human intestines of selective cultivation in substratum, so its effectiveness should be similar to human cholate very much and mean concns is 0.3% (w/v) (S.E.Gilliland and D.K.Walker (1990), J.Dairy Sci., 73:905-911; D.K.Walker and S.E.Gilliland (1993), J.Dairy Sci., 76:956-961).
Test-results shows, removes CCRC 10695 T, CCRC 14619 T, outside the CCRC 14065, no matter each test strain is containing or do not containing in the substratum of 0.3% oxgall, measured OD 660As if value (cell concentration) is all at (table 2) more than 2, and this shows the growth of 0.3% oxgall for test strain, and influence is little.
Table 2. test strain culture is cultivated in the MRS meat soup that contains or do not contain 0.3% oxgall
24 hours growth fraction
Culture Cell concentration (OD 660Value)
MRS meat soup MRS meat soup+0.3% oxgall
????B6T7 ????2.49 ????2.74
????C21T1 ????2.44 ????2.56
????B38T38 ????2.45 ????2.03
????B21T1 ????2.57 ????2.53
????B21T6 ????2.62 ????2.29
????X21B7 ????2.69 ????2.50
?CCRC?17064 ????2.91 ????2.37
?CCRC?10695 T ????2.35 ????1.09
?CCRC?14065 ????2.55 ????1.79
?CCRC?14619 T ????2.96 ????1.58
????DDS-1 ????2.87 ????2.72
But, it is also noted that these two bacterial strains of B6T7 of the present invention and C21T1 are higher than MRS meat soup at the cell concentration of MRSO meat soup, this demonstration has other interference factor formation and has influence on extinction.Therefore, further viewing test bacterial strain is cultivated the viable count of depositing after 24 hours in two groups of substratum.
Referring to Fig. 2, reduce as can be known by observed bacterium number, CCRC 14619 TThe cholate tolerance relatively poor, secondly be CCRC 10695 TAnd B38T38 bacterial strain of the present invention.And cholate tolerance the best is strain isolated B6T7 of the present invention, and the cholate tolerance of all the other each bacterial strains is close, promptly cultivates after 24 hours in the MRS meat soup that contains 0.3% oxgall, and it dies the bacterium number all in 1 to 2 log value.
In addition, observe the growth curve of part bacterial strain in containing the MRS meat soup of 0.3% oxgall, represent growth velocity with the growth curve slope, slope is big more, and the expression growth velocity is fast more, and promptly the cholate tolerance is preferable.As shown in Figure 3, the growth velocity of CCRC 17064 is the fastest, is strain isolated X21B7 of the present invention and B21T1 secondly, and CCRC 14065, CCRC 10695 TAnd the growth of DDS-1 is the slowest.
Though by the slightly little difference of result of different observed pattern gained, by these tests as can be known, the Bacterium lacticum that the present invention filtered out all has acid resistance and cholate tolerance.
Embodiment 4. the analysis of bacterial strain reducing cholesterol ability
One, experimental implementation program:
(A) cholesterol source
Test about bacterial strain reducing cholesterol ability, PPLO (BACT PPLO SERUM FRACTION) is adopted in the used cholesterol source of document and disclosed patent application in the past mostly, but this product has stopped producing at present, therefore, in present embodiment, the applicant is with reference to Huang, C.and T.E.Thomopson (1974), Methods Enzymol.32:485-489 and S.Razin etc., (1980) Biochimica Biophysica Acta, phosphatidylcholine-cholesterol the preparation method who is disclosed among the 598:628-640, adopt horse serum and phosphatidylcholine-cholesterol to originate, and prepare an equally distributed phosphatidylcholine-cholesterol solution volume and contained cholesterol concentration is a positive correlation curved line relation (Fig. 4) as cholesterol.
1. horse serum
Horse serum is soluble in water, be that the filtering membrane of 0.45 μ m is filtered the back and deposits in-20 ℃ of refrigerators with the aperture.
2. phosphatidylcholine-cholesterol [egg Yelkin TTS (Egg Lecithin)+cholesterol]
According to S.Razin etc., (1980), Biochimica Biophysica Acta, 598:628-640, egg Yelkin TTS and cholesterol are added in the frosted plug bottle of a rotary evaporation in vacuo instrument (EYELA), with the chloroform of capacity with its uniform dissolution, dry up with nitrogen again, so that form a uniform thin film on cholesterol and egg Yelkin TTS uniform mixing and the glass wall in frosted plug bottle, then covered molten (re-dissolved) with the 0.4M sucrose solution, be blown into nitrogen in this frosted plug bottle and seal up bottle cap, coat with aluminium foil again, to reduce the probability of lipid oxidation.
After shaking this frosted plug bottle under 4 ℃ with the water-bath type ultrasonator and lasting 15 minutes through sealing, be the filtering membrane 2 times of 0.22 μ m with above-mentioned mixing solutions pressurization by tool one aperture, be stored in immediately in 4 ℃ of refrigerators (must in 3 days, use).
(B) test group
Test (1): test strain is got 1% inoculum and is inoculated among the 10ml MRSS (including 8.8ml MRS and 1.2ml cholesterol solution and 0.3% oxgall) after activating twice with MRS meat soup, and in 37 ℃ anaerobism incubator (10%CO 2And 90%N 2) in cultivate and to last 24 hours, then according to described in following (C), supernatant samples and the cell precipitation ball sample of collecting the culture that inoculation has test strain to measure use for cholesterol level.
Test (2): bacterial strain with twice of MRS meat soup activation after, get 1% inoculum and be inoculated among the 10mLMRSO, cultivate down at 37 ℃ and last 20 to 22 hours, take out 1% inoculum again to be inoculated among the 10mL MRSS, and be 37 ℃ anaerobism incubator (10%CO in one 2And 90%N 2) in cultivate and to last 24 hours, then according to described in following (C), supernatant samples and the cell precipitation ball sample of collecting the culture that inoculation has test strain to measure use for cholesterol level.
(C) mensuration of cholesterol level
The mensuration of cholesterol level is to carry out (L.L.Rudel and M.D.Morris (1973), Notes on Methodology, 14:364-366 according to terephthalaldehyde (o-phthalaldehyde) method; S.E.Gilliland etc., (1985), Appl.Environ.Microbiol, 49:377-381).
With regard to above-mentioned test (1) and test (2), respectively get the test bacterium liquid of 1ml, after centrifugal 10 minutes, the 0.5ml supernatant liquor is placed a test tube with 12000rpm, this is the supernatant samples that inoculation has the culture of examination bacterial strain.
With regard to above-mentioned test (1) and test (2), respectively get the test bacterium liquid of 1ml, with 3000rpm after centrifugal 10 minutes, remove supernatant liquor, after formed cell granule (cell-pellet) covers molten with 10ml MRSO meat soup and mixes, take out 0.5ml solution and place a test tube, this is the cell precipitation ball sample that inoculation has the test strain culture.
Each specimen is added 95% ethanol of 3ml and vibration evenly, adds 2ml 50%KOH vibration more evenly, then in 60 ℃ of water-baths heating 10 minutes down, places under the room temperature again and cools off.Also vibration is even to be added the 5ml hexane through refrigerative solution, adds the H of 3ml again 2O.Formed mixture evenly left standstill under room temperature 15 minutes the back in vibration, took out the hexane layer of 2.7ml and moved on to another in vitro, utilized nitrogen that hexane is evaporated under 60 ℃.Solid after the evaporation is added into the terephthalaldehyde reagent solution (0.5mg terephthalaldehyde/1ml acetate) of 4ml, be statically placed under the room temperature after the vibration evenly and last 10 minutes, add the 2ml vitriol oil again, be statically placed under the room temperature after the vibration evenly and last 10 minutes, measure OD with spectrophotometer again 550Value.
(D) assessment of bacterial strain reducing cholesterol ability
Assess the ability of bacterial strain reducing cholesterol according to following formula:
A＝100-[(B/C)×100]
The reduced rate of A=cholesterol (%)
The B=inoculation has the interior cholesterol level (mg) of supernatant samples of the culture of test strain
C=does not inoculate the interior cholesterol level (control group) of supernatant samples of test strain culture
If the A value reaches more than 80%, promptly be considered as having obvious decline cholesterol ability.
(E) assessment of the cholesterol assimilation of test strain
A′＝100-[(B′+C′)×100]
The assimilation of A '=cholesterol (%)
B '=inoculation has the interior cholesterol level (%) of supernatant samples of test strain culture
C '=inoculation has the interior cholesterol level (%) of cell precipitation ball sample of test strain culture
If A ' value reaches more than 15%, promptly be considered as the obvious cholesterol assimilative capacity of tool.
Two, result:
The ability of I, bacterial strain reducing cholesterol
Comparison test (1) (also i.e. growth in MRS meat soup) and test (2) (also i.e. growth in MRSO meat soup), difference between the two is that the test strain of test in (2) has to be seeded to earlier in the MRSO meat soup and cultivated 20 to 22 hours, getting 1% inoculum again is seeded among the 10ml MRSS, this purpose is to be to screen again bacterial strain whether good cholate patience is arranged, and also can screen the bacterial strain with decreasing cholesterol ability simultaneously.
With the test-results demonstration (table 3) of horse serum as the cholesterol source, in the past Lactobacterium acidophilum CCRC 17064 and DDS-1 equally also have positive effect in this test, and the reducing cholesterol ability reaches 88 to 98%.And CCRC 14065, CCRC 10695 TWith CCRC 14619 TThe decreasing cholesterol ability then not obvious, this may be that the growth in MRSO meat soup is relatively poor because the cholate patience of these bacterial strains is relatively poor, so that viewed reducing cholesterol ability only reaches 30 to 43%.
And strain isolated B6T7, C21T1, B21T1, B21T6, B38T38 and the X21B7 of gained of the present invention are in two groups of tests, the ability of reducing cholesterol all reaches 91 to more than 99%, a little more than CCRC 17064 and DDS-1 had, also has the ability of good reduction serum cholesterol and demonstrate the bacterial strain that the present invention screens.
Table 3. test strain reducing cholesterol (horse serum model) aThe assessment of ability
Culture In MRS meat soup bInterior growth In MRSO meat soup cInterior growth
Cholesterol d??(μg/ml) Cholesterol reduced rate (%) Cholesterol d(μg/ml) Cholesterol reduced rate (%)
????B6T7 ????C21T1 ????B38T38 ????B21T1 ????B21T6 ????X21B7 ????CCRC?17064 ????CCRC?10695 ????CCRC?14065 ????CCRC?14619 ????DDS-1 ????Control ????6.71 ????0.69 ????2.24 ????3.10 ????2.84 ????1.34 ????1.12 ????8.00 ????11.70 ????2.84 ????5.94 ????86.06 ????92.2 ????99.2 ????97.4 ????96.6 ????96.7 ????98.4 ????98.7 ????90.7 ????86.4 ????96.7 ????93.1 ????- ????7.14 ????2.41 ????7.57 ????0.34 ????2.24 ????3.27 ????9.81 ????56.97 ????59.90 ????49.14 ????5.16 ????86.06 ????92.7 ????97.2 ????91.2 ????99.6 ????97.4 ????96.2 ????88.4 ????33.8 ????30.4 ????42.9 ????94.0 ????-
a: the MRS meat soup that is supplemented with 0.3% oxgall and 12% horse serum.
b: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRS meat soup, and cultivate under 37 ℃ and last 24 hours.
c: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRSO meat soup (MRS meat soup adds 0.3% oxgall), and cultivate under 37 ℃ and last 24 hours.
d: the initial cholesterol level in the meat soup is 86.06 μ g/ml.
Referring to table 4, compare the bacterial strain that has the reducing cholesterol ability in the horse serum test, change when originating as cholesterol with phosphatidylcholine-cholesterol, the cholesterol reduced rate (%) that is found to each bacterial strain has downtrending, this shows that lactobacterium strain can change for the reduction ability of the cholesterol of different kenels, wherein CCRC 17064 is 11 to 29% for the reduced rate of phosphatidylcholine-cholesterol, and the resulting bacterial strain of the present invention is 23 to 48% for the reduced rate of phosphatidylcholine-cholesterol, obviously be high than CCRC 17064, but a little less than DDS-1 had 50%.
Table 4. test strain reducing cholesterol (phosphatidylcholine-cholesterol model) aThe assessment of ability
Culture In MRS meat soup bInterior growth In MRSO meat soup cInterior growth
Cholesterol d??(μg/ml) Cholesterol reduced rate (%) Cholesterol d??(μg/ml) Cholesterol reduced rate (%)
????B6T7 ????C21T1 ????B38T38 ????B21T1 ????B21T6 ????X21B7 ????CCRC?17064 ????DDS-1 ????Control ????45.05 ????30.71 ????33.75 ????32.34 ????35.43 ????35.11 ????52.66 ????29.24 ????59.22 ????23.92 ????48.15 ????43.01 ????45.39 ????40.16 ????40.71 ????11.07 ????50.63 ????- ????33.70 ????43.59 ????35.16 ????32.83 ????36.25 ????39.29 ????42.01 ????29.46 ????59.22 ????43.10 ????26.40 ????40.62 ????44.57 ????38.79 ????33.65 ????29.06 ????50.26 ????-
a: the MRS meat soup that is supplemented with 0.3% oxgall and 12% phosphatidylcholine-cholesterol.
b: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRS meat soup, and cultivate under 37 ℃ and last 24 hours.
c: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRSO meat soup (MRS meat soup adds 0.3% oxgall), and cultivate under 37 ℃ and last 24 hours.
d: the initial cholesterol level in the meat soup is 59.22 μ g/ml.
II, test strain are to the assimilation of cholesterol
The mechanism of relevant milk-acid bacteria reducing cholesterol mainly is to utilize cholesterol and go bonded cholate (deconjugated bile salt) to produce co-precipitation and cholesterol got rid of (F.A.M.Kalver and R.van der Meer (1993) are with aforementioned; M.M.Brashears and S.E.Gililland (1997) are with aforementioned), and bacterial strain itself to the assimilation of cholesterol (Gilliland etc., (1985) are with aforementioned; D.O.Noh etc., (1997); With aforementioned; And open and Luo Dengren (1998), with aforementioned).
Ginseng is shown in Table 5, and can find out that CCRC 17064, resulting 6 strain isolateds of the present invention and DDS-1 all have assimilation to cholesterol, but different strains is slightly variant for the assimilative capacity of cholesterol, and viewed assimilation ratio is about about 11 to 40%.And CCRC 14065 and CCRC 10695 TAnd CCRC 14619 TThe cholesterol assimilative capacity then not obvious.
The cholesterol of table 5. test strain aThe assessment of assimilation ratio
Culture MRS meat soup bInterior growth In MRSO meat soup cInterior growth
Cholesterol in the exhausted broth culture d(％) Cholesterol (%) in the cell precipitation ball The cholesterol that assimilates (%) Cholesterol (%) in the exhausted broth culture Cholesterol (%) in the cell precipitation ball The cholesterol that assimilates (%)
????B6T7 ????C21T1 ????B38T38 ????B21T1 ????B21T6 ????X21B7 ????CCRC?17064 ????CCRC?10695 ????CCRC?14065 ????CCRC?14619 ????DDS-1 ????7.8 ????0.8 ????2.6 ????3.4 ????3.3 ????1.6 ????1.3 ????9.3 ????13.6 ????3.3 ????6.9 ????51.4 ????86.0 ????79.1 ????81.7 ????67.2 ????67.0 ????68.5 ????71.7 ????82.0 ????80.6 ????78.5 ????40.8 ????13.2 ????18.3 ????14.9 ????29.5 ????31.4 ????30.2 ????19.0 ????4.4 ????16.1 ????14.6 ????7.3 ????2.8 ????8.8 ????0.4 ????2.6 ????3.8 ????11.6 ????66.2 ????69.6 ????57.1 ????6.0 ????74.1 ????72.3 ????83.7 ????88.4 ????81.4 ????71.3 ????72.9 ????33.4 ????27.8 ????42.7 ????81.3 ????18.6 ????24.9 ????7.5 ????11.2 ????16.0 ????24.9 ????15.5 ????0.4 ????2.6 ????0.2 ????11.2
a: the MRS meat soup that is supplemented with 0.3% oxgall and 12% horse serum.
b: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRS meat soup, and cultivate under 37 ℃ and last 24 hours.
c: before test, all cultures use 1% inoculum to be gone down to posterity and are incubated in the MRSO meat soup (MRS meat soup adds 0.3% oxgall), and cultivate under 37 ℃ and last 24 hours.
d: the cholesterol concentration in the control group is defined as 100 parts.
That comprehensive above test-results, the local lactobacillus separation strains demonstration of 6 strains that the present invention filtered out have is well acidproof, the characteristic of bile tolerance and decreasing cholesterol ability.Identify in the bacterial characteristics that further carries out these strain isolateds down.
Embodiment 5. the evaluation of lactobacillus separation strains and sign
One, experimental implementation program
(1) tentative experiment
The bacterial strain of fresh culture (18 to 24 hours) is carried out preliminary evaluation, test subject comprises: gramstaining, morphologic observation, catalase reaction, motility (motility), test (O.Kandler and N.Weiss (1986) such as the long situation under aerobic and anaerobic condition, Regular, nonsporing Gram-positive rods and Cocci.In:Bergey ' s Manual ofSystematic Bacteriology. (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Hotl, J.G., ed.) Vol.II.pp.1208-1234.The Williams﹠amp; Wilkins Co., Baltimore, USA).
(2) API identification systems
This qualification test is according to G.H.Fleet etc., (1984), Appl.Environ.Microbiol.48:1034-1038, and the milk-acid bacteria evaluation is to use API 50CHL cover group (French APIBioMerieux Research laboratory, La Balme Les Grottes, Montalien, Jeraeh, France), used test event is: glycerine, erythritol, the D-pectinose, L-arabinose, ribose, the D-wood sugar, the L-wood sugar, adonitol, Beta-methyl-xyloside, semi-lactosi, D-glucose, D-fructose, the D-seminose, the L-sorbose, rhamnosyl, galactitol, inositol, mannitol, Sorbitol Powder, Alpha-Methyl-D-mannoside, Alpha-Methyl-D-glucoside, the N-acetylglucosamine, amygdaloside, arbutin, Vitamin C2, saligenin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin (inuline), melizitose, the D-raffinose, starch (amidon), glycogen, Xylitol, β-gentiobiose, the D-turanose, the D-lyxose, the D-tagatose, the D-Fucose, the L-Fucose, the D-arabitol, L-arabinose alcohol, gluconic acid, 2-ketone-gluconic acid, the product acid test of 49 kinds of carbon sources such as 5-ketone-gluconic acid.
The schedule of operation of this system is: pick bacterium colony on the flat board with aseptic cotton rod, bacterium colony evenly is suspended in 0.85% normal saline solution of 2ml, draw suspension bacteria liquid with the dropper of sterilizing, put n and drip in the 0.85% sterile physiological salt solution of another 5ml, make its concentration and McFarland No.2 (by the 1%H of 9.8ml 2SO 41%BaCl with 0.2ml 2The mixing solutions that is constituted) concentration of standard solution is suitable.Get suspension bacteria liquid this moment in addition, putting 2n drips in the Glass tubing of API 50 CHL nutrient solutions, after being mixed, add proper volume respectively to the hole (tubes of the strip) of each test strip, and add a cover mineral oil, put into then cultivate box (incubation tray) (box adds water in advance so that when cultivating nutrient solution not by evaporate to dryness), add upper cover (incubation lid) and be placed on 37 ℃ and cultivate down and last 48 hours, take out interpretation afterwards.Outcome record is got off, and compare, to identify optimal generic name and to plant name with the database of API LAB Software identification systems.
(3) microorganism computer identification systems (Micro-IS System)
This qualification test is according to M.Rogosa etc., (1971), Method for coding data onmicrobial strains for computers (edition AB), Int.J.Syst.Bacteriol.21:1A-184A, utilize the Matrix 5[lactobacilli of Micro-IS System to be suitable for], used test event comprises: motility; Temperature growth test (15 ℃, 45 ℃); Aerobic growth down; D-glucose and the test of gluconic acid aerogenesis, the product acid test of amygdaloside, L-arabinose, cellobiose, D-fructose, D-semi-lactosi, D-glucose, lactose, maltose, D-mannitol, D-seminose, melizitose, melibiose, rhamnosyl, L-rhamnosyl, D-ribose, saligenin, D-Sorbitol Powder, sucrose, trehalose, D-wood sugar etc., the growth test of the hydrolysis of Vitamin C2 and L-spermine acid deamination etc.The positive and negative result of every test is imported in the Micro-IS computer qualification program generic name that can obtain identifying or kind name.
(4) 16S rDNA sequential analysis
This qualification test is according to Rosenblum etc., (1997), Nucleic Acid Res., 25:4500-4504, fresh thalline scraped gets a little and put into the 1.5ml Eppendorf tube, after handling with Proteinase K and RNA enzyme, utilize spin tubing string (spin column) to obtain genomic dna after, carry out PCR amplification, then purifying.Product behind the pcr amplification is after electrophoresis is confirmed, with MicroSeq TM16SrDNA Gene Kit (PE Co. USA) reacts, and then utilizes ABI Prism 310 Genetic Analyzer to carry out dna sequence analysis, again with 12 dna sequence dnas of gained with MicroSeq TMSoftware (PE Co. USA) analyzes, integrates, and can obtain the 16S rDNA sequence of about 1500bp, and then with DNA database (MicroSeq TMReference Manual, 1999, PE CO. USA) compares.
Two, result
1. strain isolated B21T1 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the test result of gained is as shown in table 6, identifies that mark is 91.0 (%ID), and qualification result is a Lactobacterium acidophilum;
(iii) carry out the evaluation of strain isolated B21T1 of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 7, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.682 (ID score);
The 16S rDNA The sequencing results of strain isolated B21T1 of the present invention as shown in Figure 5, with institute's calling sequence and DNA database (MicroSeq TMReference Manual, 1999, PE CO. USA) compares, and finds that the 16S rDNA sequence (SEQ IDNO:1) of strain isolated B21T1 of the present invention and Lactobacillus gasseri [are the B in the Lactobacterium acidophilum group 1Group] 16S rDNA sequence similarity degree the highest (G.Klein etc., (1998), International Jourmal of Food Microbiology.41:103-125);
Table 6
Strain number: B21T1
The description of identification result: suspicious result
---------------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):-----------+-+?------++-++ ++--+------+---------------------------------
50 kinds of carbohydrate reaction results
0?????-?GLY???-?ERY???-?DARA??-?LARL???-?RIB????-?DXYL???-?LXYL???-?ADO????-?MDX???-?GAL????-
GLU???+?FRU???-?MNE???+?SBE???？RHA????-?DUL????-?INO????-?MAN????-?SOR????-?MDM???-?MDG????-
NAG???+?AMY???+?ARB???-?ESC???+?SAL????+?CEL????+?MAL????+?LAC????-?MEL????-?SAC???+?TRE????-
INU???-?MLZ???-?RAF???-?AMD???-?GLYG???-?XLT????-?GEN????+?TUR????-?LYX????-?TAG???-?DFUC???-
LFUC??-?DARL??-?LARL??-?GNT???-?2KG????-?5KG????-
------------significantly classification--------------------------------%Id.-------T--------at the test of bacterial classification-----
Possible bacterial strain name
Lactobacterium acidophilum 1 91.0 0.53 3
The next selection
A kind of lactobacilli lactobacillus crispatus (Lacto.crispalus) 2 5.4 0.35 4
-------------------------------------------------------------------------------------------------------------
    
Lactobacterium acidophilum 1: 3 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
Semi-lactosi (GAL) 75% D-fructose (FRU) 100%
Trehalose (TRE) 77%
Table 7
The Micro-IS system
Strain name: B21T1, B21T6, C21T1, X21B7, B6T7
-013001 motility-006001 Endospore
45 ℃ of-017013 growth 15 ℃+017017 growths
+ 016059 good gas growth-024045 glucose becomes CO 2
-028528 gluconic acid, gas+025203 amygdaloside, acid
-025181 L-arabinose, acid+025211 cellobiose, acid
+ 025193 D-fructose, acid+025194 D-semi-lactosi, acid
+ 025195 D-glucose, acid+025212 lactose, acid
+ 025213 maltose, acid-026371 D-mannitol, acid
+ 025196 D-seminose, acid-025217 tamarack sugar, acid
+ 025214 manna disaccharides, acid+025218 raffinose, acid
-025191 L-rhamnosyl, acid-025184 D-ribose, acid
+ 025210 saligenin, acid-026374 D-Sorbitol Powder, acid
+ 025215 sucrose, acid+025216 trehalose, acid
-025186 D-wood sugar, acid+028060 L MALIC ACID usability
*Check * *The ID score *Possibility *Best possibility.
1 Lactobacterium acidophilum 0.68224 0.079110108 0.0791101
2 Lactobacillus vitulinus (L.vitulinus) 0.31761 0.036828671 0.1104860
The test that suggestion is done again *Numerical value in groups *Independent numerical value
1 024029 (L+) lactic acid generates 1
According to above-mentioned qualification result, strain isolated B21T1 most probable of the present invention is the B in the Lactobacterium acidophilum group 1Group--Lactobacillus gasseri.
2. strain isolated B21T6 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the test result of gained is as shown in table 8, identifies that mark is 93.6 (%ID), and qualification result is a Lactobacterium acidophilum;
(iii) carry out the evaluation of strain isolated B21T6 of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 7, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.682 (ID score);
The 16S rDNA The sequencing results of strain isolated B21T6 (iv) of the present invention as shown in Figure 6, with institute's calling sequence and DNA database (MicroSeq TMReference Manual, 1999, PECO. USA) compares, and finds that the 16S rDNA sequence (SEQ ID NO:2) of strain isolated B21T6 of the present invention and Lactobacillus gasseri [are the B in the Lactobacterium acidophilum group 1Group] 16S rDNA sequence similarity degree the highest;
(v) according to above-mentioned qualification result, strain isolated B21T6 most probable of the present invention is the B in the Lactobacterium acidophilum group 1Group--Lactobacillus gasseri;
Table 8
Strain number: B21T6
The description of identification result: demonstrate splendid qualification result for this Pseudomonas
-----------------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):----------++--------+--+++--+--------------------------------------------
50 kinds of carbohydrate reaction results
0???????-?GLY?????-?ERY????-?DARA???-?LARL???-?RIB????-?DXYL???-?LXYL???-?ADO????-??MDX????-?GAL????-
GLU?????+?FRU?????+?MNE????-?SBE????-?RHA????-?DUL????-?INO????-?MAN????-?SOR????-??MDM????-?MDG????-
NAG?????+?AMY?????+?ARB????-?ESC????+?SAL????+?CEL????+?MAL????+?LAC????-?MEL????-??SAC????+?TRE????-
INU?????-?MLZ?????-?RAF????-?AMD????-?GLYG???-?XLT????-?GEN????+?TUR????-?LYX????-??TAG????-?DFUC???-
LFUC????-?DARL????-?LARL???-?GNT????-?2KG????-?5KG????-
------------significantly classification------------------------------%Id.--------T----------is at the test of bacterial classification-------
Possible bacterial strain name
Lactobacterium acidophilum 3 93.6 0.80 1
Lactobacillus delbrueckii subsp. Lacto.Delb.delb. 6.0 0.65 5
The next selection
Lactobacillus crispatus 0.2 0.39 4
-------------------------------------------------------------------------------------------------
Lactobacterium acidophilum 1: 1 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
D-maltose (MAL) 75%
3. strain isolated C21T1 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the test result of gained is as shown in table 9, identifies that mark is 95.6 (%ID), and qualification result is a Lactobacterium acidophilum;
(iii) carry out the evaluation of strain isolated C21T1 bacterial strain of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 7, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.682 (ID score);
The 16S rDNA The sequencing results of strain isolated C21T1 (iv) of the present invention as shown in Figure 7, with institute's calling sequence and DNA database (MicroSeq TMReference Manual, 1999, PECO. USA) compares, and finds that the 16S rDNA sequence (SEQID NO:3) of strain isolated C21T1 of the present invention and Lactobacillus gasseri [are the B in the Lactobacterium acidophilum group 1Group] 16S rDNA sequence similarity degree the highest;
(v) according to above-mentioned qualification result, strain isolated C21T1 most probable of the present invention is the B in the Lactobacterium acidophilum group 1Group--Lactobacillus gasseri.
Table 9
Strain number: C21T1
The description of identification result: good evaluation
-------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):----------++--------++-++ ++--+------+--------------------------
50 kinds of carbohydrate reaction results
0??????-GLY????-ERY????-DARA????-LARL????-RIB????-DXYL???-LXYL???-ADO????-MDX????-GAL????-
GLU????+FRU????+MNE????-SBE?????-RHA?????-DUL????-INO????-MAN????-SOR????-MDM????-MDG????-
NAG????+AMY????+ARB????-ESC?????+SAL?????+CEL????+MAL????+LAC????-MEL????-SAC????+TRE????-
INU????-MLZ????-RAF????-AMD?????-GLYG????-XLT????-GEN????+TUR????-LYX????-TAG????-DFUC
LFUC???-DARL???-LARL???-GNT?????-2KG?????-5KG????-
------------significantly--------------%Id.------------T----------is at the test of bacterial classification--------in classification
Possible bacterial strain name
Lactobacterium acidophilum 1 95.6 0.70 3
The next selection
Lactobacillus crispatus 1.5 0.53 5
----------------------------------------------------------------------------------------
Lactobacterium acidophilum 1: 3 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
Semi-lactosi (GAL) 75% D-seminose (MNE) 96%
Trehalose (TRE) 77%
4. strain isolated X21B7 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the gained test result is as shown in table 10, identifies that mark is 94.3 (%ID), and qualification result is a Lactobacterium acidophilum;
(iii) carry out the evaluation of strain isolated X21B7 of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 7, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.682 (ID score);
The 16S rDNA The sequencing results of strain isolated X21B7 (iv) of the present invention as shown in Figure 8, with institute's calling sequence and DNA database (MicroSeq TMReference Manual, 1999, PECO. USA) compares, and finds that the 16S rDNA sequence (SEQ ID NO:4) of strain isolated X21B7 of the present invention and Lactobacillus gasseri [are the B in the Lactobacterium acidophilum group 1Group] 16S rDNA sequence similarity degree the highest;
(v) according to above-mentioned qualification result, strain isolated X21B7 most probable of the present invention is the B in the Lactobacterium acidophilum group 1Group--Lactobacillus gasseri.
Table 10
Strain number: X21B7
The description of identification result: good evaluation
---------------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):----------++?--------++-++ ++--+------+---------------------------------
50 kinds of carbohydrate reaction results
0??????-?GLY????-?ERY????-?DARA???-?LARL???-?RIB????-?DXYL???-?LXYL???-?ADO????-?MDX????-?GAL????+
GLU????+?FRU????？MNE????-?SBE????-?RHA????-?DUL????-?INO????-?MAN????-?SOR????-?MDM????-?MDG????-
NAG????+?AMY????+?ARB????-?ESC????+?SAL????+?CEL????+?MAL????+?LAC????-?MEL????-?SAC????+?TRE????-
INU????-?MLZ????-?RAF????-?AMD????-?GLYG???-?XLT????-?GEN????+?TUR????-?LYX????-?TAG????-?DFUC???-
LFUC???-?DARL???-?LARL???-?GNT????-?2KG????-?5KG????-
--------significantly--------------------------------%Id.--------T--------is at the test of bacterial classification------in classification
Possible bacterial strain name
Lactobacterium acidophilum 1 94.3 0.65 2
The next selection
Harmful sheet coccus (Pediococcus Damnosus) 2 2.0 0.43 3
------------------------------------------------------------------------------------
Lactobacterium acidophilum 1: 2 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
D-seminose E (MNE) 96% trehalose (TRE) 77%
5. strain isolated B38T38 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the test result of gained is as shown in table 11, identifies that mark is 90.8 (%ID), and qualification result is a Lactobacterium acidophilum;
(iii) carry out the evaluation of strain isolated B38T38 bacterial strain of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 12, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.646 (ID score);
The 16S rDNA The sequencing results of strain isolated B38T38 (iv) of the present invention as shown in Figure 9, with institute's calling sequence and DNA database (MicroSeq TMReference Manual, 1999, PECO. USA) compares, and finds that the 16S rDNA sequence (SEQ ID NO:5) of strain isolated B38T38 of the present invention and Lactobacillus gasseri [are the B in the Lactobacterium acidophilum group 1Group] 16S rDNA sequence similarity degree the highest;
(v) according to above-mentioned qualification result, strain isolated B38T38 most probable of the present invention is the B in the Lactobacterium acidophilum group 1Group--Lactobacillus gasseri.
Table 11
Strain number: B38T38
The description of identification result: good evaluation
---------------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):----------++ ++--------+-++ +++--++------+----------------------------------
50 kinds of carbohydrate reaction results
0??????-?GLY????-?ERY????-?DARA???-?LARL???-?RIB????-?DXYL???-?LXYL???-?ADO????-?MDX????-?GAL????+
GLU????+?FRU????+?MNE????+?SBE????-?RHA????-?DUL????-?INO????-?MAN????-?SOR????-?MDM????-?MDG????-
NAG????+?AMY????-?ARB????+?ESC????+?SAL????+?CEL????+?MAL????+?LAC????-?MEL????-?SAC????+?TRE????+
INU????-?MLZ????-?RAF????-?AMD????-?GLYG???-?XLT????-?GEN????+?TUR????-?LYX????-?TAG????-?DFUC???-
LFUC???-?DARL???-?LARL???-?GNT????-?2KG????-?5KG????-
----significantly classification----------------------------------%Id.--------T--------is at the test of bacterial classification-------
Possible bacterial strain name
Lactobacterium acidophilum 1 90.8 0.93 0
The next selection
Lactobacillus delbruckii breast subspecies (Lacto.delb.lactis) 2 6.2 0.74 2
---------------------------------------------------------------------------------------------
Lactobacterium acidophilum 1: 0 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
Table 12
The Micro-IS system
Strain name: B38T38
-013001 motility-006001 Endospore
45 ℃ of-017013 growth 15 ℃+017017 growths
+ 016059 good gas growth-024045 glucose becomes CO 2
-028528 gluconic acid, gas+025203 amygdaloside, acid
-025181 L-arabinose, acid+025211 cellobiose, acid
+ 025193 D-fructose, acid+025194 D-semi-lactosi, acid
+ 025195 D-glucose, acid+025212 lactose, acid
+ 025213 maltose, acid-026371 D-mannitol, acid
+ 025196 D-seminose, acid+025217 tamarack sugar, acid
+ 025214 manna disaccharides, acid+025218 raffinose, acid
-025191 L-rhamnosyl, acid-025184 D-ribose, acid
+ 025210 saligenin, acid-026374 D-Sorbitol Powder, acid
+ 025215 sucrose, acid+025216 trehalose, acid
-025186 D-wood sugar, acid-028060 L MALIC ACID usability
*Check * *The ID score *Possibility *Best possibility
1?L.delbrueckll?ssp?lactis????0.64607?????0.145038024?????0.4351140
2 Lactobacterium acidophilums 0.35240 0.079110108 0.0791101
The test that suggestion is done again *Numerical value in groups *Independent numerical value
1 024029 (L+) lactic acid generates 2
2 029268 L-spermine acid Deamination 2
6. strain isolated B6T7 of the present invention
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a catalase, and Immobility all can be grown under aerobic and anaerobic condition;
(ii) utilize API 50 CHL of API System to identify the cover group, the test result of gained is as shown in table 13, identifies that mark is 92.4 (%ID), and qualification result is a plant lactobacillus.
(iii) carry out the evaluation of strain isolated B6T7 of the present invention with microorganism computer identification systems (Micro-IS System), the gained result is as shown in table 7, identifies bacterium Lactobacterium acidophilum by name, identifies that mark reaches 0.682 (ID score);
The 16S rDNA The sequencing results of strain isolated B6T7 (iv) of the present invention as shown in figure 10.Since API 50 CHL identify the cover group result and Micro-IS System identification systems gained result be respectively plant lactobacillus and Lactobacterium acidophilum in the lactobacillus, so 16S rDNA sequence (SEQ ID NO:6) with strain isolated B6T7 gained of the present invention, compare with the dna sequence dna (SEQ ID NO:7) of Lactobacterium acidophilum and the dna sequence dna (SEQ IDNO:8) of plant lactobacillus, the 16S rDNA sequence similarity degree of finding the 16S rDNA sequence of strain isolated B6T7 of the present invention and Lactobacterium acidophilum is the highest, and differs very big with the 16S rDNA sequence of plant lactobacillus;
Table 13
Strain number: B6T7
The description of identification result: suspicious aspect
----------------------------------------------------------------------------------------------
Test strip: API 50CHL
Result (profile):----++----++ ++-+--+---+++-++ ++ +++--+--+-----------------------------------
50 kinds of carbohydrate reaction results
0??????-?GLY????-?ERY????-?DARA???-?LARL????+?RIB????+?DXYL????-?LXYL???-?ADO????-?MDX????-?GAL????+
GLU????+?FRU????+?MNE????+?SBE????-?RHA?????+?DUL????-?INO?????-?MAN????+?SOR????-?MDM????-?MDG????-
NAG????+?AMY????+?ARB????+?ESC????-?SAL?????+?CEL????+?MAL?????+?LAC????+?MEL????+?SAC????+?TRE????+
INU????-?MLZ????-?RAF????+?AMD????-?GLYG????-?XLT????-?GEN?????+?TUR????-?LYX????-?TAG????-?DFUC???-
LFUC???-?DARL???-?LARL???-?GNT????-?2KG?????-?5KG????-
------significantly classification------------------------------------%Id.----------T----------at the test of bacterial classification-----
Possible bacterial strain name
Plant lactobacillus 1 92.4 0.59 3
The next selection
Lactobacillus salivarius 3.4 0.45 4
---------------------------------------------------------------------------------------------
Plant lactobacillus 1: 3 is at following test (annotating: test if will be asserted the part carbohydrate that Lactobacterium acidophilum advises) again
D-Sorbitol Powder (SPR) 75% Vitamin C2 (ESC) 99%
Tamarack sugar (MLE) 92%
(v) according to above-mentioned qualification result, strain isolated B6T7 most probable of the present invention is a Lactobacterium acidophilum.
Comprehensive above qualification result, the 6 strain strain isolateds that the present invention screened all belong to the Lactobacterium acidophilum strain, and wherein B6T7 is a Lactobacterium acidophilum, and B21T1, B21T6, C21T1, X21B7 and B38T38 are its B 1Group's Lactobacillus gasseri.
Referring to table 14, when comparing with the lactobacterium strain that disclosed in previous patent case and the document, the lactobacillus separation strains of gained of the present invention not only can be grown containing under the 0.3% cholate environment, after cultivating 2 hours under the sour environment of pH 2, has higher survival rate, have good reducing cholesterol effect, and simultaneously cholesterol is had co-precipitation and assimilation.Obvious thus, the strain isolated of the present invention and the offspring who cultivates of going down to posterity thereof can be good probiotic bacteriums (probioticbacteria), and can also be useful on preparation treatment and prevention of gastrointestinal and reduce the pharmaceutical composition that serum cholesterol is used for the edible product that are used to prepare such as beverage, cake, infant food, cultured milk, nutriment, animal-feed etc.
For example, can be with reference to US 5,516,684 reference examples that disclosed 1 and reference example 2 are brought manufacturing lactic drink and fermented milk drink with the lactobacillus separation strains of gained of the present invention.
Table 14. isolate of the present invention and the comparison that is disclosed in the known bacterial strain in preceding case patent and the document
Bacterial strain Separation source Feature Profit/document
Lactobacterium acidophilum GG (ATCC 53103) Human 1. bile tolerance can grow in the environment that contains 0.15% bile acide 2. acidproofly in the pH5 environment, can grow to 10 9CFU can grow to 10 in the pH3 environment 7CFU in the stomach juice of pH2.5 after 30 minutes, the bacterium number reduce 2 log values with inherent pH1～2/2hr after, bacterium number remaining 10 3CFU 3. decreasing cholesterols can reduce the excretion of cholesterol in the human feces ?US?4839281 ?US?5032399
Lactobacterium acidophilum LA16 Boar 1. decreasing cholesterol can reduce serum cholesterol 60% (in containing 0.4% oxgall and 10mg cholesterol system) animal experiment, behind the feeding 56 days, serum cholesterol can reduce by 10.5%, the LDL-cholesterol level can reduce by 9% (annotating: after LA16 is Lactobacterium acidophilum DDS-1 (human origin) the sour buttermilk feeding pig of making, again by isolating the bacterial strain with decreasing cholesterol ability in the pig manure) ?Danielson?et ?al.，1989
Lactobacterium acidophilum ATCC 43121 ATCC 4356 The pig mankind 1. grew cell concentration (OD respectively 2.93 and 7.4 hours under bile tolerance 0.3% oxgall 620nm) can increase by 0.3 unit, 2. decreasing cholesterols can reduce serum cholesterol 55.4% and 12.9% (cultivate 14 hours after) in containing 0.3% oxgall and 0.1%PPLO system ?Gilliland?and ?Walker，1990
??L.gasseri ??SBT0274 ??SBT0270 Human 1. bile tolerance can be grown 2. acidproof at pH2.5 in containing 0.3% oxgall environment, 37 ℃/3h, the bacterium number descends 2 log values at pH1.5,37 ℃/2h, 4～5 log value 3. decreasing cholesterols of bacterium number decline are cultivated after 20 hours in containing 0.3% oxgall and 100 μ g cholesterol micella/ml systems, can distinguish reducing cholesterol content 47% and 36% ?Usman?and ?Hosono，1999
L.gasseri B21T1 B21T6 C21T1 X21B7 B38T38 acidophilus strain B 6T7 Healthy infants 1. bile tolerance is containing the 24h that grows under 0.3% oxgall, the bacterial strain remaining quantity reaches 80%, be that 0.5～2 log value 2. of bacterium number minimizing is acidproof at pH2,37 ℃/2hr handle the bacterium number reduce 3～4 log values (0.85% NaCl/0.01N HCl system) 3. decreasing cholesterol in containing the MRS meat soup system of 0.3% oxgall and 12% horse serum, anaerobism is cultivated 24 hr, but reducing cholesterol content reaches more than 93% in the MRS meat soup system that contains 0.3% oxgall and 12% cholesterol micella, anaerobism is cultivated 24hr, reaches 12～40% but reducing cholesterol content reaches 25～45% pairs of cholesterol absorption assimilation effects Bacterial strain of the present invention
Annotate: ATCC 43121=CCRC 17064 ATCC 4356=CCRC 10695
PPLO (BACT PPLO SERUM FRACTION) is produced by DIFCO LABORARORIES, stops production at present
All patents and the document quoted from this specification sheets are incorporated herein for referencial use.If to some extent when conflicting, detailed description of the present invention (comprise be defined in) will be got the upper hand.
Though the present invention obtains describing with reference to above-mentioned specific concrete example, make a lot of modifications and variations down what do not deviate from scope and spirit of the present invention significantly.Therefore what be intended to is, the present invention only is subjected to as the restriction with the scope those shown of civilian appending claims.
Sequence table
＜110〉Foodstuff Industrial and Development Inst.
＜120〉has the novel acidproof and bile tolerance lactobacillus separation strains that reduces with the assimilating cholesterol ability
<160>8
<170>Microsoft?Word?2000
<210>1
<211>500
<212>DNA
＜213〉Lactobacillus gasseri B21T1
<220>
＜223〉16S rDNA
<400>1
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagcttg?cctagatgaa????60
tttggtgctt?gcaccaaatg?aaactagata?caagcgagcg?gcggacgggt?gagtaacacg????120
tgggtaacct?gcccaagaga?ctgggataac?acctggaaac?agatgctaat?accggataac????180
aacactagac?gcatgtctag?agtttaaaag?atggttctgc?tatcactctt?ggatggacct????240
gcggtgcatt?agctagttgg?taaggtaacg?gcttaccaag?gcaatgatgc?atagccgagt????300
tgagagactg?atcggccaca?ttgggactga?gacacggccc?aaactcctac?gggaggcagc????360
agtagggaat?cttccacaat?ggacgcaagt?ctgatggagc?aacgccgcgt?gagtgaagaa????420
gggtttcggc?tcgtaaagct?ctgttggtag?tgaagaaaga?tagaggtagt?aactggcctt????480
tatttgacgg?taattactta????????????????????????????????????????????????500
<210>2
<211>500
<212>DNA
＜213〉Lactobacillus gasseri B21T6
<220>
＜223〉16S rDNA
<400>2
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagcttg?cctagatgaa????60
tttggtgctt?gcaccaaatg?aaactagata?caagcgagcg?gcggacgggt?gagtaacacg????120
tgggtaacct?gcccaagaga?ctgggataac?acctggaaac?agatgctaat?accggataac????180
aacactagac?gcatgtctag?agtttaaaag?atggttctgc?tatcactctt?ggatggacct????240
gcggtgcatt?agctagttgg?taaggtaacg?gcttaccaag?gcaatgatgc?atagccgagt????300
tgagagactg?atcggccaca?ttgggactga?gacacggccc?aaactcctac?gggaggcagc????360
agtagggaat?cttccacaat?ggacgcaagt?ctgatggagc?aacgccgcgt?gagtgaagaa????420
gggtttcggc?tcgtaaagct?ctgttggtag?tgaagaaaga?tagaggtagt?aactggcctt????480
tatttgacgg?taattactta????????????????????????????????????????????????500
<210>3
<211>500
<212>DNA
＜213〉Lactobacillus gasseri C21T1
<220>
＜223〉16S rDNA
<400>3
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagcttg?cctagatgaa????60
tttggtgctt?gcaccaaatg?aaactagata?caagcgagcg?gcggacgggt?gagtaacacg????120
tgggtaacct?gcccaagaga?ctgggataac?acctggaaac?agatgctaat?accggataac????180
aacactagac?gcatgtctag?agtttaaaag?atggttctgc?tatcactctt?ggatggacct????240
gcggtgcatt?agctagttgg?taaggtaacg?gcttaccaag?gcaatgatgc?atagccgagt????300
tgagagactg?atcggccaca?ttgggactga?gacacggccc?aaactcctac?gggaggcagc????360
agtagggaat?cttccacaat?ggacgcaagt?ctgatggagc?aacgccgcgt?gagtgaagaa????420
gggtttcggc?tcgtaaagct?ctgttggtag?tgaagaaaga?tagaggtagt?aactggcctt????480
tatttgacgg?taattactta????????????????????????????????????????????????500
<210>4
<211>500
<212>DNA
＜213〉Lactobacillus gasseri X21B7
<220>
＜223〉16S rDNA
<400>4
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagcttg?cctagatgat????60
tttggtgctt?gcactaaatg?aaactagata?caagcgagcg?gcggacgggt?gagtaacacg????120
tgggtaacct?gcccaagaga?ctgggataac?acctggaaac?agatgctaat?accggataac????180
aacactagac?gcatgtctag?agtttgaaag?atggttctgc?tatcactctt?ggatggacct????240
gcggtgcatt?agctagttgg?taaggtaacg?gcttaccaag?gcaatgatgc?atagccgagt????300
tgagagactg?atcggccaca?ttgggactga?gacacggccc?aaactcctac?gggaggcagc????360
agtagggaat?cttccacaat?ggacgaaagt?ctgatggagc?aacgccgcgt?gagtgaagaa????420
gggtttcggc?tcgtaaagct?ctgttggtag?tgaagaaaga?tagaggtagt?aactggcctt????480
tatttgacgg?taattactta????????????????????????????????????????????????500
<210>5
<211>500
<212>DNA
＜213〉Lactobacillus gasseri B38T38
<220>
＜223〉16S rDNA
<400>5
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagcttg?cctagatgaa????60
tttggtgctt?gcaccaaatg?aaactagata?caagcgagcg?gcggacgggt?gagtaacacg????120
tgggtaacct?gcccaagaga?ctgggataac?acctggaaac?agatgctaat?accggataac????180
aacactagac?gcatgtctag?agtttaaaag?atggttctgc?tatcactctt?ggatggacct????240
gcggtgcatt?agctagttgg?taaggtaacg?gcttaccaag?gcaatgatgc?atagccgagt????300
tgagagactg?atcggccaca?ttgggactga?gacacggccc?aaactcctac?gggaggcagc????360
agtagggaat?cttccacaat?ggacgcaagt?ctgatggagc?aacgccgcgt?gagtgaagaa????420
gggtttcggc?tcgtaaagct?ctgttggtag?tgaagaaaga?tagaggtagt?aactggcctt????480
tatttgacgg?taattactta????????????????????????????????????????????????500
<210>6
<211>480
<212>DNA
＜213〉acidophilus strain B 6T7
<220>
＜223〉16S rDNA
<400>6
cagacgaacg?ctggcggcgt?gcctaataca?tgcaagtcga?gcgacctgaa?ccaacagatt????60
cacttcggtg?atgacgttgg?gaacgcgagc?ggcggatggg?tgagtaacac?gtggggaccc????120
tgccccatag?tctgggatac?cacttggaaa?caggtgcaat?accggataag?aaagcagatg????180
ccatgatcag?cttataaaag?gcggcgtaag?ctgtcgctat?gggatggccc?cgcggtgcat????240
tagctagttg?gtagggtaac?ggcctaccaa?ggcaatgatg?catagccgag?tttgagagac????300
tgatccggcc?acattgggac?tgagacacgg?cccaaactcc?tacgggaggg?caagcagtag????360
ggaatcctcc?acaatggacc?aaagtcctga?tggagcaacg?ccccgtgagt?tgaagaagtt????420
ttcggatcgt?aaagccctgt?tgttggtgaa?gaaggataga?ggtaagaact?ggcctttatt????480
<210>7
<211>480
<212>DNA
＜213〉Lactobacterium acidophilum
<220>
＜223〉16S rDNA
<400>7
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?agcgagctga?accaacagat????60
tcacttcggt?gatgcagttg?ggaacgcgag?cggcggatgg?gtgagtaaca?cgtggggaac????120
ctgccccata?gtctgggata?ccacttggaa?acaggtgcaa?taccggataa?gaaagcagat????180
gccatgatca?gcttataaaa?ggcggcgtaa?gctgtcgcta?tgggatggcc?ccgcggtgca????240
ttagctagtt?ggtagggtaa?cggcctacca?aggcaatgat?gcatagccga?gttgagagac????300
tgatcggcca?cattgggact?gagacacggc?ccaaactcct?acgggaggca?gcagtaggga????360
atcttccaca?atggacgaaa?gtctgatgga?gcaacgccgc?gtgagtgaag?aaggttttcg????420
gatcgtaaag?ctctgttgtt?ggtgaagaag?gatagaggta?gtaactggcc?tttatttgac????480
<210>8
<211>480
<212>DNA
＜213〉plant lactobacillus
<220>
＜223〉16S rDNA
<400>8
caggacgaac?gctggcggcg?tgcctaatac?atgcaagtcg?aacgaactct?ggtattgatt????60
ggtgcttgca?tcatgattta?catttgagtg?agtggcgaaa?tggtgagtaa?cavgtgggaa????120
acctgcccag?aagcggggga?taacacctgg?aaacagatgc?taataccgca?taacaacttg????180
gaccgcatgg?tccgagttga?aagatggcrt?cggctatcac?ttttggatgg?tcccgcggcg????240
tattagctag?atgctggggt?aacggctcac?catggcaatg?atacgtagcc?gacctgagag????300
ggtaatcggc?cacattggga?ctgagacacg?gcccaaactc?ctacgggagg?cagcagtagg????360
gaatcttcca?caatggacga?aagtctgatg?gagcaacgcc?gcgtgagtga?agaagggttt????420
cggctcgtaa?aactctgttg?ttaaagaaga?acatatctga?gagtaactgt?tcaggtattg????480

Claims (9)

1. it is identical that the strain isolated of a lactobacilli, its bacterial characteristics and Lactobacillus gasseri X21B7 are had, and described Lactobacillus gasseri X21B7 is preserved in American type culture collection, and preserving number is ATCC PTA-4480.
2. foods prods, it includes the mutant strain that edibility material and lactobacilli strain isolated as claimed in claim 1 or its go down to posterity and cultivate the offspring or derived by them.
3. foods prods as claimed in claim 2, it further comprises at least a beneficial natural disposition biology that is selected from the following group: the lactobacillus species, as Lactobacterium acidophilum, lactobacillus delbruckii breast subspecies, short lactobacillus, lactobacterium casei, plant lactobacillus, lactobacillus salivarius, bifidobacterium, lactobacillus bulgaricus, bacillus caucasicus and lactobacillus rhamnosus; The streptococcus species are as thermophilus streptococcus, streptococcus acidi lactici; Yeast is as Candida species CandidaKefyr, sieve rib yeast not; Or the combination of these bacterial classifications.
4. foods prods as claimed in claim 2, wherein said edibility material is selected from following group: fluid dairy products (milk, enriching milk), fermented dairy prod (Yogurt, yogurt, freezing yogurt, lactacidase fermenting beverage), milk powder, ice-creams, cheese, cheese, soymilk and beverage, dessert, candy, infant food, dietary product, animal-feeds such as fermenting bean milk, fruit and vegetable juice, fruit juice and sports beverages, and dietary supplement.
5. foods prods as claimed in claim 2, it is manufactured into and is the instant food that brews.
6. pharmaceutical composition, it contains lactobacilli strain isolated as claimed in claim 1 or its mutant strain that goes down to posterity and cultivate the offspring or derived by them of useful natural disposition significant quantity.
7. pharmaceutical composition as claimed in claim 6, wherein said composition is formulated into the form that the confession that is selected from following group is used for oral administration: solution, emulsion, powder, lozenge and capsule.
8. pharmaceutical composition as claimed in claim 6, wherein said composition is made into to digest the drug for controlling intestinal function agent.
9. pharmaceutical composition as claimed in claim 6, wherein said composition are made in order to reduce the medicine of serum cholesterol.
CN2005100652557A 2002-10-30 2002-10-30 Acid and choline-resistant separated strain of lactobacillus with ability of reducing and assimilating cholesterol Expired - Lifetime CN1680544B (en)

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HK06102152.4A HK1081992A1 (en) 2002-10-30 2006-02-17 Acid-and bile salt-resistant lactobacillus isolates having the ability to lower and assimilate cholesterol

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337227A (en) * 2010-07-22 2012-02-01 中国医药大学 Lactobacillus plantarum and purpose thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337227A (en) * 2010-07-22 2012-02-01 中国医药大学 Lactobacillus plantarum and purpose thereof
CN102337227B (en) * 2010-07-22 2013-03-13 中国医药大学 Lactobacillus plantarum and purpose thereof

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