EP4340641A1 - Nutraceutical composition with probiotics, prebiotics and palm date pulp - Google Patents

Nutraceutical composition with probiotics, prebiotics and palm date pulp

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Publication number
EP4340641A1
EP4340641A1 EP22730976.2A EP22730976A EP4340641A1 EP 4340641 A1 EP4340641 A1 EP 4340641A1 EP 22730976 A EP22730976 A EP 22730976A EP 4340641 A1 EP4340641 A1 EP 4340641A1
Authority
EP
European Patent Office
Prior art keywords
composition according
strains
composition
group
intestinal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22730976.2A
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German (de)
English (en)
French (fr)
Inventor
Paolo Grieco
Ettore Novellino
Gian Carlo Tenore
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Pharma Biomateck Srl
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Pharma Biomateck Srl
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Publication date
Application filed by Pharma Biomateck Srl filed Critical Pharma Biomateck Srl
Publication of EP4340641A1 publication Critical patent/EP4340641A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A23L33/22—Comminuted fibrous parts of plants, e.g. bagasse or pulp
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention concerns a nutraceutical composition based on a combination of biologically active compounds.
  • the present invention originates in the field of nutritional products and dietary supplements.
  • the present invention concerns a composition based on selected biologically active ingredients of natural origin whose combination results in an advantageous effect on the human body with benefits at the intestinal, immune, and epidermal levels.
  • lacto-fermented products is a production trend that in recent years has seen an exponential increase in the food/nutraceutical industry, thanks to the various beneficial effects of fermented product, i.e. greater digestibility, increase in vitamin content, release of bioactive molecules from the undigested food matrix, production of molecules with beneficial effects for humans (for example, butyric acid, lactic acid, etc.) by the active yeasts in the product, a non-dairy source of lactic bacteria.
  • milk is a typical growth medium for the development of lactic acid bacteria (LAB)
  • different strains have shown sufficient growth in different media and are able to maintain their content above 10 6 CFU mL 1 during the storage period (6°C/21 days).
  • the palm date is a microbiologically acceptable culture medium and, based on the sensory evaluation, is endowed with an attractive overall acceptability and represents an adequate alternative for celiac patients, people suffering from allergies to proteins of milk, or lactose intolerance.
  • CN104256648A claims a composition comprising elements of traditional Chinese medicine with multiple probiotics in addition to other components, such as peptides, prebiotics, amino acids.
  • CN107495049 describes the probiotic preparation of a drink based on fermented fruit and vegetables with a specific laxative function.
  • WQ2017126959 describes a fermented food based on starch and fermenting organisms, i.e. bacteria or other fermenting agents. The health effect of probiotics, dietary fiber and/or probiotics is described.
  • CN110973434A reports a solid drink having algae as a basic component for hair care purposes.
  • WO202Q/181096 describes a formulation with the aim of improving tannins metabolism by combining components capable of metabolizing tannins.
  • the formulation involves the use of probiotics, prebiotics, and dietary fibers combined with each other also in multilayer, but with the precise purpose of accelerating tannins metabolism.
  • US2007160589 describes the preparation of a single portion probiotic food product, substantially solid at room temperature.
  • the fermented preparation contains a dry active probiotic culture dispersed in a fully enclosing and substantially continuous fat-based coating.
  • Lactic ferments represent a ubiquitous and heterogeneous species having as common characteristic the production of lactic acid due to the metabolism of sugars, which leads to an acidification of the environment up to a pH of 3.5.
  • Lactic acid bacteria are Gram positive, non-sporulating, non-pigmented and non-motile bacteria, most of which are non-respiratory but aero-tolerant anaerobes. LABs tend to be nutritionally demanding, often requiring specific amino acids, vitamins of the B group, and other growth factors. Some of them absorb oxygen through the mediation of flavoprotein oxidase, thus producing hydrogen peroxide and/or re-oxidant NADH during dehydrogenation of sugars. Cellular energy comes from the fermentation of carbohydrates to produce the main lactic acid. Since many species of lactic acid bacteria (LAB) and other food-associated bacteria have a long historical association with human food, they are identified as generally recognized as safe (GRAS) bacteria. LAB infections are characterized as opportunistic and rely on host factors rather than inherent pathogenicity. Only rare cases of clinical infections have been reported in humans, for example in patients with endocarditis or immunodeficiency.
  • Lactobacilli are commonly found in a variety of environments, in dairy and meat products, fermented and pickled vegetables, and adhere to the human mucosa surfaces (in the gastrointestinal and vaginal tracts), as well as in soil and plants.
  • Intestinal lactobacilli (L rhamnosus, L. acidophilus, L. reuteri, L. plantarum e L. paracasei) interact with the host and have been associated with numerous health benefits.
  • L. reuteri is one of the most widely used probiotic bacteria, added to the formula of infant milk for infants with lactose intolerance or for reconstitution after diarrhea.
  • Lactobacilli are naturally present in breast milk, in particular the L. fermentum, L. rhamnosus, L. gasseri and L. salivarius species. Furthermore, L. plantarum has been frequently identified in the breast milk of healthy mothers.
  • Lactic acid bacteria can produce substances and thus create harmful conditions for unwanted bacteria, yeasts and molds which lead to an increase in the shelf life of foods.
  • the main lactic acid bacteria fermentation product is lactic acid.
  • Most of the isolated and identified antimicrobial substances produced by lactic acid bacteria are low molecular weight substances made of organic acids, reuterin, hydrogen peroxide, hydroxy fatty acids, phenolic and protein compounds.
  • Probiotics are defined as organisms and substances that contribute to the intestinal microbial balance. The most frequently cited definition is that of Fuller (1992), who defined them as “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Food and Agriculture Organization/World Health Organization (FAO/WFIO), describe probiotics as live microorganisms which, when administered in adequate amounts, confer health benefits to the host (2002).
  • FEO/WFIO Food and Agriculture Organization/World Health Organization
  • the choice of which microorganism to use as a probiotic is determined by many factors: the probiotics must be safe, non-pathogenic, and non-toxic species, survive the passage through the intestinal tract and adhere to the intestinal mucosa and to the production of organic, lactic and acetic acid.
  • the viability of probiotics in food is influenced by many factors such as pH, water activity, food redox potential, the presence of salt, sugar, hydrogen peroxide, bacteriocins, flavorings agents and coloring compounds, processing conditions, packaging, and storage.
  • the mechanisms of probiotics health properties are not yet fully understood, but their anti-carcinogenic and anti-mutagenic activity, suppression of allergies, reduction of serum cholesterol level, and reduction of blood pressure are known.
  • L. bulgaricus is able to ferment lactose, therefore it has beneficial effects for people suffering from lactose intolerance.
  • reuteri have beneficial effects against acute diarrhea caused by rotavirus, in the treatment, shortening or prevention of this disease.
  • Administration of S. boulardii as a non-pathogenic biotherapeutic yeast plays an essential role in the treatment or prevention of antibiotic-associated diarrhea caused by C. difficile.
  • rhamnosus and Bifidobacterium are able to reduce the activity of the fecal enzyme that converts procarcinogens into carcinogens (b - glucuronidase, azoreductase, urease, nitroreductase and glycolic acid reductase) due to the production of short-chain fatty acids, and can therefore help reduce the risk of colorectal cancer.
  • carcinogens b glucuronidase, azoreductase, urease, nitroreductase and glycolic acid reductase
  • Today, cereals still represent one of the most important food sources of human diet and are grown for over 73% of the total harvested area. Numerous cereals are grown in different countries, including wheat, barley, oats, maize, rye, rice, and millet, which are particularly important from an economic point of view.
  • Cereals are primarily a source of carbohydrates, and therefore a good source of energy. They form about two thirds to three quarters of the dry matter. Monosaccharides are the basic components of oligosaccharides and polysaccharides, and are mostly represented in the forms of hexoses (fructose, glucose and galactose) and pentose, arabinose and xylose. Cereals are generally good sources of protein. The proportions of essential amino acids and their digestibility mainly determine the nutritional quality of proteins. Cereals can contribute to vitamin intake due to the presence of most vitamins of the B group and appreciable amounts of vitamin E. Whole cereals also contain considerable amounts of calcium, magnesium, iron, zinc, as well as lower levels of many trace elements, for example, selenium.
  • An object of the present invention therefore consists in providing a nutraceutical composition that allows to improve the general condition of the organism.
  • the invention concerns a nutraceutical composition
  • a nutraceutical composition comprising:
  • nutraceutical composition for use in the prevention and/or treatment of an intestinal or skin disease or pathological condition is described.
  • a third aspect relates to a nutraceutical composition for use in stimulating the immune system.
  • the invention therefore concerns a nutraceutical composition
  • a nutraceutical composition comprising:
  • said one or more probiotic microorganism strains are selected from the group consisting of lactic bacteria, bifidobacteria, yeast or bacilli strains.
  • said one or more lactic bacteria strains are selected from the group consisting of L. plantarum, L. bulgaricus, L. acidophilus or L. rhamnosus strains
  • said one or more bifidobacteria strains are selected from the group consisting of B. longum, B. bifidum or B. breve strains
  • said one or more bacilli strains are a B. clausii strain.
  • said one or more lactic bacteria strains are selected from the group consisting of L. plantarum SGL07, L. bulgaricus DSMZ 20081 , L. acidophilus LA3 and L. rhamnosus ATCC 11982.
  • said one or more prebiotics are one or more cereals selected from the group consisting of wheat, barley and oats, vegetables, tubers or fruits.
  • Prebiotic fibers - such as inulin, fructo- oligosaccharides (FOS) and beta-glucans - can be found in a wide variety of plant food:
  • the composition of the invention comprises a palm date extract, wherein said palm date extract is the palm date pulp in fresh, dried, powdered, liquid or any other form.
  • the inventors have surprisingly found that the combination of elements of the composition of the invention leads to a product that finds wide application also in the diet of sports subjects, since on the one hand it is an excellent source of simple sugars (primary energy source), and on the other hand, it has a higher share of bio-fusible polyphenols which, especially in sports subjects, are very important to counteract the strong oxidative stress to which they are constantly subjected, given their apparent and proven antioxidant activity.
  • the same product may also be used as a functional food in both adults and children in particular pathological conditions.
  • composition of the invention may further comprise at least one further biologically active component selected from the group consisting of kuzu, teff, micronutrients, mineral salts, and vitamins.
  • Kuzu or Kudzu (Pueraria montana lobata) is a wild climbing plant, native to Japan and belonging to the Febaceae (leguminous) family. The most used parts are the roots and, sometimes, also the leaves.
  • Teff tef ( Eragrostis tef), is a cereal from Ethiopia and Eritrea, from which a flour (Teff flour) is obtained which provides above all a supply of complex carbohydrates.
  • composition of the invention is formulated in the form of a food bar or other nutraceutical preparation.
  • composition makes the composition easy to transport and consume.
  • said one or more probiotic microorganism strains are inoculated in said palm date extract.
  • said one or more probiotic microorganism strains have a concentration in the range from 2 x 10 9 to 6 c 10 9 , preferably from 2 x 10 9 to 5 x 10 9 , more preferably of 3 x 10 9 bacterial colonies after 48 hours of fermentation.
  • said one or more probiotic microorganism strains are one or more microorganisms selected from the group consisting of Lactiplantibacillus plantarum, Lactobacillus delbruekii subsp. bulgaricus ( L . bulgaricus), Lactobacillus acidophilus and Lactinocaseibacillus rhamnosus.
  • said composition comprises from 40 to 80% of date pulp inoculated with mixtures of probiotics, cereals from 10 to 25% and the remaining part made of one or more ingredients selected from the group comprising chocolate, fruit, dried fruit, amaranth, and cocoa butter.
  • said composition comprises from 50 to 80% of date pulp inoculated with mixtures of probiotics, cereals from 10 to 25%, and the remaining part made of one or more ingredients selected from the group comprising chocolate, fruit, dried fruit, amaranth, and cocoa butter.
  • said composition comprises from 50 to 70% of date pulp inoculated with mixtures of probiotics, cereals from 15 to 20%, and the remaining part made of one or more ingredients selected from the group comprising chocolate, fruit, dried fruit, amaranth, and cocoa butter.
  • said composition is in liquid, solid or semi-solid form suitable for oral administration and is in the form of tablets, capsules, granules, effervescent granules, pearls, drops, sticks, syrups, and sachets.
  • the composition can be in the form of a food bar comprising: from 40 to 80% of date pulp inoculated with mixtures of probiotics, from 10 to 15% of cereals, and the remaining part made of one or more ingredients as described above.
  • composition in the form of a food bar comprises from 50 to 70% of date pulp inoculated with mixtures of probiotics, from 15 to 20% of cereals, and the remaining part made of one or more ingredients selected from amaranth, almond, and Cocoa butter.
  • the food bar may comprise: 70% of dates inoculated with mixtures of probiotics (for example pulp of Phoenix dactylifera L), 20% of cereals (buckwheat, quinoa, millet, rice, corn flakes), amaranth, almond, and cocoa butter, as a total of 10%.
  • probiotics for example pulp of Phoenix dactylifera L
  • cereals buckwheat, quinoa, millet, rice, corn flakes
  • amaranth almond
  • cocoa butter cocoa butter
  • the probiotic microorganism in the probiotic mixture may be selected from the group consisting of lactic bacteria, bifidobacteria, yeast or bacilli strains, preferably it is selected from the group consisting of L. plantarum, L. bulgaricus, L. acidophilus or L. rhamnosus strains, said one or more bifidobacteria strains are selected from the group consisting of B. longum, B. bifidum or B. breve strains, and said one or more bacilli strains are a B. clausii strain.
  • said one or more strains of lactic bacteria are selected from the group consisting of L.
  • the bar may be covered with a thin layer of dark chocolate and does not contain gluten.
  • the composition may vary in the protein component, to meet the nutritional needs of the subjects it is intended for, and in the nature of the probiota, that is, in the microorganism to be delivered.
  • the bar may be prepared for the nutritional needs of a sports subject, an adult, an elderly, for a child, for a woman in gestation or post-partum state.
  • the bar may or may not be covered with chocolate, which may be dark, milk or white chocolate.
  • the bar may also be covered with toasted seeds, for example sesame seeds or other, or covered with dried fruit flakes such as coconut flakes.
  • the bars of the invention are functional snacks comprising different species of probiotics.
  • a nutraceutical composition for use in the prevention and/or treatment of an intestinal or skin disease or pathological condition is described.
  • said intestinal disease or pathological condition is selected from the group consisting of intestinal dysbiosis, intestinal irregularity, diarrhea, irritable bowel syndrome, diverticulosis, and wherein said skin disease or pathological condition is selected from the group consisting of dermatitis or eczema, and adjuvant sports activities.
  • Beta-glucans are interesting because they are polysaccharides characterized by a very low digestibility, but are favorably metabolized by the intestinal microbiota, in particular by bifidobacteria, precious allies of our large intestine. Thanks to the work done on the intestinal microbiota, these polysaccharides also affect the metabolic functions, in particular by promoting the balance of bad cholesterol levels, reducing the absorption of sugars, controlling blood sugar levels, and favorably modulating the immune system. Beta-glucans can be soluble, with a low molecular weight, or insoluble, with a high molecular weight.
  • the soluble ones are preferentially found in barley and oats, and once ingested they can be fermented in the colon, thus populating the intestinal microbiota.
  • Insoluble beta-glucans reach and pass unaltered the ileocecal valve and are metabolized by the intestinal microbiota, thus producing short-chain fatty acids, very useful for nourishment and trophism of the intestinal mucosa.
  • insoluble beta-glucans can in turn be fragmented by the microbiota giving rise to soluble beta-glucans of reduced size and low molecular weight that perform the precious immune system modulation activity.
  • beta-glucans EFSA expressed itself stating that they help reduce blood cholesterol, and consequently cardiovascular disease.
  • beta-glucans are able to activate the immune system in a gentle and balanced way, by stimulating the activity of phagocytes.
  • dates are rich in beta-glucans, in particular beta-D-glucan which, thanks to its molecular weight, has the characteristics of being both soluble and insoluble. Its solubility allows beta-D-glucan to bind to dietary cholesterol and remove it from the body through the stools.
  • beta-D-glucan helps maintain blood glucose levels by slowing the rate at which the small intestine absorbs simple sugars.
  • Beta-D-glucan being insoluble also in nature, promotes gut health and regularity by helping to produce larger, softer stools that are easy to eliminate (b-Glucan as a Food Ingredient, Asif Ahmad, Arabic Kaleem, in Biopolymers for Food Design, 2018).
  • Men and women under the age of 50 are generally advised to consume 38 and 25 grams of total fiber per day, respectively. Since most seniors require fewer calories, men and women over the age of 50 have correspondingly less fiber requirements than 30 grams and 21 grams, respectively. Dietary guidelines suggest that consuming a wide range of high-fiber foods and adhering to daily intake recommendations increase the chances of getting sufficient amounts of soluble and insoluble fiber.
  • a third aspect concerns a nutraceutical composition for use in stimulating the immune system. It has in fact been seen that debilitated subjects show benefits following administration of the composition of the invention.
  • polyphenols Health benefits exerted by polyphenols are usually attributable to their antioxidant and anti-inflammatory properties, as well as to the antitumor and antimicrobial properties that they can exert locally, when they act directly during the passage through the gastrointestinal tract, and systemically, after their absorption.
  • Biotransformation of polyphenols into their metabolites by the gut microbiota and modulation of the gut microbiota composition by polyphenols contribute to positive health outcomes.
  • polyphenols have been included in the list of compounds with prebiotic activity, as they are capable of “modeling” the intestinal microbial communities, through inhibition of pathogenic bacteria and stimulation of beneficial bacteria. Through the modulation they exert on the microbiota, they can also act on the central nervous system. To exert all these beneficial effects, their absorption in the intestinal tract and their bioavailability in the circulatory system is necessary. When not absorbed in the small intestine, polyphenols reach the colon and are extensively biotransformed by the resident microbiota. This step can improve their absorption and bioavailability (1-6).
  • Several studies have shown how polyphenols impact the intestinal microbiome. In the literature there are more than 30 studies, carried out in vitro and in vivo, in which the importance of polyphenols on the human microbiome has been shown.
  • Lactobacillus and Bifidobacterium has been shown to be significantly increased by 220% (6.90-7.40 Iog10 CFU/g of stool) and 56% (8.08- 8.27 Iog10 CFU/g of stool), respectively, in the presence of added polyphenols.
  • an increase in polyphenols entails additional benefits such as a greater presence of “good” bacteria compared to the levels of the usual diet, in particular those belonging to the Porphyromonadaceae family. This has a positive effect on the microbiota composition, creating a widespread benefit not only at the intestinal level, but also at the systemic level.
  • the combined effect of the ingredients of the present invention i.e. the use of date as a prebiotic in the presence of a suitable probiotic (for example Lactobacillus acidophilus) and additional nutritional components, has been shown to be effective in promoting, by fermentation, an increase in the production of polyphenols.
  • a suitable probiotic for example Lactobacillus acidophilus
  • composition described herein having the date as a basic component, results to be potentially effective in removing intestinal dysbiosis and in providing those nutritional components necessary to restore nutritional imbalances caused by intestinal diseases attributable to alteration of the local microbiome.
  • the preparation is an excellent source of simple sugars (primary energy source), and on the other hand it has a greater share of bio-accessible polyphenols, which are very important to counteract the strong oxidative stress to which the human body is constantly subjected in pathological or sports conditions, considering their full-blown and proven antioxidant activity.
  • nutraceutical preparation according to the present invention is also active in giving a therapeutic advantage to the body, without the need to use medicines.
  • An object of the present invention therefore consists in providing a nutraceutical composition that enables an improvement in the general condition of the organism. Examples of embodiments of the present invention, provided for illustrative purposes, are reported below.
  • Example 1 Preparation of a composition according to the invention The composition according to the invention was prepared using different lactobacilli strains.
  • L. plantarum SGL07, L. bulgaricus DSMZ 20081 , L. acidophilus LA3 and L. rhamnosus ATCC 11982 strains were reactivated by culturing them twice in MRS broth (10.0 gL 1 meat peptone; 20.0 gL 1 dextrose; 5.0 gL 1 yeast extract; 10.0 gL 1 beef extract; 2.0 gL 1 disodium phosphate; 5.0 gL 1 sodium acetate; 2.0 gL 1 ammonium citrate; 0.1 gL 1 magnesium sulfate; 0.05 gL 1 manganese sulfate; 1.0 gL 1 Tween 80) (Thermo scientific, Whalthan, Massachusetts, USA) at 37°C for 18 h to obtain 10 8 cells/mL.
  • MRS broth 10.0 gL 1 meat peptone; 20.0 gL 1 dextrose; 5.0 gL 1 yeast extract; 10.0 gL 1 beef extract; 2.0 gL
  • the bacterial cultures were centrifuged and washed in sterile physiological solution (8.5 gL 1 NaCI) and resuspended in 5 mL of the same solution.
  • Fresh date palm fruits sourced locally were deboned and ground.
  • the composition (mixture) was obtained from 200 g of ground date pulp, 100 g of cereals (variable amounts of wheat, barley, oats, corn, rye, rice, and millet) strains grown for 18 h (final concentration> 106 CFU) and sterile water, which were mixed in a mixer for 2 minutes.
  • the different mixtures were subsequently fermented.
  • the mixtures obtained were placed in plastic bags and incubated at 37°C for 48 hours. After this time, the pH was measured followed by drying the sample down to a moisture content of 15%.
  • the extrusion was performed using a laboratory single screw extruder S-45 (Metalchem Gliwice) after the fermentation time.
  • the different compositions according to the invention were used to prepare Lactofermented Food Bars (LDB).
  • LLB Lactofermented Food Bars
  • a food bar was prepared with:
  • the probiotic microorganism may be selected from the group consisting of lactic acid bacteria, bifidobacteria, yeast or bacilli strains, preferably it is selected from the group consisting of L. plantarum, L. bulgaricus, L. acidophilus or L.
  • said one or more bifidobacteria strains are selected from the group consisting of B. longum, B. bifidum or B. breve strains, and said one or more bacilli strains are a B. clausii strain.
  • said one or more lactic bacteria strains are selected from the group consisting of L. plantarum SGL07, L. bulgaricus DSMZ 20081 , L. acidophilus LA3 and L. rhamnosus ATCC 11982.
  • Bars may be covered with a thin layer of dark chocolate and do not contain gluten.
  • the chocolate coating may vary, for example, from 15 to 25% of the final weight.
  • composition may vary in the protein component to meet the nutritional needs of the subjects it is intended for, and in the nature of the probiota, that is, in the microorganism to be delivered.
  • the bar may or may not be covered with chocolate, which may be dark, milk or white chocolate.
  • CFU colony forming units
  • Serial dilutions were prepared in sterile physiological solution prior to plating on MRS agar. The plates were incubated at 37°C for 48 hours in an anaerobic system (Oxoid, Milan, Italy).
  • the samples were placed in an ultrasonic bath for an additional 10 min, before being centrifuged at 6000 rpm for 10 min. Supernatants were collected and stored in the dark at 4°C. The pellets obtained were re-extracted, as described above, with 40 mL of the same solvent mixture. Finally, the obtained extracts were filtered under vacuum, the methanolic fraction was eliminated by evaporation and the aqueous fraction was lyophilized.
  • Example 3 HPLC-MS analysis The date bars extracts were solubilized with 1% formic acid. The analyzes were performed on a Jasco Extrema LC-4000 system (Jasco Inc., Easton, MD) equipped with a photodiode array detector (DAD). The selected column was a Kinetex® C18 column (250 mm c 4.6 mm, 5 pm; Phenomenex, Torrance, CA). The analyzes were performed at a flow rate of 1 mL/min, with solvent A (2% acetic acid) and solvent B (0.5% acetic acid in acetonitrile and water 50:50, v/v).
  • Total phenol content was determined by the Folin-Ciocalteau method, using gallic acid as the standard (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 0.1 mL of samples (suitably diluted with water to obtain an absorbance value within the linear range of the spectrophotometer) were subjected to the addition of: 0.5 mL of Folin-Ciocalteau (Sigma-Aldrich, St. Louis , MO, USA) and 0.2 mL of a 7% (%w/v) Na2C03 aqueous solution, bringing to the final volume of 10 mL with water. After mixing, the samples were kept in the dark for 90 min. After the reaction period, absorbance was measured at 760 nm. Each sample was analyzed in triplicate and the total polyphenol concentration was calculated in terms of Gallic Acid Equivalents (GAE).
  • GAE Gallic Acid Equivalents
  • Antioxidant activity DPPH dosage
  • the samples antioxidant activity was measured against the radical scavenging capacity of the antioxidants present in the standard sample using the stable 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical (Sigma-Aldrich St. Louis, MO, USA).
  • the analysis was performed by adding 100 pL of each sample to 1000 pL of a methanolic solution of DPPH (153 mmol L 1 ).
  • the decrease in absorbance was determined with a UV-visible spectrophotometer (Beckman, Los Angeles, CA, USA).
  • Absorbance of the antioxidant- free DPPH radical, i.e. control was measured as a base. All determinations were in triplicate.
  • the inhibition was calculated according to the formula: [(Ai - Af) / Ac] c 100].
  • the four acid lactic bacteria strains used were able to grow in palm date bar media without external prebiotic supplementation or pH adjustment, reaching a concentration between 3.1 and 9.9 x 10 9 CFU/g after 48 hours of incubation (Table 1).
  • the shelf-life analysis carried out consisting of 4 weeks of storage at 4°C, indicated that cell survival was easily affected by storage, and the fermented product maintains a functional probiotic concentration (approximately 10 9 CFU/g) able to exert beneficial effects for those making use of it.
  • As for the pH it is decreased by about a single unit (with a slight difference depending on the bacterial strain used) and remains almost unchanged after the storage time.
  • the Folin-Ciocalteau assay was performed on hydroalcoholic extracts of LDB and control (unfermented bars). With this assay the non specific quantitative variation of polyphenolic compounds in hydroalcoholic extracts can be evaluated, so that the variation in terms of extractable polyphenols can be evaluated.
  • Uninoculated date bars showed a total phenol content of 44.86 mg GAE (gallic acid equivalent). This value increased after 48 hours of incubation with lactic acid bacteria and its positive variation changes in a specific way depending on the strain. As shown in Table 2, the bars fermented with L.
  • LDB products have, after fermentation, a concentration of probiotics well above the minimum recommended daily probiotic intake.
  • the L. rhamnosus strain has been shown to grow best in date pulp, using date components as a nutrient source for its growth and development, reaching a LDB concentration of 4.9 x 10 9 CFU/g.
  • Our results are in line with the results of other authors, who indicated the L. rhamnosus strain as the strain most able to survive in unfavorable conditions such as fruit or vegetable juice.
  • This latter probiotic potential of the formulated products is in line with another relevant nutraceutical feature thereof: the increase in the bioaccessible polyphenolic fraction.
  • This molecule can exert healthier promoting effects than its precursor and, by virtue of its smaller polarity and size, is better absorbed in the small intestine than its precursors.
  • the increase obtained in cinnamic acids i.e. ferulic and caffeic acids
  • feruloyl esterase enzymes EC 3.1.1.73
  • EC 3.1.1.73 feruloyl esterase enzymes
  • Tannase or tannin acyl-hydrolase (EC 3.1.1.20) catalyzes the hydrolysis of the ester bonds present in the hydrolysable tannins and in the esters of gallic acid, thus releasing glucose and gallic acid.
  • the fermented version was also enriched in quercetin, a non-polar polyphenol widely absorbed in the intestine. Microbial a- rhamnoside (largely produced by L.
  • rhamnosus can hydrolyze quercitrin (quercetin 3- O-rhamnoside) in its aglycone (quercetin) form, but also rutin can be hydrolyzed by a- rhamnosidase to produce quercetin-3-O-glucoside, and then further hydrolyzed by a- glucosidase (bacterial enzyme) to release quercetin.
  • quercitrin quercetin 3- O-rhamnoside
  • rutin can be hydrolyzed by a- rhamnosidase to produce quercetin-3-O-glucoside, and then further hydrolyzed by a- glucosidase (bacterial enzyme) to release quercetin.
  • quercitrin quercetin 3- O-rhamnoside
  • rutin can be hydrolyzed by a- rhamnosidase to produce quercetin-3-O-glucoside, and then further hydrolyze
  • LDB products and in particular those with L. rhamnosus, may be provided as a prototype of functional food, mainly indicated for nutrition and athletic supplementation.
  • the product thus structured is to be considered bifunctional, since after the biotransformation the rate of free and simple phenolic compounds, which are more absorbable in the intestine and at the same time act as a vector for probiotics, is increased.
  • the higher amounts of free phenolic compounds can be an unparalleled ally to counteract the strong oxidative stress to which athletes or subjects in particular pathological conditions are constantly subjected; the high level of glucose is an excellent source of energy, and the high levels of minerals can balance the loss of minerals during sports activities or following systemic diseases.
  • live probiotics may provide athletes with secondary health benefits that could positively affect athletic performance through improved recovery from fatigue, improved immune function, and maintenance of healthy gastrointestinal and upper respiratory tract function.
  • probiotic supplementation to counteract the high level of oxidative stress and to increase muscle strength and endurance.
  • composition of the present invention has proved surprisingly and advantageously suitable for use in the prevention and/or treatment of an intestinal or skin disease or pathological condition.

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EP22730976.2A 2021-05-19 2022-05-19 Nutraceutical composition with probiotics, prebiotics and palm date pulp Pending EP4340641A1 (en)

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