WO2011129693A1 - Probiotics-containing liquid fruit product - Google Patents
Probiotics-containing liquid fruit product Download PDFInfo
- Publication number
- WO2011129693A1 WO2011129693A1 PCT/NL2011/050248 NL2011050248W WO2011129693A1 WO 2011129693 A1 WO2011129693 A1 WO 2011129693A1 NL 2011050248 W NL2011050248 W NL 2011050248W WO 2011129693 A1 WO2011129693 A1 WO 2011129693A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fruit
- liquid
- juice
- nectar
- organisms
- Prior art date
Links
- 239000006041 probiotic Substances 0.000 title claims abstract description 96
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 96
- 239000007788 liquid Substances 0.000 title claims abstract description 90
- 235000013569 fruit product Nutrition 0.000 title claims abstract description 70
- 235000015203 fruit juice Nutrition 0.000 claims abstract description 82
- 230000000529 probiotic effect Effects 0.000 claims abstract description 74
- 244000005700 microbiome Species 0.000 claims abstract description 71
- 235000020400 fruit nectar Nutrition 0.000 claims abstract description 51
- 238000012258 culturing Methods 0.000 claims abstract description 47
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 36
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- 235000013572 fruit purees Nutrition 0.000 claims abstract description 7
- 235000013399 edible fruits Nutrition 0.000 claims description 21
- 244000199866 Lactobacillus casei Species 0.000 claims description 14
- 235000013958 Lactobacillus casei Nutrition 0.000 claims description 14
- 229940017800 lactobacillus casei Drugs 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 241001672694 Citrus reticulata Species 0.000 claims description 9
- 244000078534 Vaccinium myrtillus Species 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
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- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
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- 240000004749 Annona muricata Species 0.000 claims description 6
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- 235000017537 Vaccinium myrtillus Nutrition 0.000 claims description 6
- 235000002118 Vaccinium oxycoccus Nutrition 0.000 claims description 6
- 235000004634 cranberry Nutrition 0.000 claims description 6
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 claims description 5
- 229940009289 bifidobacterium lactis Drugs 0.000 claims description 5
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 4
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 4
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- 241000218588 Lactobacillus rhamnosus Species 0.000 claims description 4
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 4
- WVXRAFOPTSTNLL-NKWVEPMBSA-N 2',3'-dideoxyadenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1CC[C@@H](CO)O1 WVXRAFOPTSTNLL-NKWVEPMBSA-N 0.000 claims description 3
- 235000009434 Actinidia chinensis Nutrition 0.000 claims description 3
- 244000298697 Actinidia deliciosa Species 0.000 claims description 3
- 235000009436 Actinidia deliciosa Nutrition 0.000 claims description 3
- 244000099147 Ananas comosus Species 0.000 claims description 3
- 235000007119 Ananas comosus Nutrition 0.000 claims description 3
- 241001444063 Aronia Species 0.000 claims description 3
- 241000167854 Bourreria succulenta Species 0.000 claims description 3
- 235000004936 Bromus mango Nutrition 0.000 claims description 3
- 244000241235 Citrullus lanatus Species 0.000 claims description 3
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 claims description 3
- 235000008733 Citrus aurantifolia Nutrition 0.000 claims description 3
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- 240000002319 Citrus sinensis Species 0.000 claims description 3
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- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 3
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- 240000003768 Solanum lycopersicum Species 0.000 claims description 3
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- 240000006909 Tilia x europaea Species 0.000 claims description 3
- 235000003095 Vaccinium corymbosum Nutrition 0.000 claims description 3
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- 240000006365 Vitis vinifera Species 0.000 claims description 3
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 3
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 3
- 235000003650 acai Nutrition 0.000 claims description 3
- 235000021029 blackberry Nutrition 0.000 claims description 3
- 235000007123 blue elder Nutrition 0.000 claims description 3
- 235000021014 blueberries Nutrition 0.000 claims description 3
- 235000019693 cherries Nutrition 0.000 claims description 3
- 235000007124 elderberry Nutrition 0.000 claims description 3
- 235000008995 european elder Nutrition 0.000 claims description 3
- 239000004571 lime Substances 0.000 claims description 3
- 240000001987 Pyrus communis Species 0.000 claims 2
- 235000021559 Fruit Juice Concentrate Nutrition 0.000 claims 1
- 239000002609 medium Substances 0.000 description 30
- 241000894006 Bacteria Species 0.000 description 18
- 239000000306 component Substances 0.000 description 14
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- 241000186660 Lactobacillus Species 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
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- 239000002253 acid Substances 0.000 description 4
- 235000013361 beverage Nutrition 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- 244000116699 Lactobacillus acidophilus NCFM Species 0.000 description 1
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- 229940116298 l- malic acid Drugs 0.000 description 1
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- 229940059406 lactobacillus rhamnosus gg Drugs 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 230000002879 macerating effect Effects 0.000 description 1
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- -1 prebiotics Chemical class 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/09—Mashed or comminuted products, e.g. pulp, purée, sauce, or products made therefrom, e.g. snacks
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the invention is in the field of liquid fruit products, such as fruit beverage and fruit purees, comprising viable probiotic micro-organisms.
- the invention pertains to a method of providing a liquid fruit product with sufficient viable probiotic micro-organisms.
- Probiotics are currently defined in the art as live microorganisms which when administered in adequate amounts confer a health benefit on the host (F AO/WHO). By definition, all probiotics have a proven non-pathogenic character. In general these health benefits are associated with improving the balance of human or animal micro flora in the gastro-intestinal tract.
- microflora in the Gl-tract is important for human well being is already known since long.
- a review paper entitled "Normal Fecal Flora of Man” published in The American Journal of Clinical Nutrition (Vol. 23: pp. 1457-1465) starts with: The influence of the balance of the normal flora of the intestinal tract on the health and well-being of the host is well documented.
- Intestinal bacteria are reported to be implicated in metabolism of amongst many other components: carbohydrates (e.g. prebiotics, sugars), proteins, fats, cholesterol, nucleotides, hormones, vitamins and minerals.
- the human microflora ecology affects amongst others: the natural resistance, immunity, osteoporosis, and cancers.
- microflora appearing in the gastro-intestinal tract have an important impact on resistance against infections.
- intestinal micro flora affects amongst other aspects: gut health (e.g. epithelial functionality, mucus growth, epithelial barrier function, mucosal immunity, defecation patterns, intestinal transit), atopic disorders, vaginal infections, skin disorders, human wellbeing and human moods.
- gut health e.g. epithelial functionality, mucus growth, epithelial barrier function, mucosal immunity, defecation patterns, intestinal transit
- atopic disorders e.g. epithelial functionality, mucus growth, epithelial barrier function, mucosal immunity, defecation patterns, intestinal transit
- vaginal infections e.g. epithelial functionality, mucus growth, epithelial barrier function, mucosal immunity, defecation patterns, intestinal transit
- atopic disorders e.g. epithelial functionality, mucus growth, epithelial
- probiotic microorganisms particularly lactobacilli
- dairy products particular challenges are met when attempting to incorporate such micro-organisms in liquid fruit products such as fruit juices or fruit purees.
- liquid fruit products such as fruit juices or fruit purees.
- probiotic microorganisms are sensitive to heat treatments, acid, and oxygen.
- the liquid fruit product it is notoriously difficult for the liquid fruit product to retain an acceptable shelf life after being provided with probiotic microorganisms, i.e. the time period during which the liquid fruit product retains enough viable probiotic bacteria, and a limited number of spoilers.
- EP 166 238 refers to one method in which lactobacilli are proliferated in animal milk, and fruit juice is added thereto. This (apart from being limited to the production of milk -containing products), has a drawback in that the number of micro-organisms will elapse with time as a result of bacteriostatic components in the fruit juice, which reduces shelf- life.
- EP 166 238 seeks to solve the foregoing problems by contacting fruit juice with a treating agent such as an amide resin or an diatomaceous earth, activated carbon, or strongly basic ion exchange resins, with the aim to remove bacteriostatic components from the fruit juice prior to proliferating lactobacilli therein.
- the invention in one embodiment, presents a process for the production of a liquid fruit product containing viable probiotic micro-organisms, the method comprising (a) providing a first liquid being a liquid fruit product; (b) providing a second liquid being a fruit juice or nectar; (c) adjusting the acidity of the second liquid to a pH of from 5 to 8 so as to form a fruit -juice or nectar -based pre-culturing medium; (d) providing at least one viable probiotic micro- organism capable of being cultured; (e) culturing the probiotic micro-organism in the fruit -juice or nectar-based pre-culturing medium so as to form a probiotics-containing fruit juice or nectar; (f) combining the probiotics- containing fruit juice or nectar with the liquid fruit product.
- the invention provides the use of fruit juice or nectar, the pH of which has been adjusted to 5 to 8, as a pre-culturing medium for probiotic micro-organisms that are added to a liquid fruit product.
- the invention provides a probiotic microorganisms-containing liquid fruit product, comprising at least 10 7 per 250 ml of viable probiotic micro-organisms, and having a shelf life at 4°C -7°C of at least three months.
- the invention relates to a fruit juice or nectar, having a pH of 5 to 8, and comprising at least 10 9 viable probiotic microorganisms per 100 ml.
- the invention in a broad sense, combines the advantages of separately proliferating probiotic micro-organisms, and using a medium that constitutes a natural source for a liquid fruit product.
- the choice of a fruit juice or nectar as the pre-culturing medium distinguishes the method of the invention from using pre-culturing media, such a broths, conventional in the art.
- the pH adjustment of the fruit-juice or nectar pre-culturing medium distinguishes the method of the invention from the attempts made in the art to raise the pH of such end products for the purpose of proliferating probiotic micro-organisms therein.
- the judicious choice of fruit juice or fruit nectar as the pre-culturing medium not only serves to better address the above-mentioned deficiencies of the prior art, but also is advantageous in other respects.
- any taste- imparting components thereof such as peptone
- the ingredients of such a liquid fruit product will not include any component that is not natural to the liquid fruit product. This presents advantages from the consumers' point of view, but also from the manufacturer's point of view (no process step required to remove unwanted ingredients or no specific addition to be made to the declaration of ingredients on the package of the liquid fruit product). Further, the pre-culturing in fruit juice or fruit nectar is very convenient in terms of the logistics in a production plant.
- a single bypass of the main stream of liquid product is required to generate a side-stream to conduct the process steps in which a portion of said fruit or nectar is used as a pre-culturing medium, after which the two streams are recombined.
- the invention is generally applicable to liquid fruit products. These include fruit juices, fruit nectars, and fruit purees.
- a fruit juice is the liquid naturally contained in fruit or vegetable tissue. Juice is prepared by mechanically squeezing or macerating fresh fruits without the application of heat or solvents. A fruit juice can be freshly obtained, or can be reconstituted from a concentrate. In most jurisdictions, the term "fruit juice" reserved for beverages that (whether fresh or reconstituted) are 100% pure fruit juice.
- a nectar is a fruit juice diluted with water and, optionally, provided with added ingredients such as sugar or artificial sweeteners. Generally, a fruit nectar comprises of from 25% to 70% of fruit juice.
- a fruit puree is a ground, pressed, strained, or otherwise mashed fruit, which generally gives a thick liquid, or soft paste, which in this description is comprised in the definition of "liquid fruit product.”
- At least two different liquid fruit products play a role.
- One is the above-identified first liquid, i.e. a liquid fruit product that, upon the addition of the pre-culturing medium, is provided with viable probiotic microorganisms.
- the other is the above-identified second liquid, i.e. a fruit juice or nectar that is used as a pre-culturing medium.
- the fruit or fruits, from which the juices or nectars used in the pre- culturing medium are obtained may be the same as or different from the fruit or fruits from which the liquid fruit product is obtained prior to the addition of the pre-culturing medium.
- the fruit or fruits used to obtain the pre-culturing medium are among the fruit or fruits of the liquid fruit product prior to the addition of the pre-culturing medium.
- the intended end-product is orange juice provided with probiotic micro-organisms
- the liquid fruit product prior to the addition of the pre-culturing medium is orange juice
- the pre- culturing medium is orange juice the acidity of which has been adjusted to pH 5-8, preferably pH 5.5.
- the intended end-product is a mixed fruit juice comprising cranberry and apple juice
- the liquid fruit product prior to the addition of the pre-culturing medium is either cranberry juice, or apple juice, or already a mix of said juices
- the pre-culturing medium is either cranberry juice, or apple juice, or already a mix of said juices, the acidity of which has been adjusted to pH 5-8, preferably pH 5.5. to 7, more preferably 5.8 to 6.5.
- the method of the invention also presents great convenience for obtaining sterilized or pasteurized liquid fruit products comprising viable probiotic micro-organisms. It will be understood, that such a product cannot be sterilized or pasteurized after the addition of the probiotic micro-organisms, as the latter would thereupon no longer be viable.
- both the step of sterilization or pasteurization and the step of adding viable probiotic bacteria can be conducted in mutually beneficial way. Accordingly, the liquid fruit product, prior to the addition of the probiotic micro-organisms, is sterilized or pasteurized.
- the fruit juice or nectar that is used as a pre-culturing medium also is sterilized or pasteurized, prior to initiating the culturing.
- the probiotic micro-organisms After culturing the probiotic micro-organisms in the (otherwise non-bacterial) fruit juice or nectar, the latter, comprising the culture of probiotic micro-organisms, and the liquid fruit product are combined aseptically (i.e. under aseptic, preferably sterile conditions).
- the original stream of fruit juice or nectar will typically be heat-treated first, under sterilization or pasteurization conditions, and thereafter a side-stream will be generated that is used as a medium for the culturing of probiotic microorganisms. After the culturing has taken place, the two streams are combined again, aseptically.
- Sterilization and pasteurization are known to the skilled person.
- sterilization treatment refers to such a high-temperature treatment as will kill all microbial life.
- Pasteurization refers to various forms of elevated temperature treatment, with the temperature and time of the treatment determining the result.
- Pasteurization is generally done in a plurality of subsequent heat treatments or in a single heat treatment, normally at a temperature in the range of from 60°C to 110°C.
- the heat treatment process of pasteurization is not only dependent on the treated material but also on other conservation parameters like pH, storage temperature in stock, and water activity.
- UHT treatment Ultra High Temperature
- UHT treatments Ultra High Temperature
- Steven Nagy, Fruit Juice Processing Technology Particularly suitable UHT treatments for fruit juice are heating at for 15-60 seconds at 95-90°C.
- fruits are selected from the group consisting of apple, orange, grapefruit, lemon, lime, pineapple, banana, tomato, passion fruit. mango, grape, cherry, cranberry, guava. soursop (guanabana), pomegranate, carrot, acai, mandarin, tangerine, kiwi, strawberry, raspberry, blackberry, blueberry, pear, huckleberry, pomegranate, elderberry, chokeberry, mandarin, melon, watermelon, lychee, bloodorange, acerola, caja, and mixtures thereof.
- the fruit juice or nectar When used as the pre-culturing medium, it is imperative that the fruit juice or nectar has a pH that is sufficiently high to allow growth of probiotic micro-organisms to take place. In general, although this is dependent on the species, a pH of 5 to 8 is required to grow probiotic micro-organisms, and preferably the pH is 5.5. to 7, more preferably 5.8 to 6.5, and most preferably about 6. In order to ensure a continued bacterial growth despite the acid generally produced by the bacteria, it is preferred to add a buffer to the pre- culturing medium. The pH adjustment will generally be done with an edible base, such as sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate. The buffer will typically be an acid/base couple that has buffer capacity within the aforementioned pH range. Examples of suitable, nutritionally acceptable buffers include citrate buffers, phosphate buffers, lactate buffers ⁇
- the pre-culturing medium further comprises a source of protein, preferably protein of low molecular weight, such as peptide mixtures resulting from protein hydrolysis.
- a suitable protein source is, e.g., casitone (a pancreatic digest of casein).
- the liquid fruit product to which the probiotics-containing fruit juice or nectar is added will generally have a pH below 4.3, particularly below 4.
- the overall pH of the resulting viable probiotic microorganisms containing liquid fruit product will be sufficiently low to prevent the probiotic micro-organisms from further growth.
- the concentration of probiotic micro-organisms in the liquid fruit product can be well controlled. In one embodiment, e.g.
- the acidity of the probiotics- containing fruit juice or nectar can be brought to a value below pH 4.3, and preferably below pH 4, prior to combining the probiotics-containing fruit juice or nectar and the liquid fruit product. This can be done using an edible acid, e.g. citric acid or lactic acid.
- the fruit juice used as a pre-culturing medium can be in the form of a concentrate, i.e. juice from which water has been removed. With a view to its suitability as a culturing medium, the medium should not be so concentrated as to have too low a water activity (A w ).
- a typical minimum A w lies around 0.9, e.g. 0.93 for lactic acid bacteria.
- the ratio of addition of fruit juice or nectar comprising the cultured probiotic micro-organisms to liquid fruit product will mainly depend on the concentration of viable probiotic micro-organisms present after pre-culturing, and the concentration ultimately desired in the liquid fruit product.
- a typically desired concentration of probiotic bacteria in liquid nutritional products is 10 7 to 10 11 , preferably 10 9 to 10 10 per daily dose of the product, the preferred values depending on the type of bacteria.
- the daily amount of liquid fruit product will generally differ per type of product.
- the product may be presented as a drink, just as other fruit drinks, and be provided in packages of, typically 0.06, 0.15, 0.20, 0.25 or 0.33 1 for one dose, or in packages of 1-5 1, mostly 1-1.5 1, intended to be poured in a glass (generally 0.2-0.5 1) and drunk. Or else, e.g., the product may be packaged in small-size (e.g. 50-100 ml) containers that serve to emphasize that it is a special health addition to the normally pattern of liquids consumption. To cover the most regular presentation forms, the liquid fruit product of the invention will preferably have 10 7 -10 9 probiotic micro-organisms per 100 ml.
- the pre-cultured liquid i.e. the fruit juice or nectar after culturing the probiotic micro-organisms therein, will generally be added to the liquid fruit product in a ratio, by weight, of pre-cultured liquid to liquid fruit product of 1:10 to 1:1000, preferably 1:100 to 1:500, although smaller or larger ratio's are also conceivable.
- the pre-cultured liquid preferably contains 10 9 - 10 10 viable probiotic micro-organisms, preferably about 5 10 9 .
- said juice or nectar is provided with at least one viable probiotic micro-organism capable of being cultured.
- the fruit juice or nectar of adjusted pH is inoculated with a number of desired bacteria, generally 10 5 or more, preferably 10 6 -10 7 .
- the culturing thereupon takes place under conditions generally known to the skilled person.
- the fruit -juice or nectar- based pre-culturing medium is subjected to temperature at which bacterial growth is promoted, generally of from 20 °C to 45°C, preferably at 25°C to 35 °C for mesophilic bacteria, and preferably 35 °C to 45°C for thermophilic bacteria, most preferably at 37°C.
- the method of the invention is applicable to any micro-organisms of which it is desired that these be included in a liquid fruit product. This particularly refers to probiotic micro-organisms.
- the term 'probiotic' refers to: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host.
- Probiotic bacteria include those belonging to the genera Lactococcus, Streptococcus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Lactobacillus or Bifidobacterium.
- Bifidobacteria used as probiotics include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium thermophilum and others.
- Specific strains of bifidobacteria used as probiotics include Bifidobacterium lactis Bbl2.
- Lactobacilli used as probiotics include Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei,
- Lactobacillus cellobiosus Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarus and others.
- Lactobacillus include Lactobacillus rhamnosus GG, Lactobacillus acidophilus LA5, Lactobacillus reuteri SD2112, Lactobacillus johnsonii LAI, Lactobacillus casei Shirota, Lactobacillus casei CRL431, Lactobacillus gasseri BNR17, Lactobacillus acidophilus NCFM and
- Lactobacillus salivarius UCC 118 Lactobacillus salivarius UCC 118.
- Lactobacillus rhamnosus Goldin Gorbach LGG
- Lactobacillus GG Lactobacillus G.G., strain ATCC 53103
- Lactobacillus G.G., strain ATCC 53103 is a bacterium that occurs naturally in the human digestive tract. It is a much studied bacterium, of generally recognized health benefit. It is widely recognized as a probiotic, and consequently incorporated into many nutritional products, such as dairy products, nutritional supplements, infant formula, and the like.
- Other preferred probiotics are Lactobacillus casei CRL431,
- Lactobacillus acidophilus LA5 Lactobacillus reuteri SD2112, Bifidobacterium lactis Bbl2, and Lactobacillus plantarum.
- the invention in another embodiment of what has been substantially described hereinbefore, further pertains to the use of fruit juice or nectar, the pH of which has been adjusted to 5 to 8, preferably 6-7, as a pre-culturing medium for probiotic micro-organisms that are added to a liquid fruit product.
- the invention also pertains to the novel products that stem from the pre-culturing done in the method of the invention.
- a fruit juice comprising the desired amount of probiotic micro-organisms (e.g. LGG) of 10 7 per 100 ml, could be produced having a shelf life (refrigerated, i.e.
- the invention in this respect, in yet a further embodiment, provides a probiotic micro-organisms containing liquid fruit product, comprising at least 10 7 per 250 ml of viable probiotic microorganisms, and having a shelf life at 4-7°C of at least three months.
- the invention is not limited to liquid fruit products having a shelf life of 18 months. Factors different from the viability of the probiotic microorganisms can play a role in determining the acceptable storage period of a liquid fruit product. E.g., some fruit-juice containing drinks have a shelf life of 5-6 weeks, and other liquid fruit products may well have a shelf life of 8-12 weeks. Also, the shelf life will differ per microorganism, and will also depend on the precise nature of the product.
- the invention relates to the direct result of the pre-culturing.
- the invention relates to a fruit juice or nectar, having a pH of 6 to 8, and comprising at least 10 9 viable probiotic micro-organisms per 100 ml.
- This is in fact suitable as an intermediate for the production of any final nutritional product for which it is desired that it comprises both probiotic micro-organisms, and fruit juice or nectar.
- Juice used for this experiment is commercially available Coolbest Vitaday apple-pear with/without supplements.
- the pH of the juices was adjusted to 6.5 using NaOH (sterile) after addition of the supplements, 100 ml of each juice variant was prepared:
- the selected strains were pre-cultured anaerobic in MRS-broth for about 24h at 30°C for strain 2 and at 37°C for all the other strains.
- the different juices (about 10 ml per strain/juice combination) were inoculated with 1% of the pre- culture. Just after inoculation and after 44h at 30°C (strain 2) or 37°C (all other strains), the numbers of bacteria were determined by plating on MRSa. Results
- Lactobacillus casei Defensis strain isolated from actimel (DN-114 001)
- Juice used for this experiment is commercial available Appelsientje Multifruit mild.
- the juice was supplemented with 50 mM phosphate buffer and 0.5% casiton and the pH was adjusted to 6.5 using NaOH (sterile).
- the juice was pasteurized.
- strains were pre-cultured anaerobic in MRS-broth for about 24h at 30°C for strain 2 and at 37°C for all the other strains.
- Strains 1 and 5 were incubated for about 40h.
- the juice 50 ml per strain/juice - time combination
- the number of bacteria were determined by plating on MRSa and the pH was measured (Table 1).
- the grown culture was diluted (1:100) with fresh unsupplemented juice and stored at 4-7°C. At To and after 9 and 18 weeks of storage, the numbers of bacteria were determined by plating on MRSa and the pH was measured (Table 2).
- Table 1 Number of cfu/ml for all freshly inoculated variants juice and after 47h of growth of the culture.
- Table 2 Number of cfu/ml and pH for all freshly inoculated variants and after 9, 18 wk of storage at 4-7°C
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Abstract
Disclosed is a method of providing a liquid fruit product, such as a juice, a nectar or a fruit puree, with viable probiotic micro-organisms. The method involves the pre-culturing of the micro-organisms in fruit juice or nectar as a pre-culturing medium, and thereupon combining the fruit juice or nectar pre-culturing medium with the liquid fruit product. As a result, a liquid fruit product can be obtained with a desired concentration of probiotic micro-organisms, and yet of sufficiently long shelf life.
Description
Title: PROBIOTICS-CONTAINING LIQUID FRUIT PRODUCT
Field of the Invention
The invention is in the field of liquid fruit products, such as fruit beverage and fruit purees, comprising viable probiotic micro-organisms.
Particularly, the invention pertains to a method of providing a liquid fruit product with sufficient viable probiotic micro-organisms.
Background of the Invention
Probiotics are currently defined in the art as live microorganisms which when administered in adequate amounts confer a health benefit on the host (F AO/WHO). By definition, all probiotics have a proven non-pathogenic character. In general these health benefits are associated with improving the balance of human or animal micro flora in the gastro-intestinal tract.
The fact that microflora in the Gl-tract is important for human well being is already known since long. For example, in 1970 a review paper entitled "Normal Fecal Flora of Man" published in The American Journal of Clinical Nutrition (Vol. 23: pp. 1457-1465) starts with: The influence of the balance of the normal flora of the intestinal tract on the health and well-being of the host is well documented. Intestinal bacteria are reported to be implicated in metabolism of amongst many other components: carbohydrates (e.g. prebiotics, sugars), proteins, fats, cholesterol, nucleotides, hormones, vitamins and minerals. The human microflora ecology affects amongst others: the natural resistance, immunity, osteoporosis, and cancers. Numerous publications have shown mechanistic explanations and clinical studies show efficacy that micro flora appearing in the gastro-intestinal tract have an important impact on resistance against infections. Also the intestinal micro
flora (and also temporary colonizing probiotic species) affects amongst other aspects: gut health (e.g. epithelial functionality, mucus growth, epithelial barrier function, mucosal immunity, defecation patterns, intestinal transit), atopic disorders, vaginal infections, skin disorders, human wellbeing and human moods. In fact one can state that microflora is effective in any place in or on the human body.
In view of the exerted health benefits, many nutritional products desirably are provided with probiotic micro-organisms. However, the live nature of probiotics brings about challenges when incorporating them into nutritional products.
Whilst numerous examples are known of incorporating probiotic microorganisms, particularly lactobacilli, in dairy products, particular challenges are met when attempting to incorporate such micro-organisms in liquid fruit products such as fruit juices or fruit purees. E.g., it is difficult to provide an adequate concentration of the probiotic micro-organisms in such products, inter alia since probiotic microorganisms are sensitive to heat treatments, acid, and oxygen. Also, it is notoriously difficult for the liquid fruit product to retain an acceptable shelf life after being provided with probiotic microorganisms, i.e. the time period during which the liquid fruit product retains enough viable probiotic bacteria, and a limited number of spoilers.
Background references include WO 97/49303, EP 1 508 282, and
Champagne et al., Journal of Food Science, Volume 73, no. 5 (2008), pages M221-M226. Other references relate to making drinks of fermented fruits, in which carbon dioxide is generated, e.g. JP 2005 333 898, Herein the L-malic acid concentration of fruit or vegetable juice is adjusted so as to allow added bacteria to result in malolactic fermentation.
Several references recognize the above problems, and attempt to address them. E.g., in EP 166 238 it is confirmed as commonly accepted that many lactobacilli hardly proliferate in fruit juice. In view of the belief that this is due to the generally low pH of fruit juice, attempts have been made to elevate the
H value of the juice. This, however, is referred to as having drawbacks, and as being unsuitable for the practical utilization in the production of fruit beverages. This is mainly due to unwanted alterations in the taste of the fruit juice contained therein, but also to increasing the risk of contamination with other bacteria, or to discolorations that will be induced should the fruit juice be sterilized after increasing the pH. EP 166 238 refers to one method in which lactobacilli are proliferated in animal milk, and fruit juice is added thereto. This (apart from being limited to the production of milk -containing products), has a drawback in that the number of micro-organisms will elapse with time as a result of bacteriostatic components in the fruit juice, which reduces shelf- life. EP 166 238 seeks to solve the foregoing problems by contacting fruit juice with a treating agent such as an amide resin or an diatomaceous earth, activated carbon, or strongly basic ion exchange resins, with the aim to remove bacteriostatic components from the fruit juice prior to proliferating lactobacilli therein. Irrespective of whether this could be a suitable method, it were desired to provide a method that does not require the removal of components from fruit juice. For, particularly in the area of fruit juices, the consumer's expectation is that the juice resembles freshly squeezed juice as much as possible, which is at odds with removing components therefrom. In respect of fruit juice, the present invention seeks to provide the combined benefit of a juice that is as natural as possible, with the health benefits of probiotic microorganisms added. Similar considerations play a role in the case of fruit purees and, although to a lesser extent, fruit beverages that by law are not denoted juices, such as fruit nectars. Another reference that choose to remove bacteriostatic components from fruit juice is EP 0 113 055.
Another attempt to provide fruit juices with probiotic micro-organisms is US 2008/0206403 (which corresponds to WO 2006/131569). Herein, similar to the foregoing references, the method chosen is to remove components from the juice, in this case organic acids.
Yet another reference is WO 2009/037136. Herein an attempt is disclosed to enhance the shelf- life of probiotic compositions, or at least broaden the possible conditions for keeping the probiotic compositions. The solution presented is to add an amount of 0.1% to 99.9% by weight of gum arabic to the probiotic composition. Irrespective of whether this could have a viable contribution to prolonging the shelf- life of liquid fruit products comprising probiotic micro-organisms, it will be clear from the foregoing that it is desired to provide a method of incorporating probiotic micro-organisms in liquid fruit products such as fruit juice without adding (apart from the probiotic micro- organisms themselves), components not naturally present in such liquid fruit product.
By the same token, it is also not desired, in addition to the foregoing problems associated with shelf-life, to choose the more standard method of proliferating probiotic micro-organisms in a culture medium, and then adding the resulting culture to a liquid fruit product. Such a method is described, e.g., in Sheehan et al., Innovative Food Science and Emerging Technologies 8 (2007), 279-284. The components of regular bacterial culture media will not normally be acceptable as components in liquid fruit product, let alone in a fruit juice that is to resemble natural, freshly squeezed juice, and harvesting the micro-organisms without the culture medium components, if possible at all, requires cumbersome process steps to remove such components. It would be desirable to avoid such steps, and keep the production of the liquid fruit product as straightforward as possible.
Summary of the Invention
In order to better address one or more of the foregoing desires, the invention, in one embodiment, presents a process for the production of a liquid fruit product containing viable probiotic micro-organisms, the method
comprising (a) providing a first liquid being a liquid fruit product; (b) providing a second liquid being a fruit juice or nectar; (c) adjusting the acidity of the second liquid to a pH of from 5 to 8 so as to form a fruit -juice or nectar -based pre-culturing medium; (d) providing at least one viable probiotic micro- organism capable of being cultured; (e) culturing the probiotic micro-organism in the fruit -juice or nectar-based pre-culturing medium so as to form a probiotics-containing fruit juice or nectar; (f) combining the probiotics- containing fruit juice or nectar with the liquid fruit product.
In another aspect, the invention provides the use of fruit juice or nectar, the pH of which has been adjusted to 5 to 8, as a pre-culturing medium for probiotic micro-organisms that are added to a liquid fruit product.
In yet another aspect, the invention provides a probiotic microorganisms-containing liquid fruit product, comprising at least 107 per 250 ml of viable probiotic micro-organisms, and having a shelf life at 4°C -7°C of at least three months.
In a still further aspect, the invention relates to a fruit juice or nectar, having a pH of 5 to 8, and comprising at least 109 viable probiotic microorganisms per 100 ml.
Detailed Description of the Invention
The invention, in a broad sense, combines the advantages of separately proliferating probiotic micro-organisms, and using a medium that constitutes a natural source for a liquid fruit product. The choice of a fruit juice or nectar as the pre-culturing medium distinguishes the method of the invention from using pre-culturing media, such a broths, conventional in the art.
The pH adjustment of the fruit-juice or nectar pre-culturing medium, rather than of the ultimate liquid fruit product itself, distinguishes the method of the
invention from the attempts made in the art to raise the pH of such end products for the purpose of proliferating probiotic micro-organisms therein. The judicious choice of fruit juice or fruit nectar as the pre-culturing medium not only serves to better address the above-mentioned deficiencies of the prior art, but also is advantageous in other respects. Thus, e.g., by avoiding mixing the liquid fruit product with a conventional pre-culturing medium, any taste- imparting components thereof (such as peptone) are avoided. Moreover, by selecting juice or nectar as a tool in the preparation of a liquid fruit product, the ingredients of such a liquid fruit product will not include any component that is not natural to the liquid fruit product. This presents advantages from the consumers' point of view, but also from the manufacturer's point of view (no process step required to remove unwanted ingredients or no specific addition to be made to the declaration of ingredients on the package of the liquid fruit product). Further, the pre-culturing in fruit juice or fruit nectar is very convenient in terms of the logistics in a production plant. E.g., after a process step of obtaining juice or nectar, a single bypass of the main stream of liquid product is required to generate a side-stream to conduct the process steps in which a portion of said fruit or nectar is used as a pre-culturing medium, after which the two streams are recombined.
The invention is generally applicable to liquid fruit products. These include fruit juices, fruit nectars, and fruit purees. A fruit juice is the liquid naturally contained in fruit or vegetable tissue. Juice is prepared by mechanically squeezing or macerating fresh fruits without the application of heat or solvents. A fruit juice can be freshly obtained, or can be reconstituted from a concentrate. In most jurisdictions, the term "fruit juice" reserved for beverages that (whether fresh or reconstituted) are 100% pure fruit juice. A nectar is a fruit juice diluted with water and, optionally, provided with added ingredients such as sugar or artificial sweeteners. Generally, a fruit nectar comprises of from 25% to 70% of fruit juice. A fruit puree is a ground, pressed, strained, or otherwise mashed fruit, which generally gives a thick liquid, or
soft paste, which in this description is comprised in the definition of "liquid fruit product."
In the invention, at least two different liquid fruit products play a role. One is the above-identified first liquid, i.e. a liquid fruit product that, upon the addition of the pre-culturing medium, is provided with viable probiotic microorganisms. The other is the above-identified second liquid, i.e. a fruit juice or nectar that is used as a pre-culturing medium.
The fruit or fruits, from which the juices or nectars used in the pre- culturing medium are obtained, may be the same as or different from the fruit or fruits from which the liquid fruit product is obtained prior to the addition of the pre-culturing medium. Preferably, however, the fruit or fruits used to obtain the pre-culturing medium are among the fruit or fruits of the liquid fruit product prior to the addition of the pre-culturing medium. By way of example, which does not limit the invention: if the intended end-product is orange juice provided with probiotic micro-organisms, the liquid fruit product prior to the addition of the pre-culturing medium is orange juice, and the pre- culturing medium is orange juice the acidity of which has been adjusted to pH 5-8, preferably pH 5.5. to 7, more preferably 5.8 to 6.5. In another example, which does not limit the invention: if the intended end-product is a mixed fruit juice comprising cranberry and apple juice, the liquid fruit product prior to the addition of the pre-culturing medium is either cranberry juice, or apple juice, or already a mix of said juices, and the pre-culturing medium is either cranberry juice, or apple juice, or already a mix of said juices, the acidity of which has been adjusted to pH 5-8, preferably pH 5.5. to 7, more preferably 5.8 to 6.5.
The method of the invention also presents great convenience for obtaining sterilized or pasteurized liquid fruit products comprising viable probiotic micro-organisms. It will be understood, that such a product cannot be sterilized or pasteurized after the addition of the probiotic micro-organisms, as the latter would thereupon no longer be viable.
In the process of the invention, both the step of sterilization or pasteurization and the step of adding viable probiotic bacteria can be conducted in mutually beneficial way. Accordingly, the liquid fruit product, prior to the addition of the probiotic micro-organisms, is sterilized or pasteurized. The fruit juice or nectar that is used as a pre-culturing medium also is sterilized or pasteurized, prior to initiating the culturing. After culturing the probiotic micro-organisms in the (otherwise non-bacterial) fruit juice or nectar, the latter, comprising the culture of probiotic micro-organisms, and the liquid fruit product are combined aseptically (i.e. under aseptic, preferably sterile conditions).
In the aforementioned example of a production plant operation, the original stream of fruit juice or nectar will typically be heat-treated first, under sterilization or pasteurization conditions, and thereafter a side-stream will be generated that is used as a medium for the culturing of probiotic microorganisms. After the culturing has taken place, the two streams are combined again, aseptically.
Sterilization and pasteurization are known to the skilled person. In the context of the invention, and commensurate with the normal meaning of the term, sterilization treatment refers to such a high-temperature treatment as will kill all microbial life. Pasteurization refers to various forms of elevated temperature treatment, with the temperature and time of the treatment determining the result. Pasteurization is generally done in a plurality of subsequent heat treatments or in a single heat treatment, normally at a temperature in the range of from 60°C to 110°C.The heat treatment process of pasteurization is not only dependent on the treated material but also on other conservation parameters like pH, storage temperature in stock, and water activity.
A particular example of heat treatment is the UHT treatment (Ultra High Temperature). This can be generally referred to as ultra-pasteurization. Preferred treatments are those referred to in Steven Nagy, Fruit Juice
Processing Technology. Particularly suitable UHT treatments for fruit juice are heating at for 15-60 seconds at 95-90°C.
The choice of fruits for the liquid fruit products of the invention, as well as for the fruit juices or nectars used as a pre-culturing medium is largely unlimited. Preferred fruits are selected from the group consisting of apple, orange, grapefruit, lemon, lime, pineapple, banana, tomato, passion fruit. mango, grape, cherry, cranberry, guava. soursop (guanabana), pomegranate, carrot, acai, mandarin, tangerine, kiwi, strawberry, raspberry, blackberry, blueberry, pear, huckleberry, pomegranate, elderberry, chokeberry, mandarin, melon, watermelon, lychee, bloodorange, acerola, caja, and mixtures thereof.
When used as the pre-culturing medium, it is imperative that the fruit juice or nectar has a pH that is sufficiently high to allow growth of probiotic micro-organisms to take place. In general, although this is dependent on the species, a pH of 5 to 8 is required to grow probiotic micro-organisms, and preferably the pH is 5.5. to 7, more preferably 5.8 to 6.5, and most preferably about 6. In order to ensure a continued bacterial growth despite the acid generally produced by the bacteria, it is preferred to add a buffer to the pre- culturing medium. The pH adjustment will generally be done with an edible base, such as sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate. The buffer will typically be an acid/base couple that has buffer capacity within the aforementioned pH range. Examples of suitable, nutritionally acceptable buffers include citrate buffers, phosphate buffers, lactate buffers^
In a preferred embodiment, the pre-culturing medium further comprises a source of protein, preferably protein of low molecular weight, such as peptide mixtures resulting from protein hydrolysis. A suitable protein source is, e.g., casitone (a pancreatic digest of casein).
The liquid fruit product to which the probiotics-containing fruit juice or nectar is added, will generally have a pH below 4.3, particularly below 4. As a result, after the probiotics-containing fruit juice or nectar is combined with
said liquid fruit product, the overall pH of the resulting viable probiotic microorganisms containing liquid fruit product, will be sufficiently low to prevent the probiotic micro-organisms from further growth. Thus, the concentration of probiotic micro-organisms in the liquid fruit product can be well controlled. In one embodiment, e.g. if the ratio between the buffered probiotics-containing fruit juice or nectar and the liquid fruit product is too great for the pH of the liquid fruit product to override the buffered pH, the acidity of the probiotics- containing fruit juice or nectar can be brought to a value below pH 4.3, and preferably below pH 4, prior to combining the probiotics-containing fruit juice or nectar and the liquid fruit product. This can be done using an edible acid, e.g. citric acid or lactic acid. Based on the respective pHs of the probiotics- containing fruit juice or nectar and the liquid fruit product, as well as of the buffer capacity present in the probiotics-containing fruit juice or nectar ,the person skilled in the art will be able to simply calculate for which ratio of combining the two liquids a prior pH lowering would be advisable.
The fruit juice used as a pre-culturing medium can be in the form of a concentrate, i.e. juice from which water has been removed. With a view to its suitability as a culturing medium, the medium should not be so concentrated as to have too low a water activity (Aw). A typical minimum Aw lies around 0.9, e.g. 0.93 for lactic acid bacteria.
It will be understood that the ratio of addition of fruit juice or nectar comprising the cultured probiotic micro-organisms to liquid fruit product will mainly depend on the concentration of viable probiotic micro-organisms present after pre-culturing, and the concentration ultimately desired in the liquid fruit product. A typically desired concentration of probiotic bacteria in liquid nutritional products is 107 to 1011, preferably 109 to 1010 per daily dose of the product, the preferred values depending on the type of bacteria. The daily amount of liquid fruit product will generally differ per type of product. E.g., the product may be presented as a drink, just as other fruit drinks, and be provided in packages of, typically 0.06, 0.15, 0.20, 0.25 or 0.33 1 for one dose, or
in packages of 1-5 1, mostly 1-1.5 1, intended to be poured in a glass (generally 0.2-0.5 1) and drunk. Or else, e.g., the product may be packaged in small-size (e.g. 50-100 ml) containers that serve to emphasize that it is a special health addition to the normally pattern of liquids consumption. To cover the most regular presentation forms, the liquid fruit product of the invention will preferably have 107-109 probiotic micro-organisms per 100 ml.
The pre-cultured liquid, i.e. the fruit juice or nectar after culturing the probiotic micro-organisms therein, will generally be added to the liquid fruit product in a ratio, by weight, of pre-cultured liquid to liquid fruit product of 1:10 to 1:1000, preferably 1:100 to 1:500, although smaller or larger ratio's are also conceivable. To this end, the pre-cultured liquid preferably contains 109 - 1010 viable probiotic micro-organisms, preferably about 5 109.
In order to obtain the probiotics-containing fruit juice or nectar, said juice or nectar is provided with at least one viable probiotic micro-organism capable of being cultured. In general this means that the fruit juice or nectar of adjusted pH is inoculated with a number of desired bacteria, generally 105 or more, preferably 106-107. The culturing thereupon takes place under conditions generally known to the skilled person. Particularly, the fruit -juice or nectar- based pre-culturing medium is subjected to temperature at which bacterial growth is promoted, generally of from 20 °C to 45°C, preferably at 25°C to 35 °C for mesophilic bacteria, and preferably 35 °C to 45°C for thermophilic bacteria, most preferably at 37°C.
The method of the invention is applicable to any micro-organisms of which it is desired that these be included in a liquid fruit product. This particularly refers to probiotic micro-organisms.
According to the FAO/WHO, the term 'probiotic' refers to: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host. Probiotic bacteria include those belonging to the genera Lactococcus, Streptococcus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Lactobacillus or Bifidobacterium.
Bifidobacteria used as probiotics include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium thermophilum and others. Specific strains of bifidobacteria used as probiotics include Bifidobacterium lactis Bbl2.
Lactobacilli used as probiotics include Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei,
Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarus and others. Probiotic strains of Lactobacillus include Lactobacillus rhamnosus GG, Lactobacillus acidophilus LA5, Lactobacillus reuteri SD2112, Lactobacillus johnsonii LAI, Lactobacillus casei Shirota, Lactobacillus casei CRL431, Lactobacillus gasseri BNR17, Lactobacillus acidophilus NCFM and
Lactobacillus salivarius UCC 118.
A preferred probiotic micro-organism is Lactobacillus rhamnosus Goldin Gorbach (LGG). Lactobacillus GG (Lactobacillus G.G., strain ATCC 53103) is a bacterium that occurs naturally in the human digestive tract. It is a much studied bacterium, of generally recognized health benefit. It is widely recognized as a probiotic, and consequently incorporated into many nutritional products, such as dairy products, nutritional supplements, infant formula, and the like. Other preferred probiotics are Lactobacillus casei CRL431,
Lactobacillus acidophilus LA5, Lactobacillus reuteri SD2112, Bifidobacterium lactis Bbl2, and Lactobacillus plantarum.
The invention, in another embodiment of what has been substantially described hereinbefore, further pertains to the use of fruit juice or nectar, the pH of which has been adjusted to 5 to 8, preferably 6-7, as a pre-culturing medium for probiotic micro-organisms that are added to a liquid fruit product.
The invention also pertains to the novel products that stem from the pre-culturing done in the method of the invention. Thus, a fruit juice comprising the desired amount of probiotic micro-organisms (e.g. LGG) of 107 per 100 ml, could be produced having a shelf life (refrigerated, i.e. at 4-7°C) of 18 weeks, whilst a juice produced by the method of Sheehan only had a refrigerated shelf life of 12 weeks. The invention in this respect, in yet a further embodiment, provides a probiotic micro-organisms containing liquid fruit product, comprising at least 107 per 250 ml of viable probiotic microorganisms, and having a shelf life at 4-7°C of at least three months.
It will be understood that the invention is not limited to liquid fruit products having a shelf life of 18 months. Factors different from the viability of the probiotic microorganisms can play a role in determining the acceptable storage period of a liquid fruit product. E.g., some fruit-juice containing drinks have a shelf life of 5-6 weeks, and other liquid fruit products may well have a shelf life of 8-12 weeks. Also, the shelf life will differ per microorganism, and will also depend on the precise nature of the product.
Further, the invention relates to the direct result of the pre-culturing. In this respect, in a still further embodiment, the invention relates to a fruit juice or nectar, having a pH of 6 to 8, and comprising at least 109 viable probiotic micro-organisms per 100 ml. This is in fact suitable as an intermediate for the production of any final nutritional product for which it is desired that it comprises both probiotic micro-organisms, and fruit juice or nectar. E.g. a liquid fruit products as described above, but also the juice could be used to provide a probiotics-containing "fruit and dairy" drink or dessert in which the fruit juice as well as the probiotics are provided through the pre-cultured fruit juice or nectar.
It is to be understood that the invention is not limited to the
embodiments as described hereinbefore. It is also to be understood that in the claims the word "comprising" does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g.
"a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.
The invention will be illustrated with reference to the following, non- limiting Example.
Example 1
Experimental
Strains:
Lactobacillus acidophilus La5
Lactobacillus casei CRL431
Lactobacillus reuteri ATCC55730
Lactobacillus casei LGG
Bifidobacterium animalis BB12
Juice:
Juice used for this experiment is commercially available Coolbest Vitaday apple-pear with/without supplements. The pH of the juices was adjusted to 6.5 using NaOH (sterile) after addition of the supplements, 100 ml of each juice variant was prepared:
I: apple-pear juice
II: apple-pear juice containing 50 mM phosphate buffer pH6.5
III: apple-pear juice containing 0.5% casitone
IV: apple-pear juice containing 50 mM phosphate buffer and 0.5% casiton.
Growth in juice
The selected strains were pre-cultured anaerobic in MRS-broth for about 24h at 30°C for strain 2 and at 37°C for all the other strains. The different juices (about 10 ml per strain/juice combination) were inoculated with 1% of the pre- culture. Just after inoculation and after 44h at 30°C (strain 2) or 37°C (all other strains), the numbers of bacteria were determined by plating on MRSa.
Results
Table 1:
Number of viable cells (cfu/ml) for all freshly inoculated variants and after 44h of incubation.
Juice Strain 0 hr 44 hr
I 1 1.5E+05 <lE+05
2 1.6E+07 6.0E+07
3 1.4E+07 2.0E+07
4 1.0E+07 4.3E+07
5 2.7E+06 <lE+05
II 1 3.0E+04 4.5E+08
2 1.5E+07 2.0E+08
3 1.6E+07 3.7E+07
4 7.4E+06 2.0E+08
5 2.8E+06 1.6E+08
III 1 2.0E+04 8.0E+06
2 1.2E+07 4.7E+08
3 2.4E+07 1.1E+08
4 6.0E+06 1.1E+09
5 1.6E+06 3.1E+08
IV 1 1.0E+04 4.3E+07
2 1.7E+07 7.7E+08
3 1.4E+07 1.0E+09
4 1.2E+07 1.0E+09
5 3.2E+06 5.3E+08
The fruit juices with the resulting bacterial cultures are added as a preculture to the respective fruit juices, with a ratio of addition of 1:10 to 1:1000, resulting in a shelf life of 12-18 weeks.
Example 2
Objective: To determine the bacterial stability during the shelf life of 18 weeks after dilution of the probiotics grown in supplemented juice with undiluted juice.
Experimental
Strains:
1. Lactobacillus acidophilus La5
2. Lactobacillus casei CRL431
3. Lactobacillus reuteri ATCC55730
4. Lactobacillus casei LGG
5. Bifidobacterium animalis BB12
6. Lactobacillus casei Defensis strain isolated from actimel (DN-114 001)
Juice:
Juice used for this experiment is commercial available Appelsientje Multifruit mild. The juice was supplemented with 50 mM phosphate buffer and 0.5% casiton and the pH was adjusted to 6.5 using NaOH (sterile). The juice was pasteurized.
Growth in juice
The strains were pre-cultured anaerobic in MRS-broth for about 24h at 30°C for strain 2 and at 37°C for all the other strains. Strains 1 and 5 were incubated for about 40h. The juice (50 ml per strain/juice - time combination)
was inoculated with 1% of the pre-culture and incubated for about 47 h at 30°C for strain 2 and at 37°C for all the other strains. Just after inoculation and after 47 h, the number of bacteria were determined by plating on MRSa and the pH was measured (Table 1).
The grown culture was diluted (1:100) with fresh unsupplemented juice and stored at 4-7°C. At To and after 9 and 18 weeks of storage, the numbers of bacteria were determined by plating on MRSa and the pH was measured (Table 2).
Results
Table 1: Number of cfu/ml for all freshly inoculated variants juice and after 47h of growth of the culture.
Strain 0 h 47h
Cfu/ml pH Cfu/ml pH
1 3.2E+05 6.37 1.0E+06 3.82
2 7.4E+06 6.41 2.74E+9 3.63
3 1.47E+07 6.38 8.5E+07 4.42
4 4.9E+06 6.37 1.38E+09 3.47
5 3.16E+06 6.39 7.7E+08 4.12
6 1.0E+07 6.38 4.0E+09 3.53
Blank 6.42 6.23
Table 2: Number of cfu/ml and pH for all freshly inoculated variants and after 9, 18 wk of storage at 4-7°C
Conclusion: Using the described procedure. The target of 107 cfu/ml after 18 weeks of cold storage could be reached for 3 of the strains tested: Lactobacillus casei CRL431, Lactobacillus casei LGG and Lactobacillus casei Defensis. For the first two strains, the number of cfu/ml was stable over a period of 18 weeks for strains.
Claims
1. A process for the production of a liquid fruit product containing viable probiotic micro-organisms, the method comprising (a) providing a first liquid being a liquid fruit product; (b) providing a second liquid being a fruit juice or nectar; (c) adjusting the acidity of the second liquid to a pH of from 5 to 8 so as to form a fruit-juice or nectar-based pre-culturing medium; (d) providing at least one viable probiotic micro-organism capable of being cultured; (e) culturing the probiotic micro-organism in the fruit -juice or nectar-based pre- culturing medium so as to form a probiotics-containing fruit juice or nectar; (f) combining the probiotics-containing fruit juice or nectar with the liquid fruit product.
2. A process according to claim 1, wherein the acidity of the second liquid is adjusted to a pH of from 5.5. to 7, preferably 5.8 to 6.5.
3. A process according to claim 1 or 2, wherein the fruit juice or nectar, prior to culturing the probiotic micro-organisms therein, is provided with a buffer for the desired pH range.
4. A process according to any one of the preceding claims, wherein the fruit juice or nectar is sterilized or pasteurized prior to adding the at least one probiotic micro-organism to be cultured.
5. A process according to claim 4, wherein the liquid fruit product is sterilized or pasteurized prior to being combined with the probiotics-containing fruit juice or nectar, and the combining of said liquid fruit product and said probiotics-containing fruit juice or nectar is conducted under aseptic conditions.
6. A process according to any one of the preceding claims, wherein the liquid fruit product is selected from the group consisting of fruit juices, fruit nectars, and fruit purees.
7. A process according to any one of the preceding claims, wherein the fruit juice forming the second liquid is a fruit juice concentrate.
8. A process according to any one of the preceding claims, wherein the first and second liquids are the same juice or nectar.
9. A process according to any one of the preceding claims, wherein the fruits from which the first and or second liquids are obtained, are selected from the group consisting of apple, orange, grapefruit, lemon, lime, pineapple, banana, tomato, passion fruit, mango, grape, cherry, cranberry, guava. soursop (guanabana), pomegranate, carrot, acai, mandarin, tangerine, kiwi,
strawberry, raspberry, blackberry, blueberry, pear, huckleberry, pomegranate, elderberry, chokeberry, mandarin, melon, watermelon, lychee, bloodorange, acerola, caja, and mixtures thereof.
10. A method according to any one of the preceding claims, wherein the probiotic micro-organism is selected from the group consisting of Lactobacillus rhamnosus Goldin Gorbach (LGG), Lactobacillus casei CRL431, Lactobacillus acidophilus LA5, Lactobacillus reuteri SD2112, Bifidobacterium lactis Bbl2, Lactobacillus plantarum, and mixtures thereof.
11. The use of fruit juice or nectar, the acidity of which has been adjusted to a pH of from 5 to 8, and preferably to a pH of from 6 to 7, as a pre-culturing medium for growing probiotic micro-organisms that are added to a liquid fruit product.
12. A probiotic micro-organisms-containing liquid fruit product, comprising at least 107 per 250 ml of viable probiotic micro-organisms, and having a shelf life at 4°C -7°C of at least three months.
13. A liquid fruit product according to claim 12, obtainable by a method according to any one of the claims 1 to 11.
14. A fruit juice or nectar, having a pH of 5 to 8, preferably a pH of 6 to 7, comprising at least 109, preferably 109 to 1010, viable probiotic micro-organisms per 100 ml.
15. A fruit juice or nectar according to claim 14, wherein the fruit is selected from the group consisting of apple, orange, grapefruit, lemon, lime, pineapple, banana, tomato, passion fruit, mango, grape, cherry, cranberry. guava. soursop (guanabana), pomegranate, carrot, acai, mandarin, tangerine, kiwi, strawberry, raspberry, blackberry, blueberry, pear, huckleberry, pomegranate, elderberry, chokeberry, mandarin, melon, watermelon, lychee, bloodorange, acerola, caja, and mixtures thereof., and the probiotic microorganism is selected from the group consisting of Lactobacillus rhamnosus Goldin Gorbach (LGG), Lactobacillus casei CRL431, Lactobacillus acidophilus LA5, Lactobacillus reuteri SD2112, Bifidobacterium lactis Bbl2, Lactobacillus plantarum, and mixtures thereof.
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