Method for obtaining a fermented carob POD extract and its uses

Abstract

The present invention relates to a method for producing a fermented extract from carob pod, and to the fermented extract obtained by this method. The invention also relates to a pharmaceutical composition comprising said fermented extract and to the medical uses of the fermented extract. Finally, the invention relates to a food product or a nutritional supplement comprising the fermented extract of the invention and to the use of the fermented extract of the invention as a functional ingredient in food or as a nutritional supplement.

Description

[0001] METHOD FOR OBTAINING A FERMENTED CAROB POD EXTRACT AND ITS USES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention belongs to the field of extracts from plants, in particular, from carob pod (seedless part), and their uses for human health and nutrition.

[0004] BACKGROUND OF THE INVENTION

[0005] Carob (Ceratonia siliqua L.) is a leguminous evergreen tree widely cultivated in the Mediterranean region. The tree produces pod-like fruits known as carob pods, which have been valued for their nutritional and therapeutic properties for centuries. Carob pods (seedless part) are rich in various bioactive compounds, including dietary fiber, polyphenols, flavonoids, and particularly D-pinitol, a naturally occurring cyclitol with significant health benefits.

[0006] D-pinitol, a cyclic polyol, has attracted considerable attention due to its multiple therapeutic properties. Scientific research has demonstrated that D-pinitol exhibits antiinflammatory, anti-diabetic, and antioxidant activities. It has been particularly noted for its ability to improve insulin sensitivity, making it beneficial for managing conditions such as Type 2 diabetes and metabolic syndrome. Additionally, D-pinitol has been associated with cardiovascular health, providing protective effects against heart disease.

[0007] Traditionally, carob pods have been utilized in various forms for human consumption. Carob powder, made by grinding the dried pods, serves as a natural sweetener and chocolate substitute. Carob syrup, another popular product, is derived by boiling the pods in water. Both products are commonly used in baking, confectionery, and as health supplements due to their high nutritional value and pleasant flavor. Several methods have been developed for preparing carob extracts, primarily focusing on maximizing the yield of valuable components like polyphenols and pinitol. Conventional extraction techniques include aqueous extraction, ethanol extraction, and acid hydrolysis. Aqueous extraction involves boiling or soaking the carob pods in water to release soluble compounds. This method is straightforward but may result in lower concentrations of the desired bioactive components.

[0008] Ethanol extraction, on the other hand, uses ethanol as a solvent to dissolve and concentrate the bioactive compounds from the carob pods. This method tends to be more efficient in extracting polyphenols and flavonoids, but it requires subsequent removal of the solvent, which can be costly and time-consuming. Acid hydrolysis is another approach where acids, such as hydrochloric acid, are used to break down the pod matrix, thereby releasing the desired compounds. While effective, this method can lead to the degradation of sensitive compounds and the generation of unwanted byproducts.

[0009] Despite these existing methods, there remains a need for a more efficient, cost- effective, and environmentally friendly method for preparing carob pod extracts rich in D- pinitol. Such a method would not only enhance the yield and purity of D-pinitol but also preserve the integrity of other valuable nutrients present in the carob pods. The present invention addresses this need by providing an improved method for preparing a carob pod extract rich in pinitol, which can be utilized for various health and nutritional applications.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have designed a new method for preparing an extract from carob pod. This method combines steps of extraction of water soluble substances, clarification and pasteurization with a fermentation step. As a result of this new method, an extract characterized by its low content in sugars and high content in inositols, specially D-pinitol, is obtained. The method avoids using, such as high temperatures, so the full-spectrum is conserved.

[0012] Therefore, in a first aspect, the invention is related with a method for producing an extract from carob pod, wherein the method comprises:

[0013] (i) extracting water soluble substances from carob pod by contacting carob pod with water at a temperature from about 50eC to about 90eC, obtaining a first raw juice and a humid solid residue;

[0014] (ii) clarifying the first raw juice obtained in step (i) by microfiltration, obtaining a clarified juice,

[0015] (iii) pasteurizing the clarified juice obtained in step (ii);

[0016] (iv) subjecting the pasteurized clarified juice obtained in step (iii) to a liquid fermentation with Saccharomyces cerevisiae, obtaining a fermented extract;

[0017] (v) clarifying the fermented extract obtained in step (iv) by microfiltration, obtaining a clarified fermented extract; and

[0018] (vi) concentrating the clarified fermented extract until about 65 to about 68 degrees Brix. In a second aspect, the invention is related with a fermented extract from carob pod obtainable by the method according to any one of claims 1 to 16, wherein the fermented extract comprises D-pinitol at a concentration of least 24 % weight / weight on a dry matter basis.

[0019] In a third aspect, the invention is related with a pharmaceutical composition comprising the fermented extract of the invention.

[0020] In a fourth aspect, the invention is related with the fermented extract of the invention for its use in medicine.

[0021] In a fifth aspect, the invention is related with the fermented extract of the invention for use in the treatment and / or prevention of type 2 diabetes mellitus and metabolic syndrome.

[0022] In a sixth aspect, the invention is related with a food product or a nutritional supplement comprising the fermented extract of the invention.

[0023] In a seventh aspect, the invention is related with the use of the fermented extract of the invention as a functional ingredient in foods or in nutritional supplements.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1. Diagram of composition changes in nutrients due to fermentation

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] Method of the invention

[0028] In a first aspect, the invention related to a method for producing an extract from carob pod, wherein the method comprises:

[0029] (i) extracting water soluble substances from carob pod by contacting carob pod with water at a temperature from about 50eC to about 90eC, obtaining a first raw juice and a humid solid residue;

[0030] (ii) clarifying the first raw juice obtained in step (i) by microfiltration, obtaining a clarified juice,

[0031] (iii) pasteurizing the clarified juice obtained in step (ii);

[0032] (iv) subjecting the pasteurized clarified juice obtained in step (iii) to a liquid fermentation with Saccharomyces cerevisiae, obtaining a fermented extract;

[0033] (v) clarifying the fermented extract obtained in step (iv) by microfiltration, obtaining a clarified fermented extract; and (vi) concentrating the clarified fermented extract until about 65 to about 68 degrees Brix.

[0034] The term “carob”, as used herein, refers to the fruit of the carob tree. The term “carob tree”, refers to a plant of the species Ceratonia siliqua which belongs to the leguminosae family (Fabaceae). Carob fruit is a legume and as such comprises a pod and seeds. Although the term carob is sometimes used to refer to the whole fruit or bean, in the context of the present invention the term “carob” refers to the carob fruit without the seeds

[0035] The term “extract”, as used herein, refers to a product created from a natural source, in the context of the present invention, carob pod, by extracting and concentrating certain substances present in the natural source. The term “extract” and “carob pod extract” are used interchangeably in the context of the present invention.

[0036] In a particular embodiment, the carob pods that can be used as the starting has a humidity between 10% and 20%.

[0037] As the skilled person would understand, before proceeding with the step (i) of the method of the invention the carob pods may be processed so that they are chopped into smaller pieces. In a particular embodiment, carob pods are obtained from Carob fruit through milling and separating the carob seeds (raw material used to obtain locust bean gum). In a particular embodiment, the carob pods are chopped into pieces of between 5 and 20 mm of diameter.

[0038] In a particular embodiment, the carob pod that can be uses as the starting material for the method of the invention comprises the following composition:

[0039] Step (i) of the method of the invention: soluble extraction

[0040] Step (i) comprises extracting water soluble substances from carob pod by contacting carob pod with water at a temperature from about 50eC to about 90eC, obtaining a first raw juice and a humid solid residue;

[0041] The term “water soluble substances”, as used herein, refers to those substances that are water-miscible (miscibility with water of greater than 50 percent by weight at 25 degrees centigrade and atmospheric pressure). Example of water soluble substance that are present in carob pod are sugars (monosaccharides and disaccharides), inositols, other polyols, soluble polyphenols, complex carbohydrates, nitrogen compounds, minerals and soluble fibre.

[0042] The extraction is done by contacting carob pod, which preferably is chopped into smaller pieces, more preferably into pieces of 5-20 mm of diameter, with water at about 50eC to 90eC. In a particular embodiment, the temperature of the water is about 50eC, about 60eC, about 70eC, about 80eC or about 90eC.

[0043] The extraction can be done by different techniques, like, without limitation, counter-current flow extraction, batch extraction or a combination of both.

[0044] In a particular embodiment, step (i) is performed by counter-current flow extraction, batch extraction or a combination of both. The term “counter-current flow extraction”, as used herein, refers to an extraction model wherein the material (carob pod) and the solvent (water) are exactly flowing in opposite directions. Thus, the non-extracted carob pods (fresh material) will be extracted with the most enriched Carob Extract of the process, and the already exhausted / extracted Carob Pods (material) will be extracted with fresh solvent (water).

[0045] In a particular embodiment, the counter-current flow extraction is carried out for a period of time between about 45 minutes to about 90 minutes.

[0046] The term “batch extraction”, as used herein, refers to a method of extracting water soluble substances from carob pod by immersing the carob pod in water for a period of time and at a controlled temperature. In a particular embodiment, batch extraction is carried out for a period of about 30 to 90 minutes.

[0047] Depending on the method used for the extraction of the water soluble substances, the extraction ratio carob pod / water is from 1 to 3.5 or from 1 to 15.

[0048] In a particular embodiment, the water used in step (i) is reverse osmosis water. The term “reverse osmosis water”, as used herein, refers to water that has been purified through a process that uses a semi-permeable membrane to filter out contaminants, including certain molecules, particles, and microorganisms, such as: dissolved salts (sodium, potassium, calcium magnesium, chloride and sulfates); chemicals, such as chlorine and chloramines, fluoride, pesticides and herbicides, and volatile organic compounds (e.g., benzene and toluene); heavy metals, including lead, chromium, and copper; biological contaminants, for example, bacteria, viruses, and protozoa; particulate matter like sediment, silt, and other suspended solids; pharmaceuticals such as hormones and antibiotics and radiological elements such as radium and uranium.

[0049] In a particular embodiment, step (i) does not comprise the use of extraction and clarification coadjuvants, enzymes or acidifying agents. Examples of coadjuvants that are not used in step (i) include authorized extraction solvents such as ethyl acetate, ethanol and acetone. Examples of enzymes that are not used in step (i) include pectinases, proteinases, and amylases. Examples of acidifying agents that are not used in step (i) include citric acid.

[0050] As a result of step (i) two products are obtained:

[0051] - a liquid product called the “raw juice”, which includes the water soluble components and

[0052] - a solid product called “humid solid residue”.

[0053] In a particular embodiment, the raw juice is characterized by having between about 4 to about 19 degrees Brix. The term “degrees Brix”, as used herein, refers to the soluble solids content of an aqueous solution. Although, traditionally,eBrix refers to sugar content, meaning that, one degree Brix would be 1 gram of sucrose in 100 grams of solution; in the context of the present invention, the more general meaning of soluble solids content in an aqueous solution is used. Therefore, in the context of the present invention, one degree Brix is 1 gram of soluble solids in 100 grams of solution.

[0054] In a particular embodiment, step (i) comprises an additional step of reducing the amount of water in the humid solid residue obtained after the extraction. In this particular embodiment, the raw juice that is obtained directly after the extraction process is called “first raw juice”. The reduction of the amount of water in the humid solid residue is done by a process comprising decanting the humid solid residue, pressing the humid solid residue or a combination of both, thereby obtaining a “second raw juice” that is combined with the first raw juice.

[0055] When the amount of water in the humid solid residues is done by decantation, humidity can be reduced from approximately 85% to approximately 75-70%. When the amount of water in the humid solid residues is done by pressing the humid solid residue, the humidity can be further reduced to 50-60%.

[0056] Step (ii) of the method of the invention: clarification of the first raw juice.

[0057] Step (ii) of the method of the invention comprises clarifying the first raw juice obtained in step (i) by microfiltration, obtaining a clarified juice.

[0058] The term “microfiltration”, as used herein, refers to a pressure-driven separation process used to remove fine particles from a liquid. In this process a fluid is passed through a special pore-sized membrane filter to separate particles and substances of certain size and / or molecular weight from the liquid.

[0059] In a particular embodiment, microfiltration is performed using membranes with a pore size of about 0.8 pm (250 KDa) to about 1.2 pm (800 KDa). In a particular embodiment, the membranes are polymeric membranes, more particularly, spiral polymeric membranes.

[0060] In a particular embodiment, step (ii) does not comprise the use of extraction and clarification coadjuvants, enzymes or acidifying agents. Examples of coadjuvants that are not used in step (ii) include authorized extraction solvents such as ethyl acetate, ethanol and acetone. Examples of enzymes that are not used in step (ii) include pectinases, proteinases, and amylases. Examples of acidifying agents that are not used in step (ii) include citric acid. As a result of the microfiltration process, insoluble material, macrocoloidal material (pectins, hemicelluloses, proteins, starches, tannins and polyphenolic colloidal complexes), bacteria, moulds and yeasts are removed from the raw juices, resulting in a clarified juice.

[0061] In a particular embodiment, the pH of the clarified juiced is measured after step (ii) if the pH is lower than 4.8, it is adjusted to about 5.5.

[0062] In a particular embodiment, if during the microfiltration process the retentate reaches a relatively high viscosity making it difficult the microfiltration process, reserve osmosis water can be added to the retentate, preferably in a ratio retentate:water 1 :1 or 1 :2.

[0063] Step (Hi) of the method of the invention: pasteurization of the clarified juice

[0064] Step (iii) comprises pasteurizing the clarified juice obtained in step (ii). This step is intended to sterilize the juice that will be the subtract of the fermentation process of step (iv).

[0065] The term “pasteurization”, as used herein, refers to a heat-treatment process that destroys or inactivates microorganisms in certain media, preferably in foods and beverages. The temperature used in pasteurization is usually lower than 100eC. in a particular embodiment, the pasteurization is done at a temperature of 100eC or lower, for example, about 98eC, about 96eC, about 94eC, about 92eC, about 90eC, about 88eC, about 86eC, about 84eC or lower. In a particular embodiment, the pasteurization is done at a temperature of about 88eC to about 92eC, more particularly at a temperature of 90eC + / - 2eC.

[0066] In a particular embodiment, pasteurization is performed for a period of time of about 60 seconds to about 120 seconds, more particularly from about 45 seconds to 120 seconds, even more particularly for at least 45 seconds, preferably for about 80 seconds.

[0067] In a particular embodiment, pasteurization is performed by using a heat plate exchanger.

[0068] In a particular embodiment, the clarified juice can be diluted with reverse osmosis water before pasteurization. The dilution can be such that, if the clarified juice is 15 degrees Brix, water is added until the resulting juices is about 7-9 degrees Brix.

[0069] Step (iv) of the method of the invention: fermentation of the pasteurized clarified juice. Step (iv) comprises subjecting the pasteurized clarified juice obtained after step (iii) to a liquid fermentation process with Saccharomyces cerevisiae, obtaining a fermented extract.

[0070] The term “fermentation”, as used herein, refers to a biochemical process by which energy is extracted anaerobically from molecules such as glucose and other carbohydrates. In microorganisms, fermentation is the primary means for producing ATP by the anaerobic degradation of organic nutrients.

[0071] The term “liquid fermentation”, also known as “submerged culture”, as used herein, refers to a method for culturing a microorganism, such as a yeast, in a liquid medium or broth. Such medium includes the substrates for the fermentation.

[0072] In the method of the invention, the medium or broth for fermentation is the pasteurized clarified juice obtained after step (iii).

[0073] In the method of the invention fermentation is carried out by the yeast Saccharomyces cerevisiae.

[0074] In a particular embodiment, the yeast is Saccharomyces cerevisiae. In a more particular embodiment, the yeast is Saccharomyces cerevisiase var. bayanus.

[0075] In a particular embodiment, the yeast is hydrated with water without chlorine or with the pasteurized clarified juice obtained after step (iii). An illustrative non-limitative example of how this can be done can be found in the examples included below.

[0076] In a particular embodiment, S. cerevisiae is hydrated with water without chlorine or with the pasteurized clarified juice obtained in step (iii). For hydration with the pasteurized clarified juice, reverse osmosis water can be added to the pasteurized clarified juice until approximately 1 degree Brix. The temperature is kept at about 30- 40eC. once the pasteurized clarified juice is diluted as explained, the yeast is added, in a particular embodiment at a dose of approximately 22.5 g yeast / hL. The yeast added to the pasteurized clarified juice is kept still for approximately 10-30 minutes, and after that the hydrated yeast is added to the pasteurized clarified juice and being maintained a 30eC.

[0077] Fermentation can be started by adding the hydrated yeast to a fermentation tank containing the pasteurized clarified juice and keeping it at 30-40eC with stirring, preferably only during the first 6 hours.

[0078] In a particular embodiment, the fermentation is performed at a temperature of about 30eC to about 40eC.

[0079] In a particular embodiment, the fermentation is performed under agitation conditions only during about the first 6 hours of the fermentation process. In a particular embodiment, the fermentation is performed until the glucose in the pasteurized clarified juice is depleted. In general, glucose should be depleted approximately 30 hours after the start of the fermentation process.

[0080] The product obtained after step (iv) is called “fermented extract”. This product should not be pasteurized to avoid cell lysis

[0081] Step (v) of the method of the invention: clarification of the fermented extract.

[0082] Step (v) comprised clarifying the fermented extract obtained in step (iv) by microfiltration.

[0083] The aim of this step is obtaining a clarified fermented extract, which is free of turbidity.

[0084] The term “microfiltration” has been previously defined.

[0085] In a particular embodiment, microfiltration is performed using membranes with a pore size of about 0.8 pm (250 KDa) to about 1.2 pm (800 KDa). In a particular embodiment, the membranes are polymeric membranes, more particularly, spiral polymeric membranes

[0086] In a particular embodiment, step (v) does not comprise the use of extraction and clarification coadjuvants, enzymes or acidifying agents. Examples of coadjuvants that are not used in step (ii) include authorized extraction solvents such as ethyl acetate, ethanol and acetone. Examples of enzymes that are not used in step (ii) include pectinases, proteinases, and amylases. Examples of acidifying agents that are not used in step (ii) include citric acid.

[0087] As a result of the microfiltration process, insoluble material, macrocoloidal material (pectins, hemicelluloses, proteins, starches, tannins and polyphenolic colloidal complexes), bacteria, moulds and yeasts are removed from the fermented extract, resulting in a clarified fermented extract.

[0088] Step (vi) of the method of the invention: concentration of the clarified fermented extract.

[0089] Step (vi) comprises concentrating the clarified fermented extract obtained after step (v) until about 65 to about 68 degrees Brix.

[0090] In a particular embodiment, step (vi) comprises a first step of desolventization and a second step of concentration.

[0091] The term “desolventization” or “semi-concentration” as used herein, refers to a process for removing ethanol and other organic volatile substances produced during fermentation. In a particular embodiment, after desolventization the extract has between 20 and 35 degrees Brix. The term “concentration”, as used herein, refers to a process for removing water from a product, increasing its dry matter content.

[0092] In a particular embodiment, the desolventization and concentration is performed with a dual-effect tubular concentrator working at high vacuum, as described in the example below.

[0093] In a particular embodiment, additional steps can be carried out after step (vi), for example pasteurization, cooling and filtration.

[0094] The term “pasteurization” has been previously defined.

[0095] The cooling can be done with a plate heat exchanger until a final temperature of about 20-25eC, not more than 30eC.

[0096] In a particular embodiment, the filtration is performed with 100 pm metallic filters in order to eliminate foreign bodies.

[0097] Extract of the invention and its uses

[0098] In a second aspect, the invention relates to a fermented extract from carob pod obtainable by the method of the first aspect, hereinafter “fermented extract of the invention” or “clarified fermented extract of the invention”. In a particular embodiment, the fermented extract of the invention comprises D-pinitol at a concentration of at least 24 % weight / weight in a dry matter basis.

[0099] The terms “obtainable be”, “obtained by” or “directly obtained by” are used here interchangeably.

[0100] The term “pinitol”, “D-pinitol”, also known as “3-O-Methyl-D-chiro-inositol” or “D- (+)-ch iro- Inositol”, as used herein, refers to the compound of formula

[0101] In a particular embodiment, the fermented extract of the invention has a concentration of D-pinitol of at least 145 kg pinitol per kg of the clarified fermented extract having about 68 degrees Brix, or at least 24% weight / weight on a dry matter basis.

[0102] The concentration of pinitol can be measured with any suitable analytical techniques used by the skilled person, such as for example with TLC (thin-layer chromatography), HPLC (high performance liquid chromatography), gas chromatography or GC-MS (gas-chromatography-mass spectrometry). An example of determination of D-pinitol by HPLC is described by Tetik, N, Turhan, I, Oziyci, HR, Karhan, M. 201 1. Determination of D-pinitol in carob syrup. Int. J. Food Sci. Nutr., 62, 572-576. In a particular embodiment, the concentration of pinitol is determined by the method described by Tetik, N, Turhan, I, Oziyci, HR, Karhan, M., 2011 . Determination of D-pinitol in carob syrup. Int. J. Food Sci. Nutr., 62, 572-576.

[0103] In a particular embodiment, the clarified fermented extract of the invention is further characterized by having the following components in the indicated concentrations:

[0104] - myo-inositol: approximately 15 g / kg 68 degrees Brix (2.6 % dry matter basis); D-chyro-inositol: approximately 1.2 g / kg 68 degrees Brix (0.19 % dry matter basis);

[0105] - Total inositols: approximately 161 -162 g / kg 68 degrees Brix (26,9 % dry matter basis);

[0106] In a particular embodiment, the clarified fermented extract concentrate of the invention is characterized by the composition indicated in Tables 2 or Table 3 for the “clear fermented carob extract concentrate”.

[0107] Pharmaceutical composition of the invention

[0108] In another aspect, the invention relates to a pharmaceutical composition comprising the clarified fermented carob extract of the invention.

[0109] The term “pharmaceutical composition”, as used herein, refers to a composition comprising a therapeutically effective amount of the fermented extract according to the present invention and at least one pharmaceutically acceptable excipient.

[0110] The term “therapeutically effective amount”, as used herein, refers to the amount of the fermented extract according to the present invention, which is required to achieve an appreciable prevention, cure, delay, reduction of the severity of, or amelioration of one or more symptoms of the disease or condition, such as type 2 diabetes mellitus or metabolic syndrome. The person skilled in the art will be able to determine therapeutically effective amounts of these molecules without undue experimentation by means of conventional techniques well-known in the art.

[0111] The terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier,” refer to any compound or combination of compounds that is essentially non-toxic to the subject at the dosage and concentration employed, and is compatible with the other components of a pharmaceutical composition. This includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Thus, an excipient is an inactive substance formulated alongside the active ingredient of a pharmaceutical composition, for the purpose of bulking-up compositions that contain said active ingredients. Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. Excipients also can serve various therapeutic-enhancing purposes, such as facilitating compound (drug) absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients depends upon the route of administration and the dosage form, as well as the active ingredient and other factors. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; potassium sorbate; sodium benzoate); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, trehalose or polyols (like mannitol, sorbitol); salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

[0112] In a particular embodiment, the route of administration of the pharmaceutical composition of the invention is oral.

[0113] Medical uses of the invention

[0114] The beneficial effects of D-pinitol for controlling blood sugar levels are known, and also the potential benefits of this compound for patients with type 2 diabetes and metabolic syndrome [Antonowski T, Osowski A, Lahuta L, Gorecki R, Rynkiewicz A, Wojtkiewicz J. Health-Promoting Properties of Selected Cyclitols for Metabolic Syndrome and Diabetes. Nutrients. 2019 Sep 30;11 (10):2314)]. Since the fermented extract of the invention is significantly enriched in D-pinitol, it can be used for treating these diseases.

[0115] In another aspect, the invention relates to the fermented extract of the invention for its use in medicine. Alternatively, the invention relates to the use of the fermented extract of the invention for manufacturing a medicament.

[0116] In another aspect, the invention relates to the fermented extract of the invention for use in the treatment and / or prevention of type 2 diabetes mellitus and metabolic syndrome. Alternatively, the invention relates to the use of the fermented extract of the invention for manufacturing a medicament for the treatment and / or prevention of type 2 diabetes mellitus and metabolic syndrome. Alternatively, the invention relates to a method for treating and / or preventing type 2 diabetes mellitus or metabolic syndrome, comprising administering a therapeutically effective amount of the fermented extract of the invention to a patient in need thereof.

[0117] The term “subject”, as used herein, refers to all animals classified as mammals and includes, without limitation, domestic and farm animals, primates, and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human of any age or race.

[0118] The term “prevention”, “preventing” or “prevent”, as used herein, relates to the administration of a pharmaceutical composition according to the invention to a subject who has not been diagnosed as possibly having the disease, but who would normally be expected to develop said disease or be at increased risk for said disease. The prevention intends to avoid the appearance of said disease. The prevention may be complete (e.g., the total absence of a disease). The prevention may also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition.

[0119] The term “treatment”, as used herein, refers to any type of therapy, which is aimed at terminating, preventing, ameliorating, or reducing the susceptibility to a clinical condition as described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment (i.e., a therapy to reduce the susceptibility to a clinical condition), of a disorder or a condition as defined herein. Thus, “treatment,” “treating,” and their equivalent terms refer to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. That is, “treatment” includes (1 ) preventing the disorder from occurring or recurring in a subject, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or, at least, symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter.

[0120] The term “diabetes mellitus”, or simply “diabetes”, as used herein, refers to a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from low levels of the hormone insulin with or without abnormal resistance to insulin's effects. Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin, and / or when the body cannot effectively use the insulin it produces. Hyperglycemia, or raised blood sugar, is a common effect of uncontrolled diabetes and over time leads to serious damage to many of the body's systems, especially the nerves and blood vessels. The World Health Organization recognizes three main forms of diabetes mellitus: type 1 , type 2, and gestational diabetes (occurring during pregnancy). While ultimately all forms are due to the beta cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia, type 1 diabetes is usually due to autoimmune destruction of the pancreatic beta cells, while type 2 diabetes is characterized by insulin resistance in target tissues. This causes a need for abnormally high amounts of insulin. Gestational diabetes is similar to type 2 diabetes in that it involves insulin resistance; the hormones of pregnancy can cause insulin resistance in women genetically predisposed to developing this condition.

[0121] The term “metabolic syndrome”, as used herein, refers to a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes. Metabolic syndrome is also known as metabolic syndrome X, syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS. Symptoms and features are fasting hyperglycemia (type 2 diabetes mellitus or impaired fasting glucose, impaired glucose tolerance, or insulin resistance); high blood pressure; central obesity, overweight with fat deposits mainly around the waist; decreased HDL cholesterol; and elevated triglycerides. According to the International Diabetes Federation (IDF) definition for persons to be defined as having the metabolic syndrome as used herein they must have: central obesity (defined as waist circumference with ethnicity specific values) plus any two of the following four factors: raised triglyceride, reduced HDL cholesterol, raised blood pressure, raised fasting plasma glucose. The skilled reader will understand how to diagnose metabolic syndrome as used herein at a person by using the “IDF consensus worldwide definition of the metabolic syndrome”, 2006. In a particular embodiment, metabolic syndrome is associated with any one of the following conditions: overweight, high blood pressure, pre-diabetes, type-2 diabetes, dyslipidemia (hypercholesterolemia and / or hypertriglyceridemia) and high cardiovascular risk.

[0122] Food product and nutritional supplement of the invention and their uses

[0123] In another aspect, the invention relates to a food product or a nutritional supplement comprising the fermented extract of the invention.

[0124] As used in the present invention, the term “food product” refers to any substance or product of any nature, solid or liquid, natural or transformed, which due to its characteristics, applications, components, preparation and state of preservation, can be commonly or ideally used for any of the following purposes: a) for normal human or animal nutrition or indulgence; or b) as dietetic products in special cases of human or animal diet. The food product of the invention will preferably be a ready-to-eat food product, which is one that does not have to be diluted by means of an aqueous solution suitable for consumption, for example. In principle, the ingredients present in a ready-to- eat food product are balanced and adding additional ingredients to the food product to make it ready to eat, as is considered by a person skilled in the art, is not necessary. The food product of the invention can also be a concentrated food product, which is one in which one or more ingredients are present at a higher concentration than in a ready- to-eat food product, so to use the product it is necessary to dilute it by means of an aqueous solution suitable for consumption, for example.

[0125] The term “nutritional supplement”, as used herein, refers to nutritional products ingested by a person for generally maintaining or improving the health of that person. Nutritional supplements may be in the form of a medicine-like product, such as a capsule, tablet, chewable soft gel, syrup, or elixir. Nutritional supplements may also be in the form of a nutritional food or beverage product, such as a meal replacement bar, protein bar, milk- or soy-based beverage, protein drink, energy drink, hydration beverage, enhanced water, or mix-in powder for a beverage.

[0126] In another aspect, the invention relates to the use of the fermented extract of the invention as a functional ingredient in foods or in nutritional supplements. The term “functional ingredient”, as used herein, refers to a bioactive compound that can be used in the manufacture of functional food products.

[0127] The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0128] EXAMPLES

[0129] DESCRIPTION OF THE B4UE (AC15) PRODUCTION PROCESS

[0130] 1. Extraction process and decantation-pressing process

[0131] Extraction Process is performed with Hot Osmosis Water on (seedless) Carob Pods or Raw Material from the Overall Process, and is intended for extracting natural bioactive substances present in carob, particularly water-soluble substances such as Sugars (monosaccharides and disaccharides), Inositols, other Polyols, Soluble Polyphenols, complex carbohydrates, nitrogenous compounds, minerals, and Soluble Fiber components.

[0132] Carob Juice is obtained from traditional methods in compliance with the procedures authorized in the duly established and implemented EU Directive 2012 / 12 / EU, the Food Codex-EEC, as well as “Good Manufacturing Practice” (GMP) and the HACCP of EC Regulation No. 852 / 2004.

[0133] When the Fruit itself, as a result of its botanical and compositional characteristics, allows Juice Extraction using traditional methods (for example, juicing, cutting-crushing- pressing), it is then possible to add (reverse osmosis) water and / or (decalcified) soft water to the fruit to EXTRACT soluble compounds from the raw material.

[0134] This is therefore valid for the fruit of (seedless) Carob Pods of Ceratonia siliqua L and thus permitted and declared in EU Directive 2012 / 12 / EU (fruit juices and certain similar products intended for human consumption).

[0135] Hot water extraction can be carried out independently using two technologies or combination thereof:

[0136] 1 ) Countercurrent extraction with a Horizontal extractor-diffuser: This technology consists of at least 5 extraction steps to at most 20 extraction or enrichment steps. It is a COUNTERCURRENT process, characterized by the solvent (reverse osmosis water) coming into contact with-extracting the most depleted or exhausted-extracted raw material fraction of the process, while the carob extract richest in extracted soluble solids comes into contact with-extracts fresher (nonextracted) raw material. This allows water-soluble dry matter to be extracted in a highly optimized manner.

[0137] The Water Temperature ranges from 50 to 90eC. The mean extraction-residence time in the Diffuser often ranges from at least 45 minutes to at most 1 hour 30 minutes.

[0138] 2) BATCH extraction in an extraction tank, with controlled temperature and time, by means of vigorous stirring that allows and facilitates the suspension of Carob in the tank, with the extraction solvent (reverse osmosis water).

[0139] The carob / water extraction ratio is 1 to 3.5 or 1 to 15, with a temperature between 50eC and 90eC, based on the extraction technology used.

[0140] Extraction coadjuvants, enzymes, and acidulants are not used in the Extraction process.

[0141] Raw Carob Juice obtained contains all the native-natural water-soluble substances from (seedless) Carob Pods: carbohydrates, polyols (including inositols), minerals, organic acids, colored compounds, pectin, and other colloidal matter, soluble fiber, polyphenols, nitrogenous substances (such as proteins, amino acids), aromatic compounds, volatile substances, etc.

[0142] RAW CAROB JUICE (liquid) obtained by EXTRACTION has between 4eBrix and 19eBrix.

[0143] The Decantation-Pressing Process is performed on Post-Extraction WET SOLID CAROB RESIDUE.

[0144] The Post- Extraction WET SOLID CAROB RESIDUE is subjected to a soluble solid recovery and / or Water content reduction process by means of PRESSING and / or DECANTATION.

[0145] + Decantation by means of which the mentioned Solid Residue is subjected to liquid or Juice removal, and water loss, by gravity, reducing its Moisture (from about 85% at first to a range of 70-75%).

[0146] + Pressing by means of a continuous screw press, generating Pressed Juice. The initial Moisture of the Solid Residue is also reduced to 50-60% in the end.

[0147] This SECOND RAW Carob JUICE obtained from the Decantation-Pressing of Solid Carob Residue is pumped and pooled together with the FIRST RAW JUICE obtained during Extraction; giving rise to Total Raw Carob JUICE with aeBrix range from 8 to 21eBrix.

[0148] This Total Raw Juice (final) has a very high turbidity and suspended insoluble and colloidal matter.

[0149] The Total Raw Carob Juice, in liquid form, is intended for the production of AC15 B4UE.

[0150] 2. Raw carob juice clarification process

[0151] To be able to successfully perform the subsequent stages, it is necessary to introduce technologies which allow removing insoluble matter and suspended colloidal matter, as well as other possible turbidity-causing molecules in the Raw Carob Juice.

[0152] Raw Carob Juice Clarification follows the guidelines established in EU Directive 2012 / 12 / EU (fruit juices and similar products intended for human consumption): Annex I, Section II. Sub-section 3 (Authorized Substances and Treatments)

[0153] This technology is none other than Tangential Clarification (or Cross-Flow Filtration), with MICROFILTRATION (M-F) being chosen as the most suitable technology for the purpose at hand. In other words, only physical separation methods are used exclusively.

[0154] Organic-polymeric spiral M-F membranes, approved for feeding or Food Grade status, and with a pore size (molecular weight cut-off) of 0.8 micrometers to 1.2 micrometers (250-800 KDa) will be used.

[0155] Clarification and filtration coadjuvants, enzymes, and acidulants are not used in the CLARIFICATION process. Authorized Filtration coadjuvants (for example, perlite, diatomaceous earth, cellulose, insoluble polyamide, PVPP, polystyrene) are not used to also favor the capacity of filtration.

[0156] All the membrane materials used for spiral-wound and flat membrane configurations comply with EU Regulation (EC) 1935 / 2004, EU Regulation 10 / 201 1 and Title 21 of the FDA (CFR) regulations, and are suitable for use in food (Food Grade) and pharmaceutical product processing applications.

[0157] The microfiltration (clarification) process will remove insoluble material, macro- colloidal matter (pectin, hemicelluloses, proteins, starches, tannins, and colloidal polyphenolic complexes) that may cause turbidity-precipitations later on. Likewise are bacteria, fungi, and yeasts are removed as a result of the pore size.

[0158] Microfiltration generates TWO fractions: the clarified-filtered fraction or PERMEATE, and the non-filtered fraction or RETENTATE. The Permeate fraction is called: Clarified Carob Juice.

[0159] In the present process, the Permeate / Retentate Ratio obtained is usually: 85 / 15 to 95 / 5 (w / w).

[0160] In other words, between 850 and 950 kg of Clarified Carob Extract (Output) are usually obtained from 1 Ton of Crude Carob extract (Input). The rest corresponds to Retentate.

[0161] If the pH of the Clarified Juice is in the order of 4.8 - 6.0, NO adjustment is made; if it is below 4.8, NaOH is added until pH 5.5 (+ / -). This will facilitate the subsequent Fermentation stage.

[0162] The Retentate fraction (5-15% of the initial weight) contains all the macromolecules and microorganisms of the Input material of the Clarification process (Raw Carob Juice).

[0163] This Retentate fraction reaches a relatively high viscosity when it reaches higher P / R Ratios, making the M-F process itself difficult (gradual drop in the Filtration Flow, l / h). In such case, Reverse Osmosis Water is added to the Retentate in a 1 :1 and / or 1 :2 proportion (Retentate:Water) in order to reduce viscosity and to be able to keep filtering with a reasonable Flow (l / h); “extracting” at the same time soluble solids in the Retentate to the Permeate (Clarified Extract), thereby optimizing yields.

[0164] This process is usually known as DIAFILTRATION.

[0165] The final Retentate resulting from Diafiltration is an unused residual material that is removed the installation (scrubber).

[0166] 3. Clarified carob extract pasteurization

[0167] This is a very critical operation aiming at stabilizing the Clarified Carob Juice MICROBIOLOGICALLY SPEAKING, apart from other secondary stabilizations (enzymatic inactivation, co-pigmentation, self-aggregation, and / or biochemical condensation).

[0168] The subsequent FERMENTATION phase by means of the controlled addition of selected microbial Starters requires an absolutely “sterile” fermentation substrate, which does not cause any microbial-biological competition for the compositional ecosystem of the substrate (Clarified Carob Juice), since it would lead to an uncontrolled and erratic microbial Fermentation in its final compositional results (particularly in bioactive compounds content).

[0169] Therefore, this Pasteurization ENSURES the stability and microbiological sterility of the Clarified Carob Juice for subsequent FERMENTATION. The Pasteurized Clarified Juice will be stored in a duly sanitized Fermentation Tank. Pasteurization is performed in a Heat Plate Exchanger with suitable holding. The Pasteurization parameters used are: 90 + / -2eC, for at least 45 seconds (holding). Outlet temperature of 30-35eC (at most 40eC) for cooling performed with cooled water and with the product entering the Pasteurizer.

[0170] These conditions provide 5 log cycles of Thermal Destruction of microorganisms in the present Clarified Carob Juice.

[0171] The Cooling Temperature is adapted to subsequent needs / requirements for dealing with the subsequent technical-productive FERMENTATION process.

[0172] Preparation of the Clarified Extract before PASTEURIZATION and OPTIONAL:

[0173] For example: 20,000 I of reverse osmosis water are added to 20,000 I of Clarified Carob Juice (15eBrix) until obtaining a Clarified Carob Juice at 7-9eBrix, with a total volume of 40,000 I of Clarified Carob Juice. This (diluted) Juice is the one TO BE PASTEURIZED according to the conditions and equipment of this stage 4.3, and the substrate for FERMENTATION in the subsequent technical-productive stage.

[0174] 4. Clarified pasteurized carob juice fermentation

[0175] The purpose of this stage is to reduce-minimize or almost drastically remove (fermentable) SUGARS that are naturally present in the Clarified Carob Juice, where these sugars correspond mainly to Sucrose, Glucose, and Fructose (about 97% of all sugars).

[0176] Sugars constitute between 75 and 81 % of the Dry Matter of Clarified Carob Juice. Inositols (bioactive) with about 9.5 - 11 .5% (Dry Basis) of Clarified Carob Juice constitute the primary objective of Fermentation: to reduce-minimize-remove the Fermentable Dry Matter contents in order to increase the concentration of Inositols up to concentrations of about 25 - 27% (Dry Basis) in the Fermented Carob Extract.

[0177] The chosen Fermentation process, at the bacterial starter level, is the Saccharomyces Cerevisiae yeast. Strains of this species are duly selected and chosen for their microbial metabolism on the substrate: Clarified-Pasteurized Carob Juice.

[0178] The processes performed in this FERMENTATION stage on the Clarified- Pasteurized Carob Juice are as follows:

[0179] Note: Preparation of the Clarified Extract before PASTEURIZATION (preceding stage) and OPTIONAL: 20,000 L of reverse osmosis water are added to 20,000 I of Clarified Carob Juice (15eBrix) until obtaining a Clarified Carob Extract at 7-9eBrix and a total volume of 40,000 I of Clarified Carob Extract. This Extract is the one TO BE PASTEURIZED according to the conditions and equipment of the preceding stage 4.3, and the substrate for FERMENTATION.

[0180] 4.1. Yeast hydration:

[0181] Saccharomyces cere v / s / ae var. bayanus, is used.

[0182] If no chlorine free water is available, but only Reverse Osmosis Water is, and it contains 0.09 ppm Total Chlorine (normally 0.9 ppm), the Yeast will be Hydrated with Clarified-Pasteurized Carob Juice. The presence of residual Chlorine affects the biological activity of the yeast.

[0183] An example calculation (for 40,000 I of Clarified-Pasteurized Carob Juice to be fermented), which must be adjusted according to theeBrix of the Clarified- Pasteurized Carob Juice, is indicated below:

[0184] 1 . 840 I of Reverse Osmosis Water are added to about 60 I of 15eBrix Clarified-Pasteurized Carob Juice.

[0185] 2. Therefore obtaining 900 I of Clarified-Pasteurized Carob Juice DILUTED to 1eBrix and at a Temperature of 30-40eC.

[0186] 3. This would amount to, for a Yeast Dose of 22.5 g Yeast / HI (20-30 g / HI), of the product to be fermented, and for a Total volume to be Fermented of Clarified-Pasteurized Carob Juice of 40,000 I (or 400 HI): 9 kg of BY Yeast, which will be added to the 900 I of Clarified- Pasteurized-Diluted (1eBrix) Carob Juice.

[0187] 4. Leave to stand for 10 min after adding to the 840 I (900 I is now obtained).

[0188] 5. Stir immediately for 10 minutes.

[0189] Note: Once the Yeast is hydrated, no more than 30 minutes should pass without adding it to the Clarified-Pasteurized Carob Extract, being maintained at 30eC.

[0190] 4.2. Addition of yeasts and fermentation:

[0191] 1. Before the 10 minutes for stirring the yeast preparation have elapsed, transfer / add this mixture (900 I), by means of air pump, to the FERMENTATION Tank containing 40,000 I Clarified- Pasteurized Carob Juice, and duly maintained at 30- 40eC. 2. Keep under stirring only the first 6 hours. START OF FERMENTATION

[0192] ■ FERMENTATION Tank Samples:

[0193] Record the temperature every 12 hours, and take samples

[0194] (100 ml). Filter 10-20 ml of sample in the laboratory through

[0195] 0.2 micrometers to remove cells and stop fermentation process. Measure glucose, OD600,eBrix, pH and store in refrigeration for subsequent sugar analysis by HPLC.

[0196] 3. After about 30 hours from the start, fermentation should be finished: glucose will be analyzed in the laboratory and when it is depleted, the process (fermentation) will be extended for another 16 hours to deplete the carbon source of sugars.

[0197] 4. The FERMENTATIVE process should be completed in 3-4 days (at most 7 days).

[0198] After completion of the Fermentation Process on the Clarified-Pasteurized Carob Juice, the Fermented end product obtained is renamed from Juice to EXTRACT according to the application of criteria related to the Legislation on juices (EU Directive 2012 / 12 / Ell on fruit juices and certain similar products intended for human consumption).

[0199] The FERMENTED EXTRACT product should not be Pasteurized in order to prevent the generated biomass from experiencing massive CELL LYSIS phenomena, which would result in drastic compositional changes (complex carbohydrates, proteins, peptides, amino acids, phospholipids, etc.) due to the significant contribution of the fermentative microbial flora (alive or dead), and which would modify the functional properties of the finished product.

[0200] 5. Second tangencial filtration (Microfiltration)

[0201] Stage 4.2 Raw Carob Juice CLARIFICATION PROCESS herein already describes this technology and the purpose of performing this process in said stage.

[0202] In this productive post-Fermentation technical stage, it is necessary to reduce- minimize-remove the biomass generated in the Fermentation, of highly complex oligomeric and / or polymeric nature (complex carbohydrates and complex nitrogenous compounds, among others), and which causes significant turbidity and potential generation of sediments, by inter- and intra-reactivity between the generated substances and substances naturally present in the Clarified Carob Juice (before fermentation). Fermentation has also generated significant amounts of water-soluble substances such as Ethanol, Acetaldehyde, Glycerin, Mannitol, Organic Acids with significant reduction of pH, CO2, and volatile (aromatic) substances.

[0203] Furthermore, a primary purpose of the finished product (AC15, B4UE) is its sensory characteristic of the absence of turbidity, presenting a clarity and brightness typical of a Clarified-Filtered Extract.

[0204] In this second M-F, the Permeate / Retentate ratios can reach values of 95 / 5, including additional Diafiltration process (addition of Reverse Osmosis Water) on the Retentate of the process.

[0205] As in the 1stM-F, at microbiological level, this CLARIFIED Fermented Carob Extract (Permeate) will be free of microorganisms.

[0206] 6. Clarified fermented carob extract concentration

[0207] This stage is divided into two sub-stages:

[0208] 1. Desolventization or Semi-Concentration: Removal of EtOH / AcH and other organic volatile substances produced during fermentation. Reaching between 20 and 35eBrix.

[0209] 2. Final Concentration up to 65-68eBrix.

[0210] A DUAL-EFFECT tubular CONCENTRATOR-EVAPORATOR working at high vacuum is used, allowing work to be performed at temperatures considerably < 100eC, and therefore minimizing thermal degradation effects on components of the product.

[0211] The first effect works at a lower temperature than the second effect for the DESOLVENTIZATION of the Fermented-Clarified Carob Extract.

[0212] Fresh boiler steam is used as a thermal-heat source element for the operation of the Concentrator-Evaporator.

[0213] Water evaporated from the Fermented-Clarified Carob Extract, in the FIRST EFFECT, and which contains EtOH and other volatile substances, is recovered, after CONDENSATION (cold thermal source), for subsequent uses within the productive activity of the company.

[0214] Finally, the SECOND EFFECT achieves the purpose of reducing-removing Water and Aw, and increasing the Dry Matter contents andeBrix in the finished product: between 65 and 68eBrix.

[0215] At the CONCENTRATOR outlet, the concentrated product (outlet at about 70eC) is cooled to 25-30eC by means of a heat exchanger (cooling with cold water and with product at the evaporator-concentrator inlet). The concentration stage provides a very significant reduction in Water Activity (Aw), with a corresponding increase in the MICROBIOLOGICAL stability of the concentrated product (less water available for the development of microorganisms); which along with a reduction in pH (<3.5) and the highly significant loss of Carbon sources (e.g., sugars), converts Concentrated Fermented-Clarified Extract into a product that is very stable against microbiological alteration, both against viable microflora (molds-yeasts, acid-resistant bacteria) and particularly against pathogens.

[0216] 7. Final stages (Pasteurization, Cooling, Filtration, and Packaging).

[0217] These correspond to standard processes within the industry.

[0218] As described in the stage 4.5 above, the compositional and physicochemical characteristics of the Concentrated Fermented-Clarified Carob Extract make it a product that is reasonably stable against microbiological alteration. However, and within the concept and requirements of GMP and HACCP, final Pasteurization, as well as Filtration, are recommended to eliminate the potential presence of foreign bodies.

[0219] Pasteurization is carried out in a Plate Heat Exchanger with holding; the chosen Pasteurization parameters are:

[0220] + 95 + / -2eC for at least 45 seconds (holding).

[0221] This guarantees 5 Log cycles of Thermal Destruction of microorganisms. Considering the reference microorganisms, Molds-Yeasts and Acid-Tolerant Bacteria.

[0222] Cooling is coupled to the previous stage (Plate Heat Exchanger), being performed with cooled water and with the product entering the Pasteurizer. The product is cooled to 20-25eC (at most 30eC).

[0223] FILTRATION is carried out on the Fermented-Clarified-Pasteurized-Cooled Carob Extract by means of 100-microns METALLIC FILTERS to remove FOREIGN BODIES (for example, metal, glass, burnt bodies, wood, insects, etc.).

[0224] PACKAGING of the product Fermented-Clarified (Pasteurized-Cooled-Filtered) Carob Extract will be performed according to the chosen / selected formats.

[0225] All the components in contact with the product-ingredient in question, and provided by the PACKAGING suppliers, must be “Approved for Human Consumption” (Food Grade Approval).

[0226] The PACKAGED end product (AC15 = B4UE) can be stored at ROOM Temperature (20 - 30eC) in the time established in the Specifications of the product in question. Characterization of the final product

[0227] Table 2. Clear carob juice concentrate versus clear fermented carob juice concentrate

[0228] (*) Results expressed as “g / kg of product or g / kg of extract” (fresh weight basis).

[0229] Advantages of the Clear Fermented Carob Extract Concentrate are focused on a better Nutrition profile as well as with a very significant increase of Bioactive compounds naturally occurring in the Carob, and providing well-known and recognized Health benefits:

[0230] Reduction in sugar content: approx, a 95 % reduction from the carob juice to the fermented carob extract. Sugar reduction is an overall or global nutritional strategy or goal for any sort of new food developments.

[0231] Increase in protein content: approx. 2,0 folds higher in carob extract vs carob juice. This is also an important nutritional goal.

[0232] - No fats: the new product does not contain Fats (< 0,5 g / 100g).

[0233] - Significant increase in inositols (bioactive compounds): approx. 2,4 folds higher in the fermented carob extract vs carob juice. This is a very relevant finding due to the very important health benefits of the inositols - Significant increase of total soluble polyphenols (Bioactive compounds). Approx. 2,5 folds higher in fermented carob extract vs carob juice.

[0234] - Significant appearance of lactic acid in the fermented carob extract due to the fermentation process (approx. 30 % of the final DM). Lactic acid is present in all fermented dairy products and providing clear and known health benefits.

[0235] - Glycerol contents, in the fermented carob extract, are not worrying (approx. 33 g / Kg) because glycerol has a DL50 of 12,6g / Kg BW in rats and 18,7 g / Kg BW in guinea pigs.

[0236] - The majority of the native fiber of the carob pods has been removed because it is insoluble fiber. Only soluble fiber remains in the fermented carob extract

[0237] (Pre-biotic properties).

[0238]

[0239] Table 3. Characterization of INPUT and OUTPUT materials of the global process of AC15 Fermented Carob Extract Concentrate (B4UE).

[0240] Fiber (2 methods are applied):

[0241] + By species: sum of Cellobiose + Kestose + Arabino-Xylans + Stachyose + Raffinose

[0242] + AOAC Methods: Soluble + Insoluble Fiber = Total Dietary Fiber

[0243] Minerals: sum of Cations + Anions (no Ashes IFU Method).

[0244] ECT: Extractable Condensed Tannins. NECT : Non-Extractable Condensed Tannins

[0245] Hidrolizable Tannins are integrated within the Total Soluble Polyphenols

[0246] CONDENSED TANNINS (CT) as Insoluble Highly Polymerized Pro-Anthocyanidins are associated with fractions of Fiber (i.e., Lignine), Resistant Proteins or Resistant Starch, and they are defined as part of Total Fiber (AOACC Method). Anyway, CT are independently analyzed by specific Method.

Claims

1. CLAIMS1 . Method for producing an extract from carob pod, wherein the method comprises:(i) extracting water soluble substances from carob pod by contacting carob pod with water at a temperature from about 50eC to about 90eC, obtaining a first raw juice and a humid solid residue;(ii) clarifying the first raw juice obtained in step (i) by microfiltration, obtaining a clarified juice,(iii) pasteurizing the clarified juice obtained in step (ii);(iv) subjecting the pasteurized clarified juice obtained in step (iii) to a liquid fermentation with Saccharomyces cerevisiae, obtaining a fermented extract;(v) clarifying the fermented extract obtained in step (iv) by microfiltration, obtaining a clarified fermented extract; and(vi) concentrating the clarified fermented extract until about 65 to about 68 degrees Brix.

2. The method according to claim 1 , wherein step (i) is performed by counter-current flow extraction, batch extraction or a combination of both.

3. The method according to claims 1 or 2, wherein the water used in step (i) is reverse osmosis water.

4. The method according to any one of claims 1 to 3, wherein step (i) and (ii) do not include the use of extraction and clarification coadjuvants, enzymes or acidifying agents.

5. The method according to any one of claims 1 to 4, wherein microfiltration in step (ii) is done by using membranes with a pore size of about 0.8 pm to about 1 .2 pm.

6. The method according to any one of claims 1 to 5, wherein the method further comprises a step of reducing the amount of water in the humid solid residue obtained in step (i) by a process comprising decanting the humid solid residue, pressing the humid solid residue or a combination of both, thereby obtaining a second raw juice that is combined with the first raw juice before step (ii).

7. The method according to any one of claims 1 to 4, wherein after step (ii) the pH of the clarified juice is measured and if the pH is lower than 4.8, it is adjusted to about 5.5.

8. The method according to any one of aspects 1 to 7, wherein pasteurization in step (iii) is performed by using a heat plate exchanger at a temperature of about 88eC to about 92eC.

9. The method according to any one of claims 1 to 8, wherein the fermentation in step (iv) is done by S. cerevisiae var. bayanus.

10. The method according to any one of claims 1 to 7, wherein S. cerevisiae is hydrated with water without chlorine or with the pasteurized clarified juice obtained in step (iii).

11. The method according to any one of claims 1 to 7, wherein the fermentation is performed at a temperature of about 30eC to about 40eC.

12. The method according to any one of claims 1 to 11 , wherein the fermentation is performed under agitation conditions only during about the first 6 hours of the fermentation process.

13. The method according to any one of claims 1 to 12, wherein the fermentation is performed until the glucose in the pasteurized clarified juice is depleted.

14. The method according to any one of claims 1 to 13, wherein microfiltration in step (v) is done by using membranes with a pore size of about 0.8 pm to about 1 .2 pm.

15. The method according to any one of claims 1 to 14, wherein the concentration in step (vi) comprises a first step of desolventization and a second step of concentration.

16. The method according to any one of claims 1 to 15, further comprising a step of pasteurization, cooling and filtration after step (vi).

17. A fermented extract from carob pod obtainable by the method according to any one of claims 1 to 16, wherein the fermented extract comprises D-pinitol at a concentration of at least 24% weight on a dry matter basis.

18. A pharmaceutical composition comprising the fermented extract according to claim 17.

19. The fermented extract according to claim 17 for its use in medicine.

20. The fermented extract according to claim 17 for use in the treatment and / or prevention of type 2 diabetes mellitus and metabolic syndrome.

21. A food product or a nutritional supplement comprising the fermented extract according to claim 17.

22. Use of the fermented extract according to claim 17 as a functional ingredient in foods or in nutritional supplements.

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