WO2021234552A1 - Formulation of a fermented vegetable blend based on sprouted brown rice - Google Patents

Formulation of a fermented vegetable blend based on sprouted brown rice Download PDF

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WO2021234552A1
WO2021234552A1 PCT/IB2021/054242 IB2021054242W WO2021234552A1 WO 2021234552 A1 WO2021234552 A1 WO 2021234552A1 IB 2021054242 W IB2021054242 W IB 2021054242W WO 2021234552 A1 WO2021234552 A1 WO 2021234552A1
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Prior art keywords
formulation
mixture
cmr
weight
strains
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PCT/IB2021/054242
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French (fr)
Inventor
Andrea Buffolo
Francesco Saverio VESSIO
Pia Tonin
Cinzia Lucia RANDAZZO
Alessandra PINO
Cinzia CAGGIA
Nunziatina RUSSO
Simone RONSISVALLE
Ignazio BARBAGALLO
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Mister Bio Food Srl
Frescolat Srl
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Priority to EP21731291.7A priority Critical patent/EP4152936A1/en
Publication of WO2021234552A1 publication Critical patent/WO2021234552A1/en

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    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/36—Vegetable material
    • A21D2/38—Seed germs; Germinated cereals; Extracts thereof
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D10/00—Batters, dough or mixtures before baking
    • A21D10/002—Dough mixes; Baking or bread improvers; Premixes
    • A21D10/005—Solid, dry or compact materials; Granules; Powders
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/045—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with a leaven or a composition containing acidifying bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • 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
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • 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
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/152—Cereal germ products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei

Definitions

  • the object of the present invention is a new formulation based on sprouted brown rice, fermented through the use of lactobacilli strains, ascribed to the L paracasei species, isolated from a mixture of sprouted brown rice compositions, for use in food products, dermocosmetics and/or pharmaceutical formulations.
  • the invention further relates to formulations comprising mixtures based on sprouted brown rice and strains of lactobacilli of the L paracasei species for use in food products, dermocosmetics and/or pharmaceutical formulations.
  • the germination of rice is configured as a process of activation of vital and metabolic processes necessary for seed reproduction, which allows to obtain a naturally lactose-free and easily digestible creamy matrix.
  • the lipid fraction is modest and, unlike that of cow’s milk, cholesterol-free, very low in saturated fats and rich in polyunsaturated fatty acids.
  • Rice vegetable drink is enjoying some commercial success for many reasons, including: a pleasant taste (given by the richness in simple sugars), low allergenicity, absence of lactose, cholesterol, gluten and saturated fatty acids, a lower environmental impact, and the vegetable origin (Caceres, P. J., Martinez- Villaluenga, C., Amigo, L., & Frias, J. (2014). Maximising the phytochemical content and antioxidant activity of Ecuadorian brown rice sprouts through optimal germination conditions. Food Chemistry, 152, 407-414). Examples of rice-based vegetable drinks are described in the patent application EP3210479A1.
  • the hydrolytic enzymes determine hydrolysis of starch, non-starch polysaccharides and proteins with formation of simple sugars with a low glycemic index, such as glucose and maltose, peptides, and amino acids. Germination also involves the production of bioactive components with antioxidant properties, such as ascorbic acid, tocopherols, tocotrienols and phenolic compounds, as well as increase in the concentration of g-aminobutyric acid, dietary fiber, ferulic acid, tocotrienols, magnesium, potassium, and zinc (Kayahara H, Tsukahara K (2000) Flavor, health and nutritional quality of pre-germ inated brown rice. Presented at 2000 Int Chem Congr Pac Basin Soc in Hawaii, December 2000).
  • the formulation of a fermented product based on sprouted brown rice can allow to expand the limited range of non-dairy origin fermented products on the market, satisfying consumers’ demands for natural, gluten-free and vegan products.
  • the fermentation carried out by lactic bacteria can improve the health beneficial properties of g-aminobutyric acid, as well as increase the concentration of nutrients and bioactive compounds by improving the functional characteristics of cereals, for example in the production of vegetable drinks (Waters, D. M., Mauch, A., Coffey, A., Arendt, E. K., & Zannini, E. (2015). Lactic Acid Bacteria as a Cell Factory for the Delivery of Functional Biomolecules and Ingredients in Cereal-Based Beverages: A Review. Critical Reviews in Food Science and Nutrition, 55(4), 503-520).
  • sprouted brown rice is considered, compared to unmalted cereals, an excellent substrate for the formulation of fermented products, and especially vegetable drinks, to date scientific evidence as well as food products available on the market are obtained using other types of cereals, such as oats, maize, non-brown rice, barley and sorghum, or a mixture thereof, using different strains of lactobacilli for the fermentation, such as Lactobacillus plantarum, reuteri and acidophilus, Streptococci and commercially available yeasts (Freire, A. L., Ramos, C. L., & Schwan, R. F. (2017).
  • W02020/031143A1 describes an acetic fermentation process of sprouted brown rice, based on the use of bacteria of the Acetobacter species or lactic bacteria, such as Lactobacillus bulgaricus or Streptococcus thermophilus, to obtain acidic ingredients used as components of vegan food preparations based on cereals.
  • Patent applications CN107593837 and CN108812773A describe a method for producing bread from sprouted brown rice, where such rice is heat- treated, hydrolyzed, and micronized.
  • KR20140102001A describes a method for extracting the water-soluble GABA component from sprouted brown rice and obtaining a healthy functional composition for diabetic patients.
  • sprouted brown rice is subjected to heat treatment, hydrolyzed with amylase, and pulverized.
  • CN110917061A describes a composition comprising an ergothioneine extract, sprouted brown rice fermentation filtrate, and acetyl chitosamine, and use thereof in cosmetic applications.
  • the sprouted brown rice used during its preparation is pulverized, heated, and hydrolyzed.
  • CN104222797 describes a method of preparing rice noodles based on thermally treated, hydrolyzed, and pulverized sprouted brown rice, with particular nutritional characteristics for children.
  • Fermentation operated by lactic bacteria improves, in fact, the functional characteristics of various cereals, including brown rice, through the production of bioactive substances and metabolization of substances harmful to the human body (Waters, Mauch, Coffey, Arendt, & Zannini, (2015). Lactic Acid Bacteria as a Cell Factory for the Delivery of Functional Biomolecules and Ingredients in Cereal-Based Beverages: A Review. Critical Reviews in Food Science and Nutrition, 55(4), 503-520).
  • sprouted whole grains have recently been shown to be excellent substrates for growing lactic bacteria, better than unmalted grains (Nsogning Dongmo, Procopio, Sacher, & Becker, (2016). Flavor of lactic acid fermented malt based beverages: Current status and perspectives. Trends in Food Science & Technology, 54 37-51), to date only a few commercial fermented products based on sprouted brown rice are available on the market.
  • the bacteria strains available on the market and used in the fermentation processes of mixtures based on sprouted whole grains are able to originate the production of nutrient active substances necessary to obtain food preparations with improved organoleptic characteristics.
  • the mixtures fermented using commercially available bacterial strains usually involve maintenance of phytic acid present in the seed, a substance that is not digestible by humans and able to chelate nutrients, and therefore make some important microelements such as zinc and iron, and to a lesser extent also macro elements such as calcium and magnesium, non-absorbable.
  • strains have shown excellent technological and functional characteristics, also when used in formulations including mixtures of sprouted brown rice to obtain food preparations, dermocosmetics and/or pharmaceutical formulations, in the absence of fermentation.
  • strains isolated and selected from the mixtures produced by the inventors showed excellent technological characteristics, such as excellent proteolytic, lipolytic, acidifying and coagulant activities, the ability to produce diacetyl and exopolysaccharides, and suitability for lyophilization, characteristics that allow their use in the preparation of food preparations with optimal organoleptic characteristics for use in different dietary regimes.
  • strains showed excellent functional performances, in terms of resistance to lysozyme, resistance to low pH values, resistance to bile and gastrointestinal digestion, an antimicrobial activity, an antioxidant and anti-inflammatory activity, which allow their use in the preparation of dermocosmetic formulations and pharmaceutical formulations.
  • the components of sprouted brown rice used in the mixture of the present invention were not freeze-dried and then reconstituted, but simply micronized and partly heat-treated and/or hydrolyzed.
  • the heat treatment and hydrolysis of only some of the rice components present in the mixture has the purpose of reducing the total and unwanted microbial load present in the starting composition, thus making the product safer from a microbiological point of view and, at the same time, splitting the rice starches into simpler sugars, for a better subsequent lactic fermentation.
  • fermentation of the mixture with the strains of the present invention is able to significantly decrease or even cancel the concentration of phytic acid, which is considered an anti-nutrient, in the final product and make all nutrients and micronutrients present in sprouted rice grain, which have an important function in maintaining the microbiota and intestinal bacterial flora, more bioavailable and usable.
  • the present invention therefore consists in the development of a new vegetable mixture based on sprouted brown rice and the preparation of a formulation based on sprouted brown rice obtained by fermentation with lactic bacteria L paracasei CMR.V1 , CMR.V2 and CMR.V3, isolated from the above mixture.
  • An object of the present invention is therefore a rice mixture comprising two or more components, wherein at least one component consists of heat-treated and hydrolyzed, micronized sprouted brown rice; and at least one component consists of not heat-treated and not hydrolyzed micronized sprouted brown rice.
  • said mixture consists of two components of sprouted brown rice, the first heat-treated and hydrolyzed, the second not heat-treated and not hydrolyzed.
  • said mixture comprises three, four or more components of sprouted brown rice.
  • additional components can be either heat- treated and/or hydrolyzed, or neither heat-treated nor hydrolyzed; for example, a third component could only be heat-treated, while a fourth component could only be hydrolyzed, as appropriate.
  • the heat treatment and hydrolysis of the above components are processes that are performed before mixing them.
  • said hydrolysis is performed in the presence of the enzyme alpha-beta-amylase.
  • said enzyme is used in an amount comprised between 0.1% and 5% by weight, with respect to the weight of the rice subjected to hydrolysis.
  • from 20 to 100% of the total starches present in the composition of micronized sprouted brown rice are hydrolyzed during the hydrolysis.
  • the hydrolysis is carried out at a temperature comprised between 20 °C and 90 °C, preferably at a temperature between 16 °C and 50 °C, for a period of time comprised between 1 hour and 24 hours, preferably between 2 and 12 hours.
  • the heat treatment is selected from pasteurization, sterilization or thermization.
  • each of the above components of micronized sprouted brown rice is present in the mixture of the present invention in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
  • the micronized sprouted brown rice particles used in the mixture of the present invention have an average size comprised between 10 and 100 pm, preferably between 25 and 50 pm.
  • Micronization of sprouted rice grains according to the present invention can take place using systems known in the art, such as micronization with homogenizing pump, turbo emulsion, micronization with colloid mill, micronization with ball mill or micronization by compression with immersed rollers.
  • the heat treatment is carried out at a temperature higher than 40 °C, even more preferably higher than 55 °C.
  • the sterilization is carried out at a temperature comprised between 105 °C and 135 °C for a period comprised between 5 seconds and 30 minutes, more preferably at a temperature between 110 °C and 130 °C for a period between 20 seconds and 20 minutes, even more preferably at a temperature of about 121 °C for about 15 minutes.
  • the pasteurization is carried out at a temperature comprised between 60 °C and 95 °C for a period comprised between 10 and 60 minutes, preferably between 80 °C and 95 °C for a period between 10 and 20 minutes.
  • the therm ization is carried out at a temperature comprised between 50 °C and 75 °C for a period comprised between 30 minutes and 5 hours, preferably at a temperature between 50 and 55 °C for a period between 10 minutes and 2 hours.
  • heat treatment of the mixture components is performed following sprouting, and hydrolysis is performed after micronization of rice seeds.
  • the mixture of the present invention has a protein content comprised between 0.5-3 g/100 g of total mixture, a carbohydrate content between 10-30 g/100 g of total mixture and a fat content between 0.5-2.5 g/100 g of total mixture.
  • the mixture of the present invention has a protein content comprised between 1-2.5 g/100 g of total mixture, a carbohydrate content between 12-25 g/100 g of total mixture and a fat content between 0.5-1.5 g/100 g of total mixture.
  • the mixture of the present invention is incubated at a temperature comprised between 25 °C and 50 °C, preferably at a temperature between 30 °C and 42 °C.
  • the above mixture is incubated for a time comprised between 1 and 56 hours, more preferably between 10 and 24 hours.
  • a further embodiment of the present invention is the lactobacilli strain L paracasei CMR.V1 (deposit number DSM 33253, filed on October 1, 2019, at the DSMZ Center in Germany).
  • a further embodiment of the present invention is the lactobacilli strain CMR.V2 (deposit number DSM 33254, filed on October 1, 2019, at the DSMZ Center in Germany).
  • a further embodiment of the present invention is the lactobacilli strain CMR.V3 (deposit number DSM 33316, filed on October 1, 2019, at the DSMZ Center in Germany).
  • the above lactobacilli strains are isolated from the mixture according to the present invention.
  • a further embodiment is a formulation obtained from the mixture of the present invention fermented in the presence of a starter culture comprising at least one of the CMR.V1, CMR.V2 or CMR.V3 strains, and/or a mixture thereof.
  • a further embodiment of the present invention is a formulation obtained from the fermentation of a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice, and a starter culture comprising at least one of the CMR.V1 , CMR.V2 or CMR.V3 strains and/or a mixture thereof.
  • said formulation is obtained from a mixture fermented in the presence of a starter culture comprising at least two of the CMR.V1, CMR.V2 or CMR.V3 strains, more preferably said mixture is fermented in the presence of a starter culture comprising three of the CMR.V1, CMR.V2 and CMR.V3 strains.
  • said mixture is fermented in the presence of a starter culture comprising from 10 6 UFC to 10 11 UFC of at least one of the above strains, more preferably comprising between 10 7 UFC and 10 10 UFC of at least one of the above strains.
  • a starter culture comprising from 10 6 UFC to 10 11 UFC of at least one of the above strains, more preferably comprising between 10 7 UFC and 10 10 UFC of at least one of the above strains.
  • said mixture is fermented in the presence of a starter culture comprising from 10 6 UFC to 10 11 UFC of at least two of the above strains, more preferably comprising between 10 7 UFC and 10 10 UFC of at least one of the above strains.
  • a starter culture comprising from 10 6 UFC to 10 11 UFC of at least two of the above strains, more preferably comprising between 10 7 UFC and 10 10 UFC of at least one of the above strains.
  • said mixture is fermented in the presence of a starter culture comprising from 10 6 UFC to 10 11 UFC of each of the above strains, more preferably comprising between 10 7 UFC and 10 10 UFC of each of the above strains.
  • the above mixture is fermented at a temperature comprised between 20°C and 47°C, preferably at a temperature between 25°C and 42°C, more preferably at a temperature between 37°C and 40°C.
  • the above fermentation is carried out for a time comprised between 1 and 56 hours, more preferably for a time between 30 minutes and 24 hours, even more preferably for a time between 10 minutes and 12 hours.
  • the mixture of the present invention is fermented in the presence of further components selected from prebiotics, such as inulin, fructo- oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
  • prebiotics such as inulin, fructo- oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
  • the formulation obtained from the above fermentation can be in a solid, semi-solid or liquid form.
  • the formulation when in a solid form, it is in the form of a lyophilized powder or obtained by a spray-drying method, fluid bed or other forms of dehydration.
  • the formulation of the present invention is characterized by a phytic acid content lower than 1% by weight, with respect to the total weight of the formulation, preferably the above formulation is free of phytic acid.
  • said formulation is characterized in that at least one component of the rice mixture is not heat-treated and not hydrolyzed, micronized sprouted brown rice.
  • said heat treatment is selected from pasteurization, sterilization or therm ization.
  • each of the above components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
  • said mixtures have a protein content comprised between 0.5 and 3 g/100 g of total mixture, a carbohydrate content between 10 and 30 g/100 g of total mixture and a fat content between 0.5 and 2.5 g/100 g of total mixture.
  • the mixture according to the present invention is fermented in the presence of further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
  • prebiotics such as inulin, fructo-oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
  • a further embodiment of the present invention is a formulation comprising a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice, and at least one of the CMR.V1 , CMR.V2 or CMR.V3 strains and/or a mixture thereof.
  • said lactobacilli strains are in a lyophilized or liquid form.
  • at least one component of the rice mixture is not heat-treated and non-hydrolyzed, micronized sprouted brown rice.
  • said mixture consists of two sprouted brown rice components, the first heat-treated and hydrolyzed, the second not heat-treated and not hydrolyzed.
  • said mixture comprises three, four or more sprouted brown rice components.
  • additional components can be either heat- treated and/or hydrolyzed, or neither heat-treated nor hydrolyzed; for example, a third component could only be heat-treated, while a fourth component could only be hydrolyzed, as appropriate.
  • said heat treatment is selected from pasteurization, sterilization or therm ization.
  • the mixtures used to obtain said invention are obtained by the hydrolysis, micronization and therm ization processes described above.
  • said formulation is characterized in that each of the above components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
  • the above mixtures have a protein content comprised between 0.5 and 3 g/100 g of total mixture, a carbohydrate content between 10 and 30 g/100 g of total mixture and a fat content between 0.5 and 2.5 g/100 g of total mixture.
  • the formulation of the present invention is characterized by a phytic acid content lower than 1% by weight, with respect to the total weight of the formulation, preferably the above formulation is free of phytic acid.
  • the formulation according to the present invention contains further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto-oligosaccharides orxylitol, guar gum and/or mixtures thereof.
  • prebiotics such as inulin, fructo-oligosaccharides, galacto-oligosaccharides orxylitol, guar gum and/or mixtures thereof.
  • a further embodiment is the use of the formulations according to the present invention in the production of food preparations, dermocosmetic products and/or pharmaceutical formulations.
  • food preparations according to the present invention are selected from vegetable drinks, yogurt, puddings and sorbets, preparations and bases for flours, leavened products, desserts, snacks, biscuits, pizzas, focaccia, semifreddo, crepes, ice cream and cakes.
  • said food preparations comprise an amount by weight comprised between 0.1 and 99% of the formulations according to the present invention, preferably they comprise an amount by weight between 10 and 90% of the formulation, more preferably they comprise an amount by weight between 20 and 80% of the formulation. According to a further preferred embodiment, said food preparations consist entirely of the formulation of the present invention.
  • said food preparations comprise at least one further ingredient.
  • said further ingredient is selected from lipids derived from cereals or pseudo cereals, thickeners, colorants, oils, food excipients, food adjuvants, probiotics, emulsifiers, sugars, sweeteners, flavorings, spices, salt, vegetables, coffee, cocoa, edible marine algae, fruit, nuts, berries, legumes, fatty substances, legume seeds, oil seeds, protein seeds, edible herbs, natural or artificial flavors, medicinal herbs, seeds obtained from edible herbs, seeds obtained from medicinal herbs, proteins and amino acids isolated from legumes, fibers isolated from vegetables, functional active ingredients and any ingredient for use in the food industry, or a combination thereof.
  • said thickeners include substances selected from carrageenans, agar-agar, starches, carob seeds, guar, xanthan gum, white rice flour, brown rice flour, carob flour, baobab, inulin, pectin, glucomannan, tara root, kuzu root, konjak root, arrowot root, or a combination thereof.
  • said legumes and/or said legume seeds are selected from beans, broad beans, peas, lupins, chickpeas, peanuts, lentils, azuki beans (Vigna angularis), grass peas, fabaceae, trees such as acacia (Acacia), pagoda (Sophora), false acacia (Robinia pseudoacacia), carob tree (Ceratonia siliqua), or a combination thereof.
  • said edible marine algae belong to the genus selected from Undaria, Palmaria, Ecklonia, Porphyra, Sargassum, or a combination thereof.
  • said edible herbs and/or said officinal herbs are selected from dandelion (Taraxacum), garlic, aloe, bay leaf, chamomile, stevia, or a combination thereof.
  • said dermocosmetic products are selected from face and body creams, serums and masks for body and hair treatment, face cleansers, body cleansers, soothing cleansers and lavenders for feminine and masculine intimate hygiene, lip emollients, sun protection skin creams, creams for the treatment of skin lesions and scars, dermo-repairing creams, or creams for the treatment of breast fissures.
  • said dermocosmetic products comprise an amount between 0.1 and 99% by weight of the formulations according to the present invention, more preferably an amount between 10 and 90% by weight of the formulation, still more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation, and at least one physiologically acceptable excipient.
  • the above pharmaceutical formulations comprise an amount between 0.1 and 99% by weight of the formulations according to the present invention, more preferably an amount between 10 and 90% by weight of the formulation, still more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation, and at least one physiologically acceptable excipient.
  • the above food preparations, dermocosmetic products and/or pharmaceutical formulations may contain at least one further active ingredient.
  • Said further active ingredient has a dietary, alimentary, nutraceutical activity and are selected from probiotics, mineral salts, tonics, multivitamins and multiminerals, intestinal function adjuvants, vitamins, venotonics, trophic and adjuvant for joints, liver function adjuvants, antacids, eye health supplements, anti-hair loss products, immune function adjuvants, products for the urinary tract, memory and cognitive function adjuvants, multifunctional antioxidants, immune system stimulating products, omega 3, menopause products, peripheral neuropathies adjuvants, products for the treatment of calculosis, such as potassium citrate, or other specific food supplements.
  • probiotics mineral salts, tonics, multivitamins and multiminerals
  • intestinal function adjuvants vitamins, venotonics, trophic and adjuvant for joints
  • liver function adjuvants antacids
  • eye health supplements anti-hair loss products
  • immune function adjuvants products for the urinary tract
  • the active ingredients with dietary, alimentary and/or nutraceutical action are selected from vitamins, such as vitamin A, D, E, K, vitamins of group B, pantothenic acid, minerals, such as magnesium, calcium, phosphorus, iron, zinc, copper, manganese, fluorine, selenium, chromium, molybdenum, iodine, boron, potassium, chlorine, sodium and silicon salts, and other substances with a nutritional and/or physiological effect such as essential amino acids, branched amino acids, hydroxycaprylic acid (HICA), hyaluronic acid, conjugated linoleic acid (CLA), nervonic acid, alpha-ketoisocaproate (KIC), arabinogalactan, arabinoxylan, arginine-alpha-ketoglutarate (AAKG), astaxanthin, beta- alanine, betaine, beta-glucans, butyrate, caffeine, carnosine, citicoline, chlorophyll, coen
  • said further active ingredients are present in an amount comprised between 1 and 20% by weight, preferably between 5 and 15% by weight, with respect to the total weight of the formulation.
  • physiologically acceptable excipients usable for the formulations according to the present invention can be selected from diluents, lubricants, aggregation agents, disintegrating agents, film forming agents, coloring agents, sweeteners or flavoring agents, or antioxidant-antimicrobial agents.
  • the physiologically acceptable excipients usable in the formulations of the present invention are selected from sodium calcium, magnesium, potassium citrate, sodium, calcium, magnesium, potassium phosphate, light magnesium oxide, magnesium hydroxide, magnesium hydroxy carbonate, sodium carbonate, sodium chloride, potassium carbonate, sodium bicarbonate, potassium bicarbonate, adipic acid, citric acid, tartaric acid, alginic acid, stearic acid and salts thereof, oleic acid, I- leucine, glycerol behenate, hydroxypropylmethylcellulose, hydrogenated vegetable oils, such as palm oil, palm butter, cocoa butter, cocoa mass, cocoa powder, xylitol, maltitol, sorbitol, mannitol, sucralose, acesulfame K, sodium cyclamate, aspartame, sucrose, erythritol, citrus extract, fructose, dextrose, maltose, sprayed malt, sodium aspartate
  • said pharmaceutical formulations can be administered orally, topically, rectally, or vaginally.
  • the pharmaceutical form is selected from a tablet, capsule, granule, powder, oily pearl, solution, or suspension, and even more preferably said oral form is selected from a tablet, capsule, granule, powder, or solution.
  • the pharmaceutical form is selected from a cream, ointment, gel, paste, solution, wash (solution or suspension), drops, buffer (buffer solution), suspension, eye drops, spray, wipe or powder, patches, and preferably is selected from a cream, gel, spray, suppositories or ointment.
  • the pharmaceutical form is selected from cream, suppository, or an enema.
  • the pharmaceutical form is selected from a cream, ovule, wipe, or cannula.
  • said pharmaceutical formulations are for use in the prevention and treatment of vaginal infections, gastrointestinal infections and/or inflammation, dryness and irritation of the mucous membranes induced by vaginal infection, healing of wounds and burns, regeneration of internal and external epithelial tissues, restoration of the intestinal and epithelial microbiota, and reduction of inflammation.
  • physiologically acceptable excipient refers to a substance devoid of any pharmacological effects of its own, and that does not produce any adverse reactions when administered to a mammal, preferably a human being.
  • Physiologically acceptable excipients are well known in the art and are described, for example, in Handbook of Pharmaceutical Excipients, sixth edition (2009), incorporated herein by reference.
  • heat treatment means heating to a temperature above 40 °C, preferably above55 °C.
  • the term “sterilization” means a heating process carried out at a temperature comprised between 105 and 135 °C for a period comprised between 5 seconds and 30 minutes, more preferably at a temperature of 110-130 °C for a period between 20 seconds and 20 minutes, even more preferably at a temperature of about 121 °C for about 15 minutes.
  • the term “pasteurization” means a heating process carried out at a temperature comprised between 60 and 95 °C for a period comprised between 10 and 60 minutes, preferably between 80 and 95 °C for a period between 10 and 20 minutes.
  • thermoization means a heating process carried out at a temperature comprised between 50 and 75 °C for a period comprised between 30 minutes and 5 hours, preferably at a temperature between 50 and 55 °C for a period between 10 minutes and 2 hours.
  • the term “hydrolysis” means the treatment of micronized brown rice grains in the presence of alpha-beta- amylase and water to transform starches into malts and simple sugars.
  • micronization means a process of micromachining the rice grains until particles of less than 100 microns are obtained.
  • prebiotics means any substance present in food that is not absorbed by the body but is used by the intestinal flora.
  • FOS fructo-oligosaccharides
  • oligofructose oligosaccharides
  • oligofructans short chain oligosaccharides
  • galacto-oligosaccharides also known as oligogalactosyllactose, oligogalactose, oligolactose or transgalacto-oligosaccharides, means oligosaccharides made of galactose and glucose.
  • starter culture means a preparation containing selected strains of microorganisms useful for being inoculated and carrying out the fermentation process.
  • Brown rice seeds are subjected to sprouting in water. Sprouting takes place at temperatures between 10 °C and 50 °C in the presence of an amount of water between 30% and 80% by weight, with respect to the total weight of the composition. Once sprouted, the rice grains can be heat-treated with steam or in water, according to known techniques, at temperatures comprised between 40- 135 °C.
  • the sprouted rice grains (heat-treated or untreated) are subjected to a micronization treatment with a colloidal mill, in order to obtain particles with an average size comprised between 10 and 100 pm, preferably between 25 and 50 pm.
  • This step of the process is also carried out in water to limit overheating nutrients, and therefore thermo-degradation and/or oxidation thereof.
  • Sprouted and micronized rice grains can subsequently be subjected to a hydrolysis process, which is preferably carried out in water in the presence of the alpha-beta-amylase enzyme, to transform the starches into malts and simple sugars.
  • the rice grains placed in the container are steamed at a temperature of about 121 °C for 15 minutes, to reduce the microbial load present in the composition.
  • composition of sprouted rice obtained is subjected to micronization in a colloidal mill, in order to obtain particles with an average size comprised between 10 and 100 pm.
  • This step of the process is also carried out in water to limit overheating nutrients, and therefore thermo degradation and/or oxidation thereof.
  • the obtained composition (2kg) is incubated for 1 hour at 37 °C, in the presence of 2 g of alpha-beta-amylase enzyme/kg of composition, to transform the starches into malts and simple sugars.
  • Composition 2 (Matrix K)
  • the rice grains are subjected to micronization with a colloidal mill in water, in order to obtain particles with an average size comprised between 10 and 100 pm.
  • compositions of sprouted brown rice obtained as reported above are combined in a mixture, in a ratio of 5% by weight composition 1 and 95% by weight composition 2, and incubated at a temperature comprised between 37+4 °C for 16-20 hours in a suitable container.
  • Isolates were subjected to purification smears on agar medium as well as propagation on liquid medium. Each isolate was subjected to phenotypic characterization by microscope observation, Gram staining, catalase test. All non-spore-forming, Gram-positive, catalase negative isolates were propagated in MRS broth (2% v/v), under microaerophilic conditions, and stored at -80 °C in liquid medium supplemented with glycerol as cryoprotectant.
  • CMR.V1, CMR.V2 and CMR.V3 strains Through genotypic characterization, based on a study of the Tuf gene, it was possible to ascribe the strains to the Lactobacillus paracasei species. The strains were also subjected to 16S rDNA sequencing, thus confirming the belonging of the strains to the aforementioned species. The strains were therefore studied for technological, safety and functional performance by in vitro tests.
  • the technological characterization involved the study of the following performances: proteolytic activity, lipolytic activity, acidifying activity in BioSuRice® Cream, coagulating activity in BioSuRice® Cream, diacetyl production, exopolysaccharides production, suitability for lyophilization.
  • the three selected strains showed proteolytic and lipolytic activity, and excellent acidifying and coagulating activity in BioSuRice® Cream.
  • the strains were found to be producers of exopolysaccharides and diacetyl, showing, in addition, suitability for lyophilization.
  • the strains were also studied for functional performances by evaluating resistance to lysozyme, low pH values, bile, simulated gastrointestinal digestion, antimicrobial activity against pathogenic microorganisms, anti-inflammatory activity in liver cells and macrophages, antioxidant activity. Furthermore, in accordance with what is reported in the guidelines for probiotics and prebiotics, drawn up by the Ministry of Health, the strains were also subjected to an assessment of safety requirements.
  • the extracellular proteolytic activity was determined in plate by using the growth substrate Plate Count Agar (PCA, Oxoid) supplemented with 10% (w/v) of skim milk (Oxoid). Cell cultures in exponential growth phase (9 log cfu/mL) were spot-plated and incubated at 37 °C for 72 h. After incubation, 1% HCI was spread over the plates surface. Proteolytic activity was evidenced by the presence of a clear zone around the colonies. The results were expressed as + (presence of activity) and - (absence of activity).
  • the lipolytic activity was evaluated in plate using Tributyrin Agar culture medium (Merck, Germany). Cell cultures in exponential growth phase (9 log cfu/mL) were spot-plated and incubated at 37 °C for 72 h. Lipolytic activity was detected by a clear zone surrounding the growth. Results were expressed as + (presence of activity) and - (absence of activity).
  • bile salts bovine bile salts, Oxgall; Sigma-Aldrich
  • the ability of the strains to survive in the presence of bile salts was evaluated as follows.
  • the strains under study were revitalized in MRS broth, and the cell suspension in exponential growth phase (9 log cfu/mL) was inoculated in MRS broth containing the percentages of bile salts reported above.
  • MRS without bile salts was used as a control. Aliquots were collected immediately after inoculation (0 hours) and after 2 and 4 hours of incubation at 37 °C, and plate counting of viable cells was performed. The analysis was carried out in triplicate and the results are reported as mean and standard deviation. For each strain, % survival was assessed, and calculated taking into account the final (cfuF) and initial (cful) population density as (cfuF/cful *100), and the 80% value was considered as the minimum survival limit.
  • SGJ gastric juice
  • SIF simulated intestinal fluid
  • the cells were pre-treated with lipopolysaccharide (LPS) at a concentration of 100 ng/mL for 2 hours.
  • LPS lipopolysaccharide
  • the anti-inflammatory effect of the strains under study was evaluated by treating the differentiated cells with the bacterial strains conditioned media at a concentration of 10 pg/mL for 6 hours.
  • the cells were washed with PBS, collected by trypsinization, and then lysed for RNA extraction.
  • the quantification of IL-8 (lnterleukin-8) and IL-10 genes was performed by real-time qRT-PCR.
  • the strains under study were tested for the antioxidant activity using 1 ml_ of supernatant (cell-free) supplemented with 1 ml_ of PBS (0.1 M, pH 7.0) and 1 ml_ of linoleic acid (50 mM) in ethanol (99.5%). The oxidation was measured by determination of ferric thiocyanate. Butylated hydroxytoluene (BHT) and a-tocopherol (1 mg/mL) were used as positive controls. MRS broth was used as a negative control. The results are expressed as absorbance values at 500 nm. The analysis was carried out in triplicate. Table 13. Evaluation of the antioxidant activity
  • the strains tested showed antioxidant activity.
  • the strains under study were revitalized in MRS broth medium and incubated at 37 °C for 18-24 hours.
  • Cell cultures in exponential growth phase were transferred, by propagation smear, on to Blood Agar plates containing 5% defibrinated mutton blood (Biolife, Milan, Italy) and incubated at 37 °C for 24-48 h.
  • the hemolytic activity was visually detected and distinguished as b-hemolysis, a-hemolysis or g-hemolysis based on the presence of clear zones, green halos or no zone around colonies, respectively.
  • DNAse production was tested by transferring 5 pl_ of an exponentially growing cell culture onto DNAse agar plates (Oxoid). After incubation at 37 °C for 48 hours, the plates were covered with 1 N HCI for 5 minutes. The presence of clear zones around the colonies is considered as an indicator of positive DNase production.
  • Gelatinase production was evaluated using gelatin agar plates (30 g/L gelatin, 5 g/L peptone, 3 g/L yeast extract and 17 g/L agar). After incubation at 37 °C for 48 h, the surface of the plates was coated with saturated ammonium sulfate (Merck). The presence of clear zones around the colonies is considered as an indicator of positive gelatin activity. For both tests, the results were expressed as: - (absence of activity), + (presence of activity).
  • the CMR.V1, CMR.V2 and CMR.V3 strains were deposited at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH) (see attachment). The following codes were assigned:
  • DSM 33253 strain CMR.V1 DSM 33254: strain CMR.V2 DSM 33316: strain CMR.V3
  • EPS exopolysaccharides
  • nt not tested.
  • Coagulant activity - (clot absence); + (not very compact clot); ++ (compact clot); +++ (very compact clot).
  • Proteolytic and lipolytic activity + (activity presence); - (activity absence).
  • Diacetyl production - (no production); + (low production); ++ (medium production); +++ (high production).
  • EPS production - (non-producers); + (low production); ++ (medium production); +++ (high production).
  • Formulation 1 is obtained from a fermentation process of the mixture of the present invention in the presence of starter cultures containing the isolated strains CMR.V1 , CMR.V2 and CMR.V3.
  • composition 1 A mixture consisting of 40% by weight of composition 1 and 60% by weight of composition 2 (as obtained above) is fermented in the presence of a starter culture comprising 10 7 CFU of the three isolated strains of lactobacilli L paracasei CMR.V1, CMR.V2 and CMR.V3, according to the procedures and equipment known in the art, at a fermentation temperature equal to 40 + 2 °C and for a fermentation time of 12-15 hours.
  • a starter culture comprising 10 7 CFU of the three isolated strains of lactobacilli L paracasei CMR.V1, CMR.V2 and CMR.V3, according to the procedures and equipment known in the art, at a fermentation temperature equal to 40 + 2 °C and for a fermentation time of 12-15 hours.
  • the product obtained from this fermentation has a pH comprised between 4.3 and 4.6, a creamy consistency and a final density of lactobacilli comprised between 10 7 -10 9 CFU/g of mixture.
  • Formulation 2 is obtained from a fermentation process of the mixture of the present invention in the presence of a starter culture comprising the isolated strains CMR.V1 and CMR.V3.
  • composition 1 A mixture consisting of 30% by weight of composition 1 and 70% by weight of composition 2 (as obtained above) is fermented in the presence of a starter culture comprising 10 8 CFU of the isolated strains of lactobacilli L paracasei CMR.V1 and CMR.V3, according to the procedures and equipment known in the art, at a fermentation temperature equal to 42 + 2 °C and for a fermentation time of 12-15 hours.
  • the product obtained from this fermentation has a pH comprised between 4.3 and 4.6, a creamy consistency and a final density of lactobacilli comprised between 10 7 -10 9 CFU/g of mixture.
  • formulations obtained from fermentation of the different mixtures according to the present invention can be used to obtain different types of food preparations and dermocosmetic and pharmaceutical formulations, some examples of which are reported below.
  • Formulation 3 is obtained by mixing 40% of composition 1 and 60% of composition 2 mentioned above.
  • Formulation 2 (lyophilized powder) 24 g
  • component 1 Physico-chemical parameters and functional substances present in component 1 , component “K” and fermented product.
  • the chemical-physical analysis involved quantitative determination of pH, humidity, proteins, carbohydrates, fats, ashes, dietary fiber, energy value, dry matter, and sugars.
  • studies were carried out on components 1 and “K” as well as on the fermented product obtained by mixing the two components and using the autochthonous starter strains described above.
  • the determinations also concerned the quantification of functional substances such as: lactic acid, butyric acid, inositol, GABA, oryzanol.
  • the concentration of phytic acid was also determined.

Abstract

The object of the present invention is a new formulation based on sprouted brown rice, fermented through the use of lactobacilli strains, ascribed to the L paracasei species, isolated from a mixture of sprouted brown rice compositions, for use in food products, dermocosmetics and/or pharmaceutical formulations. The invention also relates to formulations comprising mixtures based on sprouted brown rice and strains of lactobacilli of the L paracasei species for use in food products, dermocosmetics and/or pharmaceutical formulations.

Description

TITLE
Formulation of a fermented vegetable blend based on sprouted brown rice.
DESCRIPTION
The object of the present invention is a new formulation based on sprouted brown rice, fermented through the use of lactobacilli strains, ascribed to the L paracasei species, isolated from a mixture of sprouted brown rice compositions, for use in food products, dermocosmetics and/or pharmaceutical formulations. The invention further relates to formulations comprising mixtures based on sprouted brown rice and strains of lactobacilli of the L paracasei species for use in food products, dermocosmetics and/or pharmaceutical formulations.
State of the art
The germination of rice is configured as a process of activation of vital and metabolic processes necessary for seed reproduction, which allows to obtain a naturally lactose-free and easily digestible creamy matrix. The lipid fraction is modest and, unlike that of cow’s milk, cholesterol-free, very low in saturated fats and rich in polyunsaturated fatty acids. Rice vegetable drink is enjoying some commercial success for many reasons, including: a pleasant taste (given by the richness in simple sugars), low allergenicity, absence of lactose, cholesterol, gluten and saturated fatty acids, a lower environmental impact, and the vegetable origin (Caceres, P. J., Martinez- Villaluenga, C., Amigo, L., & Frias, J. (2014). Maximising the phytochemical content and antioxidant activity of Ecuadorian brown rice sprouts through optimal germination conditions. Food Chemistry, 152, 407-414). Examples of rice-based vegetable drinks are described in the patent application EP3210479A1.
During rice germination process, the hydrolytic enzymes determine hydrolysis of starch, non-starch polysaccharides and proteins with formation of simple sugars with a low glycemic index, such as glucose and maltose, peptides, and amino acids. Germination also involves the production of bioactive components with antioxidant properties, such as ascorbic acid, tocopherols, tocotrienols and phenolic compounds, as well as increase in the concentration of g-aminobutyric acid, dietary fiber, ferulic acid, tocotrienols, magnesium, potassium, and zinc (Kayahara H, Tsukahara K (2000) Flavor, health and nutritional quality of pre-germ inated brown rice. Presented at 2000 Int Chem Congr Pac Basin Soc in Hawaii, December 2000).
In this context, the formulation of a fermented product based on sprouted brown rice can allow to expand the limited range of non-dairy origin fermented products on the market, satisfying consumers’ demands for natural, gluten-free and vegan products. It is in fact known that the fermentation carried out by lactic bacteria can improve the health beneficial properties of g-aminobutyric acid, as well as increase the concentration of nutrients and bioactive compounds by improving the functional characteristics of cereals, for example in the production of vegetable drinks (Waters, D. M., Mauch, A., Coffey, A., Arendt, E. K., & Zannini, E. (2015). Lactic Acid Bacteria as a Cell Factory for the Delivery of Functional Biomolecules and Ingredients in Cereal-Based Beverages: A Review. Critical Reviews in Food Science and Nutrition, 55(4), 503-520).
Although sprouted brown rice is considered, compared to unmalted cereals, an excellent substrate for the formulation of fermented products, and especially vegetable drinks, to date scientific evidence as well as food products available on the market are obtained using other types of cereals, such as oats, maize, non-brown rice, barley and sorghum, or a mixture thereof, using different strains of lactobacilli for the fermentation, such as Lactobacillus plantarum, reuteri and acidophilus, Streptococci and commercially available yeasts (Freire, A. L., Ramos, C. L., & Schwan, R. F. (2017). Effect of symbiotic interaction between a fructo-oligosaccharide and probiotic on the kinetic fermentation and chemical profile of maize mixtureed rice beverages. Food Research International, 100, 698-707.; Salmeron, I., Thomas, K., & Pandiella, S. S. (2015). Effect of potentially probiotic lactic acid bacteria on the physicochemical composition and acceptance of fermented cereal beverages. Journal of Functional Foods, 15, 106-115).
W02020/031143A1 describes an acetic fermentation process of sprouted brown rice, based on the use of bacteria of the Acetobacter species or lactic bacteria, such as Lactobacillus bulgaricus or Streptococcus thermophilus, to obtain acidic ingredients used as components of vegan food preparations based on cereals.
Patent applications CN107593837 and CN108812773A describe a method for producing bread from sprouted brown rice, where such rice is heat- treated, hydrolyzed, and micronized.
KR20140102001A describes a method for extracting the water-soluble GABA component from sprouted brown rice and obtaining a healthy functional composition for diabetic patients. In such method, sprouted brown rice is subjected to heat treatment, hydrolyzed with amylase, and pulverized.
CN110917061A describes a composition comprising an ergothioneine extract, sprouted brown rice fermentation filtrate, and acetyl chitosamine, and use thereof in cosmetic applications. The sprouted brown rice used during its preparation is pulverized, heated, and hydrolyzed.
CN104222797 describes a method of preparing rice noodles based on thermally treated, hydrolyzed, and pulverized sprouted brown rice, with particular nutritional characteristics for children.
Recently, Caceres and collaborators (Caceres, P.J., Penas, E., Martinez- Villaluenga, C., Garcia-Mora, P., Frias, J., Development of a multifunctional yogurt-like product from germinated brown rice, LWT - Food Science and Technology (2018)) evaluated the suitability of sprouted brown rice for producing a yogurt-like fermented product, obtained using a commercial starter culture containing lactobacilli and commercially available raw, wet or sprouted brown rice. The authors highlighted an increase in the concentration of GABA and inositol compounds in the fermented product compared to the starting composition, thus highlighting how fermentation increases the bioavailability of beneficial nutrients, naturally present in sprouted brown rice.
Fermentation operated by lactic bacteria improves, in fact, the functional characteristics of various cereals, including brown rice, through the production of bioactive substances and metabolization of substances harmful to the human body (Waters, Mauch, Coffey, Arendt, & Zannini, (2015). Lactic Acid Bacteria as a Cell Factory for the Delivery of Functional Biomolecules and Ingredients in Cereal-Based Beverages: A Review. Critical Reviews in Food Science and Nutrition, 55(4), 503-520).
Although sprouted whole grains have recently been shown to be excellent substrates for growing lactic bacteria, better than unmalted grains (Nsogning Dongmo, Procopio, Sacher, & Becker, (2016). Flavor of lactic acid fermented malt based beverages: Current status and perspectives. Trends in Food Science & Technology, 54 37-51), to date only a few commercial fermented products based on sprouted brown rice are available on the market.
Furthermore, not all the bacteria strains available on the market and used in the fermentation processes of mixtures based on sprouted whole grains, even within the different lactic bacteria species, are able to originate the production of nutrient active substances necessary to obtain food preparations with improved organoleptic characteristics. In particular, the mixtures fermented using commercially available bacterial strains usually involve maintenance of phytic acid present in the seed, a substance that is not digestible by humans and able to chelate nutrients, and therefore make some important microelements such as zinc and iron, and to a lesser extent also macro elements such as calcium and magnesium, non-absorbable.
The need to identify new formulations based on sprouted and fermented brown rice that allow to obtain preparations not only for food use, but also for dermocosmetic and/or pharmaceutical use, and that exploit the beneficial properties of sprouted brown rice fermented by autochthonous strains of lactobacilli, is therefore evident. DESCRIPTION OF THE INVENTION
It has been surprisingly observed that by using new mixtures of two or more components based on micronized sprouted brown rice, subjected to particular heat and hydrolysis treatments, it is possible to isolate new strains of Lactobacilli belonging to the L paracasei species, defined herein as CMR.V1, CMR.V2 and CMR.V3, and characterized by excellent technological and functional performances, when used in fermentation processes of mixtures based on sprouted brown rice.
These strains have shown excellent technological and functional characteristics, also when used in formulations including mixtures of sprouted brown rice to obtain food preparations, dermocosmetics and/or pharmaceutical formulations, in the absence of fermentation.
In particular, the strains isolated and selected from the mixtures produced by the inventors, showed excellent technological characteristics, such as excellent proteolytic, lipolytic, acidifying and coagulant activities, the ability to produce diacetyl and exopolysaccharides, and suitability for lyophilization, characteristics that allow their use in the preparation of food preparations with optimal organoleptic characteristics for use in different dietary regimes.
Furthermore, these strains showed excellent functional performances, in terms of resistance to lysozyme, resistance to low pH values, resistance to bile and gastrointestinal digestion, an antimicrobial activity, an antioxidant and anti-inflammatory activity, which allow their use in the preparation of dermocosmetic formulations and pharmaceutical formulations.
Therefore, the use of these autochthonous strains in fermentation processes of mixtures based on sprouted brown rice, or in association with these mixtures in the absence of fermentation, allows to obtain particularly advantageous preparations from a nutritional point of view, and having specific organoleptic, biological, and pharmacological properties, which can be used to obtain preparations for food, dermocosmetic and pharmaceutical use.
In particular, unlike the product proposed by Caceres and collaborators (2018), the components of sprouted brown rice used in the mixture of the present invention were not freeze-dried and then reconstituted, but simply micronized and partly heat-treated and/or hydrolyzed. The heat treatment and hydrolysis of only some of the rice components present in the mixture, has the purpose of reducing the total and unwanted microbial load present in the starting composition, thus making the product safer from a microbiological point of view and, at the same time, splitting the rice starches into simpler sugars, for a better subsequent lactic fermentation. Another great advantage over the fermented product obtained by Caceras and collaborators (2018) lies in the use in the fermentation process of new autochthonous strains, which are produced by the specific characteristics of the mixture used and isolated therefrom, instead of commercially available strains. The use of such strains and mixtures of the present invention allows to guarantee obtaining an entirely vegetable product with unique organoleptic properties compared to commercially available products.
In particular, fermentation of the mixture with the strains of the present invention is able to significantly decrease or even cancel the concentration of phytic acid, which is considered an anti-nutrient, in the final product and make all nutrients and micronutrients present in sprouted rice grain, which have an important function in maintaining the microbiota and intestinal bacterial flora, more bioavailable and usable.
The present invention therefore consists in the development of a new vegetable mixture based on sprouted brown rice and the preparation of a formulation based on sprouted brown rice obtained by fermentation with lactic bacteria L paracasei CMR.V1 , CMR.V2 and CMR.V3, isolated from the above mixture.
An object of the present invention is therefore a rice mixture comprising two or more components, wherein at least one component consists of heat- treated and hydrolyzed, micronized sprouted brown rice; and at least one component consists of not heat-treated and not hydrolyzed micronized sprouted brown rice.
According to a preferred embodiment, said mixture consists of two components of sprouted brown rice, the first heat-treated and hydrolyzed, the second not heat-treated and not hydrolyzed.
According to one embodiment of the invention, said mixture comprises three, four or more components of sprouted brown rice. Optional additional components, equally to or differently from each other, can be either heat- treated and/or hydrolyzed, or neither heat-treated nor hydrolyzed; for example, a third component could only be heat-treated, while a fourth component could only be hydrolyzed, as appropriate.
According to the present invention, the heat treatment and hydrolysis of the above components are processes that are performed before mixing them. Preferably, said hydrolysis is performed in the presence of the enzyme alpha-beta-amylase. Even more preferably, said enzyme is used in an amount comprised between 0.1% and 5% by weight, with respect to the weight of the rice subjected to hydrolysis.
According to one embodiment of the invention, from 20 to 100% of the total starches present in the composition of micronized sprouted brown rice are hydrolyzed during the hydrolysis.
According to one embodiment of the invention, the hydrolysis is carried out at a temperature comprised between 20 °C and 90 °C, preferably at a temperature between 16 °C and 50 °C, for a period of time comprised between 1 hour and 24 hours, preferably between 2 and 12 hours. According to one embodiment of the invention, the heat treatment is selected from pasteurization, sterilization or thermization.
According to a preferred embodiment, each of the above components of micronized sprouted brown rice is present in the mixture of the present invention in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
Preferably, the micronized sprouted brown rice particles used in the mixture of the present invention have an average size comprised between 10 and 100 pm, preferably between 25 and 50 pm.
Micronization of sprouted rice grains according to the present invention can take place using systems known in the art, such as micronization with homogenizing pump, turbo emulsion, micronization with colloid mill, micronization with ball mill or micronization by compression with immersed rollers.
According to a preferred embodiment of the present invention, the heat treatment is carried out at a temperature higher than 40 °C, even more preferably higher than 55 °C.
According to a further preferred embodiment of the present invention, the sterilization is carried out at a temperature comprised between 105 °C and 135 °C for a period comprised between 5 seconds and 30 minutes, more preferably at a temperature between 110 °C and 130 °C for a period between 20 seconds and 20 minutes, even more preferably at a temperature of about 121 °C for about 15 minutes.
According to a further preferred embodiment of the present invention, the pasteurization is carried out at a temperature comprised between 60 °C and 95 °C for a period comprised between 10 and 60 minutes, preferably between 80 °C and 95 °C for a period between 10 and 20 minutes. According to a further preferred aspect of the present invention, the therm ization is carried out at a temperature comprised between 50 °C and 75 °C for a period comprised between 30 minutes and 5 hours, preferably at a temperature between 50 and 55 °C for a period between 10 minutes and 2 hours.
According to the present invention, heat treatment of the mixture components is performed following sprouting, and hydrolysis is performed after micronization of rice seeds.
According to a further preferred embodiment, the mixture of the present invention has a protein content comprised between 0.5-3 g/100 g of total mixture, a carbohydrate content between 10-30 g/100 g of total mixture and a fat content between 0.5-2.5 g/100 g of total mixture.
Preferably, the mixture of the present invention has a protein content comprised between 1-2.5 g/100 g of total mixture, a carbohydrate content between 12-25 g/100 g of total mixture and a fat content between 0.5-1.5 g/100 g of total mixture.
In a preferred embodiment, during the isolation step of the lactobacilli strains, the mixture of the present invention is incubated at a temperature comprised between 25 °C and 50 °C, preferably at a temperature between 30 °C and 42 °C.
Preferably the above mixture is incubated for a time comprised between 1 and 56 hours, more preferably between 10 and 24 hours.
The above incubation conditions allow isolation of the lactobacilli strains of the present invention.
A further embodiment of the present invention is the lactobacilli strain L paracasei CMR.V1 (deposit number DSM 33253, filed on October 1, 2019, at the DSMZ Center in Germany).
A further embodiment of the present invention is the lactobacilli strain CMR.V2 (deposit number DSM 33254, filed on October 1, 2019, at the DSMZ Center in Germany).
A further embodiment of the present invention is the lactobacilli strain CMR.V3 (deposit number DSM 33316, filed on October 1, 2019, at the DSMZ Center in Germany).
In a preferred embodiment, the above lactobacilli strains are isolated from the mixture according to the present invention. A further embodiment is a formulation obtained from the mixture of the present invention fermented in the presence of a starter culture comprising at least one of the CMR.V1, CMR.V2 or CMR.V3 strains, and/or a mixture thereof.
A further embodiment of the present invention is a formulation obtained from the fermentation of a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice, and a starter culture comprising at least one of the CMR.V1 , CMR.V2 or CMR.V3 strains and/or a mixture thereof.
Preferably, said formulation is obtained from a mixture fermented in the presence of a starter culture comprising at least two of the CMR.V1, CMR.V2 or CMR.V3 strains, more preferably said mixture is fermented in the presence of a starter culture comprising three of the CMR.V1, CMR.V2 and CMR.V3 strains.
Preferably, said mixture is fermented in the presence of a starter culture comprising from 106 UFC to 1011 UFC of at least one of the above strains, more preferably comprising between 107 UFC and 1010 UFC of at least one of the above strains.
Preferably, said mixture is fermented in the presence of a starter culture comprising from 106 UFC to 1011 UFC of at least two of the above strains, more preferably comprising between 107 UFC and 1010 UFC of at least one of the above strains.
Even more preferably, said mixture is fermented in the presence of a starter culture comprising from 106 UFC to 1011 UFC of each of the above strains, more preferably comprising between 107 UFC and 1010 UFC of each of the above strains.
According to the present invention, the above mixture is fermented at a temperature comprised between 20°C and 47°C, preferably at a temperature between 25°C and 42°C, more preferably at a temperature between 37°C and 40°C.
Preferably the above fermentation is carried out for a time comprised between 1 and 56 hours, more preferably for a time between 30 minutes and 24 hours, even more preferably for a time between 10 minutes and 12 hours.
Preferably the mixture of the present invention is fermented in the presence of further components selected from prebiotics, such as inulin, fructo- oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
According to a preferred embodiment, the formulation obtained from the above fermentation can be in a solid, semi-solid or liquid form.
Preferably, when the formulation is in a solid form, it is in the form of a lyophilized powder or obtained by a spray-drying method, fluid bed or other forms of dehydration.
In a further preferred embodiment, the formulation of the present invention is characterized by a phytic acid content lower than 1% by weight, with respect to the total weight of the formulation, preferably the above formulation is free of phytic acid.
According to a preferred embodiment, said formulation is characterized in that at least one component of the rice mixture is not heat-treated and not hydrolyzed, micronized sprouted brown rice. Preferably, said heat treatment is selected from pasteurization, sterilization or therm ization.
According to a further preferred embodiment, in the formulation of the present invention, each of the above components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
According to a preferred aspect, said mixtures have a protein content comprised between 0.5 and 3 g/100 g of total mixture, a carbohydrate content between 10 and 30 g/100 g of total mixture and a fat content between 0.5 and 2.5 g/100 g of total mixture.
Preferably, the mixture according to the present invention is fermented in the presence of further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
A further embodiment of the present invention is a formulation comprising a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice, and at least one of the CMR.V1 , CMR.V2 or CMR.V3 strains and/or a mixture thereof. Preferably, said lactobacilli strains are in a lyophilized or liquid form. Preferably, at least one component of the rice mixture is not heat-treated and non-hydrolyzed, micronized sprouted brown rice.
According to a preferred embodiment, said mixture consists of two sprouted brown rice components, the first heat-treated and hydrolyzed, the second not heat-treated and not hydrolyzed.
According to one embodiment of the invention, said mixture comprises three, four or more sprouted brown rice components. Optional additional components, equally to or differently from each other, can be either heat- treated and/or hydrolyzed, or neither heat-treated nor hydrolyzed; for example, a third component could only be heat-treated, while a fourth component could only be hydrolyzed, as appropriate.
Preferably, said heat treatment is selected from pasteurization, sterilization or therm ization.
The mixtures used to obtain said invention are obtained by the hydrolysis, micronization and therm ization processes described above.
According to a preferred embodiment, said formulation is characterized in that each of the above components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
According to a preferred aspect, the above mixtures have a protein content comprised between 0.5 and 3 g/100 g of total mixture, a carbohydrate content between 10 and 30 g/100 g of total mixture and a fat content between 0.5 and 2.5 g/100 g of total mixture.
In a further preferred embodiment, the formulation of the present invention is characterized by a phytic acid content lower than 1% by weight, with respect to the total weight of the formulation, preferably the above formulation is free of phytic acid.
According to a further preferred embodiment, the formulation according to the present invention contains further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto-oligosaccharides orxylitol, guar gum and/or mixtures thereof.
A further embodiment is the use of the formulations according to the present invention in the production of food preparations, dermocosmetic products and/or pharmaceutical formulations.
Preferably, food preparations according to the present invention are selected from vegetable drinks, yogurt, puddings and sorbets, preparations and bases for flours, leavened products, desserts, snacks, biscuits, pizzas, focaccia, semifreddo, crepes, ice cream and cakes.
According to a preferred embodiment, said food preparations comprise an amount by weight comprised between 0.1 and 99% of the formulations according to the present invention, preferably they comprise an amount by weight between 10 and 90% of the formulation, more preferably they comprise an amount by weight between 20 and 80% of the formulation. According to a further preferred embodiment, said food preparations consist entirely of the formulation of the present invention.
In a further preferred embodiment, said food preparations comprise at least one further ingredient.
Preferably, said further ingredient is selected from lipids derived from cereals or pseudo cereals, thickeners, colorants, oils, food excipients, food adjuvants, probiotics, emulsifiers, sugars, sweeteners, flavorings, spices, salt, vegetables, coffee, cocoa, edible marine algae, fruit, nuts, berries, legumes, fatty substances, legume seeds, oil seeds, protein seeds, edible herbs, natural or artificial flavors, medicinal herbs, seeds obtained from edible herbs, seeds obtained from medicinal herbs, proteins and amino acids isolated from legumes, fibers isolated from vegetables, functional active ingredients and any ingredient for use in the food industry, or a combination thereof.
Preferably, said thickeners include substances selected from carrageenans, agar-agar, starches, carob seeds, guar, xanthan gum, white rice flour, brown rice flour, carob flour, baobab, inulin, pectin, glucomannan, tara root, kuzu root, konjak root, arrowot root, or a combination thereof.
Preferably said legumes and/or said legume seeds are selected from beans, broad beans, peas, lupins, chickpeas, peanuts, lentils, azuki beans (Vigna angularis), grass peas, fabaceae, trees such as acacia (Acacia), pagoda (Sophora), false acacia (Robinia pseudoacacia), carob tree (Ceratonia siliqua), or a combination thereof.
Preferably, said edible marine algae belong to the genus selected from Undaria, Palmaria, Ecklonia, Porphyra, Sargassum, or a combination thereof.
Preferably, said edible herbs and/or said officinal herbs are selected from dandelion (Taraxacum), garlic, aloe, bay leaf, chamomile, stevia, or a combination thereof.
In a further preferred aspect, said dermocosmetic products are selected from face and body creams, serums and masks for body and hair treatment, face cleansers, body cleansers, soothing cleansers and lavenders for feminine and masculine intimate hygiene, lip emollients, sun protection skin creams, creams for the treatment of skin lesions and scars, dermo-repairing creams, or creams for the treatment of breast fissures. According to a preferred embodiment said dermocosmetic products comprise an amount between 0.1 and 99% by weight of the formulations according to the present invention, more preferably an amount between 10 and 90% by weight of the formulation, still more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation, and at least one physiologically acceptable excipient.
According to a preferred embodiment, the above pharmaceutical formulations comprise an amount between 0.1 and 99% by weight of the formulations according to the present invention, more preferably an amount between 10 and 90% by weight of the formulation, still more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation, and at least one physiologically acceptable excipient.
According to a further preferred embodiment, the above food preparations, dermocosmetic products and/or pharmaceutical formulations may contain at least one further active ingredient.
Said further active ingredient has a dietary, alimentary, nutraceutical activity and are selected from probiotics, mineral salts, tonics, multivitamins and multiminerals, intestinal function adjuvants, vitamins, venotonics, trophic and adjuvant for joints, liver function adjuvants, antacids, eye health supplements, anti-hair loss products, immune function adjuvants, products for the urinary tract, memory and cognitive function adjuvants, multifunctional antioxidants, immune system stimulating products, omega 3, menopause products, peripheral neuropathies adjuvants, products for the treatment of calculosis, such as potassium citrate, or other specific food supplements.
Preferably, the active ingredients with dietary, alimentary and/or nutraceutical action are selected from vitamins, such as vitamin A, D, E, K, vitamins of group B, pantothenic acid, minerals, such as magnesium, calcium, phosphorus, iron, zinc, copper, manganese, fluorine, selenium, chromium, molybdenum, iodine, boron, potassium, chlorine, sodium and silicon salts, and other substances with a nutritional and/or physiological effect such as essential amino acids, branched amino acids, hydroxycaprylic acid (HICA), hyaluronic acid, conjugated linoleic acid (CLA), nervonic acid, alpha-ketoisocaproate (KIC), arabinogalactan, arabinoxylan, arginine-alpha-ketoglutarate (AAKG), astaxanthin, beta- alanine, betaine, beta-glucans, butyrate, caffeine, carnosine, citicoline, chlorophyll, coenzyme Q10, ubiquinol, choline, collagen, colostrum, chondroitin sulfate, creatine, dimethylglycine, enzymes such as alpha- galactosidase, bromeline, enzymes from fermented maltodextrin, lactase, beta-galactosidase, papain, superoxide dismutase, epigallocatechin gallate, phytosterols, flavonoids such as quercetin, quercitrin, rutin, spireoside, hesperidin, hesperitin or diosmin, phospholipids such as phosphatidylcholine, phosphatidylserine or phosphoserine, GABA, gamma- oryzanol, glycero-phosphoryl-ethanolamine, glycocyamine, glucomannan, glucosamine, glucuronolactone, glutathione, guar gum, hydroxytyrosol/olive polyphenols, inositol, isoflavones, lactoferrin, lactulose, lycopene, lutein, melatonin, N-acetyl-D-glucosamine, NADH, naringin, norvaline, nucleotides, fish oil (DHA-EPA), homotaurine, ornithine alpha-ketoglutarate (OKG) or palmitoylethanolamide (PEA).
In a further preferred embodiment of the present invention, said further active ingredients are present in an amount comprised between 1 and 20% by weight, preferably between 5 and 15% by weight, with respect to the total weight of the formulation.
The physiologically acceptable excipients usable for the formulations according to the present invention can be selected from diluents, lubricants, aggregation agents, disintegrating agents, film forming agents, coloring agents, sweeteners or flavoring agents, or antioxidant-antimicrobial agents. Preferably, the physiologically acceptable excipients usable in the formulations of the present invention are selected from sodium calcium, magnesium, potassium citrate, sodium, calcium, magnesium, potassium phosphate, light magnesium oxide, magnesium hydroxide, magnesium hydroxy carbonate, sodium carbonate, sodium chloride, potassium carbonate, sodium bicarbonate, potassium bicarbonate, adipic acid, citric acid, tartaric acid, alginic acid, stearic acid and salts thereof, oleic acid, I- leucine, glycerol behenate, hydroxypropylmethylcellulose, hydrogenated vegetable oils, such as palm oil, palm butter, cocoa butter, cocoa mass, cocoa powder, xylitol, maltitol, sorbitol, mannitol, sucralose, acesulfame K, sodium cyclamate, aspartame, sucrose, erythritol, citrus extract, fructose, dextrose, maltose, sprayed malt, sodium aspartate, neoesperidin, maltodextrin, inositol, inulin, brewer’s yeast, silica gel, vegetable fibers, such as pea fiber, chitosan, flavoring agents, such as essential oils, powders or the like, peppermint, spearmint or sweet mint, badian anethole, vanilla, sage, liver, sodium glutamate, fish flour, chicken, grapefruit, peach, lime, or mixtures thereof.
According to a further preferred embodiment of the present invention, said pharmaceutical formulations can be administered orally, topically, rectally, or vaginally.
Preferably, when the pharmaceutical formulations of the invention are administered orally, the pharmaceutical form is selected from a tablet, capsule, granule, powder, oily pearl, solution, or suspension, and even more preferably said oral form is selected from a tablet, capsule, granule, powder, or solution.
Preferably, when the administration of the pharmaceutical formulations of the invention is carried out topically, the pharmaceutical form is selected from a cream, ointment, gel, paste, solution, wash (solution or suspension), drops, buffer (buffer solution), suspension, eye drops, spray, wipe or powder, patches, and preferably is selected from a cream, gel, spray, suppositories or ointment.
Preferably, when the administration of the pharmaceutical formulations of the invention is carried out rectally, the pharmaceutical form is selected from cream, suppository, or an enema.
Preferably, when the administration of the pharmaceutical formulations of the invention is carried out vaginally, the pharmaceutical form is selected from a cream, ovule, wipe, or cannula.
Preferably said pharmaceutical formulations are for use in the prevention and treatment of vaginal infections, gastrointestinal infections and/or inflammation, dryness and irritation of the mucous membranes induced by vaginal infection, healing of wounds and burns, regeneration of internal and external epithelial tissues, restoration of the intestinal and epithelial microbiota, and reduction of inflammation.
DEFINITIONS
Unless otherwise defined, all terms of the art, notations, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this description belongs. In some cases, terms with meanings that are commonly understood are defined herein for clarity and/or ready reference; therefore, the inclusion of such definitions in the present description should not be construed as being representative of a substantial difference with respect to what is generally understood in the art.
The term “physiologically acceptable excipient” refers to a substance devoid of any pharmacological effects of its own, and that does not produce any adverse reactions when administered to a mammal, preferably a human being. Physiologically acceptable excipients are well known in the art and are described, for example, in Handbook of Pharmaceutical Excipients, sixth edition (2009), incorporated herein by reference.
The terms “comprising”, “having”, “including” and “containing” are to be intended as open-ended terms (/. e. , meaning “comprising, but not limited to”), and are to be considered as a support also for terms such as “consist essentially of”, “consisting essentially of”, “consist of”, or “consisting of”. According to the present invention, the term “sprouting” means the activation of vital and metabolic processes necessary for the reproduction of the seed.
According to the present invention, the term “heat treatment” means heating to a temperature above 40 °C, preferably above55 °C.
According to the present invention, the term “sterilization” means a heating process carried out at a temperature comprised between 105 and 135 °C for a period comprised between 5 seconds and 30 minutes, more preferably at a temperature of 110-130 °C for a period between 20 seconds and 20 minutes, even more preferably at a temperature of about 121 °C for about 15 minutes.
According to the present invention, the term “pasteurization” means a heating process carried out at a temperature comprised between 60 and 95 °C for a period comprised between 10 and 60 minutes, preferably between 80 and 95 °C for a period between 10 and 20 minutes.
According to the present invention, the term “thermization” means a heating process carried out at a temperature comprised between 50 and 75 °C for a period comprised between 30 minutes and 5 hours, preferably at a temperature between 50 and 55 °C for a period between 10 minutes and 2 hours.
According to the present invention, the term “hydrolysis” means the treatment of micronized brown rice grains in the presence of alpha-beta- amylase and water to transform starches into malts and simple sugars. According to the present invention, the term “micronization” means a process of micromachining the rice grains until particles of less than 100 microns are obtained. According to the present invention, the term “prebiotics” means any substance present in food that is not absorbed by the body but is used by the intestinal flora.
According to the present invention, the term “fructo-oligosaccharides” (FOS), also called oligofructose or oligofructans, means short chain oligosaccharides (fructans) present in various vegetable species, where they play the role of energy reserve.
According to the present invention, the term “galacto-oligosaccharides”, also known as oligogalactosyllactose, oligogalactose, oligolactose or transgalacto-oligosaccharides, means oligosaccharides made of galactose and glucose.
According to the present invention, the term “starter culture” means a preparation containing selected strains of microorganisms useful for being inoculated and carrying out the fermentation process.
EXPERIMENTAL SECTION
Some embodiments of the present invention are reported below purely for illustrative and non-limiting purposes.
MATERIALS AND METHODS
Preparation of compositions based on sprouted brown rice
The following steps are performed for preparing the compositions used in the invention.
Brown rice seeds are subjected to sprouting in water. Sprouting takes place at temperatures between 10 °C and 50 °C in the presence of an amount of water between 30% and 80% by weight, with respect to the total weight of the composition. Once sprouted, the rice grains can be heat-treated with steam or in water, according to known techniques, at temperatures comprised between 40- 135 °C.
Subsequently, the sprouted rice grains (heat-treated or untreated) are subjected to a micronization treatment with a colloidal mill, in order to obtain particles with an average size comprised between 10 and 100 pm, preferably between 25 and 50 pm. This step of the process is also carried out in water to limit overheating nutrients, and therefore thermo-degradation and/or oxidation thereof.
Sprouted and micronized rice grains can subsequently be subjected to a hydrolysis process, which is preferably carried out in water in the presence of the alpha-beta-amylase enzyme, to transform the starches into malts and simple sugars.
Composition 1
5 kg of brown rice seeds are subjected to sprouting in a container containing about 10 liters of water. Sprouting takes place at a temperature of 25 + 2 °C for 48 hours.
Once sprouted, the rice grains placed in the container are steamed at a temperature of about 121 °C for 15 minutes, to reduce the microbial load present in the composition.
Subsequently, the composition of sprouted rice obtained is subjected to micronization in a colloidal mill, in order to obtain particles with an average size comprised between 10 and 100 pm. This step of the process is also carried out in water to limit overheating nutrients, and therefore thermo degradation and/or oxidation thereof. The obtained composition (2kg) is incubated for 1 hour at 37 °C, in the presence of 2 g of alpha-beta-amylase enzyme/kg of composition, to transform the starches into malts and simple sugars.
Composition 2 (Matrix K)
5 kg of brown rice seeds are subjected to sprouting in a container containing about 10 liters of water. Sprouting takes place at a temperature of 25 + 2 °C for 48 hours.
Once sprouted, the rice grains are subjected to micronization with a colloidal mill in water, in order to obtain particles with an average size comprised between 10 and 100 pm.
Preparation of the mixture for strain isolation
The two compositions of sprouted brown rice obtained as reported above are combined in a mixture, in a ratio of 5% by weight composition 1 and 95% by weight composition 2, and incubated at a temperature comprised between 37+4 °C for 16-20 hours in a suitable container.
Once incubation of the mixture ended, 60 bacterial strains were isolated and screened for their technological and functional characteristics.
Among the 60 strains analyzed, those that did not have acidifying and coagulating capabilities, those that did not have proteolytic or lipolytic activity, and those that did not have the capability to produce diacetyl or exopolysaccharides were rejected. Some characteristics of the strains analyzed are shown in Table 17.
The process of isolation and characterization of the isolated strains is reported below in detail.
Isolation process of probiotic lactobacilli strains CMR.V1, CMR.V2 and CMR.V3
From the mixture based on sprouted brown rice obtained above, 60 strains of lactic bacteria were isolated at the Dipartimento di Agricoltura, Alimentazione e Ambiente dell’Universita degli Studi di Catania. The research was performed under the scientific responsibility of Prof. Cinzia Randazzo. The strains were studied from a phenotypic, genotypic, technological, and functional point of view, and based on the best technological, functional and safety performances, the new strains CMR.V1, CMR.V2 and CMR.V3 were selected and characterized. In detail, the De Man Rogosa and Sharpe agar (MRS) culture medium was used for isolation of the strains. Isolates were subjected to purification smears on agar medium as well as propagation on liquid medium. Each isolate was subjected to phenotypic characterization by microscope observation, Gram staining, catalase test. All non-spore-forming, Gram-positive, catalase negative isolates were propagated in MRS broth (2% v/v), under microaerophilic conditions, and stored at -80 °C in liquid medium supplemented with glycerol as cryoprotectant.
Characterization of CMR.V1, CMR.V2 and CMR.V3 strains Through genotypic characterization, based on a study of the Tuf gene, it was possible to ascribe the strains to the Lactobacillus paracasei species. The strains were also subjected to 16S rDNA sequencing, thus confirming the belonging of the strains to the aforementioned species. The strains were therefore studied for technological, safety and functional performance by in vitro tests.
Study of technological and probiotic performances of CMR.V1 (DSM n. 33253) CMR.V2 (DSM n. 33254) and CMR.V3 (DSM n. 33316) strains.
The technological characterization involved the study of the following performances: proteolytic activity, lipolytic activity, acidifying activity in BioSuRice® Cream, coagulating activity in BioSuRice® Cream, diacetyl production, exopolysaccharides production, suitability for lyophilization. The three selected strains showed proteolytic and lipolytic activity, and excellent acidifying and coagulating activity in BioSuRice® Cream.
The strains were found to be producers of exopolysaccharides and diacetyl, showing, in addition, suitability for lyophilization. The strains were also studied for functional performances by evaluating resistance to lysozyme, low pH values, bile, simulated gastrointestinal digestion, antimicrobial activity against pathogenic microorganisms, anti-inflammatory activity in liver cells and macrophages, antioxidant activity. Furthermore, in accordance with what is reported in the guidelines for probiotics and prebiotics, drawn up by the Ministry of Health, the strains were also subjected to an assessment of safety requirements.
In detail, hemolytic activity, ability to produce DNAse and gelatinase, as well as antibiotic resistance against various antibiotics for which EFSA has established the breakpoint values, were studied.
The properties of the autochthonous strains CMR.V1 (DSM 33253), CMR.V2 (DSM 33254) and CMR.V3 (DSM 33316) ascribed to the species L paracasei are listed below, and supported by the experimental results reported below. For convenience, these results are divided into:
Technological performances;
Functional performances; Safety performance.
Technological Performances a. Proteolytic Activity
The extracellular proteolytic activity was determined in plate by using the growth substrate Plate Count Agar (PCA, Oxoid) supplemented with 10% (w/v) of skim milk (Oxoid). Cell cultures in exponential growth phase (9 log cfu/mL) were spot-plated and incubated at 37 °C for 72 h. After incubation, 1% HCI was spread over the plates surface. Proteolytic activity was evidenced by the presence of a clear zone around the colonies. The results were expressed as + (presence of activity) and - (absence of activity).
Table 1. Evaluation of proteolytic activity
Figure imgf000030_0001
After incubation at 37 °C for 72 h, all tested strains exhibited proteolytic activity proven by the appearance of a clear zone around the colonial spot. b. Lipolytic Activity
The lipolytic activity was evaluated in plate using Tributyrin Agar culture medium (Merck, Germany). Cell cultures in exponential growth phase (9 log cfu/mL) were spot-plated and incubated at 37 °C for 72 h. Lipolytic activity was detected by a clear zone surrounding the growth. Results were expressed as + (presence of activity) and - (absence of activity).
Table 2. Evaluation of lipolytic activity
Figure imgf000030_0002
Figure imgf000031_0001
After incubation at 37 °C for 72 h, all tested strains exhibited lipolytic activity proven by the appearance of a clear zone around the colonial spot. c. Acidifying Activity
Cell cultures in exponential growth phase were transferred (1% v/v) into tubes containing 10 mL of BioSuRice® Cream and incubated at 37 °C. The pH was measured after 0, 2, 4, 6, 12, 24 and 48 hours of incubation using a pH meter. All experiments were performed in triplicate. The results are reported as change in pH (DrH).
Table 3. Evaluation of acidifying activity
Figure imgf000031_0002
All tested strains exhibited a good acidifying activity in BioSuRice® Cream proven by the significant reduction in pH over the time interval considered. d. Coagulating Activity
Cell cultures in exponential growth phase were transferred (1% v/v) into tubes containing 10 mL of BioSurice® Cream and incubated at 37 °C for 6 hours. The coagulating activity was evaluated taking into account the compactness of the clot generated after incubation at the optimal growth temperature. The results were reported as: - (absence of clot);
+ (not very compact clot);
++ (compact clot);
+++ (very compact clot).
Table 4. Evaluation of coagulating activity
Figure imgf000032_0001
After incubation at 37 °C for 6 hours, all tested strains exhibited good coagulating activity proven by the formation of a compact clot. e. Diacetyl Production
Cell cultures in exponential growth phase were transferred (1 % v/v) into sterile tubes containing UHT milk supplemented with a-naphthol (1 % w/v) and KOH (16% w/v) and incubated at 37 °C for 10 minutes. Diacetyl production is demonstrated by formation of a red ring in the upper part of the tubes. Based on the intensity of the red color present in the upper part of the tubes, the strains were classified as characterized by (all experiments were performed in triplicate):
- (no production);
+ (low production);
++ (medium production);
+++ (high production).
Table 5. Acetyl production
Figure imgf000033_0001
All strains tested exhibited the ability to produce diacetyl. f. Production of Exopolvsaccharides (EPS)
This was tested on plate using a loop touch test. Cell cultures in the exponential growth phase were propagated in De Man, Rogosa and Sharpe (MRS) agar culture medium (Oxoid). After incubation at 37 °C for 48 hours, EPS production was evaluated based on the presence of mucoid colonies. The strains were classified as:
- (non-producers);
+ (low production);
++ (medium production);
+++ (high production).
Table 6. Evaluation of exopolysaccharide production
Figure imgf000033_0002
After incubation at 37 °C for 48 hours, all tested strains exhibited the ability to produce EPS. q. Suitability for Freeze-drying
Suitability for freeze-drying of the strains under study was proven by the ability to maintain the population density unchanged after the treatment. All 3 strains were lyophilized at Veneto Agricoltura. Functional Performances
The autochthonous strains CMR.V1 (DSM 33253), CMR.V2 (DSM 33254) and CMR.V3 (DSM 33316), ascribed to the L paracasei species, have proved to possess:
Resistance to lysozyme,
Resistance to low pH values,
Resistance to bile,
Resistance during gastrointestinal digestion simulation Details of the functional characterizations carried out are reported below. a. Resistance to Lysozyme
The ability of the strains under study to survive in the presence of lysozyme was evaluated in MRS broth after 0, 30, and 120 minutes of incubation at 37 °C. Tolerance to lysozyme was determined by plate counting of viable cells. The analysis was carried out in triplicate and the results are reported as mean and standard deviation. For each strain, % survival was assessed, and calculated taking into account the final (cfuF) and initial (cful) population density as (cfuF/cful *100), and the 80% value was considered as the minimum survival limit.
Table 7. Evaluation of resistance to lysozyme
Figure imgf000034_0001
All tested strains showed the ability to survive in the presence of lysozyme, showing % survival greater than 80% after incubation at 37 °C for 120 minutes. The survival rate after 120 min is 87.1%, 86.6%, and 91.1% for CMR.V1, CMR.V2, and CMR.V3 strains, respectively. b. Tolerance to Low pH Values
The ability of the strains to tolerate low pH values was tested in MRS broth acidified at pH 2.0 and pH 3.0 by 1 M HCI. MRS at pH 6.2 was used as a control. The strains under study were revitalized in MRS broth and the cell suspension in exponential growth phase (9 log cfu/mL) was inoculated into the acidified culture medium. Aliquots were collected immediately after inoculation (0 hours) and after 2 and 4 hours of incubation at 37 °C. Tolerance to low pH values was determined by plate counting of viable cells. The analysis was carried out in triplicate and the results are reported as mean and standard deviation. For each strain, % survival was assessed, and calculated taking into account the final (cfuF) and initial (cful) population density as (cfuF/cful *100), and the 80% value was considered as the minimum survival limit.
Table 8. Evaluation of resistance to low pH
Figure imgf000035_0001
All tested strains showed the ability to survive at pH 3.0 and 2.0, showing % survival greater than 80% after incubation at 37 °C for 4 hours. After 4 hours at pH 3.0, the survival rate is 90.7%, 96.0% and 97.3% for CMR.V1, CMR.V2 and CMR.V3 strains, respectively.
After 4 hours at pH 2.0 the survival rate is 87.6%, 90.0% and 95.3% for CMR.V1, CMR.V2 and CMR.V3 strains, respectively. c. Tolerance to Bile Salts
The ability of the strains to survive in the presence of bile salts (bovine bile salts, Oxgall; Sigma-Aldrich), at final concentrations of 0.5% and 1.0% was evaluated as follows. The strains under study were revitalized in MRS broth, and the cell suspension in exponential growth phase (9 log cfu/mL) was inoculated in MRS broth containing the percentages of bile salts reported above. MRS without bile salts was used as a control. Aliquots were collected immediately after inoculation (0 hours) and after 2 and 4 hours of incubation at 37 °C, and plate counting of viable cells was performed. The analysis was carried out in triplicate and the results are reported as mean and standard deviation. For each strain, % survival was assessed, and calculated taking into account the final (cfuF) and initial (cful) population density as (cfuF/cful *100), and the 80% value was considered as the minimum survival limit.
Table 9. Evaluation of resistance to bile
Figure imgf000036_0001
All tested strains showed the ability to survive in the presence of bile salts at concentrations of 0.5% and 1 %.
After 4 hours of incubation, the survival rate to 0.5% bile salts is 82.1%, 88.6% and 89.4% for CMR.V1, CMR.V2 and CMR.V3 strains, respectively. After 4 hours of incubation, the survival rate to 1% bile salts is 83.0%, 85.5% and 88.9% for CMR.V1 , CMR.V2 and CMR.V3 strains, respectively. d. Survival during Simulated Gl Digestion
The ability of the strains under study to survive during gastrointestinal (Gl) transit was determined in vitro using simulated gastric juice (SGJ) and simulated intestinal fluid (SIF). In detail, SGJ (0.3% pepsin, 0.5% NaCI, adjusted to pH 2 by adding 1 M HCI) and SIF (0.1% pancreatin, 0.5% bile salt, 0.5% NaCI, 0.4% phenol, adjusted to pH 8 by adding 1 M NaOH) were prepared immediately before use and sterilized using a 0.22 pm cellulose acetate filter (Minisart filters, Sartorius, Gottingen, Germany). All chemicals were sourced from Sigma Aldrich (St. Louis, MO).
Cell cultures in exponential growth phase (9 log cfu/mL) were pelletized by centrifugation and resuspended in phosphate buffer (PBS). The cell suspension obtained was mixed with SGJ and incubated for 2 hours at 37 °C, under stirred microaerophilic conditions (200 rpm). The cells, pelleted by centrifugation, were suspended again in SIF, and incubated at 37 °C for 3 hours. Cells treated with SGJ and SGJ-SIF were subjected to plate counting of viable cells. The analysis was carried out in triplicate and the results are reported as mean and standard deviation. For each strain, % survival was assessed, and calculated, taking into account the final (cfuF) and initial (cful) population density, as (cfuF/cful *100), and the 80% value was considered as the minimum survival limit.
Table 10. Survival during simulated Gl digestion
Figure imgf000038_0001
All tested strains showed the ability to survive during Gl digestion simulation, exhibiting % survival greater than 80% after incubation at 37 °C. The survival rate after SIF treatment is 86.8%, 84.0% and 89.3% for CMR.V1, CMR.V2 and CMR.V3 strains, respectively. e. Antimicrobial Activity
Strains under study were tested for antagonistic activity using Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Listeria monocytogenes DSM 12464, and Salmonella enterica serovar typhimurium ATCC 14028 as target bacteria. The test was performed using the agar spot test. After incubation at 37 °C for 48 hours, the antimicrobial activity was evaluated by measuring the inhibition zone obtained. The results were expressed as:
- (no activity);
+ (inhibition zone of diameter <10 mm;
++ (inhibition zone of diameter comprised between 11 and 20 mm);
+++ (inhibition zone of diameter > 20 mm). Table 11. Evaluation of the antimicrobial activity
Figure imgf000039_0001
All tested strains showed antimicrobial activity against the tested pathogens.
The greatest antimicrobial activity against Escherichia coli was exhibited by the CMR.V3 strain. f. Anti-inflammatory Activity in Macrophages
Cell lines LX-2 and U937, used as an in vitro model of inflammation, were suspended in Dulbecco’s modified Eagle’s medium (DMEM), 1 g/L D- glucose (Gibco, Life Technologies, Milan, Italy) supplemented with 10% v/v bovine serum (FBS) (Invitrogen, Carlsbad, California, USA), 1% penicillin/streptomycin (Carlo Erba, Milan, Italy) and 60 mg/mL of gentamycin (Gibco). U937 cells were differentiated into macrophages by treatment with 200 nM PMA (Phorbol 12-myristate 13-acetate) for 72 h. In order to simulate the inflammatory process, the cells were pre-treated with lipopolysaccharide (LPS) at a concentration of 100 ng/mL for 2 hours. The anti-inflammatory effect of the strains under study was evaluated by treating the differentiated cells with the bacterial strains conditioned media at a concentration of 10 pg/mL for 6 hours. At the end of the treatment, the cells were washed with PBS, collected by trypsinization, and then lysed for RNA extraction. The quantification of IL-8 (lnterleukin-8) and IL-10 genes was performed by real-time qRT-PCR.
Table 12. Evaluation antimicrobial activity
Figure imgf000040_0001
The analysis carried out shows that all strains exhibit anti-inflammatory activity in macrophages, resulting in the reduction of pro-inflammatory interleukin IL-8 and an increase in anti-inflammatory interleukin IL-10. q. Antioxidant activity
The strains under study were tested for the antioxidant activity using 1 ml_ of supernatant (cell-free) supplemented with 1 ml_ of PBS (0.1 M, pH 7.0) and 1 ml_ of linoleic acid (50 mM) in ethanol (99.5%). The oxidation was measured by determination of ferric thiocyanate. Butylated hydroxytoluene (BHT) and a-tocopherol (1 mg/mL) were used as positive controls. MRS broth was used as a negative control. The results are expressed as absorbance values at 500 nm. The analysis was carried out in triplicate. Table 13. Evaluation of the antioxidant activity
Figure imgf000040_0002
Figure imgf000041_0001
The strains tested showed antioxidant activity.
3. Safety Performance
In accordance with what reported in the guidelines for probiotics and prebiotics drawn up by the Ministry of Health, the autochthonous strains CMR.V1, CMR.V2 and CMR.V3, ascribed to the L paracasei species, were subjected to evaluation for safety requirements. In detail, hemolytic activity, ability to produce DNAse and gelatinase, as well as antibiotic resistance against various antibiotics for which EFSA has established the break-point values were studied.
The strains under study meet the safety of use requirements, and in particular:
No hemolytic activity;
No ability to produce DNAse and gelatinase;
Presence of acquired antibiotic resistance excluded;
Presence of potentially transmissible antibiotic resistances excluded. Details of the characterizations carried out are provided below a. Hemolytic Activity
The strains under study were revitalized in MRS broth medium and incubated at 37 °C for 18-24 hours. Cell cultures in exponential growth phase were transferred, by propagation smear, on to Blood Agar plates containing 5% defibrinated mutton blood (Biolife, Milan, Italy) and incubated at 37 °C for 24-48 h. The hemolytic activity was visually detected and distinguished as b-hemolysis, a-hemolysis or g-hemolysis based on the presence of clear zones, green halos or no zone around colonies, respectively.
The results were expressed as + (presence of activity) and - (absence of activity).
Table 14. Evaluation of the hemolytic activity
Figure imgf000042_0001
None of the tested strains showed hemolytic activity b. Ability to produce DNase and qelatinase
DNAse production was tested by transferring 5 pl_ of an exponentially growing cell culture onto DNAse agar plates (Oxoid). After incubation at 37 °C for 48 hours, the plates were covered with 1 N HCI for 5 minutes. The presence of clear zones around the colonies is considered as an indicator of positive DNase production. Gelatinase production was evaluated using gelatin agar plates (30 g/L gelatin, 5 g/L peptone, 3 g/L yeast extract and 17 g/L agar). After incubation at 37 °C for 48 h, the surface of the plates was coated with saturated ammonium sulfate (Merck). The presence of clear zones around the colonies is considered as an indicator of positive gelatin activity. For both tests, the results were expressed as: - (absence of activity), + (presence of activity).
Table 15. Gelatinase and DNase production
Figure imgf000043_0001
None of the strains tested showed ability to produce DNAse.
Deposit of the Strains at DSMZ International Collection
The CMR.V1, CMR.V2 and CMR.V3 strains were deposited at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH) (see attachment). The following codes were assigned:
DSM 33253: strain CMR.V1 DSM 33254: strain CMR.V2 DSM 33316: strain CMR.V3
Summary of the performances obtained from the isolated strains
The results of the technological and probiotic performances of the CMR.V1 (DSM 33253), CMR.V2 (DSM 33254) and CMR.V3 (DSM 33316) strains, ascribed to the Lactobacillus paracasei, are reported below.
Table 16. Properties of the isolated strains
Figure imgf000043_0002
Figure imgf000044_0001
The characteristics of 40 of the strains analyzed are summarized in Table 17 below.
Table17. Summary of the characteristics of the isolated strains
Figure imgf000045_0001
Figure imgf000046_0001
NOTES
EPS: exopolysaccharides; nt: not tested.
Coagulant activity: - (clot absence); + (not very compact clot); ++ (compact clot); +++ (very compact clot).
Proteolytic and lipolytic activity: + (activity presence); - (activity absence).
Diacetyl production: - (no production); + (low production); ++ (medium production); +++ (high production).
EPS production: - (non-producers); + (low production); ++ (medium production); +++ (high production).
All isolated strains exhibited clotting capacity in BioSurice® Cream. 82.5% of tested strains showed coagulating activity in BioSurice® Cream. In detail, 31 out of 40 strains caused the formation of a very compact clot; 1 strain produced a compact clot; 1 strain produced a not very compact clot; 8 out of 40 strains tested did not exhibit any clotting activity in BioSurice® Cream. The proteolytic activity was exhibited by 82.5% of the tested strains (33/40). 97.5% (39/40) of tested strains showed lipolytic activity.
Only 3 strains (30%) showed diacetyl production, EPS production and suitability for freeze-drying, fundamental characteristics for the use in fermentation processes.
Examples of formulations obtained from fermented mixtures Formulation 1.
Formulation 1 is obtained from a fermentation process of the mixture of the present invention in the presence of starter cultures containing the isolated strains CMR.V1 , CMR.V2 and CMR.V3.
A mixture consisting of 40% by weight of composition 1 and 60% by weight of composition 2 (as obtained above) is fermented in the presence of a starter culture comprising 107 CFU of the three isolated strains of lactobacilli L paracasei CMR.V1, CMR.V2 and CMR.V3, according to the procedures and equipment known in the art, at a fermentation temperature equal to 40 + 2 °C and for a fermentation time of 12-15 hours.
The product obtained from this fermentation has a pH comprised between 4.3 and 4.6, a creamy consistency and a final density of lactobacilli comprised between 107-109 CFU/g of mixture.
Formulation 2
Formulation 2 is obtained from a fermentation process of the mixture of the present invention in the presence of a starter culture comprising the isolated strains CMR.V1 and CMR.V3.
A mixture consisting of 30% by weight of composition 1 and 70% by weight of composition 2 (as obtained above) is fermented in the presence of a starter culture comprising 108 CFU of the isolated strains of lactobacilli L paracasei CMR.V1 and CMR.V3, according to the procedures and equipment known in the art, at a fermentation temperature equal to 42 + 2 °C and for a fermentation time of 12-15 hours.
The product obtained from this fermentation has a pH comprised between 4.3 and 4.6, a creamy consistency and a final density of lactobacilli comprised between 107-109 CFU/g of mixture.
The formulations obtained from fermentation of the different mixtures according to the present invention can be used to obtain different types of food preparations and dermocosmetic and pharmaceutical formulations, some examples of which are reported below.
Formulation 3 Formulation 3 is obtained by mixing 40% of composition 1 and 60% of composition 2 mentioned above.
Examples of Food Preparations Example 1
Plain yogurt (125 g pot)
Formulation 1 124 g
Sweetener 1 g
Flavoring 0.1g
Fruit yogurt (125 g pot)
Formulation 1 100 g
Fruit 23 g
Preservatives 1 g
Flavorings 0.5 g
Saccharose 1 g
Example 2
Chocolate pudding (125 g pot)
Formulation 2 (lyophilized powder) 24 g
Chocolate 100 g
Preservatives 1 g
Example 3
Base for pizza, focaccia or piadina
Formulation 2 900 g
Spelled flour 80 g
Pectin 2 g
Salt q.s. Base for focaccia
Formulation 2 800 g
Soya flour 150 g
Vegetable fats 2 g
Salt q.s.
Example 4 Cake mix
Formulation 1 800 g
BioSuRice® Cream 200 g
Examples of dermocosmetic and pharmaceutical preparations Example 5
Face moisturizing cream
Formulation 1 80%
Sodium benzoate 0.5%
Ethyl palm itate 1%
PEG 5%
Aloe vera 5%
Glicerin 2%
Water q.s.
Example 6
Facial toning mask
Combination of algae 40%
Formulation 2 40%
Almond oil 5%
Water q.s. Example 7
Vial with lactobacilli oral suspension to restore intestinal bacterial flora
Formulation 1 containing 10 billion lactobacilli L. paracasei CMR.V1, CMR.V2 and CMR.V3
Water q.s.
Example 8
Ice cream
Formulation 3 60%
Sunflower oil 8%
Thickener 5%
Sweetener 7%
- 10 billion lactobacilli L. paracasei CMR.V1, CMR.V2 and CMR.V3
- Water q.s.
Example 9
Serum for cutaneous microbiota
Formulation 3, in powder, containing 10 billion lactobacilli L. paracasei
CMR.V1 , CMR.V2 and CMR.V3
Example 10
Supplements in capsules
- Formulation 3, powder 70%
- 10 billion lactobacilli L. paracasei CMR.V1, CMR.V2 and CMR.V3
Physico-chemical parameters and functional substances present in component 1 , component “K” and fermented product.
In accordance with official analytical protocols, analytical determinations were carried out in order to define the chemical-physical characteristics of component 1, component K, and a mixture of the two components fermented using the previously described autochthonous strains.
The chemical-physical analysis involved quantitative determination of pH, humidity, proteins, carbohydrates, fats, ashes, dietary fiber, energy value, dry matter, and sugars. In addition, studies were carried out on components 1 and “K” as well as on the fermented product obtained by mixing the two components and using the autochthonous starter strains described above. The determinations also concerned the quantification of functional substances such as: lactic acid, butyric acid, inositol, GABA, oryzanol. The concentration of phytic acid was also determined.
These compounds were analyzed by HPLC. In detail, the samples under study were subjected to extraction cycles with organic solvents (methanol) and water. Methanol amounts were dried and then re-solubilized in mass- grade methanol.
Table 18. Chemical-physical characteristics and nutritional substances present in component 1 , “K” and fermented product.
Figure imgf000052_0001
*g/100g; **ng/mL; N/D: below the detection limit
The results show that the fermented product has higher GABA and oryzanol values than the starting compositions. Phytic acid, notoriously classified as an anti-nutritional compound, is below the detection limit in the fermented product or absent.

Claims

1. Lactobacilli L. paracasei strain CMR.V1 (deposit number DSM
33253).
2. Lactobacilli L. paracasei strain CMR.V2 (deposit number DSM
33254).
3. Lactobacilli L. paracasei strain CMR.V3 (deposit number DSM
33316).
4. Formulation obtainable by fermenting a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice, and a starter culture comprising at least one of the CMR.V1, CMR.V2 or CMR.V3 strains according to claims 1 -3 and/or a mixture thereof.
5. Formulation according to claim 4, characterized in that at least one component of the rice mixture is not heat-treated and not hydrolyzed, micronized sprouted brown rice.
6. Formulation according to claims 4 or 5, characterized in that it has a phytic acid content lower than 1 % by weight, with respect to the total weight of the formulation, preferably said formulation is free of phytic acid.
7. Formulation according to any one of claims 4-6, characterized in that said heat treatment is selected from pasteurization, sterilization or therm ization.
8. Formulation according to any one of claims 4-7, characterized in that each of said components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
9. Formulation according to any one of claims 4-8, characterized in that the protein content is comprised between 0.5 and 3 g/100 g of total mixture, the carbohydrate content is between 10 and 30 g/100 g of total mixture and the fat content is between 0.5 and 2.5 g/100 g of total mixture.
10. Formulation according to any one of claims 4-9, characterized in that said mixture is fermented in the presence of further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto- oligosaccharides orxylitol, guar gum and/or mixtures thereof.
11. Formulation comprising a rice mixture of two or more components, wherein at least one component is heat-treated and hydrolyzed, micronized sprouted brown rice and at least one of the CMR.V1, CMR.V2 or CMR.V3 strains and/or a mixture thereof.
12. Formulation according to claim 11, characterized in that at least one component of the rice mixture is not heat-treated and not hydrolyzed, micronized sprouted brown rice.
13. Formulation according to claims 11 or 12, characterized in that it has a phytic acid content lower than 1% by weight, with respect to the total weight of the formulation, preferably said formulation is free of phytic acid.
14. Formulation according to any one of claims 11-13, characterized in that each of said components is present in the mixture in an amount comprised between 1 and 99% by weight, with respect to the total weight of the mixture, preferably in an amount between 5 and 80% by weight, with respect to the total weight of the mixture, more preferably in an amount between 20 and 70% by weight, with respect to the total weight of the mixture.
15. Formulation according to any one of claims 11-14, characterized in that it comprises further ingredients, preferably selected from prebiotics, such as inulin, fructo-oligosaccharides, galacto-oligosaccharides or xylitol, guar gum and/or mixtures thereof.
16. Use of the formulation according to claims 4-10 or according to claims 11-15 in the production of food preparations, dermocosmetic products or pharmaceutical formulations.
17. Food preparations containing from 0.1 to 99% by weight of the formulation according to claims 4-10 or of the formulation according to claims 11-15, more preferably an amount comprised between 10 and 90% by weight of the formulation, even more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation and, optionally, a further ingredient.
18. Dermocosmetic products or pharmaceutical formulations containing from 0.1 to 99% by weight of the formulation according to claims 4-10 or of the formulation according to claims 11-15, more preferably an amount comprised between 10 and 90% by weight of the formulation, even more preferably an amount between 20 and 80% by weight of the formulation, with respect to the total weight of the formulation, and at least a physiologically acceptable excipient.
19. Food preparations, dermocosmetic products, or pharmaceutical formulations according to claims 16-18, characterized in that they comprise a further active ingredient.
20. Food preparations according to claims 17 or 19, selected from vegetable drinks, yogurt, puddings and sorbets, preparations and bases for flours, leavened products, desserts, snacks, biscuits, pizzas, focaccia, semifreddo, crepes, ice cream or cakes.
21. Dermocosmetic products according to claims 18 or 19 selected from face and body creams, serums and masks for body and hair treatment, face cleansers, body cleansers, soothing cleansers and lavenders for feminine and masculine intimate hygiene, lip emollients, sun protection skin creams, creams for the treatment of skin lesions and scars, dermo-repairing creams or creams for the treatment of breast fissures.
22. Pharmaceutical formulations according to claims 18 or 19 for use in the prevention and/or treatment of vaginal infections, gastrointestinal infections and/or inflammations, dryness and irritation of the mucous membranes induced by vaginal infection, healing of wounds and burns, regeneration of internal and external epithelial tissues, restoration of the intestinal and epithelial microbiota and reduction of inflammation.
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