JP2014513058A - Indirect substrate for microorganisms that metabolize 1,2-propanediol - Google Patents

Abstract

  The present invention generally relates to improved activity of certain probiotics. Increased efficiency is obtained through the use of specific substrate components that indirectly provide a source of energy specific to probiotics. These substrate components are specifically designed to stimulate 1,2-propanediol production. Substrates are exemplified as rhamnose, fucose, pectin with a high percentage of rhamnose, and fucoidan with a high percentage of fucose.

Description

  The present invention relates generally to improving the activity of certain probiotics in mammals. The present invention further relates to a preparation comprising a substrate component and a specific probiotic, wherein the substrate component is specifically designed to improve the efficiency of the probiotic. Selecting the substrate component, most Lactobacillus reuteri strains produce 1,2-propanediol that can be used independently as an energy source and / or as an external electron acceptor.

  The United Nations Food and Agriculture Organization defines probiotics as “live microorganisms that give the host a health benefit when administered at the right dose”. Currently, several different bacteria are used as probiotics, for example lactic acid bacteria such as Lactobacillus and Bifidobacterium strains.

  The effectiveness of probiotics is strain specific and each strain can contribute to the health of the host through different mechanisms. Various probiotics prevent or inhibit the growth of pathogens, suppress the production of virulence factors by pathogens, regulate immune responses pro-inflammatory or anti-inflammatory, and in a number of other ways Can affect the host.

  Prebiotics “have a beneficial effect on the host by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the colon that can improve the health of the host. The “digestible food ingredient to give” is defined. Prebiotic targets are usually Bifidobacterium and Lactobacillus, but prebiotics are often not selective, so stimulation of only beneficial genus or probiotic strains is difficult to perform obtain. Because it is difficult to find prebiotics that are selective for a particular probiotic, we have indirectly directed energy sources and / or external electron acceptors to a particular probiotic to increase energy yield. To use specific substrate components (SSCs) that are supplied manually.

  Lactobacillus reuteri is a hetero-fermenting lactic acid bacterium that is frequently found in the gastrointestinal tract of humans and other animals. Lactobacillus reuteri is thought to be an endemic organism of the human gastrointestinal tract and is present, for example, on the mucosa of the stomach, gastric sinus, duodenum, and ileum. See, for example, U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. . When Lactobacillus reuteri cells grow under anaerobic conditions in the presence of glycerol, they produce an antibacterial substance known as reuterin (β-hydroxypropionaldehyde). The ability to generate reuterin is due to the propanediol utilization (pdu) operon. The pdu operon is a metabolic mechanism that also allows growth on 1,2-propanediol (PD). This pdu operon is a very important feature of Lactobacillus reuteri when utilizing bacterial colonization and full capacity of humans. This mechanism is rare among other Lactobacillus, so bacteria that do not have a pdu-mechanism cannot grow on 1,2-PD and they use 1,2-PD as an electron acceptor I can't do that either.

  Various Lactobacillus reuteri strains colonize the intestine, act as diarrhea drugs, regulate gastrointestinal motility, function as inhibitors of bacterial pathogens, immunomodulate the gastrointestinal mucosa, stomach It has the ability to function as an anti-inflammatory agent.

  In the patent application WO2009 / 151391, the pdu mechanism of Lactobacillus reuteri is stimulated with 1,2-PD or glycerol before lyophilizing the bacteria. In this manufacturing design, the lyophilized pdu mechanism of Lactobacillus reuteri is stimulated with the ability to produce and store reuterin.

  Emma Arskold et al., “The Phosphoketolase Pathway Dominates in Lactobacillus Reuteri ATCC55730 Containing Lactobacillus Reuteri ATCC55730” 206-212) describes that the growth ability of Lactobacillus reuteri on glucose can be improved by adding fructose as an external electron acceptor. However, in this paper, a method for selecting specific SSCs based on their ability to indirectly supply 1,2-PD, which is only available by bacteria with a pdu-mechanism, to specific probiotics. Is not taught at all.

  A common problem with oral administration of probiotic bacteria is an insufficient amount and / or activity of the probiotic bacteria at the gastrointestinal tract site where they exert their effect. This can result in increased doses of probiotic bacteria and / or may require more frequent administration, and may result in loss of activity. This leads to unnecessary costs, undesirable frequency of consumption and / or reduced health benefits. In the present invention, for example, the local amount and / or metabolic activity of Lactobacillus reuteri is increased, resulting in the possibility of reducing the dose of probiotics, for example, and further site-specific health benefits are conceivable.

  1,2-propanediol (1,2-PD) is an energy source that can be produced locally by other coexisting microorganisms, possibly combined with other sugars by certain probiotic species such as Lactobacillus reuteri Used. The inventors of the present invention surprisingly stimulated these coexisting bacteria to produce 1,2-PD by orally administering a highly selective SSC, thereby producing Lactobacillus reuteri. It has been discovered that it is possible to indirectly improve the activity of organisms utilizing 1,2-PD such as.

  Pectin is a polysaccharide derived from the plant cell wall. Various pectic polysaccharides including homogalacturonan (HG), xylogalacturonan (XGA), apiogalacturonan, rhamnogalacturonan I (RGI), and rhamnogalacturonan II (RGII) Can be detected in the cell wall. The proportion between HG, XGA, RGI and RGII varies, but typically HG is the most abundant polysaccharide comprising about 65% of pectin, while RGI comprises 20% to 35%. XGA and RGII are minor components, each constituting less than 10%. Different pectic polysaccharides are not separate molecules, but are covalently linked domains. L-rhamnose is found as a component in the pectin structures RGI and RGII. L-fucose is also found as a component in the RGII structure. Bacteria found in the GI tract that can convert L-rhamnose or L-fucose belong to the genus Bacteroides and Enterobacteria, including, for example, E. coli.

  Pectin is resistant to digestion in the human body but is broken down into sugars and then further metabolized by bacteria in the small intestine and colon, for example, 1,2-propanediol. Pectin stimulates bacterial growth in the small intestine and in the colon. Pectin used as a treatment for diarrhea is associated with an improved intestinal environment and is also known to have anticancer properties. Modified citrus pectin (MCP) is citrus pectin that has been broken down into small complex molecules and is used to support cell growth and proliferation.

  Fucoidan is a sulfated polysaccharide found mainly in various species of brown algae and brown seaweeds such as mozuku, kombu, rim, moui, hibamata, wakame and hijiki. Has been found in. Galactooligosaccharides (GOS) generally contain a series of galactose units resulting from a continuous transgalactosylation reaction with terminal glucose units and are classified as prebiotics.

  Lynch MB et al. "The effects of dietary laminaria and fucoidan on nutrient digestibility, nitrogen availability, intestinal microflora and volatile fatty acid concentrations in pigs. , Intestinal microflora and volatility fatty acid conigration in pigs) "(J Sci Food Agric. Feb 2010; 90 (3): 430-7) Proximal colon and distal I have found that it has a large amount of Lactobacillus subspecies in the intestine. Thus, it is suggested that fucoidan can be an edible means for improving intestinal health in pigs. A large amount of Lactobacillus population in stool caused by fucoidan diet is also described in J. Org. V. Discovered by O'Doherty et al. "The effect of dietary laminarine and fucoidan diet of the pe- lective pe- valently per captive and selected selenium of the stool of the stool and the fucoidan diet." ).

  However, for example, based on the amount of L-rhamnose and / or L-fucose, select pectin and fucoidan, or fractions thereof, to produce 1,2-PD by bacterial fermentation, and high deoxy sugar content Such a composition having a high amount of L-rhamnose and / or L-fucose content is used to produce large amounts of 1,2-PD, whereby most other microorganisms lack the pdu mechanism. It has not previously been known to indirectly supply energy sources and / or external electron acceptors that are not available to certain micro-organisms such as Lactobacillus reuteri.

  The patent application WO2010 / 117274 relates to carbohydrates that can induce a detectable increase in C5 and / or C6 short chain fatty acids (SCFA). SCFA has a positive effect on the health of the gastrointestinal tract to be treated. The carbohydrates used include pectin. Even when selecting pectin that can contain trace amounts of rhamnose, to indirectly supply specific energy sources and / or external electron acceptors to probiotics with a pdu mechanism, thereby improving their activity L-rhamnose or L-fucose does not disclose selection and use of specific pectin in large amounts.

  U.S. Patent No. 7,101,565 relates to a composition comprising prebiotics and probiotics. Prebiotics can include pectin or pectic polysaccharides. However, a method of selecting a specific pectin, or a combination of pectin and L-rhamnose or L-fucose, which results in high amounts of 1,2-PD in the gastrointestinal tract, which is beneficial for probiotics with a pdu mechanism Is not disclosed in this invention.

  US Pat. No. 5,902,578 discloses an invention relating to a method for preventing diarrhea associated with infectious agents such as rotavirus, or diarrhea associated with antibiotic therapy through the use of Lactobacillus. However, in this invention, Lactobacillus is not related to additional SSC for better efficiency.

  By administering SSC together with probiotics, such as Lactobacillus reuteri, supplying specific energy sources and / or external electron acceptors indirectly to such probiotics, No one has ever disclosed a method for improving health benefits. Oral administration of SSC with large amounts of L-rhamnose and / or L-fucose ensures the supply of 1,2-propanediol, thus indirectly providing energy sources and / or external electron acceptors for certain prebiotics. Supply. This locally increases the amount of health-promoting microorganisms, such as Lactobacillus reuteri, leading to better efficiency and realizing site-specific effects.

  For example, the use of pectin with probiotics has been known for some time, but for indirectly supplying specific energy sources and / or specific external electron acceptors to specific probiotics, 1 , 2-SPD selection methods based on their ability to form PD have not previously been known.

  The present invention discloses methods for enhancing the activity of certain probiotics, as well as the manufacture and use of products comprising substrate components and optionally probiotics. The product of the present invention can be used to improve the activity of Lactobacillus reuteri, for example in mammals. This product can be used to improve host health. Depending on the probiotic strain used, use the product, for example, improve gastrointestinal health, improve immune-related health, treat and / or prevent diarrhea and constipation, normalize stool viscosity Can improve gastrointestinal motility, treat and / or prevent infectious diseases, modulate inflammatory and anti-pathogenic effects.

  The high efficiency of probiotics can be obtained by stimulating the coexisting micro-organisms to produce 1,2-propanediol (1,2-PD). Coexisting micro-organisms are stimulated with a specific specific substrate component (SSC) as described herein. SSC ensures the presence of 1,2-PD in the gastrointestinal tract and indirectly supplies 1,2-PD to certain beneficial organisms.

  The ability to utilize 1,2-PD as either an energy source and / or as an external electron acceptor is unique to bacteria with a pdu-mechanism, so administration of SSC only improves the activity of certain probiotics Let

  In order to improve the activity of cobiotic probiotics, SSC can be administered together with the probiotics. It is also possible to administer SSC alone, for example to increase the activity of a previously administered probiotic.

  1,2-PD, administered alone or produced by SSC, can be further combined with galactooligosaccharides (GOS) or other galactose-containing sugars to provide an even better source of energy for the microorganism.

  Heterotype fermenting lactic acid bacteria produce lactic acid, ethanol and carbon dioxide using the phosphoketolase pathway (PKP). PKP has a poor energy yield compared to the Emden-Meyerhof pathway (EMP). This drawback can be offset by adding an external electron acceptor.

  The inventors have surprisingly ensured that the presence of 1,2-propanediol in the gastrointestinal tract simultaneously supplies the probiotic with a suitable external electron acceptor, thus probiotics. It has been discovered that the activity of tics is improved.

  The SSC of the present invention selectively increases the growth of hetero-type fermenting lactic acid bacteria such as Lactobacillus reuteri. This is because they give the bacteria an electron acceptor that is suitable to allow increased activity.

  Lactobacillus reuteri relies on an electron acceptor suitable for growth in a particular environment, and we surprisingly find that 1,2-propanediol acts as a suitable electron acceptor, It has been found that it can be supplied by administration.

  According to one aspect of the present invention, there is provided a method for improving the activity of a probiotic bacterium having a pdu-operon in the gastrointestinal tract of an individual comprising administering the substance to the individual, Methods are provided that have the ability to be metabolized to 1,2-propanediol in the gastrointestinal tract of an individual.

  In one embodiment of the method, the substance comprises a deoxy sugar, which has the ability to be metabolized to 1,2-propanediol in the gastrointestinal tract of said individual. In one embodiment, the deoxy sugar is rhamnose or fucose.

  In one embodiment of the method, the substance is (a) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, (f Including fucoidan having a high proportion of fucose, or (g) a combination of rhamnose, fucose and pectin.

  In one embodiment of the invention, a pectin having a high percentage of rhamnose is 5-15% rhamnose, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% rhamnose. Is defined as including. In one embodiment of the invention, fucoidan having a high percentage of fucose is defined as including more than 15% fucose.

  In one embodiment of the method, the substance is administered concurrently with a bacterium having a pdu operon.

  In a preferred embodiment of the invention, the substance is administered orally to the individual.

  In another preferred embodiment of the method, the bacterium having the pdu-operon comprises Lactobacillus reuteri.

  In one embodiment of the method, the substance is administered to the individual in a daily dose of 0.25 to 25 g, preferably 1-2 g.

  In one embodiment of the invention, the method further comprises co-administering a galactooligosaccharide or other saccharide comprising galactose.

  According to a second aspect of the present invention, there is provided a substance for use in enhancing the activity or increasing the proliferation of a probiotic bacterium having a pdu-operon in the gastrointestinal tract of an individual, the gastrointestinal tract of the individual A substance comprising a deoxy sugar having the ability to be metabolized to 1,2-propanediol is provided.

  In one embodiment, the deoxy sugar is rhamnose or fucose.

  In one embodiment of the invention, the substance is (a) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, ( f) fucoidan with a high proportion of fucose, or (g) a combination of rhamnose, fucose and pectin.

  In one embodiment of the invention, a pectin having a high percentage of rhamnose is 5-15% rhamnose, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% rhamnose. Is defined as including. In one embodiment of the invention, fucoidan having a high percentage of fucose is defined as including more than 15% fucose.

  In a preferred embodiment of the substance, the bacterium having the pdu-operon is Lactobacillus reuteri.

  In one embodiment of the invention, the substance is administered to the individual in a daily dose of 0.25-25 g, preferably 1-2 g.

  In one embodiment of the invention, the substance is for use in combination with galactooligosaccharides or other sugars including galactose.

  According to a third aspect of the present invention, there is provided a composition comprising (i) a bacterium having a pdu-operon and (ii) a substance containing deoxy sugar, wherein the deoxy sugar is 1,2- Compositions having the ability to be metabolized to propanediol are provided.

  In one embodiment, the composition comprises (a) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, (f Including) a fucoidan having a high proportion of fucose, or (g) a bacterium having a pdu-operon in combination with a combination of rhamnose, fucose and pectin.

  In one embodiment of the invention, a pectin having a high percentage of rhamnose is 5-15% rhamnose, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% rhamnose. Is defined as including. In one embodiment of the invention, fucoidan having a high percentage of fucose is defined as including more than 15% fucose.

  In another embodiment, the composition further comprises other sugars including galactooligosaccharides or galactose.

  In one embodiment relating to the composition, the bacterium having the pdu-operon is Lactobacillus reuteri.

  In one embodiment of the composition, the substance is present in an amount, such as an amount that provides a daily dose of 0.25 to 25 g, preferably 1-2 g.

  According to another embodiment of the present invention, the composition described above is for use in enhancing the activity or increasing the proliferation of probiotic bacteria having a pdu-operon in the gastrointestinal tract of an individual. .

FIG. 4 is a graph showing the growth of Lactobacillus reuteri DSM 17938 in modified MRS (without glucose and citrate) with 1,2-PD, galactose and combinations thereof added.

  Certain probiotics, particularly the Lactobacillus reuteri propanediol utilization (pdu) mechanism, allows growth on 1,2-propanediol (1,2-PD) as an energy source, and an external electron acceptor Enables the ability to use 1,2-PD.

  The inventors of the present invention surprisingly used 1,2-PD, which can be utilized as an energy source or as an external electron acceptor using a specific specific substrate component (SSC). We have discovered ways to improve the activity of certain probiotics by indirectly supplying them to various probiotic organisms.

  SSC is a substance. According to one embodiment of the invention, this substance is a substrate. In one embodiment, the material consists of only one component. In another embodiment, the material includes two or more components.

  Oral administration of these carefully selected SSCs stimulates coexisting microorganisms to produce 1,2-PD, thereby as an energy source for certain microorganisms having a pdu-mechanism such as Lactobacillus reuteri or external 1,2-PD is generated locally that can act as an electron acceptor. SSCs can be administered alone or together with probiotics.

  As can be seen in FIG. 1, the presence of 1,2-PD improves the growth status for certain microorganisms, and we further improve this in the presence of galactooligosaccharide (GOS) or galactose. You have found that you can.

  The SSCs of the present invention select for their ability to indirectly supply specific probiotics with 1,2-PD that can be used as an energy source or as an external electron acceptor. The inventors of the present invention have found that SSCs containing large amounts of L-rhamnose or L-fucose are most effective when used to improve the activity of certain probiotics. Therefore, the SSC used in the present invention is carefully selected with respect to the amount of L-rhamnose and L-fucose.

  Specific fractions of pectin, such as rhamnogalacturonan I and II, preferably containing a high proportion of L-rhamnose, can be used as SSC. These preferred pectin fractions preferably contain 5-15% rhamnose. These pectin fractions, when degraded, yield more rhamnose than unfractionated pectin, which can be referred to herein as pectin, normal pectin, or normal pectin. A particular daily dose, eg 2 g of such a preferred pectin fraction thus produces a large amount of 1,2-PD compared to the same daily dose (2 g) of normal pectin. Pectin can also be used normally when administered indirectly with L-rhamnose or L-fucose in the same manner that favors certain micro-organisms such as Lactobacillus reuteri. In addition to 1,2-PD, this combination also supplies other substrates such as galactose, arabinose and galacturonic acid to certain probiotics as a result of pectin degradation. The inventors of the present invention, as can be seen in FIG. 1, 1,2-PD in combination with certain pectin components, such as galactose, arabinose and galacturonic acid, are not suitable for certain probiotics such as Lactobacillus reuteri. It has been shown to produce a synergistic effect that improves the use of 1,2-PD.

  Certain fractions of fucoidan, preferably fucoidan containing a large amount of L-fucose, can be used as SSC in the present invention, and these fucoidan fractions preferably contain more than 15% fucose. These fucoidan fractions, when decomposed, usually give more fucose than fucoidan and thus produce a greater amount of 1,2-PD.

  L-rhamnose or L-fucose alone can also be used alone as the SSC of the present invention.

  Other SSCs, such as gums and other polysaccharides, can be used according to the present invention if they contain L-rhamnose, L-fucose, and the like. The gums include but are not limited to Karaya gum and gum arabic. In addition, several human milk oligosaccharides (HMOs) derived from human breast milk can be used as SSCs in the present invention.

  Administration of the SSC of the present invention, either alone or in combination with probiotics such as Lactobacillus reuteri, ensures the supply of energy sources for the particular probiotic and / or increases the energy yield. Ensures the presence of the receptor, thus improving the local activity and efficiency of the probiotic.

  In other embodiments, and to support this effect, it is further possible to add GOS or galactose to increase the ability of Lactobacillus reuteri to utilize 1,2-PD.

  Depending on the intended indication, colonize the intestine, act as a diarrhea treatment, regulate gastrointestinal motility, function as an inhibitor of bacterial pathogens, immunomodulate the gastrointestinal mucosa, stomach Several Lactobacillus reuteri strains with different abilities such as functioning as anti-inflammatory agents in can be used in the present invention herein.

  Pectin, L-rhamnose and L-fucose are resistant to digestion in the human body, but are broken down into sugars and then found in the human body to certain parts of the human GI tract, such as the stomach, stomach sinus, duodenum, and It is further metabolized, for example, to 1,2-propanediol by coexisting micro-organisms available on the small intestinal mucosa. Thus, the invention herein can also promote site-specific effects in the human GI tract. For example, by using a Lactobacillus reuteri strain selected as a probiotic, it is possible to improve the anti-inflammatory effect of this strain in the ileum.

  For example, in humans with highly disturbed microflora, which can convert SSC to 1,2-PD and lack the coexisting micro-organisms normally found, along with certain strains of Lactobacillus reuteri, Ensuring the effectiveness of the SSC to actively deliver and administer such coexisting micro-organisms such as Lactobacillus rhamnosus to the recipient when using the specific SSC system described in Is another possibility of the invention herein.

  Products comprising the SSC of the present invention alone or in combination with specific probiotics are preferably formulated as tablets, capsules, powder sachets and the like. The product may be a food supplement, a pharmaceutical product, and the like. For such products, the amount of probiotic supply must be sufficient to bring about the desired effect of a specific strain, and the improvement effect by SSC is currently considered. Such levels are not limited to 10E + 4 CFU to 10E + 11 CFU per day, but are typically in this range, and preferably in the Lactobacillus reuteri range of 10E + 6 CFU to 10E + 9 CFU.

  The amount of SSC should be in the range of 0.25 to 25 grams (g) per day when using pectin with high amounts of L-rhamnose and / or L-fucose. When using another L-rhamnose and / or L-fucose and normal pectin combination, the total amount of SSC should be in the range of 0.25-25 g per day. In any case, the daily dose of 0.25 to 25 g is for example 0.25, 0.5, 1, 1.5, 2, 5, 10, 15, 20 or 25 grams, preferably 1-2 grams. 1, 1.25, 1.5, 1.75 or 2 grams, etc. The ratio between normal pectin and any one of L-rhamnose or L-fucose, or combinations thereof is 95: 5 to 0: 100, but preferably 80:20 to 20:80, and even more preferably A range of 70:30 is better.

  Another option in using the invention herein is that SSC and probiotics may be one or more times after the initial supply, supplying only SSCs in a type of shuttle program to reduce treatment costs. Are alternately supplied together.

  It is essential for the present invention that the probiotics used have a pdu-mechanism. This is because the ability to use 1,2-PD as an energy source and / or as an external electron acceptor is essential. Thus, in another embodiment of the invention, during the generation of a probiotic strain, the probiotic pdu mechanism is stimulated with 1,2-PD for improved efficiency. This is done by adding 1,2-PD or glycerol and possibly cobalt or vitamin B-12 at the start of the fermentation step during bacterial culture (vitamin B-12 and cobalt are important for producing reuterin. For). With this manufacturing design, lyophilized bacteria for use in the next step are appropriately prepared to activate the pdu-mechanism more quickly. This improved efficiency of the pdu mechanism thus increases the efficiency of the administered SSC of the present invention. Another way to improve the efficiency of the administered SSCs is to combine them with GOS or galactose, and the inventor has shown that the combination of 1,2-PD and galactose is expected to grow Lactobacillus reuteri. It shows that there are benefits outside.

  One skilled in the art would readily conceive of numerous modifications and changes, and thus does not wish to limit the present invention to the exact construction shown and described, and thus all suitable modifications and equivalents are also It is within the scope of the present invention as defined by the appended claims.

(Example 1. Production of a sachet containing a composition of Lactobacillus reuteri and pectin, rhamnose and galactooligosaccharides)
The composition consists of:
Lactobacillus reuteri DSM 17938: 10E + 8 CFU / sachet pectin (GENU® pectin (citrus) type USP / 200, CP Kelco France SARL, France): 840 mg / sachet L-rhamnose: (rhamnose monohydrate L-(+ ), Kaden Biochemicals GmbH, Hamburg, Germany) 360 mg / sachet GOS15 (VIVINAL®, Friesland Campina Domo, Netherlands) 800 mg / sachet

  The composition is at ambient temperature on a LAF bench (Heto-Holten A / S, Holten Laminair Model S-2010 1.2, Denmark) with desiccant (10 cm × 12 cm, packaging material PET12 / PE / ALU12 / PE / PE + Use desiccant / Alcan PE) and fill aluminum foil bags known in the art. To each bag, 2 g of powder containing Lactobacillus reuteri, pectin, L-rhamnose and galactooligosaccharide is added using Denver Instrument GmbH, German Balance XP-600. The filled aluminum foil bag was then emptied by Ketttenbaum Folienschweistechnik GmbH & Co. Heat seal using KG, German film sealing device model F460 / 2.

(Example 2. Production of a sachet containing a composition of Lactobacillus reuteri and rhamnose.)
The composition consists of:
2 g L-rhamnose: (rhamnose monohydrate L-(+), Kaden Biochemicals GmbH, Hamburg, Germany), 10E + 8 CFU Lactobacillus reuteri DSM 17938 per sachet Fill the bag.

(Example 3. Production of a sachet containing a composition of Lactobacillus reuteri and galactooligosaccharide and rhamnose.)
The composition consists of:
1 g GOS15 (VIVINAL®, Friesland Campina Domo, Netherlands) and 1 g L-rhamnose (rhamnose monohydrate L-(+), Kaden Biochemicals GmbH, Hamburg, Germany), 10E + 8 CFU Lactobacillus reuteri per sachet The composition containing DSM 17938 is filled into an aluminum foil bag as in Example 1.

(Example 4. Production of Lactobacillus reuteri strain subjected to initial stimulation)
This example describes the preparation of a lyophilized powder of Lactobacillus reuteri with an activated pdu-mechanism. Therefore, during the production of the sachets of Examples 1 to 3, the Lactobacillus reuteri strain subjected to the initial stimulation can be used.
Composition of fermentation medium Dextrose monohydrate 60g / l
Yeast extract KAV20g / l
Peptone type PS (from pig) 20g / l
2ammonium hydrogen citrate 5g / l
Sodium acetate (× 3H ₂ O) 4.7 g / l
Dipotassium hydrogen phosphate 2g / l
Tween 80 0.5g / l
Silivion (antifoaming agent) 0.14 g / l
Magnesium sulfate 0.10 g / l
Manganese sulfate 0.03g / l
Zinc sulfate heptahydrate 0.01g / l
Water, sufficient amount Centrifugation medium (derived from pig) Peptone O-24 Ortana
Cryoprotectant (bovine derived) lactose 33%
Gelatin hydrolyzate (from bovine) 22%
Sodium glutamate 22%
Maltodextrin 11%
Ascorbic acid 11%

Steps for generating lyophilized Lactobacillus reuteri powder 1. Inoculate 20 ml of fermentation medium with 0.6 ml of lyophilized Lactobacillus reuteri powder from a working cell mass vial. Fermentation is carried out in a vessel at 37 ° C. for 18-20 hours without stirring or pH control, ie in a static state.
2. Inoculate two 1 liter flasks of fermentation medium with 9 ml of cell slurry (from step 1) per liter of medium. Fermentation is carried out at 37 ° C. for 20-22 hours without stirring or pH control, ie static conditions.
3. Two 1 liter cell slurries from step number 2 are used to inoculate a 600 liter container containing 600 liters of fermentation medium. Fermentation is carried out at 37 ° C. for 13 hours with stirring and pH control. The pH is 6.5 at the start of fermentation. pH control is initiated using 20% sodium hydroxide solution when the pH drops below 5.4. The pH control is set to pH 5.5.
4). The fourth and final fermentation step is performed in a 15,000 liter container with inoculation from step number 3. Fermentation is carried out at 37 ° C. for 9-12 hours with stirring and pH control. The pH is 6.5 at the start of fermentation. pH control is initiated using 20% sodium hydroxide solution when the pH drops below 5.4. The pH control is set to pH 5.5. 100 mM glycerol is added at the end of the fermentation, just before the culture reaches the stationary phase. Fermentation is terminated when the culture reaches stationary phase, which can be seen by a decrease in the amount of sodium hydroxide solution added. About 930 liters of sodium hydroxide solution is added to 10,200 liters of medium and 600 liters of inoculum during fermentation.

  The cell slurry from the final fermentation (step 4) is separated twice at 10 ° C. in an Alfa Laval continuous centrifuge. The first centrifugation reduces the cell slurry volume from about 11,730 liters to 1200 liters. This volume is washed with 1200 liters of peptone (Peptone 0-24, Orthona) solution in a 3000 liter container and separated again before mixing with the cryoprotectant (see below). A washing step with peptone is performed to avoid any freezing point depression during the lyophilization process.

  The second centrifugation reduces the cell slurry volume to 495 liters. This volume is mixed with 156 kg of cryoprotectant solution to make about 650 liters of cell slurry.

  The cell slurry is pumped into a 1000 liter container. The container is then moved to the lyophilization plant.

  In a lyophilization plant, exactly 2 liters of cell slurry is poured onto each plate in the lyophilizer. The maximum capacity of the lyophilizer is 600 liters, removing all excess cell slurry volume.

  Lactobacillus reuteri cell slurry has a dry matter content of 18% and is lyophilized for 4-5 days.

  During the lyophilization process, the pressure during the process is between 0.176 mbar and 0.42 mbar. When the pressure reaches 0.42 mbar, the vacuum pump operation is started. A PRT (pressure test) is used to determine when the process is finished. If after 120 seconds the PRT or pressure increase is less than 0.02 mbar, the process is stopped.

(Example 5.1, The combination of 1,2-PD and galactose provides a synergistic effect that improves the activity of Lactobacillus reuteri)
Grow Lactobacillus reuteri DSM 17938 in modified MRS broth supplemented with 1,2-PD (0.3%), galactose (0.3%) or a combination of the two (without glucose and citrate) I let you. Bacteria were grown for 24 hours at 37 ° C. As seen in FIG. 1, Lactobacillus reuteri grown in the presence of both 1,2-PD and galactose surprisingly showed significantly higher growth than growth in another material.

(Example 6. Example of shuttle program to be supplied)
Due to the improved activity of Lactobacillus reuteri induced by SSC as described in this application, the sachet of Example 1 (Sachet A) and Lactobacillus reuteri were removed in the shuttle program, but others were prepared according to Example 1. It is possible to replace the sachet (sachet B). This shuttle program does not reduce the efficiency of Lactobacillus reuteri and can reduce treatment costs.

  Sachet A is administered to the recipient on day 1 and sachet B is given to the recipient on days 2 and 3. This dosing scheme is repeated throughout the treatment period.

(Example 7. In vivo colonization in humans)
A comparison between intestinal colonization with Lactobacillus reuteri alone and intestinal colonization with the same Lactobacillus reuteri administered with SSC is made in clinical trials. Twelve healthy volunteers are divided into two groups (A and B) of 6 participants in each group. Group A is given the powder sachet of Example 1 containing a composition of Lactobacillus reuteri and SSC, rhamnose and galactooligosaccharides in the form of pectin. Group B is given the same Lactobacillus reuteri strain without the aforementioned SSC. Both groups receive 10E + 8 CFU of Lactobacillus reuteri per day for 60 days. Quantitative assessment of intestinal colonization by strains given alone or together with SSC is performed by stool sample examination at the start of the study and after 30 and 60 days of treatment. Count the Lactobacillus reuteri in the stool and compare the amounts in Group A and B stool.

As seen in Table 1, a significant increase in the amount of Lactobacillus reuteri in the stool was observed in patients who received Lactobacillus reuteri together with SSC compared to patients who received Lactobacillus reuteri alone. Seen in Values are given as the average log ₁₀ CFU ± SEM per gram of stool.

Claims (17)

  A method for enhancing the activity of a probiotic bacterium having a pdu-operon in an individual's gastrointestinal tract, comprising administering the substance to the individual, wherein the substance is in the gastrointestinal tract of the individual, The above process having the ability to be metabolized to 2-propanediol.   (A) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, (f) a high proportion of fucose. The method of claim 1 comprising fucoidan having, or (g) a combination of rhamnose, fucose and pectin.   The method according to claim 1 or 2, wherein the substance is administered simultaneously with a bacterium having a pdu operon.   The method according to any one of claims 1 to 3, wherein the bacterium having a pdu-operon comprises Lactobacillus reuteri.   5. A method according to any one of claims 1 to 4, wherein the substance is administered to the individual in a daily dose of 0.25 to 25 g, preferably 1-2 g.   6. The method according to any one of claims 1 to 5, further comprising simultaneously administering a galactooligosaccharide or other saccharide comprising galactose.   A substance for use in enhancing the activity or increasing the growth of a probiotic bacterium having a pdu-operon in the gastrointestinal tract of an individual, which is metabolized to 1,2-propanediol in the gastrointestinal tract of the individual A substance as described above, which contains a deoxy sugar having capacity.   (A) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, (f) fucoidan with a high proportion of fucose, Or (g) a substance according to claim 7, comprising a combination of rhamnose, fucose and pectin.   The substance according to claim 7 or 8, wherein the bacterium having a pdu-operon is Lactobacillus reuteri.   10. A substance according to any one of claims 7 to 9 which is administered to an individual in a daily dose of 0.25 to 25 g, preferably 1-2 g.   11. A substance according to any one of claims 7 to 10 for use in combination with galactooligosaccharides or other sugars including galactose.   A composition comprising a bacterium having a pdu-operon and a substance comprising a deoxy sugar, wherein the deoxy sugar has the ability to be metabolized to 1,2-propanediol in the gastrointestinal tract of an individual. (A) rhamnose, (b) fucose, (c) pectin with a high proportion of rhamnose, (d) rhamnose in combination with pectin, (e) fucose in combination with pectin, (f) fucoidan with a high proportion of fucose, Or (g) a combination of rhamnose, fucose and pectin,
13. The composition of claim 12, comprising a bacterium having a pdu-operon together with.   14. The composition of claim 12 or 13, further comprising a galactooligosaccharide or other saccharide comprising galactose.   15. The composition according to any one of claims 12 to 14, wherein the bacterium having a pdu-operon is Lactobacillus reuteri.   16. A composition according to any one of claims 12 to 15, wherein the substance is present in an amount which provides a daily dose of for example 0.25 to 25 g, preferably 1-2 g.   17. A composition according to any one of claims 12 to 16 for use in enhancing the activity or increasing the proliferation of a probiotic bacterium having a pdu-operon in the gastrointestinal tract of an individual.