WO2005089560A1 - Shelf stable product with living micro-organisms - Google Patents

Shelf stable product with living micro-organisms Download PDF

Info

Publication number
WO2005089560A1
WO2005089560A1 PCT/EP2005/003089 EP2005003089W WO2005089560A1 WO 2005089560 A1 WO2005089560 A1 WO 2005089560A1 EP 2005003089 W EP2005003089 W EP 2005003089W WO 2005089560 A1 WO2005089560 A1 WO 2005089560A1
Authority
WO
WIPO (PCT)
Prior art keywords
micro
organisms
product
milk
living
Prior art date
Application number
PCT/EP2005/003089
Other languages
French (fr)
Inventor
Sylvie Tournade
Frédéric AYMES
Roberto Reniero
Original Assignee
Nestec S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=34924524&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2005089560(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Nestec S.A. filed Critical Nestec S.A.
Priority to DE602005005032T priority Critical patent/DE602005005032T2/en
Priority to CA002560947A priority patent/CA2560947A1/en
Priority to BRPI0509195-0A priority patent/BRPI0509195A/en
Priority to MXPA06010751A priority patent/MXPA06010751A/en
Priority to AU2005224031A priority patent/AU2005224031B2/en
Priority to US10/598,908 priority patent/US8399238B2/en
Priority to CN2005800091616A priority patent/CN1953667B/en
Priority to EP05716322A priority patent/EP1729590B1/en
Publication of WO2005089560A1 publication Critical patent/WO2005089560A1/en
Priority to HK07106261A priority patent/HK1101532A1/en

Links

Classifications

    • 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
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A23D7/0053—Compositions other than spreads
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A23V2400/169—Plantarum
    • 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
    • A23V2400/21—Streptococcus, lactococcus

Definitions

  • the present invention relates to a liquid product, which is water- or milk-based and which comprises living micro-organisms, and which is basically shelf stable.
  • the present invention further relates to a method for preparing a liquid, water- or milk-based delivery system for micro-organisms, and a method for providing to a consumer living micro-organisms in a water- or milk based liquid.
  • the bacterial population of the human or animal gastro-intestinal tract constitutes an enormously complex ecosystem that includes many types of microorganisms.
  • microbiota bacterial micro-flora
  • the bacterial population of the human or animal gastro-intestinal tract constitutes an enormously complex ecosystem that includes many types of microorganisms.
  • bacterial micro-flora the total population of which is approximately 10 cells. From the stomach to the colon the number of bacteria increases throughout the gastro-intestinal tract. The highest bacterial counts are found in the colon with up to 10 11 colony forming units (cfu) per ml gut content.
  • microorganisms that have a beneficial effect on their host, including bacteria, are generally referred to as probiotic micro-organisms or probiotics.
  • a generally applied approach to obtain a stable preparation of probiotics comprises the steps of conducting a fermentation to obtain high cfu of bacteria, adding a carrier or protective material, such as carbohydrates or milk powder, and, drying the mixture, for example by freeze-drying. In this way, a bacterial preparation with low Aw and prolonged shelf life may be obtained.
  • a disadvantage of a hot drying or a freeze-drying process as applied according to this principle is, however, that significant losses of living bacteria must be accounted for. Furthermore, only dried products are obtained according to this kind of procedure. It would be advantageous to have more convenient forms of consumption.
  • Another way of delivering a probiotic is the preparation of a material, which was fermented by the probiotic. This is the case, for example, with yoghurts that were obtained from fermenting milk with micro-organisms.
  • the advantage of these products is that they are relatively stable when chilled, due to the low pH of the product after fermentation.
  • the acid produced by the fermenting activity of the probiotic does not correspond to every consumer's taste. In addition, these products still have to be chilled.
  • sweet acidophilus milks which are commercially available have to be chilled in order to prevent deterioration and fermentation of the product (see
  • EP 0 154 614 discloses a method of cultivating, in milk, organisms that have a slow growth capacity. Basically, a bulk starter is prepared together with milk, which is kept at optimum growth conditions until a pH of 4.5- 5 is reached.
  • probiotics in the form of a product that has a prolonged shelf life and a convenient way of being consumed.
  • probiotic micro-organisms may be kept stable in a water- or milk-based liquid at room temperature for a prolonged time, such as several months. This surprising finding could be obtained, for example, when the probiotics were provided in a liquid that does not contain carbohydrates that may be digested by the micro-organism.
  • the present invention provides, in a first aspect a liquid product, which is water- or milk-based and which comprises living micro-organisms, characterised in that the liquid product has a shelf-life of at least 1 month at 10°, during which the pH of the product decreases less than 2 points and/or the amount of living bacteria decreases less than 2 log-units.
  • the present invention provides a method for manufacturing a liquid, water- or milk-based delivery system for micro-organisms, which is shelf stable for at least 1 month at 10°C, the method comprising the steps of fermenting a medium to obtain a biomass of living micro-organisms, and adding the biomass to a water-based liquid which is free of carbohydrates that can be metabolised by the micro-organism.
  • the present invention provides a method for providing to a consumer living micro-organisms in a water-or milk-based liquid, the method comprising the step of administering to a consumer the product according to the invention.
  • the present invention provides the use of micro-organisms in water- or milk based liquid, shelf-stable products.
  • the present invention provides a liquid product, providing benefits of living probiotics, whereby the product can be stored at temperatures above 10°C for at least one month.
  • the present invention provides a method for providing to an individual benefits of living probiotics, the method comprising the step of providing to the individual the product according to the invention.
  • the present invention provides a method for distributing living micro-organisms in a consumable form, the method comprising the steps of distributing the product according to the invention.
  • Figure 1 shows pH and acidity in °Dornic over two months a product according to the present invention inoculated with micro-organisms at 2-5x10 7 cfu/ml.
  • Figure 2 shows cfu losses of bacteria stored at 25°C in a liquid product according to the present invention over a period of 98 days.
  • the products according to the invention comprising living micro-organisms may also be referred to as delivery systems for said micro-organisms.
  • water- or milk-based liquid refers to any liquid, which is not hydrophobic.
  • the liquid product according to the invention has a Brookfield viscosity of below 200 Pa s "1 . More preferably, the liquid product according to the invention has a Brookfield viscosity of below 100 Pa s "1 , most preferably, the liquid product has a Brookfield viscosity of below 20 Pa s "1 .
  • the Brookfield viscosity is established at a shear rate of 5 RPM and at a temperature of 8°C. These values were established with a Brookfield DN-II+ viscometer.
  • shelf-stable generally means that the product does not need to be chilled at 8°C or below, but can be stocked in an un-chilled shelf of a super- market.
  • the temperature in a super market is usually in the range between 16-
  • the "shelf life" of a product is taken to start at the moment on which the manufacture of the product is completed, that is, after filling and sealing of the product.
  • the further time that it may take until the product is on the shelf in a super market is not taken into consideration for the starting point of the "shelf life" according to the present invention. This is due to the high variability of the time between manufacture and appearance in the super market, which in practice may depend on many, inconstant factors.
  • the products of the present invention are characterised in that they comprise milk proteins, lactose and/or other milk or milk derived solids.
  • milk solids include dairy products or products containing ingredients that are derived from milk, for example milk protein, such as whey proteins, casein protein, milk carbohydrates, such as lactose, or milk lipids, such as cream, for example.
  • milk protein such as whey proteins, casein protein, milk carbohydrates, such as lactose, or milk lipids, such as cream, for example.
  • the product according to the present invention comprises milk protein.
  • the product may be a whole, skimmed, or half-skimmed milk, buttermilk, or a reconstituted milk, based on a milk powder, for example.
  • the liquid product is a milk that was sterilised, for example by ultra heat treatment (UHT).
  • the products are shelf-stable, liquid dairy products.
  • the products according to the present invention may be a syrup, a soft drink, a carbonated soft drink, a tea, such as a cold tea, a juice, such as an apple, orange or generally fruit juice, for example.
  • the products may also be a soy-based product.
  • it may be a soy- milk or a soy-drink.
  • the liquid product may be a soy-based replacement for milk.
  • the soy-based product may be free of lactose.
  • the product according to the present invention preferably has a water activity of 0.4 or above, more preferably 0.6 or above, even more preferably 0.7 or above.
  • the product according to the invention has a water activity of 0.8 or above.
  • the product according to the present is a non-fermented product.
  • the product according to the present invention may be envisaged to be a fermented product, which was obtained, for example, by fermenting a medium, heat treating or pasteurising the medium to reduce bacterial load, and, at the same time, kill the fermenting bacteria. Then the fermented, optionally pasteurised product could be supplemented with a micro-organism according to the present invention, which will not further grow on the fermented medium.
  • the product may be a yoghurt, which is heat-treated and to which microorganisms which are not able to grow on the fermented, heat-treated product are added, in order to obtain a product which fulfils the features of the present invention.
  • the product according to the present invention may be a stirred or set yoghurt, which is natural or which has additional flavours or ingredients, for example fruits.
  • the product according to the invention could also be a shelf stable fresh cheese.
  • log unit refers to the logarithm on the base of ten. Therefore, a decrease of one log-unit cfu refers to, for example a decrease from 10 9 to 10 8 cfu. In other words, a decrease of one log unit means a decrease to 10% of the starting value of 100%, for example.
  • a decrease in one pH point may be illustrated at the example of a pH decrease of from 7.0 to 6.0, or from 6.5 to 5.5.
  • a decrease in 2 pH points may be a decrease from pH 6.7 to pH 4.7, for example.
  • the products of the present invention are free of carbohydrates that can be metabolised by the micro-organisms.
  • the micro-organism is preferably a bacterium that cannot metabolise lactose.
  • the micro-organisms cannot use lactose as a nutrient.
  • lactose examples include Lactobacillus paracasei (CNCM 1-2116), or Lactobacilhis rhamnosus (ATCC53103).
  • the microorganism cannot use sucrose as a nutrient.
  • Lactobacilhis delbruecki subsp bidgaricus (ATCC 11842), and Lactobacilhis helveticus (ATCC 15009) are strains that cannot use sucrose as a substrate.
  • the product of the present invention may be free of another carbohydrate source that could be metabolised by the micro-organism.
  • the micro-organism comprised in the product may be able to metabolise or use as a macro-nutrient only the missing carbohydrates, such as sucrose, for example.
  • the product according to the present invention is preferably free of one or more of the carbohydrates selected from the group consisting of fructose, glucose and sucrose. Preferably, it is free of glucose and/or fructose, more preferably they are free of sucrose.
  • the term "free of a carbohydrate”, for example “free of sucrose”, refers to liquid products in which the carbohydrate is present in amounts smaller than 0.2 g per 100 ml of the liquid product.
  • the carbohydrate source is present in amounts smaller than 0.1 g /100ml, more preferably less than 0.05g/100ml, most preferably less than 0.01 g/lOO ml.
  • the product may be originally free of potentially fermentable carbohydrates, due to the selection of its ingredients. Alternatively, all potentially fermentable carbohydrates may have been removed from the product or its ingredients.
  • the liquid product is preferably free of any of these three carbohydrates.
  • the micro-organisms is naturally incapable of using carbohydrates that are present in the liquid product.
  • it may be a strain which naturally cannot use lactose.
  • it can be a natural mutant.
  • the micro-organism may be genetically engineered to be unable to grow on specific carbohydrates.
  • the micro-organism is a bacterium. More preferably, it is a lactic acid bacterium.
  • the micro-organism is a probiotic.
  • the micro-organism is a strain selected from the group of genera consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enteroccus and mixtures of these.
  • the micro-organism is a Lactobacillus-strai . More preferably it is selected from the group consisting of L. c ⁇ sei, L. p ⁇ r ⁇ c ⁇ sei, L. ⁇ cidophilus, L. pl ⁇ nt ⁇ rum, and mixtures of these.
  • the micro-organism is a L. p ⁇ r ⁇ c ⁇ sei-strain, for example, the micro-organism is Lactobacillus paracasei (CNCM 1-2116).
  • the liquid product according to the present invention comprises at least 10 4 cfu of the micro-organism per ml at the beginning of shelf life. More preferably, at least 10 5 , even more preferably, at least 10 7 , most preferably at least 10 8 cfu/ml.
  • the liquid product comprises at least 10 6 cfu /ml.
  • the liquid product according to the present invention contains 10 5 -10 9 , more preferably 10 6 -10 8 cfu of the micro-organism per ml during the whole shelf life as defined according to the invention.
  • the liquid products has shelf life of at least 1 month at 20°C during which the product experiences a decrease in pH of less than 2 points and during which the loss of living micro-organisms is less than 2 log-units.
  • the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
  • the liquid product according to the present invention preferably has a shelf-life of at least 2 months at 20°C during which the pH of the products decreases less than 2 points and the amount of living micro-organisms decreases less than 2 log units.
  • the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
  • the liquid product according to the present invention has a shelf life of at least 2 months at 20°C during which the pH decreases less than 2 points and the amount of living micro-organisms decreases less than 1.5 log units.
  • the pH decreases less than 1 log unit, more preferably less than 0.5 unit.
  • the liquid product according to the invention has a shelf live of at least 2 months at 20°C during which the pH decreases less than 1 point and the amount of living micro-organisms decreases less than 2 log units.
  • the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
  • the liquid product according to the invention has a shelf live of at least 2 months at 20°C during which the pH decreases less than 0.7 point and the amount of living micro-organisms decreases less than 2 log units.
  • the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
  • the product may have a shelf life of at least 2 months at 20°C during which the pH decreases less than 0.7 points and the amount of living bacteria decreases less than 1, preferably less than 0.5, more preferably less than 0.3 log units.
  • the liquid product according to the present invention has shelf life of at least 1 month at 25°C during which the product experiences a decrease in pH of less than 2 points and during which the loss of living micro-organisms is less than 2 log-units.
  • the decrease in pH is less than 1 unit, more preferably less than 0.7 unit.
  • the liquid product according to the present invention preferably has a shelf-life of at least 2 months at 25°C during which the pH of the products decreases less than 2 points and the amount of living micro-organisms decreases less than 2 log units.
  • the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
  • the liquid product according to the present invention has a shelf life of at least 2 months at 25°C during which the pH decreases less than 2 points and the amount of living micro-organisms decreases less than 1.5 log units.
  • the pH decreases less than 1 log unit, more preferably less than 0.5 unit.
  • the liquid product according to the invention has a shelf live of at least 2 months at 25°C during which the pH decreases less than 1 point and the amount of living micro-organisms decreases less than 2 log units.
  • the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
  • the liquid product according to the invention has a shelf live of at least 2 months at 25°C during which the pH decreases less than 0.7 point and the amount of living micro-organisms decreases less than 2 log units.
  • the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
  • the product may have a shelf life of at least 2 months at 25°C during which the pH decreases less than 0.7 points and the amount of living bacteria decreases less than 1, preferably less than 0.5, more preferably less than 0.3 log units.
  • all the above-indicated parameters of shelf life of the product according to the present invention, as defined by storage temperature (10, 20, or 25°C), decrease in pH (2, 1, 0.5, 0.3 points) and cfu (2, 1.5, 1, 0.5, 0.3 log units) may be obtained for a shelf-life of as much as 3 months or more.
  • the product may have any pH in the range of 3.4 - 8, preferably 4 - 7.5, most preferably 6 - 7, as long as the specific pH in this range remains stable during shelf life as defined according to the present invention.
  • the pH of the product according to the present invention remains in the range of 6 - 7 during a shelf life of 2 months at 20°C.
  • the pH of liquid product according to the present invention, before shelf life is 3 or above. More preferably, the pH is 4 or above, even more preferably 5 or above, still more preferably, it is 6 or above. For example, the pH of the product is 5.5 or above. Most preferably, the pH of the product at the beginning of shelf life is in the range of 6.1 - 7.8.
  • the step of femienting a medium is conducted for at least 7 hours at above 30°C, more preferably at least 8 hours above 35°C, for example at 37°C.
  • the method for manufacturing the liquid, water- or milk based delivery system for micro-organisms may further comprise the step of separating the biomass from the fermented medium and/or, as a further option, washing the biomass, before adding to the water- or milk based liquid.
  • the biomass may be frozen, dried or freeze-dried before adding it to the water- or milk based liquid.
  • the consumer preferably is in need or wants to consume the living micro-organisms, and the micro-organisms are administered in sufficient amounts.
  • Sufficient amounts usually means that at least 10 5 -10 8 , preferably 10 6 -10 7 living micro-organisms are administered per serving. Preferably, at least one serving is consumed per day.
  • An aspect of the present invention provides liquid products or a method for providing benefits of living probiotics. There are many known benefits of living micro-organisms and many more may be discovered in the future.
  • Examples of such benefits may are relieving gut pain, providing gut comfort, providing general well-being, increasing mineral absorption, preventing or reducing constipation and/or diarrhoea, protecting from infection, preventing infection, immunomodulation, increasing immune responses against pathogens or parasites, and prevention of cancer.
  • One or more micro-organisms may be selected and combined or mixed in a single preparation to alleviate one or more of these exemplary benefits.
  • the product providing benefits of living probiotics may be stored at temperatures above 15°C for at least 2 months.
  • the storage time starts with the shelf life as defined above.
  • the product providing benefits of probiotics may be stored at temperatures and for time periods corresponding to those according to the other products according to the present invention.
  • the product may be stored at temperatures up to or above 25°C for up to 3 months or more.
  • the present invention provides a method for distributing living micro-organisms in a consumable form.
  • the distribution corresponds to the distribution of shelf stable products.
  • Shelf-stable and chilled products have usually different distribution patterns, in that chilled distribution channels require chilling at usually 6°C or below during storage, transport and subsequent shelf life in a super market, for example.
  • the distribution comprising transport, and optionally, storage in a store-house and/or in the shelf of a super-market, may be performed at temperatures above 10°C, preferably at temperatures in the range of 10°C - 25°C, more preferably at temperatures between 14-20°C.
  • the living micro-organisms in consumable form correspond to the liquid product according to the invention.
  • a shelf stable, milk-based liquid comprising a probiotic strain was prepared according to the following procedure.
  • Lactobacillus paracasei (CNCM 1-2116) was chosen. This strain is ⁇ -galactosidase-deflcient and cannot, therefore, use lactose as a substrate of carbohydrates.
  • the experiment was independently conducted with a frozen concentrate as well as a starter preparation.
  • the starter preparation the following medium was used: - 1% yeast extract - 2% glucose - 1% calcium carbonate - 0.05% manganese sulfate - 95.05% water
  • the medium was heat treated at 95°C for 30 min and then cooled at 4°C (not longer than 24 hours) or at 37°C for immediate inoculation.
  • Inoculation was carried out with 0.05% by weight of a partly defrosted can of a frozen concentrate of the selected strain, which is characterised by 1-2 xlO 10 cfu of bacteria per gram of concentrate.
  • UHT half-skimmed milk was purchased in a supermarket. It was completed with a sweetener (0.01% by weight of sucralose) in order to give a sweet taste to the liquid product.
  • Sucralose was added in the form of a sterile solution prepared by filtration: 5g of sucralose were dissolved into 95g of water. This 5% sucralose solution was sterilised by filtration with vacuum driven disposable filter system (membrane diameter 0.22 ⁇ m. From this sterile solution, 20g (20ml) were removed and added into 980g of UHT purchased milk. The UHT-milk so obtained was thus free of any carbohydrate that could be metabolised by CNCM 1-2116.
  • the milk was then inoculated with the strain so as to obtain a starting concentration of 2 - 5 xlO 7 cfu of CNCM 1-2116 per ml.
  • the standard MRS medium Man- Rogosa-Sharpe
  • the milk bottle on storage was mixed well and 1 ml was taken to carry out the analysis.
  • Appropriate dilutions were carried out on a diluent containing 1.5% tryptone, 0.85% NaCl and 0.01% of an antifoaming agent.
  • One ml portions of the dilution were mixed with the MRS agar in a Petri-dish.
  • Figure 1 shows pH-development (square-symbols) and Domic Degree (rhombus- symbols) development in the course of the storage period.
  • the Domic acidity is a very sensitive value, which theoretically may change during shelf life even at constant pH. Even though this value has not the same importance as pH for the purpose of the present invention, the acidity values are of the shelf stable product are indicated for illustration of the stability of the product during a prolonged shelf life at 25°C.
  • Figure 2 shows the decrease in log cfu/ml over a storage time of 98 days at 25°C. As can be seen, the mortality of bacteria remains very low during the first 70 days, resulting in a loss of cfu of less than 0.2 log.
  • a liquid such as a milk
  • a shelf-stable way such as at 25°C for two months without substantial loss of cell counts and without substantial decrease in pH.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Health & Medical Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Dairy Products (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to shelf-stable liquid products, for example diary products, comprising micro-organisms, especially probiotics. The micro-organisms are able to survive for several months at room temperature basically due to their inability to metabolise nutrients contained in the liquid product. The invention further relates to a method for manufacturing the liquid product according to the invention.

Description

Shelf Stable Product with Living Micro-organisms
The present invention relates to a liquid product, which is water- or milk-based and which comprises living micro-organisms, and which is basically shelf stable. The present invention further relates to a method for preparing a liquid, water- or milk-based delivery system for micro-organisms, and a method for providing to a consumer living micro-organisms in a water- or milk based liquid.
The bacterial population of the human or animal gastro-intestinal tract constitutes an enormously complex ecosystem that includes many types of microorganisms. There are more than 400 different species of bacteria in an individual's microbiota (= bacterial micro-flora), the total population of which is approximately 10 cells. From the stomach to the colon the number of bacteria increases throughout the gastro-intestinal tract. The highest bacterial counts are found in the colon with up to 1011 colony forming units (cfu) per ml gut content.
Depending of the kind of a specific bacterium that is part of the microbiota it may live in a parasitic, commensal or symbiotic relationship with its host. Many beneficial effects may be exerted by symbiotic bacteria, for example prevention of constipation and/or diarrhea, enhancement of mineral absorption, protection from infection, prevention of cancer, immunomodulation and others. Microorganisms that have a beneficial effect on their host, including bacteria, are generally referred to as probiotic micro-organisms or probiotics.
In order to provide to a consumer the beneficial functionalities of probiotics, huge efforts have been made to prepare probiotics in sufficient quantity in an orally consumable form. These efforts, however, were repeatedly confronted with several technical problems linked to the production and stabilisation of probiotic preparations.
For example, the fact that many probiotic bacteria possess an anaerobic metabolism imposes specific technical requirements on all process and product levels between a starting culture and a consumable product suitable to deliver said bacterium in sufficiently high concentration to a human or animal.
Actually, the mere fact that living bacteria are metabolically active - even at chilled temperatures - imposes problems: ingestible carriers of probiotics often sustain degradation by the bacterial activity, which may render the carrier completely unpalatable.
It is another pertinent problem to warrant that sufficient amounts of probiotic arrive in a living state in the small and large intestines. As a matter of fact, the acidity of the stomach as well as the exposure to bile acids drastically reduce survival of bacteria during the passage of the gastro-intestinal tract.
In the prior art many possibilities for providing probiotic bacteria are reported. A generally applied approach to obtain a stable preparation of probiotics comprises the steps of conducting a fermentation to obtain high cfu of bacteria, adding a carrier or protective material, such as carbohydrates or milk powder, and, drying the mixture, for example by freeze-drying. In this way, a bacterial preparation with low Aw and prolonged shelf life may be obtained. A disadvantage of a hot drying or a freeze-drying process as applied according to this principle is, however, that significant losses of living bacteria must be accounted for. Furthermore, only dried products are obtained according to this kind of procedure. It would be advantageous to have more convenient forms of consumption.
Another way of delivering a probiotic is the preparation of a material, which was fermented by the probiotic. This is the case, for example, with yoghurts that were obtained from fermenting milk with micro-organisms. The advantage of these products is that they are relatively stable when chilled, due to the low pH of the product after fermentation. However, the acid produced by the fermenting activity of the probiotic does not correspond to every consumer's taste. In addition, these products still have to be chilled.
Similarly, "sweet acidophilus milks" which are commercially available have to be chilled in order to prevent deterioration and fermentation of the product (see
EP 0 154 614B1, Col. 2, lines 27-33). This holds true even though the product itself has not been fermented.
EP 0 154 614 discloses a method of cultivating, in milk, organisms that have a slow growth capacity. Basically, a bulk starter is prepared together with milk, which is kept at optimum growth conditions until a pH of 4.5- 5 is reached.
Thereafter the bulk starter is added to a process milk batch and cultivated therein, again at optimum growth conditions, until a bacterial content of 108 cfu per ml is obtained. This is an acidified product and thus has the same disadvantages as reported above.
In view of the prior art it is an objective to provide other means or another delivery system for probiotic micro-organisms.
It is another objective to find a way of providing probiotics without the losses during a drying step.
Furthermore, it is an objective to provide viable probiotics while avoiding the costs and energy expenditures associated with the cooling or chilling of a probiotic preparation.
In addition, it is an objective of the present invention to provide probiotics in the form of a product that has a prolonged shelf life and a convenient way of being consumed.
Remarkably, it has been found that probiotic micro-organisms may be kept stable in a water- or milk-based liquid at room temperature for a prolonged time, such as several months. This surprising finding could be obtained, for example, when the probiotics were provided in a liquid that does not contain carbohydrates that may be digested by the micro-organism.
Accordingly, the present invention provides, in a first aspect a liquid product, which is water- or milk-based and which comprises living micro-organisms, characterised in that the liquid product has a shelf-life of at least 1 month at 10°, during which the pH of the product decreases less than 2 points and/or the amount of living bacteria decreases less than 2 log-units.
In a further aspect, the present invention provides a method for manufacturing a liquid, water- or milk-based delivery system for micro-organisms, which is shelf stable for at least 1 month at 10°C, the method comprising the steps of fermenting a medium to obtain a biomass of living micro-organisms, and adding the biomass to a water-based liquid which is free of carbohydrates that can be metabolised by the micro-organism. In another aspect, the present invention provides a method for providing to a consumer living micro-organisms in a water-or milk-based liquid, the method comprising the step of administering to a consumer the product according to the invention.
In still another aspect, the present invention provides the use of micro-organisms in water- or milk based liquid, shelf-stable products.
In another aspect, the present invention provides a liquid product, providing benefits of living probiotics, whereby the product can be stored at temperatures above 10°C for at least one month.
In yet another aspect the present invention provides a method for providing to an individual benefits of living probiotics, the method comprising the step of providing to the individual the product according to the invention.
In still a further aspect, the present invention provides a method for distributing living micro-organisms in a consumable form, the method comprising the steps of distributing the product according to the invention.
In the figures,
Figure 1 shows pH and acidity in °Dornic over two months a product according to the present invention inoculated with micro-organisms at 2-5x107 cfu/ml.
Figure 2 shows cfu losses of bacteria stored at 25°C in a liquid product according to the present invention over a period of 98 days.
Within the context of this specification the word "comprises" is taken to mean "includes, among other things". It is not intended to be construed as "consists of only".
In the context of the present invention, the products according to the invention comprising living micro-organisms may also be referred to as delivery systems for said micro-organisms. The term "water- or milk-based liquid" refers to any liquid, which is not hydrophobic. For example, the term excludes lipid- or oil based liquids. The term includes, however, emulsion-type of liquids containing lipid droplets in a hydrophilic base, for example.
Preferably, the liquid product according to the invention has a Brookfield viscosity of below 200 Pa s"1. More preferably, the liquid product according to the invention has a Brookfield viscosity of below 100 Pa s"1, most preferably, the liquid product has a Brookfield viscosity of below 20 Pa s"1. The Brookfield viscosity is established at a shear rate of 5 RPM and at a temperature of 8°C. These values were established with a Brookfield DN-II+ viscometer.
The term "shelf-stable" generally means that the product does not need to be chilled at 8°C or below, but can be stocked in an un-chilled shelf of a super- market. The temperature in a super market is usually in the range between 16-
25°C.
For the purpose of the present invention, the "shelf life" of a product is taken to start at the moment on which the manufacture of the product is completed, that is, after filling and sealing of the product. For simplicity and for giving a clear moment, the further time that it may take until the product is on the shelf in a super market is not taken into consideration for the starting point of the "shelf life" according to the present invention. This is due to the high variability of the time between manufacture and appearance in the super market, which in practice may depend on many, inconstant factors.
In an embodiment, the products of the present invention are characterised in that they comprise milk proteins, lactose and/or other milk or milk derived solids.
In particular, milk solids include dairy products or products containing ingredients that are derived from milk, for example milk protein, such as whey proteins, casein protein, milk carbohydrates, such as lactose, or milk lipids, such as cream, for example. Preferably, the product according to the present invention comprises milk protein.
Accordingly, the product may be a whole, skimmed, or half-skimmed milk, buttermilk, or a reconstituted milk, based on a milk powder, for example. Preferably, the liquid product is a milk that was sterilised, for example by ultra heat treatment (UHT). Preferably, the products are shelf-stable, liquid dairy products.
Alternatively, the products according to the present invention may be a syrup, a soft drink, a carbonated soft drink, a tea, such as a cold tea, a juice, such as an apple, orange or generally fruit juice, for example.
The products may also be a soy-based product. For example, it may be a soy- milk or a soy-drink. For example, the liquid product may be a soy-based replacement for milk. The soy-based product may be free of lactose.
The product according to the present invention preferably has a water activity of 0.4 or above, more preferably 0.6 or above, even more preferably 0.7 or above. For example, the product according to the invention has a water activity of 0.8 or above.
Preferably, the product according to the present is a non-fermented product.
However, the product according to the present invention may be envisaged to be a fermented product, which was obtained, for example, by fermenting a medium, heat treating or pasteurising the medium to reduce bacterial load, and, at the same time, kill the fermenting bacteria. Then the fermented, optionally pasteurised product could be supplemented with a micro-organism according to the present invention, which will not further grow on the fermented medium. For example, the product may be a yoghurt, which is heat-treated and to which microorganisms which are not able to grow on the fermented, heat-treated product are added, in order to obtain a product which fulfils the features of the present invention. Accordingly, the product according to the present invention may be a stirred or set yoghurt, which is natural or which has additional flavours or ingredients, for example fruits. The product according to the invention could also be a shelf stable fresh cheese.
The term "log unit" refers to the logarithm on the base of ten. Therefore, a decrease of one log-unit cfu refers to, for example a decrease from 109 to 108 cfu. In other words, a decrease of one log unit means a decrease to 10% of the starting value of 100%, for example. A decrease in one pH point may be illustrated at the example of a pH decrease of from 7.0 to 6.0, or from 6.5 to 5.5. A decrease in 2 pH points may be a decrease from pH 6.7 to pH 4.7, for example.
In an embodiment, the products of the present invention are free of carbohydrates that can be metabolised by the micro-organisms. For example, if the liquid product comprises lactose, the micro-organism is preferably a bacterium that cannot metabolise lactose.
Therefore, in a further embodiment of the present invention, the micro-organisms cannot use lactose as a nutrient. Examples for micro-organisms that cannot use lactose are Lactobacillus paracasei (CNCM 1-2116), or Lactobacilhis rhamnosus (ATCC53103).
According to another example according to the present invention, the microorganism cannot use sucrose as a nutrient. Lactobacilhis delbruecki subsp bidgaricus (ATCC 11842), and Lactobacilhis helveticus (ATCC 15009) are strains that cannot use sucrose as a substrate.
Alternatively, the product of the present invention may be free of another carbohydrate source that could be metabolised by the micro-organism.
For illustrating the principle of the present invention it is theoretically assumed that if any of the products of the present invention is free of sucrose, but contains other carbohydrates, the micro-organism comprised in the product may be able to metabolise or use as a macro-nutrient only the missing carbohydrates, such as sucrose, for example.
The product according to the present invention is preferably free of one or more of the carbohydrates selected from the group consisting of fructose, glucose and sucrose. Preferably, it is free of glucose and/or fructose, more preferably they are free of sucrose.
For the purpose of the present invention, the term "free of a carbohydrate", for example "free of sucrose", refers to liquid products in which the carbohydrate is present in amounts smaller than 0.2 g per 100 ml of the liquid product. Preferably, the carbohydrate source is present in amounts smaller than 0.1 g /100ml, more preferably less than 0.05g/100ml, most preferably less than 0.01 g/lOO ml.
The product may be originally free of potentially fermentable carbohydrates, due to the selection of its ingredients. Alternatively, all potentially fermentable carbohydrates may have been removed from the product or its ingredients.
Accordingly, if the micro-organism is capable of using sucrose, fructose and glucose, the liquid product is preferably free of any of these three carbohydrates.
Preferably, the micro-organisms is naturally incapable of using carbohydrates that are present in the liquid product. For example, it may be a strain which naturally cannot use lactose. For example, it can be a natural mutant. Alternatively, the micro-organism may be genetically engineered to be unable to grow on specific carbohydrates.
Preferably, the micro-organism is a bacterium. More preferably, it is a lactic acid bacterium.
In a preferred embodiment, the micro-organism is a probiotic.
In an embodiment of the present invention, the micro-organism is a strain selected from the group of genera consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enteroccus and mixtures of these.
Preferably, the micro-organism is a Lactobacillus-strai . More preferably it is selected from the group consisting of L. cαsei, L. pαrαcαsei, L. αcidophilus, L. plαntαrum, and mixtures of these. Preferably, the micro-organism is a L. pαrαcαsei-strain, for example, the micro-organism is Lactobacillus paracasei (CNCM 1-2116).
Preferably, the liquid product according to the present invention comprises at least 104 cfu of the micro-organism per ml at the beginning of shelf life. More preferably, at least 105, even more preferably, at least 107, most preferably at least 108 cfu/ml. For example, the liquid product comprises at least 106 cfu /ml. Preferably, the liquid product according to the present invention contains 105-109, more preferably 106-108 cfu of the micro-organism per ml during the whole shelf life as defined according to the invention.
In an embodiment of any of the liquid product according to the present invention, the liquid products has shelf life of at least 1 month at 20°C during which the product experiences a decrease in pH of less than 2 points and during which the loss of living micro-organisms is less than 2 log-units. Preferably, the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
The liquid product according to the present invention preferably has a shelf-life of at least 2 months at 20°C during which the pH of the products decreases less than 2 points and the amount of living micro-organisms decreases less than 2 log units. Preferably, the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
Preferably, the liquid product according to the present invention has a shelf life of at least 2 months at 20°C during which the pH decreases less than 2 points and the amount of living micro-organisms decreases less than 1.5 log units. Preferably, the pH decreases less than 1 log unit, more preferably less than 0.5 unit.
Preferably, the liquid product according to the invention has a shelf live of at least 2 months at 20°C during which the pH decreases less than 1 point and the amount of living micro-organisms decreases less than 2 log units. Preferably, the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
Preferably, the liquid product according to the invention has a shelf live of at least 2 months at 20°C during which the pH decreases less than 0.7 point and the amount of living micro-organisms decreases less than 2 log units. Preferably, the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
For example, the product may have a shelf life of at least 2 months at 20°C during which the pH decreases less than 0.7 points and the amount of living bacteria decreases less than 1, preferably less than 0.5, more preferably less than 0.3 log units.
Preferably, the liquid product according to the present invention has shelf life of at least 1 month at 25°C during which the product experiences a decrease in pH of less than 2 points and during which the loss of living micro-organisms is less than 2 log-units. Preferably, the decrease in pH is less than 1 unit, more preferably less than 0.7 unit.
The liquid product according to the present invention preferably has a shelf-life of at least 2 months at 25°C during which the pH of the products decreases less than 2 points and the amount of living micro-organisms decreases less than 2 log units. Preferably, the decrease in pH is less than 1 unit, more preferably less than 0.5 unit.
Preferably, the liquid product according to the present invention has a shelf life of at least 2 months at 25°C during which the pH decreases less than 2 points and the amount of living micro-organisms decreases less than 1.5 log units. Preferably, the pH decreases less than 1 log unit, more preferably less than 0.5 unit.
Preferably, the liquid product according to the invention has a shelf live of at least 2 months at 25°C during which the pH decreases less than 1 point and the amount of living micro-organisms decreases less than 2 log units. Preferably, the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
Preferably, the liquid product according to the invention has a shelf live of at least 2 months at 25°C during which the pH decreases less than 0.7 point and the amount of living micro-organisms decreases less than 2 log units. Preferably, the amount of living micro-organisms decreases less than 1 log unit, more preferably less than 0.5 log units.
Preferably, the product may have a shelf life of at least 2 months at 25°C during which the pH decreases less than 0.7 points and the amount of living bacteria decreases less than 1, preferably less than 0.5, more preferably less than 0.3 log units. Preferably, all the above-indicated parameters of shelf life of the product according to the present invention, as defined by storage temperature (10, 20, or 25°C), decrease in pH (2, 1, 0.5, 0.3 points) and cfu (2, 1.5, 1, 0.5, 0.3 log units) may be obtained for a shelf-life of as much as 3 months or more.
The product may have any pH in the range of 3.4 - 8, preferably 4 - 7.5, most preferably 6 - 7, as long as the specific pH in this range remains stable during shelf life as defined according to the present invention.
For example, the pH of the product according to the present invention remains in the range of 6 - 7 during a shelf life of 2 months at 20°C.
In an embodiment, the pH of liquid product according to the present invention, before shelf life, is 3 or above. More preferably, the pH is 4 or above, even more preferably 5 or above, still more preferably, it is 6 or above. For example, the pH of the product is 5.5 or above. Most preferably, the pH of the product at the beginning of shelf life is in the range of 6.1 - 7.8.
In an embodiment of the method for manufacturing the step of femienting a medium is conducted for at least 7 hours at above 30°C, more preferably at least 8 hours above 35°C, for example at 37°C.
Optionally, the method for manufacturing the liquid, water- or milk based delivery system for micro-organisms, the method may further comprise the step of separating the biomass from the fermented medium and/or, as a further option, washing the biomass, before adding to the water- or milk based liquid. According to an additional option, the biomass may be frozen, dried or freeze-dried before adding it to the water- or milk based liquid.
In the method for providing to a consumer living micro-organisms, the consumer preferably is in need or wants to consume the living micro-organisms, and the micro-organisms are administered in sufficient amounts. Sufficient amounts usually means that at least 105-108, preferably 106-107 living micro-organisms are administered per serving. Preferably, at least one serving is consumed per day. An aspect of the present invention provides liquid products or a method for providing benefits of living probiotics. There are many known benefits of living micro-organisms and many more may be discovered in the future. Examples of such benefits may are relieving gut pain, providing gut comfort, providing general well-being, increasing mineral absorption, preventing or reducing constipation and/or diarrhoea, protecting from infection, preventing infection, immunomodulation, increasing immune responses against pathogens or parasites, and prevention of cancer. One or more micro-organisms may be selected and combined or mixed in a single preparation to alleviate one or more of these exemplary benefits.
Preferably, the product providing benefits of living probiotics may be stored at temperatures above 15°C for at least 2 months. The storage time starts with the shelf life as defined above. More preferably, the product providing benefits of probiotics may be stored at temperatures and for time periods corresponding to those according to the other products according to the present invention. For example, the product may be stored at temperatures up to or above 25°C for up to 3 months or more.
According to a further aspect, the present invention provides a method for distributing living micro-organisms in a consumable form. Preferably, the distribution corresponds to the distribution of shelf stable products. Shelf-stable and chilled products have usually different distribution patterns, in that chilled distribution channels require chilling at usually 6°C or below during storage, transport and subsequent shelf life in a super market, for example. According to the present invention, the distribution, comprising transport, and optionally, storage in a store-house and/or in the shelf of a super-market, may be performed at temperatures above 10°C, preferably at temperatures in the range of 10°C - 25°C, more preferably at temperatures between 14-20°C. This way of distributing living micro-organisms in a consumable form entails the advantage of lower energy expenditures during distribution, because basically chilling is necessary. Preferably, the living micro-organisms in consumable form correspond to the liquid product according to the invention. Example:
A shelf stable, milk-based liquid comprising a probiotic strain was prepared according to the following procedure.
MATERIAL AND METHODS
As a probiotic micro-organism, Lactobacillus paracasei (CNCM 1-2116) was chosen. This strain is β-galactosidase-deflcient and cannot, therefore, use lactose as a substrate of carbohydrates.
The experiment was independently conducted with a frozen concentrate as well as a starter preparation.
For the starter preparation the following medium was used: - 1% yeast extract - 2% glucose - 1% calcium carbonate - 0.05% manganese sulfate - 95.05% water
The medium was heat treated at 95°C for 30 min and then cooled at 4°C (not longer than 24 hours) or at 37°C for immediate inoculation.
Inoculation was carried out with 0.05% by weight of a partly defrosted can of a frozen concentrate of the selected strain, which is characterised by 1-2 xlO10 cfu of bacteria per gram of concentrate.
Fermentation was carried out at 37°C during 11 hours under stirring conditions, followed by cooling at 4°C.
At the end of fennentation, a starter culture having a pH of 4.6 and containing 109 cfu of X. paracasei per ml of fermented medium was obtained.
UHT half-skimmed milk was purchased in a supermarket. It was completed with a sweetener (0.01% by weight of sucralose) in order to give a sweet taste to the liquid product. Sucralose was added in the form of a sterile solution prepared by filtration: 5g of sucralose were dissolved into 95g of water. This 5% sucralose solution was sterilised by filtration with vacuum driven disposable filter system (membrane diameter 0.22μm. From this sterile solution, 20g (20ml) were removed and added into 980g of UHT purchased milk. The UHT-milk so obtained was thus free of any carbohydrate that could be metabolised by CNCM 1-2116.
The milk was then inoculated with the strain so as to obtain a starting concentration of 2 - 5 xlO7 cfu of CNCM 1-2116 per ml.
In parallel, the same strain was added to another sample of half-skimmed milk directly in the form of the frozen concentrate discussed above, to obtain the same starting concentration of 2 - 5 xlO cfu of CNCM 1-2116 per ml.
The milks inoculated with the starter preparation and the frozen concentrate, respectively, was then mixed well before filling into sterile glass bottles.
Products were then stored for 2 months at 25°C and analysed along the storage.
Analysis included measurements of pH, Domic acidity and cell-counts (cfu).
For establishing cell counts of CNCM 1-2116, the standard MRS medium (Man- Rogosa-Sharpe) for plating of lactobacilli was used. Before each cell-count, the milk bottle on storage was mixed well and 1 ml was taken to carry out the analysis. Appropriate dilutions were carried out on a diluent containing 1.5% tryptone, 0.85% NaCl and 0.01% of an antifoaming agent. One ml portions of the dilution were mixed with the MRS agar in a Petri-dish.
Plates were examined after anaerobic incubation at 37°C for 48 hours and the number of colony forming units par ml were calculated.
RESULTS
Cfu-losses of CNCM 1-2116 in UHT skimmed and half-skimmed milk, when stored at 25°C remained, below 0.3 log and reached final value of 107 cfu/ml after a shelf life of two months. In the same period, pH decreased from 6.50 - 6.40 to a final level of 6.30. The Domic acidity increased very slightly along the storage at 25°C from 18°D to21 to a final level below 25°D.
Figure 1 shows pH-development (square-symbols) and Domic Degree (rhombus- symbols) development in the course of the storage period.
The Domic acidity is a very sensitive value, which theoretically may change during shelf life even at constant pH. Even though this value has not the same importance as pH for the purpose of the present invention, the acidity values are of the shelf stable product are indicated for illustration of the stability of the product during a prolonged shelf life at 25°C.
Figure 2 shows the decrease in log cfu/ml over a storage time of 98 days at 25°C. As can be seen, the mortality of bacteria remains very low during the first 70 days, resulting in a loss of cfu of less than 0.2 log.
The experiment surprisingly showed that bacterial preparations can be kept in a liquid, such as a milk, in a shelf-stable way, such as at 25°C for two months without substantial loss of cell counts and without substantial decrease in pH. This makes the water- or milk based liquid a valuable, easy-to-handle delivery system or carrier for probiotic micro-organisms, which are unable to metabolise carbohydrates contained in the water- or milk based liquid.
TRAITE DE BUDAPEST SUR LA RECONNAISSANCE INTERNATIONALE DU DEPOT DES MICRO-ORGANISMES AUX FINS DE LA PROCEDURE EN MATIERE DE BREVETS FORMULE INTERNATIONALE
DDEESTINATAIRE ~l RECEPISSE EN CAS DE DEPOT INITIAL, delivre en vertu de la regie 7.1 par 1'AUTORITE DE DEPOT INTERNATIONALE identifiee an bas de cette page
SOCIETE DES PRODUITS NESTLE S.A. Patents department Avenue Nestlό 55 CH-1800 Vevey NOM ET ADRESSE I — DU DEPOSANT
IDENTIFICATION DU MICRO-ORGANISME
Reference d' identification donnee par le Numero d ' ordre attribue par DEPOSANT : 1 ' AUTORITE DE DEPOT INTERNATIONALE NCC 2461 1 - 2116
II. DESCRIPTION SCIENTIFIQUE ET/OU DESIGNATION TAXONOMIQUE PROPOSEE
Le micro-organisme identifie sous chiffre I etait accompagne : d'une description scientifique d'une designation taxonomique proposes
(Cocher ce qui convient)
III. RECEPTION ET ACCEPTATION
La presente autorite de depot internationale accepte le micro-organisme identifie sous chiffre I, qu'elle a reςu le 12 JANVIER 1999 (date du depot initial)1
IV. RECEPTION D'UNE REQUETE EN CONVERSION
La presente autorite de depot internationale a retju le micro-organisme identifie sous chiffre 1 le (date du depot initial) et a recu une requete en conversion du depot initial en depot conforme au Traite de Budapest le (date de reception de la requete en conversion)
AUTORITE DE DEPOT INTERNATIONALE
Nom Signature ( s ) de la ( des ) personne ( s ) CNCM coτnpetente ( s ) pour representer 1' autorite Collection Natioπale de depot internationale ou de l ' ( de s ) de Cultures de Microorganismes employe ( s ) la CNCM
Adresse INSTITUT PASTEUR 28, Rue du Docteur Roux F-75724 PARIS CEDEX 15 Date
Figure imgf000017_0001
1 En cas d'application de la regie 6.4.d), cette date est la date a laquelle le sta-tut d' autorite de depot Internationale a etέ acquis.

Claims

Claims
1. A liquid product, which is water- or milk-based and which comprises living micro-organisms, characterised in that the liquid product has a shelf-life of at least 1 month at 10°C, during which the pH of the product decreases less than 2 points and/or the amount of living bacteria decreases less than 2 log-units.
2. The product according to Claim 1, which is characterised in that it comprises milk proteins, lactose and/ or other milk or milk-derived solids.
3. The liquid product according to Claim 1, which is free of carbohydrates that can be metabolised by the micro-organisms.
4. The product according to Claim 1 or 2, wherein the micro-organisms cannot use lactose as a nutrient.
5. The product according to Claim 1, wherein the micro-organism is a strain selected from the group of genders consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus and mixtures of these.
6. The liquid product according to Claim 1, wherein the shelf life of the product is at least 1 month at 20°C during which the product experiences a decrease in pH of less than 2 points and during which the loss of living bacteria is less than 2 log-units.
7. The product according to any of the preceding Claims, wherein trie microorganism is a probiotic.
8. The liquid product according to any of the preceding Claims, which has, at the beginning of shelf life, a pH of 4 or above.
9. A method for manufacturing a liquid, water- or milk-based delivery system for micro-organisms, which is shelf stable for at least 1 month at 10°C, the method comprising the steps of: fermenting a medium to obtain a biomass of living micro-organisms, adding the biomass to a water-based liquid which is free of carbohydrates that can be metabolised by the micro-organism.
10. The method according to Claim 8, wherein the step of fermenting a medium is conducted for at least 7 hours at above 30°C.
11. A method for providing to a consumer living micro-organisms in a water- or milk based liquid, the method comprising the step of administering to a consumer the product according to any of Claims 1-3.
12. Use of micro-organisms in water- or milk based liquid, shelf-stable products.
13. A liquid product, providing benefits of living probiotics, whereby the product can be stored at temperatures above 10°C for at least one month.
14. A method for providing to an individual benefits of living probiotics, the method comprising the step of providing to the individual the product according to any of Claims 1- 8 or 13.
15. A method for distributing living micro-organisms in a consumable form, the method comprising the steps of distributing the product according to any of Claims 1-8 or 13.
PCT/EP2005/003089 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms WO2005089560A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
DE602005005032T DE602005005032T2 (en) 2004-03-24 2005-03-23 STORABLE PRODUCT WITH LIVING MICROORGANISMS
CA002560947A CA2560947A1 (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms
BRPI0509195-0A BRPI0509195A (en) 2004-03-24 2005-03-23 shelf stable living microorganism product
MXPA06010751A MXPA06010751A (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms.
AU2005224031A AU2005224031B2 (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms
US10/598,908 US8399238B2 (en) 2004-03-24 2005-03-23 Shelf-stable product with living micro-organisms
CN2005800091616A CN1953667B (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms
EP05716322A EP1729590B1 (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms
HK07106261A HK1101532A1 (en) 2004-03-24 2007-06-12 Shelf stable product with living micro-organisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04007040.1 2004-03-24
EP04007040 2004-03-24

Publications (1)

Publication Number Publication Date
WO2005089560A1 true WO2005089560A1 (en) 2005-09-29

Family

ID=34924524

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/003089 WO2005089560A1 (en) 2004-03-24 2005-03-23 Shelf stable product with living micro-organisms

Country Status (19)

Country Link
US (1) US8399238B2 (en)
EP (1) EP1729590B1 (en)
CN (1) CN1953667B (en)
AR (1) AR048189A1 (en)
AT (1) ATE387097T1 (en)
AU (1) AU2005224031B2 (en)
BR (1) BRPI0509195A (en)
CA (1) CA2560947A1 (en)
CR (1) CR8605A (en)
DE (1) DE602005005032T2 (en)
ES (1) ES2302187T3 (en)
HK (1) HK1101532A1 (en)
MX (1) MXPA06010751A (en)
MY (1) MY143693A (en)
PT (1) PT1729590E (en)
RU (1) RU2006137373A (en)
TW (1) TW200601977A (en)
WO (1) WO2005089560A1 (en)
ZA (1) ZA200608809B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2494645C2 (en) * 2008-03-18 2013-10-10 Фризлэнд Брэндз Б.В. Probiotic food product with extended storage life
WO2023057370A1 (en) 2021-10-04 2023-04-13 Chr. Hansen A/S Method for producing fermented milk products for ambient storage

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2140772A1 (en) * 2008-07-03 2010-01-06 Nestec S.A. Temperature-induced delivery of nutrients by micro-organisms in the gastrointestinal tract
US20100247712A1 (en) * 2009-03-26 2010-09-30 Rudolph Marvin J Food compositions compromising dried probiotics, methods of manufacture and uses thereof
EP3823457A1 (en) * 2018-07-17 2021-05-26 DuPont Nutrition Biosciences ApS New food products stable at ambient temperature
WO2023060509A1 (en) * 2021-10-14 2023-04-20 Dupont Nutrition Biosciences Aps New food products with improved properties

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63309138A (en) * 1987-06-10 1988-12-16 Yakult Honsha Co Ltd Liquid yoghurt containing bacteria of genus bifidobacterium
EP0667106A1 (en) * 1994-01-13 1995-08-16 Societe Des Produits Nestle S.A. Acidic amylaceous fermented composition for reducing the fermented milk and/or fruit content in foodstuffs

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2224096A1 (en) 1973-04-09 1974-10-31 Dumonteaux Genevieve Fermented milk products and yoghourts prepn - from ultra-filtered milk having high protein and low lactose contents
US4477471A (en) 1982-04-15 1984-10-16 Microlife Technics, Inc. Preservation of foods with non-lactose fermenting Streptococcus Lactis subspecies diacetilactis
US4886664A (en) 1982-06-18 1989-12-12 Rhone-Poulenc, S.A. Low-water-activity inocula for biological control
US4839281A (en) 1985-04-17 1989-06-13 New England Medical Center Hospitals, Inc. Lactobacillus strains and methods of selection
CA2002796C (en) 1988-11-21 1999-02-16 Stanley E. Mainzer Production of fermented food products
US4956186A (en) 1989-10-25 1990-09-11 Borden, Inc. Process for the production of low calorie yogurt
US5071763A (en) 1990-04-23 1991-12-10 The United States Of America As Represented By The Secretary Of Agriculture Lactose hydrolysis by mutant streptococcus thermophilus
IT1244691B (en) 1991-01-29 1994-08-08 Sitia Yomo Spa MILK MOUSSE, ITS USE FOR PREPARING DESSERTS AND DESSERTS SO OBTAINED
GB9107305D0 (en) 1991-04-08 1991-05-22 Unilever Plc Probiotic
DK0518096T3 (en) * 1991-06-14 1996-01-29 Nestle Sa Yogurt containing live microorganisms
ATE151714T1 (en) * 1992-07-06 1997-05-15 Nestle Sa SEALED FLEXIBLE BAG AND METHOD FOR MAKING SAME
JP2824821B2 (en) 1993-11-04 1998-11-18 カルピス株式会社 Lactic acid bacteria and fermented milk products
US6033691A (en) * 1996-06-13 2000-03-07 Sancor Cooperativas Unidas Limitada Process for manufacturing a biologically active fermented milk product and product obtained by the process
FR2778921B1 (en) 1998-05-22 2001-05-11 Gervais Danone Sa MUTANT STRAINS OF LACTOBACILLUS BULGARICUS WITHOUT BETA-GALACTOSIDASE ACTIVITY
EP0965643B1 (en) 1998-06-17 2003-11-05 Societe Des Produits Nestle S.A. Mobile genetic elements as tools for genetic modification of L. delbrueckii or L. helveticus
US6787348B1 (en) * 1998-08-26 2004-09-07 Chr. Hansen Liquid starter cultures having improved storage stability and use thereof
CA2260673A1 (en) 1999-02-04 2000-08-04 Natrel Inc. A process for making a lactose-free milk without negatively affecting the organoleptic properties thereof; and milk so processed
EP1034787A1 (en) 1999-03-11 2000-09-13 Société des Produits Nestlé S.A. Lactobacillus strains preventing diarrhea caused by pathogenic bacteria
BR0008920A (en) * 1999-03-11 2001-12-18 Nestle Sa Lactobacillus strains capable of preventing diarrhea caused by pathogenic bacteria erotavirus
DE69922195T2 (en) * 1999-08-31 2005-05-04 Société des Produits Nestlé S.A. Device for extracting a substance for the preparation of a beverage
AU2001262250A1 (en) 2000-05-15 2001-11-26 Societe Des Produits Nestle S.A. Probiotic lactic acid bacteria, unable to utilize lactose
EP1294851A2 (en) 2000-06-19 2003-03-26 Regents Of The University Of Minnesota Siderophores-producing bifidobacteria thereby and uses thereof
EP1315423B1 (en) 2000-08-22 2010-03-31 Encoate Holdings Limited A thermo-stable bio-matrix
AR025644A1 (en) 2000-09-12 2002-12-04 Sintesis Quimica S A I C A WATERPROOF BASED COMPOSITION FOR SEEDS, AND A METHOD FOR PRESERVING COMPOSITION.
AU2100402A (en) 2000-11-30 2002-06-11 Bio Balance Corp Bacterial strain, processed plant extracts, compositions containing same, processes for their preparation and their therapeutic and industrial applications
JP5186077B2 (en) 2000-12-18 2013-04-17 プロバイオヘルス・エルエルシー Probiotic compound obtained from Lactobacillus casei KE01 strain
US20030060496A1 (en) 2002-08-15 2003-03-27 Merritt Douthitt Pruitt Inoculant tolerant fungcidal compositions
US20030017192A1 (en) * 2001-06-19 2003-01-23 Hanny Kanafani Process for producing extended shelf-life ready-to-use milk compositions containing probiotics
EP1384483A1 (en) 2002-07-23 2004-01-28 Nestec S.A. Probiotics for treatment of irritable bowel disease (IBS) through improvement of gut neuromuscular function
US7780970B2 (en) * 2002-08-06 2010-08-24 Danisco A/S Composition
CN1867258A (en) 2003-08-18 2006-11-22 生物平衡公司 Stable liquid probiotic composition, preparation and applications thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63309138A (en) * 1987-06-10 1988-12-16 Yakult Honsha Co Ltd Liquid yoghurt containing bacteria of genus bifidobacterium
EP0667106A1 (en) * 1994-01-13 1995-08-16 Societe Des Produits Nestle S.A. Acidic amylaceous fermented composition for reducing the fermented milk and/or fruit content in foodstuffs

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE FSTA [online] 1999, O'BRIEN J: "Sugar profiles of cultured dairy products in the UK.", XP002292993, Database accession no. 2000-00-p1138 *
DATABASE FSTA [online] 2003, KOKSOY A ET AL: "Effects of water and salt level on rheological properties of ayran, a Turkish yoghurt drink.", XP002292992, Database accession no. 2004-00-p0572 *
DATABASE WPI Section Ch Week 198905, Derwent World Patents Index; Class D13, AN 1989-035915, XP002292994 *
KOKSOY, A, INTERNATIONAL DAIRY JOURNAL, vol. 13, no. 10, 2003, CORRESPONDENCE (REPRINT) ADDRESS, M. KILIC, DEP. OF FOOD ENG., FAC. OF CHEM. & METALLURGICAL ENG., ISTANBUL TECH. UNIV., MASLAK ISTANBUL 80626, TURKEY. FAX +90-212-285-2925. E-MAIL MERAL.KILIC(A)IT, pages 835 - 839 *
O'BRIEN, JOURNAL OF HUMAN NUTRITION AND DIETETICS, vol. 12, no. 3, 1999, FOOD SAFETY RES. GROUP, SCH. OF BIOL. SCI., UNIV. OF SURREY, GUILDFORD GU2 5XH, UK. TEL. +44 (0) 1483 259222. FAX +44 (0) 1483 300374. E-MAIL J.O'BRIEN(A)SURREY.AC.UK, pages 245 - 250 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2494645C2 (en) * 2008-03-18 2013-10-10 Фризлэнд Брэндз Б.В. Probiotic food product with extended storage life
WO2023057370A1 (en) 2021-10-04 2023-04-13 Chr. Hansen A/S Method for producing fermented milk products for ambient storage

Also Published As

Publication number Publication date
MY143693A (en) 2011-06-30
AU2005224031B2 (en) 2011-02-24
BRPI0509195A (en) 2007-09-18
EP1729590A1 (en) 2006-12-13
CA2560947A1 (en) 2005-09-29
ZA200608809B (en) 2008-06-25
HK1101532A1 (en) 2007-10-18
DE602005005032D1 (en) 2008-04-10
TW200601977A (en) 2006-01-16
CN1953667B (en) 2010-12-01
ATE387097T1 (en) 2008-03-15
PT1729590E (en) 2008-06-04
CN1953667A (en) 2007-04-25
ES2302187T3 (en) 2008-07-01
AR048189A1 (en) 2006-04-05
MXPA06010751A (en) 2006-12-15
CR8605A (en) 2007-06-06
US20070280909A1 (en) 2007-12-06
DE602005005032T2 (en) 2009-03-12
AU2005224031A1 (en) 2005-09-29
US8399238B2 (en) 2013-03-19
EP1729590B1 (en) 2008-02-27
RU2006137373A (en) 2008-04-27

Similar Documents

Publication Publication Date Title
Krasaekoopt et al. Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT-and conventionally treated milk during storage
CN1317985C (en) Production of pure soya-bean lactacidase fermenting beverage
EP1169925B1 (en) Foodstuffs containing a fermented vegetable matrix and relevant preparation process
ES2425580T3 (en) Novel lactic acid bacteria
CN104839332B (en) A kind of preparation method of high activity peptide acidified milk
EP1189517B1 (en) Method for the production of an immunostimulant milk product and uses thereof
CN102105065A (en) Improvement of growth of bifidobacteria in fermented milk products
US6156353A (en) Strains of lactobacillus helveticus for forming exclusively L(+) lactic acid in milk
WO2011129693A1 (en) Probiotics-containing liquid fruit product
EP1729590B1 (en) Shelf stable product with living micro-organisms
WO2011114182A1 (en) Dried fermented dairy product containing a high density of living bifidobacteria
WO1996010627A1 (en) Streptococus thermophilus strain, fermentation process using such strain and product obtained
WO2019121698A1 (en) Fermented dairy compositions and methods of preparing the same
JPS6041441A (en) Multi-culture yogurt, solid spread, cottage cheese and production thereof
MXPA05012399A (en) Method and inoculum for acidifying lactic fermentation.
JP2965281B2 (en) Bifidobacterium culture and method for producing the same
JP7131913B2 (en) Method for producing fermented milk and lactic acid bacteria starter
EP3738438A1 (en) A method for producing milk and dairy supplements with a unique fatty acid composition by restoring the commensal natural microbiota
JP6966275B2 (en) How to improve the survival of lactic acid bacteria
US20240023591A1 (en) Acetobacter for use in preparation of fermented products
JP4051353B2 (en) Lactic acid fermented food containing intestinal inhabiting lactic acid bacteria at a high concentration
WO2017108126A1 (en) Compositions and methods for increasing or maintaining faecalibacterium prausnitzii populations
US20240032568A1 (en) Fermented beverages and processes of preparing the same
Sharar Lactic acid fermentation of pasteurized and powdered milk and optimizing the factors affecting the fermentation process
Phetsomphou et al. Dairy Foods: Microbiology

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: CR2006-008605

Country of ref document: CR

WWE Wipo information: entry into national phase

Ref document number: 2005716322

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 12006501800

Country of ref document: PH

WWE Wipo information: entry into national phase

Ref document number: 2005224031

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: PA/a/2006/010751

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 06095815

Country of ref document: CO

Ref document number: 5536/DELNP/2006

Country of ref document: IN

Ref document number: 2560947

Country of ref document: CA

Ref document number: 200580009161.6

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Ref document number: DE

ENP Entry into the national phase

Ref document number: 2005224031

Country of ref document: AU

Date of ref document: 20050323

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 2005224031

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2006/08809

Country of ref document: ZA

Ref document number: 200608809

Country of ref document: ZA

WWE Wipo information: entry into national phase

Ref document number: 2006137373

Country of ref document: RU

WWP Wipo information: published in national office

Ref document number: 2005716322

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10598908

Country of ref document: US

ENP Entry into the national phase

Ref document number: PI0509195

Country of ref document: BR

WWP Wipo information: published in national office

Ref document number: 10598908

Country of ref document: US

WWG Wipo information: grant in national office

Ref document number: 2005716322

Country of ref document: EP