HK1118213B - Method of improving immune function in mammals using 3-hpa producing lactobacillus strains in combination with medium chain triglyceride oil - Google Patents

Description

Method for improving immune function in mammals using 3-HPA producing lactobacilli in combination with medium chain triglyceride oil

Background

Technical Field

The present invention relates to the use of MCT (medium chain triglyceride) oils in combination with specific strains of lactobacillus in products for mammals to achieve an immune enhancing effect.

Description of the Related Art

In nutrition, a substance commonly referred to as fat or oil is one of several substances classified as lipids by chemists and dieticians. Fats are characterized by being insoluble in water and soluble only in specific solvents such as ethanol. Fats have a similar composition to carbohydrates but provide a higher energy per gram of fat. They contain 2 to 2.5 times more energy than saccharides. Unfortunately, fats utilize oxygen in metabolism uneconomically and as a result are difficult to burn. The body requires fat for various processes, for example they are used to build cell walls and they provide a transport mechanism for fat soluble vitamins (vitamins A, D, E and K).

There are three types of fat: triglycerides, cholesterol and phospholipids. Fats and oils found in foods are primarily triglycerides. Triglycerides consist of glycerol, an alcohol triglyceride, and three fatty acid chains, which make up the majority of the fat we consume and store in the body. They are divided into three distinct groups: saturated, monounsaturated, and polyunsaturated. The classification of fats is linked to their chemical composition. Saturated fatty acids have a close chemical structure with their carbon atoms "saturated" with hydrogen atoms, making it impossible for other compounds to bind to them. Monounsaturated fatty acids have a single double bond and two free carbon atoms, to which other chemical bonds of a hydrogen atom can react. Finally, polyunsaturated fatty acids have two or more double bonds and have multiple free carbon atoms to which bonding can be made. These double bonds make unsaturated fatty acids more bioactive than nearly inert saturated fatty acids.

The fatty acids that are components of triglycerides are composed of chains of carbon, oxygen and hydrogen atoms of varying lengths. The length of the carbon chain results in the separation of these chains into short, medium and long chain triglycerides. Medium Chain Triglycerides (MCT) have unique properties that make them more biologically available than other forms of fatty acids (Osbom h.t.et al, comprehensive reviews in Food Science and Food Safety, VoI 1, 2002, page 93-103). MCTs bypass the usual route of fat acquisition, which is used by the body to acquire available energy. Unlike other fats, which require the body to store them before use, MCTs require limited processing and are quickly available for use in the body's energy system. Another obvious feature of medium chain triglycerides is that they cannot be stored as body fat regardless of how much body fat is consumed.

MCT oil occurs naturally, and the most abundant source is coconut oil. Most MCT oils are refined from coconut oil. MCT oil is a clear, tasteless, low viscosity liquid. MCT oil is of interest because it behaves more like a sugar than a fat when metabolized in the body. The body's preferred fuel is sugars, and the body will run out of its sugar storage before using other fuels. The sugar works quickly, the athletes take glucose tablets to provide energy, the body gets hot quickly when we drink wine, and the sugar is usually used within a few hours after eating, which is why we eat it so frequently. In contrast, the basic role of fat is to store energy and the animal deposits fat ready for wintering. Long chain fats (i.e., a common species) are converted by the digestive system to chemicals called chylomicrons, and they are subsequently transported throughout the body through the lymphatic system before entering the circulatory system. This is a relatively slow process, and fat is therefore metabolized much more slowly than sugars. Unlike other fats, MCT oil does not enter the lymphatic system; instead, it is transported directly to the liver where it is metabolized, thus releasing energy very quickly, like a carbohydrate, and producing many ketones in the process. MCTs are commonly used in a variety of nutraceuticals, including medical nutraceuticals, such as parenteral or enteral nutraceuticals.

for example, Tufano M.A. et al report the effect of Total Parenteral Nutrition (TPN) with long- (LCT) and Medium Chain Triglycerides (MCT) on host resistance in "overview tolipopolysaccharide, cytokine release and pharmacological functions both on microorganism and with differential total nutrient number nutrients" (immunopharmacological Immunotoxicol.1995 Aug; 17 (3): 493-509.) the in vivo and in vitro production of tumor necrosis factor α (TNF- α) and interleukin-6 (IL-6) was studied after blood clearance of E.coli (Escherichia coli) stimulated by lipopolysaccharide (LPS.) in BALB/c mice with a 25% reduction in LCTs and a 50% reduction in the mortality of the TPN mixture.

Careful clinical control of triglyceride injection concentrations, particularly MCT concentrations, has the potential to provide anti-tumor effects and maintain a normal immune system (Kimoto Y. et al.; anti-tumor effect of medium-chain and its infection on the body; cancer dose Prev.1998; 22 (3): 219-24). MCT oil is also described as part of nutritional formulas per se, for example in european patent EP0756827 and US patent US6589576, but not in combination with any lactobacillus. Furthermore, European patent EP1344458A1 discloses a method for protecting probiotic organisms such as lactobacilli, by making them into pellets, preferably with a minimum volume of 0.02cm³this work is not at the core of the present invention, which relates to combining a selected lactobacillus strain producing 3-HPA (β -hydroxy-propionaldehyde) with MCT oil in a liquid product to enhance immunity.

According to a generally accepted definition, probiotics are a live microbial feed supplement that provides a beneficial effect to a host animal by improving the microbial balance in the intestine of the host animal. Although originally referred to as animal feed supplements for farm animals, this definition is also readily accepted as applicable to human situations. The main probiotic bacteria for human consumption are food products in the form of dairy products comprising intestinal lactobacilli and bifidobacteria species. It is implied in the definition that consumption of probiotic affects the composition of the intestinal microflora. This effect of probiotics on the intestinal ecosystem has been suggested to be beneficial to the ingester. Various benefits of altering the intestinal microflora through the action of probiotics have been described in the literature including: improving resistance to infectious diseases, especially of the intestine, reducing the duration of diarrhea, lowering blood pressure, reducing blood cholesterol levels, reducing allergies, stimulating phagocytosis by peripheral blood leukocytes, regulating cytokine gene expression, assisting in the regression of tumors, and reducing carcinogens or the production of co-carcinogens.

Lactobacillus reuteri is a naturally occurring resident in the gastrointestinal tract of animals and is commonly found in the intestine of healthy animals, including humans.it is known to have antimicrobial activity see U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. when Lactobacillus reuteri cells are grown in the presence of glycerol anaerobic conditions, they produce an antimicrobial substance known as β -hydroxy-propionaldehyde (3-HPA).

3-HPA is a metabolic intermediate that is secreted from cells by several species of Lactobacillus. Those known as 3-HPA-exporting bacteria include Lactobacillus reuteri, Lactobacillus corynebacterium, Lactobacillus thalamus, Lactobacillus hilgardii, etc. (Claisse O et al, JFood prot.2001 Jun; 64 (6): 833-7) (Sauvageot N et al, Int J Food Microbiol.2000 Apr 10; 55 (1-3): 167-70). 3-HPA has long been known to have antimicrobial properties, which in part explains the ability of producer strains to kill pathogenic bacteria. Lactic acid bacteria, including Lactobacillus reuteri and Lactobacillus corynebacterium, have also been shown to have an effect on the immune system of their host organism. See, e.g., Wagner RD et al, "biotherapeutic effects of probiotics on Candida in immunological chemicals" (InfectImmune 1997Oct 65: 4165-72); however, there is a difference in effect between strains and methods to increase the effect are needed, for example the method of selecting strains that recruit CD4+ cells and bind toxins as provided in WO 2004/034808. The exact mechanism by which lactobacilli stimulate or modify host immune cell activity remains unclear. Many studies have shown that specific substances from selected lactobacilli released into the growth substrate are responsible for modulating the immune response of the host cell. These substances are generally considered to be proteins, polypeptides and nucleic acids. See, for example, Pena et al, CellMicrobiol.2003 Apr; 5(4): 277-85.

The present invention herein provides a novel method for enhancing the immunomodulatory effects of specific lactobacilli in some animals, including humans, by combining selected 3-HPA producing lactobacilli with MCT oil. The effect of this combination includes the antimicrobial action of those lactobacilli in addition to the previously known immunomodulation of those lactobacilli. It brings about the effect that 3-HPA is produced and acts directly on the proliferation of lymphocytes by this substance. 3-HPA or its precursor glycerol, or its metabolites, 1, 3-propanediol and 3-hydroxy-propionic acid, have not previously been disclosed as potential modulators of the mammalian immune system.

Although the possible antimicrobial activity effects of several lactobacilli are known and their specific immunomodulating effects are also known, it was not previously known that the immunomodulating effect could be significantly improved by combining 3-HPA producing strains with MCT oil.

It is therefore an object of the present invention to provide strains of lactobacillus known to produce 3-HPA under appropriate conditions and to bind them with MCT oil to enhance immune regulation. It is another object of the present invention to provide products comprising said strains and MCT oil for administration to animals, including humans.

Other objects and advantages will become apparent from the following description and claims.

Summary of The Invention

The present invention is herein a method for enhancing immune function in a mammal using lactobacillus producing selected 3-HPA in combination with MCT oil. The effect of this combination is not only the immunomodulatory and antimicrobial effects of those lactobacilli previously known. It brings about the effect that 3-HPA is produced and acts directly on the proliferation of lymphocytes by this substance. Other objects and advantages will become apparent from the following description and claims.

Drawings

FIG. 1 shows the results of phagocyte detection: a, cell control; b1, Lactobacillus reuteri lysate (800. mu.g/ml); b2, Lactobacillus reuteri lysate (400. mu.g/ml); b3, Lactobacillus reuteri lysate (200. mu.g/ml); b4, Lactobacillus reuteri lysate (100. mu.g/ml); b5, Lactobacillus reuteri lysate (50. mu.g/ml); c1, Listeria monocytogenes lysate (800. mu.g/ml); c2, Listeria monocytogenes lysate (400. mu.g/ml); c3, Listeria monocytogenes lysate (200. mu.g/ml); c4, Listeria monocytogenes lysate (100. mu.g/ml); d, lactobacillus reuteri supernatant (MRS); e, lactobacillus reuteri supernatant (glycerol solution); f, Lactobacillus reuteri + Listeria monocytogenes lysate.

FIG. 2 shows the results of lymphocyte proliferation assays: b, ConA (6.25. mu.g/ml); c, PWM (5 mug/ml); d, PHA (6.25. mu.g/ml); e5, LPS (2.5. mu.g/ml); f1, Lactobacillus reuteri lysate (400. mu.g/ml); f2, Lactobacillus reuteri lysate (200. mu.g/ml); f3, Lactobacillus reuteri lysate (100. mu.g/ml); f4, Lactobacillus reuteri (50. mu.g/ml); g1, Lactobacillus reuteri supernatant (MRS 100. mu.l); g2, Lactobacillus reuteri supernatant (MRS 50. mu.l); h1, Lactobacillus reuteri supernatant (100. mu.l glycerol solution); h2, Lactobacillus reuteri supernatant (glycerol solution 50. mu.l).

Figure 3 shows the number of bacteria 3 days after administration of listeria monocytogenes (6d) in RL (lactobacillus reuteri) and PC (positive control) treatment.

FIGS. 4a-4g show the results of an analysis of the major immune cells 3 days (6d) and 2 weeks (17d) after inoculation with Listeria monocytogenes. Specifically, fig. 4a shows (beta) TCR + CD3-T cells, fig. 4b shows CD4+ CD25+ T cells, fig. 4c shows CD8+ T cells, fig. 4d shows CD4/CD8 ratio, fig. 4e shows (gamma, delta) TCR + CD25+ T cells, fig. 4f shows NK + CD28+ cells, and fig. 4g shows NK + cells.

FIG. 5 is a flow chart of one embodiment of a process for preparing a product of the present invention.

Detailed description of the invention and preferred embodiments

It has surprisingly been found that the substances or metabolites produced and exported by the lactobacillus when incubated in glycerol can stimulate important cellular components of the immune system and overall boost the host immune system and resist infection. Feeding substances (e.g., oils, fats and lipids) and is expected to stimulate the host's immune system by combining conditions that metabolize glycerol in the gastrointestinal tract with lactobacilli that produce 3-HPA from glycerol. MCT oil is the best additive herein for the present invention, as follows. Therefore, it is advantageous to deliver lactobacilli to a host in such a manner.

A preferred embodiment of the invention is a product comprising MCT oil and 3-HPA producing lactobacillus prepared and used for oral or intubation feeding to the gastrointestinal tract of a person suffering from lipodystrophy, such as most normal infants and young children, but also including adults suffering from lipodystrophy. The reason is the fat metabolism of the MCT-oil of the invention, e.g. normally not glycerol but monoacylglycerols (glycerol still with one fatty acid) are produced in the gastrointestinal tract. These monoacylglycerols are absorbed through the wall of the digestive tract and processed into new lipids in the liver and circulatory system; however, for example, bile salt stimulated lipase secreted in human milk was found to lack positional specificity, i.e. it hydrolyses emulsified triacylglycerols to glycerol and fatty acids. It also hydrolyzes micronized sn-2 monoacylglycerols ("sn" refers to the position of the fatty acid on the glycerol backbone of acylglycerols, e.g., the sn-2 position is the central position of the fatty acid on the glycerol backbone). This is in contrast to pancreatic lipase, which hydrolyses sn-1 and sn-3 ester bonds with a significant preference. When two enzymes are used together, such as in the intestines and stomach of breast-fed infants, the sn-2 monoacylglycerols formed by pancreatic lipase become excellent substrates for bile salt-stimulated lipase. (pediatic Research, VoI 16, 882-885, digest of human milk lipids: physiologic design of sn-2 monoacylglycerol hydrosis by biolesalt-stimulated lipid, O.Hemell and dL.Blackberg). Thus, the end product of triacylglycerol hydrolysis is glycerol and fatty acids rather than sn-2 monoacylglycerol and fatty acids. Bile salt stimulated lipase also catalyzes the incorporation of fatty acids into acylglycerols, but to a much lesser extent than pancreatic lipase. Together, these two effects of bile salt stimulated lipase promote the entire process of intraluminal lipolysis. In newborn infants, bile salts in the duodenum are at low concentrations and glycerol and fatty acids should also be more readily absorbed than monoacylglycerol and fatty acids. Thus, as a supplement to pancreatic lipase, bile salt-stimulated lipase may ensure that infants and other persons with an immature fat digestion and absorption mechanism make full use of, for example, milk lipids. As a result, for example, infants have different systems for obtaining lipids from their breast milk (or formula) because their fat metabolism is immature, and thus 3-HPA-producing lactobacilli as a supplement to nursing infants have a rather abundant source of glycerol to work together in such cases, but in order to ensure that fats are available that can be metabolized to 3-HPA-producing secretions and to improve the immune function of the invention, the present invention proposes that a source of fat, such as MCT oil, should be delivered together with the 3-HPA-producing lactobacilli. This helps to improve the effect of the immune system of e.g. an infant.

In the preferred embodiments above, the products of the invention are delivered into the gastrointestinal tract to raise the immune system, either wholly or locally, in the recipient. The reason why the 3-HPA produced in the gastrointestinal tract may affect the entire immune system is the abundant immune function in the gastrointestinal tract. This is by, for example, cells such as macrophages and lymphocytes growing on the mucosa and intestinal wall. The gastrointestinal tract is considered by many experts in the field to be the largest immune organ in the human body ("Overview of gutfloranduotics", Holzapfel, w.h.et.al., International Journal of food Microbiology, May 1998).

In another preferred embodiment of the invention a similar product may be used, e.g. comprising MCT-oil and 3-HPA producing Lactobacillus, but topical application is used to boost the immune system and combat skin diseases. By dispersion of the skin epithelium and interaction with the subcutaneous immune cells, it is possible to increase the immune effect against pathogenic bacteria while reducing inflammatory reactions and treatments such as eczema, acne and wounds.

MCT oils are unusual lipids that cleave rapidly and glycerol occurs rapidly, which is why MCT is used in the present invention to enhance immune effects. The probiotic, prophylactic and pharmaceutical formulation products according to the invention may comprise any additives and excipients acceptable for nutritional or pharmaceutical use. Medium chain triglyceride oil is an oil-containing medium chain triglyceride, i.e. a medium chain fatty ester of glycerol. Medium chain fatty acids are well known in the art and contain 6 to 12 carbon atoms, preferably 8 to 10 carbon atoms such as octanoic acid and decanoic acid. Akomed R from Karshong is an example of an MCT oil useful according to the present invention. It comprises caprylic/capric triglyceride.

As can be seen from the following examples, MCT oils for use in the present invention are readily available on the market. The 3-HPA producing Lactobacillus can be isolated using different methods, for example as described by Rodriguese E.et al (Letters in applied microbiology Volume 37 Issue 3 Page259-September 2003). One of the preferred species is Lactobacillus reuteri and one preferred strain is Lactobacillus reuteri ATCC 55730.

The features of the present invention will be more clearly understood by reference to the following examples, which should not be construed as limiting the invention.

Example 1: increased phagocytic activity in macrophages

SUMMARY

Lactobacillus reuteri supernatant was taken from Lactobacillus reuteri ATCC 55730 grown in the presence and absence of glycerol (50 mM). These supernatants were used for pretreatment of abdominal macrophages (taken from 6 week old SPF B ALB/c mice) for overnight incubation. Macrophages were then inoculated with listeria monocytogenes to determine their phagocytic capacity. After 2 hours of incubation, the number of listeria monocytogenes surviving in macrophages was determined.

Control incubation in the absence of lactobacillus reuteri supernatant showed significant survival of listeria monocytogenes from macrophages under these conditions. Macrophages were pretreated with supernatant of lactobacillus reuteri cells grown in the absence of glycerol and did not affect phagocytic phagocytosis compared to control incubations (figure 1). However, surprisingly, pretreatment of macrophages with supernatant from lactobacillus reuteri cells grown in the presence of glycerol resulted in macrophages effectively killing all listeria monocytogenes. Thus, lactobacillus reuteri grown in the presence of glycerol is able to stimulate the phagocytic activity of the cells. The medium with glycerol alone (control) did not have such an effect (control "H" is the same as the other controls).

Pretreatment of macrophages with lactobacillus reuteri or listeria monocytogenes zymolyte induces stimulation of similar but less pronounced macrophage phagocytic activity in a dose dependent manner. Glycerol-induced metabolism in Lactobacillus reuteri results in more effective stimulation of phagocytic activity than induction of antigen exposure.

Detailed description of the experiments

Research method

1. Phagocytic cell assay and lymphocyte proliferation assay. Lymphocytes and abdominal macrophages were isolated from specific pathogen-free BALB/c mice (6 weeks old) and used to test for any immune re-enhancement effect in the host. Phagocytosis utilizes abdominal macrophages.

Isolation of abdominal macrophages. BALB/c mice were sacrificed by cervical pulling, followed by harvesting5ml of IMDM medium (Iscove's modified Dulbecco's medium) was soaked and rubbed. The impregnated IMDM was recovered and viable cells were counted using the phtalol blue display technique and at an adjusted final concentration of 1x10⁶The/ml was used in the experiment.

-preparation of bacterial lysates. Lactobacillus reuteri SD2112 and listeria monocytogenes HPB3 serotype 4b were incubated in MRS +0.02M glucose medium and pancreatin soybean medium (TSB) for 24 hours, respectively, and centrifuged at 8,000rpm for 20 minutes to collect cell debris. The cell debris was washed twice with 0.9% NaCl solution. Followed by heating at 60 ℃ for 3 hours for inactivation, and the concentration of the protein was calculated by the bicinchoninic acid (BSA) protein detection method and adjusted to 5 mg/ml.

Preparation of Lactobacillus reuteri supernatant. Lactobacillus reuteri was incubated in MRS +0.02M glucose medium for 24 hours and centrifuged to obtain MRS medium supernatant. The supernatant was suspended in a 0.05M glycerol solution and incubated for 6 hours, followed by recovering the glycerol solution supernatant. The number of cell debris recovered was as high as 2g/30ml (wet weight). All recovered supernatant was used after adjusting the pH to 7.3.

-phagocytosis assay. Abdominal macrophages suspended in IMDM + 10% horse serum, 4X10⁵Each cell per well and treated with various concentrations of lactobacillus reuteri, listeria monocytogenes cell lysate and lactobacillus reuteri supernatant overnight incubation at the following concentrations and conditions:

TABLE 1 treatment conditions for phagocytosis assay

Numbering	Treatment of	(μg/ml)	Numbering	Treatment of	(μg/ml)
						A	Cell controls	-	D1	Lactobacillus reuteri supernatant (MRS)	100
B1	Lactobacillus reuteri lysate	800	D2	Lactobacillus reuteri supernatant (MRS)	50
						B2	Lactobacillus reuteri lysate	400	E1	Lactobacillus reuteri supernatant (glycerol solution)	100
B3	Lactobacillus reuteri lysate	200	E2	Lactobacillus reuteri supernatant (glycerol solution)	50
						B4	Lactobacillus reuteri lysate	100	F1	Lactobacillus reuteri + listeria monocytogenes lysate	400+400
B5	Lactobacillus reuteri lysate	50	F2	Lactobacillus reuteri + listeria monocytogenes lysate	200+200
						C1	Listeria monocytogenes lysate	800	F3	Listeria monocytogenes lysate	100+100
C2	Listeria monocytogenes lysate	400	G	MRS control	100
						C3	Listeria monocytogenes lysate	200	H	Glycerol solution control	100
C4	Listeria monocytogenes lysate	100	I	IMDM controls	-

MRS control and glycerol solution control were employed by adjusting PH in the culture medium, which was not incubated with lactobacillus reuteri.

Abdominal macrophages treated with the above conditions were incubated in TBS and incubated with 4x10⁴Listeria monocytogenes (HPB3 serotype 4b, gentamicin sensitive) was inoculated and washed with 0.9% NaCl solution. After incubation for 2 hours at 37 ℃, macrophages were washed and subsequently treated with 200 μ g/ml gentamicin to kill extracellular bacteria. Cells were washed three times with Phosphate Buffered Saline (PBS) and lysed with 0.5 ml/well PBS + 0.5% Triton X-100 to expose intracellular microorganisms. Then, the number of bacteria was counted by a thin layer method using Palcam agar (Oxoid me, Ogdensburg, n.y., USA) and pancreatin soybean agar (Oxoid Inc, Ogdensburg, n.y., USA) by serial dilution.

Example 2: increased lymphocyte proliferation

SUMMARY

Lymphocytes were prepared from spleens and thymus of BALB/c mice and incubated in the supernatant of Lactobacillus reuteri ATCC 55730 grown in the presence and absence of glycerol (50 mM). Lactobacillus reuteri ATCC 55730 was chosen because it is a good producer of known 3-HPA and therefore meets the selection element of the present invention. Control incubations used glycerol alone, media alone, and a known stimulator of lymphocyte proliferation.

Known stimulants (ConA, PWM, PHA abd LPS) elicit significant lymphocyte proliferation stimulation. Lysate from lactobacillus reuteri resulted in a small, dose-dependent stimulation of lymphocyte proliferation, and incubation with supernatant from lactobacillus reuteri grown in the absence of glycerol did not result in a significant increase in lymphocyte proliferation. Surprisingly, the supernatant of Lactobacillus reuteri grown in the presence of glycerol resulted in an increase in the level of proliferation of lymphocytes to a level significantly higher than that seen with known stimulants (see FIG. 2).

Detailed description of the experiments

Lymphocyte proliferation assay

Isolation of splenic and thymic lymphocytes. BALB/c mice were sacrificed by cervical pulling, their thymuses isolated, dipped in Hanks' balanced salt solution (HBSS) and gradually crushed to obtain single cells. Individual cells were centrifuged through the bilayer in Histopaque-1083(Sigma) to obtain lymphocytes. These cells were washed twice with IMDM and the viable cells were counted by the orcinol blue visualization technique and at an adjusted final concentration of 1x10⁷the/mL was used in the experiment.

-preparation of bacterial lysates. As described in the phagocytosis assay, lactobacillus reuteri and listeria monocytogenes lysates were prepared.

Preparation of Lactobacillus reuteri supernatant. The lactobacillus reuteri supernatant was prepared as described in the phagocytosis assay.

-lymphocyte proliferation assay. Lymphocyte proliferation assays were performed using 4x106 cells per well with lymphocytes suspended in IMDM + 10% horse serum. Lymphocytes were treated with different concentrations of Lactobacillus reuteri, Listeria monocytogenes cell lysate and Lactobacillus reuteri supernatant and a reference stimulant at the following concentrations and conditions:

TABLE 2 treatment conditions for lymphocyte proliferation assays

Numbering	Treatment of	(μg/ml)	Numbering	Treatment of	(μg/ml)
						A	Cell controls	-	G1	Lactobacillus reuteri supernatant (MRS)	100
B	ConA	6.25	G2	Lactobacillus reuteri supernatant (MRS)	50
						C	PWM	5	H1	Lu's scaleLactobacillus supernatant (Glycerol solution)	100
D	PHA	6.25	H2	Lactobacillus reuteri supernatant (glycerol solution)	50
						E	LPS	2.5	1	IMDM controls	-
F1	Lactobacillus reuteri lysate	400	J1	MRS control	100
						F2	Lactobacillus reuteri lysate	200	J2	MRS control	50
F3	Lactobacillus reuteri lysate	100	K1	Glycerol solution control	100
						F4	Lactobacillus reuteri lysate	50	K2	Glycerol solution control	50

In the results reported in table 2, MRS control and glycerol solution control were employed by adjusting pH in a medium not incubated with lactobacillus reuteri, and the following symbols were employed:

ConA: concanavalin a, PWM: pokeweed mitogen, PHA: phytohemagglutinin, LPS: lipopolysaccharide from Salmonella typhimurium.

According to each condition, a stimulant comprising ConA was added to the wells containing lymphocytes 3 hours before alamar blue was added at a concentration of 10%. 72 hours after addition of each stimulant, lymphocyte proliferation levels were determined using the values of OD570nm-OD600 nm.

Lymphocyte proliferation results are depicted as Stimulation Index (SI), which is the ratio of (OD treatment-OD medium control)/OD cell control, however, in the case of lactobacillus reuteri supernatant treatment group, the values of the J and K treatment groups, rather than IMDM + 10% horse serum, were used as OD medium control values.

Stimulation and alteration of immune cell distribution rates in mice

Specific sterile BALB/c mice (6 weeks old) were divided into 4 groups. Lactobacillus reuteri and Listeria monocytogenes were prepared as 5x10⁸Mice and oral administration. 0.25M 10% sodium bicarbonate was administered 30 minutes prior to administration of Listeria monocytogenes to enhance survival of Listeria monocytogenes.

TABLE 3 rat treatment group

Group of	Treatment of	Sample(s)
			Negative Control (NC)	PBS was administered during the study	6d，17d，31d
R	Lactobacillus reuteri was administered during the study period	6d，17d
			RL	L. reuteri 3d administration during study in Listeria monocytogenes	6d，17d
Positive Control (PC)	PBS at 3d administration of Listeria monocytogenes during study	6d，17d，31d

To investigate the protective effect of lactobacillus reuteri against listeria monocytogenes, 8 mice in each group were sacrificed 3 days (6d) and 14 days (17d) after application of listeria monocytogenes, respectively, and blood, liver and spleen were removed. In addition, to investigate the role of lactobacillus reuteri itself in the immune mechanism, 8 mice in both NC and R groups were further tested at 31d, respectively.

1) Calculation of Listeria monocytogenes in liver and spleen

Liver and spleen from each mouse group were weighed and homogenized in a blender with 10ml of PBS (pH 7.0). Viable bacteria were counted in serial dilutions using Palcam agar.

2) Detection of immune cells using flow cytometry

-separating leukocytes from blood. Blood was drawn from 2-3 animals and centrifuged bilaterally at 1,500rpm in Histopaque-1083 for 30 minutes, after which lymphocytes were collected from the lymphocyte layer. They were washed three times with Phosphate Buffered Saline (PBS) and suspended in RPMI-1640 medium, and then the viable cells were counted using the orcinol blue display technique and adjusted to a final concentration of 1X10⁷Used in the experiment per ml.

Flow cytometry assays use leukocyte surface monoclonal antibodies. The isolated leukocytes were suspended in a first washing buffer (PBS 450ml, ACD 50ml, 20% NaN 35 ml, gamma globulin (gamma globulin) -free horse serum 10ml, 250mM EDTA 20ml, 0.5% phenol red 1ml) containing 20% goat serum and sensitized at 4 ℃ for 10 minutes. After centrifugation, 100. mu.l of leukocyte-surface antigen-specific monoclonal antibody and 1X10 of the antibody were added to each well of a V-bottomed 96-well microplate⁷50 μ l/ml of leukocytes isolated from blood, previously hypersensitized at 4 ℃ for 30 minutes. Binary staining was performed using the same reagents as in table 4. After washing three times with the first washing buffer, the remaining leukocytes were washed three times with a second buffer at 4 ℃ which was substantially the same as the first buffer except that horse serum was removed and mixed with a 2% formalin solution. More than 2,000 stained cells were examined by flow cytometry and data analysis was performed by means of CellQuest program (Becton Dickinson).

TABLE 4 monoclonal adsorption for immune cell detection

Marking	(alpha beta)TCR/CD3	CD4/CD25	CD8a/CD25	(alpha beta)TCR/CD25	NK(CD49b)/CD28
						Fluorescence	FITC paired mouse anti-mouse (alpha beta) TCR	FITC Pair mouse anti-mouse CD4	FITC Pair mouse anti-mouse CD8a	FITC paired hamster anti-mouse (gamma delta) TCR	FITC Pair mouse anti-mouse CD49b/PANNK
Fluorescence	PE paired mouse anti-mouse CD3	PE paired mouse anti-mouse CD25	PE paired mouse anti-mouse CD25	PE paired mouse anti-mouse CD25	PE paired hamster anti-mouse CD28

Results and discussion

1) Detection of phagocytosis Using abdominal macrophages

The results of phagocytosis using abdominal macrophages are shown in FIG. 1. As shown in the figure, the results of the abdominal macrophage phagocytosis assay proved to be highest in the case of treatment with the glycerol supernatant of Lactobacillus reuteri and the lysate of Lactobacillus reuteri + Listeria monocytogenes (P < 0.05). In other words, the treatment of the supernatant of the glycerol solution of Lactobacillus reuteri, whether 100. mu.l or 50. mu.l, had the same effect and almost completely killed the Listeria monocytogenes phagocytosed by macrophages. Each of the mixed treatment solutions of Lactobacillus reuteri + Listeria monocytogenes lysate of 400, 200, and 100. mu.l/ml partially kills Listeria monocytogenes phagocytosed by macrophages. The group treated with the Lactobacillus reuteri lysate alone had lower phagocytic activity than the above two treatment groups and Listeria monocytogenes survived even after phagocytosis, although the phagocytic activity of the Lactobacillus reuteri lysate treatment group (B) was seen to be higher than that of the Listeria monocytogenes lysate treatment group (C) (P < 0.05). Inspired by these results, it was recognized that lactobacillus reuteri lysate itself and its metabolites can enhance phagocytic activity of abdominal macrophages, particularly in the presence of pathogenic bacterial lysates, such as listeria monocytogenes. Considering that the group treated with MRS medium supernatant was found not to enhance the phagocytic activity of abdominal macrophages; while the group treated with the supernatant of glycerol solution showed high phagocytic activity, the substance enhancing the activity of phagocytic cells was assumed to be a glycerol metabolite and the possibility of being a non-proteobacteria with high antibiotic activity could not be assumed to be excluded. This event is believed to require further investigation by subsequent testing.

The group treated with the listeria monocytogenes lysate was found to have some enhanced phagocytic activity, although its activity was lower than that of the lactobacillus reuteri lysate treated group. It should be considered that this result was not obtained by treatment with live listeria monocytogenes, and a special note was that phagocytic activity was confirmed to be reduced in the panel treated with the high concentration listeria monocytogenes lysate. This appears to indicate that macrophage activity is inhibited by several virulence factors present in high concentrations of listeria monocytogenes lysate.

2) Lymphocyte proliferation assay

The results shown in FIG. 2 are lymphocyte proliferation assays using lymphocytes isolated from spleen and thymus. The proliferation of lymphocytes isolated from spleen and thymus was confirmed to be highest in the Lactobacillus reuteri supernatant (glycerol solution) treated group (P < 0.05).

One method used in experiments to detect proliferation is the alamar blue assay, which is based on the extent of reagents reduced due to cell proliferation. It cannot be excluded that its value may be higher than the reagent reduction degree due to aldehyde oxidation because the lactobacillus reuteri supernatant (glycerol solution) contains 3HPA, an aldehyde. However, since the cell proliferation of the lactobacillus reuteri supernatant (glycerol solution) treated group was also considered to be the highest under microscopic observation, the lymphocyte proliferation ability of the lactobacillus reuteri supernatant (glycerol solution) could be confirmed.

The group treated with lactobacillus reuteri lysate alone was also confirmed to have a certain degree of lymphocyte proliferation activity, although this activity was lower than the reference stimulant.

Example 3: administration of Lactobacillus reuteri in MCT oil to human young children to study reduction of eczema

This study was conducted to determine whether dietary supplementation of lactobacillus reuteri alone and MCT oil could affect the treatment of atopic eczema in young children currently undergoing standard treatment. Cases studied in the children's hospital of lund showed that at least one child had persistent eczema, and the problem of eczema was solved by supplementing lactobacillus reuteri and MCT oil with a 30 day diet. This study clearly demonstrates that standard therapeutic supplementation with lactobacillus reuteri demonstrated such effects under controlled study conditions.

The primary endpoint of this study was a reduction in the extent and severity of eczema as determined by the available scoring System (SCORAD) for lactobacillus reuteri and MCT oil administration over a 12 month treatment period. The scores were normalized at the start of treatment. The score reduction of the lactobacillus reuteri-MCT group at 3, 6, 9 and/or 12 months of treatment was significantly considered as a primary endpoint compared to the placebo group.

This is a double blind randomized study that is performed continuously. Patients diagnosed with moderate atopic dermatitis and with parental consent between the ages of 3 months and 3 years (i.e., up to but not including the 4 year old birthday) were included in the study. The diagnosis need not be new, e.g. children who are already under standard therapy may also be included.

The patients enrolled were from a population of patients who looked at allergy. The researcher provides information about the study to the parents of the potential participants, and if the inclusion criteria are met, written consent is obtained and the patient is included. Patients were randomized into two groups, each group containing 20 patients. One group received a lactobacillus reuteri supplement in MCT oil and the other group received a placebo formulation containing MCT oil alone. The study product was taken for 12 months.

The investigator filled out an eczema score based on the patient's examination and photographed the eczema. The home environment is recorded from a questionnaire to determine risk factors in the home environment. The study product was handed to the parent and started to be taken daily.

Both groups continued to their prescription for conventional drug treatment of eczema. To test steroid usage, the patient's parents are asked to carry all of their steroid prescriptions to the hospital and the drugs are weighed and recorded.

Parents of patients received a telephone call to monitor progress one month after receiving study products and patients returned to the hospital for a full examination with the photographic files of eczema and updated eczema scores completed after 3, 6, 9 and 12 months. The treatment was completed in 12 months.

Blood samples were taken at the beginning of the study to determine total IgE levels and gm allotypes for the patients, and total IgE levels were again determined at the completion of the study. Skin puncture tests were performed at two time points for eggs, milk, fish, cats and peanuts.

The container of study product was retrieved, weighed and recorded to ensure that the requirements were met. Dosage form

In the whole study, L.reuteri in MCT oil was measured at 1X10⁸CFU/day dose was administered, which is equivalent to 10 drops of Lactobacillus reuteri-MCT oil. Equal doses of MCT oil were administered in placebo.

Medicament/intervention/diet

All forms of steroids are permissible and special diets can be taken if desired. Patients should completely avoid all other kinds of foods with added lactobacilli and probiotics during the study.

Definition of

The severity and extent of eczema is defined by the SCORAD index (Kunz B, Oranje AP, Labreze L, Stalder J F, Ring J, Taieb A. Dermatology.1997; 195 (1): 10-9.Clinical validations and guidelities for the SCORAD index: consensus report of the European task force on adaptive Descriptions).

Procedure for each visit

The study required parents to take a total of 5 clinical examinations with the patient and to communicate with one parent at a time.

The first visit (0 day)

After written consent, the patients were included in the study. The home environment of the patient is recorded according to a questionnaire to determine dermatitis risk factors in the home environment. The investigator filled out an eczema score based on the patient's findings, and the eczema was photographed. Blood was sampled to analyze total IgE as well as gm allotype (genetic determinant of allergy). Skin puncture tests were performed to determine allergic reactions to eggs, milk, fish, peanuts and cats.

Each patient received a random number and handed the corresponding study product (two bottles) to their parents for daily dosing. All patients continued with their prescription for conventional eczema medication.

To test steroid usage, the patient's parents are asked to carry all of their prescribed steroids to the hospital and the drugs are weighed and recorded.

Telephone access (1 month)

A telephone contact was made with the patient's parent 1 month after the first visit to monitor progress, ensure that the study product was taken and identify any problems the parent/patient encountered in complying with the study protocol.

The second visit (3 months)

The patient returns to the hospital to carry out comprehensive examination, takes a picture of the eczema and completes the update of the eczema scoring table. The patient's parents are asked to carry all prescribed steroids to the hospital and the drugs are weighed and recorded. The first two bottles of study product were weighed and recorded. One bottle should be empty and retained by the investigator. The other bottle should be emptied and left for continued use in the study. Two additional bottles of study product were handed to parents at this visit.

The third visit (6 months)

The patient returns to the hospital to carry out comprehensive examination, takes a picture of the eczema and completes the update of the eczema scoring table. The patient's parents are asked to carry all prescribed steroids to the hospital and the drugs are weighed and recorded. The bottles of study product were weighed and recorded. Both bottles should now be empty and retained by the investigator. The third bottle was almost full and was kept on for use in the study. Two additional bottles of study product were handed to the parent in this observation.

The fourth visit (9 months)

The patient returns to the hospital to carry out comprehensive examination, takes a picture of the eczema and completes the update of the eczema scoring table. The patient's parents are asked to carry all prescribed steroids to the hospital and the drugs are weighed and recorded. The bottles of study product were weighed and recorded. One bottle should be empty and retained by the investigator. The second bottle should be half empty and the third bottle full. These two bottles were kept for further use in the study.

The fifth and last visit (12 months)

The patient returns to the hospital to carry out comprehensive examination, takes a picture of the eczema and completes the update of the eczema scoring table. Blood samples were taken for analysis of total IgE amounts. Skin puncture tests were performed to determine allergic reactions to eggs, milk, fish, peanuts and cats.

The patient's parents should carry all prescribed steroids to the hospital and the drugs are weighed and recorded. The bottles of study product were weighed and recorded. The remaining two bottles should now be empty and retained by the investigator.

Statistical method and sample size determination

Patients were randomized into two groups, each group consisting of 25 people. Generally for each study, based on the previous results, it was determined that treatment was 50% effective (as in earlier studies) and 80% effective, with an estimated 20 per group being sufficient to show the effect. Each group includes 5 additional children ready for a case of halfway exit.

Overview and discussion of results

TABLE 5 oil group of Lactobacillus delbrueckii ATCC 55730+ MCT for eczema at 0, 3, 6, 9 and 12 months

Rand nr	0 month	3 months old	6 months old	9 months old	12 months old
						2	58	0	16	37	36
6	34	6	0	0	0
						7	48	6	4	0	4
10	35	7	0	8	7
						11	34	7	0	0	4
12	30	2	1	1	4
						15	50	14	8	12	10
17	28	11	4	10	11
						19	26	0	0	0	0
21	28	10	20	13	4
						23	23	10	8	4	8
25	26	4	4	4	4
						27	31	13	27	18	10
29	44	19	17	14	13
						30	56	18	38	18	18
31	44	4	19	11	10
						35	48	14	14	7	20
36	28	6	4	4	4
						39	44	4	0	0	0
40	35	4	0	4	4

Mean value 37.507.959.208.258.55

MCT-oil compositions

1	41	40	18	29	20
						3	38	11	16	15	17
4	28	22	12	12	39
						5	34	18	4	12	12
8	19	14	21	10	18
						9	68	68	60	51	60
13	54	48	29	26	48
						14	26	60	42	42	48
16	56	8	64	48	31
						18	54	30	48	31	54
20	12	12	4	12	13
						22	22	0	0	0	0
24	30	10	31	48	34
						26	34	13	22	11	18
28	56	4	7	10	11
						32	39	32	30	28	18
33	28	31	22	22	31
						34	31	48	31	42	34
37	35	31	22	22	12

Mean value 37.6526.5025.7024.4527.30

Lactobacillus reuteri ATCC 55730, a good producer of 3-HPA, combined with MCT oil, is a very important improvement on eczema.

Example 4: preparation of products comprising selected strains

In this example, a product comprising 3-HPA producing Lactobacillus and MCT was prepared. The product is preferably an oil-based composition comprising lactobacillus reuteri SD2112, with good stability and shelf life.

Description of the preparation Process

A flow chart of a preferred manufacturing process is shown in fig. 5. One such possible process that may be used in the present invention is as follows.

1. Mixing of the ingredients

Medium chain triglycerides such as Akomed R (Karlshamns AB5 Karshamn, Sweden) were mixed with silica, Cab-o-sil M5P, (M5P, Cabot) in a BoIz mixer (Manual Alfred BOLZ apparatus ebau GmbH, Wangen im Allgau, Germany).

2. Homogenization

A Sine Pump and dispax mill (Sine Pump, Arvada, Colorado, United States) were connected to the BoIZ mixer and the mixture was homogenized.

3. Vacuum drying

The mixture was dried in a BoIz jar under a 10mBar vacuum for 12 hours to remove water from the oil and extend the shelf life of the product.

4. Adding lactobacillus reuteri

Approximately 20kg of the dried oil compound was transferred to a 50 liter stainless steel vessel. Lactobacillus reuteri powder, e.g., lyophilized cells, is suspended in oil in the desired amount. For example, 0.2 is addedActivity of 10 Kg¹¹CFU/g culture. It was mixed slowly until homogenized.

5. Mixing

The premix with Lactobacillus reuteri was returned to the BoIz mixer.

6. Discharging

The suspension was poured into a 200 l glass vessel and blanketed with nitrogen. The suspension was kept in a container until it was filled into product bottles.

The products formulated herein are intended for oral administration. Alternatively, products formulated for tube feeding may be supplemented with enteral nutrition as known in the art, or adapted for topical application to the skin using standard ingredients as known in the art.

While specific embodiments have been described herein, it will be apparent to those skilled in the art that various modifications can be made without departing from the spirit and scope of the invention.

Claims (7)

1. A product for enhancing immune function in a mammal comprising 3-HPA producing lactobacillus in combination with medium chain triglyceride oil, wherein the medium chain triglyceride oil is an oil containing carbon chain triglycerides of 8 to 10 carbon atoms, the lactobacillus being selected from the group consisting of lactobacillus reuteri strains; the Lactobacillus reuteri strain is strain ATCC 55730.

2. A product according to claim 1 wherein the medium chain triglyceride oil is caprylic/capric triglyceride.

3. The product of claim 1, wherein the product is formulated for oral administration.

4. The product of claim 1, wherein the product is formulated for catheter feeding as an additive to enteral nutrition.

5. Use of a product according to any one of claims 1 to 4 in the preparation of a product for enhancing immune function in a mammal, wherein the medium chain triglyceride oil is an oil containing carbon chain triglycerides of 8 to 10 carbon atoms, and the lactobacillus is selected from the group consisting of Lactobacillus reuteri strains; the Lactobacillus reuteri strain is strain ATCC 55730.

6. Use according to claim 5, wherein the medium chain triglyceride oil is caprylic/capric triglyceride.

7. Use according to claim 5, wherein the product is administered orally.