HK1068519A - Process for producing extended shelf-life ready-to-use milk compositions containing probiotics - Google Patents

Description

Ready-to-use milk composition containing probiotics and having an extended shelf life

Background

This application claims the benefit of U.S. provisional application serial No. 60/299,288 (filed on 6/19/2001), which is incorporated herein by reference.

Technical Field

The present invention relates generally to a method for the preparation of milk compositions, and in particular to a method for the preparation of a ready-to-use milk composition containing probiotics and having an extended shelf life.

Description of the Prior Art

Probiotics

Probiotics have been reported to confer different health benefits to consumers, such as inhibition of bacterial pathogens, reduction of the risk of colon cancer, stimulation of immune responses and reduction of serum cholesterol levels. Although there are several ways to administer probiotics to consumers, one convenient way is to simply add the probiotics to commonly consumed food products, such as milk and yogurt. However, in order to obtain the desired health benefits, these probiotics must be carefully selected and added to the food in sufficient amounts to ensure the recommended dose of probiotic is consumed. Also, these food products must be processed and manipulated in such a way that: the viability of these probiotic microorganisms is maintained during processing and during the time that these food products are wasted on the shelf while awaiting sale and consumption. Unfortunately, many probiotics added to food products are killed during processing or simply die when the product is left on the shelf for an extended period of time.

The probiotics used in the milk composition must be carefully selected to ensure that these probiotics are compatible with the milk composition. Several probiotics are known that can be used with milk compositions, such as lactobacillus and bifidobacterium. Among them, bifidobacterium is the most controversial research and is the most important. Bifidobacteria are bacteria that are mainly present in the faeces of milk-fed infants. The unique flora bacteriology of the genus bifidobacterium protects the fed infants against intestinal and systemic disorders caused by bacterial pathogens such as coliform and streptococcus. Coliforms and streptococci are common microflora causing newborn infant infections such as gastroenteritis. To combat these infections, bifidobacteria metabolize lactose to produce acetate and lactate and increase intestinal acidity. It is believed that increased intestinal acidity inhibits the growth of these pathogens and increases the resistance of the suckling infant to newborn infant diseases such as infectious gastroenteritis.

However, the beneficial effects of bifidobacteria and other probiotics are only possible when these probiotics are viable and have an affinity that enables them to be transplanted onto the human intestine. To produce a therapeutic effect, the minimum recommended level of viable bifidobacteria cells in the yogurt at consumption is about 10⁶Colony forming units ("cfu")/g product. In Japan, the fermented milk and lactic acid bacteria drink Association requires a minimum of 10 per ml⁷Viable Bifidobacteria cells are present in fresh dairy products that are required to contain Bifidobacteria (Ishibashi, N. and S.Shimamura.1993. Bifidobacteria: research and development in Japan (Bifidobacterium: research and development in Japan). Food Technology 47: 126, 129-34). Swiss food management and international standards FIL/IDF require that dairy products containing bifidobacteria contain not less than 10⁶Growth of cfu/ml or g product (Shin, H-S, Lee, J-H., Pestka, J.and Ustunol, Z.2000. sub.Bifidobacterium Industrial in skim milk containing oligosaccharides and inulin andsurvival (Growth and viability of commercial Bifidobacterium sp in skinning consistent oligosaccharides and vitamins) J.food Sci 65 (5): 884-887). Probiotic organisms often show poor viability in these products. Commercially available pasteurized dairy products containing live probiotics have only a typical shelf life of about 2 weeks. Furthermore, evaluation of bifidobacteria viability in industrial unfermented milk sold in the us within 18 days showed a 71% reduction in bifidobacteria population by the product shelf life and a bifidobacteria count only slightly greater than 10⁶cfu/ml products (Shin, H-S, Lee, J-H., Pestka, J. and Ustunol, Z.2000. survival of bifidobacteria in industrial dairy products during the cold storage period (vitality of Bifidobacterium in commercial dairy products) J.food Protection63 (3): 327-. Several studies have been carried out to increase the viability of probiotics in dairy products such as milk, yoghurt and cheese by controlling the species or strains of the probiotic used, oxygen, acidity, pH, temperature and using bifidogenic factors which will increase the viability of the probiotic. However, none have succeeded in producing a product with an extended shelf life.

The awareness of the health benefits of bifidobacteria and other probiotics to infants has led to the development of bifidobacteria-containing infant products currently marketed in some parts of the world, such as europe and asia. However, these infant products are powdered infant products, which are not convenient ready-to-use milk compositions. These powdered infant products have several problems. These probiotics are often ineffective because they die during processing and storage and are therefore not available to the infant to produce the desired health benefits. Moreover, commercially available sterilized ready-to-use infant products are often preferred in powder form because of the convenience and safety of the powder to the consumer. Reconstitution of powdered infant products with water that has not been properly sterilized can pose a health risk to the infant. Similarly, improper reconstitution of the powdered product may result in concentrations of nutrients and probiotics that are beneficial to the infant or lower or higher.

Milk composition

Industrial milk compositions are prepared using pasteurization. Pasteurization requires that the milk be heated to a specific temperature and for a specific time. The pasteurization process kills all pathogens and most spoilage-causing microorganisms. The milk produced according to this method has a refrigerated storage life of about 15 days. Industrial milk compositions containing probiotics can be made by adding the probiotics to the milk prior to pasteurization, but the pasteurization process will kill many of the probiotic microorganisms and thus prevent the consumer from getting a sufficient dose of probiotics when the milk is consumed. Similarly, a milk composition containing probiotics may be prepared by adding the probiotics to milk after pasteurization. For example, U.S. patent No. 5,902,575 discloses an industrial milk composition containing probiotics prepared by adding a probiotic mixture to 1% pasteurized and vitaminized low fat milk and then filling the milk into its containers.

In the preferred method, the industrial milk composition containing probiotics is prepared by adding the probiotics to the milk after pasteurization. These probiotics must compete with other microorganisms growing in the composition. Studies have shown that milk compositions containing probiotics, such as sub-species of Bifidobacterium and sub-species of Lactobacillus, have a typical shelf life of about 20 days when stored at about 4 ℃ and that only about 30% of the probiotics remain viable at the end of the shelf life (Shin, H-S, Lee, J-H., Pestka, J. and Usutenol, Z.2000. Viability of bifidobacteria in commercial products during the cold storage period (Viability of Bifidobacterium in commercial products frozen storage.) J.food Protection63 (3): 327). Methods for improving shelf life and increasing viability include finding improved strains and adding various compounds such as ascorbic acid and growth factors to the compositions. As a result of these limitations, current industrial milk compositions containing probiotics are unlikely to have the number of probiotic microorganisms required to impart the desired health benefits. Also, these compositions deteriorate in about 20 days; cannot be sold and consumed and must be discarded as waste.

Therefore, there is a need for new and improved milk compositions having an extended shelf life, e.g. more than 90 days, and containing probiotics which remain viable for such a long time.

Summary of The Invention

It is therefore an object of the present invention to provide a method for the preparation of a ready-to-use milk composition containing probiotics with an extended shelf life.

It is another object of the present invention to provide a ready-to-use milk composition containing probiotics with an extended shelf life.

It is a further object of the present invention to provide a ready-to-use milk composition containing probiotics and having an extended shelf life, which probiotics remain viable during the extended shelf life.

It is another object of the present invention to provide a ready-to-use milk composition containing probiotics in an amount sufficient to provide the recommended dose of live probiotics to the consumer and having an extended shelf life.

It is another object of the present invention to provide a ready-to-use milk composition containing probiotics in an amount sufficient to benefit the health of the consumer and having an extended shelf life.

These and other objects are achieved using a novel method wherein a milk composition is ultrapasteurized, cooled to about 20-30 ℃, and inoculated with a probiotic culture prepared under aseptic conditions. The resulting milk composition is ready-to-use, has an extended shelf life, and surprisingly contains sufficient probiotic bacteria to be beneficial to the consumer even after an extended shelf life of more than 90 days. The probiotic bacteria are added to the composition in specific amounts so that the consumer receives a beneficial dose of probiotic bacteria. In a preferred embodiment, the method is used to prepare an infant formula containing probiotics. These infant formulas may be fed to infants to increase the microflora in the infant's gut.

Other and further objects, features and benefits of the present invention will be apparent to those skilled in the art.

Detailed Description

Definition of

The term "extended shelf life" as used herein refers to the time during which a product can be stored without quality degradation below some minimum acceptable level. The minimum acceptable level of the milk composition of the present invention requires that the composition substantially maintains the same physical and chemical properties, such as taste, flavor, color, viscosity, sedimentation, etc., for at least 90 days, and that the composition contains live probiotic bacteria in an amount of at least 80% inoculum when stored under refrigerated conditions, i.e. at about 4 ℃.

The term "ready-to-use" as used herein refers to a liquid milk composition or infant formula which is easy to consume and has no added other ingredients or added water.

The term "probiotic" as used herein refers to cultures of living microorganisms that beneficially affect humans or animals by enhancing the performance of the intestinal resident microbial flora.

The term "aseptic conditions" as used herein refers to an environment substantially free of microorganisms and includes filling the industrial sterilized cold milk composition into a pre-sterilized container, followed by aseptic, gas-tight sealing with the pre-sterilized closure in a substantially microorganism-free environment.

The term "pasteurizing" as used herein refers to a process of heating a milk composition to (1)145 ° F for 30 minutes, (2)161 ° F for 15 seconds, (3)191 ° F for 1 second, (4)204 ° F for 0.05 seconds, or (5)212 ° F for 0.01 seconds.

As used herein, the term "ultrapasteurized" refers to a process of heating a milk composition to at least 280 ° F for at least 2 seconds to produce a milk composition having an extended shelf life under refrigerated conditions.

The term "industrial sterilization" as used herein refers to conditions obtained by: (1) the heat is applied so that there is no: (a) microorganisms capable of regeneration in food products under normal non-refrigerated conditions of storage and distribution; and (b) live microorganisms (including spores) of public health importance; or (2) controlling water activity and applying heat such that the food product is free of microorganisms that can regenerate under normal non-refrigerated conditions of storage and distribution in the food product.

As used herein, the term "infant formula" refers to a composition that replaces human milk to meet the nutritional needs of an infant. In the united states, the contents of infant formula are regulated by federal regulations under 21 CFR parts 100, 106 and 107. These regulations define macronutrient, vitamin, mineral and other component levels to mimic the nutritional and other properties of human milk.

The invention

In one aspect, the present invention is a method of preparing a ready-to-use milk composition containing probiotics with an extended shelf life. The method comprises the following steps:

an ultrapasteurized milk composition;

cooling the ultrapasteurized milk composition to a temperature of about 20-30 ℃ while maintaining aseptic conditions;

preparing a probiotic culture selected from the group consisting of bifidobacterium, lactobacillus, and combinations thereof under sterile conditions; and

inoculating the probiotic culture under aseptic conditions into an ultrapasteurized cold milk composition in an amount sufficient to produce a probiotic concentration composition of at least 1 x 10⁸Individual probiotic micro-organisms per ml of milk composition.

The resulting milk composition containing the probiotic is placed in a sterile container under aseptic conditions and sealed with a sterile closure. In a preferred embodiment, the ultrapasteurized cold milk composition is filled into an aseptic container, the probiotic bacteria are inoculated into the composition within the container under aseptic conditions, and the container is sealed under aseptic conditions with an aseptic closure. In one embodiment, these containers are purged under sterile conditions with a sterile inert gas, typically nitrogen, to remove air (oxygen) from the container just prior to sealing. The removal of air prevents many anaerobic microorganisms such as bifidobacteria from dying due to oxygen toxicity. Oxygen toxicity, if air is retained, can result in a significant reduction in the concentration of probiotics during preparation and storage.

Milk compositions useful in the present invention are milk obtained from mammals such as humans, cows, sheep, horses, etc. Typical animals include cows, sheep, goats, buffalos, camels, llamas, mares and deer. The milk compositions of the present invention also include soy milk. Soymilk as used herein means soymilk prepared by grinding dehulled soybeans, mixing the ground soybeans with water, boiling the mixture and recovering the dissolved soymilk from the soybeans. The soy milk can be formed into a milk-like product having a taste, texture and appearance similar to animal milk. Similarly, these milk compositions may be whey hydrolysate-based milk compositions or casein hydrolysate-based milk compositions. These milk compositions may be derived from a single substance or from a mixture of milk or soy from one or more species, such as a mixture of human and bovine milk or a mixture of soy and bovine milk. In a preferred embodiment, the milk composition is selected from the group consisting of whole milk, skim milk, lactose-free milk, soy-based milk, whey hydrolysate-based milk, casein hydrolysate-based milk, and mixtures thereof.

In a preferred embodiment, the milk composition is an infant formula. The infant formula may be ready-to-feed (ready-to-use), i.e., a formula that can be consumed without additional compositional changes, such as the addition of water, preferably sterile water, prior to consumption, or a reconstituted powdered infant formula made by mixing water with powdered products such as those available from Mead Johnson & Company (Enfamil  infant formula) or ross laboratories (simmilact  infant formula).

The probiotic bacteria useful in the present invention are any probiotic bacteria that are compatible with typical milk compositions, including infant formula. Preferably, the probiotic bacteria are selected from the genera bifidobacterium and lactobacillus, such as lactobacillus acidophilus and bifidobacterium bifidum. Most preferably the probiotic is selected from the group consisting of bifidobacterium lactis.

The probiotic is inoculated into the ultrapasteurized milk composition in an amount sufficient to provide a probiotic dosage recommended by the health professional for the particular probiotic added to the composition. Generally the probiotic is sufficient to provide the milk composition with at least 1 x 10⁸The probiotic micro-organisms are added to the milk composition in an amount of concentration per ml. Preferably, to yield at least 1X 10⁸Bifidobacteria lactobacillus subspecies is added to the composition in an amount of probiotic micro-organisms per ml concentration.

The milk composition of the invention may contain one or more different probiotics or different types of probiotics.

The probiotic cultures of the present invention may be prepared aseptically using various methods known to those skilled in the art. One preferred method involves aseptically weighing the desired amount of microorganisms into sterilized glass vials to make a probiotic suspension, sealing the vials with sterilized caps, sterilizing the reverse osmosis water in an autoclave at 121 ℃ for 20 minutes, aseptically mixing the sterile water into the glass vials containing the probiotics, and covering the vials with sterile containers. If desired, the probiotic may be dispersed by shaking. Similarly, the probiotic bacteria may be inoculated into the sterile cold milk composition using methods known to those skilled in the art. Typically, the probiotic is simply added under sterile conditions using a sterile injection device such as a pipette or needle by injecting the required amount of the probiotic solution into the composition.

The milk composition of the invention has an extended shelf life of at least 90 days, preferably at least 120 days. Indeed, regression analysis of the stability data of the milk compositions of the invention showed that they were stable for about 3000 days.

Oxygen scavengers may be added to the composition to prevent loss of probiotic viability. Ascorbic acid or any oxygen scavenger known in the food industry and compatible with the composition may be used. In the study of improving the activity of probiotics in yogurt, a product with an ascorbic acid level of 250mg/kg is beneficial to improving the activity of lactobacillus delbrueckii subsp. (live, R.I. and Shah, N.P., 1997. yogurt and probiotics Viability in yogurt made from industrial starter cultures.) Int Dairy journal 7: 31-41. Similarly, any growth factor that is beneficial in protecting the viability of the probiotic may be added to the composition. Any growth factor known in the food industry and compatible with the composition may be used, such as fructooligosaccharides, galactooligosaccharides and inulin. The growth factor is added to the composition in an amount necessary to prevent loss of viability, typically in an amount of up to about 5% by weight. The viability of bifidobacterial subspecies in skim milk was maximal in the presence of fructo-oligosaccharides, galacto-oligosaccharides and inulin (in descending order), at a maximum level of 5% w/v (Shin, H.S., Lee, J.H., Pestka, J. and Usutenol, Z.2000. Growth and viability of Bifidobacterium industrial subspecies in skim milk containing oligosaccharides and inulin) (J FoodScience 65 (5): 884-.

Any container and closure capable of maintaining a sterile environment during processing and storage may be used. Glass bottles, cartons and plastic bottles are all acceptable. Preferably, these containers have low oxygen permeability, are resistant to light transmission, and maintain their integrity during handling, such as glass bottles or laminated aluminum packaging.

In another aspect, the present invention provides a ready-to-use milk composition containing probiotics prepared according to the method described herein and having an extended shelf life.

The milk compositions of the present invention are useful because they provide a convenient and economical supply to the consumer of the amount of live probiotic bacteria required to benefit the health of the consumer.

Having generally described this invention, the following examples are given as particular embodiments of the invention and to demonstrate the feasibility and benefits thereof. It should be understood that these examples are given by way of illustration and are not intended to limit the specification or the claims that follow in any manner.

Example 1

Preparation of large-volume ready-to-use baby products

The water (27,000g) was heated to about 120 deg.F (110 deg.F) and 130 deg.F. Liquid skim milk, liquid whey, lactose, mineral preparation (prepared by dissolving calcium phosphate, potassium citrate, sodium citrate and calcium chloride in 6,120 g of 150 DEG F water), mineral/trace mineral preparation (prepared by dissolving ferrous sulfate, sodium chloride and trace mineral premix in 120g of 100 DEG F water) were added. An oil formulation is prepared by heating the fat mixture, lecithin and vitamin ADEK to 160-. The mono-and diglycerides and carrageenan are then added to the oil formulation and mixed thoroughly. The milk/mineral preparation and oil preparation are then mixed together. The mixture was then heated to about 250F (245F. 255F.) for 45 seconds using direct steam injection and cooled to about 160F (150F. 170F.). The mixture was homogenized 2 times at 160F (150F) and 170F, with a total second stage pressure of 500psig and 3000 psig. The mixture was cooled to 40 ° F (35-50 ° F). The total solids were determined (should be about 18%). The amount of water (water proper) added to obtain 12.4% solids of the final product was calculated. The dry vitamin premix and nucleotide premix are dissolved in the appropriate amount of water and then added to the mixture. The final formulation was stored in a lidded jar. The result was a 120-liter batch of ready-to-use baby products containing the components shown in table 1.

TABLE 1

Composition of	Volume (gram)
		Liquid whey	6412.04 g
Fat mixture	4193.1
		Liquid skim milk	2294.81
Lactose	2273.39
		Potassium citrate	93.56
Mono-and diglycerides	86.80
		Calcium phosphate	50.22
Dry vitamin premix	45.19
		Lecithin concentrates	44.33
Carrageenan	33.91
		Calcium chloride	31.80
Sodium chloride	16.92
		Nucleotide premixes	8.35
Ascorbic acid	8.11
		Ferrous sulfate	7.30
Citric acid sodium salt	5.46
		Vitamin A, D, E, K1Concentrate	3.89
Trace mineral premix	3.65
		Water in an amount sufficient to	120 liters of water

Example 2

Preparation of liquid suspension of bifidobacteria

The infant formula should contain not less than 1 × 10 during its entire storage period⁷Viable bifidobacteria per ml. In order to compensate for potential loss of vitality during its storage period, the initial dose is 110⁸Viable bifidobacteria per ml infant formula.

Bifidobacterium lactis was used in this study since it is one of the strains cited in most literature and one of the most studied in probiotic studies. Many clinical studies have been performed on the use of bifidobacterium lactis in infants and children.

By mixing 0.85g of Bifidobacterium lactis BB12^TM(Chr Hansen BioSystems, Milwaukee, W1) (containing 1X 10)¹⁰Live bifidobacterium lactis/g) was aseptically placed into an empty 3-oz sterilized glass bottle and the bottle was capped with a sterilized cap. The empty glass bottles and lids were sterilized by placing them in an autoclave at 250 ° F for 20 minutes. Glass vials containing 85ml of reverse osmosis water were sterilized in an autoclave at 250 ℃ F. for 20 minutes. The glass vial was aseptically emptied of sterile water (85ml) into a glass vial containing 0.85g of Bifidobacterium lactis BB12, capped and shaken to disperse well. 3ml of this inoculum, when mixed with 3-ounces of baby formula (or other milk composition), will result in a formula containing 1X 10⁸Viable bifidobacteria per ml of product.

Example 3

Preparation of a ready-to-use infant formula containing viable bifidobacteria

The large quantities of ready-to-use infant products prepared according to example 1 were ultrapasteurized by direct injection of steam using a MicroThermics UHT/HTST Lab 25 apparatus (MicroThermics, Inc., Raleigh, NC). Prior to ultrapasteurization, the apparatus was sterilized by holding the steam at 270 ° F for 30 minutes in the inlet and outlet tubes. The covered area was disinfected by washing all contact surfaces with 200ppm chlorine sanitizer. The covered area is also equipped with an air filter. A positive airflow is also maintained to prevent outside air from entering the sterilized coverage area.

The infant formula was heated at 280 ℃ F. (138 ℃) for 8 seconds and cooled to 73-80 ℃ F. (23-27 ℃). The baby product was placed in a sterilized 3-ounce glass bottle and capped with a sterilized cap. Glass bottles were chosen as packaging materials because they are impermeable to gases, such as oxygen, which may interfere with viability studies. 3ml of a bifidobacteria suspension prepared according to example 2 was aseptically inoculated into glass vials and purged with nitrogen (10 seconds at about 5psi pressure) to remove headspace oxygen. These vials were capped with a sterile closure. To avoid contamination of the baby product, careful attention is paid.

To determine adequate sterilization conditions for glass bottles and lids, a microbiological (scrub) test of the glass bottles and lids was performed. This test yielded less than 10 cfu/total plate count for both vial and cap.

It is important that the infant formula prior to inoculation with bifidobacteria does not contain other viable microorganisms that compete with bifidobacteria. The presence of other microorganisms that will compete with bifidobacteria will shorten the shelf life of the product. Bifidobacteria are scarcely competitive in the presence of other vitamins and are therefore liable to be exceeded. (Gomes, A, M.P. and Malcata, F.X., 1999. Bifidobacterium subspecies are related to lactic acid bacteria: biological, biochemical, technical and therapeutic properties associated with the use as probiotics (Bifidobacterium spp and Lactobacillus: biological, biochemical, technical and therapeutic As probiotics.) Food Science and Technology 10: 139-.

To determine the ultrapasteurization conditions at 270 ° F (138 ℃) for 8 seconds to kill microorganisms (possibly competing with bifidobacteria), a sample of the product subjected to the heating conditions was microbiologically tested. These samples gave a total plate count of < 10 cfu/ml.

To determine the presence of contaminants in the ultra-pasteurized infant product inoculated with bifidobacteria, possibly accessible by filling, venting and capping, a negative control sample (ultra-pasteurized infant product without bifidobacteria) was also prepared.

Example 4

Monitoring of storage and viability

The test sample (with bifidobacteria) and the control sample (without bifidobacteria) from example 3 were stored under refrigerated conditions (4-7 ℃) for 91 days. Two vials were removed per test and control sample per week. The bifidobacteria are listed below: these samples were placed in Lactobacillus MRS agar (Difco # 0882-17-0). Under anaerobic conditions (5% CO)₂、5％H₂And 90% N₂) The inoculated plates were then incubated at 98 ℃ F. (37 ℃) for 72 hours in a Forma Scientific Model 1024(Thermo Forma, Marietta, OH) chamber. Duplicate bottles were also assayed, exhibiting acidity determined by titration and pH determined with an Orion Research digital pH meter (Orion Research, Cambridge, MA). The viability of the test and control samples is shown in table 2.

Table 2.

Viability of Bifidobacterium lactis in infant formulas ready for use during refrigeration

(average of two log cfu/ml readings)

Cultivation time (sky)	Test sample (with Bifidobacterium lactis)	Anaerobic plate count/ml control sample (average of two readings)
			0	7.83	＜10
7	8.02	＜10
			10	7.82	＜10
14	8.03	＜10
			22	8.24	＜10
29	7.93	＜10
			35	8.11	＜10
42	8.00	＜10
			49	8.05	＜10
56	8.16	＜10
			63	7.90	＜10
79	7.70	＜10
			91	7.95	＜10

Referring to table 2, the control samples did not obtain any anaerobic viable microorganisms based on less than 10cfu/ml readings on all samples throughout the storage period. This confirms that there are no contaminants during sample preparation. The test specimen remained not less than 1X 10 throughout the 91-day storage period⁷Target levels of viable bifidobacteria. It is very likely that this level will be a maintenance regime for more than 91 days. Viability values (average of two log cfu/ml readings) are plotted on the y-axis and time (days) on the x-axis. The curve yields a linear regression equation with y-0.0008 x + 8.0097. If the graph assumes a linear regression for 91 days storage, the product will take approximately 2,500 days to 1X 10⁶The level of viable bifidobacteria per ml of product, which suggests a minimum level of bifidobacteria therapeutic effect. Thus, the data show that the present invention can significantly extend the shelf life of a milk composition containing probiotics sufficiently over 100 days, most likely several hundred days. In contrast, commercially available non-fermented milk products (claim 1 × 10)⁶Viable probiotics/ml) has a very limited shelf life of only about 21 days under refrigerated conditions (Shin, H-S, Lee, J-H., Pestka, J. and Ustunol, z.2000a. Viability of bifidobacteria in industrial dairy products during refrigerated periods (viatility of bifidobacteria in commercial dairyproducts). Journal of food protection63 (3): 327-331). These commercially available products are made by adding a probiotic culture mixture to sterilized and vitaminized cow milk and then filling into cartons (see us patent No. 6,194,578).

Table 3 shows the acidity and pH of the test and control samples monitored during storage.

Table 3.

pH and acidity of a ready-to-use infant formula with viable Bifidobacterium lactis

Cultivation time (sky)	Test sample (with Bifidobacterium lactis)		Control sample
						pH	Acidity (% w/w)	pH	Acidity (% w/w)
0	6.14	0.01875	6.96	0.00485
					7	6.21	0.00489	6.92	0.00040
10	6.92	0.00411	6.98	0.00185
					14	6.58	0.00467	6.96	0.00277
22	6.85	0.00513	7.06	0.00279
					29	6.61	0.00544	6.98	0.00323
35	6.39	0.00588	6.86	0.00274
					42	6.54	0.00541	7.08	0.00272
49	6.43	0.00636	6.94	0.00316
					56	6.46	0.00542	7.10	0.00366
63	6.47	0.00808	7.03	0.02697
					79	6.34	0.00715	6.87	0.00364
91	6.45	0.01083	6.87	0.003200

Referring to table 3, neither pH nor acidity changed significantly in the control and test samples during storage. The increase in acidity and subsequent decrease in pH will indicate the metabolic activity of the bifidobacteria. The acidity produced by the fermentation of lactose by bifidobacteria will cause acid shock and kill the bifidobacteria. Thus, the data show that the present invention can significantly extend the shelf life of milk compositions containing probiotics. Sterilizing the milk composition to kill the microorganisms that will compete with the bifidobacteria prior to bifidobacteria inoculation and cold storage can preserve the viability of the bifidobacteria and thereby significantly extend its shelf life.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims (25)

1. A method for preparing a ready-to-use milk composition containing one or more probiotics and having an extended shelf life, the method comprising the steps of:

an ultrapasteurized milk composition;

cooling the ultrapasteurized milk composition under aseptic conditions;

preparing a probiotic culture; and

the probiotic culture is inoculated under aseptic conditions into the ultrapasteurized cold milk composition.

2. The method of claim 1, wherein the probiotic culture is selected from the group consisting of Bifidobacterium, Lactobacillus, and combinations thereof.

3. The method of claim 1, wherein more than one probiotic culture is prepared and inoculated into the ultrapasteurized cold milk.

4. The method of claim 1 wherein the composition in an amount sufficient to produce a probiotic concentration is at least 1 x 10⁸The individual probiotic micro-organisms per ml of milk composition are inoculated with the probiotic culture.

5. The method of claim 1, wherein the probiotic culture is a bifidobacterium lactis.

6. The method of claim 1, wherein the ultrapasteurized milk composition is cooled to a temperature of about 20 ℃ to about 30 ℃.

7. The method of claim 1, further comprising placing the ready-to-use milk composition containing the probiotic in a sterile container in a substantially microorganism-free environment and sealing the container with a sterile closure.

8. The method of claim 7, wherein the container is purged with a sterile inert gas under sterile conditions and then sealed.

9. The method of claim 1, wherein the milk composition is selected from the group consisting of whole milk, skim milk, lactose-free milk, soy-based milk, whey hydrolysate-based milk, casein hydrolysate-based milk, and mixtures thereof.

10. The method of claim 1, wherein the milk composition is an infant formula.

11. A method for preparing a ready-to-use milk composition containing one or more probiotics and having an extended shelf life, comprising the steps of:

an ultrapasteurized milk composition;

cooling the ultrapasteurized milk composition to a temperature of about 20 ℃ to about 30 ℃ while maintaining aseptic conditions;

preparing at least one probiotic culture selected from the group consisting of bifidobacterium, lactobacillus, and combinations thereof under aseptic conditions; and

inoculating the probiotic culture under aseptic conditions into an ultrapasteurized cold milk composition in an amount sufficient to produce a probiotic concentration composition of at least 1 x 10⁸Individual probiotic micro-organisms per ml of milk composition.

12. The method of claim 11 wherein the probiotic is bifidobacterium lactis.

13. The method of claim 11, further comprising placing the ready-to-use milk composition containing the probiotic in a sterile container in a substantially microorganism-free environment and sealing the container with a sterile closure.

14. The method of claim 13, wherein the container is purged with a sterile inert gas under sterile conditions and then sealed.

15. The method of claim 11 wherein the milk composition is selected from the group consisting of whole milk, skim milk, lactose-free milk, soy-based milk, whey hydrolysate-based milk, casein hydrolysate-based milk, and mixtures thereof.

16. The method of claim 11, wherein the milk composition is an infant formula.

17. A ready-to-use milk composition containing one or more probiotics and having an extended shelf life, the milk composition prepared according to the method of claim 1.

18. A method for preparing a ready-to-use infant formula containing one or more probiotics and having an extended shelf life, the method comprising the steps of:

an ultra pasteurized infant formula;

cooling the ultrapasteurized infant product to a temperature of about 20 ℃ to about 30 ℃ while maintaining aseptic conditions;

preparing a probiotic culture selected from the group consisting of Bifidobacterium, Lactobacillus, and combinations thereof under sterile conditions; and

inoculating the probiotic culture under aseptic conditions into an ultrapasteurized cold infant product in an amount sufficient to produce a probiotic concentration composition of at least 1 x 10⁸Individual probiotic micro-organisms per ml of infant formula.

19. The method of claim 18, wherein the infant product in the ultra-pasteurization step is a ready-to-use infant product.

20. The method of claim 18, wherein more than one probiotic culture is prepared and inoculated into the ultra pasteurized cold infant formula.

21. The method of claim 18, wherein the infant product in the ultra-pasteurization step is a reconstituted powdered infant product.

22. The method of claim 18 wherein the probiotic is bifidobacterium lactis.

23. The method of claim 18, further comprising placing the probiotic-containing ready-to-use infant formula in a sterile container in a substantially microorganism-free environment and sealing the container with a sterile closure.

24. The method of claim 23, wherein the container is purged with a sterile inert gas under sterile conditions and then sealed.

25. A ready-to-use infant formula containing one or more probiotics and having an extended shelf life, prepared according to the method of claim 18.