RU2821998C2 - Compositions containing secretory iga and probiotics - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method for preparing a composition containing secretory IgA and a probiotic, involving: a) providing a source of SIgA in the form of milk, which is not exposed to a temperature higher than 70 °C after expression, or its fraction; b) optionally microfiltration of milk; c) milk pasteurisation at 61–200 °C for 0.1–200 s; d) optional spray drying at temperature of liquid concentrate less than 70 °C; e) mixing with probiotic at 12–45 °C for 10–360 min; and f) optionally spray drying at temperature of liquid concentrate less than 70 °C.

EFFECT: invention widens the range of methods for producing SIgA, having the required level of sterility for baby food, while preserving its bioactivity and ability to enhance the beneficial effect of probiotics after association with them.

9 cl, 10 dwg, 7 tbl, 7 ex

Description

Scope of the invention

The present invention relates to methods for preparing compositions containing secretory IgA and one or more probiotics. In particular, the invention relates to methods for preparing compositions in which secretory IgA and one or more probiotics are associated. The invention also relates to compositions prepared by such methods and uses of such compositions, for example, for the attenuation or prevention of non-viral infection and/or inflammation.

Prerequisites for creating the invention

Infections in general are unwanted colonization of a host by foreign species. Typically, the infecting organism attempts to use the resources of the host organism for its own reproduction. Thus, the infecting organism, or pathogen, can disrupt the normal functioning of the host and can lead to additional infection-related disorders that can vary in severity and, in the worst case, can lead to death.

Inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. In general, inflammation is a protective attempt by the body to remove harmful stimuli and also to initiate the repair process of damaged tissue. However, dysregulation of inflammation can lead to serious diseases regardless of the age of the subject.

One way to achieve the goals of preventing and treating infection and/or inflammation is by administering a composition containing probiotics.

Probiotic microorganisms are known to have beneficial effects on the health and general condition of the host. The use of probiotic bacteria has attracted considerable attention as a safe and accessible form of prevention or treatment of, for example, digestive system diseases (Isolauri E, et al., Dig Dis Sci 1994,39:2595-2600). Typical probiotic bacteria used for this purpose belong to the genera Lactobacillus or Bifidobacterium.

The effectiveness of probiotics, first, depends in part on their ability to withstand the conditions of the digestive tract and attach to the intestinal epithelium. Additional critical aspects of the potential benefit of probiotics to the host are their effects on epithelial barrier function and their interaction with the host mucosal immune system.

Although some probiotics have already achieved impressive results in stimulating or mitigating the host's immune system response, it is desirable to further enhance the efficiency with which probiotic microorganisms can interact with the host mucosa and thereby exert beneficial effects on the host.

Antibodies are often glycoproteins that specifically recognize their antigens. Five classes of immunoglobulins have been described in vertebrates, including immunoglobulins IgG, IgM, IgA, IgD, and IgE, all of which vary in their function in the immune system.

One of the most characteristic features of the mucosal immune system in most mammals is the dominant presence of secretory antibodies, particularly secretory IgA (SIgA), a class of antibodies unique to mucosae. Biosynthesis of polymeric IgA (pIgA) occurs in the lamina propria of the mucosa, and its transport through the epithelium lining the surface of the mucosa is ensured by the polymeric Ig receptor (pIgR), expressed on the basolateral side of the crypts and columnar epithelial cells. During transport of the pIgR-pIgA complex to the apical surface of cells, the extracellular portion of pIgR, called the secretory component (SC), is shed off and remains associated with polymeric IgA, thereby releasing SIgA into the mucosal lumen.

Some of the authors of the present invention have previously identified SIgA antibodies that can associate and form complexes with strains of commensal bacteria. In particular, SIgA antibodies, when associated with probiotics, have been found to stimulate the interaction and subsequent mutual influence of probiotics on the host, which helps to enhance the health benefits of probiotics, such as triggering an immune-stimulating effect against infections or preventing any potentially harmful inflammatory process (WO2009/156301 and WO2009/156307).

It is very difficult to produce infant formulas containing bioactive ingredients such as SIgA while meeting current quality standards and regulations regarding microbiological contamination. Indeed, currently commonly used methods for preparing commercial powdered infant formulas involve high temperature processing (such as, for example, 145°C for 6 s), which is absolutely detrimental to bioactive SIgA. Thus, it would be desirable to provide a method that would produce SIgA having the required level of sterility for infant formula while maintaining its bioactivity and thus the ability to enhance the health benefits of probiotics once associated with them. It is also desirable to optimize the method for producing nutritional compositions containing bioactive SIgA associated with probiotics. Indeed, SIgA is not present in existing infant formulas (IF) due to the heat treatment steps involved in the production of infant formulas.

There is also a need to improve the conditions for the association of SIgA and probiotics to form complexes, in particular to obtain complexes with optimized bioactivity. Indeed, the complexity of the matrix environment and temperature can have a major impact on the dynamics of association of probiotics and SIgA and thereby the efficiency of the complexes.

Summary of the invention

The present inventors have developed an improved method for preparing compositions containing bioactive SIgA and one or more probiotics, in particular a method that results in optimized association of SIgA and probiotic. This method results in compositions containing probiotic-SIgA complexes with optimized bioactivity to enhance the health benefits of probiotics and with a level of microbiological safety suitable for use in products for sensitive consumers, such as infant formulas.

In one aspect, the invention provides a method for preparing a composition containing secretory IgA and a probiotic, the method comprising the steps of:

(a) providing a source of SIgA in the form of milk or a fraction thereof;

(b) optionally microfiltration of milk;

(c) pasteurization of milk at a temperature of 61–200°C;

(d) optionally spray drying at a temperature of the liquid concentrate less than 70°C;

(e) mixing with a probiotic, wherein mixing is performed at a temperature of 1-45°C for 10-1440 minutes; And

(f) optionally spray drying at a temperature of the liquid concentrate less than 70°C.

In another aspect, the invention provides a composition prepared or prepared using the method of the present invention.

In a further aspect, the invention relates to compositions prepared or prepared using the method of the present invention for use in the modulation, attenuation, prevention and/or treatment of infections and/or inflammation.

In another embodiment, the present invention provides a product containing a composition obtained using the method of the present invention.

Description of graphic materials

Fig. 1 is a schematic representation of the proposed mechanism leading to improved effects of probiotics from SIgA when associated with them, due to increased interaction with the intestinal mucosa of the host. Possible and documented pathways of interaction of SIgA associated with commensal bacteria with the intestinal mucosa of the host organism are shown.

Fig. 2 shows the results of a validation trial designed to test the ability of the thymic stromal lymphopoietin excretion (TSLP) bioassay to evaluate the improvement in the efficacy of probiotics when associated with SIgA, as outlined in the introductory part of the example section. Compared to normal epithelial cells (without probiotics), supplementation with probiotics alone stimulated the production of thymic stromal lymphopoietin (TSLP) by epithelial cells. Association of increasing amounts of purified intact SIgA with the same amount of probiotics increases TSLP production by epithelial cells in a dose-dependent manner.

Fig. 3 - SIgA yield results according to ELISA (Fig. 3A) and logarithmic reduction in microbiological load (Fig. 3B) in milk after microfiltration with different membranes at a temperature of 52 to 55°C, as described in example 2.

Fig. 4 - results of quantitative measurement by semi-quantitative ELISA and biological analysis of TSLP excretion in milk after heat treatment under various conditions of duration and temperature, as described in example 3, allowing for a relative comparison between quantitative ELISA and bioactivity data for various samples in comparison with the untreated RO method milk (standard sample).

Fig. 5 - results of biological analysis of TSLP excretion after the association stage by incubation of RO skim milk and BL818 formulas under various conditions of duration and temperature, as described in example 4, compared with BL only (BL818, not associated with SIgA) and with control from normal cells, and neither BL nor SIgA is added to the bioassay.

Fig. 6 shows the results of a biological assay for TSLP excretion after performing the association step with various concentrations of BL818 as described in Example 5.

Fig. 7 shows the results of a biological assay for the excretion of TSLP complexes formed between SIgA and liquid or spray-dried biomass as described in Example 6.

Fig. 8 - results of measurements using the enzyme-linked immunosorbent spot (ELISPOT) method, allowing to assess the effect on the formation of the immune system both at the level of the mucous membrane (PP; Fig. 8A) and at the systemic level (blood; Fig. 8B) with additional consumption at an early age prototypes of various infant formulas containing probiotics only, SIgA only, or a combination thereof, as described in Example 7.

Detailed Description of the Invention

As used herein, the terms “comprising,” “comprising,” and “consisting of” are synonymous with the terms “including” or “includes”; or “comprising” or “comprising”, and they are inclusive, or non-limiting, and do not exclude additional non-exclusive terms, elements or stages. The terms “comprising,” “comprising,” and “consisting of” also include the term “comprising.”

Way

The invention provides a method for producing a composition containing secretory IgA and a probiotic, the method comprising the steps of:

(a) providing a source of SIgA in the form of milk or a fraction thereof;

(b) optionally microfiltration of milk;

(c) pasteurization of milk at a temperature of 61–200°C;

(d) optionally spray drying at a temperature of the liquid concentrate less than 70°C;

(e) mixing with a probiotic, and mixing is performed at a temperature of 10-45°C for 10-1440 minutes; And

(f) optionally spray drying at a temperature of the liquid concentrate less than 70°C.

Providing a source of SIgA

Immunoglobulin A (IgA) is an antibody that plays an important role in mucosal immune function. IgA can be divided into serum IgA and secretory IgA.

Secretory IgA (SIgA), which is the predominant and more stable immunoglobulin in intestinal mucosal secretions, has been found to be particularly effective for the present purpose.

Secretory IgA can consist of 2–4 IgA monomers, which can be linked by two additional chains. One of the additional chains, called the J (junction) chain, is rich in cysteine and is structurally different from the other immunoglobulin chains and is formed in cells that secrete IgA.

Preferably, the secretory IgA is bioactive secretory IgA (ie, secretory IgA capable of performing its natural functions, such as epitope binding; for example, bioactive secretory IgA is not denatured).

The source of SIgA is in the form of milk or milk fraction. In some embodiments, the milk is cow's milk, sheep's milk, goat's milk, human breast milk, horse's milk, or camel's milk. In preferred embodiments, the milk is cow's milk. In a preferred embodiment, the milk is skim milk. In some embodiments, the milk is colostrum. In some embodiments, the milk fraction is whey.

In one embodiment, the milk used as the source of SIgA is concentrated milk. In a preferred embodiment, the milk is first defatted by centrifugation and then concentrated by reverse osmosis.

In some embodiments, the milk provided in step (a) is not exposed to temperatures above 70°C, preferably 58°C, after expression. In preferred embodiments, the milk provided in step (a) is not exposed to temperatures greater than 15°C after expression. In one embodiment, the milk or milk fraction used as the source of SIgA has not been pasteurized.

In some embodiments, the milk provided in step (a) contains 9-30%, preferably 18-26%, of total solids.

In some embodiments, the mixture provided in step (e) contains 15-50% total solids.

Microfiltration

Microfiltration (MF) is a physical separation process in which a feed liquid is passed through a membrane of defined pore sizes to separate microorganisms and suspended particles from the process fluid.

Microfiltration is commonly used in the dairy industry, particularly for the processing of milk and whey. For example, microfiltration helps remove bacteria and spores from milk.

Microfiltration allows you to increase the shelf life of the product. Additionally, microfiltration can also be used to separate casein from whey proteins. This results in a casein-enriched concentrate that can be used to make cheese, as well as a whey/whey protein stream that can be further processed to produce whey protein concentrate.

Microfiltration methods are well known in the art. For example, microfiltration can be carried out using a polymer membrane or ceramic membranes.

The present inventors have found that the pore size of the microfiltration membrane affects the retention of SIgA in the composition. Small pore sizes provide the greatest reduction in microbial contamination but result in the greatest loss of SIgA, while larger pores are less effective in reducing microbial contamination but result in the retention of more SIgA in the composition. Thus, it is preferable to use a pore size that provides a good balance between reducing microbiological contamination and SIgA retention, resulting in a composition that has a microbial content acceptable for use in "sensitive" products, such as, for example, infant formula, and has sufficient amounts of SIgA. Thus, it is preferable to use membranes with a pore size greater than 500 nm, preferably between 1000 and 1400 nm.

In some embodiments, microfiltration step (b) is performed at a temperature of 10-70°C. In preferred embodiments, the microfiltration in step (b) is performed at a temperature of 10-62°C.

In cases where milk or milk fraction is concentrated, for example by reverse osmosis (RO), the microfiltration step can be performed before or after the concentration step. In embodiments in which whole milk skimmed by centrifugation and concentration is used as the source of SIgA, the microfiltration step can be performed before or after the concentration step (such as RO), preferably after the concentration step (such as RO).

Pasteurization

Pasteurization is a widely used method in which products such as foods (such as milk and fruit juice) are heat treated to eliminate pathogens. This process is essentially designed to make food safer by destroying or inactivating organisms that may be pathogens or otherwise contribute to food spoilage, including vegetative bacteria, but not bacterial spores.

Pasteurization is widely used in the dairy and other food processing industries to preserve and ensure food safety.

Various methods for performing pasteurization are well known in the art and include the use of indirect heat exchangers and live steam injection (DSI).

In some embodiments, the pasteurization in step (c) is performed at a temperature of 61-120°C for 0.1-200 seconds. In preferred embodiments, the pasteurization in step (c) is carried out at a temperature of 70-90°C for 2-70 seconds, preferably 75-85°C for 5-15 seconds. In preferred embodiments, pasteurization in step (c) is performed at a temperature of 82-84°C for 5-7 seconds.

In other embodiments, the pasteurization in step (c) is performed at a temperature of 120-200°C.

In some embodiments, pasteurization in step (c) is performed at a temperature of 120-200°C for 0.3-2 seconds.

Spray drying

Spray drying is a frequently used method of producing dry powder from a liquid or slurry by rapid drying with hot gas, and is regularly used to dry heat-sensitive materials such as food and pharmaceutical materials. Air can be used as a heated drying medium; although inert gases such as nitrogen can also be used. In spray dryers, a liquid or slurry is typically dispersed using an atomizer or spray nozzle to produce a stream of droplets of controlled size.

Spray drying is widely used in the food and pharmaceutical industries, and equipment suitable for spray drying is widely available.

In preferred embodiments, the spray drying in step (d) and/or step (f) is performed at a liquid concentrate temperature of less than 15°C.

In some embodiments, spray drying in step (d) and/or step (f) is performed at an air flow temperature of 40-90°C.

Association of probiotics and SIgA

In some embodiments, mixing in step (e) is performed at a temperature of 12-45°C, preferably 15-45°C for 10-360 minutes, preferably for 10-200 minutes.

In some embodiments, mixing in step (e) is performed at a temperature of 30-38°C, preferably 36-38°C.

In some embodiments, mixing in step (e) is performed for 25-60 minutes, preferably 25-40 minutes, more preferably 28-32 minutes.

In preferred embodiments, mixing in step (e) is performed at a temperature of 36-38°C for 28-32 minutes.

All probiotic microorganisms can be used in accordance with the invention. As used herein, the term “probiotic” means cells and preparations of microorganisms or components of cells of microorganisms that have a beneficial effect on the health or condition of the host (Salminen S, Ouwehand A. Benno Y. et al “Probiotics: how should they be defined » Trends Food Sci. Technol. 1999:10 107–10).

In some embodiments, the probiotic is selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus and Saccharomyces, or contains a combination thereof.

In some embodiments, the probiotic is selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Enterococcus faecium, Saccharomyces boulardii, and Lactobacillus re uteri, or contains a combination of both.

In preferred embodiments, the probiotic is selected from the group consisting of Lactobacillus johnsonii (NCC533; CNCM I-1225), Bifidobacterium longum (NCC490; CNCM I-2170), Bifidobacterium longum (NCC2705; CNCM I-2618), Bifidobacterium lactis (2818; CNCM I-3446), Lactobacillus paracasei (NCC2461; CNCM I-2116), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus (NCC4007; CGMCC 1.3724) and Enterococcus faecium SF 68 (NCIMB10415), or contains a combination thereof.

In preferred embodiments, the probiotic is Bifidobacterium longum, preferably Bifidobacterium longum subsp. infantis. In other preferred embodiments, the probiotic is Bifidobacterium lactis, preferably Bifidobacterium lactis CNCM I-3446.

The combination of probiotics and SIgA is especially effective if SIgA and probiotics are combined before administration. This has the advantage that the complexes that provide the beneficial effect do not need to be formed after consumption of the product, but are already present in the product. The preparation methods of the present invention provide optimal complex formation.

In some embodiments, SIgA and at least one probiotic are associated in the composition. Preferably they are associated in the form of a complex. In such a complex or association, SIgA is associated with probiotic bacteria.

In some embodiments, the amount of SIgA per 2E7 CFU of probiotic in the mixture in step (e) is from 0.34 ng to 20 μg. In preferred embodiments, the amount of SIgA per 2E7 CFU of probiotic in the mixture in step (e) is from 3.4 ng to 10 μg, preferably from 34 ng to 5 μg, more preferably from 340 ng to 2 μg. In a preferred aspect, such amounts of SIgA are bioactive SIgA. Preferably, bioactive SIgA is defined as SIgA that increases probiotic-induced TSLP production, upon association with the probiotic, to a level above that induced by the probiotic alone, as measured using the bioassay described in the presented examples.

In some embodiments, the probiotic concentration in the mixture in step (e) is 1E6-1E11 CFU/ml. In preferred embodiments, the concentration of probiotic in the mixture in step (e) is 1E8-1E10 CFU/ml.

Specific aspects of the method

In preferred embodiments, the method includes the steps of:

(a) providing a source of SIgA in the form of milk or a fraction thereof;

(b) optionally microfiltration of milk at a temperature of 10–65°C;

(c) pasteurization of milk at a temperature of 82–84°C for 5–7 s;

(d) optionally spray drying at a temperature of the liquid concentrate less than 15°C;

(e) mixing with a probiotic, and mixing is performed at a temperature of 36-38°C for 28-32 minutes; And

(f) optionally spray drying at a temperature of the liquid concentrate less than 15°C.

Composition

The present invention provides a composition obtained or obtained using the method of the present invention as described above.

The invention also relates to a composition containing SIgA and at least one probiotic microorganism. SIgA and the probiotic microorganism may preferably be combined in compositions. SIgA and the probiotic microorganism may preferably be present in a quantitative ratio (number of SIgA molecules per CFU of bacteria) of at least 10:1, preferably at least 100:1, most preferably at least 2000:1 to 100,000:1. The more SIgA molecules are attached to the surface of the probiotic microorganism, the more effective this combination will be. The upper limit of SIgA saturation is determined by the surface of the probiotic microorganism and the number of available binding sites for SIgA.

In preferred embodiments, SIgA and the probiotic are at least partially associated in the composition.

In some embodiments, the infant formula contains a probiotic: secretory IgA complex.

In some embodiments, the composition meets the requirements for infant formula, such as those described by the Food and Agriculture Organization (FAO) in Annexes I and/or II of the Code of Hygienic Practice for Powdered Formulas for Infants and Young Children CAC/RCP 66 -2008. This result is achieved, in particular, as a result of a pasteurization step, which is optionally preceded by microfiltration.

Product

In one embodiment, the invention provides a product containing a composition obtained using the method of the present invention as described above. The composition obtained by the method of the present invention may be a food product, an animal nutrition product or a pharmaceutical composition. For example, the product may be a nutritional composition, nutraceutical, beverage, nutritional supplement, fortifier, or drug.

In some embodiments, the composition is a nutritional composition or a pharmaceutical composition.

In a preferred embodiment, the food product is selected from an infant formula, a follow-on or weaning formula, a 1 to 3 year old formula, an infant cereal, an infant food, a supplement, or a fortifier.

In some embodiments, the composition is for human or non-human animal consumption, preferably the composition is for human consumption.

In some embodiments, the composition is intended for consumption by infants, adolescents, adults, or the elderly.

Newborns, in whom endogenous SlgA antibodies are difficult to detect, depend on maternal IgG transferred through the placenta and exogenous SlgA, which is present in large quantities in breast milk. Together, this provides passive immunization of the digestive tract, which is important for protecting the host during the building and formation phase of the gastrointestinal immune system.

In one embodiment, the composition of the present invention is intended for consumption by newborn children and infants (up to 2 years), preferably non-breastfed newborns and infants (up to two years). Indeed, the maximum health benefits of administration of compositions containing optimized probiotic-SIgA complexes are achieved in situations where natural SIgA is absent or present in limited quantities. This is the situation, for example, in the case of non-breastfed newborns and infants up to two years of age.

In preferred embodiments, the composition is an infant formula, infant nutritional supplement, or milk fortifier.

The nutritional supplement or drug may be presented, for example, in the form of tablets, capsules, lozenges or liquid. The nutritional supplement or drug is preferably provided in the form of sustained release formulations, providing a continuous supply of SIgA and probiotics over a long period of time.

The composition is preferably selected from the group consisting of milk powder based products; instant drinks; ready-to-drink drinking formulations; powdered nutritional supplements; liquid nutritional supplements; milk-based products, in particular yoghurts or ice cream; cereal products; drinks; water; coffee; cappuccino; malt drinks; chocolate-flavored drinks; culinary products; soups; tablets; and/or syrups.

The composition may additionally contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, auxiliary compounds, dispersing agents, wetting agents, processing aids (solvents), anti-caking agents, taste masking agents, weighting agents, gelling agents, gelling agents, antioxidants and antimicrobial agents.

The composition may also contain conventional pharmaceutical additives and excipients, excipients and diluents, including, without limitation, water, gelatin of any origin, vegetable gums, lignin sulfonate, talc, sugars, starch, acacia, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, dyes, wetting agents, fillers, etc.

In addition, the composition may contain an organic or inorganic carrier material suitable for oral or enteral administration, as well as vitamins, minerals, trace elements and other micronutrients in accordance with the recommendations of government authorities, such as the US Recommended Dietary Allowances (RDA).

The composition of the invention may contain a protein source, a carbohydrate source and/or a lipid source.

Any suitable edible protein may be used, for example animal proteins (such as milk proteins, meat proteins and egg whites); plant proteins (such as soy protein, wheat protein, rice protein and pea protein); mixtures of free amino acids; or combinations thereof. Particularly preferred are milk proteins such as casein and whey proteins, and soy proteins.

If the composition includes a fat source, the fat source preferably provides from 5% to 40% of the energy content of the composition; for example 20% to 30% of the energy value. Docosahexaenoic acid (DHA) may be added. A suitable fat profile can be achieved using a mixture of canola oil, corn oil and high oleic acid sunflower oil.

The carbohydrate source preferably provides from 40% to 80% of the energy content of the composition. Any suitable carbohydrate may be used, such as sucrose, lactose, glucose, fructose, corn syrup powder, maltodextrins, and mixtures thereof.

The composition may also contain prebiotics. Supplementation with prebiotics provides health benefits because when combined with probiotics, they can produce synergistic effects on health benefits. A composition containing a combination of prebiotics and probiotics is generally referred to as a symbiotic composition.

As used herein, the term “prebiotic” means nutritional substances that promote the growth of beneficial bacteria, such as bifidobacteria or lactobacilli, and/or probiotics in the intestines. Prebiotics are not broken down in the stomach and are not absorbed in the gastrointestinal tract of the person taking them orally, but are fermented by the microflora of the gastrointestinal tract and/or probiotics.

Prebiotics that can be used in accordance with the invention are not particularly limited and include all nutrients that promote the growth of probiotics in the intestine. Preferably, the prebiotic is selected from the group consisting of oligosaccharides, optionally containing fructose, galactose, mannose; dietary fiber, in particular soluble fiber, soy fiber; inulin; or mixtures thereof. The preferred prebiotics are human milk oligosaccharides (HMO), bovine milk oligosaccharides (BMO), fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides, xylooligosaccharides, soy oligosaccharides, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), saccharides (PAO), maltooligosaccharides, pectins and/or their hydrolysates.

The compositions may also contain at least one other type of other food-grade bacteria, preferably selected from the group consisting of lactic acid bacteria, bifidobacteria, enterococci, or mixtures thereof. Such other food-grade bacteria may help promote healthy intestinal microflora and thereby further further the object of the present invention.

The amount of SIgA required to achieve an effect is not limited. In general, it is preferred that the product contains from 0.0001 mg SIgA to 100 mg SIgA, for example from 0.0001 mg SIgA to 10 mg SIgA per daily dose. In preferred embodiments, the composition contains 0.001-0.1 mg of secretory IgA per daily dose.

Generally, probiotics will be effective in a wide range of amounts. In some embodiments, the composition contains from 1E2 to 1E12, preferably from 1E2 to 1E10 probiotic cells per daily dose.

In preferred embodiments, the product is an oral composition for infants.

As used herein, the term “infant oral composition” means a composition suitable for consumption by an infant. Examples of compositions for oral administration to infants include, but are not limited to, infant nutritional supplements and infant formulas.

During the first four months of life, infants usually cannot eat solid foods. Accordingly, in preferred embodiments, the oral composition for infants may be provided and/or consumed in liquid form. In some embodiments, the infant oral composition is for feeding infants who are unable to eat solid foods.

In preferred embodiments, the oral composition for infants is an infant nutritional supplement. The supplement may be offered in addition to breast milk and/or infant formula. Thus, in preferred embodiments, the infant oral composition is an infant nutritional supplement suitable for consumption by infants aged 0-12 months, 6 weeks to 12 months, 0-6 months, 6 weeks to 6 months , 4–6 months, preferably 0–4 months or 6 weeks to 4 months.

The children's supplement may further contain one or more of the following: protein; fat (lipids); carbohydrates; and essential vitamins and minerals. The children's supplement may further contain sweetening, flavoring and/or coloring agents.

In other embodiments, the infant oral composition does not contain additional protein; fat; carbohydrates; and/or essential vitamins and minerals.

In other embodiments, the oral composition for infants is an infant formula or formula for feeding infants. The term "infant formula" means a food product intended for consumption by infants during the first four to six months of life and which itself meets the nutritional needs of this category of people. The term “infant formula” means a food product intended for consumption by infants over four months of age and forming the main liquid component in the diet of this category of persons, which involves gradually increasing variety.

The requirements for infant formula are well known to specialists. For example, recommendations and requirements are offered by the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), for example, in Koletzko, B., et al., 2005. “Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group ", Journal of pediatric gastroenterology and nutrition, 41(5), pp. 584–599. Typically, infant formula in ready-to-feed liquid form (e.g. dissolved from powder) provides 60–70 kcal/100 ml. Infant formula typically contains per 100 kcal: about 1.8–4.5 g of protein; about 3.3–6.0 g fats (lipids); about 300–1200 mg linoleic acid; about 9–14 g of carbohydrates selected from the group consisting of lactose, sucrose, glucose, glucose syrup, starch, maltodextrins and maltose and combinations thereof; and essential vitamins and minerals.

In some embodiments, the oral composition for infants further comprises one or more carriers. As used herein, the term "carrier" means any substance useful as an excipient, filler, bulking agent, diluent, coloring agent, stabilizer, thickener, binder, flavoring agent, or the like. Preferably one or more carriers contain skim milk powder and/or lactose. Most preferably, the carrier is skim milk powder and/or lactose.

The oral composition for infants may be in powder form, which powder form may be reconstituted into a liquid form before use. In preferred embodiments, the infant oral composition is reconstituted with water prior to use. Preferably, the composition is reconstituted with water to produce a single serving.

In other preferred embodiments, the composition is in a ready-to-drink drinkable form. The ready-to-drink drinking composition may be offered in a bottle.

First and second medical uses

First medical use

In one embodiment, the invention provides a composition for use in therapy.

Second medical use against infection

In another aspect, the invention provides a composition for use in the prevention and/or mitigation of a non-viral infection, wherein the composition is or can be prepared using the method of the invention.

In some embodiments, the non-viral infection is a bacterial infection, a parasitic infection, or a fungal infection.

The non-viral infection may be a bacterial infection selected from Escherichia coli infection, Vibrio cholerae infection, Salmonella infection, Clostridium infection, Shigella infection, parasitic infection including Giardia lamblia, Entamoeba histolytica and Cryptosporidium spp, or mixtures thereof.

Typical bacterial infectious diseases for the treatment or prevention of which the invention can be used include salmonellosis, shigellosis, typhoid fever, bacterial meningitis, anthrax, botulism, brucellosis, campylobacteriosis, cat scratch disease, cholera, diphtheria, epidemic typhus, gonorrhea, impetigo, legionellosis, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, melidoidosis, rheumatic arthritis, staphylococcal infection (methicillin-resistant Staphylococcus aureus (MRSA)), nocardiosis, whooping cough (whooping cough), plague, pneumococcal pneumonia, psittacosis, Q rickettsiosis, American spotted fever (RMSF), scarlet fever, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhus and/or genitourinary tract infections.

Typical parasitic infectious diseases for the treatment or prevention of which the invention can be used include African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, clonorchiasis, cryptosporidiosis, cysticercosis, diphyllobothriasis, draconulosis, echinococcosis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, infection free-living amoebae, giardiasis, gnathostomiasis, hymenolepidosis, isosporosis, dum-dum fever, leishmaniasis, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, enterobiasis, scabies, schistomiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, trichinosis, trichiuriasis, omoniasis and /or trypanosomiasis.

Typical fungal infectious diseases for the treatment or prevention of which the invention can be used include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis and/or tinea pedis.

In another aspect, the invention provides an infant formula of the invention for use in mitigating or preventing a non-viral infection.

In another aspect, the invention provides a method of reducing or preventing a non-viral infection, comprising the step of administering a composition or infant formula of the invention to a subject in need thereof.

Second medical use against inflammation

In another aspect, the invention provides a composition for use in the prevention and/or alleviation of inflammation, wherein the composition is or can be prepared using the method of the invention.

Typical inflammatory conditions that the invention may be used to treat or prevent include, but are not limited to, acute inflammation such as sepsis, infections, burns, and chronic inflammation such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, necrotizing enterocolitis, skin inflammations such as UV- or chemical-induced skin inflammation, eczema, sensitive skin, psoriasis, vitiligo, acne, liver inflammation, alcoholic cirrhosis, allergies, atopy, bone inflammation, rheumatoid arthritis, systemic lupus, Gougerot-Sjögren's syndrome, Reiter's disease, polio, dermatomyositis, thyroiditis, Basedow's disease, Hashimoto's disease, type I diabetes, Addison's disease, autoimmune hepatitis, celiac disease, Biermer's disease, multiple sclerosis, myassetnia, encephalomyelitis, ocular inflammation, obesity-related inflammation, low-grade age-related inflammation , Blau's disease, Alzheimer's disease, cardiovascular diseases, atherosclerosis, metabolic syndrome, ginginitis, periodontitis and their combinations.

The composition of the invention is particularly effective for the prevention and/or treatment of inflammatory conditions selected from chronic inflammatory conditions such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, necrotizing enterocolitis, eczema, allergy and atopy.

The composition of the present invention can also be used to control and/or alleviate the inflammatory response of the body.

The composition of the present invention can further be used to produce, improve or enhance homeostasis and oral tolerance.

In another aspect, the invention provides an infant formula of the invention for use in alleviating or preventing inflammation.

In another aspect, the invention provides a method of reducing or preventing inflammation, comprising the step of administering a composition or infant formula of the invention to a subject in need thereof.

Other Medical Uses

In some embodiments, the composition produces, improves, or enhances homeostasis and oral tolerance.

In some embodiments, the composition provides control and/or alleviation of the inflammatory response.

In some embodiments, the composition provides an increase in immune function in a subject.

In another aspect, the invention provides a composition for use in enhancing immune function in a subject, which composition can be prepared using the method of the invention.

In another aspect, the present invention provides an infant formula of the invention for use in enhancing immune function in a subject.

In another aspect, the present invention provides a method of enhancing immune function in a subject, comprising the step of administering a composition or infant formula of the invention to a subject in need thereof.

Method of treatment

It should be understood that all references to treatment herein include curative, palliative and prophylactic treatment. Preference is given to treating mammals, particularly humans. Methods for treating humans and animals are included within the scope of the invention.

As used herein, the term “prevention” includes preventing and reducing the risk of a condition.

Subject

The term "subject" refers to either a human or a non-human animal.

The composition can be administered to humans or non-human animals, in particular companion animals or livestock. The composition has positive effects for any age group. Preferably the composition is intended for use in infants, adolescents, adults or the elderly. However, it can be administered to mothers during pregnancy and breastfeeding to treat the infant.

Examples of non-human animals include vertebrates, such as mammals such as non-human primates (particularly great apes), dogs, rodents (eg mice, rats or guinea pigs), pigs and cats.

The term “companion animal” means any domestic animal, including (without limitation) cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like.

Preferably the subject is a human.

Those skilled in the art will appreciate that all elements of the invention described herein can be freely combined without departing from the described scope of the invention.

Preferred elements and embodiments of the invention are described below by way of non-limiting examples.

In the practice of the present invention, unless otherwise indicated, standard methods used in chemistry, biochemistry, molecular biology, microbiology and immunology known to those skilled in the art will be used. Such methods are described in the literature. See, for example, J. Sambrook, E. F. Fritsch and T. Maniatis, (1989), Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic additions) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley &Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

All publications identified in the description above are incorporated herein by reference. Various modifications and variations of the described methods, compositions and uses will be apparent to one skilled in the art without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, it is intended that the following claims include various modifications to the described embodiments of the invention that will be apparent to those skilled in the art.

Examples

Introductory part: description of quantitative determinations of SIgA by ELISA (presence of SIgA) and biological analysis of TSLP excretion (bioactivity of SIgA)

The presence and quantity of SIgA molecules (or at least portions of SIgA molecules) were measured by ELISA using a commercially available bovine IgA kit (Bovine IgA ELISA Quantitation Set, Bethyl Laboratories Inc., cat no E10-131). The recommended protocol for the kit with adaptation was used in that the kit's standard IgA sample was replaced with native bovine SIgA (purified by classical gel permeation chromatography under native conditions, as described, for example, in Favre et al., 2003, J. Chromatogr. B Analyt Technol Biomed Sci Mar 25;786(1–2):143–51) to ensure correct quantification of SIgA (not just IgA). A standard curve for SIgA was prepared to typically cover a range of SIgA concentrations from 1000 to 10 ng/mL. Kit validation tests have shown that the milk matrix does not interfere with quantitation at the sample dilutions used (typically 1:1000 to 1:20,000).

It should be noted that the detection of SIgA molecules by ELISA is not at all an indicator of the integrity of SIgA molecules, and, therefore, their biological activity. For example, a degraded SIgA molecule will be detected if it is still recognized by the polyclonal detection antibodies used in the ELISA kit. Moreover, degraded or even cleaved SIgA molecules may expose epitopes not normally accessible to detector antibodies when the SIgA molecules are in their native form. All this can lead to artificially high amounts of detectable SIgA (as shown in example 3 and in Fig. 5 below).

To address this issue, and consistent with the original concept of increasing the effectiveness of probiotics by associating with native bioactive SIgA molecules, a bioassay was developed to evaluate the bioactivity of SIgA molecules (for a detailed description of the experimental procedure, see Mathias et al, 2010, JBC, vol. 285, no. 44, pp. 33906–33913). Briefly, Caco-2 human colon epithelial adenocarcinoma cells (HTB 37, American Tissue Culture Collection) were grown at 37°C in complete Dulbecco's modified Eagle's medium (DMEM) (C-DMEM) consisting of DMEM-Glutamax medium (Sigma ) with the addition of 10% fetal bovine serum (FBS, Sigma), 1% nonessential amino acids (Sigma), 10 mmol HEPES buffer (pH 7.0, Sigma), 0.1% transferrin (Invitrogen AG, Basel, Switzerland) and 1% streptomycin/penicillin (Sigma), and were used at passages between 23 and 37. Cells cultured to 80% confluency were plated on Transwell filters (12 mm diameter; 0.4 μm pore size; Corning Costar, CA). Cambridge, Massachusetts, USA) with a density of 0.8E5 cells/ ^cm2 . The culture medium was changed every 2 days. The formation of a polarized monolayer of Caco-2 cells at week 3 was detected by morphology and monitoring of transepithelial electrical resistance (TER; Ohm x ^cm2 ) using a Millicell-ERS instrument (Millipore, Bedford, Massachusetts, USA). The TER values of well differentiated monolayers were in the range of 380–450 Ohm x ^cm2 . It should be noted that this process results in the creation in vitro of dense epithelium with separated apical and basolateral culture compartments, corresponding to the luminal and mucosal compartments in the intestine, respectively. Before exposure to the probiotic or probiotic-SIgA complexes, the apical portion of Caco-2 cell monolayers was washed twice with phosphate-buffered saline (PBS; 116.3 mmol NaCl, 10.4 mmol Na ₂ HPO ₄ , 3.2 mmol KH ₂ PO ₄ , pH 7.4), and the C-DMEM medium was replaced with, as a rule, 500 μl of the same medium without FBS and antibiotics (DMEM-A ^- ).

Addition of probiotics to the apical compartment of the culture system (see detailed protocol below) results in the secretion of several immune factors in the basolateral compartment, including thymic stromal lymphopoietin (TSLP), which is known to play an important role in maintaining a non-inflammatory environment in the intestine by promoting dendritic maturation. cells - key agents of any immune response. Thus, after overnight incubation with probiotics, TSLP release is detected by ELISA using a commercially available human protein kit.

The fresh probiotic biomass preparation was typically obtained by overnight cultivation of the bacterial strain Bifidobacterium lactis CNCM I-3446, hereinafter referred to as BL818, in de Man-Rogosa-Sharp nutrient medium ((MRS) Difco Laboratories, Detroit, Michigan, USA) with the addition of 0.05% L-cysteine, at 37°C and without stirring. Estimation of the number of colony-forming units (CFU) per milliliter obtained under these culture conditions was made by correlating optical density measurements at 600 nm with plating of serial dilutions of overnight incubations.

To assess the bioactivity of SIgA, probiotics (typically 2E7 CFU of fresh biomass or 2E8 CFU of spray-dried bacteria) and SIgA were mixed in a final volume of 100–500 μL of PBS in a 1.5 mL low protein binding tube and incubated for 30 minutes. at 37°C unless otherwise stated. In an alternative embodiment, probiotics alone (standard sample) or preformed probiotic-SIgA complexes were simply diluted in PBS to obtain the required amount of probiotics in a final volume of 100–500 μl. Probiotic-SIgA complexes formed during this incubation step and pre-existing ones were then pelleted by centrifugation (typically 6000 g for 5 minutes), and the supernatant containing unbound SIgA was discarded. Probiotic-SIgA complexes were resuspended in 500 μl of DMEM- ^A culture medium and then added to the apical compartment of Caco-2 cell culture for overnight incubation (typically 16 hours) at 37°C in a cell culture incubator.

After overnight incubation, culture medium was collected from the basolateral compartment of polarized Caco-2 cells and stimulated TSLP production was measured (concentration expressed as picograms per ml of culture medium) by ELISA using commercially available human TSLP kits (Biolegend, San Diego, CA , USA).

Preassociation of SIgA with probiotics increased TSLP production by Caco-2 cells in a dose-dependent manner compared with probiotics alone. Therefore, a standard curve obtained with a fixed amount of probiotic associated with increasing amounts of native bovine SIgA (the same as that used for quantitative determinations by ELISA). In an alternative embodiment, the bioassay was performed without a standard curve, allowing only semi-quantitative comparison of SIgA bioactivity between different test samples. Bioassay tests were performed in at least two technical replicates, preferably in triplicate.

Example 1: Method according to the present invention

Raw milk was obtained from a Swiss farm and centrifuged at 15°C to obtain raw skim milk. Raw skim milk was concentrated to 24% dry matter at 15°C using a reverse osmosis (RO) concentrating unit (Tetra Alcross RO with spiral membrane Alfa Laval RO98pHt-3838/30 - TetraPak, Lund, Sweden). RO 24% skim milk was preheated in a plate heat exchanger to 55°C and then heated to 83°C by live steam injection. After passing through a tubular cooler with an average residence time of 6 seconds, the RO skim milk was flash cooled to 55°C using a flash vessel as is common in the dairy industry. The RO skim milk was further cooled to 5°C in a plate heat exchanger and then spray dried under mild conditions without preheating in an open top spray dryer as described in US2353495 to form RO skim milk powder. The air flow temperature and relative humidity in the Egron dryer were 65°C and 12%. The RO skim milk powder contained 139 μg SIgA/g powder by ELISA and 25 μg bioactive SIgA/g powder by bioassay.

Parallel fermentation produced BL818 probiotics, centrifuged them to concentrate the bacteria, and obtained a liquid fresh biomass at 5°C with 1.4E11 CFU/g BL818 as measured by flow cytometry, later confirmed by plate counting.

RO skim milk powder was dissolved in warm water and a liquid suspension of BL818 was added to form a 34% solids suspension containing 5E8 CFU/g BL818 and 340 ng of bioactive SIgA per 2E7 CFU of BL818. The suspension was kept at 37°C for 30 minutes and cooled to 5°C in a plate heat exchanger and then spray dried in a spray dryer under mild conditions as described above. This produced a powder containing probiotics with higher bioactivity than spray-dried probiotics alone as determined in bioassay tests (136.7 ± 1.8 vs. 77.7 ± 4.8 pg/mL TSLP production, respectively).

Example 2. Effect of microfiltration of skim milk on SIgA content

Raw skim milk (8.0–8.7% total solids) and reverse osmosis (RO) skim milk (about 24% total solids) were used for these experiments. Both types of milk were subjected to microfiltration (MF) using different membranes at temperatures ranging from 52 to 55°C. The SIgA content of milk before filtration and after filtration in different phases was measured by ELISA as described above. The percentage of SIgA in the permeate after microfiltration (SIgA yield [%]) was calculated compared to the amount of SIgA in the milk before filtration. Microbiological contamination of the milk was also assessed and the logarithmic reduction in the number of bacteria in the milk (reduction in total plate counts [log]) was calculated. The results obtained are presented in Table 2 below and FIG. 3.

The best performing membrane test was repeated at 14°C to evaluate the effect of temperature on SIgA retention and microbiological contamination reduction.

Both types of milk were heated to processing temperature on a plate heat exchanger and microfiltrated in a microfiltration unit using different membranes.

A more detailed description of the membranes used and key processing parameters is presented in Table 1 below.

Table 1. Membrane supplier and type, processing parameters for microfiltration test

Membrane Treatment Provider Type Material Over-
ness
m ² Pore size, nm Pace.
°C TMD
Bar Consumption, l/h Tami 0.14 µm Spiral module Tami
Ø 25 mm, 8 channels, 19 modules, length 1178 mm Ceramics 2 x 3.8 140 55 1.5 2000–6000 Alfa-Laval MFP05–3838
(fluoropolymer) Polymer 2 x 4.7 500 52 0.9 12,000 Microdyn – Nadir MV020 - 3838 (PVDF) Polymer 2 x 5.7 200 52 0.9 12,000 Tami 1.4 µm Spiral module Tami
Ø 25 mm, 23 channels, length 1178 mm Ceramics 19 x 0.35 1400 55 0.14 2000–6000

Before use, the device was disinfected to assess the effect of the method on the bacterial load in the retentate and permeate. To do this, the device was washed with 1% P3-Oxonia (Ecolab, St. Paul, MN, USA) overnight.

The results of microfiltration experiments are presented in the table below.

Table 2. Summary of processing conditions and results of microfiltration experiments.

Membrane Milk solids [%] Microfiltration temperature [°C] SIgA in milk (up to) [µg/ml] SIgA in permeate (after) [µg/ml] SIgA output
[%] Decrease in quantity on cups [log] Tami 0.14 µm 8.3 55 254 142 55.9 3.3 MFP05 8.4 52 294 133 45.2 4.2 MV020 8.7 52 375 124 33.1 4.7 MMS/Tami 1.4 µm 23.9 55 1537 1058 68.8 1.8 MMS/Tami 1.4 µm 24 14 1441 1010 70.1 2.0

The results indicate that small pore sizes provide the greatest reduction in microbiological contamination but result in the greatest loss of SIgA during the microfiltration process. Larger pore sizes are less effective in reducing microbiological contamination but allow more SIgA to be retained (higher SIgA yield) in the milk after microfiltration. In particular, microfiltration using a membrane with a pore size of 500 nm or less results in such low yields of SIgA in milk after microfiltration that milk becomes an uneconomical source of SIgA for use in the method according to the invention. In contrast, the 1400 nm pore size ceramic membrane (MMS) had little effect on SIgA content, showing a 69% SIgA yield in milk after microfiltration. The reduction in microbiological load, although less than the other membranes tested, was close to 2 log. This reduction in microbiological load is sufficient for the use of the resulting milk in the method of the present invention. In particular, after having subsequently undergone a heat treatment step such as that described in the present application and in particular in Example 3 below, the milk obtained by microfiltration with an MMS membrane is suitable for use in "sensitive" products such as infant formula. The 2 log reduction in microbiological load achieved with the MMS membrane is sufficient to allow the use of mild conditions during the heat treatment step to retain bioactive SIgA and still produce an infant formula grade composition starting with any milk of standard microbiological quality. Thus, a membrane with a pore size of 1400 nm provides the best balance between reducing microbiological load and preventing SIgA loss.

Example 3: Comparison of Different Conditions for Heat Treatment of Reverse Osmosis (RO) Skim Milk

The raw milk was centrifuged at 15°C to obtain raw skim milk. Raw skim milk was concentrated from 9% dry matter to 22.9% dry matter at 15°C using a reverse osmosis (RO) concentrating unit (see Example 1). Four different heat treatment processes were performed on RO skim milk using steam injection and flash cooling:

- Sample 1: 74°C / 103 s

- Sample 2: 83°C / 6 s

- Sample 3: 87°C / 2 s

- Sample 4: 90°C / 0.96 s

The liquid RO pasteurized skim milk was cooled to 5°C and the cold concentrate was spray dried.

The presence of SIgA molecules in the original RO-skim milk (standard sample), heat-treated RO-skim milk (samples 1–4), and in the finished powders obtained from samples 1–4 was assessed by ELISA. Quantification of SIgA bioactivity in heat-treated RO skim milk and finished powders was performed using a TSLP excretion bioassay as described above. Correlated results of the ELISA and TSLP excretion bioassay are presented in FIG. 4.

The results of SIgA quantification by ELISA show that heat treatment at 90°C for 0.96 s (sample 4) resulted in the highest amounts of SIgA in both heat-treated milk and powder. The SIgA bioactivity assay (TSLP excretion bioassay) surprisingly showed that intermediate conditions were more favorable for maximizing the retention of bioactive SIgA, and heat treatment at 83°C for 6 s (sample 2) was considered the preferred choice. This shows that the portion of SIgA detected by ELISA in high temperature treated samples is in fact inactive, and that the TSLP excretion biological assay used herein appears to be a more reliable method for determining the amount of truly effective SIgA in the composition. Such data indicate that quantification of SIgA by ELISA does not correlate with the integrity of SIgA molecules and thus their bioactivity.

In microbiological terms, all heat treatments described above resulted in a total plate count (TPC) reduction of 2.0–2.1 log, while providing at least a 7 log reduction in pathogens that are more heat sensitive than other microorganisms. The spray dried powder was shown to be completely free of Enterobacteriaceae, Enterobacter Sakazakii and Salmonella.

Example 4. Effect of temperature and time on the formation and bioactivity of SIgA-BL818 complexes

Association of SIgA and BL818 in RO skim milk was tested at a ratio of 34 ng SIgA per 2E7 CFU of BL818 under different time and temperature conditions (37°C, 20°C and 12°C, for 60, 30 and 15 minutes at each temperature ). It should be noted that this experiment did not test temperatures above 37°C, as such temperatures would be harmful to the probiotic, nor temperatures below 12°C, since preliminary tests performed at 4°C showed no association (data not shown) .

The experiment was carried out using three identical samples of RO skim milk mixed with liquid biomass BL818. For each sample, 41.9 g of skim milk powder, heat treated according to the preferred conditions identified in Example 3 (Sample 2), containing 285 μg of bioactive SIgA per gram of powder (as determined by TSLP excretion bioassay) was dissolved in 65 ml of distilled water . 23.3 mL by volume of BL818 probiotic biomass (thawed frozen stock biomass) containing 3E11 CFU/mL was then added to achieve the required ratio of SIgA to BL818. Immediately after adding BL818 biomass, the three mixtures were incubated at 37°C, 20°C or 12°C, respectively, with slow mixing. After 15, 30 or 60 minutes of incubation, during which association of probiotics with SIgA could occur, two samples (repeated) were taken from each mixture and centrifuged at 6000 g for 5 minutes at 4°C to precipitate the already formed probiotic-probiotic complexes. SIgA and to stop the association process. After removal of the unbound SIgA-containing supernatant, the pellet was resuspended in cell culture bioassay medium and bioassayed for the equivalent of 2E7 CFU from each sample as described above.

The bioactivity of SIgA complexes, assessed by bioassay of TSLP excretion after the association step for each sample, provided an indirect estimate of the level of association of SIgA with BL818 in the form of complexes.

Shown in FIGS. 5 results show that the formation of probiotic-SIgA complexes increased with increasing temperature during the association stage. The best temperature for the association stage was 37°C. The formation of complexes was less effective when the temperature was reduced to 20°C and further to 12°C. However, complexation at 12°C was still sufficient to achieve bioactivity greater than that of BL818 alone, as indicated by the horizontal gray line in FIG. 5.

This test also showed that the selected association stage durations, ranging from 15 to 60 minutes, resulted in successful complex formation, even though complexation was slightly reduced in the 15 minute duration trials.

Taking into account all the results obtained, the optimal conditions for carrying out the stage of association of probiotics with SIgA were 30 minutes at a temperature of 37°C.

Example 5. Effect of BL818 concentration (in CFU/ml) on the formation and bioactivity of SIgA-BL818 complexes

A study was conducted to examine the effect of BL818 concentration on the efficiency of probiotic-SIgA complex formation. To do this, laboratory association tests were carried out with a constant ratio of SIgA to BL818 (34 ng SIgA/2E7 CFU BL818), constant total solids and constant association conditions, which were determined to be best in example 4 (30 minutes at 37°C), but with BL818 concentrations of 5.4E10, 5.4E9 and 5.4E8 CFU/ml, respectively. Test samples were prepared as described in Table 3.

Table 3. Composition of samples used to test the effect of BL818 concentration on the formation and bioactivity of SIgA-BL818 complexes

Distilled water [ml] RO skim milk powder with 285 µg bioactive SIgA/g [g] RO skim milk powder without bioactive SIgA [g]* Biomass BL818 with 3.05E11 CFU/g [g] Control 1 (5.4E10 CFU/ml BL818 without SIgA) 65.2 0.0 41.9 22.9 Control 2 (5.4E8 CFU/ml BL818 without SIgA) 87.9 0.0 41.9 0.229 Test 1 (5.4E10 CFU/ml with SIgA) 65.2 41.9 0.0 22.9 Test 2 (5.4E09 CFU/ml with SIgA) 86.0 4,169 37.53 2.29 Test 3 (5.4E08 CFU/ml with SIgA) 87.6 0.417 41.75 0.229

* RO skim milk heat treated at 145°C for 6 seconds resulting in no detectable SIgA signals at either the ELISA or bioassay level.

After incubation, samples were centrifuged to stop the association process as described in Example 4 and subjected to a TSLP excretion bioassay (samples were normalized to 2E07 CFU in the bioassay). The bioactivity of the complexes thus obtained reflected the efficiency of complex formation.

Shown in FIGS. 6 results show that the concentration of BL818 during the association stage influences the efficiency of complex formation, with a tendency for it to increase as the concentration of BL818 decreases. Indeed, the best bioactivity was observed at a BL818 concentration of 5.4E8 CFU/ml (142 ± 4 pg/ml TSLP), then at a concentration of 5.4E9 (135 ± 5 pg/ml TSLP) and then at 5.4E10 CFU/ml (126 ± 3 pg/ml TSLP). However, it was found that all concentrations of BL818 produced significantly increased bioactivity of the BL818-SIgA complexes compared to BL818-only controls.

Example 6. Effect of the used form of BL818 (liquid biomass or spray-dried culture) on the formation and bioactivity of SIgA-BL818 complexes

To compare the effect of probiotic form on the efficiency of probiotic-SIgA complex formation, laboratory association tests were performed with a constant ratio of SIgA to BL818 (34 ng SIgA/2E7 CFU BL818), constant total solids and constant association conditions, which were determined to be best in the examples 4 and 5 (30 minutes at 37°C, 5.4E08 CFU BL818/ml), but using BL818 either from liquid biomass form or from powder form (spray dried on a Mini spray-drier B-290, Büchi, Flaville, Switzerland). Samples were prepared as described in Table 4.

Table 4. Composition of samples used to test the effect of the used form of BL818 (liquid biomass or spray-dried culture) on the formation and bioactivity of SIgA-BL818 complexes

Distilled water [ml] RO skim milk powder with 285 µg bioactive SIgA/g [g] RO skim milk powder without bioactive SIgA [g]* Biomass BL818 with 3.05E11 CFU/g [g] Spray dried BL818 powder with 9.45E + 11 CFU/g [g] Control (5.4E08 CFU/ml BL818 without SIgA) 87.9 0.0 41.9 0.229 0.0 Analyzed biomass (5.4E08 CFU/ml with SIgA) 87.6 0.417 41.75 0.229 0.0 Test powder (5.4E08 CFU/ml with SIgA) 87.1 0.417 41.75 0.0 0.740

After incubation, samples were centrifuged to stop the association process as described in Example 4 and subjected to a TSLP excretion bioassay (samples were normalized to 2E07 CFU in the bioassay). The bioactivity of the complexes thus obtained reflected the efficiency of complex formation.

Shown in FIGS. 7 results show that the form of BL818 used for the association step influences the efficiency of complexation, with a tendency to decrease when BL818 is used in spray-dried powder form. However, it was found that both forms of BL818 resulted in significantly increased bioactivity of BL818-SIgA complexes compared to BL818-only controls.

Example 7 In Vivo Effect of the Compositions of the Present Invention on the Prevention and Treatment of Infections and Inflammation

The physiological effect of a starter formula containing BL818-SIgA complexes prepared using the method of the present invention compared to a starter formula with probiotics alone was tested in vivo in a mouse model of neonatal immune system development.

In this model, the study involved pups born within a 3-day time window to synchronized germ-free female C3H-HEN mice. On day 5 of life, litters were matched for size as well as sex and date of birth of the pups. The cubs were kept in sterile conditions with their dams until one week of age. The entire litter was then transferred to a pathogen-free environment and treated with mouse fecal material containing intestinal bacteria to initiate natural microbial colonization. At this time, pups were started on 20 μl per day of one of the infant formulas tested (see Table 5 below for details) by oral instillation for 14 days until weaned at 21 days of age. It should be noted that keeping litters under sterile conditions until day 7 before the start of microbiological colonization and feeding served two purposes. First, it mimicked the human immune status after birth as closely as possible. Indeed, human babies are born with a slightly more mature immune system than baby mice, but studies have shown that this difference is leveled out after a week of life. Secondly, animals deprived of bacterial flora have an underdeveloped immune system, characterized in part by the absence of mucosal IgA production, since such IgA is part of the female milk secretion. Therefore, maintaining female mice under sterile conditions for as long as possible allowed for minimal side effects of endogenous milk SIgA compared to SIgA present in probiotic-SIgA complexes, allowing unbiased comparisons between probiotics alone and probiotics pre-associated with SIgA.

Table 5. Test groups for in vivo experiments

Group Group Description Reason for use 1) Only a prototype of infant formula (FC) Comparative Negative control for DS matrix Checking that the DS matrix does not distort the planned measurements 2) DS prototype with BL only
(1E4 CFU/day) Comparative Standard sample for BL in DS matrix Standard pattern for the effect of BL+SIgA prototypes 3) Prototype DS with only SIgA ¹⁾ (500 pg/day) Comparative Standard Sample for SIgA in DS Matrix Standard pattern for the effect of BL+SIgA prototypes 4) Prototype DS with BL (1E4 CFU/day) and SIgA (500 pg/day), dry mixture Comparative Prototype DS with BL and SIgA without association before administration Testing the effectiveness of SIgA + BL administered without association compared to BL alone 5) Prototype DS with BL (1E4 CFU/day) and SIgA (500 pg/day) complexed using the optimized method of Example 1 Invention Prototype DS with BL and SIgA with association before administration Efficiency test of the optimized SIgA + BL complexation method according to the invention.

1) SIgA dosages refer to bioactive SIgA; the amounts of SIgA are determined using the TSLP bioassay in cow's milk heat treated at 83°C for 6 seconds, and the required dosage of bioactive SIgA is obtained using the corresponding amount of such milk in the prototype infant formula (see Table 6 below).

To obtain DS prototypes, the compositions described in Table 6 below were mixed dry in the laboratory. DS prototypes containing BL818 alone or BL818 in combination with bioactive SIgA [groups 5), 6) and 7)] were targeted at BL818 2E7 CFU BL818/g formula, which is the standard target probiotic concentration in infant formula containing BL818 probiotics.

Table 6. Compositions of DS prototypes 4)–8)

Dry Mixing Ingredients Quantity in group 1 [g] Quantity in group 2 [g] Quantity in group 3 [g] Quantity in group 4 [g] Quantity in group 5 [g] Infant formula base matrix (mostly whey, not hydrolyzed) 300.00 299.72 295.89 295.61 295.62 Probiotic Pre-Blend (Spray Dried BL818 in Maltodextrin; 2.11E10 CFU BL818/g) - 0.2844 - 0.2844 - Processed cow's milk (24.84 µg bioactive SIgA per g milk) - - 4.106 4.106 - BL818-SIgA powder prepared in Example 1 (340 ng bioactive SIgA, 2E7 CFU BL818,
1.4E9 CFU of BL818 per gram of powder) - - - - 4,380 Total 300 300 300 300 300

The powdered infant formula itself was prepared as follows. Used all the typical ingredients for a non-hydrolyzed, probiotic-free infant formula, the majority of which is whey.

Table 7. Infant Formula Base for In Vivo Experiments

Ingredient Solids [%] Liquid skim milk, 18% solids, no fat 10,807 Lactose powder 36,986 Liquid cream with fat content 36% 8,996 Demineralized whey powder 20,641 Vegetable oil mixture 16,759 Liquid soy lecithin 0.705 Blend of oils with LCPUFA 0.686 Dry tricalcium citrate 0.585 Choline bitartrate 0.395 Potassium chloride 0.277 Ready-made mixture of vitamins and minerals 0.235 Tripotassium citrate 0.149 Trisodium citrate 0.121 Disodium phosphate 2 dihydrate 0.101 Magnesium chloride 0.056 Moisture 2.5 Total ingredients used (solids) 97.5

All tested infant formula compositions were reconstituted by thoroughly mixing until the powder was completely dissolved. They were diluted 40 times with water heated to 37°C to obtain the equivalent of 1E4 CFU of probiotic in 20 μl of the daily dosage (probiotic dosage adapted for newborn mice). The test infant formulas were given to the pups no later than 5 minutes after reconstitution from the powder to, firstly, simulate the actual practice of bottle feeding and, secondly, to prevent association of probiotics and SIgA prior to administration in the test infant formula 4.

On the 21st day of life, feeding with the mixture was stopped and the cubs were taken away from their mother, placing males and females in separate cages. The cubs were then kept under normal conditions and killed on the 54th day of life. To evaluate the differential effects of pre-weaning formula feeding on the development of the mucosal and systemic immune systems, Peyer's patches (PB, sites of induction of the intestinal immune system) and blood were collected in ice-cold DMEM (PAA Laboratory, cat. no. E15-883). containing 10% inactivated fetal calf serum (FCS, Amimed 2-01F10-I), and into heparinized tubes (Milian, cat no. HEP-75PP), respectively, and stored at 4°C until further use.

To separate immune cells from collected PBs, the latter were first incubated for 1 h 30 min at 37°C in 1 ml DMEM containing 5 mmol CaCl2 (Merck, cat. no. 1.02382.0250) and 2 U/ml Liberase™ Research Grade (Roche, cat. no. 05401127001). The PB and incubation medium were transferred to a 15 mL conical tube and washed with 12 mL of DMEM. After centrifugation at 400 g for 4 minutes at 4°C, the supernatant was discarded and the pellet was resuspended in 500 μl of ice-cold DMEM and transferred to a 70 μm cell sieve (Becton Dickinson, cat. no. 352350) pre-installed over a 50-volume conical tube. ml. The tissues were ground on a cell sieve using a syringe plunger (black part). The sieve and plunger were washed with 10 ml of ice-cold DMEM. The cell suspension was then passed through a 40 µm cell sieve (Becton Dickinson, cat. no. 352340) pre-positioned over another 50 ml conical tube. The cell sieve was washed again with 5 mL of ice-cold DMEM. The cell suspension was transferred into a 15 ml conical tube and centrifuged at 400 g for 5 minutes at 4°C. The supernatant was discarded and the isolated cell pellet was resuspended in 1 ml of ice-cold DMEM/10% FCS as described above. Cell number and viability (usually >80%) were determined by trypan blue staining using microscopy. Cells were stored at 4°C until further use.

To isolate leukocytes, blood samples were centrifuged at 700 g for 10 minutes at 4°C. The supernatant (plasma) was discarded and the pellet was resuspended in 1 ml of red blood cell lysis buffer (NH4Cl 0.15 mol/l (Sigma, cat. no. A 9434), KHCo3 10 mmol (Sigma Aldrich, cat. no. 431583) and EDTA 0.1 mmol (Promega, cat. no. V4231)). The cell solution was then directly transferred into a 15 mL conical tube and incubated for one minute at room temperature. The lysis reaction was then stopped by filling the tube with DMEM/10% FCS and mixing. The cell suspension was centrifuged for five minutes at 400 g and the supernatant was discarded. If the cell pellet was still red, the lysis step was repeated until the pellet became white because it consisted primarily of leukocytes. Finally, cells were resuspended in DMEM/10% FCS, cell number and viability were determined as described for PB cells above, and stored at 4°C until further use.

Measurement of endogenous IgA production was used as a generally accepted marker for monitoring the formation of the immune system (Macpherson AJ, Mc Coy KD, Johansen FE, Brandtzaeg P.; The immune geography of IgA induction and function; Mucosal Immunol 2008; 1:11–22). In the present study, the IgA ELISPOT technique was used for this purpose, which made it possible to accurately count the number of IgA-secreting cells in PB and blood cell preparations. The wells of 96-well cellulose sterile “multi-Screen-HA” plates (Millipore, cat. no. MAHA S45 10) were incubated for 2 hours at 37°C (dry incubator) with 100 μl of a 1 : 200 dilution of goat anti-mouse IgA antiserum (specific to alpha chain) (Sigma, cat. no. 8769) in sterile PBS. After washing the wells 4 times with sterile PBS in a volume of 200 μl/well, the wells were filled with 200 μl of RPMI-1640 culture medium (Invitrogen, cat. no. 61870-010) supplemented with 5% inactivated fetal calf serum (FCS, Amimed 2-01F10- I) and again incubated for 2 h at 37°C in a humidified cell culture incubator. Previously prepared suspensions of PB and blood cells were centrifuged (400 g for 5 minutes at 4°C), the supernatant was discarded, and the cell sediment was resuspended in RPMI / 5% FCS medium heated to 37°C to a concentration of 2x1E6 cells/ml. The wells of a 96-well plate were emptied and filled with freshly prepared suspensions of PB and blood cells in a volume of 100 μl. Each cell suspension sample was tested in triplicate. The plate was then incubated for 18 hours at 37°C in a humidified cell culture incubator. After washing the wells 4 times with sterile PBS in a volume of 200 μl/well, the wells were filled with 100 μl of a freshly prepared 1:500 dilution of goat anti-mouse IgA antiserum (alpha chain specific) conjugated to biotin (KPL, cat, no. 16-18-01 ), in PBS supplemented with 0.05% Tween 20 (Bio-Rad, cat. no. 170-6531) and incubated for 1 hour 30 minutes at room temperature. After another 4-fold washing of the wells with sterile PBS in a volume of 200 μl/well, the wells were filled with 100 μl of a freshly prepared 1:500 dilution of streptavidin-alkaline phosphatase (Sigma, cat. no. E-2636) in PBS supplemented with 0.05% Tween 20 and incubated for 1 hour at room temperature. After another washing step as described above, the wells were filled with 100 μl of a freshly prepared dilution of one tablet of BCIP/NBT substrate (Sigma, cat. no. B-5655) in distilled water and incubated for ≤ 5 minutes at room temperature. The plate was then washed with distilled water. The plastic bottom of the plate was then removed and allowed to dry at room temperature for at least 6 hours in the dark. The number of spots (1 spot = 1 IgA-secreting cell) was then automatically counted for each well using the AID ELISPOT reading system (Autoimmun Diagnostika GmbH) and reported as the number of IgA-secreting cells per 1E6 total number of cells.

The results of the IgA ELISPOT assay (presented in Fig. 8) showed that feeding infant formula containing pre-associated probiotic-SIgA complexes (group 5) to newborn pups produced the greatest enhancement of immune system formation among all groups tested. This occurred both at the mucosal level (PB, Fig. 8A) and at the systemic level (blood, Fig. 8B). Pre-association of probiotics and SIgA in accordance with the method of the present invention was more effective than simultaneously given probiotics and SIgA, but not in the pre-associated form (group 4). Further, pre-association of probiotics and SIgA in infant formula had a synergistic effect on endogenous IgA production compared with the corresponding effects of probiotics or SIgA alone added to the infant formula matrix (groups 2 and 3, respectively) using the basal infant formula as a standard (group 1 ).

These in vivo data confirm the value of the present industrial process for the production of a new biologically active ingredient for infant formula.

Claims (15)

1. A method for producing a composition containing secretory IgA and a probiotic, including the steps: (a) providing a source of SIgA in the form of milk or a fraction thereof, wherein the milk provided in step (a) is not exposed to temperatures above 70°C, preferably 58°C, more preferably 15°C after expression; (b) optionally microfiltration of milk; (c) pasteurizing the milk at a temperature of 61–200°C, wherein the pasteurization in step (c) is carried out for 0.1–200 s, preferably 5–15 s; (d) optionally spray drying at a temperature of the liquid concentrate less than 70°C; (e) mixing with a probiotic, and mixing is carried out at a temperature of 12-45°C for 10-360 minutes; And (f) optionally spray drying at a temperature of the liquid concentrate less than 70°C. 2. The method according to claim 1, in which pasteurization in stage (c) is performed at a temperature of 82–84°C for 5–7 s. 3. The method according to any preceding claim, wherein the mixing in step (e) is carried out at a temperature of 36-38°C for 28-32 minutes. 4. The method according to any preceding claim, wherein the amount of secretory IgA per 2E7 CFU of the probiotic in the mixture in step (e) is from 0.34 ng to 20 μg, preferably from 3.4 ng to 2 μg. 5. The method according to any preceding claim, wherein the concentration of the probiotic in the mixture in step (e) is 1E6-1E11 CFU/ml, preferably 1E8-1E10 CFU/ml. 6. The method according to claim 5, in which the probiotic, if Bifidobacterium lactis or Bifidobacterium longum subspecies infantis. 7. The method of any preceding claim, wherein the secretory IgA and the probiotic are at least partially associated in the composition. 8. The method according to any preceding claim, wherein the composition contains 0.0001-10 mg of secretory IgA per daily dose. 9. The method according to any preceding claim, wherein the composition contains from 1E2 to 1E12, preferably from 1E2 to 1E10 probiotic cells per daily dose.