WO2026013243A1

WO2026013243A1 - Process for inoculation of live bacterial cultures into a liquid food product

Info

Publication number: WO2026013243A1
Application number: PCT/EP2025/069861
Authority: WO
Inventors: Tina Malling THORSEN
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2024-07-12
Filing date: 2025-07-11
Publication date: 2026-01-15
Anticipated expiration: 2027-01-12

Abstract

The present invention relates to a process for producing a liquid food product comprising live bacteria.

Description

PROCESS FOR INOCULATION OF LIVE BACTERIAL CULTURES INTO A LIQUID FOOD PRODUCT

Technical field of the invention

The present invention relates to a method for inoculation of concentrated live bacteria cultures into a liquid food product, the concentrated live cultures requiring neither incubation nor preculture and which have no potential health risk.

Background of the invention

Probiotics, which are live bacterial cultures that provide health benefits when consumed, can be added to dairy products or other food products at various stages of production. Commonly used probiotic strains in the dairy products include Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus easel, Bifidobacterium bifidum, and others.

The addition of probiotics to dairy products involves careful consideration of factors such as the type of probiotic strain, concentration levels, fermentation conditions, and post-fermentation processing. The goal is to ensure the viability and stability of the probiotic cultures throughout the production process and during the shelf life of the final product.

Various methods can be used to incorporate probiotics into dairy, including direct inoculation, use of starter cultures containing probiotics, or post-fermentation addition. Careful monitoring of factors such as pH, temperature, oxygen levels, and storage conditions is essential to maintain the viability and functionality of the probiotic cultures.

In the dairy industry, adding probiotics to products like yogurt, kefir, fermented milk, and cheese has been studied extensively due to the potential health benefits associated with consuming these probiotic-rich dairy products. These benefits may include improved gut health, immune modulation, and overall well-being.

The food product to be inoculated with concentrated live cultures means that the manufacturer using them often needs to work in batchwise mode for the inoculation and fermentation phases.

Probiotics can be added at different stages of the dairy production process to ensure their viability and survival. Common points in the process where probiotics can be added include:

Adding probiotics after the food product such as milk has been pasteurized and cooled but before fermentation is a common practice. This allows the probiotic cultures to be introduced into a favorable environment for growth without being exposed to high temperatures that could compromise their viability.

Probiotics can be added during the fermentation stage, especially in products like yogurt and kefir, where the fermentation process provides an ideal environment for the growth and activity of probiotic cultures.

In some cases, probiotics can be added after the fermentation process, such as when preparing probiotic-fortified dairy drinks or when formulating probiotic-enriched products.

Regardless of the stage at which they are added, it is crucial to monitor and control factors such as temperature, pH, oxygen levels, and processing conditions to ensure the viability and stability of the probiotic cultures throughout the production process and during the shelf life of the final product.

Probiotics are often distributed in bags. The bags are often small in volume such as less than 1 liter (L) for easier handling. Challenges associated with larger bags for storing probiotics include difficulties in maintaining temperature and moisture control, ensuring uniform distribution of contents, and handling and storage logistics. Larger bags pose a higher risk of contamination, require more extensive space and infrastructure, and can lead to increased product waste and reduced shelf life. Additionally, quality control processes are more complex and less efficient in larger volume contexts, potentially compromising the viability and efficacy of the probiotics.

Adding probiotics in batches or from smaller bags can also have several drawbacks and may result in uneven distribution of the probiotic cultures within the product, potentially leading to variability in the concentration of live bacteria throughout the batches. Furthermore, the viability and activity of probiotic cultures may be affected during storage and handling in bags, especially if not maintained under optimal conditions or if the bags are not suitable for aseptic inoculation. This could lead to a decrease in the number of viable probiotics by the time they are introduced into the food product stream. Furthermore, it can be challenging to control the flow rate of probiotics from bags into the food stream, potentially leading to varying concentrations of probiotics being introduced at different times. Handling probiotics in bags or during batchwise addition introduces opportunities for contamination and compromises sterility, which is crucial for maintaining the quality and safety of the product. Also, batchwise addition may not align with continuous production processes, potentially impacting the efficiency of the overall manufacturing operation.

Summary of the invention

The above objects are achieved with the present invention, which among others, is directed to a process of inoculating live bacterial cultures into a liquid food product.

In one aspect the present invention relates to a process of producing a liquid food product comprising live bacteria at a predetermined concentration, said process comprising the following steps of: a) providing the liquid food product in a product stream moving from at least one food production tank via a first pipe to an aseptic tank and optionally further through a second pipe to a dosing unit, wherein said product stream has a flow rate ql, b) providing at least one container comprising a suspension of live bacteria, the container further comprising at least one outlet port equipped with opening means for regulating a flow of the bacteria suspension q2 and further wherein the concentration of the live bacteria in the suspension is at least 10E8 cfu/g; c) aseptically and operatively connecting the at least one container to said product stream either before or after or to the aseptic tank via a sterile transfer tube allowing for a flow rate q2 of the live bacteria suspension into said product stream, the transfer tube having a first end coupled to an outlet port of the container or aseptic tank (109) and a second end coupled to an inlet port of the first or the second pipes or the aseptic tank, d) opening the opening means of the at least one container to allow the bacteria suspension to flow into the sterile transfer tube; e) using a pump or gravity feed system or manually applying force by squeezing the container to facilitate the flow of the bacteria suspension from the at least one container through the sterile transfer tube and into the first or second pipes or the aseptic tank and thereby inoculate the live bacteria suspension into the product stream with an inoculation rate q2/ql*100, wherein the total volume of the bacterial suspension must be greater than IL and wherein q2 is adjusted to the concentration of the live bacteria suspension, the flow rate ql of the liquid food product and the predetermined concentration of bacteria in the liquid food product. f) optionally conduct the product stream from dosing unit for dosing the product and packaging.

The volume of the container must be greater than 1 Liter (L). The size of the container designed to contain a bacteria suspension great than 1 L may be determined by a combination of factors, including but not limited to the total volume of suspension required for storage, the intended purpose and frequency of bacteria suspension usage, compliance with regulatory and safety standards, the physical space available for container installation, the materials of construction compatible with the suspension, temperature control needs, pressure requirements, allowances for thermal expansion and contraction, cost considerations, maintenance and cleaning requirements, environmental conditions, and potential for future expansion or changes in usage patterns.

The total volume of the bacteria suspension needed for depends at least on the flow rate of the bacteria suspension q2 and the total time for production of the liquid food product comprising the live bacteria. When adding the live bacteria suspension by using one or more containers having a volume greater than 1 L and aseptically connected to the food system after any possible heat treatment of the food product allows for a continuous production of the liquid food product comprising live bacteria rather than batch production. Also, such a process allows for higher product flow rates (q 1). It is required that the bacteria of the present invention possess the ability to survive for an extended period of time within a larger volume. This ensures the viability and functionality of the bacteria are maintained over the specified duration, facilitating their intended application within the system. The survival capability of the bacteria in a larger volume is critical to achieving the desired outcomes and maintaining the efficacy of the process.

The bacteria may be stored in the container for 1, 5, 10, 50, 72 hours or more prior to inoculation into the liquid food product.

The viability of the bacteria may be at least 60% after inoculation into a liquid food product as calculated based on the concentration of CFU (colony forming units) of the concentrated culture and the concentration of CFU of the inoculated material, such as 75%, 80%, 90%, 95% or 99%.

In one embodiment of the invention, the flow rate of the bacteria suspension may be different from 0.5 L/h (q2 < 0.5 L/h or q2 >0.5 L/h) at a density of the bacteria suspension of 1 g/ml.

The ratio of the flow rate of bacteria q2 to the flow rate of the product stream ql is a measure of the concentration of probiotics relative to the overall flow of the product. This ratio provides insight into the relative abundance of probiotics within the product stream and is denoted the inoculation rate. Furthermore, the inoculation rate can be useful in determining the level of probiotic supplementation or inoculation in the product stream, which is important for consistency, quality control, and achieving desired probiotic levels in the final product. The inoculation rate is also dependent on the concentration of bacteria in the end product, i.e., the predetermined concentration, and the concentration of the bacteria in the suspension to be inoculated into the liquid food product and may be estimated by the ratio of the concentration of bacteria in the end product and the concentration of the bacteria in the suspension to be inoculated into the liquid food product.

In one embodiment the concentration of the live bacteria in the end product may typically be in the range 10E+05 to 10E+09 CFU/g, such as 10E+05, 10E+06, 10E+07, 10E+08 or 10E+09 CFU/g.

The concentration of live bacteria comprised in the container may be at least 10E+8 or in the range of 10E+8 to 10E+ 12 such as 10E+8, 10E10+9, 10E+10, 2xl0E+ 10, 5X10E+ 10, 10E+11, 2X10E+ 11, 5X10E+ 11 or 10E+ 12 CFU/g.

In one embodiment of the invention, the inoculation rates may be greater than 0.0001%, such as 0.0001%, 0.001%, 0.0045%, 0.005%, 0.007%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.5% or 1.0%. In a preferred embodiment the inoculation rate is between 0.001%-0.2%. In one embodiment the inoculation rate may be 0.009%.

Table 1 : Examples of inoculation rates based on the ratio-% between the concentration of the bacteria in the liquid food product and the bacteria suspension to be added to the liquid food product.

A method and system for controlling the flow rate of bacteria may involve selecting a suitable pump to achieve desired dosing. Pumps capable of lower flow rates, such as the Watson Marlow 530/520R.ET pump head paired with a 3.2 mm pump tube, offering a minimum flow rate of 14.4 ml/h, equivalent to 0.0144 L/h, may be used. Some implementations position the pump within the aseptic tank alongside the bags. Additionally, integrating weighing cells or flow cells to regulate the pump allows for enhanced dosing control, albeit at an increased equipment cost. Weighing cells can measure the mass of the substance being dispensed, while flow cells can monitor the flow rate of the substance. By integrating these components, the system gains the ability to adjust dosing in real-time based on accurate measurements, leading to more precise and consistent dosing of the substance. This enhanced control helps optimize dosing accuracy and ensures that the desired amount of the substance is delivered, contributing to improved overall process efficiency and product quality.

In one embodiment of the invention the flow rate of the bacteria suspension may be in the range of 0.01 to 20.0 L/h, such as 0.01, 0.025, 0.1, 0.15, 0.20, 0.25, 0.30, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, 0.90, 2.0, 2.3, 3.0, 4.0, 4.5, 10.0, 20.0 L/h.

The flow rate for the liquid product may be in the range of 1,000 to 50,000 L/h depending on the product and system used. The maximum flow rates for canning operations typically range from 30,000 to 40,000 L/h, while bottling operations may achieve a flow rate of up to 13,000 L/h. It is noted that the flow characteristics are influenced by factors such as temperature, carbonation levels, pressure, and viscosity. Additionally, it is observed that beer exhibits a propensity to foam, unlike soda and juice. A person skilled in the art is knowledgeable in adjusting operational parameters, such as temperature, carbonation levels, pressure, and viscosity, to optimize flow rates during canning and bottling processes, taking into account the specific properties of different beverages. It is understood that practitioners skilled in beverage packaging techniques are capable of making appropriate adjustments to accommodate variations in flow behaviour, thereby maintaining operational efficiency and product quality.

In a preferred embodiment, the flow rate ql of the liquid product stream is greater than 5,000 L/h or in the range of 5,000-20,000 L/h, such as 5,000, 10,000, 15,000 or 20,000 L/h.

In some embodiments, the live bacteria, such as the probiotic live bacteria as described herein are introduced or inoculated to the liquid food product after the fermentation of said liquid food product, such as dairy liquid food product or a beverage, for example after the fermentation to obtain yogurt, buttermilk, kefir, quark, tvorog, creme fraiche or sour cream. In such embodiments, the live bacteria may thus be added using the process described herein to the final/end liquid product.

The process of addition of the live bacteria to the liquid product described herein surprisingly maintains the cell count stability and final product quality, such as the post-acidification of the product during storage, as demonstrated in the Examples herein.

In another embodiment, the one or more food production tank is one or more fermentation tank.

In yet another embodiment, a heating unit may be inserted before the container or the aseptic tank. This is to ensure that the bacteria are added after fermentation, pasteurization or heating of the product.

The at least one container may be a bag, the bag comprising a flexible, biocompatible material or a tank, the tank may be a culture tank or a dosing tank or a combination of at least one bag and at least one tank. In an embodiment with at least two containers, the at least two containers may be connected to allow a flow of live bacteria through the containers. The at least two containers may be a combination of a bag and a culture tank. The total volume of the container or combination of containers must great than 1 liter (L) to comprise a bacteria suspension of greater than IL. The container is characterized by being aseptically connectable to a food production system. The container comprises means for establishing and maintaining an aseptic connection with the food production system, thereby ensuring sterility during the transfer of contents between the container and the food production system.

In one embodiment where the container is a culture tank, said culture tank is prefilled with live bacterial suspension 1 h up to 72 hours prior to dispensing into the liquid food product.

In one embodiment, the first and the second pipes are provided with mixing means when connected to the container, and wherein the process comprises a further step of activating the mixing means in the first or the second pipe to ensure uniform distribution of the bacteria within the product.

All containers or tanks described in the present invention such as the food or fermentation tank, the culture tank or the aseptic tank may comprise mixing means. Also, the container being a bag may have mixing means. This mixing means may include, but is not limited to, mechanical, manual, or automated mixing mechanisms such as stirrers, agitators, or other suitable mixing devices. This mixing speed of the bacteria before transferred to the liquid food product may be in the range of 50-250 rpm, such as 50 rpm or 100 rpm and may be referred to as gently stirring.

In one embodiment, the live bacteria is a freeze-dried culture, wherein the freeze-dried culture may be suspended in a suitable diluent solution such as peptone water, milk, water, saline or the liquid food product to be inoculated prior to inoculation. Further, the live bacteria may not comprise any added additives. The live bacteria may be frozen in the presence of a cryoprotectant in order to stabilize the bacterial cells during and after freezing or freeze-drying.

The viability of the bacteria may be at least 60% after inoculation into a liquid food product as calculated based on the concentration of CFU (colony forming units) of the concentrated culture and the concentration of CFU of the inoculated material.

The viability of the bacteria may be at least 80% after storage for five months at a temperature of -20°C for a freeze-dried culture or -55°C for a frozen culture followed by direct inoculation into a liquid food product as calculated based on the concentration of CFU (colony forming units) of the bacteria before freezing and the concentration of CFU of the inoculated liquid food product.

The bacterial strain may be selected from the group consisting of Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus, and here "Lactobacillus" represents the grandfathered term referring to all bacteria classified in LactobaciHaceae prior to 2020 (including but not limited to Lactobacillus acidophilus, Lacticaseibacillus easel subsp. easel, Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum , Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius'), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum'), as well as suitable combinations of the foregoing.

The live bacterial culture may comprise one or more strains belonging to the genus Lactobacillus. In one embodiment, the bacteria is Lacticaseibacillus rhamnosus GG. The nucleotide sequence of the Lacticaseibacillus rhamnosus GG may be at least 99.00% identical to SEQ ID NO. 1 as defined herein.

The liquid food product may be a dairy product, or a beverage and it may be a fermented product. The dairy product may be a yogurt, buttermilk, kefir, quark, tvorog, creme fraiche or sour cream. The liquid food product may be a water, juice, smoothie, yogurt drink, kefir drink or fermented tea.

Brief description of the figures and drawings

Figure 1 : System for production of a liquid food product where the live bacteria is added from a container to a culture tank and where the culture tank is connected to a first pipe between the product tank and aseptic tank and where the first pipe comprises mixing means.

Figure 2: System for production of a liquid food product where the live bacteria is added directly from a container to the aseptic tank comprising mixing means.

Figure 3: System for production of a liquid food product where the live bacteria is added from a bag to a culture tank and where the second pipe connecting the aseptic tank to the dose tank comprises mixing means.

Figure 4: The CFU count of the different sampling points is shown.

Figure 5: Flow diagram a process according to the invention.

Detailed description of the invention

Prior to outlining the present invention in more details, a set of terms and conventions is defined :

To describe the correlation between the start concentration of live bacteria or probiotics, i.e., the bacteria comprised in the container, the food stream rate, the inoculation rate of the bacteria, and the end concentration of bacteria in the final product in a liquid food production system, mathematical modeling or statistical analysis may be used.

Some bacterial strains may continue to multiply in the final product depending on the genus/species/strain and the food product. This will allow for a lower concentration of the bacteria suspension to be added to the liquid food product.

One approach to determine the final concentration of the live bacteria in the end product is to use a mass balance equation to represent the dynamics of bacterial concentration in the system. This equation can at least account for the start concentration of bacteria, the food stream rate (inflow and outflow), and the inoculation rate of the bacteria. By solving or simulating this equation, it may be analyzed how these factors influence the end concentration of live bacteria in the final product.

Another approach is to conduct experimental studies to measure the end concentration of bacteria under different combinations of start concentration of bacteria, product stream rate, and inoculation rate of the bacteria. Statistical analysis can then be used to identify correlations and quantify the relationship between these variables.

Overall, understanding the correlation between these factors may require a combination of mathematical modeling, experimental data, and statistical analysis to accurately describe their impact on the end concentration of probiotics in the final product.

In general, if the inoculation rate is kept constant across different concentrations of the bacteria, the resulting concentration of bacteria in the end product (measured as CFU/g) would decrease with each dilution of the bacteria suspension due to the reduction in the start bacterial concentration. However, the relationship between the inoculation rate and the resulting concentration of bacteria may not follow a straightforward correlation such as a linear or exponential correlation, as it depends on factors such as the viability of the bacteria, the accuracy of dilution techniques, and the growth conditions.

The addition of probiotics or live bacteria to liquid product streams may affect dosing precision in different ways:

- The uniformity and consistency of the probiotic culture can significantly impact the precision of the dosing or inoculation. Variations in the concentration or viability of the probiotic cells within the culture can lead to inconsistent dosing.

- The precision and accuracy of the dispensing equipment used to add probiotics to the product stream can influence dosing precision. Factors such as calibration, maintenance, and design of the equipment play a crucial role.

- The effectiveness of mixing and distribution within the culture tank and product stream can impact dosing precision. Inadequate mixing can result in uneven distribution of probiotics, leading to variability in the final product.

Environmental factors such as temperature, pH, and exposure to oxygen can affect the viability and stability of probiotics, which in turn can influence dosing precision.

Consistent flow rates and stable process conditions are essential for precise dosing. Fluctuations in flow rates or process stability can lead to variations in probiotic dosing.

- Accurate sampling and testing protocols are important for verifying the concentration and viability of probiotics. Inaccurate testing methods or inadequate sampling can lead to imprecise dosing.

- Adherence to quality control measures, including regular monitoring, validation of dosing procedures, and corrective actions, is essential for maintaining dosing precision.

- The specific characteristics of the product, such as viscosity, density, and compatibility with probiotics, can impact dosing precision and the ability of the probiotics to disperse uniformly.

Overall, achieving dosing precision when adding probiotics requires careful attention to these factors and may involve a combination of process optimization, equipment selection, and quality assurance measures.

The skilled person in the field of liquid food production and probiotics will understand how to conduct the analyses by utilizing their expertise in microbiology, food science, and statistical methods. They will possess the knowledge and skills to apply mathematical modeling techniques, such as mass balance equations, to represent the dynamics of bacterial concentration in the liquid food production system. Additionally, they will be adept at designing and conducting experimental studies to measure the end concentration of probiotics under varying conditions of start concentration, product stream rate, and inoculation rate.

Furthermore, the skilled person will have a deep understanding of statistical analysis methods, including regression analysis, ANOVA (analysis of variance), and correlation analysis, which are essential for identifying and quantifying the relationships between the different variables. They will also be proficient in using statistical software to analyze experimental data and draw meaningful conclusions regarding the correlation between the start concentration of probiotics, the food stream rate, the probiotic inoculation rate, and the end concentration of probiotics in the final product. Overall, the skilled person will possess a combination of theoretical knowledge, practical experience, and analytical skills to effectively conduct and interpret these analyses in the context of liquid food production with probiotics.

The term batch production involves processing products such as dairy products in discrete, separate quantities or "batches". Each batch goes through the production process independently of the others. This method is often used for products that require specific recipes, variations, or small-scale production runs.

The term continuous production involves a non-stop process where products such as dairy products are produced continuously without interruption. This method is suitable for high-volume production and standardized products.

In the context of the present invention, bags are utilized for storing and handling biological materials, including microorganisms. The bag must be suitable for holding concentrated microorganisms without contamination or degradation. The material may be sterile plastic, polyethylene, polypropylene. The bag is sterile or can be sterilized to maintain the viability of the microorganisms and prevent contamination. The bag may be sealed by heat or zip-lock to ensure the contents are securely contained. The bag may be a flexible, biocompatible material, a secure sealing mechanism to prevent contamination and leakage, and a port for introducing and extracting the microorganisms. The bags may be connected together to increase the volume of microorganisms added to the food stream. Bags are flexible and can be easily manipulated or stored in various spaces. They can be produced in different sizes to accommodate varying volumes of microorganisms. Bags are often more cost-effective than rigid containers, especially for large-scale applications. The bags may be used for transporting live bacteria between different locations, the microorganisms may be in a frozen format. Thus, the bags may be suitable for temporary storage of microbial live cultures. Also, the bags must be suitable for aseptic inoculation. Freeze-dried cultures may be stored in special bags protecting against oxidation of the product such as flexible bags comprising aluminium. In some contexts, specific terms like "bioreactor bag," "sterile sampling bag," or "storage bag" might be used to highlight the intended purpose and specific features of the bag.

In the context of a patent application or technical description, the term "liquid" may encompass pourable substances. A liquid is a state of matter characterized by having a definite volume but no fixed shape, allowing it to flow and take the shape of its container. This definition includes substances that are pourable, such as water, beverages, oils, yogurts and various solutions and milk products.

The term "genus" means genus as defined on the website: www.ncbi.nlm.nih.gov/taxonomv. A bacterial "strain" as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied.

In connection with strains of the genus Lactobacillus and here "Lactobacillus" represents the grandfathered term referring to all bacteria classified in Lactobacillaceae prior to 2020, the term "CFU" means colony forming units as determined by growth (forming a colony) on an MRS agar plate (pour plating) incubated at anaerobic conditions at 37 °C for 3 days. The MRS agar has the following composition (g/l) :

Bacto Proteose Peptone No. 3: 10.0

Bacto Beef extract: 10.0

Bacto Yeast extract: 5.0

Dextrose: 20.0

Sorbitan Monooleate Complex: 1.0

Ammonium Citrate: 2.0 Sodium Acetate: 5.0

Magnesium Sulfate: 0.1

Manganese Sulfate: 0.05

Potassium Phosphate Dibasis: 2.0

Bacto Agar: 15.0

Milli-Q water: 1000 ml. pH may be adjusted to 5.4 or 6.5: pH is be adjusted to 6.5 for L. rhamnosus, L. casei and L. paracasei. For all other Lactobacillus species the pH may be adjusted to 5.4. In particular, pH may be adjusted to 5.4 for i., delbrueckii subsp. bulgaricus,- L. acidophilus and L. helveticus. pH is adjusted to 6.5 for L. rhamnosus, L. casei and L. paracasei.

Surface plating may be used instead of the pour plating mentioned above. In this case, reading of the plate is after 2 days of anaerobic incubation.

The composition of ambient live bacteria to be added to the liquid food product of the present invention may be provided in several forms. It may be a powder, pellets or tablets. It may be a frozen form, dried form, freeze dried form, or liquid form. Thus, in one embodiment the composition is in frozen, dried, freeze-dried or liquid form. The live bacteria must be thawed and in case of providing the bacteria in a dry format, these must be suspended in solution suitable for the end product such as a peptone water, milk or the liquid product to obtain a concentrated solution of bacteria prior to transfer from the bags. The solution of bacteria may be diluted according to the specific use and concentration of live bacteria desired in in the end product.

Peptone water may serve as a diluent for the preparation of dilutions of the bacteria or suspension of the dried microorganisms into a concentrated solution. It is composed of peptone (a partially digested protein), sodium chloride, and water. The composition of peptone water can vary depending on the microorganisms and its intended use and may compose of physiological saline 0.9%, buffer pH 7-5, water and milk. If milk is used, it is important that the culture is lactose-negative, otherwise the bacterial culture will start utilizing the lactose in the milk. The composition of peptone water may vary depending on the microorganisms and its intended use.

The composition of the present invention may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and/or nutrients, more preferably cryoprotectants, lyoprotectants and/or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectantare known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate).

In one embodiment, the composition according to the present invention may comprise one or more cryoprotective agent(s) selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine -5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine- 5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and/or flavorants.

In one embodiment of the invention the cryoprotective agent is an agent or mixture of agents, which in addition to its cryoprotectivity has a booster effect.

The expression "booster effect" is used to describe the situation wherein the cryoprotective agent confers an increased metabolic activity (booster effect) on to the thawed or reconstituted culture when it is inoculated into the medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercial frozen or freeze-dried cultures may retain their viability, although they may have lost a significant portion of their metabolic activity e.g. cultures may lose their acid-producing (acidification) activity when kept stored even for shorter periods of time. Thus, viability and booster effect has to be evaluated by different assays. Whereas viability is assessed by viability assays such as the determination of colony forming units, booster effect is assessed by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity" refers to the oxygen removal activity of the cultures, its acid-producing activity, i. e. the production of e. g. lactic acid, acetic acid, formic acid and/or propionic acid, or its metabolite producing activity such as the production of aroma compounds such as acetaldehyde, (a-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)). In one embodiment, the live bacteria do not comprise any added additives or cryoprotective agents.

In one embodiment the composition of the invention contains or comprises from 0.2% to 20% of the cryoprotective agent or mixture of agents measured as % w/w of the material, such as 0.2% to 15%, from 0.2% to 10%, from 0.5% to 7%, and from 1% to 6% by weight, including within the range from 2% to 5% of the cryoprotective agent or mixture of agents measured as % w/w of the frozen material by weight. The culture may comprise approximately 3% of the cryoprotective agent or mixture of agents measured as % w/w of the material by weight. The amount of approximately 3% of the cryoprotective agent corresponds to concentrations in the 100 mM range. It should be recognized that for each aspect of embodiment of the invention the ranges may be increments of the described ranges.

In the present context the term "from x% to y%" means to include the end-points, thus equal to the term "from and including x% to and including y%"

In a further aspect, the composition of the present invention contains or comprises an ammonium salt (e.g., an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g., growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells. The term "ammonium salt", "ammonium formate", etc., should be understood as a source of the salt or a combination of the ions. The term "source" of e.g., "ammonium formate" or "ammonium salt" refers to a compound or mix of compounds that when added to a culture of cells, provides ammonium formate or an ammonium salt. In some embodiments, the source of ammonium releases ammonium into a growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, ammonium is not provided by the dairy substrate. It should of course be understood that ammonia may be added instead of ammonium salt. Thus, the term ammonium salt comprises ammonia (NH3), NH4OH, NH4⁺, and the like.

In one embodiment the composition of the invention may comprise thickener and/or stabilizer, such as pectin (e.g. HM pectin, LM pectin), gelatin, CMC, Soya Bean Fiber/Soya Bean Polymer, starch, modified starch, carrageenan, alginate, and guar gum.

In one embodiment wherein the microorganism produces a polysaccharide (such as EPS) which causes a high/ropy texture in the acidified milk product the acidified milk product is produced substantially free, or completely free of any addition of thickener and/or stabilizer, such as pectin (e.g. HM pectin, LM pectin), gelatin, CMC, Soya Bean Fiber/Soya Bean Polymer, starch, modified starch, carrageenan, alginate, and guar gum. By substantially free should be understood that the product comprises from 0% to 20% (w/w) (e.g. from 0% to 10%, from 0% to 5% or from 0% to 2% or from 0% to 1%) thickener and/or stabilizer.

The composition of the present invention may comprise probiotic bacteria. Probiotic bacterial strains must be added after fermentation and/or heating or step of pasteurization of the liquid product.

The term "probiotic bacteria" refers to viable bacteria which are administered in adequate amounts to a consumer for the purpose of achieving a health-promoting effect in the consumer. Probiotic bacteria may be capable of surviving the conditions of the gastrointestinal tract after ingestion and and may be able to colonize the intestine of the consumer.

It will be appreciated that the Lactobacillus genus taxonomy was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if not otherwise indicated. For the purpose of the present invention, the table below presents a list of new and old names of some "Lactobacillus" species relevant to the present invention, and here "lactobacillus" represents the grandfathered term referring to all bacteria classified in Lactobacillaceae prior to 2020.

Table 2. New and old names of some Lactobacillus species relevant to the present invention.

In a particular embodiment of the invention the probiotic strain according to the present invention is selected from the group consisting of bacteria of the genus Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus easel, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii, the genus Bifidobacterium, such as the Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis, and the like.

In a particular embodiment of the invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus easel, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii.

In a particular embodiment of the invention, the probiotic Lactobacillus strain is selected from the group consisting of a Lacticaseibacillus rhamnosus strain and a Lacticaseibacillus paracasei strain. In a particular embodiment of the invention, the probiotic strain is Lacticaseibacillus rhamnosus strain GG deposited as ATCC 53103.

In a particular embodiment of the invention, the probiotic Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis. In a particular embodiment of the invention, the probiotic Bifidobacterium probiotic strain is Bifidobacterium animalis subsp. lactis BB-12® deposited as DSM 15954.

In certain embodiments, the composition comprises the probiotic Lacticaseibacillus rhamnosus GG. It is preferred that the probiotic is LGG® produced and sold by Chr. Hansen A/S or its successor in title. It is preferred that the nucleotide sequence of the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1, preferably at least 99.10%, at least 99.20%, at least 99.30%, at least 99.40%, at least 99.50%, at least 99.60%, at least 99.70%, at least 99.80% identity to SEQ ID NO.l, more preferably at least 99.90%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98% identity to SEQ ID NO. 1, even more preferably at least 99.99% identity to SEQ ID NO. 1 or is identical to SEQ ID NO. 1

In certain embodiments, the composition comprises the probiotic Lacticaseibacillus rhamnosus GG and the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the nucleotide sequence of SEQ ID NO.l. Preferably, the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15 nucleotides from the nucleotide sequence of SEQ ID NO.l.

The nucleotide sequence of SEQ ID NO. 1 as used herein is available under reference number GCF_028475085.1 in the ncbi database (https://www.ncbi.nlm.nih .gov/) as Lacticaseibacillus rhamnosus GG genome assembly ASM2847508vl. Reference is made to the database in the version accessible on 07 March 2024.

The term "sequence identity of [a certain] %" in the context of two or more nucleotide sequences refers to a relationship between the sequences of two polynucleotides, as determined by sequence comparison (alignment). As used herein, "sequence identity" is determined across the entire length of a sequence. "Sequence identity" means that the two or more sequences have nucleotides in common in the given percentage when compared and aligned. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithms, or computer program.

Percent sequence identity of nucleotide sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. In preferred embodiments, the "percent identity" of two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et aL, J. Mol. Biol. 215:403-10, 1990. Where gaps exist between two sequences, Gapped BLAST ® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17) :3389-3402, 1997. When utilizing BLAST and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

Another local alignment technique which may be used, for example, is based on the Smith- Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol. 147: 195-197). A general global alignment technique which may be used, for example, is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453), which is based on dynamic programming.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The above mixtures or kit-of-parts may be further combined with other lactic acid bacteria such as but not limited to probiotic bacteria. In one embodiment the at one or more lactic acid bacteria is selected from the group consisting of Bifidobacterium such as Bifidobacterium animalis subsp. lactis (e.g. BB-12®), Lactobacillus acidophilus (LA-5®), Lacticaseibacillus rhamnosus (e.g. LGG®) and any combinations thereof. Which Bifidobacterium, Lactobacillus acidophilus and/or Lacticaseibacillus rhamnosus to apply depend on their application and food to be produced.

In a particular embodiment the bags comprises from >10⁸, 10⁸ to 10¹³ CFU/g of the Lactobacillus strain, from 10⁸ to 10¹² CFU/g, from 10⁸ to 10¹¹ CFU/g, or from 10⁷ to 10¹⁰ CFU/g of the Lactobacillus strain.

In a particular embodiment the composition further comprises from >10⁸ (10⁸ equals 10E+08), 10⁸ to 10¹³ CFU/g of the Lactobacillus strain, from 10⁸ to 10¹² CFU/g, from 10⁸ to 10¹¹ CFU/g, or from 10⁷ to IO¹⁰ CFU/g of the Lacticaseibacillus rhamnosus GG strain.

The composition culture may be frozen, spray-dried, freeze-dried, vacuum-dried, air dried, tray dried or in liquid form. Typically, if the culture is freeze-dried, the storage stability of the composition and/or starter culture can be extended by formulating the product with low water activity. By controlling the water activity (Aw), it is possible to predict and regulate the effect of moisture migration on the product. Therefore, it may be preferred that the water activity (Aw) of the dried compositions herein is in the range from 0.01-0.8, preferably in the range from 0.05-0.4.

The live bacteria are typically thawed under carefully controlled conditions to prevent them from becoming metabolically active. The concentrated live bacteria may be thawed at >0 - 30°C, such as 15°C until completely thawed prior to the transfer to the culture tank or aseptic tank. This may be by means of a temperature-controlled chamber between 0 to 30°C or by rehydrating freeze-dried concentrated live cultures until a temperature of 0 to 30°C such as 15°C. The live bacteria may be thawed at even higher temperatures than 30°C but then the culture is typically cooled afterwards to 5-15 °C or 5-10°C.

"Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

Fermentation processes to be used in production of fermented milk products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected so as to support the achievement of the present invention, i.e. to obtain a fermented product such as a dairy or dairy analogue product in solid or liquid form (fermented milk product).

Even though the fermented product and/or the food product itself comprise acid and flavor generated during fermentation it may be desired that fermented product and/or the dairy product comprises an ingredient selected from the group consisting of a fruit concentrate, a syrup, a probiotic bacterial strain or culture, a coloring agent, a thickening agent, a flavoring agent, a preserving agent and mixtures thereof.

The food product may be a fermented product. The fermented product may be a dairy or dairy analogue product. In another embodiment the fermented product may be in the form of liquid food product such as a stirred type product, a set type product or a drinkable product.

It will be appreciated that aspects and embodiments disclosed in the parts termed "System" and "Method of producing a liquid food product" may be equally relevant to the present part of the specification.

The use of the terms "a” and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The potency of a culture refers to the strength or effectiveness of the live microorganisms within the culture. In the context of probiotics, potency typically refers to the viable count of live bacteria present in the product at the time of consumption. This is often expressed as the number of colony-forming units (CFUs) per serving or per gram of the product. The potency of a culture is an important factor in determining its potential health benefits and effectiveness in delivering the desired probiotic effects to the consumer.

In one embodiment of the invention the process for producing a liquid food product comprising live bacteria at a predetermined concentration, said process comprises the following steps of: a) providing the liquid food product in a product stream moving from at least one food production tank 106 via a first pipe 107 to an aseptic tank 109 and optionally further through a second pipe 108 to a dosing unit 113, wherein said product stream has a flow rate ql, b) providing at least one container 100, 101 comprising a suspension of live bacteria, the container further comprising at least one outlet port 105a equipped with opening means for regulating a flow of the bacteria suspension q2 and further wherein the concentration of the bacteria suspension is at least 10E8 cfu/g; c) aseptically and operatively connecting the at least one container 100, 101 to said product stream before the aseptic tank via a sterile transfer tube 110, 111 allowing for a flow rate q2 of the live bacteria suspension into said product stream, the transfer tube 110, 111 having a first end coupled to an outlet port 104, 105a, 105b of the container or aseptic tank 109 and a second end coupled to an inlet port 102, 103a, 103b of the first or the second pipes 107, 108 or the aseptic tank 109, d) opening the opening means of the at least one container 100, 101 to allow the bacteria suspension to flow into the sterile transfer tube 110, 111; e) using a pump 112a, 112b or gravity feed system or manually applying force by squeezing the container to facilitate the flow of the bacteria suspension from the at least one container through the sterile transfer tube 110, 111 and into the first or second pipes 107, 108 or the aseptic tank 109 and thereby inoculate the live bacteria suspension into the product stream with an inoculation rate q2/ql*100, wherein the bacterial suspension has a total volume greater than IL and wherein q2 is adjusted to the concentration of the live bacteria suspension, the flow rate ql of the liquid food product, and the predetermined concentration of the bacteria in the liquid food product, f) optionally conducting the product stream from dosing unit (113) for dosing the product and packaging.

The food production system upon which this embodiment is based is depicted in Fig. 1.

A flow diagram of the food production system is exemplified in Fig. 5. The food production system according to the invention is not limited to the flow diagram in Fig. 5.

In another embodiment of the invention the process for producing a liquid food product comprising live bacteria, the at least one container 100 is connected directly to the aseptic tank. The food production system upon which this embodiment is based is depicted in Fig. 2.

In yet another embodiment of the invention the process for producing a liquid food product comprising live bacteria comprise the at least one container 100, 101 is connected to the pipe 108 after the aseptic tank. The food production system upon which this method is based is depicted in Fig. 3.

In one embodiment there is one container which has a volume greater than 1 L.

In another embodiment of the invention, there are two connected containers, the containers being a bag and a tank, respectively, wherein the bag has a volume of 1 L and the tank has a volume greater than 1 L.

In yet another embodiment, the flow rate of the bacteria suspension from the container to the food production system may be q2 < 0.5 L/h or q2 >0.5 L/h at a density of the bacteria suspension of 1 g/ml.

The inventors have demonstrated herein that using the process of the present invention, the cell count of the live bacteria in its container (100, 101) was very stable, even when the live bacteria was held for example up to 72hrs, such as 24hrs, after thawing, as liquid product at low temperature, such as 10 °C or lower, before its transfer or inoculation to the liquid food product.

The inventors have also demonstrated herein that using the process of the present invention, the cell count of the live bacteria in the liquid product was very stable, even when the live bacteria was held for example up to 72hrs, such as 24hrs, after thawing, as liquid product at low temperature, such as 10 °C or lower, before its transfer or inoculation to the liquid food product.

In addition, in embodiments where the liquid product is a fermented dairy product, such as yogurt, the inventors have shown herein that using the process of the present invention, both cell count and post-acidification of the final product when stored for up to 150 days, such as 28 days, such as 14 days, such as 6 days, at 25 °C or 37 °C was not affected by the holding time of the live bacteria after thawing and before its transfer or inoculation to the liquid food product. This was shown at least up to 72hrs after thawing, such as 24hrs after thawing.

The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the live microbial culture and all its features, which may readily be part of the final composition or product obtained by the method as described herein. Embodiments and features of the present invention are also outlined in the following items.

Items

1. A process for producing a liquid food product comprising live bacteria at a predetermined concentration, the process comprising the steps of: a) providing the liquid food product in a product stream moving from at least one food production tank (106) via a first pipe (107) to an aseptic tank (109) and optionally further through a second pipe (108) to a dosing unit (113), wherein said product stream has a flow rate ql, b) providing at least one container (100, 101) comprising a suspension of live bacteria, the container further comprising at least one outlet port (104, 105a, 105b) equipped with opening means for regulating a flow of the bacteria suspension q2 and further wherein the concentration of the live bacteria in the suspension is at least 10E8 cfu/g; c) aseptically and operatively connecting the at least one container (100, 101) to said product stream either before or after or to the aseptic tank via a sterile transfer tube (110, 111) allowing for a flow rate q2 of the live bacteria suspension into said product stream, the transfer tube (110, 111) having a first end coupled to an outlet port (104, 105a, 105b) of the container or aseptic tank (109) and a second end coupled to an inlet port (102, 103a, 103b) of the first or the second pipes (107, 108) or the aseptic tank (109), d) opening the opening means of the at least one container (100, 101) to allow the bacteria suspension to flow into the sterile transfer tube (110, 111); e) using a pump (112a, 112b) or gravity feed system or manually applying force by squeezing the container to facilitate the flow of the bacteria suspension from the at least one container through the sterile transfer tube (110, 111) and into the first or second pipes (107, 108) or the aseptic tank (109) and thereby inoculate the live bacteria suspension into the product stream with an inoculation rate q2/ql*100, wherein the bacterial suspension has a total volume greater than IL and wherein q2 is adjusted to the concentration of the live bacteria suspension, the flow rate ql of the liquid food product, and the predetermined concentration of the bacteria in the liquid food product, f) optionally conducting the product stream from dosing unit (113) for dosing the product and packaging.

2. The process according to item 1, wherein the flow rate of product stream ql is in the range of 5,000-20,000 L/h and/or wherein q2 < 0.5 L/h or q2 >0.5 L/h at a density of the bacteria suspension of 1 g/ml.

3. The process according to items 1 or 2, wherein the one or more food production tank is one or more fermentation tank.

4. The process according to any of items 1-3, wherein a heating unit (114) is inserted before the container or the aseptic tank.

5. The process according to any one of items 1-4, wherein the at least one container is a bag, the bag comprising a flexible, biocompatible material, or a tank, the tank is a culture tank or a dosing tank or a combination of at least one bag and at least one tank.

6. The process according to item 5, wherein at least two or more containers are connected to allow a flow of live bacteria through the containers. The process according to item 6, wherein the at least two containers are a combination of a bag and a culture tank. The process according to any one of items 1-7, wherein the concentration of live bacteria comprised in the container is at least 10E+8 or in the range of 10E+8 to 10E+12 such as 10E+8, 10E10+9, 10E+10, 2xl0E+ 10, 5X10E+10, 10E+11, 2X10E+11, 5X10E+ 11 or 10E+12 CFU/g. The process according to any of the preceding items, wherein the first (107) and the second pipes (108) are provided with mixing means when connected to the container, and wherein the process comprises a further step of activating the mixing means in the first (107) or the second pipe (108) to ensure uniform distribution of the bacteria within the pipes; The process according to any one of the preceding items, wherein the live bacteria are a freeze-dried culture, wherein the freeze-dried culture is suspended in a milk, water, saline or peptone-water solution or the liquid food product to be inoculated prior to inoculation. The process according to any one of the preceding items, wherein the live bacteria do not comprise any added additives. The process according to any of the preceding items, wherein the live bacteria are frozen in the presence of a cryoprotectant in order to stabilize the bacterial cells during and after freezing or freeze-drying. The process according to any of the preceding items, wherein the viability of the bacteria are at least 60% after inoculation into a liquid food product as calculated based on the concentration of CFU (colony forming units) of the concentrated culture and the concentration of CFU of the inoculated material. The process according to any of the preceding items, wherein the bacterial strain is selected from the group consisting of Lactococcus lactis subsp. lactis biovar. Diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lacticaseibacillus easel subsp. easel, Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum , Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius'), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum)' , or any suitable combinations of the foregoing. The process according to any one of the preceding items, wherein the live bacterial culture comprises one or more strains belonging to the genus Lactobacillus.

16. The process according to any of the preceding items, wherein the bacteria is Lacticaseibacillus rhamnosus GG.

17. The process according to item 16, wherein the nucleotide sequence of the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1 as defined herein.

18. The process according to any one of the preceding items, wherein the liquid food product is a dairy product or a beverage.

19. The process according to item 18, wherein the dairy product is yogurt, buttermilk, kefir, quark, tvorog, creme fraiche or sour cream.

20. The process according to item 18, wherein the liquid food product is a water, juice, smoothie, yogurt drink, kefir drink or fermented tea.

List of elements: container (100, 101) inlet port (102, 103a, 103b) outlet port (104, 105a, 105b) food production tank (106) first pipe (107) second pipe (108) aseptic tank (109) sterile transfer tube (110, 111) pump (112a, 112b) dosing unit (113) heating unit (114) mixing means in a pipe (115a, 115b)

Examples

EXAMPLE 1

Requirements and constraints:

Example of the correlation between the concentration of Lactobacillus rhamnosus GG (LGG®) culture, the culture flow rate when inoculating said strain into a product stream of pasteurized fermented milk at different product flow rates to achieve a concentration of 1E7 cfu/g in the final milk product.

Target inoculated cell count in final product: 1E7 cfu/g

Culture bag : 1kg Culture density: lg/ml

Min possible culture flow: 0.5 L/h

Max acceptable change of bag : 1 bag/2h

Table 3.

PPY is Post Pasteurized Yogurt.

Too low culture flow rates (marked in bold) : Too short time between change of culture bag

Acceptable culture flow rate: Acceptable time (assuming 2h is acceptable)

Conclusion: High product flows (>5000 L/h) require a higher culture flow at a given concentration of bacteria. This means that with a culture flow of more than 0.5 at a concentration of 5,00E+10 of the bacteria, the number of bags required to end up with the desired concentration in the end product will be too high for the system to process. In the example the maximum change of bag is 1 bag per 2 hours. Increasing the bacteria concentration, however, may not solve the problem since the inoculation rate is lowered accordingly causing the culture flow to be too low or number of bags/hour required will be too high.

EXAMPLE 2 -.Testing stability at 10°C of Lactobacillus rhamnosus GG (LGG®) after thawing.

The goal of the trial was to investigate the stability of the strain of Lactobacillus rhamnosus GG over time (up to 72hrs), when stored as a liquid product in a clean and sterilized tank.

The frozen product bags (940g/bag - 40 bags in total) were thawed by partly submerging in a water bath at 28°C, for up to 90min - or until the product was completely thawed. The liquid product was transferred aseptic from the bags via a sterile tubing and a pump to an already clean and sterilized 40L fermentation tank. Automatic regulation of agitation, temperature, overpressure and nitrogen gas flow were initiated directly after transfer, to control the conditions for the product during the trial (set points: 10-ll°C, 125 rpm, 70L/min nitrogen gas flow, overpressure 0.25bar). The liquid product comprising the live culture was left in the tank for up to 72 hours prior to sampling. When surface plating is applied the colonies are counted already after 2 days, to avoid that the colonies grow together.

Hereafter samples were taken via a sampling port at different timepoints, from T=0 and up to T= 74 hours. Samples were distributed into tubes and placed directly in a -55°C freezer and analyzed for cell count (CFU). Cell count (CFU) of Lactobacillus rhamnosus GG (LGG®) was counted by using MRS agar, surface plating method, with anaerobic incubation at 37 °C for 2 days.

The CFU count of the different sampling points is shown in Figure 4.

Table 4: CFU results of the different sampling points.

Conclusion: The experiment shows a very stable CFU count after storage in the tank after 72 hours at 10°C. Thus, based on the results it is possible to dose Lactobacillus rhamnosus GG (LGG®) into a liquid food product, using a production line which potentially could operate for up to 72 hours, as long as the above mentions conditions are applied. The culture product cell count, the food product flow rate and flow rate of the culture product into the food product will determine the cell count in the final product. EXAMPLE 3 - Yoflex® Acidifix® yogurt culture + Lactobacillus rhamnosus GG (LGG®) - Effect of storage of L. rhamnosus GG after thawing before inoculation in final product

Milk base was prepared and fermented with Yoflex® Acidifix® 1.0 to pH 4.5 followed by heat treatment at 75 °C for 20 sec to provide a yogurt. The L. rhamnosus GG strain was thawed and kept at different conditions with respect to temperature and storage time before aseptic inoculation into the heat-treated yogurt to test the robustness of the solution. The product was kept at 25 °C for 3 days for initial growth of the L. rhamnosus GG strain and product was hereafter kept at 25°C and 37°C for 3 months. Cell count and post acidification was checked at different interval.

Material and method :

Table 5. Content of the milk base.

Cultures:

F-DVS Yoflex®Acidifix 1.0 (Chr. Hansen A/S) (yogurt culture)

F-DVS nu-trish® LGG® (Chr. Hansen A/S) (LGG®)

Process:

Milk base was prepared according to the table above and pasteurized at 134 °C for 4 seconds.

Milk base was fermented with 100U Yoflex® Acidifix 1.0 /T milk base at 43 °C until pH 4.5.

Heat treatment - at 75 °C for 20 seconds. The Lactobacillus rhamnosus GG strain was thawed at 28 °C for 99 min. One bag was kept at 25 °C for 2h to simulate the time it can take to empty a bag in filling line at room temp. A second bag was kept at 25 °C for 24h and another bag was kept at 4 °C for 48h to mimic time kept in refrigerator for 2 days, followed by 2h at 25 °C. L. rhamnosus GG strain was added at 0.009 % and cell count was measured on bags right after inoculation.

The products were initially stored at 25 °C for 3 days and hereafter stored at 25 °C and 37 °C for 3 months. Cell count and post acidification was measured during storage. Cell count of the Lactobacillus rhamnosus GG strain is counted by using Difco MRS agar, pour plate method with anaerobic incubation at 37 °C for 3 days.

Results:

Table 6.

Table 7. Post acidification results (pH) Conclusion: The experiment shows a very stable CFU count after storage for 3 months at 25 °C and 37 °C in the milk-based product (Table 6) and acidification values comparable to the blank control (Table 7). Thus, based on the results it is possible to add Lactobacillus rhamnosus GG (LGG®) into a liquid food product where Lactobacillus rhamnosus GG is viable after long-time storage at temperatures in the ambient range.

EXAMPLE 4 - Effect of holding time after thawing of Lactobacillus rhamnosus GG (LGG®) on the final cell count stability in ambient yogurt.

The goal of the trial was to investigate if different holding time after thawing would affect the stability of the strain of Lactobacillus rhamnosus GG in the final ambient yogurt.

Milk base was prepared and fermented with Yoflex®Acidifix® 1.0 to pH 4.45 followed by heat treatment at 75 °C for 20 sec to provide a yogurt. Thawed L. rhamnosus GG strain kept at different holding times, was inoculated aseptically into the heat-treated yogurt to test the robustness of the solution. The final product was placed at 25 °C and 37 °C. The initial growth of the L. rhamnosus GG strain was analysed after 6 days and cell count and pH was hereafter followed up to 150 days.

Material and method :

Table 8. Content of the milk bases 1 and 2

Cultures:

F-DVS Yof lex® Acid if lx 1.0 (Chr. Hansen A/S) (yogurt culture)

F-DVS nu-trish® LGG® Al (Chr. Hansen A/S) (LGG®) Process:

Milk base was prepared according to the table above and pasteurized at 134 °C for 4 seconds. Milk base was fermented with 100U Yoflex® Acidifix 1.0 /T milk base at 43 °C until pH 4.45. Yogurt was heat treated at 75 °C for 20 seconds.

The frozen LGG® product bags - nu-trish® LGG® Al - (940g/bag - 40 bags in total) were thawed by partly submerging in a water bath at 28°C, for up to 90min - or until the product was completely thawed.

The liquid product was transferred aseptic from the bags via a sterile tubing and a pump to an already clean and sterilized 40L fermentation tank. Automatic regulation of agitation, temperature, overpressure and nitrogen gas flow were initiated directly after transfer, to control the conditions for the product during the trial (set points: 10-ll°C, 125 rpm, 70L/min nitrogen gas flow, overpressure 0.25bar). The liquid product comprising the live culture was left in the tank for up to 72 hours prior to sampling.

Samples were taken via a sampling port at different timepoints, T=0, T=24 and T=72 hours, and samples were inoculated at 0.009 % into the pasteurized yogurt. The LGG® cell count and pH was measured in the yogurt after inoculation.

The product was placed at 25 °C and 37 °C. The initial growth of the L. rhamnosus GG strain was analysed after 6 days and cell count and pH was hereafter followed up to 150 days.

Cell count of the Lactobacillus rhamnosus GG strain is counted by using Difco MRS agar, pour plate method with anaerobic incubation at 37 °C for 3 days.

Results:

Table 9. Cell count stability of LGG® in ambient yogurt stored at 25 °C

Table 10. Cell count stability of LGG® in ambient yogurt stored at 37 °C

Similar growth and stability are obtained in the sample with LGG® inoculated right after thawing and samples with LGG® which have been kept at 10 °C for 24h and 72h before inoculation, i.e. it is possible to keep LGG® in thawed condition for up to 72h without any negative effect on stability. This is the case at both 25 °C and 37 °C . LGG® stability is affected by the milk base. Table 11. Post acidification of ambient yogurt with and without LGG® stored at 25 °C (pH)

Table 12. Post acidification of ambient yogurt with and without LGG® stored at 37 °C (pH)

The samples with LGG® inoculated at T=0, T=24h and T=72h have similar pH during storage. Differences are observed between the milk bases due to differences in citrate metabolism, which result in increased pH (milk base 1) at day 150, compared to milk base 2. The citrate metabolism happens faster at 37 °C. Conclusion: The experiment shows that holding time up to 72h after thawing of LGG® gives a similar Lactobacillus rhamnosus GG (LGG®) performance (cell count) and post acidification (pH) in the final product when stored for up to 150 days at 25 °C or 37 °C.

EXAMPLE 5 - Effect of holding time after thawing of Lactobacillus rhamnosus GG (LGG®) culture bags on the cell count of the culture.

The goal of the trial was to investigate the stability of Lactobacillus rhamnosus GG culture after thawing when kept at 10 °C for up to 72hrs.

Three frozen product bags (940g/bag) were thawed by partly submerging in a water bath at 28°C, for up to 90min - or until the product was completely thawed. Cell count was made on a bag right after thawing. Two bags with thawed LGG® were placed at 10 °C and cell count was made after 24h and 72h.

Cell count (CFU) of Lactobacillus rhamnosus GG (LGG®) was counted by using MRS agar, pour plating method, with anaerobic incubation at 37 °C for 3 days.

Conclusion:

Results show that LGG® cell count is stable after thawing when kept in culture bags at 10 °C for up to 72h.

Claims

2. The process according to claim 1, wherein the flow rate of product stream ql is in the range of 5,000-20,000 L/h and/or wherein q2 < 0.5 L/h or q2 >0.5 L/h at a density of the bacteria suspension of 1 g/ml.

3. The process according to claims 1 or 2, wherein the one or more food production tank is one or more fermentation tank.

4. The process according to any of claims 1-3, wherein a heating unit (114) is inserted before the container or the aseptic tank.

5. The process according to any one of claims 1-4, wherein the at least one container is a bag, the bag comprising a flexible, biocompatible material, or a tank, the tank is a culture tank or a dosing tank or a combination of at least one bag and at least one tank.

6. The process according to claim 5, wherein at least two or more containers are connected to allow a flow of live bacteria through the containers.

7. The process according to claim 6, wherein the at least two containers are a combination of a bag and a culture tank.

8. The process according to any one of claims 1-7, wherein the concentration of live bacteria comprised in the container is at least 10E+8 or in the range of 10E+8 to 10E+12 such as 10E+8, 10E10 + 9, 10E+10, 2xl0E+10, 5X10E+10, 10E+11, 2X10E+11, 5X10E+ 11 or 10E+12 CFU/g.

9. The process according to any of the preceding claims, wherein the first (107) and the second pipes (108) are provided with mixing means when connected to the container, and wherein the process comprises a further step of activating the mixing means in the first (107) or the second pipe (108) to ensure uniform distribution of the bacteria within the pipes.

10. The process according to any one of the preceding claims, wherein the live bacteria are a freeze-dried culture, wherein the freeze-dried culture is suspended in a milk, water, saline or peptone-water solution or the liquid food product to be inoculated prior to inoculation.

11. The process according to any one of the preceding claims, wherein the live bacteria do not comprise any added additives.

12. The process according to any of the preceding claims, wherein the live bacteria are frozen in the presence of a cryoprotectant in order to stabilize the bacterial cells during and after freezing or freeze-drying.

13. The process according to any of the preceding claims, wherein the viability of the bacteria are at least 60% after inoculation into a liquid food product as calculated based on the concentration of CFU (colony forming units) of the concentrated culture and the concentration of CFU of the inoculated material.

14. The process according to any of the preceding claims, wherein the bacterial strain is selected from the group consisting of Lactococcus lactis subsp. lactis biovar. Diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lacticaseibacillus easel subsp. easel, Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum , Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius'), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum)' , or any suitable combinations of the foregoing.

15. The process according to any one of the preceding claims, wherein the live bacterial culture comprises one or more strains belonging to the genus Lactobacillus.

16. The process according to any of the preceding claims, wherein the bacteria is Lacticaseibacillus rhamnosus GG.

17. The process according to claim 16, wherein the nucleotide sequence of the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1.

18. The process according to any one of the preceding claims, wherein the liquid food product is a dairy product or a beverage.