WO2025224073A1

WO2025224073A1 - Use of lactobacillus bacteria for reducing adipose and promoting muscle tissue

Info

Publication number: WO2025224073A1
Application number: PCT/EP2025/060877
Authority: WO
Inventors: Dr. Jürgen SCHREZENMEIR; Helmut ESSL
Original assignee: Slimbiotics GmbH
Current assignee: Slimbiotics GmbH
Priority date: 2024-04-25
Filing date: 2025-04-22
Publication date: 2025-10-30
Anticipated expiration: 2026-10-25

Abstract

The present disclosure relates to compositions containing live and vital or inanimated Lactobacillus bacteria for reducing body weight and adipose tissue and promoting muscle tissue in living organisms, in particular for treating obesity in humans, and/or supporting the buildup of muscle mass and/or improving muscle tone in humans and/or animals, and/or improving the ability to concentrate in humans, where the Lactobacillus bacteria are selected from one or several of the strains L. plantarum K4, L. fermentum K7, L. fermentum K8 and/or L. fermentum K4. Moreover, it relates to methods of preventing the buildup or reducing body weight and the amount of adipose and promoting growth of muscle tissue in living organisms, in particular for preventing or treating obesity in humans, wherein the compositions are administered orally.

Description

Use of Lactobacillus bacteria for reducing adipose and promoting muscle tissue

FIELD

The present disclosure relates to methods and compositions for preventing the buildup or reducing the amount of fat tissue, supporting the buildup of muscle tissue and/or improving muscle tone in living organisms of the animal kingdom, including humans, or improving the ability to concentrate in humans, in particular methods and compositions for treating or reducing the risk of obesity by administering certain strains of live or inanimated/devi- talized Limosilactobacillus (formerly and for brevity’s sake hereinafter still Lactobacillus (Zheng et aL, Int. J. Syst. Evol. Microbiol. 2020)) fermentum and/or Lactiplantibacillus (formerly and, again, for the sake of brevity hereinafter still Lactobacillus') plantarum bacteria or compositions containing these strains, as medicationns or dietary food supplements or as additives to food for both humans and animals, specifically pets.

BACKGROUND

It is known that human and animal health can be improved by administering probiotics, i.e. live and vital bacteria, whether in the form of a medication, such as a liquid in which the living and active bacteria are dissolved or suspended or powders, pills or capsules containing bacteria in an inactive, but viable state. The bacteria can be administered by themselves or, typically, as part of a composition, such as mixed into food stuff, e.g., solid food or beverages. In particular, probiotic beverages and yoghurt, but also supplements, have been known and been commercially successful for some time.

Usual types of probiotic bacteria are taken from the lactic acid bacteria families Bifidobacteriaceae and Lactobacillaceae, and in these families, particularly the genera Bifidobacterium and Lactobacillus respectively, which both are among the major genera of the bacteria that make up the mammalian microbiota at a number of body sites, including the digestive system. Among the Lactobacillus and Bifidobacterium-species known or suggested to promote human and animal health when administered in sufficient quantities are L. acidophilus, L. plantarum, L. easel sub. easel, L. easel sub. rham- nosus, L. zeae, L. salivarius, L. lactis and subspecies, L. helveticus, L. reuteri, L. amylovorus, L. crispatus, L. curvatus, L. delbrueckii and all its known subspecies, L. gasseri, L johnsonii, L. fermentum, L. brevis, B. longum, B. breve, B. bifidum, B. infantis and B. lactis (taxonomy before 2020). The oral administration of particular strains of these bacteria is thought, and in some cases has been proven, to improve intestinal health by preventing multiplication of unwanted bacteria and thereby having an equilibrating effect on the intestinal microbiome, with the positive consequences of improving nutrient uptake, preventing cramps and diarrhea and helping with accute and chronic diseases.

Some of the effects of probiotics are necessarily linked to introducing the beneficial bacteria into the target location, such as the intestinal tract of a human being or animal, in a live and vital form, where they can directly aid in restoring a possible diminished population, in competing with less favourable microorganisms or by exerting effects through direct contact with the intestinal tissue or by releasing active metabolites and signaling molecules, respectively. It has been found, however, that in some cases positive effects on the health of the host can also be observed when administering so called postbiotic compositions where most or, typically, all bacteria have been inanimated, sometimes also referred to as ‘devitalized’, i.e. treated such that their metabolism ceases to function, which can e.g. be achieved by a pressure treatment, a heat-treatment, freezing or intense (UV-)irradiation and x- ray or gamma-ray irradiation, or at least rendered incapable of reproduction, such as can be achieved, e.g., by inactivating the bacteria by means of a less intense UV-irradiation. Probiotics and postbiotics of some or all the aforementioned bacteria as well as other probiotically utilized bacteria such as Streptococcus thermophilus and methods of their use are disclosed in the publication of an international patent application WO 03/071883 A1.

Intestinal health and a balanced intestinal microbiome have also been linked to the immune defence, An intensively studied example of enhancing immune defense by probiotics is respiratory tract infection, such as common cold, influenza-like infections and other viral infections. Meta-analyses of numerous studies in thousands of children and adults demonstrated a significant protective effect against respiratory tract infections (Wang et al, 2016. Probiotics for prevention and treatment of respiratory tract infections in children - A systematic review and meta-analysis of randomized controlled trials. Medicine 95: 31 ; Nour et aL, 2023. Effect of probiotics on common cold, influenza, and influenza-like illness A systematic review and meta-analysis. Top Clin Nutr 38: 196-210).

The use of certain L. plantarum and L. fermentum strains, among these the strains L. fermentum K7-Lb1 , L. fermentum K8-Lb1 , L. fermentum K11-Lb3 and L. plantarum K4-Lb6, as a probiotic to modulate the human immune system has been proposed in the German patent application publication DE 10 2009 037089 A1 (also published in English as US 2016/0074442 A1). On the basis of an observed anti-inflammatory effect due to a suppression of the cytokines Th1 and Th2 and an enhancement of the release of defen- sin, it has therein been claimed that the strains K7-Lb1 , K8-Lb1 , K6-Lb2 and K6-Lb4 might be effective in favorably influencing and treating, respectively, among other abnormal conditions, obesity and metabolic syndrome.

Through promoting or suppressing the production of interleukins, some postbiotics have been proposed to treat inflammation. Interleukins are cytokines, i.e. secreted proteins and signal molecules, that are expressed by white blood cells as well as some other body cells and have various functions in the body depending on the specific interleukin. Among them, inter- leukin-10 (IL-10) inhibits the synthesis of a number of other cytokines and therefore exhibits a pleiotropic effect in immunoregulation and inflammation. Since it enhances B-cell survival and proliferation and antibody production, while inhibiting synthesis of inflammatory cytokines, it can have regulating effect on the immune response against pathogens, while checking inflammatory overreaction. Interleukin-12 (IL-12) is involved in the differentiation of naive T-cells into Th1 helper cells and is therefore known as a T cell stimulating factor. It activates killer cells and T lymphocytes and also has an anti-angiogenic activity, i.e. it blocks the formation of new blood vessels, which stems from IL-12’s stimulatory effect on the production of interferon gamma, a key cytokine for upregulating inflammation response. With these functions, it has been shown to sometimes worsen the symptoms of autoimmune diseases and, in conjunction with IL-23, be effective against inflammatory bowel disease. It has been proposed as a drug for preventing or slowing tumor growth, however without a substantial effect being observed so far.

Compositions containing L. plantarum, specifically the L. plantarum strain OLL2712, and methods using these to increase the IL-10 and IL-12 production and specifically promote IL-10 over IL-12 with an associated anti-inflammatory effect, is proposed in WO 2021/261423 A1 . As was already the case in DE 10 2009 037089 A1 , where it is also suggested, that through the anti-inflammatory effect the composition might also be effective against obesity since obesity is linked to systemic inflammation factors. The bacteria are proposed to preferably be heat-killed before adding them to the composition. The composition may in particular comprise or substantially consist of milk, either raw or preferably sterilized and pre-homogenised.

Further uses for Lactobacillaceae-species, specifically L. plantarum, are suggested in the Japanese patent application JP 2017-048244 A, where a composition containing L. plantarum is disclosed as promoting collagen production and collagen absorption in humans as well as being effective in treating obesity. The bacterial cells are preferably to be added to the composition in powder form, which is obtained by subjecting the bacterial cells to a drying or extraction treatment/process. To back up the claimed antiobesity effect a study in mice was performed.

In their recent article “Effect of a Probiotic and Synbiotic on Body Fat Mass, Body Weight and Traits of Metabolic Syndrome in Individuals with Abdominal Overweight: A Human, Double-Blind, Randomised, Controlled Clinical Study”, published in Nutrients, vol. 15, page 3039, Laue et al. have shown that administering probiotics of the L. fermentum strains K7-Lb1 , K8- Lb1 and K11-Lb3 is effective in reducing body fat mass and connected to this, body weight, waist circumference, waist-to-height-ratio, visceral adipose tissue (SAD) and liver steatosis grade (LSG). The effect of the administration of said strains in postbiotic form was not studied.

While the effectiveness of both pro- and postbiotics has been suggested and to some degree proven for some Lactobacillaceae strains, it is not possible to generalize this effect. The (exact mechanism of) action of the substances produced by the live strains of certain bacteria or contained in the remains of theseinanimated bacteria on a system as complex as a human or animal body, despite the continually growing body of knowledge about and ability to model them, is still not sufficiently understood and thus their effects cannot be predicted with any degree of confidence. Notably, it has been observed that while some bacterial species and strains show beneficial effects, other species or even other strains of the same species have no or the opposite effect. The pertinent strains of t. fermentum clearly differed from other /., fermentum strains in their immunomodulatory activity: whereas some strains showed a stimulation of Th1 and Th2 response of human PBMC, the strains K7 and K8 suppressed Th1 and Th2 response and only strain K11 induced a defensin release by human enterocytes, whereas other strains of the same species did not (s. Patent claiming the anti-inflammatory effect of the pertinent living strains; Ghadimi et al, 2011. Mol. Nutr. Food Res. 55: 1533-1542 and Ghadimi et aL, 2014. Immunobiology 219: 208- 217). Also, the observed effects are, in general, rather small and it is challenging to confirm their presence with statistical significance. In view of the high prevalence of overweight and associated diseases and in view of the complex pathogenesis implying more than hundred genes involved in the disposition to obesity and associated diseases, there is a need for identifying other effective bacterial species and strains showing positive effects on human and animal health. Particularly, it would be desirable to find other bacterial species and/or strains which are effective in the treatment or prevention of obesity in particular and in reducing the amount or the buildup of adipose or fatty tissue in human beings as well as animals in general, as well as promote further beneficial effects to human and/or animal health and well being such as supporting build-up of muscle mass and/or improvement in muscle tone and or improve humans ability to concentrate

SUMMARY

It is therefore an object of the present invention to find alternative and preferably more effective pro- and postbiotics which show the afore described positive effects in regulating the amount or buildup of fatty and/or muscle tissue in living organisms, including humans and animals, specifically mammals and/or in promoting human mental concentration ability.

In particular, it is an object to identify suitable bacterial species and/or strains for use as pro- and/or postbiotics and in pro- and postbiotic compositions.

It is a further object to find methods for preparing postbiotics containing the identified bacterial species and/or strains.

Finally, it is also an object to find methods and ways to control the population size of nematodes such as Caenorhabditis elegans.

These and other objects are achieved by a pharmaceutical or dietary composition according to one of the claims 1 - 9, a method for making such a composition according to one of the claims 10 - 18 and a method of using such a composition to reduce the buildup or amount of fatty tissue and/or to treat obesity and/or to support the buildup of muscle mass and/or improve muscle tone in living organisms and/or improve the ability to concentrate in humans according to one of the claims 19 - 23.

According to this invention, the pharmaceutical composition exhibiting the aforementioned positive and desired effects, is made up of or contains either exclusively or together with other substances or bacteria, live or inanimated strains of bacteria belonging to the genus Lactobacillus and the species L. plantarum or L. fermentum, wherein the bacteria are specifically selected from one or more of the strains L. fermentum K7-Lb1 (deposited at the German Collection of Microorganisms and Cell Cultures or DSMZ located in Braunschweig, Germany, under the number DSM 22831), L. fermentum K8- Lb1 (DSM 22832), L. fermentum K11-Lb3 (DSM 22838) and L. plantarum K4- Lb6 (DSM 22830).

The composition may be a probiotic, i.e. it may contain - either exclusively or partially - live and vital bacteria, i.e. bacteria with intact metabolism and cell structure capable of growing and reproducing in the right conditions.

The composition of the invention may also be a postbiotic, i.e. comprise inanimated bacteria, i.e., some or all of the bacteria it contains have been inanimated by a suitable technique to prevent them from further reproducing even in conditions favourable to non-inanimated, i.e. live and vital bacteria. The inanimation/devitalization may be achieved by various means, such as heat-treatment, cold treatment, high pressure treatment or UV-, x-ray or gamma-ray irradiation.

Some preferred inanimation techniques will be explained in the context of the method of making a composition according to this invention described in detail further below.

In preferred embodiments of the composition of the invention the concentration of the bacteria - either live and inanimated or both - lies between 10⁶ and 10¹² cfu/g, in particular between 10⁸ and 10¹¹ cfu/g, particularly preferably between 10⁹ and 10¹⁰ cfu/g. In some embodiments, in the postbiotic composition all or most, i.e. 95 % or more in particular 99% or more of the bacteria it contains have been inanimated. In some embodiments the postbiotic contains less then 5000, preferably less than 3000, more preferably less than 1000 live bacteria per gramm of composition.

The composition of the invention may consist exclusively of live or inanimated bacteria, either in hydrated form, such as dissolved in water, or in dehydrated form, such as a dried, in particular freeze- or spray-dried, powder.

In other embodiments, the composition may comprise other substances. These can be substances that act as a growth medium for the live bacteria or acted as growth medium for the bacteria before they were inanimated, such as a sugar, e.g. glucose solution.

The other substances may also be of such type as to increase the effectiveness of the composition and/or to be beneficial for the living organism, to which the composition is to be administered. Such other substances may in particular be one or more of prebiotics, such as dietary fibres, specifically natural fibres, minerals salts, in particular sodium or potassium, phosphorous, iron, zinc, magnesium, manganese, and/or vitamins, in particular A, B1 , B2, B6, B12, niacin, C, D3, E and/or folic acid. The composition may be a liquid containing Lactobacillus bacteria and other substances in a dissolved or suspended form. Alternatively, the composition may be a mixture of dry powders.

In some embodiments the composition is based on a carrier food substance that is mixed or inoculated with live and/or inanimated Lactobacillus bacteria to a desired concentration. The carrier food substance may be a liquid such as water, milk, whey, yoghurt, a fruit and/or vegetable preparation such as a juices, , liquid shots or a smoothie or a syrup or solid such as cereal, candy, including gummy candy, chocolate, hard fat, wax, shortening, cookies, cakes, protein powder, sausages, cheese and other dairy products. To treat animals such as pets or farm animals/livestock, the carrier substance may also be an animal feed substance, such as a livestock feed or fodder comprising hay, silage, soy, corn, oats, sorghum and/or barley. The carrier substance may alternatively also consist of or comprise meat, including cartilage, or fish in raw or dried and/or powdered form, such as is the case for cat and or dog food.

The composition used in connection with a carrier food substance may be a probiotic or a postbiotic. If a probiotic is used, the combination with chocolate is particularly advantageous since the fat of the chocolate will protect the live bacteria from moisture while at the same time that absence of moisture arrests their reproduction. Both effects help to increase the shelf life of the probiotic. Furthermore, the chocolate, while it melts already in the mouth or during the passage through the esophagus, does not fully dissolve until the small intestine and therefore most of the live bacteria will be protected during their passage during the stomach from the low pH conditions there.

A further aspect of the invention is a method for making, i.e. producing, a pro- or postbiotic with the afore-described features. This method involves culturing live Lactobacillus bacteria of one or more desired strains, i.e. L. fermentum K7-Lb1 (DSM 22831), L. fermentum K8-Lb1 (DSM 22832), L. fermentum K11-Lb3 (DSM 22838) and L. plantarum K4-Lb6 (DSM 22830), in suitable condition. This culturing may comprise the following steps:

1. Setting up a fresh plate of each desired Lactobacillus strain on MRS agar by streaking an aliqot from a frozen stock using a sterile inoculation loop;

2. Incubating the streaked plates at 30°C for between 36 h and 40 h to allow growth of single colonies;

3. For each strain, picking one or more of the single colonies of the bacteria from the respective plate using a sterile inoculation loop and inoculating a desired volume of MRS broth, for instance 25 ml, in a sterile falcon tube of sufficient size, preferably around twice the volume of the MRS broth, for instance 50 ml,

4. Incubating the falcon tubes prepared in step c in a shaker at 30°C for between 16 and 18 h at 250 rpm, ensuring the possibility of gas exchange with the ambient air, in particular in case of a lidded shaker by leaving with the lid slightly open, 5. Centrifuging the falcon tubes at 4000 rpm for 10 minutes at ambient temperature to pellet the bacterial cells;

6. Discarding the supernatant from each of the falcon tubes and adding an amount of liquid Nematode Growth Medium (NGM) corresponding to between 1/4 and ¹/₂ the volume of the discarded supernatant of the respective falcon tube, for instance 10 ml;

7. Vortexing the falcon tubes to resuspend the bacterial cells in the liquid NGM;

8. Centrifuging the falcon tubes at 4000 rpm for 10 minutes to pellet the bacterial cells;

9. Discarding the supernatant from each falcon tube and determining the weight of each of the bacterial pellets, for instance by weighing the falcon tube on a scale after taring the scale with an empty falcon tube of the same kind;

10. Resuspending each of the bacterial pellets in liquid NGM to a concentration of between 30 mg and 50 mg, in particular 40 mg of bacteria per ml of NGM.

In some embodiments of the method for making a composition according to the invention the Lactobacillus bacteria are devitalized/inanimated in a inan- timation- or devitalization treatment, or inanimation respectively devitalisaton for short, which is one or a combination of an irradiation treatment and a temperature treatment, namely a heat treatment or a cold, i.e. freezing, treatment or high-pressure treatment or irradiation treatment.

The method may also comprise more than one inanimation stage, with each stage corresponding to an inanimation treatment. For instance, the inanimation may comprise a first or pre-inanimation stage or treatment, a second or main inanimation treatment/stage and, possibly also a third, fourth, etc. inanimation treatment/stage. The inanimation treatments of the different stages may be of the same type (heat, cold, radiation) and/or technique (specific treatment parameters) or they may be of different type and/or technique. For instance, in some embodiments the first inanimation stage, or first inanimation for short, is a cold treatment followed by a heat treatment as the second inanimation (stage) or vice versa.

A third, fourth and so on inanimation stage comprising one of the first or a different type of inanimation treatment may be performed subsequent to the main inanimation.

The advantage of having more than one inanimation stage is the increased effectiveness of the inanimation, i.e. the fraction of bacteria inanimated, where typically a fraction of 1 ,00 or 100% is desired. Moreover, it ensures effectiveness of the inanimation not only for the Lactobacillus bacteria which are the desired microbial component of the composition according to the invention, but also of other, undesired bacteria which may be present in trace amounts after the culturing stage, e.g. one following the aforede- scribed protocol.

This goal is achieved even more when two or more different inanimation types are combined, since bacteria which have a natural resistance to one technique typically are weak against another. Thermophilic bacteria for instance are typically not very cold resistant and vice versa.

The term “pre-inanimation” and “main inanimation (stage)” as used herein imply, that the latter is aimed at devitalisting the primary microbial content of the composition according to the invention, i.e. the (mixture of) Lactobacillus-bacteria belonging to the above-mentioned four strains, while the preinanimation is aimed at other, possibly unwanted, bacteria which may be present among the bacteria. In contrast, the terms “first”, “second”, “third” inanimation/devitalization (stage)as used here are not intented to convey such a meaning.

The temperature treatment for a inanimation stage as part of the method of making a composition according to the invention may be a heat treatment comprising heating the live or pre-inanimated Lactobacillus bacteria to a temperature no less than 70°C for no less than 60 minutes, in particular to a temperature of 75°C for 90 minutes. The temperature treatment for a inanimation stage as part of the method of making a composition according to the invention may be a freezing or cold treatment, in particular comprising cooling the live or pre-inanimated Lactobacillus bacteria to a temperature of less than 0°C, in particular less than - 5°C, for instance between -5°C and -50°C at a cooling rate of no more than 0,5°C/min, in particular no more than 0,1 °C/min. This slow cooling or freezing rate ensures the formation of large ice crystals which pierce and rupture the cell membrane of the bacteria, which kills them more effectively than a fast cooling/freezing, where the ice crystals formed may be two small to appreciably damage the bacteria.

The cooling may take place at ambient or elevated pressure. The cooling may be repeated once or several times, with the bacteria in between being warmed to a temperature above 0°C, in particular above 5°C, for instance room temperature for between 1 to 30 minutes, for instance 10 minutes. Repeating the freezing treatment increases its effectiveness in devitalizing the Lactobacillus bacteria, and any other bacteria which might be present, even further.

The cold or freezing treatment described above may or may not be part of a freeze-drying process. The cold treatment may involve cooling the bacteria to a temperature of less than -5°C, such as -10°C, -15°C, -20°C, -25°C or - 30°C. In this case, it in preferred embodiments, the bacteria are heated to a temperature closer to 0°C, such as -2°C or -1°C, in order to speed up the freeze-drying process, since the sublimation of water molecules from the ice crystals in the bacteria and the frozen solution surroundung them happens faster at higher temperatures.

An alternative way of devitalizing the bacteria is a high pressure and ultra- high pressure treatment wherein pressures of more than 200 Mpa, in particular between 200 and 1000, such as between 400 - 700 Mpa are applied for at least 5 min, in particular for at least 10 min, such as between 15 and 30 min. A third aspect of the invention is a method for preventing buildup or reducing the amount of fatty tissue in a living organism. This method involves administering a composition according to the invention to the living organism which is to be (prophylactically) treated, such as a human being or an animal, in particular a (non-human) mammal.

The standard way of administering the composition is orally. Alternatively or additionally, it may be administered directly to the intestinal tract, for instance anally.

In preferred embodiments, the administered composition according to the invention has a suitably high concentration of bacteria, such as, for the probiotic formulation, between 10⁶ and 10¹² cfu/g, particularly preferably between 10⁹ and 5*10¹¹ cfu/g, or, for the postbiotic formulation, between 10⁶ and 10¹² cells/g, particularly preferably between 10¹⁰ and 10¹² cells/g

In some embodiments of the method of the third aspect of the invention, the composition is a probiotic containing either only bacteria of L. fermentum K8- Lb1 or the three L. fermentum strains K7-Lb1 , K8-Lb1 and K11-Lb3 to equal parts. In these embodiments, the composition is administered at a daily dose of 40 mg /day corresponding to 6*10^A9 cfu /day.

In other embodiments the composition is a postbiotic containing either only inanimated bacteria of L. fermentum K8-Lb1 or the three L. fermentum strains K7-Lb1 , K8-Lb1 and K11 -Lb3 to equal parts. In these embodiments, the composition is administered at a daily dose of 20 mg, corresponding to a minimum of 6*10^A9 inanimated cells.

The daily dose is preferrably administered in one or two servings. It may be administered in the form of a pill, a dragee or coated tablet or a capsule. Here and throughout, the cell count may be determined by prior CFU, microscopically or by flow cytometry.

Further details and features of embodiments of the present disclosure are described below with reference to the figures of preferred exemplary embodiments. These are only intended to illustrate the various embodiments, and in no way to limit the scope of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 : A table detailing the nematode worm growth conditions for study trials testing the effectiveness of both the pro- and post- biotics according to embodiments of the invention with a non- obesogenic diet.

Fig. 2: A diagram showing histograms of the combined fluorescence intensity measurement results of fluorescent microscopy measurements performed on fixed and Nile-Red-stained nematodes. Fluorescence represented lipid accumulation in the nematodes. The nematodes were fed for 48h a non-obe- sogenic diet as set out in the table of Fig. 1 . The diet included, for six sets of groups, one of six different probiotic compositions containing one or more of the stated L. fermentum and/or L. plantarum strains according to the invention and, for comparison, a control group receiving a known inhibitor of fat digestion (orlistat) as well as a no-treatment control group.

Fig. 3: A diagram similar to Fig. 2, however showing the results of trials with six postbiotic compositions containing the same L. fermentum and/or L. plantarum strains but inanimated by heattreatment.

Fig.4: A table detailing the nematode worm incubation conditions for study trials testing the effectiveness of postbiotic compositions according to embodiments of the invention with an obe- sogenic diet. Fig. 5: A graph showing the aggregate fluorescence intensity measurement results of fluorescent microscopy measurements on fixed and Nile-Red-stained nematodes for assessing fat accumulation. The nematodes werefed an obesogenic diet as set out in the non-grayed rows of table of Fig. 4 for 48h. The diet included, for six groups, one of six different probiotic compositions containing L. fermentum and/or L. plantarum strains singly or as a mixture and, for comparison, a control group receiving a known inhibitor of fat digestion (orlistat) as well as a no-treatment control group.

Fig.6A: A table listing nematode growth conditions for study trials investigating the acute glucose-induced toxicity effect exhibited by some Lactobacillus strains under the growth conditions of the grayed-out rows of the table shown in FIG. 4.

Fig.6B: A table showing the change in pH after 0, 24 and 48 hours of on-chip culturing of C. elegans with diets consisting of standard NGM and four Lactobacillus strains with and without adding 2% glucose.

Fig.7A: A table giving the genera identified by DNA-sequencing samples of pro- and postbiotics of the four Lactobacillus strains of the invention.

Fig.7B: A table setting out the nematode growth conditions for study trials comparing the effectiveness of freeze-dried postbiotics produced by the Centro Sperimentale die Latte (CSL), India, and non-freeze-dried postbiotics prepared by the company nemalife, Texas, USA, according to embodiments of the invention with an obesogenic diet. Fig. 8: A diagram showing the aggregate fluorescence intensity measurement results of fluorescent microscopy measurements on the fixed and Nile-Red-stained nematodes after being fed an obesogenic diet as set out in the table of Fig. 7B for 48h.

Fig. 9A Participant demographic information of a placebo-controlled blind study to test the efficacy of an L. fermentum K8-Lb1 post- biotic in humans.

Fig. 9B: Body Composition, Body Weight and Blood Pressure at Baseline.

Fig. 10: Body Weight in postbiotic and placebo group over 12 weeks.

Fig. 11 A-C: Fat Mass, fatt mass ITT and fat mass PP in postbiotic and the placebo group over 12 weeks.

Fig.12: Waist circumference in postbiotic and placebo group.

Fig. 13: Visceral Fat Mass in postbiotic and placebo group over 12 weeks.

Fig. 14: Muscle Mass in postbiotic and placebo group over 12 weeks.

Fig. 15A: Systolic Blood Pressure in postbiotic and placebo group over

12 weeks.

Fig. 15B: Diastolic Blood Pressure in postbiotic and placebo group over

12 weeks.

Fig. 15C: Resting Heart Rate in postbiotic and placebo group over 12 weeks.

Fig. 16: Liver Function Biomarkers in postbiotic and placebo group.

Fig. 17: Inflammation Biomarker CRP in postbiotic and placebo group.

Fig. 18A: Metabolic Biomarkers in postbiotic and placebo group.

Fig. 18B: Metabolic Biomarkers of the stratum with normal glucose and the stratum with impaired glucose levels (fasting glucose BG > 100 mg/dl (IFG + T2D)) in postbiotic and placebo group. Fig. 19: Lipid Profile in postbiotic and placebo group.

Fig. 20A: Changes in Validated Questionnaires (Perceived Stress Scale

(PSS), Anxiety GAD-7 Questionnaire (GAD-7) and Three Factor Eating Questionnaire (TFEQ) in Postbiotic and Placebo Group at Week 4 (A). Week 8 (B). and Week 12 (C).

Fig. 20B Subject Specific Parameters in postbiotic and placebo group at week 4.

Fig. 20C Subject Specific Parameters in postbiotic and placebo group at week 8.

Fig. 20D Subject Specific Parameters in postbiotic and placebo group at week 12.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the following, illustrative embodiments and example implementations of the methods and compositions of this invention are presented making reference to the figures. These are intended merely to illustrate the invention without in any way limiting its scope.

Example 1 : Probiotics in non-obesoqenic condition

In a first example, the fat tissue reducing effect of probiotics containing Lactobacillus bacteria belonging to the four Lactobacillus strains L. fermentum K7-Lb1 (DSM 22831), L. fermentum K8-Lb1 (DSM 22832) L. fermentum K11-Lb3 (DSM 22838) and L. plantarum K4-Lb6 (DSM 22830) for use in the method of the invention were prepared and their effectiveness tested in a comparative study using a nematode model, specifically the nematode Cae- norhabditis elegans (in the following C. elegans).

In this study, age-synchronous populations of N2 (wild type) animals from eggs to the L4 larval stage were raised using standard plate culture techniques. During this developmental period, all animals were fed a diet of live E. coll OP50 to standardize their growth. At the L4 stage, animals were loaded into microfluidic devices (chips) and maintained in liquid nematode growth media (NGM) buffer supplemented with live E. coll OP50 at 20 mg/ml at 20°C overnight. After 24 hours on-chip, the worms reached the 1st day of adulthood and had their media exchanged for the conditions listed in the table of FIG. 1.

As can be seen, among these diets, two (groups 1 and 2) involved continued feeding of OP50, with one of them (group 2) being given the known anti-obesity medicament Orlistat® (Tetrahydrolipstatin, a lipase inhibitor) at 6pg/ml while the other served as no-treatment control. The remaining six cohorts were fed probiotic, i.e. live and vital, Lactobacillus bacteria of one of the aforementioned four strains also at a concentration of 20 mg/ml. All experiments were performed at 20(+-1)°C.

Four groups were each fed only a single one of the aforementioned Lacto- bacillus-strains while the other two were used to test the effectiveness of two mixtures containing more than one of the strains. Specifically, one of the two compositions tested (group 7) contained equal parts live bacteria of all three L. fermentum strains of the study (K7-Lb1 , K8-Lb1 and K11-Lb3), while the other (group 8) was given live bacteria of all four Lactobacillus strains in the study (the L. fermentum strains plus L. plantarum K4-Lb6) in equal parts.

The live Lactobacillus bacteria of the strains under study were prepared using the following protocol:

1. Set up a fresh plate of each Lactobacillus strain on MRS agar by streaking an aliquot from a frozen stock using a sterile inoculation loop.

2. Incubate the streaked plates at 30°C for 36 - 40 hours to allow growth of single colonies.

3. For each strain, pick a single colony of the bacteria from the MRS agar plate using a sterile inoculation loop and inoculate 25 ml of MRS broth in a sterile 50 ml falcon tube.

4. Incubate the falcon tube in a shaker at 30°C for 16-18 hours at 250 rpm with the lid slightly loose to allow gas exchange.

5. Centrifuge the falcon tubes at 4000 rpm for 10 minutes at ambient temperature to pellet bacterial cells.

6. Discard the supernatant from each tube and add 10 mL of liquid NGM media to the pellet.

7. Vortex the tubes to resuspend the bacterial cells in the liquid NGM.

8. Centrifuge the falcon tubes at 4000 rpm for 10 minutes at ambient temperature to pellet bacterial cells.

9. Discard the supernatant from each tube and determine the weight of the bacterial pellet by using an empty 50 ml falcon tube to tare the scale.

10. Resuspend the pellet in liquid NGM to a concentration of 40 mg of bacteria per ml of media. This concentration forms a 2X stock of each culture.

Alternatively, the following protocol may be used:

1. Prepare and autoclave 100 ml of MRS broth;

2. Prepare sterile MRS agar plates;

3. Set up a fresh plate of the respective strain on MRS agar by streaking an aliquot from a frozen stock using a sterile; 4. inoculation loop;

5. Incubate the streaked plate at 30°C for 36 - 40 hours to allow for the growth of single colonies;

6. Pick a single colony of the bacteria from the agar plate and inoculate it into 25 ml MRS broth in a sterile 50 ml falcon tube;

7. Incubate the falcon tube in a shaker at 30°C for 16-18 hours at 250 rpm with the lid slightly loose to allow gas exchange.

Other standard techniques for preparing a sufficient solution containing a sufficient number of (live) bacteria could alternatively also be used.

The nematodes of each group were maintained on their respective diet for 48 h before being fixed and stained with Nile Red, a fluorescent dye that binds lipids. Then, fluorescence microscopy was used to image the brightness of the lipid staining in the intestine of the worms, which correlates to the levels of fat tissue in each animal.

This test procedure was carried out a total of four times to increase the statistical significance of the results. The combined results of all four trials are depicted in FIG. 2, which shows a diagram with the histograms of the measured fluorscence intensity wherein each dot represents the measurment result for one individual animal. When combining the results of the trials, the results of each trial were normalized to the mean fluorescence of the no treatment control group 1 of each trial.

As can be seen, the OP50 control (no-treatment control group 1) clearly showed the highest average (mean) and median fluorescence intensity, corresponding to the highest content of fatty tissue in these worms. While the histogram has a rather long tail, the distribution is clearly unimodal with bunching around the median value of 0,9 (the mean is normalized to 1.0 with the mean values of the other groups being normalized by the same factor). Likewise, the histrogram for the OrlistatO-control group 2 also shows a unimodal distribution with clearly lower mean and median values compared to the no-treatment control group.

Interestingly, all the probiotic diets did better than the Orlistat® control group, with all of them showing median and most also having lower mean values. Notably, all the probiotic groups showed greater variation and distributions which are not clearly unimodal, with two- group 3 (L. fermentum K7) and group 6 (L. plantarum K4) - being clearly multimodal. It should be noted that at least for group 6 this is not an effect of the aggregation of the results of the four trials, but that a multimodal histogram was observed even within at least some trials. The overall best performing diets are that of group 4 (pure L. fermentum K8) and the two groups that were fed equal- parts-mixtures of strains, i.e. groups 7 and 8.

Since the basic metabolism of C. elegans is comparable to that of mammals, in particular humans, these results are strong evidence that probiotic compositions containing the tested strains can cause a reduction and/or reduce the buildup of fatty tissue in mammals, specifically humans, as well.

Example 2: Postbiotics in non-obesoqenic conditions

As a second example, we present a study similar to the first one, where, however, the fat tissue reducing effect of postbiotics was studied. The diets that were compared in this study are given in the bottom eight rows of the table of FIG. 1. As a comparison with example 1 , i.e. the first eight rows, shows, the diets for the eight groups involved in this study (groups 9 - 16) were the same as those of the groups of the probiotic-study (groups 1 - 8) with the only difference that all the bacteria, including those for the two control groups (group 9 and 10), were heat-killed before being fed to the nematodes.

As in the study of example 1 , at first, live Lactobacillus bacteria of the strains under study were prepared using one of the protocols detailed above for example 1. The live bacteria thus obtained were inanimated by heat-killing them according to the following protocol:

1. Take the tubes containing the desired strain and incubate them at 75°C for 90 minutes to kill the bacteria.

2. Allow the solutions to cool down to room temperature.

3. Take a small aliquot of the heat-killed culture using a sterile inoculation loop and streak on a fresh MRS agar plate. Incubate the plate at 30°C for 36 - 40 hours to confirm that there is no growth of bacterial colonies.

4. Store the heat-killed culture at 4°C until it is needed for the feeding the worms.

Again, as in example 1, the nematodes of each group were maintained on their respective diet for 48 h before being fixed and stained with Nile Red, after which the brightness of the lipid staining in the intestine of the worms, which correlates to the levels of fatty tissue, was imaged by a fluorescence microscopy system.

This procedure was again carried out a total of four times. The aggregated results of the four trials are depicted in FIG. 3. In the histograms shown there each dot represents the measured fluorescence intensity (on the y- axis) for one individual animal. When combining the results of the four trials, the results of each trial were normalized to the mean fluorescence of the no treatment control (group 9) of each trial.

As can be seen, the OP50 control (no-treatment control group 1) again showed the highest mean and median fluorescence intensity, i.e. the worms fed the standard diet of 20 mg/ml/day of E. coli had the highest amount of measured intestinal fat. The histogram of the OP-50 control (group 9 of FIG. 1) is again unimodal, being bunched around the median value of 0.97 (the mean being again normalized to 1.00) which - within the statistical significance - is notably the same as the one seen in the study of example 1 (group 1 of FIG. 1). Thus, it appears that the buildup of fatty tissue by the tested nematodes is independent of whether they are fed live or heat-killed E. coll.

The only other histogram with a unimodal distribution is that of group 13 (which received a diet of t. fermentum K11 in addition to NGM). All other histograms show signs of more or less pronounced multimodality, with those of groups 12, 14, 15 and 16 being bi- and even trimodal (group 15, which shows three clusters at intensities 0.5, 0.85 and 1.1).

Notably, in this study the histogram for the OrlistatO-control group 10 also shows signs of multimodality with three peaks being just discernible. This not an effect of the aggregation of the four trials: while in one trial the measurement results for all specimen from group 10 where unimodally bunched very closely around the (untypically low) median of 0.5x10⁶, each of the histograms of the other trials individually already showed multimodality.

Comparing the mean and median values, all the probiotic diets performed approximately as well as the Orlistat® control group, with some of them outperforming that control, notably group 12 (on a diet of NGM+L. fermentum K&) and group 16 (which was fed an equal-parts-mixture of all four strains).

This is good evidence that postbiotic compositions can be at least as effective as a state-of-the-art fat-reducing medicament when administered to animals or human beings alone or as part of a composition and in some cases might even show higher effectiveness.

Example 3: Obesogenic conditions

The studies presented in the first two examples above correspond to testing the effectiveness of preventing build-up of fatty tissue when the nematodes receive a normal diet. To also test the effectiveness of the four Lactobacillus strains of this invention (L. fermentum K7-Lb1 , L. fermentum K8-Lb1 , L. fermentum K11-Lb3 and L. plantarum K4-Lb6) as pro- and postbiotics in obesogenic conditions, the diet of the previous studies was supplemented by a 2 % glucose solution. The growth conditions for the study/ies of this example are shown in FIG. 4. As can be seen by comparing that table to the one in FIG. 1 , except for the mentioned supplementing of 2% glucose growth conditions were the same as in the previous two studies.

The study settings were the same also in other respects, with the live bacteria being cultured according to the above-described protocol and, in case of the postbiotics-test, heat-killed according to the protocol given in example 2 before being fed to the nematodes.

However, as is indicated by the first eight rows of the table in FIG. 4 being grayed out, the probiotic study could not be completed. This was because, in the presence of 2% glucose the live Lactobacillus bacteria of the tested strains produced a substance that killed or at least severely harmed the nematodes. In a separate study performed to investigate this acute glucose toxicity effect, changes in pH alone could be ruled out, since the observed lethality of the strains, which ranked K7>K8>K11 >K4, did not correlate with the pH changes they induced, with, e.g., the least lethel strain, K4, causing the greatest change (namely drop) of pH. The details of the studies undertaken to investigate this acute glucose toxicity are given in Example 4 below.

In contrast, the postbiotic trials (groups 10 - 17 in FIG. 4) could be successfully concluded and the results are shown in FIG. 5. It should be noted that a further control group was introduced with group 9, being fed only NGM, serving as non-obesogenic no-treatment control and group 10 as obe- sogenic no-treatment control by being fed NGM+2% glucose but neither medication nor postbiotic bacteria.

As FIG. 5 shows, the nematodes of the obesogenic control group 10 showed a significantly higher intestinal fat content than those of non-obe- sogenic control group 9. The lowest intestinal fat was found in the worms of group 11 , which was the positive control group receiving the Orlistat® medication. Notably, group 13 receiving a diet of heat-killed L. fermentum K8 performed almost as well as the Orlistat® group, with the second best postbiotic being again the mixture of equal parts of all strains (group 17). While not performing as well as the previously mentioned two postbiotics, the histograms of L. plantarum K4 and the equal parts mixture of all L. fer- mentum strains are clearly bimodal and the lower mode of the bimodal histogram by itself outperforms not just L. fermentum K8 but even Orlistat®. This suggests that these postbiotics hold the potential to be even more effective against fatty tissue, if the underlying cause of the bimodality could be uncovered and thereby the conditions which allowed the nematodes of the lower halves of the respective bimodal histograms to show such remarkably low fat accumulation. This is true even more of group 17 (equalparts mixture of all four strains), which is also bimodal with a lower part better than K8 and Orlistat®, but, unlike K4 and the L. fermentum-mixture, an upper part that is small and low enough for the (mean of the) whole histogram to not be far behind (those of) the lipostatic performance of L. fermentum K8.

Lastly, it should also be mentioned that except for the group receiving a diet with (only) L. fermentum K7, all postbiotics tested significantly reduced the measured intestinal fat of the nematodes compared to that of the obe- sogenic no-treatment control group 10 (“OP50 control” in FIG. 3).

In summary, the results suggest that compositions containing the four strains of the invention when administered to animals or humans, could be effective in lowering the buildup or amount of fatty tissue. With the possible exception of L. fermentum K7-Lb1 , this is also true for the strains when administered in isolation, with the best performing strain, L. fermentum K8- Lb1 , coming close to the fat-reduction potential of the known and effective anti-obesity drug Orlistat®. Also, L. plantarum K4-Lb6, the three-strain-mix- ture K7+K8+K11 show potential and should be further studied. This is even truer of the all-strain-mixture of K7+K8+K11+K4, which, however, is already quite effective in and of itself.

Example 4: Acute glucose toxicity study In this study the phenomenon mentioned in example 3, i.e. that in the presence of glucose the four Lactobacillus strains of the invention produce a substance or substances harmful and even lethal to nematodes, was further investigated.

This phenotype was only observed in the probiotic studies, as the postbiot- ics in glucose media did not cause any toxicity. The primary objective of this study was to determine the underlying cause of the observed toxicity in C. elegans when exposed to the probiotics in the presence of 2% glucose. The first approach was to ascertain whether the probiotics were metabolizing glucose in a way that reduced the pH of the media to a level that could induce toxicity in C. elegans. This involved monitoring pH changes in the media after incubating it with the probiotics for periods of 24 and 48 hours. Following the pH monitoring, our second phase focused on investigating the purity of the probiotics, which were manufactured by the commercial supplier CSL according to our instructions.

In a second phase, deep genetic 16S sequencing was conducted to identify the bacterial strains present in the probiotics and detecting any pathogenic contaminants. This step helped to understand the composition of the probiotic cultures and any potential external influences on the observed toxicity. Finally, in a third phase it was examined whether the glucose-induced toxicity was a trait exclusive to the four strains L. fermentum K7-Lb1 , L. fer- mentum K8-Lb1, L. fermentum K11-Lb3 and L. plantarum K4-Lb6 or a broader characteristic shared among various L. fermentum strains. We examined the effects of four other L. fermentum isolates on C. elegans survival in the presence or absence of 2% glucose in their media. In addition, we used the standard bacterial food source E. coll OP50 for the no-treat- ment and glucose no-toxicity controls. The L. fermentum K7 strain, which exhibited the highest glucose-induced toxicity, was used as additional control. For assessing pH levels, the probiotic samples were first suspended in NGM media with and without 2% glucose. These samples were then incubated at a constant temperature of 20°C. The pH of the media was measured at two intervals: once after 24 hours and again at the 48-hour mark.

In terms of bacterial composition analysis, a quantity of 200 mg for each of the probiotic and postbiotic samples (specifically strains K7, K8, K11 , and K4) were prepared. These samples were then dispatched to Azenta Life Sciences for detailed 16S sequencing. The purpose of this sequencing was to meticulously analyze the bacterial profiles present in each of the samples provided.

For non-glucose control populations, age-synchronous N2 (wild type) animals from eggs were raised to the L4 larval stage using standard plate culture techniques. Age-synchronous N2 animals were grown using agar plates supplemented with 2% glucose for the glucose-treated conditions. During this developmental period, all animals were fed a diet of live E. coli OP50 to standardize their growth. At the L4 stage, non-glucose control animals were loaded into our microfluidic devices and maintained in liquid NGM buffer supplemented with live E. coli OP50 at 20 mg/ml at 20°C overnight. Similarly, the glucose-treated diet worms were loaded into chips and maintained in liquid NGM buffer supplemented with 2% glucose and live E. coli OP50 at 20 mg/ml. After 24 hours on-chip, the worms reached the 1st day of adulthood. At this point, we recorded videos of the animals in each chip to determine the population size and get baseline population counts. After obtaining the baseline data, the chips had their media exchanged for the conditions listed in the table of FIG. 6A and were placed in a 20°C incubator for 24 hours. The next day we washed the chips with liquid NGM to stimulate the worms and recorded videos to determine their survival. We then replaced the media with the appropriate condition and returned the chips to the incubator for another 24 hours. Finally, at the 48-hour mark, we washed the chips once again to stimulate the worms and capture the number of animals alive. The growth conditions as well as the strains studied for the acute glucose toxicity trials are given in the table of FIG. 6A. The assay conditions were:

• Single-trial

• Three technical replicates

• Overall population size >100 animals per condition

• Experiments conducted at a temperature of 20°C ± 1 °C (Standard)

• Food concentration: 20 mg/ml for all strains

The results of the pH measurements are shown in FIG 6B. From these it was apparent that the pH changes alone were not a sufficient explanation for the observed impact. The severity of the effect on the C. elegans worms varied among the strains, with K7 showing the greatest impact, followed by K8, K11 , and K4. Interestingly, the K4 strain, despite causing the most significant pH change in the presence of glucose, resulted in the least worm sickness. Conversely, K8, which ranked second in terms of lethality to worms, did not alter the media pH. Notably, the K7 and K11 strains exhibited similar pH changes in the presence of 2% glucose as the OP50 control. These observations suggested that the detrimental effects on worm health are influenced by factors beyond mere pH alterations in the media, indicating the need for further investigation to unravel the underlying mechanisms.

The results of the deep sequencing of the postbiotic samples produced by CSL is shown in FIG. 7A. The results indicated that the predominant taxon in the K7, K8, and K11 samples, both prebiotic and postbiotic, was Lactobacillus. In contrast, the K4 samples exhibited a higher presence of the unclassified taxon, potentially due to outdated classification systems not reflecting the recent Limosilactobacillus taxon category. Additionally, a variety of bacterial strains were present in the samples, with postbiotics showing a greater diversity of bacteria compared to probiotics. However, there was no notable presence of pathogenic strains that could be linked to the observed high lethality in the worms. The L. fermentum strains DSM 20049, DSM 20052, DSM 20055 and DSM 20391 were tested against the most lethal out of the four strains of the invention, L. fermentum K7-Lb1, according to the growth conditions stated in the table of FIG. 6A. Our findings revealed that four of these five strains caused toxicity when exposed to glucose. Notably, two strains (DSM 20052 and DSM 20391) exhibited no toxic effects in the presence of glucose, suggesting a variation in response to glucose among L. fermentum strains. Furthermore, there was one particular strain, DSM 20055, that displayed toxicity independently of glucose exposure, indicating an inherent toxic potential within that strain that is not influenced by glucose.

The study of this example aimed to investigate the toxicity of C. elegans worms when exposed to the four probiotic Lactobacillus strains of the invention in the presence of 2% glucose. pH measurements indicated that the probiotics did not significantly alter the media's pH in a way that could explain the toxicity. Deep sequencing of the probiotics and postbiotics revealed Lactobacillus as the predominant bacterial taxon, with no pathogenic strains identified that could be linked to the high worm mortality. An acute glucose toxicity test across various L. fermentum strains showed diverse responses; some strains induced toxicity in the presence of glucose, while others did not, and one strain exhibited inherent toxicity regardless of glucose. These results suggest a complex interaction between probiotics, glucose, and worm health, indicating that the observed toxicity might be influenced by factors beyond simple pH changes or bacterial composition. It also suggests that C. elegans population sizes and densities can be controlled, notably reduced, by exposure to L. fermentum strains, in some cases even without having to add a further substance in the form of glucose.

Example 5: Comparison of freeze-dried and heat-killed L. fermentum K8- Lb1 postbiotics The goal of this study was to compare the effectiveness of L. fermentum K8-Lb1-postbiotics in preventing fat accumulation caused by an obesogenic diet where the live bacteria had been inanimated in two different ways: either by heat-treatment as described above in the context of example 2 or alternatively by a heat treatment in combination with a freezing treatment as part of a freeze-drying process.

In the following, the preparation of the two types of L. fermentum K8-postbi- otics to be compared is explained in detail.

A sample containing heat-killed K8-Lb1 was produced according to the following protocol also stated in the context of example 1 :

1. Prepare and autoclave 100 ml of MRS broth;

2. Prepare sterile MRS agar plates;

3. Set up a fresh plate of the respective strain on MRS agar by streaking an aliquot from a frozen stock using a sterile;

4. Inoculation loop;

This was followed by concentration of the initial culture to 20 mg/ml, heatkilling and enumeration according to the following steps:

1. Centrifuging the falcon tube containing the overnight culture at 4000 rpm for 10 minutes at ambient temperature to pellet bacterial cells;

2. Discarding the supernatant from the tube and add 10 mL of liquid NGM media to the pellet;

3. Vortexing the tube to resuspend the bacterial cells in the liquid NGM;

4. Centrifuging the falcon tube at 4000 rpm for 10 minutes at ambient temperature to pellet bacterial cells; 5. Discarding the supernatant from the tube and determine the weight of the bacterial pellet by using an empty 50 ml falcon tube to tare the scale;

6. Resuspending the pellet in liquid NGM to a concentration of 20 mg of bacteria per ml of media (this dose is equivalent to the concentration that was fed to the worms in the previous examples);

7. Taking the 20 mg/ml tube and vortex briefly to resuspend the K8 bacteria;

8. Placing the tube in an oven and incubate at 75°C for 90 minutes to kill the bacteria;

9. Allowing the heat-killed solution to cool down to room temperature;

10. Taking a small aliquot of the heat-killed culture using a sterile inoculation loop and streak on a fresh MRS agar plate and Incubate the plate at 30°C for 36 - 40 hours to confirm that there is no growth of colonies;

11. Filling an aliquot 2 ml of the heat-killed bacteria into a 15 ml Falcon tube. Wrap the tube with parafilm to ensure it is well sealed.

12. In addition, to the thus-produced K8 postbiotic, weighing out 2 grams of the CSL-produced K8 postbiotic and placing into a 15 ml Falcon tube.

13. Wrapping the tube with parafilm to ensure it is well sealed.

14. Sending both postbiotic samples to BioForm Solutions using overnight shipping.

In the above protocol, the heat-killing step 8 may involve other temperatures and times, The temperature should, however, be no less than 70°C for no less than 60 min.

Freeze-dried postbiotics were supplied by the company CSL and there were prepared from Lactobacillus batches cultured according to the protocol given above which, in a first devitalistion stage, were then heat-killed. This was followed by a second inanimation stage as part of the following freeze-drying procedure:

1. Filling the prepared live Lactobacillus samples in a glass dish;

2. placing the glass dish on a cooling plate of a freeze-drying chamber at ambient pressure; 3. freezing the bacteria by cooling the dish using the cooling plate to a temperature below 0°C, in particular between -5°C and -1°C, preferably at a rate of less than 0.5°C / min, in particular less than 0.1 °C / min;

4. Performing primary drying of the bacteria by creating a partial vacuum in the freeze-drying chamber by reducing the pressure from ambient (~1 atm = 1013 mbar) to less than 100 mbar, in particular less than 10 mbar at a rate of between 1 mbar/s and 100 mbar/s and exposing the bacteria to the vacuum while providing sufficient heat to the dish to keep its temperature constant and while removing water wapor from the freeze drying chamber using known methods, such as connecting the chamber to a second chamber containing freezer plate or coil that is kept at a very low temperature, until the moisture content does not change further,

5. Performing secondary drying by raising the temperature according to the adsorption isotherms of cell tissue while lowering the pressure in the freeze-drying chamber further to less than 5 mbar, in particular less than 1 mbar until the residual water content of the microbial material is measured to be below 5 %.

The freezing step 3 may also involve cooling the bacteria to other minimum temperatures or at other cooling rates. The cooling rate should be sufficiently small to cause the formation of large ice crystals, which pierce and rupture the cell walls of the bacteria.

Such a slow freezing treatment is also suitable as the first or only inanimation stage when devitalising Lactobacillus strains for producing a postbiotic. Moreover, between step 2 and 3 the standard N₂-O₂-atmosphere of the chamber may be replaced by a pure inert-gas atmosphere such as a pure N₂-, Ne- or Ar-atmosphere. Also, at the end of the freeze-drying procedure, the vacuum may be broken by introducing such an inert-gas atmosphere.

When the growth media in which the bacteria are cultured is not removed before the freeze-drying process, the freeze dried postbiotic may contain significant amounts of sugar, as was the case in the CLS-produced postbi- otics. To ensure these sugars do not lead to fat accumulation, the CLS-pro- duced postbiotic was washed with liquid NGM twice to dissolve the sugars. After the second wash, the CSL-produced postbiotic was resuspended in NGM + 2% glucose to match the produced sample.

After obtaining the two types of postbiotics to be compared, a diet-based method to increase intestinal fat deposition (IFD) in the worms by supplementing the liquid media with 2% glucose (W/V) was used similar to the one detailed in the context of example 2. Here also, this resulted in animals raised on this diet having an increased IFD phenotype akin to a high sugar diet in humans. For non-obesogenic control, age-synchronous populations of N2 (wild type) animals from eggs were raised to the L4 larval stage using standard plate culture techniques. For the obesogenic conditions, age-synchronous N2 animals were raised using agar plates supplemented with 2% glucose. During this developmental period, all animals were fed a diet of live E. coli OP50 to standardize their growth. At the L4 stage, non-obesogenic control animals were loaded into microfluidic devices and maintained in liquid NGM buffer supplemented with live E. coli OP50 at 20 mg/ml at 20°C overnight. Similarly, the obesogenic diet worms were loaded into chips and maintained in liquid NGM buffer supplemented with 2% glucose and live E. coli OP50 at 20 mg/ml. After 24 hours on-chip, the worms reached the 1st day of adulthood and had their media exchanged for the conditions listed in the table of FIG. 7B.

Flow cytometry data was used to ensure that an equivalent number of AFUs/ml were used for each sample. In addition to the two K8 samples, controls included a no-treatment, glucose-induced fat gain, and an OrlistatO-induced fat loss controls. Orlistat® at a concentration of 6 pg/ml has been shown to reduce fat levels in C. elegans. The worms were washed daily with liquid NGM to remove progeny and waste and have their specific diet replenished. Finally, all animals were fed their specific diets for 48 hours, after which they underwent methanol fixation and Nile Red staining. Fat levels in each group were measured by the intensity of the fluorescent signal from the Nile Red staining in the intestine of the worms using epifluorescence microscopy.

The assay conditions were:

• Single-trial

• Three technical replicates

• Overall population size >100 animals per condition

• Experiments conducted at 20°C ± 1°C (Standard)

Two trials were conducted to assess the efficacy of the purely heat-treated K8 postbiotic and the CSL-produced, freeze-dried K8 postbiotic formulations. The second trial was conducted to compensate for the low population size in the first trial. In order to combine the results of the two trials, the results of each trial were normalized to the mean fluorescence of the no treatment control of each trial.

The combined data of both trials are shown in FIG. 8. The analysis affirms that both the purely heat-treated K8 postbiotic and the freeze-dried postbi- otics significantly reduced fat levels compared to the heat killed OP50 glucose diet. Furthermore, they outperformed Orlistat® in fat reduction. Notably, while the difference in the fat-reducing effects of the heat-killed and the freeze-dried postbiotics is (slightly) below the significance threshold, the data of FIG.8 suggests that the freeze-dried L. fermentum K8-postbiotic outperforms its heat-killed counterpart.

In fact, the performance of the freeze-dried postbiotic prepared for the inventor by CSL are on par with the non-obesogenic control (“No Treatment Control” in FIG. 8). This suggests that a freezing or cold treatment when devitalizing the live Lactobacillus bacteria, in particular the strain L. fermentum K8-Lb1 , but likely also the other three strains L. fermentum K7-Lb1 , L. fermentum K11-Lb3 and L. plantarum K4-Lb6, improves their efficacy in reducing fatty tissue of living organisms, in particular animals and humans. Example 6: Study of t. fermentum K8-Lb1 dietary supplement in humans

The applicant performed a placebo-controlled blind study to determine the efficacy of a postbiotic of t. fermentum K8-Lb1 (DSM 22832) in humans.

The postbiotic was administered in the form of a capsule with each capsule containing 33 mg devitilized bacteria corresponding to about 10^A10 cells. The exact content of the capsules is listed in the table below.

Participants

A total of 60 male and female participants aged 18 or over were recruited for this study with 58 of the 60 participants completing the study. Participant demographics are reported in Table 1. All participants satisfied the following inclusion and exclusion criteria.

Inclusion criteria:

• Women & Men,

• Age 18+,

• BMI between 25-32 (average BMI of subjects, shall not exceed 30),

• Generally healthy - don't live with any uncontrolled chronic disease,

• Own a sleep-tracking device (smart watch etc.).

Exclusion criteria:

• Any pre-existing chronic conditions that would prevent participants from adhering to the protocol, including oncological and psychiatric disorders.

• Anyone with known severe allergic reactions. • Women who are pregnant, breastfeeding or attempting to become pregnant.

• Unwilling to follow the study protocol.

• Subjects currently enrolled in another clinical study.

• Subjects having finished another clinical study within the last 4 weeks before inclusion.

• Hypersensitivity, allergy or intolerance against any compound of the test products (e.g. acacia gum).

• Condition after implantation of a cardiac pacemaker or other active implants.

• Sulfonylurea treatment.

• Any disease or condition that might significantly compromise the hepatic (ascites), hematopoietic, renal, endocrine, pulmonary, central nervous, cardiovascular, immunological, dermatological, gastrointestinal or any other body system with the exception of the conditions defined by the inclusion criteria.

• History of or present liver deficiency as defined by Quick < 70%.

• Regular medical treatment, including OTC, which may have an impact on the study aims (e.g. probiotics containing supplements, laxatives, steroids etc.).

• History of hepatitis B, C, HIV.

• Subjects who are scheduled to undergo any diagnostic intervention or hospitalization which may cause protocol deviations.

• Simultaneous study participation by members of the same household;

• History of ascites;

• Any diet to lose body weight;

• Eating disorders or vegan diet;

• Anorexic drugs;

• Present drug abuse or alcoholism

Study Design and Intervention Procedure

This hybrid study required participants to complete questionnaires at home, attend their local Quest Diagnostics Center for blood tests, and complete at-home measurements. Consent forms describing the study process, instructions, evaluation methods, and bill of rights were provided to participants before study onboarding. Following the consent process, participants attended in-person blood draws and completed the Baseline questionnaire, which included answering validated questionnaires about their gastrointestinal health, eating patterns, stress, and anxiety. The validated questionnaires were:

■ Three-Factor Eating Questionnaire (TFEQ)12,

■ Perceived Stress Scale (PSS) 13,

■ Generalized Anxiety Disorder (GAD-7)14.

Participants were randomized into either the test product group or the placebo control and blinded to which group they were in. They were instructed to take one capsule of the test product/placebo daily with a meal for 12 weeks.

In-person visits were required at Baseline and Week 12 to obtain blood draws from participants for biomarker measurement. Virtual assessments were conducted weekly (digital tracking of weight, muscle mass, fat mass, blood pressure, and resting heart rate), and study questionnaires were completed on Week 4, Week 8, and Week 12. At the time of both blood draws, the waist circumference of each participant was also measured.

Data Analysis and Statistics

Data from individual questions about sleep quality, physical exercise, energy, and control of body weight and eating habits were collected using a textual 5-point Likert scale. The textual Likert data was transformed into numerical values from 1 to 5 (the best outcome being 1 or 5 was dependent on the question and is indicated in the relevant figures). Data from the PSS was collected using a textual 5-point Likert scale. The textual Likert data was transformed into numerical values from 1-5. This was then tallied for each participant to obtain a total PSS score. A lower score indicated a reduction in stress symptoms. Data from the GAD-7 questionnaire was collected using a textual 4-point Likert scale. The textual Likert data was trans- formed into numerical values from 1-4. This was then tallied for each participant for a total GAD-7 score. A lower score indicated a reduction in anxiety symptoms. Data from the TFEQ was collected using a textual 4-point Likert scale. The textual Likert data was transformed into numerical values from 1- 4. This was then tallied for each participant. A lower score indicated a reduction in experiencing food control symptoms.

Changes over the weeks relative to the Baseline were compared between the two groups.

The data were checked for normality using the Pearson Test and analyzed based on the normality of the data, utilizing a Welch’s test for normally distributed parametric data and a Mann-Whitney test for non-parametric data. To compare changes in participants’ responses between the two groups, data for each participant at each time point was normalized to their respective Baseline response, and normalized data were compared at each time point. Statistical analyses were performed in GraphPad Prism 10.0, and the significance level was set at p<0.05 and p-values between 0.05 and 0.10 were referred to as a “trend toward significance.”

The alteration of body fat mass (BFM) during intervention, expressed as %(Endline/Baseline value x 100) and as assessed by Withings scale, was defined as the primary parameter. All other parameters were regarded as exploratory.

Compliance with the ITT (Intention-to-treat) principle would necessitate complete follow-up of all randomized subjects for study outcomes. As this cannot be achieved in most studies, a full analysis set (FAS) was planned to be analysed to provide evidence of an effect. This is as complete as possible and as close as possible to the ITT set (FAS) including all randomized individuals. The elimination of individuals was considered to be justified according to the ICH E9 guideline in the following cases:

• Violation of an essential and, before randomization, objectively measurable inclusion criterion.

• Not taking a single dose of the test substance (without knowledge of the assigned test group). • Lack of any dates for the assessment of effectiveness after randomization.

Evaluation in the ITT and PP (Per Protocol) populations served as sensitivity analysis with regard to the primary and secondary parameters. ITT was defined as all individuals randomized and having taken at least one dose of the test products (at V1). Subjects were evaluated in the planned treatment regimen rather than the actual treatment given.

The PP set was defined as all randomized individuals who had no major protocol deviation. Per-protocol analysis was used for checking the robustness of the product effect.

Results

As intended, n = 60 individuals could be randomised and allocated to the Pobiotic and Placebo group, with 30 individuals each. Imputations were only made for the primary parameter BFM in the ITT population. Of the Baseline ITT population, 41 ,67% were male and 58,33% were female. The distribution between races of the total ITT population and the mean age, and the distribution between sex and race in the postbiotic and the placebo group are shown in the Table of FIG. 9A.

Body weight was 84.60±10.42 kg in the postbiotic and 86.72±16.10 in the placebo group. There was no significant difference in body weight, body fat mass, visceral fat mass, muscle mass and vital parameters between the groups at baseline (FIG. 9B).

Starting from baseline values 84.60±10.42 kg in the postbiotic and 86.72±16.10 in the placebo group, body weight decreased by 1.79% in the postbiotic and by 0.12% in the placebo group. The difference was significant (p=0.047, cp. Table of FIG. 10).

Waist circumference decreased by 3.25% in the postbiotic and increased by 1.24% in the placebo group during intervention. The difference between the groups was significant (p=0.034, Table of FIG. 12)

Body fat mass (primary parameter) showed a 1 .85% decrease in the probiotic group and a 0.41% increase in the placebo group, and the difference between these changes was statistically significant (p=0.016; Tables of FIG. 11A). This significant difference was also seen in the ITT (p=0.011) and the PP population (p=0.027, T ables of FIGs 11 B and 11 C)

Visceral fat mass tended (p=0.053) to be decreased in the postbiotic group compared to the placebo group (Table in FIG. 13).

Muscle mass tended (p=0.062) to be increased in the postbiotic compared to the placebo group (Table in Fig. 14).

Vital parameters showed no significant difference between Postbiotic and Placebo group by the 12-week intervention (Tables in FIGs. 15A - 15C).

ALT, AST and yGT tended to be decreased in the postbiotic group compared to the placebo group during intervention (Table of FIG. 16). Highly sensitive CRP as biomarker for inflammation showed a nominal, but not significant decrease in the postbiotic compared to the placebo group (Table of Fig. 17). The reduction of HbA1 c was close to a trend in the postbiotic group compared to the placebo group (p=0.1486, Table of FIG. 18A). In the subpopulation with normal glucose levels (< 100 mg/dl), eAG differed significantly (p<0.046) between the groups (cp. Table of FIG. 18B)

Serum lipids did not significantly differ between postbiotic and placebo group (Table in FIG. 19).

As part of the overall questionnaire, participants were asked to complete three validated questionnaires, including the Perceived Stress Scale, Generalized Anxiety Disorder-7, and the Three Factor Eating Questionnaire, which allowed for the analysis of their stress and anxiety levels, as well as their relationship with food. The answers were transformed using a textual Likert scale, and the sum of each participant’s answers was calculated to find an overall score for each validated questionnaire. For each questionnaire, lower scores represented better outcomes, higher scores worse outcomes. The alterations during intervention did not significantly differ between the groups (Table in FIG 20A).

Participants were asked to answer individual questions at the end of Week 4, Week 8, and Week 12 (FIGs. 20B - 20D9. These questions measured control over body weight, eating habits, physical exercise, several sleep parameters, overall mood, energy, and the frequency and intensity of gastrointestinal symptoms. At endline assessment, the postbiotic group tended (p=0.066) to feel in higher control of my body weight (Table in FIG. 20D). At Week 12, for the frequency / intensity of poor concentration / focus, there was a 12.18% decrease in the test product group and a 22.99% increase in the placebo group - the difference between these changes was statistically significant (p=0.014; Table in Fig. 20D). This suggests an improvement in the test product group and a worsening in the placebo group. The feeling of nausea tended to be improved (p=0.055, Table in FIG. 20D).

Discussion

This study investigated the efficacy of the test product in improving various health-related parameters over 12 weeks. The investigation covered a comprehensive array of measures, including quantification of blood biomarkers, validated questionnaires assessing stress, anxiety, and eating behaviours, specific health-related questions targeting body weight control and other lifestyle factors, and objective measurements such as body composition and vital signs.

The drop-out rate was acceptable with 4 to 12% for the objective data and evenly distributed between the postbiotic and placebo group. This makes a bias based on drop-out rate unlikely.

After 12-week intervention body fat mass (primary parameter) was significantly (p=0.016) reduced in the postbiotic group (98.15±3.32% of baseline) compared to the placebo group (100.41 ±3.39%). In line with this, body weight (p=0.047) and waist circumference (p=0.034) were significantly reduced and visceral fat tended to be reduced (p=0.053). Again in line with this, the postbiotic group tended (p=0.066) to feel in higher control of my body weight. In spite of the decrease in body weight and body fat, muscle mass tended (p=0.062) to be increased. ALT (p=0.098), AST (p=0.072) and GGT (p=0.086) tended to be reduced indicating a reduction in liver steatosis. In line with this, HbA1 c (p=0.149) and eAG were close to a trend for reduction by the postbiotic product compared to the placebo and eAG even differed significantly between the groups in individuals with normal fasting glucose levels (< 100 mg/dl) before intervention. The ability to concentrate, which can be impaired in overweight, was significantly (p=0.014) improved.

The significant reduction of BFM was more than expected in this exploratory study with data from only N=58 individuals. Based on the results of a DB-RCT with the pertinent viable strains (Laue et aL, 202315) and assuming a 30% higher effect size for the postbiotic versus the probiotic product (based on data in C. elegans), a sample size of N=112 was calculated for BFM aiming at a probability of 80% for attaining a significant result (power 0.8).

Conclusion

The 12-week study of this example explored the effects of a postbiotic formulation according to present invention on weight management and metabolic health. After 12-week intervention body weight, waist circumference, body fat mass and the ability to concentrate was significantly improved in the postbiotic group compared to the placebo group. According to this, visceral fat, ALT, AST and yGT tended to be reduced and a reduction of HbA1 c and eAG was observed, which was significant in case of eAG in individuals with normal glucose levels before intervention.

Although the embodiments of the present disclosure have been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the present disclosure.

Claims

1. A pharmaceutical or dietary composition for preventing buildup or reducing the amount of fatty tissue and/or body weight and/or supporting the buildup of muscle mass and/or improving muscle tone in living organisms, specifically human beings and/or animals, such as pets and/or livestock, and/or improving the ability to concentrate in humans, the composition containing live or inanimated strains of Lactobacillus bacteria, wherein the Lactobacillus bacteria are selected from one or more of the four following strains:

- L. plantarum K4-Lb6 (DSM 22830),

- L. fermentum K7-Lb1 (DSM 22831),

L. fermentum K8-Lb1 (DSM 22832), and/or

- L. fermentum K11 -Lb3 (DSM 22838).

2. The composition of claim 1 , wherein it comprises a mixture of more than one of the four Lactobacillus strains, in particular a mixture of equal parts of only the three L. fermentum strains or a mixture of equal parts of all four Lactobacillus strains.

3. The composition of one of the preceding claims, wherein it is a probiotic, i.e. contains live and vital bacteria.

4. The composition of claim 1 or 2, wherein the composition is a postbiotic, i.e. comprises inanimated bacteria.

5. The composition of the preceding claim, wherein the bacteria it contains have been inanimated by a temperature treatment and/or by an irradiation and/or by a high pressure treatment.

6. The composition of claims 4 or 5, wherein all or substantially all, i.e. more than 95 %, preferably more than 99 % of the bacteria have been inanimated, and/or wherein the composition contains less than 5000, preferably less than 3000, most preferably less than 1000 live bacteria per gramm of composition.

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7. The composition of one of the preceding claims, wherein a concentration of live and/or inanimated bacteria together is between 10⁶ and 10¹² cells/g, in particular between 10⁸ and 5*10¹¹ cells/g, preferably between 10⁹ and 5*10¹¹ cells/g, most preferably 3*10^A11 cells/g.

8. The composition of one of the preceding claims, comprising other substances which increase the effectiveness of the composition, in particular one or more of prebiotics such as dietary fibres, specifically natural fibres, are included, minerals salts, in particular sodium or potassium, phosphorous, iron, zinc, magnesium, manganese, and/or vitamins, in particular A, B1 , B2, B6, B12, niacin, C, D3, E and/or folic acid.

9. The composition of one of the preceding claims, further comprising as a carrier

■ a food substance such as milk, whey, yoghurt and/or a fruit and/or vegetable preparation, protein powder, a cereal bar or a candy bar, chocolate or cheese or another dairy product or a gummy candy, or

■ a beverage substance such as water, tea, coffee, fruit juice, sports drinks, energy drinks, carbonated beverages, or alcoholic beverages, or

■ an animal or pet feed substance, which may be further comprise one or more of hay, silage, corn, soy, sorghum, oats, barley, meat, cartilage and/or fish.

10. A method for making a composition according to one of the preceding claims comprising a. culturing live Lactobacillus bacteria of one or more desired strains among the strains of claim 1 and in particular subsequently separating the bacteria from the growth medium, b. optionally submitting the cultured bacteria to an inanimation treatment, c. optionally, mixing the bacteria with further substances such as prebiotics, mineral salts or vitamins, d. optionally, adding the bacteria to a carrier food substance such as a fruit and/or vegetable preparation, milk, whey or yoghurt, protein powder, a gummy candy, a cereal bar or a candy bar, a chocolate bar, a cookie, a cake, a sausage, a cheese or another dairy product and/or an animal feed substance, further comprising one or more of hay, corn, soy, sorghum, oats, barley, meat, cartilage and/or fish.

11. The method of the preceding claim, whereby the culturing comprises a. Setting up a fresh plate of each desired Lactobacillus strain on MRS agar by streaking an aliquot from a frozen stock using a sterile inoculation loop; b. Incubating the streaked plates at 30°C for between 36 h and 40 h to allow growth of single colonies; c. For each strain, picking one or more of the single colonies of the bacteria from the respective plate using a sterile inoculation loop and inoculating a desired volume of MRS broth, for instance 25 ml, in a sterile falcon tube of sufficient size, preferably around twice the volume of the MRS broth, for instance 50 ml, d. Incubating the falcon tubes prepared in step c in a shaker at 30°C for between 16 and 18 h at 250 rpm, ensuring the possibility of gas exchange with the ambient air, in particular in case of a lidded shaker by leaving with the lid slightly open, e. Centrifuging the falcon tubes at 4000 rpm for 10 minutes at ambient temperature to pellet the bacterial cells; f. Discarding the supernatant from each of the falcon tubes and adding an amount of liquid Nematode Growth Medium corresponding to between 1/4 and ¹/₂ the volume of the discarded supernatant of the respective falcon tube, for instance 10 ml; g. Vortexing the falcon tubes to resuspend the bacterial cells in the liquid Nematode Growth Medium; h. Centrifuging the falcon tubes at 4000 rpm for 10 minutes to pellet the bacterial cells; i. Discarding the supernatant from each falcon tube and determining the weight of each of the bacterial pellets, for instance by weighing the falcon tube on a scale after taring the scale with an empty falcon tube of the same type.

12. The method according to one of the preceding method-claims, wherein the Lactobacillus bacteria are inanimated by an inanimation treatment comprising one or more stages.

13. The method of the preceding claim, where one of the one or more stages of the inanimation is one of: a temperature treatment, in particular one or a combination of a heat treatment and or freezing treatment, and/or an irradiation treatment, such as a UV-irradiation treatment, and / or a pressure treatment.

14. The method of one or both of the two preceding claims, wherein the inanimation treatment includes a heat treatment comprising heating the live or pre-inanimated Lactobacillus bacteria to a temperature no less than 70°C for no less than 60 minutes, in particular to a temperature of 75°C for 90 minutes.

15. The method of one or all of the three preceding claims, wherein the inanimation treatment includes a freezing treatment, in particular comprising cooling, once or multiple times after intermediate warming, the live or preinanimated Lactobacillus bacteria to a temperature of less than 0°C, in particular less than -5°C, for instance between -5°C and -30°C at a cooling rate of no more than 0,5°C/min, in particular no more than 0,1 °C/min.

16. The method of the preceding claim wherein the freezing treatment is part of a freeze-drying process.

17. The method of any of the preceding claims 12 - 16, wherein at least one stage of the inanimation treatment is a pressure treatment involving subjecting the bacteria to a pressure of at least 200 MPa for at least 5 min, specifically at least 400 MPa for at least 10 min, more specifically between 400 and 800 MPa for between 15 and 30 min.

18. The method of one or all of claims 12 - 17, wherein the inanimation treatment includes two or more stages, with one of the two or more stages being a heat treatment, in particular one according to claim 11 , and another one of the two or more stages being a pressure treatment or a freezing treatment, in particular one according to claim 14 wherein the heat treatment may come before the freezing treatment or vice versa, the freezing treatment before the heat treatment.

19. A method for preventing buildup or reducing the amount of fatty tissue and/or reducing body weight and/or supporting the buildup of muscle mass and/or improving muscle tone in a living organism, in particular a human being or an animal, such as a pet or livestock, and/or improving the ability to concentrate in humans, the method involving administering the living organism a composition according to one of the above composition-claims.

20. The method of the preceding claim wherein the composition is administered orally and/or anally and/or topically.

21. The method of one of the two preceding method-claims, wherein a composition according to claims 6 and 7 is administered.

22. The method of one of the two preceding method-claims, wherein the composition contains live or inanimated bacteria of only the strain L. fermen- tum K8-Lb1 or of the three L. fermentum strains K8-Lb1 , K7-Lb1 and K11- Ib3 to equal parts.

23. , The method of one of the two preceding method-claims, wherein the composition is a probiotic administered at a dose of at least 40 mg per day corre- spending to 6°10^A9 CFU per day, or a postbiotic and is administered at a dose of at least 20 mg or at least 30 mg, specifically 33 mg, per day, corresponding to at least 6*10^A9 or at least 9*10^A9, specifically about 10^A10, cells per day.

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