HK1172928A - Probiotic derived non-viable material for allergy prevention and treatment - Google Patents

Description

Probiotic-derived non-viable material for the prevention and treatment of allergies

Technical Field

The present invention relates to a method for harvesting non-viable bioactive material from the probiotic strain Lactobacillus rhamnosus Goldin Gorbach (LGG). In particular, the invention relates to a method for preparing an antiallergic probiotic material obtainable by said harvesting method and to a diet or nutritional product comprising said probiotic material.

Background

Lactobacillus GG (lactobacillus g., strain ATCC 53103) is a bacterium that naturally occurs in the human digestive tract. It is a bacterium that has undergone many studies and is generally recognized as having health benefits. It is recognized as a probiotic and is therefore incorporated into many nutritional products, such as dairy products, nutritional supplements, infant formulas, and the like.

It is currently defined in the art that probiotics are viable microorganisms that when administered in appropriate amounts provide health benefits to the host. However, the live nature of probiotics presents challenges when incorporated into nutritional products. These challenges can vary in magnitude, depending, among other things, on the type of probiotic strain used, the health status of the individual receiving the product, or both. Moreover, from a processing technology point of view, incorporation of living microorganisms into products requires considerable obstacles to be overcome. This is particularly useful if the probiotic bacteria are to be incorporated into long-term storage products, for example powdered products such as infant formula. Moreover, as the complexity of the nutritional product matrix increases, the challenge increases.

In particular with regard to the dietary products of pregnant women, infants and children, the safety and efficacy of the use of probiotic bacteria is further evaluated. Specific safety issues relate to possible effects on nutrient utilisation, elimination of antibiotic resistance transfer, and short and long term effects on intestinal colonization, immune response and infection. While this does not mean that probiotics are not available in such products, the complexity of the procedure for using live or viable bacteria is increased.

On the other hand, there is an important need to provide probiotic benefits, especially in the case of infant and children's diet products. Moreover, it is particularly challenging to ensure the stability and viability of viable bacteria in nutritional products that are available through retail or hospital outlets and exposed to ambient temperatures. The use of bacterial products by applying (processed) culture supernatants will provide considerable advantages in this respect.

It is known that the intestinal microbiota of infants is much less developed than that of adults. Although the population of adult is greater than 10¹³The microbial composition of the strain is close to 500,some harmful and some beneficial, whereas the microbiota of infants contains only a fraction of those microorganisms, not only in absolute numbers but also in species diversity. Infants are born with a sterile gut, but acquire gut flora from the birth canal, their original environment and their intake. Because the gut microbiota is very unstable in the early neonatal period, it is often difficult for the infant gut to maintain a delicate balance between harmful and beneficial bacteria, thus reducing the ability of the immune system to function properly.

Maintaining this balance is particularly difficult in formula-fed infants, because formula feeding differs in the bacterial species in the gut from breast-fed infants. The faeces of breast-fed infants contain predominantly bifidobacteria, usually less streptococci and lactobacilli. In contrast, the microbiota of formula-fed infants is more diverse, containing bifidobacteria and bacteroides, and more pathogenic species, staphylococci, escherichia coli, and clostridia. The species of bifidobacteria in the faeces of breast-fed and formula-fed infants are also different. Various factors have been proposed as the cause of the different fecal flora of breast-fed and formula-fed infants, including low protein content and different composition in breast milk, low phosphorus content in breast milk, various oligosaccharides in breast milk, and the humoral and cellular mediators in breast milk that have many immune functions. Agostoni et al, biological Bacteria in digital Products for Infants: anaerobic by the ESPGHAN Committee on Nutrition (Probiotics in infant food products: reviews by the ESPGHAN Nutrition Committee), J.Peditar.Gastro.Nutr.38: 365-.

The establishment of normal intestinal flora is of great significance for health and disease. The main functions of the intestinal microbiota, from the host point of view, are to prevent intestinal colonization by pathogenic organisms and to inhibit proliferation of potentially pathogenic microorganisms, increasing the natural resistance of the intestine to infectious diseases. Probiotics exert this effect by preventing the binding of pathogenic bacteria to intestinal cells, either directly by the production of antimicrobial compounds or indirectly by altering the pH of the intestinal lumen through the synthesis of short chain volatile fatty acids.

In addition, the normal flora stimulates gastrointestinal immunity, increases mucosal IgA production, stimulates local production of anti-inflammatory cytokines and reduces the production of pro-inflammatory cytokines characteristic of allergic inflammation. Reviews of most relevant studies on the use of Probiotics in primary prevention of food allergy, atopic dermatitis and atopy have shown that probiotic therapy alleviates allergic inflammation, as evidenced by control of clinical symptoms and reduction of local and systemic inflammatory markers (Miraglia delGiudice M, De Luca MG. the role of Probiotics in the clinical management of food allergy and atopic dermatitis J clean Gastroenterol 2004; 38 (Zen 6): S84-5; Preslott S, Bjorksten B, Probiotics for the prevention or treatment of the allergic disease probiotic J.

In view of the above, it will be appreciated that it is generally desirable to provide nutritional products, dietary products, and in particular infant formulas, with probiotics. The term "infant formula" refers to a composition that meets the nutritional needs of an infant as a substitute for breast milk.

It will also be appreciated that it is desirable to incorporate probiotics without having to incorporate live or viable bacteria, particularly in infant formulas. In fact, this is called "non-viable probiotic".

Research on such non-viable probiotics is ongoing. However, as pointed out by Tao et al (Am J. physiol Cell physiol.290: C1018-C1030 (2006)), although probiotics appear to improve the course of many diseases, little is known about their mechanism of action. In particular low molecular weight fractions (MW < 10kDa) are used in the treatment of in particular Inflammatory Bowel Disease (IBD) instead of live bacteria. Other references to non-viable probiotic bacteria are for example US 2004/208863, which relates mainly to the anti-inflammatory action of bacteria, and products secreted therefrom, and US 7052896, which relates to the anti-inflammatory and anti-allergic properties of peptides produced by lactobacillus rhamnosus species.

US 6,506,389 describes proteins obtainable from a non-pathogenic microorganism, said proteins having mucosal binding activity and a molecular weight of 20-40kD, preferably 20-30kD, or equivalent polypeptides thereof. The gist of this disclosure is to provide screening methods for identifying proteins and polypeptides capable of specific binding to mucosa.

None of the prior art documents suggest a suitable straightforward fermentation and collection method in order to obtain from LGG a non-viable probiotic material supporting anti-allergic activity.

Moreover, the collection of secreted bacterial products poses problems, and undesired components cannot be easily stripped from the culture medium. This particularly relates to nutritional products for relatively fragile subjects, such as infant formulas or clinical nutrition. This problem does not arise if the specific components of the culture supernatant are first isolated, purified and then applied to the nutritional product. However, it is desirable to use a more complete (complete) culture supernatant. This will serve to provide a composition that better reflects the natural action of the probiotic (i.e. LGG). However, at present, it is not possible to use only the culture supernatant itself as a basis for non-viable probiotic material, particularly for infant formula and the like. It would therefore be more desirable to provide a solution to this problem.

Summary of The Invention

In order to better meet one or more of the above desires, the present invention proposes, in one aspect, a composition comprising a mixture of proteins, said composition being obtainable from a culture supernatant in the exponential phase after the LGG batch culture process, for use in the treatment or prevention of allergic diseases.

In another aspect, the invention relates to a method of harvesting a composition having anti-allergic activity from an LGG culture medium, the method comprising growing LGG in a suitable medium, determining a post-exponential phase of LGG population growth during which a culture supernatant is isolated from the bacterial culture.

In a further aspect, the present invention provides a diet product comprising a non-viable probiotic composition obtainable from a culture supernatant in the exponential phase after LGG batch cultivation process, and the use of the above composition as an additive in a nutritional product.

In a further aspect, the present invention provides a method of treating or preventing allergic disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising a non-viable probiotic material obtainable from a culture supernatant in the exponential phase after an LGG batch culture process.

Brief Description of Drawings

FIG. 1 shows a graph in which the LGG population increases with the passage of time after culture; where FIG. 1a is depicted as a function of the optical density (OD600) of the medium and the pH change, and FIG. 1b presents the bacterial count determined by the plate culture technique.

FIG. 2 shows the production of cytokine IL-10 at different stages (MJ1, MJ2, MJ3) of LGG culture harvest;

FIG. 3 depicts a timeline for Ovalbumin (OVA) sensitization in vivo; a neonatal mouse model for testing the compositions of the invention;

figure 4 depicts microscope images of stained lung tissue of mice receiving the OVA model and orally treated with different compositions including viable LGG and LGG supernatant;

FIG. 5 shows a graph representing the infiltration of allergic cells in the lungs of OVA-allergic animals; it was revealed that there was a decrease in allergic (eosinophilic) cells in lung lavage fluid of both LGG and LGG supernatant treated mice.

FIG. 6(a-c) shows graphs representing the in vitro response of 3 different cytokines associated with inflammatory processes in allergic diseases; the data show that LGG supernatant predominantly stimulated inhibitory (IL-10) cytokine production.

FIG. 7 presents a flow chart of an in vivo study of perinatal administration of LGG culture supernatants of the present invention;

fig. 8 shows the results of anaphylaxis, showing that there was a decrease in allergic (eosinophilic) cells in lung lavage fluid following perinatal administration of LGG culture supernatant of the invention.

Detailed description of the embodiments

In a broad sense, the present invention is based on the insight that culture supernatant (which may also be referred to as "spent medium") having antiallergic activity can be collected from LGG batch culture. In the context of the present invention, the term "antiallergic" includes "allergy-preventing activity as well as antiallergic-therapeutic activity".

While not wishing to be bound by theory, the inventors believe that this activity may be attributed to a mixture of components, including proteinaceous material, possibly including (epi) polysaccharide material, as found released into the medium at a later stage of the LGG batch culture index (or "log") phase. The composition is hereinafter referred to as "culture supernatant of the present invention".

LGG is a probiotic strain isolated from the intestinal flora of healthy humans. It is disclosed in U.S. Pat. No. 5,032,399 to Gorbach et al, which is incorporated herein by reference in its entirety. LGG is resistant to most antibiotics, stable in the presence of acids and bile, and adheres earnestly to mucosal cells of the human intestinal tract. It survives for 1-3 days in most individuals and for up to 7 days in 30% of subjects. In addition to its colonization ability, LGG also has a beneficial effect on mucosal immune responses. LGG was maintained at the storage authority American type Culture Collection (American type Culture Collection) accession number ATCC 53103.

The skilled worker knows the accepted phases of batch cultivation of bacteria. These are the "lag", "log" ("log" or "exponential"), "rest" and "death" (or "log decline") phases. At all stages where viable bacteria are present, the bacteria metabolize the nutrients from the medium, secreting (draining, releasing) material into the medium. The composition of the material secreted at a given point in time during the growth phase is generally unpredictable.

In the present invention, the secreted material is collected from the late exponential phase. The late exponential phase occurs at a time after the mid-exponential phase (the mid-exponential phase is half the time during the exponential phase, so the late exponential phase is the second half of the time between the lag phase and the stationary phase). In particular, the term "late exponential phase" as used herein refers to the latter quarter of the time between the lag phase and the stationary phase of the LGG batch process. According to the present invention, it is preferred that the time point be 75% to 85% during the exponential phase, most preferably about the lapse of the exponential phase⁵/₆The time of (3) was measured by collecting the culture supernatant.

The term "culturing" or "culturing" refers to the propagation of a microorganism (in this case, LGG) on or in a suitable medium. Such media can be of various types, in particular liquid broths, as is conventional in the art. For example, a preferred broth is MRS broth typically used for culturing lactobacilli. MRS broth generally contains polysorbate, acetate, magnesium and manganese, which are known to act as specific growth factors for lactobacilli as well as a nutrient rich matrix. Typical compositions comprise (in g/litre) the following: casein peptone 10.0; 8.0 parts of meat extract; yeast extract 4.0; d (+) -glucose 20.0; dipotassium phosphate 2.0;801.0, respectively; triammonium citrate 2.0; 5.0 of sodium acetate; 0.2 of magnesium sulfate; manganese sulfate 0.04.

A preferred use of the culture supernatant of the invention is infant formula. Here, in order to fully use the present invention, it is desirable to ensure a composition collected from an LGG cultureDo not contain components that are not desired or legally permitted to be present in such formulations (as may be present in the culture medium). With respect to polysorbates commonly found in MRS broth, the culture medium used to culture the bacteria may include emulsifying nonionic surfactants, such as those based on polyethoxylated sorbitan and oleic acid (typically asPolysorbate esters such as80 obtained). While these surfactants are often found in foods such as ice cream and are generally recognized as safe, they are not all considered advisable on a judicial basis, or even unacceptable for use as nutritional products for relatively fragile subjects such as infant formula or clinical nutrition.

The invention therefore also relates in a preferred embodiment to the use of a medium in which the abovementioned polysorbates can be avoided. For this purpose, the preferred medium of the invention does not contain tween 80 and may comprise an oily component selected from the group consisting of oleic acid, linseed oil, olive oil, rapeseed oil, sunflower oil and mixtures thereof. It will be appreciated that the full benefit of the oily component is achieved if the presence of polysorbate surfactants is substantially or completely avoided.

Most preferably, the MRS medium is free of Tween (Tween)80, and comprises, in addition to one or more of the above oils, peptone (typically 10g/L), meat extract (typically 8g/L), yeast extract (typically 4g/L), D (+) glucose (typically 20g/L), dipotassium hydrogen phosphate (typically 2g/L), sodium acetate trihydrate (typically 5g/L), triammonium citrate (typically 2g/L), magnesium sulfate heptahydrate (typically 0.2g/L) and manganese sulfate tetrahydrate (typically 0.05 g/L).

The cultivation is generally carried out at a temperature of 20 ℃ to 45 ℃, preferably 35 ℃ to 40 ℃, most preferably 37 ℃.

The preferred time point for harvesting culture supernatant during culture, i.e., during the latter exponential phase described above, can be determined, e.g., based on OD600nm and glucose concentration. OD600 refers to the optical density at 600nm, which is a known density measurement directly related to the concentration of bacteria in the medium.

In addition to the above, it should be noted that batch culture of lactobacillus including LGG is the general knowledge available to those skilled in the art. And therefore further elucidation of these methods is not required here.

Preferably, the compositions of the present invention are prepared by large scale fermentation (e.g., in fermentors of greater than 100L, preferably about 200L or more).

The compositions of the invention can be collected by any known technique for separating a culture supernatant from a bacterial culture. Such techniques are well known in the art and include, for example, centrifugation, filtration, sedimentation, and the like.

The supernatant may be used immediately or stored for future use. In the latter case, the supernatant is typically refrigerated, frozen or lyophilized. The supernatant may be concentrated or diluted as desired.

The composition collected according to the invention is believed to comprise a protein composition. The term "protein" is known to the skilled person and indicates that the composition comprises one or more peptides, proteins or other compounds comprising amino acid residues.

With respect to chemicals, the composition of the culture supernatant of the present invention is believed to be a mixture of various amino acids, oligopeptides and polypeptides and proteins of various molecular weights. The composition is further believed to comprise a polysaccharide structure.

In contrast to the prior art, it is emphasized that the present invention preferably relates to a whole, i.e. unfractionated, culture supernatant. The judicious choice of collection in the above-mentioned late exponential phase, and the retention of virtually all the components of the supernatant, are believed to contribute to the surprising results obtained therewith, in particular in terms of antiallergic activity, more particularly in terms of such activity after administration to pregnant and lactating women, respectively, in infants and newborns, and perinatal periods.

The complete culture supernatant is more particularly defined as substantially excluding low molecular weight components, typically below 6 kDa. This relates to the fact that the composition preferably does not comprise lactic acid and/or lactate. The preferred supernatant of the present invention therefore has a molecular weight of greater than 6kDa, as this is a typical supernatant obtained after removal of lactic acid and lactate. This usually involves filtration or column chromatography. In fact, this filtered retentate represents a molecular weight range greater than 6kDa (in other words, components below 6kDa are filtered out).

The composition of the supernatant of the invention will generally not only be proteinaceous, but will also comprise polysaccharides, in particular exopolysaccharides (such as high molecular weight polymers made up of sugar residues produced by LGG). While not wishing to be bound by theory, the inventors believe that the ratio between the amount of proteinaceous material and the amount of carbohydrate material taken from the later exponential phase, as discussed above, contributes to the anti-allergic properties compared to compositions taken from other stages, such as the middle exponential phase or the resting phase.

The culture supernatant collected according to the invention can be used in various ways in order to benefit from the discovered antiallergic activity. Such use will generally involve some form of administration of the compositions of the present invention to a subject in need thereof. In this regard, the culture supernatant may be used as such, e.g., incorporated into a capsule for oral administration, or in a liquid nutritional composition such as a beverage, or it may be processed prior to further use. The latter is preferred.

Such processing typically involves separating the protein composition from the generally liquid continuous phase of the supernatant. This is preferably done by drying methods such as spray drying or freeze drying (lyophilization). Spray drying is preferred. In a preferred embodiment of the spray drying process, a carrier material, such as maltodextrin DE29, may be added prior to spray drying. This is believed to be advantageous in view of also producing a dry powder under conditions where lactic acid (produced from LGG and present in the spent medium) is a liquid.

The compositions of the present invention have been found to possess anti-allergic (prophylactic and/or therapeutic) activity. The anti-allergic activity can be determined, as in the newly developed neonatal mouse model of allergic sensitization and lung inflammation. This model is in fact a variation of the so-called OVA model, which is widely used for the study of the immunopathology of allergic diseases and asthma and for the identification of compounds with antiallergic activity. Allergic diseases include, but are not limited to, asthma (possibly based on allergy), atopic eczema (also possibly based on allergy), food allergy and allergic rhinitis/conjunctivitis.

In order for the composition of the invention to exert its beneficial anti-allergic effect, it is digested by a subject, preferably a human subject. In particular, in preferred embodiments, the subject is a pregnant woman, a lactating woman, a neonate, an infant or a child. As mentioned above, the advantages of using materials that can be considered as "non-viable probiotics" will mostly benefit from the infant's diet product. The term "infant" refers to a postnatal human less than about 1 year of age.

It will be understood that digestion by a subject will require oral administration of the compositions of the present invention. The form of administration of the compositions of the present invention is not critical. In some embodiments, the composition is administered to the subject via a tablet, pill, capsule, caplet, soft capsule (gel cap), capsule, oil droplet, or cachet. In another embodiment, the composition is encapsulated in a sugar, fat or polysaccharide.

In yet another embodiment, the composition is added to a food or beverage product for consumption. The food or beverage product may be a children's nutritional product such as follow-on formula, growing-up milk, beverage, milk, yogurt, fruit juice, fruit-based beverage, chewable tablet, cookie, biscuit or milk powder. In other embodiments, the product may be an infant nutritional product, such as an infant formula or breast milk fortifier.

The compositions of the present invention, whether incorporated in a separate dosage form or a nutritional product, are generally administered in an amount effective to treat or prevent allergy. The effective amount is preferably equal to 1X 10⁴About 1X 10¹²An equivalent of viable probiotic per kilogram of body weight per day, more preferably 10⁸-10⁹. The back calculation of cell equivalents is well within the knowledge of the skilled person.

If the compositions of the present invention are administered via an infant formula, the infant formula may be nutritionally complete and contain the appropriate types and amounts of lipids, carbohydrates, proteins, vitamins and minerals. The amount of lipid or fat typically can vary from about 3 to about 7g/100 kcal. The lipid source may be any known or used in the art, such as vegetable oils such as palm oil, soybean oil, palm olein (palmolein), coconut oil, medium chain triglyceride oil, high oleic sunflower oil, high oleic safflower oil, and the like. The amount of protein typically can vary from about 1 to about 5g/100 kcal. The protein source may be any known or used in the art, such as skim milk, whey protein, casein, soy protein, (partially or extensively) hydrolyzed protein, amino acids, and the like. The amount of carbohydrate typically can vary from about 8 to about 12g/100 kcal. The carbohydrate source can be any known or used in the art, such as lactose, glucose, corn syrup solids, maltodextrin, sucrose, starch, rice syrup solids, and the like.

Conveniently, commercially available prenatal, preterm, infant and child nutritional products may be used. For example, can beThe formula of the premature infant is prepared,(available from Mead Johnson&Company, Evansville, ind., u.s.a.) was supplemented with appropriate levels of the compositions of the present invention and used to practice the methods of the present invention.

In one embodiment, the present composition may be combined with one or more viable probiotics. Any viable probiotic known in the art may be acceptable in this embodiment, provided that the desired result is obtained.

If viable probiotic bacteria are administered in combination with the composition of the invention, the amount of viable probiotic bacteria may correspond to about 1X 10⁴-1×10¹²Colony forming units (cfu) per kg body weight per day. In another embodiment, viable probiotic bacteria may comprise about 1 x 10⁶-1×10¹²cfu per kg body weight per day. In yet another embodiment, viable probiotic bacteria may comprise about 1 x 10⁹cfu per kg body weight per day. In yet another embodiment, viable probiotic bacteria may comprise about 1 x 10¹⁰cfu per kg body weight per day.

In another embodiment, the present composition may be combined with one or more prebiotics. "prebiotic" refers to a non-digestible food ingredient that stimulates the growth and/or activity of probiotics. Any prebiotic known in the art may be acceptable in this embodiment, provided that the desired result is obtained. Prebiotics useful in the present invention may include lactulose, gluco-oligosaccharides, inulin, polydextrose, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soy oligosaccharides, lactosucrose (lactosucrose), xylooligosaccharides and gentiooligosaccharides.

In yet another embodiment of the invention, the infant formula may contain other active agents such as LCPUFA. Suitable LCPUFAs include, but are not limited to, [ alpha ]]-linoleic acid, [ gamma ]]-linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), arachidonic acid (ARA) and/or docosahexaenoic acid (DHA). In an embodiment, the present composition is administered in combination with DHA. In another embodiment, the composition of the invention is administered in combination with an ARA. In yet another embodiment, the present composition is administered in combination with both DHA and ARA. Commercially available infant formulas containing DHA, ARA, or combinations thereof may be supplemented with the present compositions for use in the present invention. For example,which contains effective levels of DHA and ARA, are commercially available and can be supplemented with the compositions of the present invention and used in the present invention. If it includesAn effective amount of ARA in embodiments of the invention is typically from about 5mg per kg of body weight per day to about 150mg per kg of body weight per day. In one embodiment of the invention, the amount varies from about 10mg per kg of body weight per day to about 120mg per kg of body weight per day. In another embodiment, the amount varies from about 15mg per kg of body weight per day to about 90mg per kg of body weight per day. In yet another embodiment, the amount varies from about 20mg per kg of body weight per day to about 60mg per kg of body weight per day. If infant formula is used, the amount of DHA in the infant formula may vary from about 5mg/100kcal to about 80mg/100 kcal. In one embodiment of the invention, DHA varies from about 10mg/100kcal to about 50mg/100 kcal; in another embodiment, from about 15mg/100kcal to about 20mg/100 kcal. In a particular embodiment of the invention, the amount of DHA is about 17mg/100 kcal. If infant formula is used, the amount of ARA in the infant formula may vary from about 10mg/100kcal to about 100mg/100 kcal. In one embodiment of the invention, the amount of ARA varies from about 15mg/100kcal to about 70mg/100 kcal. In another embodiment, the amount of ARA varies from about 20mg/100kcal to about 40mg/100 kcal. In a particular embodiment of the invention, the amount of ARA is about 34mg/100 kcal. If an infant formula is used, the infant formula can be supplemented with oils containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA can be added to the formulation by replacing an equal amount of oil normally present in the formulation, such as high oleic sunflower oil. As another example, oils containing DHA and ARA may be added to the formula by replacing the remainder of an equivalent amount of the total fat blend normally present in a formula without DHA and ARA. If used, the source of DHA and ARA may be any source known in the art such as marine oil (marine oil), fish oil, single cell oil, egg yolk lipid, brain lipid, etc. In some embodiments, the DHA and ARA are derived from single cell Martek oil,or a variant thereof. DHA and ARA may be in natural form, provided that the residue of the LCPUFA source does not produce any substantial detrimental effect on the infant. Alternatively, refined forms of DHA and ARA may be used. In one embodiment of the invention, DHA andARA sources are single cell oils as described in us patent No. 5,374,567; 5,550,156; and 5,397,591, the disclosures of which are incorporated herein by reference in their entirety. However, the present invention is not limited to such oils. In one embodiment, a source of LCPUFA containing EPA is used in combination with at least one composition of the present invention. In another embodiment, a source of LCPUFA that is substantially free of EPA is used in combination with at least one composition of the present invention. For example, in one embodiment of the invention, an infant formula containing less than about 16mg EPA/100kcal is supplemented with a composition of the invention. In another embodiment, an infant formula containing less than about 10mg EPA/100kcal is supplemented with a composition of the invention. In yet another embodiment, an infant formula containing less than about 5mg EPA/100kcal is supplemented with a composition of the invention.

Another embodiment of the invention comprises infant formulas supplemented with the composition of the invention even without trace amounts of EPA. It is believed that providing a combination of the present composition with DHA and/or ARA provides a complementary or synergistic effect in the anti-allergic properties of formulations containing these agents.

In a further preferred embodiment, the diet product of the invention comprises one or more bioactive materials normally present in human breast milk, such as proteins or polysaccharides.

The composition of the present invention is preferably used for preventing, alleviating, ameliorating or treating allergy and/or symptoms thereof.

Allergy is defined as "an abnormal hypersensitivity reaction to a normally tolerated and generally regarded as harmless substance". The symptoms of allergy can range from runny nose to anaphylactic shock. Approximately 5 million americans suffer from allergic diseases and the incidence of these diseases is increasing.

The allergic reaction involves two basic stages. The first stage involves the early development of immediate hypersensitivity to the allergen. The allergen reaches the immune system for the first time and no allergic reaction occurs. Instead, the immune system itself is ready for future encounters with allergens. Macrophages, which are cell-scavenging, and so-called dendritic cells surround and destroy invading allergens. The cells then display the allergen fragments on their cell wall to the T lymphocytes, which are the main coordinator of the immune response of the body (immunocator). This cognitive signal plus several non-cognitive signals (e.g., cytokines) activate naive T cells, indicating that T cells differentiate into T cell effector subsets. A key role in the allergic cascade is the Th-2 phenotype of T cells (TH-2). TH-2 type T cells are characterized by secretion of several cytokines including interleukin-4 (IL-4), IL-5, and IL-13. The cytokines IL-4 and IL-13 then activate B lymphocytes to produce antibodies (IgE) directed against subclass E of a particular allergen. The interaction of specific IgE antibodies on the surface of effector cells (mast cells and basophils) with allergens triggers the early phase of immediate hypersensitivity reactions.

This mast cell activation typically occurs within minutes after a second or additional exposure to the allergen. IgE antibodies on mast cells, which are constituted during sensitization, recognize the allergen and bind to the invader. Once the allergen binds to the receptor, the granules in the mast cell release their contents. These contents, or mediators, are pro-inflammatory substances such as histamine, platelet activating factor, prostaglandins, cytokines, and leukotrienes. These mediators actually trigger allergic episodes. Histamine stimulates mucus production and causes redness, swelling and inflammation. Prostaglandins constrict airways and dilate blood vessels.

The second phase of the allergic immune response is characterized by infiltration of inflammatory cells, such as eosinophils, into the airways following allergen exposure. An important link between sensitization and inflammation is represented by T cells, which secrete mediators not only involved in IgE synthesis, but also responsible for eosinophil recruitment, activation and survival. Tissue mast cells and neighboring cells generate chemical messengers that signal circulating basophils, eosinophils, and other cells to migrate into the tissue and help fight foreign material. Eosinophils secrete their own chemicals to sustain inflammation, cause tissue damage, and recruit more immune cells. This phase may occur anywhere from hours to days after allergen exposure, and may last hours or even days.

Respiratory allergies are a specific type of allergy that affects the respiratory tract. The inner layers of the airway from the nose to the lungs are structurally similar and are often similarly affected by allergic processes. Thus, allergens affecting the nose or sinuses may also affect the lungs.

For example, allergic rhinitis, also known as hay fever, is caused by allergic reactions of the mucous membranes in the nose and airways to allergens in the air. Symptoms of allergic rhinitis often include nasal itching, pharynx itching and eye itching and excessive sneezing. Nasal congestion or discharge is often followed.

Because allergens in one region of the respiratory tract can affect other regions of the respiratory tract, rhinitis in the nasal passages can lead to asthma, a much more severe disease that occurs in the lower airways of the lungs. Asthma is characterized by the development of airway hyperresponsiveness, breathlessness, expiratory wheezing, dry cough and chest tightness. Repeated allergen exposure can sustain an inflammatory immune response in the airways, leading to airway remodeling, commonly referred to as chronic asthma. Not every person with allergic rhinitis develops asthma symptoms, but a significant number, especially those with relapsed, untreated allergies, will show changes in pulmonary inflammation. About 40% of people with allergic rhinitis will indeed develop well-developed asthma.

If the nasal inflammation accompanying allergic rhinitis reaches the sinuses, the result may be an uncomfortable infection called sinusitis, or naso-sinusitis, in which the sinuses themselves cannot empty of bacteria. Symptoms include nasal congestion, runny nose, sore throat, fever, headache, fatigue and cough, as well as forehead, back cheek pain, and even dental and chin pain.

Respiratory allergies are among the most common afflictions in childhood. As for adults, respiratory allergies in the heavy child are most likely manifested in the form of allergic rhinitis and asthma.

Prevention of respiratory allergies is particularly important in infants and young children, as early allergic sensitization to allergens appears to be associated with delayed maturation of normal immune responses. In addition, allergic sensitization is generally considered as the first step in the development of atopic diseases. Baena-Cagnani, Role of Food allergy in Childhood Asthma, allergy, clin, immun.1 (2): 145-149(2001). Frequently, early onset asthma is associated with atopy, so early allergic sensitization appears to play an important role in persistent asthma as well. Martinez, F., Development of Wheezing Disorders and Ashma in Preschool Children (Development of Wheezing disease and Asthma in preschool Children), Pediatr.109: 362-367(2002).

Not only is there a strong association between allergic sensitization and asthma, but the association appears to be age dependent. Although few children are sensitized with allergens during the first years of life, the vast majority of sensitized children develop asthma-like symptoms later in life during this period. Martinez, f., Viruses and Atopic Sensitization in the First Years of Life, am.j.respir.crit.care med., 162: S95-S99 (2000). Therefore, it is important to find a method for preventing early allergen sensitization, anaphylaxis and preventing respiratory allergy later in life.

There is increasing evidence that many aspects of health and disease are determined not only during infancy but also during pregnancy. This is especially true for allergic diseases, where the immune response at birth involves intrauterine exposure as a primary sensitization event. For example, allergen-specific T cells are already present at birth and early sensitization to food allergens is identified as a predictor of later development of respiratory allergies. IHi et al, The Natural court of Atopic Dermatitis from Atopic Dermatitis to Age 7 Years and The Association with The Association of Asthma, clin. exp. allergy 27: 28-35(1997). Furthermore, the lungs begin to develop very early after fertilization and last at least 2 or 3 years after birth. Therefore, both prenatal and postpartum airway development is important for the development of respiratory allergies in infants and children.

It has also been shown that in early gestation the human fetus develops IgE-producing B-cells, capable of producing IgE antibodies in response to appropriate antigenic stimuli in a manner similar to the well-established IgM response seen in various prenatal infections. Weil, g, et al, advanced allogenic sensing to helminthes in Offspring of Parasite-induced Mothers (Prenatal Allergic Sensitization to helminth Antigens in the progeny of Mothers Infected with parasites), j.ci i.invest.71: 1124-1129(1983). This also illustrates the importance of preventing prenatal and postpartum allergic sensitization to respiratory allergens.

Traditional medications for respiratory allergies include antihistamines, topical nasal steroids, decongestants and cromolyn solutions. As an alternative to traditional drugs, probiotics have emerged as a possible treatment for certain types of allergies. With respect to the above-mentioned disadvantages of using live or viable probiotics, the present invention is particularly advantageous for replacing such probiotics in products for preventing, alleviating, ameliorating or treating allergic diseases and/or symptoms thereof. To achieve this, the composition is preferably administered via a diet or nutritional product, more preferably a prenatal, infant or children's formula or nutritional composition, a medical food, or a food for a specific medical purpose (i.e. a food whose label defines a medical purpose), most preferably an infant formula, or a perinatal nutrition for pregnant women or lactating women, substantially as discussed above. Furthermore, the present invention can also provide probiotics in an improved manner. Because the non-viable probiotic derived material of the present invention can be prepared in an industrial environment in a standardized and reproducible manner, those problems inherent to live probiotics are avoided. Moreover, because of their non-viable nature and particularly when provided as a dry powder, they can be fully incorporated and dosed into nutritional compositions for the prevention or treatment of allergic reactions or diseases.

The invention will be illustrated hereinafter with reference to the following non-limiting examples and the accompanying drawings.

Example 1

In a batch fermentation process, LGG is grown under physiological conditions. By adding 33 percentNaOH kept the pH constant at 6 and the temperature at 37 ℃. Stirrer speed 50rpm, N for headspace₂And (5) flushing. The following medium (modified MRS broth) is provided (table 1).

TABLE 1

Bacterial growth is depicted in fig. 1(a) and (b).

Figure 1a shows the evolution of pH, titrated NaOH (33%) (DM base-dose monitoring base) and OD600 during LGG fermentation. Reference is made to the legend given in the figures. pH and OD600 measurements enable determination of bacterial growth in the fermenter; where the addition of NaOH to maintain pH at 6 is correlated with lactate production (i.e., a measure of bacterial metabolic activity), OD600 is a density measurement correlated with the number of bacteria in the fermentor.

In FIG. 1b the vertical axis shows the bacterial count in the culture medium on a logarithmic scale. The horizontal axis represents time.

Samples of culture supernatant were taken at 3 time points (indicated in the figure as MJ1, MJ2, and MJ 3).

Example 2

In analogy to example 1, LGG cultures were carried out on the basis of modified medium. Tween was not present, oil was added as follows:

(a) oleic acid was present at concentrations of 1g/kg, 2g/kg and 4 g/kg.

(b) Linseed oil is used at concentrations of 1g/kg, 2g/kg and 4 g/kg.

(c) The olive oil is present at concentrations of 1g/kg, 2g/kg and 4 g/kg.

(d) Rapeseed oil was used at concentrations of 1g/kg, 2g/kg and 4 g/kg.

(e) Sunflower seed oil was used at concentrations of 1g/kg, 2g/kg and 4 g/kg.

In all these tests (a) - (e), successful LGG growth was observed, comparable to growth in tween-containing medium. In addition, it was surprising that LGG was successfully cultured without adding Tween or any oil.

Example 3

In this example, supernatants obtained as in example 1 were screened for anti-allergic and anti-inflammatory activity using RAW 264 cells (mouse macrophage cell line) in an in vitro model recognized in the art. RAW cell cultures showed a substantial increase in the production of the regulatory cytokine IL-10 during culture with MJ2 supernatant sample harvest of LGG cultures compared to other supernatant sample harvests. See fig. 2.

Example 4

A comparison was made between the culture supernatant MJ2 obtained in example 1 and viable LGG bacteria in an Ovalbumin (OVA) sensitization model. This in vivo model, as recognized in the art, applies normally to adolescent or adult mice. In this example, the conventional model was modified to allow the study of allergies early in life.

Thus, neonatal Balb/C mice received LGG or whole LGG culture supernatant every other day for 6 weeks by intragastric administration. Animals were sensitized with Ovalbumin (OVA) 2 times on days 42 and 56, followed by later challenge and exposure to OVA aerosol on days 61, 62 and 63. This schedule is shown in figure 3. Parameters of experimental bronchial asthma were assessed by lung function analysis, histology and bronchoalveolar lavage (BAL). Systemic anaphylaxis was assessed by antibody levels and cytokine response. The latter was measured in bronchoalveolar lavage (BAL) and in restimulated draining lymph node cell cultures.

Exposure to viable LGG as well as LGG supernatant was found to reduce airway inflammation and goblet cell proliferation. Histological staining of lung tissue sections (see fig. 4) showed increased inflammatory cell infiltration and goblet cell proliferation in OVA-allergic animals, whereas LGG or LGG supernatant treated animals showed almost normal lung architecture and histology. In FIG. 4, "negative control" means: no challenge, nor LGG or LGG supernatant; "Positive control" refers to: (ii) is subjected to OVA challenge, not to LGG or LGG supernatant; "vigor LGG" refers to: (ii) is subjected to excitation and viability LGG; "LGG supernatant" refers to: subjected to excitation and LGG supernatant treatment of the present invention.

In addition, the presence of infiltrating inflammatory cells (eosinophils) was determined. Figure 5 shows that increased eosinophil infiltration in the lungs of OVA-allergic animals was significantly reduced by treatment with viable LGG or LGG supernatant. In fig. 5, the vertical axis represents the percentage of increase in allergic cells. The following samples are represented on the horizontal axis: "negative control": no OVA excitation; no treatment is carried out; "positive control": activating by OVA; no treatment is carried out; "LGG all": OVA challenge followed by treatment with viable LGG; "LGG supernatant": OVA challenge was followed by treatment with LGG supernatant of the invention.

Example 5

This example reflects the determination of in vitro cultures of cells isolated from lymph nodes (performed in a known manner). The typical Th2 cytokine profile in restimulated lymph node cell cultures from OVA-allergic mice showed increased IL-5 and low IL-10 and IFN- γ responses. Treatment with either whole (viable) LGG or LGG supernatant of the invention showed antiallergic effects, as shown by reducing IL-5 response and strongly stimulating IFN-. gamma.and IL-10 production in these cultures. This is depicted in fig. 6.

Example 6

This example shows perinatal administration of LGG culture supernatant of the present invention in an Ovalbumin (OVA) allergy model in Balb/C mice. Pregnant and lactating mothers were given intragastric administration receiving LGG supernatant on alternate days. Their progeny were sensitized and challenged with OVA. The flow of this test is illustrated in fig. 7.

Parameters of experimental bronchial asthma were assessed by lung function analysis, histology and bronchoalveolar lavage (BAL). Systemic anaphylaxis was assessed by antibody levels and cytokine response. The latter was measured in BAL and in draining lymph node cultures. Fig. 8 shows the results, and it is self-evident that infiltration of inflammatory cells (eosinophils, macrophages) was reduced in LGG supernatant-treated animals.

Claims (13)

1. A composition comprising a mixture of proteins, said composition being obtainable from a culture supernatant in the late exponential phase of an LGG batch culture process, for use in the treatment or prevention of an allergic disease.

2. The composition for use in the treatment or prevention of allergic diseases according to claim 1, obtainable by a process comprising the steps of: (a) culturing LGG in a suitable medium using a batch process; (b) collecting a culture supernatant in a late exponential phase of the culturing step, wherein the phase is defined as a second half period between a lag phase and a stationary phase of the LGG batch culture method; (c) optionally removing low molecular weight components from the supernatant to retain components above the 6kDa molecular weight; (d) the liquid content is removed from the culture supernatant to obtain the composition.

3. The composition according to claim 1 or 2, wherein the late exponential phase is defined as the last quarter of the time between the lag phase and the stationary phase of the LGG batch culture process, preferably the time of 0.75 to 0.85 elapsed in the exponential phase.

4. The composition according to any one of the preceding claims, wherein the LGG batch cultivation is carried out in a medium free of tween 80, preferably a medium containing an oily component selected from the group consisting of oleic acid, linseed oil, olive oil, rapeseed oil, sunflower oil and mixtures thereof.

5. The composition according to any one of the preceding claims, wherein the LGG batch cultivation is performed at neutral pH, preferably pH6, at physiological temperature, preferably 37 ℃.

6. The composition of any of the preceding claims, which is in a dried form, preferably spray-dried or freeze-dried.

7. The composition of claim 6, wherein a pharmaceutically acceptable carrier material, such as maltodextrin DE29, is added to the supernatant, followed by spray drying.

8. An infant or child formula which is nutritionally complete in terms of the presence of lipids, carbohydrates, proteins, vitamins and minerals, further comprising a composition according to any one of claims 1-7.

9. The composition according to any one of claims 1 to 7 in the form of a dietary product, preferably a nutritional product, or in the form of an additive to such a product, preferably a nutritional product.

10. The composition of claim 9, wherein the diet product further comprises one or more polyunsaturated fatty acids (PUFAs), preferably long chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid (ARA) or docosahexaenoic acid (DHA).

11. The composition of claim 9 or 10, wherein the diet product further comprises one or more bioactive ingredients normally present in human breast milk, such as proteins or polysaccharides.

12. The composition according to any of claims 9 to 11, wherein the diet product further comprises one or more prebiotics, preferably selected from the group consisting of non-digestible oligosaccharides, non-digestible polysaccharides, and mixtures thereof.

13. The composition according to any of claims 9 to 12, wherein the dietary product is a prenatal, infant or children's formula or a nutritional composition or supplement, a medical food or a food for special medical purposes.