HK1081992A1 - Acid-and bile salt-resistant lactobacillus isolates having the ability to lower and assimilate cholesterol - Google Patents

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 beused as the medicine for curing the gastrointestinal sickness and decreasing the cholesterol of the serum.

Description

Novel acid-resistant and cholate-resistant lactobacillus isolated strain with cholesterol reducing and assimilating capability

The present application is a divisional application of Chinese patent application 02146947.4 entitled "novel acid-and bile salt-resistant Lactobacillus isolates having the ability to reduce and assimilate cholesterol", filed on 30.10.2002.

Technical Field

The present invention relates to a novel lactobacillus isolate having bile salt and gastric acid resistance and simultaneously having the ability to reduce serum cholesterol and its use.

Background

"lactic acid bacteria" are a group of bacteria that ferment carbohydrates and use lactic acid as the main product, and have widely accepted morphological and physiological characteristics: (1) gram-positive bacteria; (2) the appearance is cocci or bacilli; (3) the catalase test was negative; (4) more than 50% of lactic acid can be generated from the glucose metabolized by the lactic acid; (5) no endogenous spores are formed; and (6) it has no mobility and can grow under microaerobic conditions.

General lactic acid bacteria known to the public until 1980 include 4 genera (W.C) such as Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Leuconostoc (Leuconostoc), Pediococcus (Pediococcus), and the like.Frazier and D.C.Westhoff, 1978Food Microbiology, 3rd ed.mcgraw-Hill, inc., New York, USA), while lactic acid bacteria in the broader sense also include the two genera Bifidobacterium (Bifidobacterium) and lactobacillus (Sporolactobacillus). In recent years, microorganisms have been identified as a taxonomic group based on DNA homology and rDNA sequence alignment analysis, and assigned taxonomic positions. To the best of the applicant's knowledge, the lactic acid bacteria family has been expanded to contain 16 genera and 223 species by 12 months 1999.

Single or mixed species that improve the intestinal microbial balance of a human or animal host upon ingestion by the human or animal host are the so-called Probiotics (O' cullivan et al (1992), Trends in Food Sci. technol., 3: 309-; Fuller, R., P.J.Heidt, V.Rush and D.van der Waaij. (eds.) (1995), Probiotics: Probiotics of use in an immobilized microbial inoculation. old Herborn University semiconductor Monograph No.8, pp.1), of which the most well-known and used Probiotics are Lactobacillus (Lactobacillus) and Bifidobacterium (Bifidobacterium).

Since The theory that Eli Metchnikoff in 1908 proposed that "eating Lactobacillus-containing yogurt could replace The toxigenic bacteria normally present in The intestine, but not only could benefit health but also could prolong Life" (EliMetchnikoff, 1908, The proliferation Of Life, Ed.P.Chalmers Mitchell, G.P.Putnam's Sons, The Knicherbocker Press, New York & London), recent research and clinical test results also showed "Lactobacillus has an important relationship with health". Lactobacillus has therefore received considerable attention.

Lactic acid bacteria not only play an important role in the complex ecosystem of the intestinal tract, but also have health benefits to the host. Lactic acid bacteria have been found to have effects including enhancing the nutritional quality of food ingested by the host and promoting vitamin synthesis and enzyme production (J.Denter and B.Bispitng, 1994, int.J.food Microbiol., 22: 23-31), inhibiting the growth of pathogenic bacteria in the gut and stabilizing the normal bacterial phase balance in the gut (Hose, H. and Sozzi, T. (1991), J.chem.technology.and Biotechnology.51: 540-544), producing antibody substances increasing the resistance of the host (H.Majamaa et al, (1995), Journal of gastrointestinal surgery-gastroenterology and Nutrition, 20: 333-338) and reducing the risk of large bowel cancer and inhibiting tumors (E.J.Schiffrin et al, 1997, am.J.Clin.Nutr, 66: 515S-S). Meanwhile, researches show that the content of serum cholesterol can be reduced by taking the milk product fermented by lactobacillus.

Cardiovascular disease is listed as a major cause of death for people in the industrial world. More deaths occur in the United states due to cardiovascular disease than cancer and other diseases, three-quarters of which are due to atherosclerosis and its complications, while high serum cholesterol is responsible for cardiovascular disease and atherosclerosis (Kannel et al, (1979), Ann. Intern. Med., 90: 85-91; Pekkanen et al, (1990), New England J. Med., 322: 1700-. In taiwan, cerebrovascular and cardiac diseases have been named as antecedent causes of ten death in recent years, and are the leading causes and causes of death of the elderly, so hypercholesterolemia is a non-negligible cause.

Mann and Spoerry discovered in 1974 that drinking yoghurt fermented by Lactobacillus can reduce the serum cholesterol concentration of human body, while eating fresh milk has no influence (am.J.Clin.Nutr., 27: 464-. Therefore, the treatment and efficacy of lactic acid bacteria to reduce cholesterol have been studied extensively in recent years.

In 1982, k.k.grunewald, j.of Food Science, 47: 2078 and 2079, it was reported that serum cholesterol levels in rats significantly decreased when the rats were fed with food containing 10% yoghurt fermented with Lactobacillus acidophilus for 4 weeks. In 1989, Danielson et al, j.anim.sci., 67: 966-974 reports that when fed to pigs on a high cholesterol diet for 56 days, the feed of acidophilic cow milk (acidophilus yogurt) fermented with Lactobacillus acidophilus LA16 isolate reduced serum cholesterol and Low Density Lipoprotein (LDL) in the pigs, while being ineffective against serum triglycerides and High Density Lipoprotein (HDL).

The above paper on the isolate of lactobacillus acidophilus LA16 is one of the studies on the characteristics and biological activities of lactobacillus acidophilus conducted by the research group led by dr. In particular, the research group led by dr. khem m.shahai has conducted considerable research on the properties and biological activities of a lactobacillus acidophilus DDS-1 isolate of human origin, including studies on the reduction of serum cholesterol levels, which DDS-1 isolate was subsequently patented (see Nebraska Cultures, inc. published on the internet for DDS-1)^TMThe data of (a) the data of (b),http://www.nebraskacultures.com.benefits.html)。

in 1997, Akalin et al compared the effects of normal yogurt [ fermented from Streptococcus thermophilus and Lactobacillus delbrueckii species ] with acidophilic milk [ fermented from Streptococcus thermophilus and Lactobacillus acidophilus ] on serum cholesterol in mice, and found that acidophilic milk has significantly higher ability to reduce serum cholesterol concentration than normal yogurt (A.S. Akalin et al, (1997), J.Dairy Sci., 80: 2721-2725).

In 1998 Smet et al in British J.of Nutrition, 79: 185- > 194: the pig has obviously increased excretion amount of bile salt in excrement in 3 to 7 weeks after the pig eats lactobacillus acidophilus, and has obviously reduced concentration of total serum cholesterol in the pig body in 3 to 7 weeks. Therefore, the enzymatic activity of BSH possessed by Lactobacillus may be one of the mechanisms responsible for the reduction of serum cholesterol in the test pigs.

Cholesterol in the human body can be synthesized by the liver itself, and can also be taken in animal food. The excretion pathway of cholesterol is divided into two pathways: (1) bile acids are formed by metabolism of the liver, and then are combined with glycine or taurine to be soluble in water, and then are combined with potassium or sodium ions to form bile salts such as glycocholate (glycocholate) or taurocholate (taurocholates), and the salts can be discharged out of the body through feces; and (2) formation of steroid hormones, which are excreted in the urine by metabolic action of the hormones, but this pathway is only a small part.

Bile salts are water-soluble end product substances in the course of cholesterol metabolism, and these bile salts may enter the enterohepatic circulation because of the presence of intestinal bacteria [ including: enzymatic action of bile salt hydratase (BSH, e.c.3.5.1.24) of lactobacillus, Enterococcus (Enterococcus), Peptostreptococcus (streptococcus), bifidobacterium, Clostridium (Clostridium) and bacteroides (bacterial) and the like ] separates bile acid from glycine or taurine to produce unbound bile salts. These unbound bile salts are insoluble in water and are easily co-precipitated with serum cholesterol and excreted.

In addition to the above, the metabolic mechanism of cholesterol has been reported to have anabolic and coprecipitative effects.

In 1985, s.e. gilliland et al in appl.environ.microbiol, 49: 377-381 reports that Lactobacillus acidophilus has the functions of absorbing and assimilating cholesterol, and has better cholesterol-reducing ability in the environment of 0.3% oxgall. Likewise, Noh et al, j.dairy Sci, (1997), 82: 3107-3113 it has been reported that the cell membrane of Lactobacillus acidophilus binds cholesterol, and the adsorbed cholesterol is further assimilated and metabolized into substances required by the cell.

On the other hand, f.a.m.kalver and r.van der Meer in appl.environ.microbiol (1993), 59: 1120-1124 reports that lactobacilli and bifidobacteria do not assimilate cholesterol but, at pH values below 6.0, because of the increased conjugate activity of the bacteria on bile salts (which may also be related to the BSH activity possessed by the bacteria), cause the cholesterol to co-precipitate with the bile salts, thereby reducing the cholesterol content of the medium.

In 1997, M.M.Brashears and S.E.Gililind indicate that without pH control (i.e. normal pH 4.5 to 5.5), lactobacilli have good cholesterol co-precipitation, whereas if pH is controlled to be maintained around 6.0, the cholesterol-lowering capacity of the bacteria is significantly reduced (M.M.Brashears and S.E.Gililind (1997), fluences of pH reducing growth on removal of cholesterol from MRS broth by Lactobacillus casei and Lactobacillus acidophilus, Animal Science Research Report, pp.32-37; http:// www.ansi.okstate.edu/Research/1997rr/006. htm).

In 1998, Zhang Jia Cheng and P.E. were tested with high fat milk and edible oil, and it is considered that "assimilation" is the main function of lactobacillus to reduce cholesterol. (Zhang Jia Cheng and P.E. (1998) ' study of cholesterol removing effect of lactic acid bacteria on food-screening of lactic acid bacteria strain (strain) ' food science (China) ' 19: 20-22).

1999, Usman and Hosono in j.dairy sci, 82: 243-248, a newly discovered species of Lactobacillus gasseri (Lactobacillus gasseri) absorbs cholesterol under bile salt-free culture conditions.

Biological methods for reducing serum cholesterol levels in humans are an economical and effective way. As can be seen from the above literature data, lactic acid bacteria may exhibit potent cholesterol-lowering effects both in vivo and in vitro, wherein possible mechanisms of action include the unbinding action of bile salt hydrolase, co-precipitation of cholesterol with unbound bile salts at acidic pH, and assimilation of cholesterol by the lactic acid bacteria cells.

However, after being ingested, the lactic acid bacteria face the pressure of the gastrointestinal environment of the human body and the specificity of intestinal absorption, so that the lactic acid bacteria must overcome the severe environment of the digestive tract and can settle (colonize) in the intestinal tract to exert the efficacy in the intestinal tract. Furthermore, lactobacillus acidophilus is a group of strains with complex nutritional requirements, and besides being stable in sour milk products, other commercially available lactobacillus are produced by dry powder or other processing forms (powder, granule and pastille), and the bacteria are not easy to survive for a long time at room temperature or under refrigeration storage, so that the number of bacteria is not easy to maintain at the initial storage number. Therefore, the actual number of bacteria contained in the commercial non-sour milk product is often less than the number of bacteria marked on its packaging. Therefore, in the production of lactic acid bacteria products, how to maintain the number of lactic acid bacteria during the distribution and storage is an extremely important issue. Screening of strains with the characteristics of acid resistance, cholate resistance, good serum cholesterol reduction and the like is an important target for developing excellent lactobacillus products.

From the above, if the strains can be screened directly aiming at the characteristics of acid resistance, bile salt resistance, storage stability and the like of the strains during strain screening, the cost of the subsequent process can be saved, and the screened strains can be more widely applied.

At present, of 171 specific health foods approved by the japan's ministry of health, 36 (about 21% of all) are probiotic strains, but the production value accounts for 82% of the total. In europe, probiotic species have produced values in the food market as high as $ 10 billion. In the United states, the sales of yogurt in 2000 were as high as $ 18.6 billion. In taiwan, in 2000, the market of probiotic strains has grown to 42 billion yuan, and the application forms of products are also expanded year by year from fermented milk products, milk, ice cream, candies, dietary supplements, etc., and the application subjects further include adults, infants, and various poultry and livestock. It is thus expected that the market for probiotic bacteria has a wide growing space.

Until now, many studies on the development of lactic acid bacteria have been made around the world, and among them, many of the patents and publications relating to acid resistance and cholesterol-lowering ability of lactobacillus strains are mainly directed to lactobacillus acidophilus strains, and mainly disclose bile salt tolerance and acid resistance strains, or only a portion of which is strongly regulated in cholesterol-lowering ability and bile salt tolerance. The current study on the bile salt tolerance of lactobacilli emphasizes that the strains can grow in the presence of 0.3% glycocholate, while the acid resistance is tested in the gastric acid tolerance state at pH2, which occurs in the early stages of gastric secretion.

In the published patent, it is found that the serum cholesterol-lowering ability of lactobacillus is about 20%, and there is a difference of about 10 to 80% in the published documents depending on the method of use.

In US 4,839,281, US 5,032,399 reference is made to Lactobacillus acidophilus GG (ATCC 53103) isolated from human feces, which is capable of growing in 0.15% bile salts and which has a residual count of 103CFU after 2 hours of cultivation in an acidic environment at a pH of 1-2.

S.e.gilliland d.k.walker in j.dairy Sci, (1990), 73: 905-911 it was reported that Lactobacillus acidophilus ATCC 43121 (corresponding to CCRC 17064) derived from pig and Lactobacillus acidophilus ATCC4356 (corresponding to CCRC 10695) derived from human both grow in the presence of 0.3% bile salts and have the ability to lower serum cholesterol.

Also, Danieloson et al, j.anim.sci. (1989), 67: 966-974, and a lactobacillus acidophilus DDS-1 isolate (an endogenous human strain) of human origin that was studied by dr. khem m.shahani et al and became a proprietary product of Nebraska Cultures, inc. (see for reference the description of lactobacillus acidophilus LA16 strain isolated from pigs and dr. khem m.shahani et al), showed the ability to reduce serum cholesterol (see for reference the description of endogenous human strains)http://www/nebraskacultures.com./benefits.html)。

Usman and Hosono in j.dairy Sci. (1999), 82: 243- & 248 reports that a separated lactobacillus species lactobacillus gasseri has the capabilities of resisting acid, resisting bile salt and reducing cholesterol.

Yoshio Saito and Jun Mizutani disclose in US 5,516,684 and US 5,707,584 two strains of lactobacillus acidophilus, lactobacillus acidophilus FERM-P-14204 and lactobacillus acidophilus FERM-P-14205, which do not exhibit the de-binding effect of bile acids, do not inhibit nutrient absorption, and reduce cholesterol in blood and liver.

If the above mentioned non-indigenous lactobacillus strains can be locally isolated and screened to obtain indigenous lactobacillus strains having acid resistance, bile salt resistance and serum cholesterol reduction ability, the adaptability to the intestinal environment of the country can be ensured, and such indigenous lactobacillus strains can be used as primers or added to processed products to ensure the intestinal inhabitation and colonization after eating, thereby improving the functionality of the products.

Disclosure of Invention

Accordingly, a first aspect of the present invention is to screen native human intestinal samples for lactobacillus isolates (isolates) having multiple properties of acid resistance, bile salt resistance, and good serum cholesterol lowering capacity.

The applicant screened the isolated strains with respect to gastrointestinal environment acid resistance, bile salt resistance and cholesterol-lowering ability by using taiwan indigenous healthy infant feces as a strain isolation source, and obtained 6 novel lactobacillus isolates having these multiple properties, i.e., lactobacillus gasseri B21T1, B21T6, C21T1, X21B7 and B38T38, and lactobacillus acidophilus B6T7, which were deposited in the "center for biological resource preservation and research" (BCRC, abbreviated as "life center") of the institute for food industry development and research on 6 month 18 of 2002 in taiwan (taiwan province 300 new bamboo city food route 331), and deposited as CCRC 910195, CCRC 910196, CCRC910198, CCRC 910199, CCRC 910197 and CCRC 910194, respectively. These isolates were also deposited under the Budapest treaty at 21.6.2002 in the American type culture Collection (ATCC, P.O. Box 1549, Manassas, VA 20108, USA) under the accession numbers PTA-4483, PTA-4484, PTA-4479, PTA-4480, PTA-4481 and PTA-4482, respectively.

Compared with the prior strains of lactobacillus acidophilus ATCC 43121, ATCC4356 and DDS-1, the novel lactobacillus isolate of the invention is proved to have better cholesterol-reducing capability.

In a second aspect of the present invention, there is provided a composition comprising at least one novel lactobacillus isolate or subcultured progeny thereof or mutant strains derived therefrom according to the present invention and suitable excipients for the preparation of food products such as beverages, pastries, baby food, yogurt, nutritional supplements, animal feed and the like.

A third aspect of the present invention provides a pharmaceutical composition comprising a probiotic effective amount of at least one novel lactobacillus isolate according to the invention or subcultured progeny thereof or mutant strains derived therefrom for use in the treatment and prevention of gastrointestinal disorders and for lowering serum cholesterol.

Drawings

The above and other objects and features of the present invention will become more apparent by referring to the following description, appended claims and accompanying drawings, when taken in conjunction with the accompanying drawings and detailed description of the present invention, in which:

FIG. 1 is a graph showing the comparison of acid resistance of the novel Lactobacillus isolate of the present invention with that of a conventional Lactobacillus strain, wherein the acid resistance is expressed as Δ log (a-b), a is the number of viable cells after 2 hours of treatment with physiological saline (pH 7), and b is the number of viable cells after 2 hours of treatment with physiological saline (pH 2);

FIG. 2 is a graph showing a comparison of the bile salt tolerance of the novel Lactobacillus isolate of the present invention with that of a previous Lactobacillus strain, where the bile salt tolerance is expressed as Δ log (c-d), c is the number of viable cells after 24 hours of treatment with MRS broth, and d is the number of viable cells after 24 hours of treatment with MRS broth supplemented with 0.3% oxgall;

FIG. 3 is a graph showing a comparison of the growth of the novel Lactobacillus isolate of the present invention and previous Lactobacillus strains in MRS broth with and without 0.3% oxgall added;

fig. 4 is a graph showing the cholesterol-lowering ability of strains according to the invention and is described in s.razin et al, (1980), Biochimica Biophysica Acta, 598: 628-640 the prepared phosphatidylcholine-cholesterol volume is related to the cholesterol concentration.

FIG. 5 shows the nucleotide sequence of the 16S rDNA of an isolated strain of Lactobacillus gasseri B21T1 of the present invention;

FIG. 6 shows the nucleotide sequence of the 16S rDNA of an isolated strain of Lactobacillus gasseri B21T6 of the present invention;

FIG. 7 shows the nucleotide sequence of the 16S rDNA of an isolated strain of Lactobacillus gasseri C21T1 of the present invention;

FIG. 8 shows the nucleotide sequence of the 16S rDNA of an isolated strain of Lactobacillus gasseri X21B7 of the present invention;

FIG. 9 shows the nucleotide sequence of the 16S rDNA of an isolated strain of Lactobacillus gasseri B38T38 of the invention; and

FIG. 10 shows the 16S rDNA nucleotide sequence differences between a Lactobacillus acidophilus B6T7 isolate and Lactobacillus acidophilus and Lactobacillus plantarum (Lactobacillus plantarum) isolated according to the present invention, where "boxed" is the nucleotide difference address between the B6T7 isolate and Lactobacillus acidophilus and "boxed" is the nucleotide difference address between the B6T7 isolate and Lactobacillus plantarum.

Detailed Description

To obtain indigenous lactobacilli meeting the indigenous habit to ensure adaptation to the intestinal environment of the native population, applicants screened suspected lactobacilli strains (selected strains) using a series of Rogosa agar-based selective media to obtain 828 isolates from 43 samples using feces of healthy infants aged 1 to 6 years who live in the new bamboo region of taiwan as a source for screening for the desired strains, and found 400 suspected strains. These suspected strains were further tested for the properties described in the section "summary of the invention" above, namely:

1. the stability of the aqueous dispersion to an acid,

2. stability to bile salts, and

3. has the capability of reducing the cholesterol level of the blood,

these suspected strains were compared with the public strains with respect to the above-mentioned properties, and 6 novel isolates of Lactobacillus which meet the above requirements and have good ability were selected.

The 6 isolates obtained were further identified by using API identification System, microbial computer identification System (Micro-IS System) and 16S rDNA sequence analysis, and the 6 isolates were classified and named Lactobacillus gasseri B21T1, B21T6, C21T1, X21B7 and B38T38, and Lactobacillus acidophilus B6T7, respectively, according to the identification results, and were deposited in the institute for food industry development (bamboo food road 331, New City, 300, Taiwan province) 6.18 days in 2002, and the deposit numbers are CCRC 910195, CCRC 910196, CCRC910198, CCRC 910199, CCRC 910197 and CCRC 910194, respectively. These strains are also under the provisions of the Budapest treaty and are deposited in the American type culture Collection (ATCC, P.O. Box 1549, Manassas, VA 20108, USA) under the terms of the Budapest treaty on day 21 6/2002, with the respective accession numbers PTA-4483, PTA-4484, PTA-4479, PTA-4480, PTA-4481 and PTA-4482.

Based on the advantageous properties described above, the novel lactobacillus isolate according to the invention is suitable as a probiotic. For example, these isolates can be formulated in a wide variety of edible materials, including: fluid milk (milk, concentrated milk), fermented milk (yogurt, frozen yogurt, lactobacillus fermented beverage), milk powder, ice cream, cheese, soy milk, fermented soy milk, vegetable juice, fruit juice, sports beverage, dessert, candy, infant food, dietetic product, animal feed, dietary supplement, etc. The bacteria content of each product may be about 10 per gram or milliliter⁶To 10⁹Colony Forming Units (CFU).

It will be apparent to those skilled in the art that the novel lactobacillus isolates of the present invention may be used as food additives, either added at the time of raw material preparation by conventional methods or added after the fermentation process without participating in the fermentation, to be formulated into any suitable form for absorption by humans and non-human animals. Preferably, the novel lactobacillus isolates of the present invention may be formulated within the edible material, alone or in combination with at least one other probiotic organism, including: lactobacillus species, such as Lactobacillus acidophilus, Lactobacillus delbrueckii subsp (Lactobacillus lactis), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarum, Lactobacillus salivarius (Lactobacillus salivarius), bifidobacterium bifidum (Lactobacillus bifidus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus caucasicus (Lactobacillus caucasicus), and Lactobacillus rhamnosus (Lactobacillus rhamnosus); streptococcus species, such as Streptococcus thermophilus, Streptococcus lactis (Streptococcus lactis); yeasts, such as Candida species Kefyr, Trichosporon florentinus (Saccharomyces florentinus); or a combination of these species.

Another form of application is to prepare the novel Lactobacillus isolates of the invention as such or the above-mentioned products containing them as freeze-dried powders or spray-dried powders, so that each product contains about 10⁸To 10⁹The above active lactobacillus cells. The product can be made into tablet or capsule by adding yeast powder, saccharide or other filler, such as digestive intestinal agent containing lactobacillus or instant food and thallus powder for direct consumption.

In addition, the present invention also contemplates the use of the above novel lactobacillus isolates, alone or in combination with other active ingredients, as a medicament for controlling the accumulation of undesirable intestinal microorganisms in the digestive tract of a mammal to alleviate the gastrointestinal discomfort symptoms caused by the undesirable intestinal microorganisms. The composition may be formulated as a solution, emulsion, powder, lozenge, capsule or other suitable form for oral administration.

Furthermore, based on the cholesterol-lowering ability of the novel lactobacillus isolates of the present invention, the present invention also contemplates the use of these lactobacillus isolates for the preparation of health foods and over-the-counter pharmaceuticals for lowering serum cholesterol.

In view of the above, it is contemplated within the scope of the technical concept of the present invention that strains having bacteriological characteristics with the novel lactobacillus isolate of the present invention, subcultured progeny of the novel lactobacillus isolate of the present invention, or mutant strains derived from the novel lactobacillus isolate of the present invention fall.

As used herein, the term "mutant" means a strain having a genetic composition that differs (e.g., by substitution, insertion, or deletion of nucleotides) by at least one nucleotide relative to a reference strain or parent strain. In addition to natural mutations, the mutants of the present invention can be generated by various methods. For example, these mutants can be obtained by random mutagenesis of the parent strain, for example by chemical mutagens, transposons or irradiation. Furthermore, the mutant strains of the invention may comprise recombinant nucleic acid sequences. For example, a mutant strain can be a strain with additional nucleic acid sequences (e.g., sequences that are transformed, transduced, or otherwise inserted into the cells of a parent strain). The additional nucleic acid sequence may be encoded as a normally or conditionally expressed polypeptide. Alternatively, the additional nucleic acid sequence may be encoded as a nucleic acid sequence that alters cell physiology, such as an antisense, ribozyme or other nucleic acid sequence. In another example, the inserted nucleic acid is inserted into an endogenous gene and alters (e.g., enhances or disrupts) the function of the endogenous gene. For example, the inserted nucleic acid may be a knock-out construct (knock-out construct) that inactivates an endogenous gene; or an artificial enhancer or promoter that increases transcription of an endogenous gene.

The invention will be further described in the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limiting the practice of the invention.

Example 1Isolation and screening of Lactobacillus isolates

The material and the method are as follows:

firstly, culture medium and diluent:

1. screening a culture medium:

(A) agar culture medium for tomato juice

Casein enzymatic hydrolysate (Sigma, Louis, Mo, USA) 10g

Skimmed milk 10g

Tomato juice 400ml

Agar 12g

Distilled water to 1L

(B) Rogosa agar medium

Rogosa agar (Merck, Darmstadt, Germany) 74.5g

Distilled water 1L

Heating with microwave to dissolve, cooling to 55 deg.C, adjusting pH to 5.5 with acetic acid

(C) Lactobacillus selective medium (LBS)

Rogosa agar 8.4g

Tomato juice 40ml

Distilled water 60ml

Heating with microwave to dissolve, cooling to 55 deg.C, adjusting pH to 5.5 with acetic acid

(D) Rogosa + X-glu agar medium

80mg of 5-bromo-4-chloro-3-indolyl 1-. beta. -D-glucopyranoside (X-glu) (Sigma, Louis, MO, USA) was added to 100ml of 0.2M Tris buffer (pH 8.5) and dissolved by ultrasonic oscillation. 10mL of the resulting solution was added to 190mL of Rogosa agar at 55 ℃ to obtain Rogosa + X-glu agar containing X-glu at a final concentration of 40. mu.g/mL.

2. Bacteria activation medium:

(A) lactobacillus Bacto MRS broth (Difco Laboratories, Detroit, MI, USA)

(B) MRS agar: bacto Lactobacillus MRS broth was supplemented with bacterial agar (agar) (manufactured by Scharlau Chemie S.A., Barcelona, Spain, European Union) (15 g/L).

3. Ribose-utilizing medium:

composition of basal medium:

Bactopeptone No. 310g

Bacto yeast extract 5g

(the manufacturer of the two components is Difco Laboratories, Detroit, MI, USA)

Tween 801 g

Ammonium hydrogen citrate 2g

Sodium acetate 5g

Magnesium sulfate 0.1g

Manganese sulfate 0.05g

Dipotassium hydrogen phosphate 2g

Chlorophenol red 0.05%

Distilled water 1L

The pH was adjusted to pH 6.3 with HCl

The prepared basal medium is subpackaged into test tubes, each tube contains 4.5ml, and after sterilization at 121 ℃ for 15 minutes, 0.5ml of 10% ribose solution after filtration and sterilization is added.

4. Diluent liquid

5.1% peptone Water

(BACTO^TM Peptone，Difco Laboratories，Detroit，MI，USA)

Secondly, separating and screening the strains:

1. taking the middle section of feces of an infant of 1-6 years old, taking out a sample with the size of about a small section of finger head by using a long bamboo stick, adding the sample into a test tube containing 9ml of diluent (0.1% peptone water) to prepare a test solution diluted by 10 times, fully and uniformly stirring the sample in the test tube, and standing for several minutes to release microorganisms contained in the sample into the diluent;

2. taking out 1ml of the diluted test solution, placing the diluted test solution into another test tube containing 9ml of the diluted test solution, and repeating the serial dilution until 10% is reached⁴-fold dilution.

3. For the serial dilutions made in step 2 above, 0.2ml of each of the dilutions had been taken 10²-、10³-、10⁴The test solutions were diluted in duplicate and the individual test solutions were placed on various screening media (Rogosa agar, tomato juice agar, lactobacillus selection medium and Rogosa + X-glu agar) and, after spreading them evenly with an L-shaped glass rod, the screening media were placed in an anaerobic incubator set at 37 ℃ (containing a mixed gas of 5% H₂、10％CO₂And 85% N₂) Internal culture for 2-4 days;

4. selecting bacillus colonies which are blue in color on Rogosa + X-glu agar and are microscopically immobile or are translucent in appearance on other screening media, selecting the bacillus colonies which are microscopically immobile, picking the selected bacillus colonies with bamboo sticks, placing the picked bacillus colonies into a test tube containing MRS broth and a fermentation inner tube, culturing for 1 to 2 days at 37 ℃, and collecting strains which grow but do not produce gas and are gram-stained to show positivity.

5. After activating the collected strain in step 4 above twice in MRS broth, growth temperature and ribose availability tests were performed as follows:

(i) inoculating 1% inoculum into a MRS broth, and subjecting the medium to static culture at 15 ℃ for 14 days, wherein growth of the strain at 15 ℃ is indicated if any, and growth of the strain at 15 ℃ is not indicated if the strain still grows aseptically at 14 days;

(ii) inoculating 1% inoculum into a MRS broth, and subjecting the medium to standing culture at 45 deg.C for 2 days, wherein growth of the strain at 45 deg.C is indicative of the strain being able to grow, and growth of the strain at 45 deg.C is indicative of the strain not being able to grow, if the strain grows aseptically;

(iii) inoculating 1% of the inoculum to a ribose-utilizing culture medium, and standing and culturing for several days at 37 ℃, wherein if the culture solution turns yellow from purple, the bacteria can grow by utilizing ribose and generate acid; if the culture solution is purple after 7 days, then the static culture is continued, and if the culture solution is not changed into yellow at 14 days, the strain can not utilize ribose to grow and produce acid.

Thirdly, obtaining a result:

in this example, the screening medium for Lactobacillus was based primarily on Rogosa agar, with tomato juice or X-glu added in some cases. Tomato juice is rich in vitamin B group, which is beneficial to the growth of lactobacillus, and acetic acid in Rogosa agar not only reduces the pH value to inhibit bacteria, but also has the effect of inhibiting the growth of microorganisms due to high-concentration acetate ions.

The appearance of lactobacillus acidophilus growing on Rogosa agar appears as white colonies, not readily distinguishable from other lactobacilli. If X-glu is added to Rogosa agar, the appearance of the Lactobacillus acidophilus colonies turns blue because the Lactobacillus acidophilus has beta-D-glucosidase activity, which breaks the beta-D-glycosidic bond of X-glu to generate blue chromophores (chromogens), which make the Lactobacillus acidophilus colonies appear blue. However, there are not a few microorganisms having the enzyme in their cells, and therefore, further microscopic examination, gram staining, and physiological and biochemical tests such as the above-mentioned saccharides and growth temperature are required, and suspected strains having desired properties are selected.

The applicant collected 43 samples from feces of healthy infants living in the area of 1 to 6 years old in the city of new bamboo, Taiwan, each sample was diluted and smeared in each selective culture medium, after the bacterial strain grew out, colonies different in color and morphology were picked out from the selective culture medium and subjected to microscopic examination, if the microscopic examination result is bacillus, then gram staining was performed, gram positive bacillus was picked out and will be referred to as isolate.

Referring to table 1, the applicant selected a total of 828 isolates among the 43 samples collected, of which 400 were suspected. Regarding the strain numbering, taking B6T7 as an example, B refers to the type of medium (LBS), 6 refers to the sample origin number, T refers to the strain color (transparent appearance), and 7 refers to the running water number of the colony.

TABLE 1 number of samples collected and their isolates

Sample number	Number of isolated plants	Suspected strain	Sample number	Number of isolated plants	Suspected 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

Sample number	Number of isolated plants	Suspected strain	Sample number	Number of isolated plants	Suspected strain
						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	Total number of	823	400

^*: sample source: feces of young children aged 1 to 6 years in the Taiwan New bamboo City

^**: sample Source number of the isolate of the present invention

These suspected strains were then tested for acid resistance, bile salt resistance and cholesterol lowering ability as described in the examples below, of which 6 isolates tested to show good properties, i.e. B21T1, B21T6, C21T1, X21B7, B38T38 and B6T7, were taken to compare these properties with the following prior strains:

1. lactobacillus acidophilus CCRC 17064 (corresponding to ATCC 43121, isolated from porcine), which is a bacterium with serum cholesterol lowering ability (S.E.Gilliland et al, (1985), appl.Environ.Microbiol., 49: 377-381; 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.311Noh et al, (1997), J.Dairy Sci., 82: 3107-3);

2. lactobacillus acidophilus CCRC 10695^T(corresponding to ATCC4356, isolated from human), which is a standard strain of Lactobacillus acidophilus from human (D.K.Walker and S.E.Gilliland (1993), J.Dairy Sci., 76: 956-flac 961),

3. lactobacillus acidophilus DDS-1 (isolated from human), a currently commercially available product (Nebraska Cultures, Inc.);

4. lactobacillus acidophilus CCRC 14065[ corresponds to CSCO 2401, commercially available from Commonwelh Scientific Research Organization (CSIRO), Canberra, Australia ]; and

5. a Lactobacillus gasseri CCRC14619 as a storage material of Lactobacillus species^T(corresponding to ATCC 33323, isolated from human) (int.J.Syst.Bacteriol. (1980), 30, 601; E.Lauer and O.Kandler, Bakteriol.Parasitenkd.Infektionskr.Hyg.Abt.1Orig.Reihe C，1980，1，75-78)。

Example 2Acid resistance test

Firstly, experimental operation procedures:

reference to j.e.holcomb et al (1991), cult.dairy prod.j., 26 (3): 4-5 and US 5,711,977 to y.s. yang et al, tested the number of viable bacteria in a neutral (pH 7) and simulated gastric acid environment (pH 2).

After activating each test strain twice with MRS broth, taking the bacterial liquid, centrifuging (3000rpm for 10 minutes), removing the supernatant, adding 1ml of physiological saline solution (0.85% NaCl, pH 7), stirring the bacterial body with a bamboo stick, oscillating uniformly, then respectively taking 0.5ml of the scattered bacterial liquid, placing the bacterial liquid in physiological saline solution with pH 7 and pH2, standing in a constant-temperature incubator at 37 ℃ for 2 hours, and respectively measuring the number of the survived bacterial. The smaller the value of Δ log [ (-) log (number of cells at pH 7-number of cells at pH 2) ], the more excellent acid resistance was considered to be a strain.

Secondly, the result is:

most microorganisms are not tolerant in acidic environments. Lactic acid bacteria themselves are acid-producing bacteria, but their growth environment can only reach pH 3.2 to 4.5, and therefore, the very low pH environment (pH2.0 to 3.2) in the stomach also becomes a major factor affecting its survival. The pH of gastric acid varied from 1.5 to 4.5 depending on the time and type of gastric contents entering, and the average duration was 2 hours, so that pH2 was used as a representative in the test, and since the acidic substance was similar to hydrochloric acid, the number of viable bacteria was measured after 2 hours of treatment with normal saline (0.85% NaCl/0.01N HCl system) adjusted to pH2 with hydrochloric acid at 37 ℃ and compared with the control group of pH 7.

The test results show that the acid resistance of Lactobacillus acidophilus CCRC 17064 is the best (figure 1) after the treatment of 37 ℃/2h at different pH values, the number of bacteria receiving the treatment is only reduced by 1.9 log values, and the Lactobacillus acidophilus CCRC 10695^TWith Lactobacillus gasseri CCRC14619^TOfThe acidity is poor, and the bacterial count is respectively reduced by 4.3 log values and 4.4 log values.

The new isolate B21T1 (classified as Lactobacillus gasseri by bacteriological characterization as shown in the examples below) obtained in example 1 of the present invention has similar acid resistance to CCRC 14065, and the number of bacteria is reduced by 3.9 log values. The acid resistance of 5 other isolates (B21T6, C21T1, X21B7, B38T38 and B6T7) in the present invention were similar and all achieved an acid resistance selection index with a Δ log of less than 4 (see U.S. Pat. No. 3, 5,711,977 to Y.S. Yang et al).

Example 3.Bile salt tolerance test

Firstly, experimental operation procedures:

after each test strain was activated twice with MRS broth, 1% of the inoculum was inoculated into 10mL of MRS broth and 10mL of MRS broth (MRSO) containing 0.3% oxgall, respectively, and after culturing at 37 ℃ for 24 hours, the cell concentration (OD) was measured with a spectrophotometer₆₆₀). In addition, the number of residual bacteria was counted, wherein a Δ log (number of bacteria in MRS broth — number of bacteria in MRSO broth) indicates that the test strain is more resistant to bile salts as the Δ log is smaller.

Secondly, the result is:

the survival rates of different strains of lactic acid bacteria treated with bile salts vary greatly, and therefore, the selection of strains of lactic acid bacteria with bile salt tolerance is of importance in the selection of probiotic bacteria (S.E.Gilliland et al, (1984), J.Dairy Sci., 67: 3045-. In previous studies, bovine bile was commonly used in culture media for the selective culture of human intestinal bacteria and therefore should be very similar in potency to human bile salts and at an average concentration of 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).

The test result shows that the CCRC 10695 is removed^T、CCRC 14619^TAnd CCRC 14065, all the test strains were foundOD measured in a medium with or without 0.3% oxgall₆₆₀Values (cell concentration) were all above 2 (table 2), which shows that 0.3% oxgall did not seem to have a significant effect on the growth of the test strains.

TABLE 2 growth comparison of test strain cultures cultured in MRS broth with or without 0.3% oxgall for 24 hours

However, it was noted that the two strains B6T7 and C21T1 of the present invention had higher biomass concentrations in MRSO broth than MRS broth, indicating that there may be other interference factors formed that affect absorbance. Therefore, the number of surviving bacteria of the test strain after 24 hours of culture in both groups of the medium was further observed.

Referring to FIG. 2, from the observed reduction in the number of bacteria, CCRC14619^TIs less well tolerated by bile salts, followed by CCRC 10695^TAnd the B38T38 strain of the invention. The best bile salt tolerance is the isolate B6T7 of the invention, and the bile salt tolerance of other strains is similar, namely the number of killed bacteria is within 1 to 2 log values after being cultured in MRS broth containing 0.3 percent oxgall for 24 hours.

In addition, the growth curve of a part of the strains in MRS broth containing 0.3% oxgall was observed, and the growth rate was represented by the slope of the growth curve, and the larger the slope, the faster the growth rate was, i.e., the bile salt tolerance was better. As can be seen from FIG. 3, the growth rate of CCRC 17064 is the fastest, and the isolates X21B7 and B21T1 of the present invention are the second best, while CCRC 14065 and CCRC 10695 are the second best^TAnd DDS-1 grows the slowest.

Although the results obtained from different observation methods are slightly different, it can be seen from these experiments that the selected lactobacilli have acid resistance and bile salt tolerance.

Example 4Reduction of bile by the StrainAnalysis of sterol Capacity

Firstly, experimental operation procedures:

(A) sources of cholesterol

For the cholesterol-lowering ability of the strain, the cholesterol source used in the prior literature and published patent applications is mostly PPLO (BACT PPLO SERUM FRACTION), but the production of this product has been stopped, so in this example, the applicant refers to Huang, C.and T.E.Thomopson (1974), Methods enzymol.32: 485- "and S.razin et al, (1980) Biochimica Biophysica Acta, 598: 628-640, the method of preparing phosphatidylcholine-cholesterol uses horse serum and phosphatidylcholine-cholesterol as cholesterol source, and prepares a positive correlation curve relationship between the volume of uniformly distributed phosphatidylcholine-cholesterol solution and the concentration of cholesterol (FIG. 4).

1. Horse serum

Horse serum was dissolved in water, filtered through a 0.45 μm pore size filter and stored in a-20 ℃ freezer.

2. Phosphatidylcholine-cholesterol [ Egg Lecithin (Egg Lecithin) + cholesterol ]

According to S.Razin et al, (1980), Biochimica Biophysica Acta, 598: 628-.

After shaking the sealed flask with a water bath type ultrasonic shaker at 4 ℃ for 15 minutes, the mixed solution was passed through a filter membrane having a pore size of 0.22 μm under pressure for 2 times, and then stored in a refrigerator at 4 ℃ for 3 days.

(B) Test group

Test (1): after the test strains were activated twice with MRS broth, 1% of the inoculum was inoculated into 10ml of MRSS (containing 8.8ml of MRS and 1.2ml of cholesterol solution with 0.3% oxgall) and incubated in an anaerobic incubator (10% CO) at 37 deg.C₂And 90% N₂) The internal culture was carried out for 24 hours, and then a supernatant sample of the culture inoculated with the test strain and a cell pellet sample were collected for use in the determination of cholesterol content as described in the following item (C).

Test (2): after two activations of the strain with MRS broth, 1% of the inoculum was inoculated in 10 mM MRSO, incubated at 37 ℃ for 20 to 22 hours, and 1% of the inoculum was taken out again and inoculated in 10mL of MRSS in an anaerobic incubator (10% CO) at 37 ℃₂And 90% N₂) The internal culture was carried out for 24 hours, and then a supernatant sample of the culture inoculated with the test strain and a cell pellet sample were collected for use in the determination of cholesterol content as described in the following item (C).

(C) Determination of the Cholesterol content

The cholesterol content was determined according to the terephthalaldehyde (o-benzaldehyde) method (L.L.Rudel and M.D.Morris (1973), Notes on method, 14: 364-.

For each of the above tests (1) and (2), 1ml of the test bacterial suspension was centrifuged at 12000rpm for 10 minutes, and 0.5ml of the supernatant was placed in a test tube, which was a sample of the supernatant of the culture inoculated with the test strain.

For each of the above tests (1) and (2), 1ml of the test cell suspension was centrifuged at 3000rpm for 10 minutes, the supernatant was removed, and the resulting cell pellet (cell-pellet) was dissolved in 10ml of MRSO broth and mixed well, and 0.5ml of the solution was taken out and placed in a test tube, which was a cell pellet sample inoculated with the culture of the test strain.

3ml of each test sample was added95% ethanol, 2ml 50% KOH, and then heating in water bath at 60 deg.C for 10 min, and cooling at room temperature. The cooled solution was added with 5ml of hexane and shaken well, and then 3ml of H was added₂And O. The resulting mixture was left to stand at room temperature for 15 minutes after shaking it to homogeneity, and a 2.7ml layer of hexane was taken out and transferred to another tube, and hexane was evaporated with nitrogen at 60 ℃. Adding 4ml of terephthalaldehyde reagent solution (0.5mg of terephthalaldehyde/1 ml of acetic acid) into the evaporated solid, shaking, standing at room temperature for 10 minutes, adding 2ml of concentrated sulfuric acid, standing at room temperature for 10 minutes, and measuring OD with spectrophotometer₅₅₀The value is obtained.

(D) Evaluation of Cholesterol lowering ability of Strain

The ability of the strain to reduce cholesterol was evaluated according to the following formula:

A＝100-[(B/C)×100]

percent reduction of cholesterol (%)

B-Cholesterol content (mg) in the supernatant sample of the culture inoculated with the test strain

Cholesterol content in supernatant samples not inoculated with test strain cultures (control)

If the A value is more than 80%, the cholesterol lowering ability is considered to be obvious.

(E) Evaluation of cholesterol assimilation by test Strain

A′＝100-[(B′+C′)×100]

Assimilation of cholesterol (%)

B' (% cholesterol content in supernatant sample inoculated with culture of test strain)

C' ═ Cholesterol content (%)

If the A' value is more than 15%, the cholesterol assimilation ability is considered to be obvious.

Secondly, the result is:

I. cholesterol lowering ability of strains

The difference between test (1) (i.e.growth in MRS broth) and test (2) (i.e.growth in MRSO broth) was compared in that the test strains in test (2) were inoculated into MRSO broth for 20 to 22 hours and then 1% of the inoculum was inoculated into 10ml of MRSS, which was intended to screen again the strains for good bile salt tolerance and also for cholesterol lowering ability.

The results of the experiments with horse serum as the source of cholesterol (Table 3) show that the previous Lactobacillus acidophilus CCRC 17064 and DDS-1 also have significant effect, and the cholesterol-lowering ability reaches 88-98%. And CCRC 14065, CCRC 10695^TAnd CCRC14619^TThe cholesterol-lowering ability of these strains was not evident, probably because the strains were poorly tolerant to bile salts and grew poorly in MRSO broth, so that the cholesterol-lowering ability was observed to be only 30 to 43%.

In two experiments, the cholesterol-reducing capacity of the isolates B6T7, C21T1, B21T1, B21T6, B38T38 and X21B7 obtained by the invention is more than 91-99 percent, which is slightly higher than that of CCRC 17064 and DDS-1, and the strains screened by the invention also have good serum cholesterol-reducing capacity.

TABLE 3 cholesterol lowering by test strains (horse serum model)^aAssessment of competency

^a: MRS broth supplemented with 0.3% oxgall and 12% horse serum.

^b: all cultures were subcultured in MRS broth using 1% inoculum and cultured at 37 ℃ for 24 hours prior to the experiment.

^c: prior to the experiment, all cultures were subcultured in MRSO broth (MRS broth plus 0.3% oxgall) using 1% inoculum and incubated at 37 ℃ for 24 hours.

^d: the initial cholesterol content in the broth was 86.06 μ g/ml.

Referring to table 4, when compared with strains having cholesterol-lowering ability in horse serum test, it was found that the cholesterol-lowering rate (%) of each strain was decreased when phosphatidylcholine-cholesterol was used as a cholesterol source, which indicates that the lactobacillus strains had a variation in cholesterol-lowering ability for different types, wherein the decrease rate of CCRC 17064 for phosphatidylcholine-cholesterol was 11 to 29%, whereas the decrease rate of the strains obtained by the present invention for phosphatidylcholine-cholesterol was 23 to 48%, which was obviously higher than that of CCRC 17064, but slightly lower than that of DDS-1 by 50%.

TABLE 4 cholesterol lowering by test strains (Phosphatidylcholine-Cholesterol model)^aAssessment of competency

^a: MRS broth supplemented with 0.3% oxgall and 12% phosphatidylcholine-cholesterol.

^b: all cultures were subcultured in MRS broth using 1% inoculum and cultured at 37 ℃ for 24 hours prior to the experiment.

^c: before the experiment, all cultures were subcultured in MRSO broth (MRS broth plus 0.3% oxgall) using 1% inoculum and cultured at 37 ℃For 24 hours.

^d: the initial cholesterol content in the broth was 59.22 μ g/ml.

II. Assimilation of cholesterol by test strains

The mechanism by which lactic acid bacteria reduce cholesterol is primarily the elimination of cholesterol by co-precipitation of cholesterol with de-bound bile salts (F.A. M. Kalver and R.van der Meer (1993), supra; M.M. Brasheers and S.E. Gililland (1997), supra), and the assimilation of cholesterol by the strain itself (Gilliland et al (1985), supra; D.O.Noh et al (1997); supra; and Zhang and Holo et al (1998), supra).

As shown in Table 5, it was found that CCRC 17064, the 6 isolates obtained according to the present invention, and DDS-1 all had an assimilating effect on cholesterol, but the assimilation efficiency was about 11 to 40% because the assimilation ability was slightly different among the different strains. And CCRC 14065 and CCRC 10695^TAnd CCRC14619^TThe cholesterol assimilating ability was not significant.

TABLE 5 Cholesterol of the test strains^aEvaluation of assimilation Rate

^a: MRS broth supplemented with 0.3% oxgall and 12% horse serum.

^b: all cultures were subcultured in MRS broth using 1% inoculum and cultured at 37 ℃ for 24 hours prior to the experiment.

^c: prior to the experiment, all cultures were subcultured in MRSO broth (MRS broth plus 0.3% oxgall) using 1% inoculum and incubated at 37 ℃ for 24 hours.

^d: the cholesterol concentration in the control group was defined as 100 parts.

Combining the test results, the 6 indigenous lactobacillus isolates screened by the invention have the characteristics of good acid resistance, bile salt resistance and cholesterol reducing capability. The bacteriological characterization of these isolates was further performed below.

Example 5.Identification and characterization of Lactobacillus isolates

First, experiment operation procedure

(1) Preliminary test

The strains freshly cultured (18 to 24 hours) were subjected to preliminary characterization, and the experimental items included: gram stain, morphological observation, catalase reaction, motility (motility), growth under aerobic and anaerobic conditions (O.Kandler and N.Weiss (1986), Regula, non-radioactive Gram-positive rods and Cocci.in: Bergey's Manual of systematic Bacteriology. (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holl, J.G., ed.) Vol.II.pp.1208-1234. Williams & Wilkins Co., Baltimore, USA).

(2) API authentication system

This identification test was performed according to g.h. fleet et al, (1984), appl.environ.microbiol.48: 1034 and 1038, and the identification of lactic acid bacteria using API 50CHL kit (APIBIOMerieux Research laboratory, La Balme Les Grottes, Montalien, Jeareh, France) using the following test items: glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, beta-methyl-xyloside, galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin (inuline), melezitose, D-raffinose, starch (amidin), glycogen, xylitol, beta-gentiobiose, D-turanose, D-lyxose, And (3) acid production tests of 49 carbon sources such as D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconic acid, 2-keto-gluconic acid, 5-keto-gluconic acid and the like.

The operating procedure of the system is as follows: the bacterial colony on the plate was picked up with a sterile cotton swab, suspended evenly in 2ml of 0.85% normal saline, the suspension was aspirated with a sterilized dropper, and placed n drops in another 5ml of 0.85% sterile normal saline to a concentration similar to McFarland No.2 (9.8 ml of 1% H)₂SO₄With 0.2ml of 1% BaCl₂The constituted mixed solution) was equivalent in the standard solution concentration. At this time, another suspension was dropped into a glass tube containing API 50CHL culture medium 2n times, mixed uniformly, added to the holes of each test strip with a suitable volume, covered with mineral oil, placed in an incubation box (in which water was added in advance so that the culture medium was not evaporated during incubation), covered with a lid (incubation lid), incubated at 37 ℃ for 48 hours, and then taken out for interpretation. The results are recorded and compared to databases of the API LAB Software identification system to identify the most appropriate genus and species names.

(3) Microorganism computer identification System (Micro-IS System)

This identification test is based on M.Rogosa et al, (1971), Method for coding data on microbiological strains for computers (edition AB), int.J.Syst.bacteriol.21: 1A-184A, Matrix 5[ applicable to Lactobacillus species ] using the Micro-IS System, the test items used included: mobility; temperature growth test (15 ℃, 45 ℃); growing under aerobic condition; gas production tests of D-glucose and gluconic acid, acid production tests of amygdalin, L-arabinose, cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melezitose, melibiose, rhamnose, L-rhamnose, D-ribose, salicin, D-sorbitol, sucrose, trehalose, D-xylose, and the like, and growth tests of hydrolysis of esculin and deamination of L-arginine. And inputting the positive and negative results of each test into a Micro-IS computer identification program to obtain the identified genus name or species name.

(4)16S rDNA sequence analysis

This identification test is based on rosenblium et al, (1997), Nucleic Acid res, 25: 4500-. After the products after PCR amplification were confirmed by electrophoresis, the products were analyzed by MicroSeq^TM16SrDNA Gene Kit (PE Co., USA), followed by DNA sequence analysis using ABI Prism 310Genetic Analyzer, and 12 DNA sequences obtained were sequenced in MicroSeq^TMThe 16S rDNA sequence of about 1500bp can be obtained by analyzing and integrating software (PE Co., USA), and then the sequence is matched with a DNA database (MicroSeq)^TMReference Manual, 1999, PE co., USA).

Second, result in

1. Isolate B21T1 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) using the API 50CHL identification kit of the API System, the obtained test results are shown in table 6, the identification score is 91.0 (% ID), and the identification result is lactobacillus acidophilus;

(iii) the isolate B21T1 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 7, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.682(ID score);

the result of the 16S rDNA sequence analysis of the isolate B21T1 of the present invention is shown in FIG. 5, and the obtained sequence was compared with a DNA database (MicroSeq)^TM Reference Manual, 1999, PE co., USA), found the 16S rDNA sequence of isolate B21T1 of the present invention (SEQ id no: 1) with Lactobacillus gasseri [ B of the Lactobacillus acidophilus group₁Group of]The 16S rDNA sequence of (S) has the highest similarity (G.Klein et al (1998), International journal of Food microbiology.41: 103-125);

TABLE 7

Micro-IS system

The strain name: B21T1, B21T6, C21T1, X21B7, B6T7

-013001 motility-006001 endospores

-017013 growth 15 ℃ + 017017 growth 45 ℃

+ 016059 aerobic growth-024045 glucose to CO₂

-028528 gluconic acid, gas + 025203 amygdalin, acid

-025181L-arabinose acid + 025211 cellobiose acid

+ 025193D-fructose acid + 025194D-galactose acid

+ 025195D-glucose, acid + 025212 lactose, acid

+ 025213 maltose, acid-026371D-mannitol, acid

+ 025196D-mannose, acid-025217 larch sugar, acid

+ 025214 xylomelibiose, acid + 025218 raffinose, acid

-025191L-rhamnose, acid-025184D-ribose, acid

+ 025210 salicin, acid-026374D-sorbitol, acid

+ 025215 sucrose, acid + 025216 trehalose, acid

-025186D-xylose, acid + 028060L-malic acid availability

^*Examination of^{* **}ID scoring^**Possibility of^**The best possible.

1 Lactobacillus acidophilus 0.682240.0791101080.0791101

2 Lactobacillus caldarius (L.vitulinus) 0.317610.0368286710.1104860

Recommended to do the test again^**Set of values^**Individual numerical values

1024029(L +) lactic acid to 1

According to the above identification results, the isolate B21T1 of the present invention is most likely to be B in the Lactobacillus acidophilus group₁Group-lactobacillus gasseri.

2. Isolate B21T6 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) using the API 50CHL identification kit of the API System, the obtained test results are shown in table 8, the identification score is 93.6 (% ID), and the identification result is lactobacillus acidophilus;

(iii) the isolate B21T6 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 7, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.682(ID score);

(iv) the result of the 16S rDNA sequence analysis of the isolate B21T6 of the present invention is shown in FIG. 6, and the obtained sequence was compared with a DNA database (MicroSeq)^TMReference Manual, 1999, peco., USA), found the 16S rDNA sequence of isolate B21T6 of the present invention (SEQ ID NO: 2) with Lactobacillus gasseri [ B of the Lactobacillus acidophilus group₁Group of]The 16S rDNA sequence has the highest similarity;

(v) according to the above identification results, the isolate B21T6 of the present invention is most likely to be B in the Lactobacillus acidophilus group₁Group-lactobacillus gasseri;

3. isolate C21T1 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) using the API 50CHL identification kit of the API System, the obtained test results are shown in table 9, the identification score is 95.6 (% ID), and the identification result is lactobacillus acidophilus;

(iii) the isolated strain C21T1 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 7, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.682(ID score);

(iv) the result of the 16S rDNA sequence analysis of the isolate C21T1 of the present invention is shown in FIG. 7, and the obtained sequence was compared with a DNA database (MicroSeq)^TMReference Manual, 1999, peco., USA) toBy comparison, the 16S rDNA sequence (SEQ ID NO: 3) of the isolate C21T1 of the present invention was found to be associated with Lactobacillus gasseri [ B of the Lactobacillus acidophilus group ]₁Group of]The 16S rDNA sequence has the highest similarity;

(v) according to the above identification results, the isolate C21T1 of the present invention is most likely to be B in the Lactobacillus acidophilus group₁Group-lactobacillus gasseri.

4. Isolate X21B7 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) using the API 50CHL identification kit of the API System, the obtained test results are shown in table 10, the identification score is 94.3 (% ID), and the identification result is lactobacillus acidophilus;

(iii) the isolate X21B7 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 7, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.682(ID score);

(iv) the result of the 16S rDNA sequence analysis of the isolate X21B7 of the present invention is shown in FIG. 8, and the obtained sequence was compared with a DNA database (MicroSeq)^TMReference Manual, 1999, peco., USA), found that the 16S rDNA sequence of isolate X21B7 of the present invention (SEQ ID NO: 4) with Lactobacillus gasseri [ B of the Lactobacillus acidophilus group₁Group of]The 16S rDNA sequence has the highest similarity;

(v) according to the above identification results, the isolate X21B7 of the present invention is most likely to be B in the Lactobacillus acidophilus group₁Group-lactobacillus gasseri.

5. Isolate B38T38 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) using the API 50CHL identification kit of the API System, the obtained test results are shown in table 11, the identification score is 90.8 (% ID), and the identification result is lactobacillus acidophilus;

(iii) the isolated strain B38T38 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 12, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.646(ID score);

(iv) the result of the 16S rDNA sequence analysis of the isolate B38T38 of the present invention is shown in FIG. 9, and the obtained sequence was compared with a DNA database (MicroSeq)^TMReference Manual, 1999, peco., USA), found that the 16S rDNA sequence of isolate B38T38 of the present invention (SEQ ID NO: 5) with Lactobacillus gasseri [ B of the Lactobacillus acidophilus group₁Group of]The 16S rDNA sequence has the highest similarity;

(v) according to the above identification results, the isolate B38T38 of the present invention is most likely to be B in the Lactobacillus acidophilus group₁Group-lactobacillus gasseri.

TABLE 12

Micro-IS system

The strain name: B38T38

-013001 motility-006001 endospores

-017013 growth 15 ℃ + 017017 growth 45 ℃

+ 016059 aerobic growth-024045 glucose to CO₂

-028528 gluconic acid, gas + 025203 amygdalin, acid

-025181L-arabinose acid + 025211 cellobiose acid

+ 025193D-fructose acid + 025194D-galactose acid

+ 025195D-glucose, acid + 025212 lactose, acid

+ 025213 maltose, acid-026371D-mannitol, acid

+ 025196D-mannose acid + 025217 larch sugar acid

+ 025214 xylomelibiose, acid + 025218 raffinose, acid

-025191L-rhamnose, acid-025184D-ribose, acid

+ 025210 salicin, acid-026374D-sorbitol, acid

+ 025215 sucrose, acid + 025216 trehalose, acid

-025186D-xylose, acid-028060L-malic acid availability

^*Examination of^{* **}ID scoring^**Possibility of^**Best possibility

1L.delbrueckll ssp lactis 0.64607 0.145038024 0.4351140

Lactobacillus acidophilus 0.352400.0791101080.0791101

Recommended to do the test again^**Set of values^**Individual numerical values

1024029(L +) lactic acid to 2

2029268L-arginine deamination 2

6. Isolate B6T7 of the present invention

(i) According to the result of the preliminary test, the isolate is gram-positive bacillus, does not have catalase and mobility, and can grow under aerobic and anaerobic conditions;

(ii) the test results obtained using the API 50CHL identification kit of the API System are shown in table 13, and the identification score was 92.4 (% ID), and the identification result was lactobacillus plantarum.

(iii) The isolate B6T7 of the present invention was identified by a microorganism computer identification System (Micro-IS System), and the results are shown in Table 7, wherein the identification bacterial name IS Lactobacillus acidophilus and the identification score IS 0.682(ID score);

(iv) the result of the 16S rDNA sequence analysis of the isolate B6T7 of the present invention is shown in FIG. 10. Since the results obtained in the API 50CHL identification kit and the Micro-IS System identification System are Lactobacillus plantarum and Lactobacillus acidophilus in Lactobacillus, respectively, the 16S rDNA sequence (SEQ ID NO: 6) obtained from the isolate B6T7 of the present invention was compared with the DNA sequence of Lactobacillus acidophilus (SEQ ID NO: 7) and the DNA sequence of Lactobacillus plantarum (SEQ ID NO: 8), and it was found that the 16S rDNA sequence of the isolate B6T7 of the present invention has the highest similarity with the 16S rDNA sequence of Lactobacillus acidophilus and IS very different from the 16S rDNA sequence of Lactobacillus plantarum;

(v) according to the above identification results, the isolate B6T7 of the present invention is most likely Lactobacillus acidophilus.

In summary of the above identification results, the 6 isolates selected in the present invention all belong to Lactobacillus acidophilus, wherein B6T7 is Lactobacillus acidophilus, and B21T1, B21T6, C21T1, X21B7 and B38T38 are B thereof₁A group of lactobacillus gasseri.

Referring to table 14, when compared with the lactobacillus strains disclosed in the prior patent and documents, the lactobacillus isolate obtained in the present invention can grow in an environment containing 0.3% of bile salts, has a high survival rate after 2 hours of culture in an acidic environment of pH2, has a good cholesterol-lowering effect, and has a coprecipitation effect and an assimilation effect on cholesterol. It is thus clear that the isolates of the invention and their subcultured progeny will be excellent probiotics (probiotics) and can be used for the preparation of food products such as beverages, cakes, baby food, yogurt, nutritional supplements, animal feed, etc. and for the preparation of pharmaceutical compositions for the treatment and prevention of gastrointestinal disorders and for the reduction of serum cholesterol.

For example, the lactobacillus isolate obtained in the present invention can be used to produce lactic acid beverages and yogurt beverages, as described in reference examples 1 and 2 disclosed in US 5,516,684.

TABLE 14 comparison of the isolates of the present invention with known strains disclosed in the prior patents and literature

Note: ATCC 43121 ═ CCRC 17064 ATCC4356 ═ CCRC 10695

PPLO (BACT PPLO SERUM FRACTION) is produced by DIFCO LABORARRIES and has been stopped in production

In case of conflict, the present specification, including definitions, will control.

While the invention has been described with reference to the specific embodiments described above, it will be apparent that numerous modifications and variations can be made without departing from the scope and spirit of the invention. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Sequence listing

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Claims (34)

1. An isolate of a Lactobacillus species (Lactobacillus sp.) which is Lactobacillus gasseri X21B7, said Lactobacillus gasseri X21B7 being deposited with the american type culture collection with accession number ATCC PTA-4480.

2. A food product comprising edible material and an isolate of a lactobacillus species as claimed in claim 1, or a subculture progeny thereof, wherein said subculture progeny exhibits the following bacteriological characteristics:

the bacterial count is reduced by 0.5-2 log values after the growth of 24 hours in an environment containing 0.3% of oxgall;

treating the mixture in a 0.85% NaCl/0.01N HCl system for 2 hours at the pH of 2 and the temperature of 37 ℃, wherein the bacterial count is reduced by 3-4 log values;

anaerobic culturing in MRS broth culture system containing 0.3% oxgall and 12% horse serum for 24 hr to reduce cholesterol content to above 93%;

anaerobic culture is carried out for 24 hours in an MRS broth culture system containing 0.3 percent of oxgall and 12 percent of cholesterol microcells, and the content of the cholesterol can be reduced to 25 to 45 percent; and

the absorption and assimilation rate of cholesterol reaches 12-40%.

3. The food product of claim 2, further comprising at least one probiotic organism selected from the group consisting of: a lactobacillus species; streptococcus species (streptococcus sp.); a yeast; or a combination of these species.

4.The food product of claim 3, wherein the Lactobacillus species is selected from the group consisting of: lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus delbrueckii subsp (Lactobacillus lactis), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivarius), bifidobacterium bifidum (Lactobacillus bifidus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus caudatum (Lactobacillus caucasicus), and Lactobacillus rhamnosus (Lactobacillus rhamnosus);

5. the food product of claim 3, wherein the Streptococcus species is selected from the group consisting of: streptococcus thermophilus (Streptococcus thermophilus) and Streptococcus lactis (Streptococcus lactis);

6. the food product of claim 3 wherein the yeast is selected from the group consisting of: candida species Candida Kefyr and Trichosporon florentinus (Saccharomyces florentinus).

7. The food product of claim 2, wherein the edible material is fluid dairy.

8. The food product of claim 2, wherein the edible material is fermented milk.

9. The food product of claim 2, wherein the edible material is milk powder.

10. The food product of claim 2, wherein the edible material is ice cream.

11. The food product of claim 2, wherein the edible material is cheese.

12. The food product of claim 2, wherein the edible material is cheese.

13. The food product of claim 2, wherein the edible material is soy milk.

14. The food product of claim 2, wherein the edible material is fermented soy milk.

15. The food product of claim 2, wherein the edible material is a juice.

16. The food product of claim 2, wherein the edible material is fruit juice.

17. The food product of claim 2, wherein the edible material is a sports beverage.

18. The food product of claim 2, wherein the edible material is a dessert.

19. The food product of claim 2, wherein the edible material is a candy.

20. The food product of claim 2, wherein the edible material is an infant food.

21. The food product of claim 2, wherein the edible material is a dietetic product.

22. The food product of claim 2, wherein the edible material is an animal feed.

23. The food product of claim 2, wherein the edible material is a dietary supplement.

24. The food product of claim 7, wherein the fluid dairy is milk.

25. The food product of claim 7, wherein the fluid dairy is concentrated milk.

26. The food product of claim 8, wherein the fermented dairy product is yogurt.

27. The food product of claim 8, wherein the fermented dairy product is yogurt.

28. The food product of claim 8, wherein the fermented dairy product is frozen yogurt.

29. The food product of claim 8, wherein the fermented dairy product is a lactic acid bacteria fermented beverage.

30. The food product of claim 2, manufactured as an instant brew.

31. A pharmaceutical composition comprising a probiotic effective amount of the lactobacillus species isolate of claim 1 or subcultured progeny thereof, wherein said subcultured progeny exhibit the following bacteriological characteristics:

the bacterial count is reduced by 0.5-2 log values after the growth of 24 hours in an environment containing 0.3% of oxgall;

treating the mixture in a 0.85% NaCl/0.01N HCl system for 2 hours at the pH of 2 and the temperature of 37 ℃, wherein the bacterial count is reduced by 3-4 log values;

anaerobic culturing in MRS broth culture system containing 0.3% oxgall and 12% horse serum for 24 hr to reduce cholesterol content to above 93%;

anaerobic culture is carried out for 24 hours in an MRS broth culture system containing 0.3 percent of oxgall and 12 percent of cholesterol microcells, and the content of the cholesterol can be reduced to 25 to 45 percent; and

the absorption and assimilation rate of cholesterol reaches 12-40%.

32. The pharmaceutical composition of claim 31, wherein the composition is formulated for oral administration in a form selected from the group consisting of: solutions, emulsions, powders, lozenges, and capsules.

33. The pharmaceutical composition of claim 31, wherein the composition is formulated as a digestive, intestinal-regulating agent.

34.The pharmaceutical composition of claim 31, wherein the composition is formulated as a medicament for lowering serum cholesterol.