GB2606050A

GB2606050A - Sourdough food and food supplement compositions

Info

Publication number: GB2606050A
Application number: GB2201102.7A
Authority: GB
Inventors: Hector Ameye Serge
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-10-26
Also published as: GB202201102D0

Abstract

A composition comprising a sourdough and a mixture of microorganisms consisting of a Lactobacilli acidophilus strain, a Bifidobacterium infantis strain, an Enterococcus faecium strain and a Bifidum bifidum strain. The composition may further comprise vitamin(s) and/or mineral(s), particularly vitamin D3 and K2, calcium carbonate, calcium citrate, tricalcium phosphate and silicon dioxide. The composition may further comprise an enzyme such as a phytase, particularly a bacterial phytase from Lactobacillus reuteri or Buttiauxella. A food product or supplement is also claimed, which may be a sourdough or a beverage. The composition or food product may be used in human or veterinary medicine. The composition or food product may be used in the prevention and/or treatment of intestinal disease, improvement of intestinal health and promoting healthy gut flora, the prevention and/or treatment of bone disorders and the improvement of bone health, in particular for the prevention and/or treatment of osteoporosis; it is disclosed that this may be due to the addition of a further phytase.

Description

SOURDOUGH FOOD AND FOOD SUPPLEMENT COMPOSITIONS

FIELD OF THE INVENTION

The present invention relates to food compositions comprising sourdough and applications thereof in the manufacture of food supplement products, human food products and / or animal feed products. The sourdough food compositions according to the invention is characterized in comprising besides sourdough, a complex of bacteria. In one embodiment such food compositions were shown to have a beneficial effect on the intestinal microflora. In another embodiment, the food supplement compositions further comprise a complex of minerals, enzymes (e.g. phytase), vitamins On particular vitamins 83 and K2) and calcium (e.g. calcium carbonate, tricalcium phosphate), and found particularly useful to improve minerals bioavailability for Bone Formation.

BACKGROUND TO THE INVENTION

The use of sourdough for the manufacture of bread is a very old bakery practice, currently widespread in those countries that use wheat and rye flour to produce products,. Since ancient times the sourdough has been known for its ability to improve the leavening quality and preservation of bread. The organic acids produced, mainly lactic acid and acetic acid, affect and degrade the Fermentable Oligo-, Di-, Mono-saccharides and Polyols (FODMAP) present in wheat, including the fructans, gluten and starch fractions. Additionally, the decrease in pH associated with the production of acids causes an increase in the activity of proteases and amylases, favoring the reduction of aging and generating a large amount of amino acids that, through the metabolism of yeast and / or lactic bacteria, they will become alcohols and ketones with high aromatic value. In addition to improving the texture of the bread, from the nutritional point of view, the fermentation of the sourdough results in an increase in the bioavailability of the minerals present and a reduction in the phytate content.

As supported by an abundant literature, other functional/nutritional aspects of the sourdough fermentation have emerged in recent years. The focus of research has been on the use of sourdough fermentation in baked goods, for management of irritable bowel syndrome (IBS) and irritable bowel disease (IBD), synthesis/release of bioactive compounds and exploitation of the potential of ancient grains (einkorn, emmer. Gamut) and milling by-products (bran and germ). Part of the research has been on the micro-organisms involved in the sourdough fermentation process. Cultures of lactic acid bacteria are e.g. used in the manufacturing of sourdough-bread, a process that involves the inoculation of a mixture of flour and water followed by fermentation, sometimes for several days, freshing with flour and water and finally mixing into the bread dough which is kneaded and allowed to rise slowly before baking. In comparison with bread types produced with cultivated yeasts, it usually has a mildly sour taste because of the lactic acid produced by the Lactobacilli. The pH value in the dough is stepwise decreased to 4.0 -4.3 by the acidification. Lactobacilli and yeast species are responsible for the characteristic taste and flavour of the sourdough bread. In particular Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus kefiri, and strains of Lactobacillus sanfranciscensis are known to be present in sourdough starters and to metabolise hexose sugars to produce lactic acid, acetic acid and CO2.

Lactobacillus strains isolated from sourdough starters and their use in food products, including sourdough, were shown to have benefits, a in "healthy gut flora" and therefore in the treatment of "intestinal diseases" and by extension antibacterial activity and even immune modulation (see for example PCT publications W02014122119 & W02014072408. Even in combination with other bacterium and / or yeast, in particular selected from a Lactobacillus sanfranciscensis strain, Lactobacillus rossiae strain, Lactobacillus plantarum strain, Lactobacillus brevis strain, Lactobacillus amyolyticus strain, Lactobacillus amylovarus strain, Lactobacillus lactus lactus, lactis lactus, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus strain Gluconobacter wrydans strain, Candida humilis strain, Candida milleri strain, Candida krusei strain, Saccharomyces exiguus strain, Saccharomyces barnetti strain, Saccharomyces cerevisiae strain and / or Saccharomyces minor strain.

It has been an objective of the present invention to further improve the known beneficial effects of sourdough or of the micro-organisms derivable thereof in new food supplement compositions based on the combination of sourdough with a complex of further microorganisms, found to have a synergistic effect in enhancing the beneficial effects of the individual components.

SUMMARY OF THE INVENTION

In a first embodiment the present invention provides the combination of a sourdough composition with a mixture of micro-organisms consisting of a L. acidophilus strain, a B. infantis strain, a E. faecium strain and a Bifidum Bifidum strain. As detailed in the examples hereinafter, it has surprisingly been found that such combination has a strong positive and long-lasting effect on the eubiofic index of an individual consuming such combination of a sourdough with a mixture of these bacteria strains. Thus, in one embodiment the present invention provides a composition comprising a sourdough and a mixture of microorganism consisting of a L. acidophilus strain, a B. infantis strain, a E. faecium strain and a Bifidum Bifidum strain.

Sourdough fermentation is known to rely on "yeast' and lactic acid bacteria; in particular lactic acid bacteria that are naturally present in flour to leaven the bread. Hence the composition may further comprise a culture medium or nutrients suitable for cultivation of lactic acid bacteria and/or yeasts, in particular a culture brew, a concentrate of the culture brew and/or a dry matter of the culture brew. The culture medium can be any medium known in the art for the cultivation of Lactobacilli. The culture medium can be a solid culture medium like an agar-based growth medium, a dough, in particular sourdough, or any other solid media suitable for the cultivation of microorganisms, in particular Lactobacilli. The culture medium may also be a liquid culture medium wherein the bacteria are suspended in a culture brew or a liquid nutrient medium. The composition may comprise nutrients suitable for the cultivation of Lactobacilli strains, such as carbon sources, e. g. glucose or maltose, nitrogen sources, e. g. peptone or meat extract, phosphorus sources, e. g. potassium dihydrogenphosphate, essential metal salts for bacterial growth and optionally growth-promoting substances like amino acids and/or vitamins. The composition preferably has a pH in the acidic range, in particular below pH 6.

The composition may be a liquid, paste or solid composition (e.g. lyophilized, pulverized or powdered); preferably in solid or liquid dosage forms, such as for example tablets, coated tablets, capsules, solutions, suspensions, emulsions, pellets, syrups and so on and are prepared in the usual manner by mixing the active ingredient with excipients and/or carriers such as e. g. natural mineral flours (kaolin, talc) or synthetic mineral flours (e. g. silicates), optionally adding adjuvants (e. g. glycol) and/or dispersing agents (e. g. methyl cellulose).

The compositions comprising a sourdough and the aforementioned mixture of microorganism, may further comprising at least one vitamin and/or mineral; in particular at least one vitamin or mineral selected from vitamin D3 and K2, calcium carbonate, calcium citrate, tricalcium phosphate, and silicon dioxide. The combination of a sourdough with this particular mix of micro-organisms has a positive effect on the bio-availability on the vitamins and minerals present in such composition. Adding a further phytase to the composition enhances said effect, making the present compositions particularly useful in the prevention and/or treatment of bone disorders and the improvement of bone health. In one embodiment the compositions according to the invention further comprise at least one vitamin and/or mineral; in particular at least one vitamin or mineral selected from vitamin D3 and K2, calcium carbonate, calcium citrate, and silicon dioxide; more in particular vitamin D3 and K2 and at least one mineral selected from calcium carbonate, calcium citrate, and silicon dioxide. Optionally enzymes, such as phytase can be added. Hence, in another embodiment the compositions according to the invention further comprise a phytase and at least one vitamin and/or mineral; in particular at least one vitamin or mineral selected from vitamin D3 and K2, calcium carbonate tricalcium phosphate, calcium citrate, silicon dioxide and silicon combinations; more in particular a phytase, vitamin D3 and K2 and at least one mineral selected from calcium carbonate, calcium citrate, tricalcium phosphate, and silicon dioxide The phytase enzyme used in the compositions according to the invention is in particular a bacterial phytase; more in particular a bacterial phytase sourced from L. reuteri or Butfiauxella.

The compositions in the different embodiments herein described and comprising a sourdough in combination with the aforementioned mixture of micro-organisms can be applied to an edible food or feed product, in particular as a starter culture and/or fermentation starter in the manufacture of fermented products such as beverages (e.g. kvass, water kefir, rejuvelac, darassun, pozol and boza), cheese, yoghurt (e.g. kefir, ayran, and ryazhenka) or bread.

Accordingly, in one embodiment the present invention provides a food product, or a food supplement comprising a composition according to the invention, comprising at least a sourdough in combination with a mixture of micro-organisms consisting of a L. acidophilus strain, a B. infanfis strain, a E. faecium strain and a Bifidum Bifidum strain.

In a preferred embodiment the food product is a sourdough or a beverage, in particular a fermented beverage such as kvass, water kefir, rejuvelac, darassun, pozol and boza.

Besides its application in food or feed products, the invention also relates to the use of the compositions or food products according to the invention in human or veterinary medicine. For the lactic acid bacteria present in sourdough, it is known that these bacterial strains have an anti-oxidative effect caused by the property of certain bacterial metabolites to neutralize reactive oxygen species (ROS). Under certain conditions the sourdough is also able to stimulate the immune system by enhancing the activity of defence cells. These beneficial effects of sourdough comprising compositions is also confirmed in the examples hereinafter. Unfortunately there is no sustained effect, so that the positive effect on the intestine eubiofic index in an individual is quickly lost when treatment is stopped. Complementing a base sourdough culture with the mixture of bacterial strains, not only enhances the positive effect on the intestine eu biotic index, but also converts it in a sustained effect even till 14 days after treatment is stopped. Thus in a particular embodiment the present invention provides the compositions according to the invention; or a food product according to the invention; for use in the prevention and/or treatment of intestinal disease and the improvement of intestinal health as well as promoting healthy gut flora.

When further comprising a vitamin and/or a mineral; optionally in combination with a phytase as defined herein before, the present invention also provides for said compositions for use in the prevention and/or treatment of bone disorders and the improvement of bone health.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 -Comparison of the intestinal eubiotic index at the three sampling times between the control group (0-tentic Drink) and the Experimental group (0-tentic drink with probiotics).

DETAILED DESCRIPTION OF THE INVENTION

Flour naturally contains a variety of wild yeast and Lactobacilli. When mixed with water the naturally occurring bacterial enzyme amylase breaks down the starch into the sugars glucose and maltose, that can be metabolized by the yeast. By maintaining such mixture and regularly adding fresh dough (flour) a stable fermentation culture is formed known as 'Sourdough' . For centuries Sourdough has been used as a natural leavening ingredient in the manufacture of bread, also known as bread starter, levain, masa madre, lievito naturale or Sauerteig.

The most common Lactobacilli, Lactic acid bacteria, in sourdough are L. delbrueckii, L. acidophilus, L casei, L. plantarum, L. fermentum, L. brevis, L. kefiri, and L. sanfranciscensis; In particular L. fermentum, L brevis, L. kefiri, and strains of L. sanfranciscensis.

The most common yeast species in sourdough are Kazachstania exigua (Saccharomyces exiguous), Saccharomyces cerevisiae, Candida milleri, Candida krusei strain, Saccharomyces exiguus strain, Saccharomyces barnetti strain, Saccharomyces cerevisiae strain and / or Saccharomyces minor strain; in particular Candida hum//is strain, Candida miller/ strain Candida krusei strain.

As used herein, the term "fermented" or "fermentation product" refers to a product made by adding a sourdough and a mixture of micro-organisms consisting of a L. acidophilus strain, a B. infanfis strain, a E. faecium strain and a Bifidum Bifidum strain to nutrients suitable for fermentation by the microorganisms. Specifically, the term refers to foods and feeds made by adding said combination of sourdough and mixture of micro-organisms as a starter, hereinafter also referred to as "fermentation starter" to food or feed bases which are then incubated. A fermentation starter comprises, as an active ingredient, at least such combination of sourdough and mixture of micro-organisms or a culture brew of the fermentation product, and/or a concentrate of the culture brew, and/or a dry matter of the culture brew.

The bifidum Bifidum strain, as used herein is also known as Bifidobacterium bifidum and belongs to the group of lactic bacteria typically found in the intestine and are in the form of irregular rods in size (1.5 -3 pm long by 0.9 pm wide). Depending on the environmental conditions, they can be individualized or grouped in clusters of several tens of cells. In one embodiment the Bifidum Bifidum strain used in the context of the present invention is characterised in having a 16S rRNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 168 rDNA of the strain THT 010101 (of 1453 base pairs, deposited in the Genbank database under the reference "THT-010101, complete sequence of the 16S rRNA: EF370998"). In another embodiment the Bifidum Bifidum strain used in the context of the present invention is characterised in having a 16S-23S rRNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 16S-23S rRNA of the strain THT 010101 (of 374 base pairs, deposited in the Genbank database under the reference "THT-010101, complete sequence of the 16S-23S rRNA: EF370995"). In another embodiment the Bifidum Bifidum strain used in the context of the present invention is selected from the Bifidum Bifidum strain deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under deposit number LMG 11041 or the Bifidum Bifidum strain deposited with the American Type Culture Collection (ATCC) under deposit number ATCC 29521.

The Bifidobacteriom Infantis strain, as used herein is also known as Bifidobacterium Ion gum subsp. Infantis and belongs to the group of lactic bacteria typically found in the intestine and are non-mobile Gram-positive bacteria. It is usually in the form of irregular rods in size (1 pm x 3.0 - 5.0 pm). These are individualized or grouped in pairs. In one embodiment the Bifidobacteriom Infantis strain used in the context of the present invention is characterised in having a 16S rRNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 16S rRNA of the strain THT 010201 (of 1455 base pairs, deposited in the Genbank database under the reference "THT-010201"). In another embodiment the Bifidobacteriom Infantis strain used in the context of the present invention is characterised in having a 16S-23S rRNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 16S-23S rRNA of the strain THT 010201 (of 388 base pairs, deposited in the Genbank database under the reference "THT-010201"). In another embodiment the Bifidobacteriom Infantis strain used in the context of the present invention is selected from the Bifidobacteriom Infantis strains deposited with the Belgian Coordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG 13197 or LMG 8811; more in particular the Bifidobacteriom Infantis strains deposited with the Belgian Coordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG 8811.

The Lactobacillus acidophilus strain, as used herein belongs to the group of lactic bacteria typically found in the intestine and are Gram-positive bacteria. They are usually in the form of irregular rods in size (0,6-0,9 pm x 1,6-6 pm). These are individualized or grouped in pairs, or in shod chains. In one embodiment the Lactobacillus acidophilus strain used in the context of the present invention is characterised in having a 16S rDNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 163 rDNA of the strain deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG 8151. In a particular embodiment Lactobacillus acidophilus strain used in the context of the present invention is the Lactobacillus acidophilus strain deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG 8151.

The Enterococcus faecium strain, as used herein belongs to the group of lactic bacteria typically found in the intestine and are Gram-positive bacteria. In one embodiment the Enterococcus faecium strain used in the context of the present invention is characterised in having a 16S rDNA sequence with at least 97% sequence homology, in particular at least 98% sequence homology, more in particular at least 99% sequence homology with a fragment of the 16S rDNA of the strain deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG S-28935. In a particular embodiment Enterococcus faecium strain used in the context of the present invention is the Enterococcus faecium strain deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under deposit numbers LMG 828935.

As used herein, the term "food product" means foods of vegetal or animal origin which provide the required nutrients to the organisms. The food product may be a milk-based product, a vegetable product, a meat product, a fruit juice, a wine and a bakery product or other unpasteurised open-fermented foods, preferably such as alcoholic and non-alcoholic beverages, breads, kimchi, salted-fermented sea foods, soy bean paste, cheese, yoghurt, wort and the like. More preferably, the food product is a sourdough or a beverage, in particular sourdough bread.

A food supplement product as used herein is a preparation intended to supplement the diet and provide nutrients, such as vitamins, minerals, fibres, fatty acids, amino acids and/or enzymes, that may be missing or may not be consumed in sufficient quantities in a persons diet. Other supplement may contain important regulatory components for human health such as R-glucans from yeast or multiple polyphenols from sources such as green tea, fermented tea, red wine extracts, grape seed extracts, cranberry or aronia extracts or juice etc. Further typical food supplement products are fermented concentrates.

The term "feed product" as used herein means substances or food products, as well as additives, which, depending on their level of processing, are intended for oral feeding of animals, e.g. cattle, pigs or deer.

Useful feed products starting materials include any material which is conventionally subjected to lactic acid bacterial fermentation step such as silage, e.g. grass, cereal material, peas, alfalfa-or sugar-beat leaf where the bacterial culture is inoculated in a feed crop to be ensiled in order to obtain preservation hereof, or in protein-rich animal waste products such as slaughtering offal and fish offal, also with aims of preserving this offal for animal feeding purposes.

EXAMPLE

Effect of Sourdouqh food composition on eubiotic index The effect of a sourdough food composition according to the invention on the maintenance of microflora was studied in the SIRIUS project -a 120-days isolation experiment reproducing the main characteristics of a real space flight to the Moon, including a subsequent circumlunar mission to search for a landing site, the landing of four crew members for operations on the Moon surface, a flight on the orbit of the Moon, remote control of the lunar rover to prepare the base and return to Earth.

The sourdough food composition tested, was based on a Sourdough drink -200 ml per person per day -(0-Tentice of Puratos), in combination with a mixture of microorganism consisting of L. acidophilus, B. infantis, E. faecium and Bifidum Bifidum, according to the below composition; Table 1. Composition of probiotics, used in SIRIUS experiment Preparation Strains of microorganisms in Quantity of microorganisms preparation in preparation (CFU per 1 g) 0-tentic® Saccharomyces cerevisiae 5 x 107(CFU per 1 ml) Further Microorganisms L. acidophilus 4,5 x 107 B. infanfis 3,0 x 107 E. faecium 4,5 x 107 Bifidum bifidum 5 x 107 Of the 64 volunteers participating in the SIRIUS program, three groups (n=18 / group) received either a placebo drink (Control Group), only the 0-tentice (04entic Group); or the 0-tentice drink in combination with the mixture of microorganisms (Experimental Group). Each group consumed the drink from the 151to the 15th day of the mission.

As a readout for the experiment, the specific and quantitative composition of the intestinal microflora of the crew in the SIRIUS program was sampled 26 days before the start of the mission to determine the background (Baseline). At day 15 to determine the effect during treatment, and at day 30 to see whether there is a lasting effect. Intestinal microflora was assessed by the presence of the following species: Bifidobacterium spp., Lactobacillus spp., Enterococcus spp., Escherichia coli, Enterobacter spp. Staphylococcus spp., S. aureus, Clostridium, Pseudomonas aeruginosa, Bacillus spp., Candida.

To assess the effectiveness of the dynamics of changes in quantitative and species characteristics of the microflora, we have developed and used an "eubiofic index". The "eubiofic index" is the ratio of positive changes in the quantitative and species composition of microflora to negative ones; Positive changes include; 1. Reducing the number of opportunistic pathogenic microorganisms; 2. Stabilization of quantitative characteristics of opportunistic pathogenic microorganisms at an acceptable low level; 3. Increasing the number of microorganisms of protective groups; 4. Stabilization of the number of protective groups at an acceptable high level. 10 Negative changes include; 1. Increasing the number of opportunistic pathogenic microorganisms; 2. Stabilization of quantitative characteristics of opportunistic pathogenic microorganisms at an unacceptable high level; 3. Reducing the number of microorganisms of protective groups; 4. Stabilization of the number of protective groups at an unacceptable low level.

Results: When analysing the state of microflora in the background period, it was found that for most of the evaluated microorganisms for all crew members, their number was at a level that is generally consistent with the norm. Presence of opportunistic pathogenic microorganisms, for example, Candida, Pseudomonas aeruginosa, was noted in organisms of some subjects at a level slightly above the normal value.

By the 30th day, a decrease in the number of Bifidobacterium spp. and Lactobacillus spp., which are the main components of resistant microflora, was observed in the control and 0-tentic group who hadn't not used probiotics. The number of opportunistic pathogenic microorganisms either remained at the pre-experimental level or increased. In some samples previously undetectable species, such as Candida, Bacillus spp., were found, which is probably related to the exchange of microflora, which inevitably occurred after the crew's stay in a confined compartment with an artificial habitat.

In the experimental group, a different picture was observed. Immediately after finishing drinking the 0-tentice drink with the probiotic preparation on the 15th day, the number of representatives of the protective microflora stabilized, and in some cases, even slightly increased. Opportunistic pathogenic microorganisms, which had been observed earlier in some of the crew members, eradicated or diminished (for example, one of the subjects had P.aeruginosa presented in content of his microflora, on the 15th day this microorganism was not detected).

By the 30th day, after approximately two weeks, when the prophylaxis course had been finished, the number of representative microorganisms of protective microflora either stabilized or decreased slightly. It is also interesting that opportunistic pathogenic organisms noted while baseline tesfings, had been eradicated after taking the course of prophylaxis, and hadn't been found on the 15th day, were not detected at the 30th either, which indicates the formation of sufficient resistance, which is preserved even after two weeks after stopping drinking the 0-te Mice with probiofics (Table 2).

Expressed differently, where the 0-tentic drink in isolation has the ability to maintain the eubiotic index at base level during the experiment and up to 15 days thereafter, in combination with the mixture of microorganism a strong positive and lasting read-out is present instead (Fig. 1-Eubiotic index of changes in the intestinal microflora in the SIRIUS experiment -side to side comparison between the 0-tentic Group (Control in Fig.1) and the Experimental group). This clearly shows how this combination excels not only in maintaining, but in improving the microflora of the individuals.

Table 2. Quantitative and special content of intestinal microflora of some volunteers in experiment SIRIUS Volunteers, Microbial groups Control values * -tentic group Experimental group : aseline 15th day 30th day Baseline 15th day 30th day Volunteer N° 1902 1901 E. coli 106,107 106 - 106 108 - 106 Enterococcus spp. 106,107 106 106 106 108 106 108 Bifidobacterium spp. 108,109 106 106 106 108 106 107 Lactobacillus spp. 106,107 106 107 107 108 107 107 Staphylococcus spp. <105 - 105 103 - 105 S. aureus <105 103 - 103 -Enterobacter spp. 105 106 - 105 105 - 106 Clostridium <105 105 105 105 - - -P. aeruginosa <104 Bacillus spp. <104 105 Candida <104 103 103 Volunteer N° 1904 1903 E. coli 106,107 106 106 - 105 105 - Enterococcus spp. 106,107 105 - 105 105 -Bifidobacterium spp. 108,109 106 106 106 107 108 107 Lactobacillus spp. 106,107 106 107 104 107 107 107 Staphylococcus spp. <105 105 105 S. aureus <105 105 Volunteers, Microbial groups Control values 0-tentic group Experimental group Baseline 15th day 30th day Baseline 15th day 30th day Enterobacter spp. 105 108 106 Clostridium <105 105 105 P. aeruginosa <104 - - - - -Bacillus spp. <104 105 Candida <104 103 103 Volunteer N° 1905 1906 E. coli 106,107 - - 106 106 - 106 Enterococcus spp. 106,107 106 106 - - 106 -Bifidobacterium spp. 108,109 106 108 108 105 108 108 Lactobacillus spp. 106,107 107 107 105 105 107 107 Enterobacter spp. 105 106 108 103 105 Staphylococcus spp. <105 S. aureus <105 105 108 108 Clostridium <105 105 105 105 105 105 P. aeruginosa <104 106 - - - - - Bacillus spp. <104 - 105 - - 105 - Candida <104 - - - - - -We have analyzed the current trends in microflora changes in experiments with isolation. The results of this analysis are presented in the table 3 and are expressed in eubiotic indices. Here we compared the indices of the intestinal microflora, integumentary tissues and mucous membranes of the upper respiratory tracts of the operators in the first 30 days of isolation. The results indicate significant negative shifts in the composition of the microflora of operators who did not rece;ive any prophylactic agents.

Table 3. Eubiofic index in isolation experiments without prophylactic measures and in SIRIUS experiment.

Period Tissue Baseline Intestine High respiratory tract Covering tissues -151h day Isolation experiments without prophylaxis -Control Group (n 18,64 volunteers) 0,9 1,2 0,5 SIRIUS experiment -0-tentic Group (n 18, 64 volunteers) 1,2 1,0 0,9 SIRIUS experiment -Experimental Group (n 18,64 volunteers) 3,5 1,1 0,6 Comparing these indicators with those of the "Sirius" experiment, one can be convinced that eubiosis is achieved already with the use of the 0-tentice drink. In combination with probiotic preparations, the values of the eubiofic index for intestinal microflora reach very significant values (3,5 versus 1,2). At the same time, neither the 0-tentice drink, nor the 0-tentice drink with probiotic preparations had any optimizing effect on the upper respiratory tract and integumentary tissues.

Thus, it can be concluded that the use of probiotic preparations according to the invention together with a sugar-based fermentation drink helps to optimize the quantitative and specific composition of the intestinal microflora to a greater extent than the intake of the fermentation drink itself without the adding of any probiotics. This is very important during the first period of adaptation, from the 7th to the 15th days of isolation, when the preparation was taken. The fall in the "eubiotic index" indicator, which reflects the prevalence of negative changes in the composition of the microflora recorded on the 30th day of the experiment, suggests that the positive effect achieved with the combined intake of fermentation drink and probiotic has a temporary effect. Still, and as mentioned above, the use of the probiotic preparations according to the invention have a lasting effect on the presence of opportunistic pathogenic organisms, which is preserved even alter two weeks after stopping drinking the 0-tentice with probiotics

Conclusion

1. Consumption of 0-tentice drink leads to stabilization of the intestinal microflora at the level corresponding to the initial characteristics. This is a positive trend, since usually in the first period of adaptation there is an increase in the activity of pathogenic microflora.

2 0-tentice in combination with probiotics leads to improvement of special and quantitative characteristics of intestinal microflora during acute adaptation period.

3. 0-tentice in combination with probiotics leads to a lasting decrease on the presence of opportunistic pathogenic organisms in the intestinal microflora.

Claims

CLAIMS1. A composition comprising a sourdough and a mixture of microorganism consisting of a L. acidophilus strain, a B. infantis strain, a E. faecium strain and a Bifidum Bifidum strain.
2. The composition according to claim 1, further comprising at least one vitamin and/or minerals; in particular at least one vitamin and/or mineral selected from vitamin D3 and K2, calcium carbonate, calcium citrate, tricalcium phosphate and silicon dioxide.
3. The composition according to claims 1 or 2, further comprising an ezyme, such as a phytase, in particular a bacterial phytase; more in particular a bacterial phytase sourced from L. reuteri or Butfiauxella.
4. A food product, or a food supplement comprising a composition according to any one of claims 1 to 3
5. The food product according to claim 4, wherein the food product is a sourdough or a beverage.
6. A composition according to any one of claims 1 to 3; or a food product according to claims 4 or 5; for use in human or veterinary medicine.
7. A composition according to any one of claims 1 to 3; or a food product according to claims 4 or 5; for use in the prevention and/or treatment of intestinal disease and the improvement of intestinal health as well as promoting healthy gut flora.
8. A composition according to claim 2, for use in the prevention and/or treatment of bone disorders and the improvement of bone health
9. The composition for use according to claim 8, said composition further comprising a phytase, in particular a bacterial phytase; more in particular a bacterial phytase sourced from L. reuteri or Buttiauxella.
10. A composition according to claim 2 or as defined in claim 9, for use in the prevention and/or treatment of bone loss, in particular for use in the prevention and/or treatment of osteoporosis.