Improved fermentation process using a steel tank whose inner walls are coated with gold nanoparticles

Abstract

Fermentation process characterized in that it uses a stainless steel tank whose inner walls are coated with a layer of gold nanoparticles with a thickness between 0.3 and 20 microns in order to improve the quantity, diversity, stability and efficiency of the metabolites from this fermentation.

Description

Title of the invention: Improved fermentation process using a steel tank whose inner walls are coated with gold nanoparticles

[0001] The present invention relates to a fermentation process for improving the quantity, diversity, stability, and efficacy of the metabolites produced, improving their molecular structure, and increasing proteolytic stability. It is characterized in that the process uses a stainless steel tank whose inner walls are coated with a layer of gold nanoparticles with a thickness between 0.3 and 20 microns. The present invention also relates to a cosmetic, food, or pharmaceutical composition containing the metabolites produced by the fermentation process for their properties in modulating the diversity of the microbiota.

[0002] The microbiota is known to be the collection of microorganisms, including bacteria, yeasts, fungi, and viruses, living in a specific environment called the microbiome in a host. The microbiota is located in the intestines, skin, urogenital tract, and lungs. The most important are the intestinal and skin microbiotas. Approximately 100 trillion microorganisms exist in the intestines alone. In total, microbiotas represent more than 1,000 different species and weigh approximately 2 kg. Microbiotas are acquired at birth and remain largely unchanged throughout life. However, changes in diet or lifestyle, pollution, and antibiotic use can alter the composition of the microbiota and cause an imbalance called dysbiosis, which can have adverse effects on the host.

[0003] The microbiota functions on the principle of mutualism, that is, cooperation between different organisms. This mutualism occurs not only between microorganisms, but there is also a symbiotic relationship between the microbiota and the host. First, the microbiota digests foods that the body is unable to digest on its own, such as the fiber contained in certain fruits and vegetables. The microbiota is also capable of modulating the body's health by modulating the immune system and by modulating gene expression in cells. Studies have shown that many diseases result from an imbalance in the microbiota, such as obesity, diabetes, depression (Simon Carding, Kristin Verbeke, Daniel T. Vipond, Bernard M. Corfe, and Lauren J. Owen. Dysbiosis of the gut microbiota in disease. Microbial ecology in health and disease.se) and even autism.

[0004] Microbiota bacteria communicate with host cells or with each other through metabolites produced by these bacteria. These metabolites are absorbed in the intestine through the intestinal wall by passive diffusion or by transporters. The absorbed metabolites are transported via the bloodstream to cells where they will modify gene expression. These metabolites also serve as a means of communication between different strains of bacteria, through a mechanism called quorum detection (a bacterial detection system that allows bacteria to react to a certain cell density), enabling changes in the composition and functionality of the microbiota.

[0005] In particular, bacteria produce antibacterial peptides (AMPs) called bacteriocins. These AMPs are small cationic peptides that inhibit the growth of a certain group of bacterial strains. The production of these bacteriocins by the microbiota helps protect the host against infection by pathogenic bacteria, protozoa, viruses, and fungi. (Loris Riccardo Lopetuso, Maria Ernestina Giorgio, Angela Saviano, Franco Scaldaferri, Antonio Gasbarrini, and Giovanni Cammarota. Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases. International Journal of Molecular Sciences).

[0006] Modulating microbiomes and their communication, both between microorganisms and with the host, is a promising solution for preventing or resolving diseases. Solutions for modulating the microbiota and its impact already exist on the market. Consuming probiotics (live bacteria) introduces bacteria into the microbiota, which will transiently modulate its balance. Fermenting bio-based ingredients by lactic acid bacteria outside the body also produces various types of metabolites: proteins, amino acids, fatty acids, polyphenols, minerals, organic acids, peptides, amides, polysaccharides, and vitamins. Consuming these fermented products thus provides metabolites that will either be directly absorbed by the body in the intestine or will act on microbiota modulation through quorum sensing.

[0007] The fermentation of bio-based ingredients has existed for a long time; however, its yield and the control of the metabolites produced are limited. Therefore, more industrial fermentation methods have been developed. These allow for manufacturing in a controlled environment, with a specific temperature, pressure, bacterial mixture, and substrate. This regulated production makes it possible to control the number and type of metabolites produced in order to produce a solution (fermented product) tailored to each problem and disease.

[0008] In particular, the production of antibacterial peptides is of particular interest, as it makes it possible to combat infections that would be harmful to human health. For example, studies show that infection with the bacterium Klebsiella pneumoniae is the cause of hepatic steatosis (Jing Yuan et al. Fatty Liver Disease Caused by High-Alcohol-Producing Klebsiella pneumoniae. Cell Metabolism).

[0009] Lactic fermentation processes already exist (FR3062396 Al), however, productivity is limited and the metabolites have low proteolytic stability, i.e., they can be easily digested by intestinal enzymes after ingestion.

[0010] It was while studying the improvement of the viability and yield of lactic acid bacteria in different containers that the applicant discovered quite unexpectedly that the yield of fermentation as well as the effectiveness of the bacteriocins produced could be improved by the composition and nature of the container itself.

[0011] The applicant describes below a fermentation process where the fermentation is carried out in a stainless steel fermentation tank whose inner and / or outer walls are coated with a layer of gold nanoparticles.

[0012] To optimize fermentation, the cylindrical tank is made of stainless steel and has a capacity of 1 to 200 liters; a circumference of 8 to 100 centimeters and a height of 15 to 150 centimeters. The tank preferably has an opening at the top for filling. The tank may also have an opening at the base for draining. The tank may be equipped with wheels to facilitate movement. The tank may be equipped with an agitator, as well as a pressure gauge, a thermometer, a viewing window, and a tube connected to the sterilization machine.

[0013] The walls coated with gold nanoparticles can be either the inner walls, the outer walls, or both. The layer of gold nanoparticles is deposited by electrolysis, by immersing the tank in an electrolytic bath containing a solution of gold nanoparticles. The tank is attached to two electrodes; when an electric current passes between these two electrodes, the gold nanoparticles are deposited evenly on the surface of the tank.

[0014] In order to adapt the fermentation process to the desired efficiency of the finished product, various alloys can be used for coating the tank walls. The layer of gold nanoparticles can be an alloy of gold and another precious metal included in the group: silver, copper, cobalt, nickel, aluminum, indium. The proportion of gold is between 10 and 100% pure gold.

[0015] The fermentation process is carried out in said tank in several stages. The ingredients to be fermented used in the fermentation process are fruits and vegetables, plants, algae, and certain compounds of animal origin. Bio-based ingredients may include, for example, but are not limited to: soybeans, olive leaves and fruit, rosemary, sage, thyme, green, black, and white tea, melon, watermelon, pomegranate fruit, fig fruit, leaves, flowers, and peel, and Cherry, seaweed, and sugarcane are among the materials selected for their pesticide-free cultivation using biodynamic methods. The choice of fermentation substrate influences the final metabolite composition and concentration. Depending on the desired effect of the final product, the substrate is chosen for its ability to produce the specific metabolites needed for a particular effect. The resulting fermentation products are used in cosmetic and pharmaceutical formulations. The bio-based substrates are either sterilized or not beforehand to eliminate any bacteria already present on their surface.

[0016] The bio-based ingredients are placed in the tank described above, with or without a substrate, of the polysaccharide type and preferably algal polysaccharides, and in the presence of an association of lactobacilli. The lactobacilli may be, for example, but not limited to: Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus jensenii, Lactobacillus paracasei subsp. paracasei, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helbeticus, Lactococcus lactis, Lactobacillus casei subsp. casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. delbrueckii, Enterococcus faecium and Steptcoccus thermophiles.This complex of one or more lactobacilli can be added manually or be naturally present on the bio-based substrate.

[0017] The bio-based ingredients, the substrate, and the lactobacilli complex are fermented at a temperature between 30 and 40°C for a period of between 1 and 120 hours. The fermentation process described above produces a variety of metabolites, such as peptides, polysaccharides, amino acids, and vitamins.

[0018] Fermentation in a tank whose walls are coated with a layer of gold nanoparticles increases the proteolytic stability of the peptides produced during fermentation. Indeed, during fermentation, the peptides interact with the structure of the gold nanoparticles present on the tank walls. Peptides have a flexible conformation, and the interaction with the nanoparticles on the tank walls affects the secondary and tertiary structure of peptides covalently linked by cyclization or disulfide bonds. This modification of the peptide structure confers proteolytic stability. Peptides are chains comprising between 2 and 50 amino acids linked together by peptide bonds. These bonds can be broken by peptide enzymes, particularly during digestion. Ingested proteins and peptides play an important role in human health.For example, the tripeptide (peptide composed of 3 amino acids) GSH has significant antioxidant activity. However, the GSH peptide is subject to degradation. Proteolytic degradation occurs when peptide bonds are cleaved by an enzyme in the intestine, leading to a degradation of the peptide structure and a loss of function. Preserving the peptide structure is therefore important for maintaining their activity. Tests have shown that metabolites produced by the process described in the present invention undergo less degradation. This demonstrates that the present invention allows peptides produced during fermentation to improve their structure, thereby preserving their structure and thus their activity, even if ingested. In particular, this allows antibacterial peptides to improve their efficacy. Peptides produced by the process described in the present invention have demonstrated a greater antibacterial effect against Candida albicans than metabolites produced by conventional fermentations.

[0019] Having already been fermented by a complex of bacteria, the metabolites do not undergo further degradation in the intestine and are absorbed directly through the intestinal wall into the bloodstream and are distributed to the cells where they can exert their effects.

[0020] In response to injury, irritation, infection, or harmful stimuli, the body develops an inflammatory reaction, allowing it to clear damaged tissues, eliminate pathogens, and activate healing. Inflammation results in the mobilization of immune system cells that produce various substances called inflammatory mediators, such as histamines, cytokines, and chemokines.

[0021] However, sometimes the inflammatory response occurs when it should not, for example, due to chronic stress, obesity, autoimmune diseases, or the inflammatory response does not subside even after the damaged tissue or infection has been removed. In this situation, the inflammation persists and can damage healthy tissues, thus contributing to a wide range of chronic diseases. Examples of these include metabolic syndrome, type 2 diabetes, heart disease, obesity, arthritis, depression, Alzheimer's disease, inflammatory bowel disease, psoriasis, eczema, atopic dermatitis, and acne.The metabolites produced by the process described above have the capacity to modulate the inflammatory response, notably by modulating the gene expression of inflammatory mediators, and in particular the gene expression of cytokines and specialized resolution mediators that enable the resolution of inflammation and the restoration of homeostasis. Microbiota have a significant effect on the modulation of inflammation. Indeed, their metabolites are capable of modulating the gene expression of certain mediators. For example, butyrate produced by the gut microbiota inhibits the expression of pro-inflammatory cytokines via the inhibition of [missing information]. NFkB activation. The metabolites produced by the process described in the present invention act as replacements for metabolites produced by a healthy and balanced microbiota and allow modulation of inflammatory gene expression.

[0022] The structure of peptides not only improves their stability but also their functionality. For example, star-shaped polypeptides are capable of more efficient complexation with nucleic acids compared to linear polypeptides, thereby enhancing their anti-inflammatory effects. Atomic force microscopy analysis of the structure of polylysine peptides present in the metabolites produced during the process described in this invention demonstrated the presence of numerous star-shaped structures that are not found in conventional fermentation. Star-shaped polylysine peptides demonstrated a superior anti-inflammatory effect compared to linear polylysine peptides. This clearly shows that the present invention makes it possible to enhance the anti-inflammatory effect of metabolites by producing star-shaped polylysine bonds.

[0023] The present invention also makes it possible to improve fermentation yield by increasing the quantity and diversity of metabolites produced during fermentation. Lactobacilli are sensitive to a variety of parameters that can affect their growth and fermentation efficiency. The best-known of these parameters are temperature and substrate concentration. However, other parameters can affect bacteria, such as vibrations and electromagnetic fields. Gold particles have insulating properties and are capable of reflecting radiation and electromagnetic waves. The layer of gold nanoparticles is able to protect the contents of the tank, and in particular the bacteria, from electromagnetic fields. Tests have shown that the final quantities of certain metabolites, including citrulline, GABA, and leucine, increased by 7.5%, 10.9%, and 18%, respectively.2% respectively with the process described in the present invention, compared with a conventional process. Analysis by a mass spectrometry method demonstrated the presence of metabolites in the finished product fermented with the process described in the present invention, which were not detected during conventional fermentation. This clearly shows that the process described in the present invention makes it possible to increase the quantity and diversity of the metabolites produced.

[0024] For the sake of clarity in the explanations that follow, we decide to call Goldbiota a cosmetic, food, pharmaceutical composition containing the metabolites produced by the fermentation process described above.

[0025] Goldbiota compositions have several cosmetic, pharmaceutical, or food applications. Goldbiota metabolites act on the composition of the intestinal and skin microbiota, modulate inflammation, and help maintain cellular homeostasis.

[0026] The bacteria of the microbiota communicate with each other by secreting metabolites that are used as molecular signals. These metabolites are detected by receptors located on the outer membrane of the bacteria. This mechanism, called quorum sensing, allows bacteria to monitor their environment, detect other bacteria nearby, and modulate their activity accordingly, such as their growth or peptide production. The metabolites produced during the fermentation process described above reproduce this quorum sensing mechanism. The resulting effects are a modulation of the number and diversity of different bacterial strains. A balanced microbiota, comprising a significant diversity of bacterial strains, is important for the good health of the organism.

[0027] The metabolites contained in Goldbiota mimic the bacterial communication system and can therefore modulate the composition of the microbiota, leading to an improvement in the individual's overall health. Individuals consuming Goldbiota have thus experienced an improvement in dysbiosis symptoms, such as stomach aches, constipation, or diarrhea, as well as an improvement in skin quality, a reduction in acne, and an improvement in depressive symptoms. This demonstrates that the Goldbiota compositions produced during the process described in the present invention improve the diversity and quantity of the microbiota, resulting in an improvement in the individual's overall health.

[0028] The final product obtained after filtration is in liquid form. It can be either dried or used as is. It can be used directly on its own for high-concentration applications or mixed with other liquids or additives in single-phase or multi-phase products, sometimes in the presence of surfactants. It can also be used to create mixtures containing various types of emulsions, depending on the steric and electromagnetic properties of the surfactant(s) in solution. If the final product is dried, a powder is obtained. This powder can be used either as is or mixed into dry formulations. These properties allow Goldbiota-type compositions to be formulated in a wide variety of ways, depending on the desired use.The cosmetic or pharmaceutical composition containing Goldbiota is presented as an oil-in-water or water-in-oil emulsion, multiple emulsion, microemulsion, nanoemulsion, twin-phase emulsion, PIT emulsion, stable dispersion of two immiscible phases by means of a gelling agent, stable dispersion of two immiscible phases by means of one or more surfactants, liquid, aqueous gel, oily gel, hydro-alcoholic gel, oily phase, suspension, foaming or non-foaming solution, gel, lyophilized emulsion or powder.

[0029] Depending on the chosen format and the properties of the final formulation, Goldbiota Goldbiota can be included in various cosmetic and pharmaceutical compositions for the uses mentioned above. It is available in the form of liquids, foams, pastes, ready-to-use drinks, lotions, emulsions, oils, gels, syrups, solids, powders, masks, sticks, tablets, capsules, sprays, aerosols, softgels, jellies, syrups, creams, patches, shower gels, and shampoos.

[0030] These compositions are either taken orally or applied topically. Furthermore, the oral and topical routes can be used jointly, successively, or separately, depending on the desired use.

[0031] The applicant will now provide, by way of example, studies on compositions according to the subject matter of the invention, as well as examples of formulations, without these examples being limiting. For the sake of clarity in the explanations that follow, we have decided to call Goldbiota a cosmetic or pharmaceutical composition containing metabolites produced by the process described herein, resulting from the fermentation of one or more bio-based ingredients in a fermentation tank whose walls are coated with a layer of gold nanoparticles.

[0032] Example 1: Anti-inflammatory activity

[0033] A study was conducted to demonstrate that supplementation with Goldbiota metabolites derived from the fermentation of black soybeans inhibits inflammation and that the fermentation process described in the present invention enhances the anti-inflammatory effect of the metabolites produced during this fermentation process. This study was conducted with 60 healthy volunteers of unisex age, ranging from 40 to 60 years. The participants were randomly assigned to three groups of 20 people. The first group (called the Goldbiota group) ingested a single 100 mg dose of Goldbiota, the second group (called the SNF group) ingested a 100 mg dose of conventionally fermented black soybeans, and the third group (called the Placebo group) ingested a placebo daily for 30 days.Participants underwent blood sampling at the beginning and end of the study to analyze the blood concentration of the inflammatory markers IL-6 and CRP. The results for groups 1, 2 and 3 are presented in the table below (results are shown as mean ± standard deviation):

[0034] [Tables 1] Goldbiota Group SNF Group Placebo Group IL-6 (pg / L) Day 0 Day 30 7.45 + 1.03 5.58 + 1.10 7.93 + 0.95 6.72 + 0.91 7.81 + 0.90 7.74 + 0.85 hs-CRP (mg / L) Day 0 Day 30 5.56 + 0.72 4.19 + 0.65 5.78 + 0.90 4.73 + 0.76 5.62 + 0.92 5.50 + 0.95

[0035] It is observed that blood levels of IL-6 and CRP decrease significantly at the end of the study. It is also observed that in the Goldbiota group, the decrease in inflammatory markers is greater than in the SNF group, which consumed conventionally fermented soy. For example, the inflammation marker IL-6 decreased by 25.10% in the Goldbiota group and by only 15.51% in the SNF group. It can be concluded that, given that the bio-based ingredient is the same, the fermentation process of the present invention has increased the anti-inflammatory capacity of the metabolites.

[0036] Example 2: Resistance to peptide degradation

[0037] The peptides contained in Goldbiota were isolated by UV chromatography, as were peptides from conventional fermentation (called SNF). In this test, the isolated peptides are placed in the presence of serum, which naturally contains peptidase enzymes that cleave the peptide bonds.

[0038] The peptides are mixed with serum to a final concentration of 40 pmol / L. Aliquots are taken after 0 and 60 minutes. The peptides are analyzed by RP-HPLC in the presence of 0.1% formic acid and detected by absorbance at 214 nm. Their quantities are quantified by the peak area compared to the initial peak (0 minutes). All tests were performed in triplicate. The relative peak areas compared to time 0 are presented in the table below:

[0039] [Tables2] Goldbiota SNF Relative peak area (%) 75% 42%

[0040] It is observed that the Goldbiota peptides are present in greater quantity after 60 minutes than the peptides isolated from SNF. The Goldbiota peptides have therefore been less degraded. It can thus be concluded that the fermentation process described in the present invention increases the stability of the peptides compared to peptide degradation.

[0041] Example 3: Anti-redness facial lotion

[0042] - Water........................................................86.2% - Carbomer.........................................................5% - Glycerin............................................................3% - Cetyl alcohol..........................................2% - Glyceryl Stearate SE........................................1.5% - Triethanolamine..........................................0.9% - Stearic acid..................................................0.8% - Goldbiota..........................................................0.5% - Propanediol..................................................0.1%

[0043] This aqueous lotion, applied twice a day, helps to calm redness and irritation due to inflammation.

[0044] Example 4: Weight loss capsules

[0045] - Goldbiota......................................................50 mg

[0046] - Microcrystalline cellulose......................................68 mg

[0047] - Calcium stearate............................................2.0 mg

[0048] - Vitamin E (tocopherol 50%)....................................60 mg

[0049] This food supplement comes in the form of capsules to be taken once a day, two capsules. Users have noticed weight loss and increased energy after one month of use.

Claims

Demands

1. A process for fermenting bio-based ingredients in association with one or more lactobacilli, characterized in that it uses a stainless steel tank whose inner walls are coated with a layer of gold nanoparticles of thickness between 0.3 and 20 microns in order to improve the quantity, diversity, stability and efficiency of the metabolites from this fermentation, in which the bio-based ingredients and the association of lactobacilli are placed in said tank and fermented between 30 and 40°C for a period of between 1 hour and 120 hours; in which the association of one or more lactobacilli is taken from the following list: Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus jensenii, Lactobacillus paracasei subsp.paracasei, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helbeticus, Lactococcus lactis, Lactobacillus casei subsp. casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. delbrueckii, Enterococcus faecium and Stepcoccus thermophiles; and in which the bio-sourced ingredients are taken from the following list: soybeans, olive leaves and fruits, rosemary, sage, thyme, green, black and white tea, melon, watermelon, pomegranate fruit, fig fruits, leaves, flowers and peels, cherry, algae, sugar cane.

2. Fermentation process according to claim 1, characterized in that the gold nanoparticle layer is an alloy of gold and another metal included in the group: silver, copper, cobalt, nickel, aluminum, and indium.

3. Use of a tank whose walls are coated with a layer of gold nanoparticles with a thickness between 0.3 and 20 microns in a fermentation process to improve the molecular structure and proteolytic stability of peptides produced during fermentation, to improve the efficacy of antibacterial peptides produced during fermentation, to increase the quantity and diversity of metabolites produced during fermentation, and / or to increase the anti-inflammatory effect of polylysine peptides produced by the process by forming poly(1-lysine) star-shaped bonds.

4. Cosmetic, food or pharmaceutical composition, characterized in that it contains the metabolites from the fermentation process according to claims 1 to 2.

5. Cosmetic, food or pharmaceutical composition according to claim 4, characterized in that it is in the form of an oil-in-water or water-in-oil emulsion, multiple emulsion, microemulsion, nanoemulsion, twin-phase emulsion, PIT emulsion, stable dispersion of two immiscible phases by means of a gelling agent, stable dispersion of two immiscible phases by means of one or more surfactants, liquid, aqueous gel, oily gel, hydroalcoholic gel, oily phase, suspension, foaming or non-foaming solution, gel, lyophilized emulsion or powder.

6. Cosmetic or pharmaceutical composition according to claim 4, characterized in that the composition is in the form of liquids, foams, pastes, ready-to-use drinks, lotions, emulsions, oils, gels, syrups, solids, powders, masks, sticks, tablets, capsules, sprays, aerosols, softgels, jellies, syrups, creams, patches, shower gels, shampoos.

7. Pharmaceutical composition according to claim 4, for its use in improving the diversity and quantity of an individual's microbiota.