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 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.

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 10 ⁹ 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 (A _w ). A typical minimum A _w 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 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. 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 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. To this end, the pre-cultured liquid preferably contains 10 ⁹ - 10 ¹⁰ viable probiotic micro-organisms, preferably about 5 10 ⁹ .

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 10 ⁵ or more, preferably 10 ⁶ -10 ⁷ . 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 10 ⁷ 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 10 ⁷ 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 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. 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 10 ⁷ 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.