FI95193C - New Lactobacillus acidophilus strain and its use in food preparation, especially dairy products - Google Patents

Description

! 95193

New Lactobacillus acidophilus strain and its use in the manufacture of foodstuffs, in particular dairy products This application has been separated from the application FI 860194.

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The invention relates to a new strain of Lactobacillus acidophilus and its use in the preparation of foodstuffs, in particular dairy products.

L. acidophilus is present in the normal intestinal flora of most healthy people. The genus Lactobacillus is widespread in nature. Many species, such as L. bulgaricus and L. casei, are found in dairy products as well as in fruits and vegetables. Other species of Lactobacillus-la are found in the intestinal tract of mammals and insects. 15 For many centuries, many species of Lactobacillus have been used in milk and dairy products as fermentation organisms, in the manufacture of yoghurt and various types of used and fermented milk, curds and creams. The Lactobacillus species used in the dairy industry is usually L. bulgaricus, although L. acidophilus strains have also been used very widely.

In recent years, many studies have been performed using L. acidophilus strains isolated at North Carolina State University (nNSCU). These studies 25 have shown that NSCU strains, when ingested by humans or animals, have beneficial effects on various gastrointestinal functions. chemical carcinogens including activation of the suppression of the large intestine, secretion of cholesterol reduction (which engages the TYY-30 colon cancer in a reduced hazard) and antibiotic therapy against gastro-intestinal effects of reduction of area.

It is an object of the present invention to provide a novel strain of L. acidophilus which can be used in the manufacture of foodstuffs, in particular dairy products. In a preferred embodiment 2 95193, the bacteria of the L. acidophilus strain ATCC 53103 are added in an amount of 109 to ΙΟ10 organisms per 10 to 500 ml of milk.

according to the invention of the L. acidophilus strain is 5 characterized in that on average at least 50, preferably at least 100 bacteria can attach to the human small intestinal mucosal cell after the bacteria have been incubated with the cells for five minutes. Preferably, the bacteria are further characterized in that they produce at least 3.5 milliequivalents (meq) of lactic acid per 1010 CFU (colony forming unit) in the medium at 37 ° C per day; they are able to distribute at 37 ° C in a medium at pH 3.0; they are able to distribute in a nutrient medium containing 0.1% bovine bile-15 bladder bile; their cell division time is one hour in medium at 37 ° C. "Culture medium" means any culture medium containing the nutrients necessary for the distribution of L. acidophilus.

Em. the strain can be isolated from a sample containing many bacterial strains by a method comprising the following steps in isolating bacteria of L. acidophilus species, which can be performed in any order: combining the bacteria with gastrointestinal cells and selecting bacteria that remain stable; adhere to the cells at an average rate of at least 50, and preferably at least 100 bacterial cells per gastrointestinal cell during a five minute preincubation. The selection method also preferably comprises the steps of incubating the bacteria in the sample in a nutrient medium at a pH of less than 5.5 (preferably less than 3.5), as well as selecting the bacteria that are distributed in this medium; incubating the bacteria in the sample in a medium containing at least 0.1% bile and selecting the bacteria that are distributed in the medium; and incubating the sample bacteria 35 with the medium and selecting the bacteria, 3 95193 having a distribution time of less than one hour at about 37 ° C.

Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention, and from the claims.

Isolation of the strain

Healthy humans have bacteria of L. acidophilus species as part of their normal lora, usually at a concentration of about 109 Lactobacillus bacteria per gram of intestinal-10 content; thus, healthy people have an excess of lactobacillus in their intestinal tract. The first step in the method is to examine fecal samples obtained from humans and select those with high concentrations of Lactobacillus.

These samples are next examined using a method designed to isolate strains having the properties set forth above in the Summary of the Invention, which may be briefly mentioned as follows: (1) acid resistance 20 (2) bile resistance (property by the Depart ment of Food Science, North Carolina State University, has identified as a desirable property) (3) the ability to sensitively adhere to human intestinal mucosal cells 25 (4) lactic acid production (5) sustained in vitro growth.

Acid-bile resistance

Screening for bile resistance as well as stability in a somewhat acidic environment is performed as follows. Prepare LBS agar (described in J. Bacteriol. 62 (1951) 132) having the following composition per liter: 4 95193

Tryptose and peptone 10.0 g

Yeast extract 5.9 g

Ammonium citrate 2.0 g

Monopotassium phosphate 6.0 g 5 Dextrose 20.0 g

Polysorbate-PO 4 1.0 g

Sodium acetate hydrate 25.0 g

Magnesium sulphate 0.575 g

Manganese sulphate 0.12 g Ferrous sulphate 0.034 g

Agar 15.0 g To 84 g of the dehydrated powder of the above composition are added 30 ml of tomato juice and 800 ml of distilled water, followed by stirring with 1.32 ml of acetic acid. 1.5 g of bovine gallbladder bile is then added and the pH is adjusted to 5.5 using 1 N HCl. The plates are stored in an anaerobic chamber for 1-3 days before use and then used without autoclaving. Stool samples from healthy individuals are homogenized in water under anaerobic conditions and serial dilutions are incubated on plates for 48 hours in an anaerobic chamber. Bacteria that divide on the plates are selected and further screened. This step removes many of the microorganisms present in the sample.

Screening for resistance under acidic conditions is performed using MRS broth (Difco, described in J. Applied Bacteriol. 23 (1960) 130) having the following composition: 95193 5

Bacto protease peptone # 3 10.0 g

Bacto meat extract 10.0 g

Bacto yeast extract 5.0 g

Dextrose 20.0 g 5 Tween 20 1.0 g

Ammonium citrate 2.0 g

Sodium acetate 5.0 g

Magnesium sulphate 0.1 g

Manganese sulphate 0.05 g Disodium phosphate 2.0 g 55 g of the above composition, anhydrous, are mixed with 1000 ml of distilled water and sterilized by autoclaving for 15 minutes at 6.89 kPa. The final pH of the medium is 6.5 at 25 ° C. The pH is then adjusted using 1.0 N HCl and, if necessary, 1.0 N NaOH, and the medium is sterilized by filtration. The bacteria are incubated in the medium for 48 hours in an anaerobic chamber. The selected bacteria may preferably divide at a pH below 3.5, more preferably at pH 3.0. 20 Screening of L. acidophilus

The next screening step is to isolate bacteria of the species L. acidophilus from bacteria that are able to divide in acid and bile. This step is performed using standard microbiological methods.

25 Infectivity

The next step is to study the ability of acid- and bile-resistant L. acidophilus bacteria to adhere to gastrointestinal cells. First, human gastrointestinal cells are isolated by any suitable method. To a technology included in the assembly out of the flow of a fluid from a patient, who has a functional opening of the ileum, which is flushed with saline.

The effluent saline solution is collected in cold buffer I (0.1% gelatin, 1% glucose, 500 mM sodium phosphate, pH 7.4) in a 1/10 volume. Then this 6 95193

the solution is filtered through a No. 25 silk to remove clumps and -jäänteiden, and ileal mucosa cells collected by centrifugation at 100 x g for 10 minutes. The cells are then washed in buffer II (0.45% NaCl, 0.1% glucose, 0.01% gelatin, 50 mM sodium phosphate, pH

7.4) using the same centrifugation conditions. Finally, the cells are resuspended in buffer II, 1/3 volume of tissue culture medium NCTC 135 is added, and the cells are stored at 4 ° C until use. 10 ileal cell number and viability was determined by Trypan Blue of staining. The human ileal cells can be cooled with liquid nitrogen, followed by the addition of 10% (vol./vol.) Glycerol.

The bacteria are grown for 18 hours at 37 ° C in 15 MRS medium, 6 μl of 3 H-alanine and 3 H-leucine added with η. The bacteria are then washed with 5 volumes of phosphate buffered saline, resuspended in buffer II and filtered 2.0 μ: the plexiglas-tisuodattimien through 20 to remove clumps of bacteria was then mixed with the human small intestinal cells, incubated at 37 ° C. and samples are taken 5 minutes later, the samples on 2.0 μ. the polycarbonate-by, and the excess solution is removed by aspiration with care The filters are then rinsed, first with 2 ml, then with 5 ml of phosphate buffered saline (pH

7.4). Using these filters intestinal cells and the bacteria remain attached to abstain, but the free bacteria go through. Controls include the reaction mixture without the human small intestinal cells. Retained radioactivity 30 is determined with a liquid scintillation counter. The specific activity of bacterial origin is determined from the colony-forming units of the optical density at 550 nm and from the radioactivity retained by the 0,2 μ filters. Bacterial-specific activity is used to count the number of bound bacteria.

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Bacteria that bind according to the above method at a rate of at least 50 and preferably 100 bacteria per gastrointestinal cell (representing about 10% of the bacteria in contact with the cells) are the desired bacteria.

Production of lactic acid

Lactic acid production is measured by incubating the bacterium in MRS medium or Scott Peptone Yeast Broth broth under anaerobic conditions and measuring the lactic acid after 8 or 24 hours using gas-liquid chromatography. The screened bacteria produce at least 3.5 meq. and preferably 4 meq. lactic acid per 1010 CFU at 37 ° C for 24 hours.

Sustainable Growth The final screening step concerns the selection of bacteria that have sustained growth in vitro, as measured by cell proliferation. 5 x 104 CFU of bacteria are inoculated into a culture medium, for example MRS broth, and the CFU is measured after 1, 2, 3, 4, 5, 6, 7 and 8 hours by plating 0.1 ml dilutions on LBS agar and incubating the plates. For 48 hours at 37 ° C under anaerobic conditions. Bacteria with a distribution time of less than one hour or preferably about 45 minutes at 37 ° C are selected.

Principles of Screening 25 Each step of the above screening method is designed to isolate a strain of L. acidophilus that has properties suitable for the purposes of the invention.

Acid and sapenkestävyys are important, because the bacteria are ingested and must pass through the stomach and small intestine Happa-30 systems, as well as the bile-containing conditions, and they must be able to remain alive and to divide these conditions.

Adherence is important because this property allows L. acidophilus to settle and attach to the gastrointestinal tract.

β 95193

The production of lactic acid is important because it is believed that the lactic acid produced by the bacteria is partly responsible for the beneficial effects of the establishment of the strain in question in the gastrointestinal tract.

5 Sustainable growth in vitro is an important property because bacteria must be prepared on a large scale; sustainable growth is also an indicator that bacteria will credibly grow well in vivo as well.

Properties of Strain GG The strain of the invention, designated GG, has the following properties.

Haponkestävvvs

Strain GG can grow at pH 3.0 up to a concentration of 1 x 107 CFU. At higher pH values, the increase is even greater, reaching 109 CFU at pH 5.0.

In normal human gastric fluid, the GG strain, inoculated with 108 CFU inocula, can live with a reduction in inoculum size of less than 2 log for at least 30 minutes at pH 2.5.

20 GG strain bacteria (109 CFU) were inoculated

Along the Levine tube directly into the stomach of a healthy volunteer. At a pH of 1.0 to 2.0 in the gastric contents, at least 100 GG organisms survived for at least two hours.

Bile-resistant GG strain can grow in bile gallbladder bile, 0.15%, on LBS agar.

Adhesion of the intestinal mucosal cells

The below table indicates the position of ileum GG-30 intestinal mucosal cells by the extent of adherence.

The contact time of the intestinal cells bind a number of bacteria command element 35 for 5 minutes 119 10 minutes 235 15 minutes 333 i; lU: i mi! i l iti 9 95193

Production of lactic acid

Lactic acid production was measured by gas-liquid chromatography after incubation of the GG strain in liquid medium. Subsequent concentrations of lactic acid-5 were measured at the given incubation times.

Incubation time Lactic acid yield

8 hours 1.53 meq / 108 CFU

24 hours 4.44 meq / 1010 CFU

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Cell proliferation time The GG strain showed sustained growth in vitro. Using an initial inoculum of 5 x 10 4 CFU in MRS broth, the strain showed a 40-fold increase in growth over a four-hour 15 period, reaching a density of 2 x 10 6 CFU. Thus, during this four-hour period, 5.2 cell divisions occurred, so the division time became 45 minutes.

Use

The strain of the present invention can be grown on a large scale and can be added to foods, especially dairy products such as milk and yoghurt, as has been the case with other L. acidophilus strains for many years. The bacteria of the strain in question can be ingested orally, for example, 106 to 1010 bacteria per person per day. The cultivation conditions are as follows.

The stock solution comprises sterile skim milk containing 1010 CFU of living organism. 1 ml of this stock solution is added to two liters of MRS broth. Vessel 30 is placed in an anaerobic incubation chamber and incubated overnight for 14 hours with agitation at 37 ° C. The meat broth is centrifuged at 10,000 x g for 20 minutes and the pellet is resuspended in 50-100 ml of sterile skim milk and stored at -80 ° C.

10 95193

In a particular mode of administration, 10 to 1010 organisms are added to the milk, either in a volume of 10 to 20 ml or in larger volumes, for example 250 to 500 ml. The milk is administered once daily with a single dose of 5,109 to 1010 CFU. For the animals, 0.2 to 1.0 ml of the stock milk solution is mixed with their food, so that they receive 10 to 10 CFU daily.

Side effects of antibiotic treatment

Diarrhea is the major complication of antibiotic therapy, occurring in approximately 10% of patients receiving antibiotics, especially ampicillin, amoxacillin, cephalosporins, clindamycin, erythromycin, and tetracycline. The strain of the present invention can be used to treat antibiotic-induced diarrhea in the same manner as other L. acidophilus preparations have been used in the past. In addition, the strain can be used before or concurrently with antibiotic treatment, for prophylaxis. Such prophylactic administration should occur 24 hours prior to antibiotic treatment.

20 Constipation

Slow bowel action and chronic constipation are the main problems in older people. Constipation leads to bloating, convulsions, and general malaise. The public interest on the promotion of intestinal 25 good activity is reflected in the product line, which is available in pharmacies in this state in order to facilitate tamiseen. These products range from raw excipients to short chain fatty acids, various natural fiber products such as pectins, lignins, gums and mucilages.

Cultured dairy products, yogurts and soured milk have long been used to relieve chronic constipation.

The bacteria of the L. acidophilus 35 strain of the present invention can be used alone, as described above.

Il! IKK 1111 M-i JE ». 95193, or may be administered in admixture with a non-therapeutic dose of lactose, a synthetic disaccharide used to treat constipation. Bacterulose is metabolized by bacteria, which increases their beneficial effects without the side effects that are sometimes associated with therapeutic doses of lactulose.

Resistance to colonization

In recent years, it has become apparent that the establishment of bacteria in the human gastrointestinal tract provides light for the spread of pathogenic microorganisms, especially in hosts with an impaired immune response. The intestinal microflora of healthy individuals can resist the attachment of unwanted microorganisms through a number of internal control mechanisms. Although these control mechanisms are poorly understood, the overall result is the relative Stability of the human intestinal lobe, so that the same bacterial types and the same concentrations are maintained for long periods of time.

Through the effect of antibiotic treatment, undesirable microorganisms are able to settle in the gastrointestinal tract. Among the bacteria, these undesired bacteria include Pseudomonas, Enterobacter, Serratia, Klebsiella, Citrobacter, and Proteus-Providensia. Fungi can also settle in the gut, especially hii-25 vat, such as Candida.

Although healthy individuals may resist the introduction of bacteria into the gut even during antibiotic treatment, hosts with impaired immune resistance are at serious risk in these conditions. These non-active microorganisms can penetrate the mucosa, causing sepsis as well as life-threatening disease.

Maintaining a natural resistance to the overgrowth of unwanted microorganisms is known in the 35 most recent scientific literature as the term "colo- 12 95193 nization resistance" (CR). the increase is achieved by the use of antibiotics which produce only minimal effects on the composition of the normal intestinal flora, in particular by maintaining normal anaerobic components which appear to be responsible for controlling the overgrowth of unwanted microorganisms. all new entrants to the microflora.This method produces a "sterile" microflora, at least for a short time.

15 Although each of these anti-colonization methods has its advantages, they may not be effective. Relatively few therapeutic antibiotics are able to maintain CR, so this procedure is often roe-free. Another method associated with the administration of oral antibiotics 20 produces many of its own side effects and is often ineffective because highly resistant microorganisms can settle in the gut even in the presence of antimicrobials. Thus, the colonization of unwanted microorganisms in either adult or pediatric hosts with an impaired immune response remains a major problem in modern medicine.

The bacteria of the L. acidophilus strain of the present invention can be administered as described above, in a manner useful for inducing CR, since the bacteria do not have an inherent pathogenicity. Colonization of such a natural organism in the intestine could prevent other pathogenic forms from entering the flora, while at the same time maintaining relatively healthy intestinal conditions.

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Estrogen excretion

Improvements in estrogen measurement technology have expanded data on these important sex hormones. Estrogens are highly formed in the ovariums, to a lesser extent in adipose tissue and other tissues. Estrogens are excreted mainly in the urine.

An important development in recent years concerns the understanding of the enterohepatic circulation of estrogens. It has been shown that half of the circulating estrogen circulates through the bile in the intestinal tract, where it is reabsorbed into the body. Only 5% of estrogens are excreted in the faeces.

To be excreted in the bile, estrogens are conjugated, usually as glucuronides or sulfonates. These conjugated estrogens pass through the bile into the intestine, where they are deconjugated by the action of intestinal bacteria and then reabsorbed.

The importance of the enterohepatic circulation was demonstrated in studies by Adlercreutz et al., Which examined fecal and urinary estrogens in women who received ampicillin against a minor infection. This oral antibiotic caused a 30% decrease in urinary estrogen excretion and an associated increase in faecal estrogen excretion. The explanation is that the antibiotic suppressed the bacteria in the gut that were responsible for the deconjugation of estrogens. This resulted in decreased small intestinal reabsorption and increased estrogen excretion.

The bacteria of the L. acidophilus 30 strain of the present invention can be administered as described above to menstruating women to reduce the amount of estrogen secreted; Ingestion of L. acidophilus affects the same deconjugating enzymes that affect estrogen reabsorption.

14 95193

Deposits L. acidophilus strain GG has been deposited with the American Type Culture Collection, Rockville, MA, under accession number ATCC 53103.

Other embodiments are included in the following claims.

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Claims (2)

95193 1. Lactobacillus acidophilus-stam ATCC 53103 for therapeutic use. 1. Lactobacillus acidophilus ATCC 53103 strain for non-therapeutic use. 2. Use of Lactobacillus acidophilus strain ATCC 53103 for the manufacture of foodstuffs, in particular dairy products. 10 2. An invention for Lactobacillus acidophilus strain ATCC 53103 which contains a specific product.