Coffee-based beverages

Abstract

The present invention relates to a coffee-based beverage containing a probiotic, the probiotic having a probiotic viable cell count greater than 6.0 log CFU / mL. The present invention also relates to a method for preparing a coffee-based beverage having a probiotic viable cell count greater than 6.0 log CFU / mL, the method comprising the steps of: combining coffee brew with sugar and an inactivated yeast derivative to form a mixture; adding a probiotic to the mixture to form an inoculated mixture; and fermenting the inoculated mixture for a predetermined period of time to prepare the beverage. The probiotic may include Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus fermentum, Bifidobacterium lactis, Saccharomyces boulardii, Saccharomyces cerevisiae, or a combination thereof.

Description

[Technical Field]

[0001] The present invention relates to coffee-based beverages and methods for their preparation. [Background technology]

[0002] background As consumers become more health conscious, functional foods and beverages are becoming increasingly popular. This trend has led to strong innovation in the probiotic food market, with numerous probiotic delivery formats emerging, including dairy, grains, soy, fruits, vegetables, and meat.

[0003] Coffee is also a widely consumed beverage worldwide. Consumer concerns about sugar and growing interest in beverages with fewer additives and natural sensory properties have led to a demand for coffee-based functional beverages. However, the development of probiotic-fermented coffee beverages presents several challenges. First, coffee brew lacks fermentable substrates, making it difficult to grow probiotics. However, excessive nutrient addition leads to the accumulation of lactic acid, which can adversely affect the sensory and physicochemical properties of coffee.

[0004] Therefore, there is a need for an improved coffee brew that can be considered a functional beverage. Summary of the Invention

[0005] The present invention aims to solve these problems and / or provide a coffee-based beverage.

[0006] According to a first aspect, the present invention provides a coffee-based beverage comprising a probiotic, wherein the probiotic has a probiotic viable cell count of 6.0 log CFU / mL or more. In particular, the beverage may be a fermented beverage.

[0007] According to a particular embodiment, the probiotics contained in the beverage have a probiotic viable cell count of 6.0 log CFU / mL or more after 3 months of storage, in particular the beverage has a probiotic viable cell count of 7.0 log CFU / mL or more.

[0008] The probiotics contained in the beverage may be any suitable probiotics. In particular, the probiotics may include, but are not limited to, probiotic bacteria, probiotic yeast, or a combination thereof. In certain embodiments, the probiotics may include lactic acid bacteria, bifidobacteria, Saccharomyces yeast, non-Saccharomyces yeast, or a combination thereof.

[0009] In particular, the lactic acid bacteria may be, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or combinations thereof.

[0010] In particular, the Saccharomyces yeast may be, but is not limited to, Saccharomyces (S.) boulardii, S. cerevisiae, or a combination thereof.

[0011] According to certain embodiments, the probiotics include, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, Saccharomyces (S.) boulardii, S. cerevisiae, or combinations thereof.

[0012] The beverage may further comprise an additive, which may be any suitable additive, such as, but not limited to, a sweetener, a stabilizer, a flavoring, or a combination thereof.

[0013] According to a second aspect, the present invention provides a method for preparing a coffee-based beverage comprising probiotics having a viable cell count of 6.0 log CFU / mL or more, the method comprising the steps of: - mixing the coffee brew with sugar and an inactivated yeast derivative to form a mixture; - adding probiotics to the mixture to form an inoculated mixture; and - fermenting the inoculated mixture for a predetermined period of time to prepare the beverage; The compound comprises:

[0014] The mixing step includes mixing an appropriate amount of sugar and an inactivated yeast derivative. According to a specific embodiment, the mixing step includes mixing the sugar at a concentration of 0.01 to 10% w / v with respect to the total volume of the mixture.

[0015] According to a particular embodiment, the mixing step comprises mixing the inactivated yeast derivative at a concentration of 0.005 to 5% w / v relative to the total volume of the mixture.

[0016] The step of adding a probiotic may comprise adding any suitable probiotic, for example the probiotic may be as described above in relation to the first aspect of the invention.

[0017] According to a particular embodiment, the adding step comprises adding the probiotic to provide an initial probiotic viable count of at least 6 log CFU / mL, particularly, the adding step comprises adding the probiotic to provide an initial probiotic viable count of at least 7 log CFU / mL.

[0018] The fermentation step may be performed for an appropriate predetermined period of time, for example, 4 to 100 hours.

[0019] The fermentation step may be carried out at any suitable temperature, for example, at a temperature of 15 to 45°C.

[0020] The method can further include adding an additive to the mixture. The additive can be any suitable additive. For example, the additive can be, but is not limited to, a sweetener, a stabilizer, a flavoring agent, or a combination thereof.

[0021] In order that the invention may be fully understood and readily put into practice, embodiments thereof will now be described, by way of non-limiting example only, with reference to the accompanying illustrative figures, in which: [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1 shows the effect of supplementing different levels of glucose with 0%, 0.03%, and 0.3% (Figure 1A) Optiwhite®, (Figure 1B) Optired®, and (Figure 1C) Noblesse® on the growth of L. rhamnosus GG after 24 hours. Different lowercase letters indicate significant differences (P<0.05) between glucose levels at the same IYD level. Initial inoculum size: approximately 7 Log CFU / mL. [Figure 2] Figure 2 shows the effect of supplementing with different types of IYD on the growth of L. rhamnosus GG after 24 hours. Different lowercase letters indicate significant differences (P<0.05) between IYD types at the same IYD level. Initial inoculum size: approximately 7 Log CFU / mL. [Figure 3] Figure 3 shows the effect of supplementing different levels of (Figure 3A) Optiwhite® and (Figure 3B) Noblesse® on the growth of L. rhamnosus GG after 24 hours. Different lowercase letters indicate significant differences (P<0.05) between IYD levels. Initial inoculum size: approximately 7 Log CFU / mL. [Figure 4] Figure 4 shows the effect of supplementing with different levels of (Figure 4A) Optiwhite® and (Figure 4B) Noblesse® on pH. Different lowercase letters indicate significant differences (P<0.05) between IYD levels. [Figure 5] Figure 5 shows the growth and survival of L. rhamnosus GG, L. plantarum 299v, L. paracasei Lpc-37, or L. acidophilus NCFM during fermentation and storage in (Figure 5A) supplemented coffee at 4° C., (Figure 5B) unsupplemented coffee at 4° C., (Figure 5C) supplemented coffee at 25° C., and (Figure 5D) unsupplemented coffee at 25° C. Values ​​are the means of triplicate experiments (n=3), and error bars represent the standard deviation of the means. [Figure 6] Figure 6 shows the changes in headspace volatile levels for (Figure 6A) 3-methylbutanoic acid, (Figure 6B) diacetyl, and (Figure 6C) acetoin. Mean values ​​with different lowercase letters indicate statistically significant differences (P<0.05) between different time points within the same probiotic strain. # indicates not detected. [Figure 7] Figure 7 shows the growth and survival of monocultures and mixed cultures of (Figure 7A) L. rhamnosus GG, and (Figure 7B) S. boulardii CNCM-I745 during coffee brew fermentation and storage at 4°C and 25°C. Values ​​are the means of triplicate experiments (n = 3), and error bars represent the standard deviation of the means. [Figure 8]Figure 8 shows the changes in selected alkaloids and phenolic compounds during fermentation and storage of coffee brews using monocultures and mixed cultures of L. rhamnosus GG or S. boulardii CNCM-I745—(Figure 8A) caffeine, (Figure 8B) trigonelline, (Figure 8C) caffeic acid, and (Figure 8D) chlorogenic acid. Mean values ​​with different lowercase letters indicate statistically significant differences (P<0.05) between different fermentation setups at the same time point. * indicates trace levels. [Figure 9A-B] Figure 9 shows the changes in antioxidant capacity during fermentation and storage of coffee brews using monocultures and mixed cultures of L. rhamnosus GG or S. boulardii CNCM-I745 - (Figure 9A) total phenolic content, (Figure 9B) 2,2-diphenyl-1-picrylhydrazyl, and (Figure 9C) oxygen radical scavenging assay. Mean values ​​with different lowercase letters indicate statistically significant differences (P<0.05) between different fermentation setups at the same time point. [Figure 9C] Figure 9 shows the changes in antioxidant capacity during fermentation and storage of coffee brews using monocultures and mixed cultures of L. rhamnosus GG or S. boulardii CNCM-I745 - (Figure 9A) total phenolic content, (Figure 9B) 2,2-diphenyl-1-picrylhydrazyl, and (Figure 9C) oxygen radical scavenging assay. Mean values ​​with different lowercase letters indicate statistically significant differences (P<0.05) between different fermentation setups at the same time point. [Figure 10A-B] Figure 10 shows the growth and survival of monocultures and mixed cultures of (Figure 10A) L. plantarum 299v, (Figure 10B) L. acidophilus NCFM, (Figure 10C) L. fermentum PCC, (Figure 10D) L. gasseri LAC-343, and (Figure 10E) S. boulardii CNCM-I745 at 4°C. Values ​​are the means of triplicate experiments (n=3), and error bars represent the standard deviation of the means. [Figure 10C-E]Figure 10 shows the growth and survival of monocultures and mixed cultures of (Figure 10A) L. plantarum 299v, (Figure 10B) L. acidophilus NCFM, (Figure 10C) L. fermentum PCC, (Figure 10D) L. gasseri LAC-343, and (Figure 10E) S. boulardii CNCM-I745 at 4°C. Values ​​are the means of triplicate experiments (n=3), and error bars represent the standard deviation of the means. [Figure 10F-H] Growth and survival of monocultures and mixed cultures of (Figure 10F) L. plantarum 299v, (Figure 10G) L. acidophilus NCFM, (Figure 10H) L. fermentum PCC, (Figure 10I) L. gasseri LAC-343, and (Figure 10J) S. boulardii CNCM-I745 at 25°C. Values ​​are the average of triplicate experiments (n=3), and error bars represent the standard deviation of the mean. [Figure 11A-C] Figure 11 shows the pH at 4°C of monocultures and mixed cultures of (Figure 11A) L. plantarum 299v, (Figure 11B) L. acidophilus NCFM, (Figure 11C) L. fermentum PCC, (Figure 11D) L. gasseri LAC-343, and (Figure 11E) S. boulardii CNCM-I745. Values ​​are the average of triplicate experiments (n=3), and error bars represent the standard deviation of the mean. [Figure 11D-F] Figure 11 shows the pH at 4°C of monocultures and mixed cultures of (Figure 11A) L. plantarum 299v, (Figure 11B) L. acidophilus NCFM, (Figure 11C) L. fermentum PCC, (Figure 11D) L. gasseri LAC-343, and (Figure 11E) S. boulardii CNCM-I745. (Figure 11F) pH at 25°C of monocultures and mixed cultures of (Figure 11G) L. acidophilus NCFM, (Figure 11H) L. fermentum PCC, (Figure 11I) L. gasseri LAC-343, and (Figure 11J) S. boulardii CNCM-I745. Values ​​are the average of triplicate experiments (n = 3), and error bars represent the standard deviation of the average. [Figure 11G-I]pH at 25°C of monocultures and mixed cultures of (Figure 11F) L. plantarum 299v, (Figure 11G) L. acidophilus NCFM, (Figure 11H) L. fermentum PCC, (Figure 11I) L. gasseri LAC-343, and (Figure 11J) S. boulardii CNCM-I745. Values ​​are the average of triplicate experiments (n = 3), and error bars represent the standard deviation of the mean. [Figure 11J] pH at 25°C of monocultures and mixed cultures of (Figure 11F) L. plantarum 299v, (Figure 11G) L. acidophilus NCFM, (Figure 11H) L. fermentum PCC, (Figure 11I) L. gasseri LAC-343, and (Figure 11J) S. boulardii CNCM-I745. Values ​​are the average of triplicate experiments (n = 3), and error bars represent the standard deviation of the mean. [Figure 12] Figure 12 shows the changes in lactic acid during fermentation and storage of coffee brew with monocultures and mixed cultures of probiotic LAB and S. boulardii CNCM-I745. * indicates a statistically significant difference (P<0.05) compared to the blank at the same time point. [Figure 13-1] Figure 13 shows the changes in trigonelline, caffeine, and chlorogenic acid during fermentation and storage of coffee brew with monocultures and mixed cultures of probiotic LAB and / or S. boulardii CNCM-I745. * indicates a statistically significant difference (P<0.05) compared to the blank at the same time point. [Figure 13-2] Figure 13 shows the changes in trigonelline, caffeine, and chlorogenic acid during fermentation and storage of coffee brew with monocultures and mixed cultures of probiotic LAB and / or S. boulardii CNCM-I745. * indicates a statistically significant difference (P<0.05) compared to the blank at the same time point. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description As discussed above, there is a need for functional coffee-based beverages. The present invention provides a method for preparing a functional coffee-based beverage.

[0024] In general terms, the present invention provides coffee-based beverages with value-added functional properties. Specifically, the present invention provides coffee-based beverages with high probiotic viable cell counts; the viable cell counts can be maintained for a certain period of time at appropriate temperatures, allowing for long-term transport or storage. Furthermore, endogenous coffee bioactive components, such as, but not limited to, caffeine, trigonelline, and chlorogenic acid, are retained in the beverage. Thus, the beverages of the present invention offer additional therapeutic benefits compared to conventional coffee-based beverages.

[0025] According to a first aspect, the present invention provides a coffee-based beverage containing probiotics, having a probiotic viable cell count of 6.0 log CFU / mL or greater. The provided probiotic viable cell count may be the number of live and active probiotic cells. The provided probiotic viable cell count may be the cell count at the time the beverage is prepared.

[0026] The beverage may be a fermented beverage. In particular, the beverage is a fermented probiotic beverage. In the present invention, the term probiotic beverage refers to a beverage containing live, active, vegetative probiotic cells. In particular, the probiotic cells are metabolically active.

[0027] In the present invention, probiotics include live, active microorganisms that, when ingested in certain numbers, exert health benefits beyond their general nutritional nature. The health benefits of probiotics are thought to be primarily due to their ability to colonize the gastrointestinal tract and contribute to the establishment of a healthy, balanced intestinal flora.

[0028] At any time during its shelf life from the preparation of the beverage, the beverage can contain an appropriate amount of probiotics. For example, the probiotics can have a viable cell count of 5.0 log CFU / mL or more. In particular embodiments, the probiotics have a viable cell count of 6.0 log CFU / mL or more, or 7.0 log CFU / mL or more. More particularly, the probiotics have a viable cell count of 8.5 log CFU / mL or more.

[0029] In particular, the probiotics contained in the beverage may have a viable cell count of 5.0-9.0 log CFU / mL, 5.5-8.5 log CFU / mL, 6.0-8.0 log CFU / mL, 6.5-7.5 log CFU / mL, 7.0-7.3 log CFU / mL, and more particularly, the probiotics contained in the beverage have a viable cell count of about 6.0-9.0 log CFU / mL.

[0030] According to a specific embodiment, the beverage can be stable even after a certain period of storage. For example, the probiotics contained in the beverage can have a probiotic viable cell count of 6.0 log CFU / mL or more even after three months of storage. Specifically, the probiotic viable cell count can be 6.0 to 9.0 log CFU / mL, 6.5 to 8.5 log CFU / mL, 7.0 to 8.0 log CFU / mL, or 7.2 to 7.5 log CFU / mL. More specifically, the probiotic viable cell count is 6.0 to 8.0 log CFU / mL. As a result, it can be seen that the beverage can still provide health benefits to consumers even after a certain period of time has passed since its production. Therefore, the beverage can have a suitable shelf life.

[0031] The probiotics contained in the beverage may be any suitable probiotics. For example, the probiotics may be, but are not limited to, probiotic bacteria, probiotic yeast, or a combination thereof. In a specific embodiment, the probiotics contained in the beverage may be at least one type of probiotic bacteria. In another specific embodiment, the probiotics contained in the beverage may be at least one type of probiotic yeast. In another specific embodiment, the probiotics contained in the beverage may be at least one type of probiotic bacteria and at least one type of probiotic yeast. For example, probiotics may include, but are not limited to, lactic acid bacteria, bifidobacteria, Saccharomyces yeast, non-Saccharomyces yeast, or a combination thereof.

[0032] Lactic acid bacteria can be any suitable lactic acid bacteria.For example, lactic acid bacteria can be, but not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or combinations thereof.In particular, lactic acid bacteria can be Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, or combinations thereof.

[0033] The Saccharomyces yeast may be any suitable Saccharomyces yeast. For example, the Saccharomyces yeast may be, but is not limited to, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof. In particular, the Saccharomyces yeast may be, but is not limited to, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0034] According to certain embodiments, probiotics include, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or combinations thereof. In particular, probiotics include, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, or a combination thereof. In particular, probiotics can be Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, B. lactis BB-12, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0035] According to a specific embodiment, the probiotic can comprise a combination of Saccharomyces yeast and at least one probiotic bacterium.The probiotic bacterium can be as described above.In particular, the probiotic can comprise a combination of Saccharomyces yeast and at least one of Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, or a combination thereof. For example, the probiotic comprises a combination of Saccharomyces yeast with at least one of Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, and B. lactis BB-12. The Saccharomyces yeast may be Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof. In particular, the Saccharomyces yeast may be, but is not limited to, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0036] The beverage may further comprise an additive. The additive may be any suitable additive. The additive may be any suitable additive for providing a more complete consumer product, enhancing the flavor profile of the beverage, and / or enhancing the sensory characteristics of the beverage. For example, the additive may be, but is not limited to, a sweetener, a stabilizer, a flavoring, or a combination thereof.

[0037] According to a second aspect, the present invention provides a method for preparing a coffee-based beverage comprising probiotics having a viable cell count of 6.0 log CFU / mL or more, the method comprising the steps of: - mixing the coffee brew with probiotic nutrients to form a mixture; - adding probiotics to the mixture to form an inoculated mixture; and - fermenting the inoculated mixture for a predetermined period of time to prepare the beverage; The compound comprises:

[0038] The method may be a method for preparing a coffee-based beverage according to the first aspect described above.

[0039] The method may be a method for preparing a coffee-based beverage containing probiotics having a viable cell count of 7.0 log CFU / mL or greater.

[0040] The probiotic nutrient may be any suitable nutrient that provides a suitable environment for the growth of probiotic cells, for example, probiotic nutrients may include, but are not limited to, sugars, inactivated yeast derivatives, yeast extracts, or combinations thereof.

[0041] According to certain embodiments, the mixing step may include mixing the coffee brew with sugar and an inactivated yeast derivative.

[0042] The coffee brew may be any suitable coffee brew. The inactivated yeast derivative (IYD) may be any suitable IYD. In the present invention, the IYD may include a thermally or enzymatically inactivated yeast extract. IYDs include, but are not limited to, yeast cell walls and yeast autolysates.

[0043] The mixing step includes mixing an appropriate amount of an inactivated yeast derivative. According to a specific embodiment, the mixing step includes mixing the inactivated yeast derivative at a concentration of 0.005 to 5% w / v relative to the total volume of the mixture. In particular, the inactivated yeast derivative to be mixed may be at a concentration of 0.01 to 5.0% w / v, 0.02 to 3% w / v, 0.03 to 2.5% w / v, 0.04 to 2.0% w / v, 0.05 to 1.5% w / v, 0.06 to 1.0% w / v, 0.07 to 0.9% w / v, 0.08 to 0.8% w / v, 0.09 to 0.7% w / v, 0.1 to 0.6% w / v, 0.2 to 0.5% w / v, or 0.3 to 0.4% w / v relative to the total volume of the mixture. More particularly, the inactivated yeast derivative is mixed at a concentration of 0.03 to 0.06% by volume relative to the total volume of the mixture.

[0044] The sugar may be any suitable sugar. For example, the sugar may be a fermentable sugar. According to a particular embodiment, the sugar is glucose.

[0045] The mixing step includes mixing an appropriate amount of sugar. According to a specific embodiment, the mixing step includes mixing the sugar at a concentration of 0.01 to 10% w / v relative to the total volume of the mixture. In particular, the sugar may be mixed at a concentration of 0.05 to 9% w / v, 0.1 to 8% w / v, 0.2 to 7% w / v, 0.25 to 6% w / v, 0.3 to 5% w / v, 0.4 to 4% w / v, 0.45 to 3% w / v, 0.5 to 2% w / v, 0.6 to 1.0% w / v, 0.7 to 0.9% w / v, or 0.75 to 0.8% w / v relative to the total volume of the mixture. More specifically, the sugar is mixed at a concentration of 0.25 to 0.5% by volume relative to the total volume of the mixture.

[0046] According to certain aspects, the mixing step may be by any suitable means. For example, the mixing step may include stirring the mixture.

[0047] The method may further comprise cooling the mixture prior to the step of adding the probiotics. In particular, cooling comprises cooling the mixture to ambient temperature, for example, about 25°C.

[0048] The step of adding probiotics can include adding any suitable probiotics to the mixture.For example, probiotics include, but are not limited to, probiotic bacteria, probiotic yeast, or a combination thereof.According to a specific embodiment, the probiotics added to the mixture can be at least one type of probiotic bacteria.According to another specific embodiment, the probiotics added to the mixture can be at least one type of probiotic yeast.According to another specific embodiment, the probiotics added to the mixture can be at least one type of probiotic bacteria and at least one type of probiotic yeast.For example, the probiotics added can include, but are not limited to, lactic acid bacteria, bifidobacteria, Saccharomyces yeast, non-Saccharomyces yeast, or a combination thereof.

[0049] The lactic acid bacteria added can be any suitable lactic acid bacteria.For example, the lactic acid bacteria can be, but not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or combinations thereof.In particular, the lactic acid bacteria can be Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, or combinations thereof.

[0050] The added Saccharomyces yeast may be any suitable Saccharomyces yeast. For example, the Saccharomyces yeast may be, but is not limited to, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof. In particular, the Saccharomyces yeast may be, but is not limited to, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0051] According to certain embodiments, the added probiotics include, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or combinations thereof. In particular, probiotics include, but are not limited to, Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, or a combination thereof. In particular, probiotics can be Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, B. lactis BB-12, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0052] According to a specific embodiment, the added probiotics can comprise a combination of Saccharomyces yeast and at least one probiotic bacterium. The probiotic bacterium can be as described above. In particular, the added probiotics can comprise a combination of Saccharomyces yeast and at least one of Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, or a combination thereof. For example, the probiotics added include a combination of Saccharomyces yeast and at least one of Lb. rhamnosus GG, Lb. paracasei Lpc-37, Lb. plantarum 299v, Lb. acidophilus NCFM, Lb. gasseri Lac-343, Lb. fermentum PCC, and B. lactis BB-12. The Saccharomyces yeast can be Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof. In particular, the Saccharomyces yeast can be, but is not limited to, S. boulardii CNCM-I745, S. cerevisiae CNCM I-3856, or a combination thereof.

[0053] When the adding step comprises adding a combination of probiotics, two or more probiotics can be added simultaneously or sequentially into the mixture.According to a particular embodiment, two or more probiotics are added sequentially.In particular, the adding step of probiotics comprises adding a first probiotic to the mixture, and then adding a second or subsequent probiotic after a predetermined period of time from adding the first probiotic.

[0054] According to certain embodiments, two or more probiotics may be added to the mixture simultaneously, in particular, a first probiotic and a second or subsequent probiotic are all added at the same time.

[0055] The step of adding probiotics includes adding an appropriate amount of probiotics. According to a specific embodiment, the step of adding probiotics includes adding probiotics so that the initial probiotic viable cell count is at least 1 log CFU / mL. For example, the amount of probiotics added may be at least 4 log CFU / mL. In particular, the amount of probiotics added may be about 5 to 7 log CFU / mL, 5.5 to 6.5 log CFU / mL, or 5.7 to 6 log CFU / mL. More particularly, the amount of probiotics added is 4.5 to 7.0 log CFU / mL.

[0056] According to certain embodiments, the adding step may comprise adding the probiotic to provide an initial probiotic viable count of at least 6 log CFU / mL. In particular, the adding step comprises adding the probiotic to provide an initial probiotic viable count of at least 7 log CFU / mL.

[0057] The step of adding the probiotics can be carried out under suitable conditions, for example, the step of adding the probiotics can be under aseptic conditions.

[0058] The method can further include incubating the mixture at a suitable temperature prior to the step of adding the probiotics. In particular, the temperature can be a temperature at which fermentation occurs. In this way, homogeneous growth of the probiotics can occur in the mixture.

[0059] The fermentation step can be carried out under any suitable conditions. For example, the fermentation step can be for a predetermined period of time. The predetermined period can be any period suitable for the purposes of the present invention. The predetermined period can depend on the probiotics added in the probiotic addition step. In certain embodiments, the predetermined period can be 4 to 100 hours. In particular, the predetermined period can be 4 to 96 hours, 5 to 72 hours, 6 to 60 hours, 12 to 54 hours, 18 to 48 hours, 24 to 42 hours, or 30 to 36 hours. More particularly, the predetermined period is about 12 to 14 hours.

[0060] The fermentation process may be at a predetermined temperature. The predetermined temperature may be any temperature suitable for the purposes of the present invention. According to a specific embodiment, the predetermined temperature may be 15 to 45°C. In particular, the predetermined temperature may be 20 to 40°C, 25 to 37°C, or 30 to 35°C. More particularly, the predetermined temperature is about 30°C. The temperature may be changed at any time during the fermentation period.

[0061] The method may further comprise adding an additive to the mixture. The additive may be any suitable additive. In particular, the additive may be for enhancing the flavor profile of the beverage and / or for enhancing the sensory properties of the beverage. For example, the additive may be, but is not limited to, a sweetener, a stabilizer, a flavoring, or a combination thereof.

[0062] According to certain embodiments, the prepared coffee-based beverage can be stored at a suitable temperature after fermentation. For example, the beverage can be stored at a temperature of 30°C or less. In particular, the beverage can be stored at a temperature of about 25°C or less, 1-25°C, 2-20°C, 4-15°C, 5-12°C, or 7-10°C. More particularly, the beverage is stored at a temperature of about 4-25°C.

[0063] Having generally described the invention above, the invention will be more readily understood by reference to the following embodiments; these embodiments are provided by way of example and are not intended to be limiting. [Example]

[0064] Example 1 The effect of nutrient supplementation of coffee brew on probiotic growth was investigated.

[0065] Specifically, for the purposes of this example, nutrients in the form of glucose and inactivated yeast derivatives were selected. Specifically, glucose was added as a common carbon source to provide energy in the form of ATP, which is necessary for the growth of the probiotics. Three different types of inactivated yeast derivatives (IYD) were used for the purposes of this example: Optiwhite®, Optired®, and Noblesse® (all manufactured by Lallemand Pty.), which provide the medium with peptides, amino acids, vitamins, minerals, and yeast cell wall components that can support the growth of probiotics.

[0066] method Coffee brew in 250 mL glass-capped bottles was supplemented with glucose (0%, 0.25%, 0.5%, 1%) and Optiwhite®, Optired®, or Noblesse® (0%, 0.03%, 0.06%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%). The supplemented coffee brew was then inoculated with GG (Chr. Hansen A / S) (approximately 7 Log CFU / mL), followed by dispensing 40 mL aliquots into 50 mL polypropylene centrifuge tubes. Fermentation then proceeded at 30°C for 24 hours; this was performed in triplicate.

[0067] result Figure 1 shows the effect of various levels of glucose on GG growth. In unsupplemented coffee (0% glucose, 0% IYD), no probiotic growth was observed (initial inoculum of approximately 7 Log CFU / mL). Increasing glucose levels at the 0% IYD level did not significantly increase probiotic growth. However, in the presence of IYD (0.03% or 0.3% Optiwhite®, Optired®, or Noblesse®), the addition of glucose (0.25%, 0.5%, and 1%) significantly increased probiotic biomass. These results suggest that the presence of both glucose and IYD is necessary to enable probiotic growth, reaffirming the lack of fermentable substrates in coffee brew and the need for nutrient supplementation.

[0068] It was also observed that in the presence of IYD (0.03% or 0.3% Optiwhite®, Optired®, or Noblesse®), increasing glucose levels above 0.25% did not further significantly increase probiotic growth, and therefore this level of glucose supplementation was utilized in subsequent examples.

[0069] The effects of three different types of IYD (Optiwhite®, Optired®, or Noblesse®) on the growth of GG after 24 hours of fermentation were investigated. The results are shown in Figure 2.

[0070] No significant differences in GG cell counts were observed at IYD concentrations of 0.03% and 0.3-0.6%. However, at IYD levels of 0.06%, 0.1%, and 0.2%, coffee brew supplemented with Optired® exhibited significantly lower probiotic cell counts compared to Optiwhite® and Noblesse®. Given the premise of achieving maximum probiotic biomass with minimal nutrient supplementation, Optired® was eliminated from subsequent testing. The effects of different levels of Optiwhite® and Noblesse® supplementation on L. rhamnosus GG growth and pH levels were then investigated.

[0071] Because Optiwhite® and Noblesse® enhanced L. rhamnosus GG growth to similar extents in Figure 2, their levels were varied in Figures 3 and 4 in an attempt to identify the minimum Optiwhite® / Noblesse® level required for maximum L. rhamnosus GG growth. As shown in Figure 3, a significant increase in probiotic biomass was observed with Optiwhite® supplementation when increasing from 0% to 0.06%, reaching a final cell count of 7.99 Log CFU / mL (a 0.85 Log increase). Further addition of Optiwhite® did not result in a significant further increase in biomass. With Noblesse®, increasing the usage level from 0% to 0.2% significantly increased probiotic cell count to 8.15 Log CFU / mL (a 1.1 Log increase). Supplementing Optired® above 0.2% did not result in any further cell count increase.

[0072] A 0.06% Optiwhite® dosage level may be advantageous because it results in raw material cost savings compared to using a higher dosage of 0.2% Noblesse®. Furthermore, increased IYD dosage was visually observed to increase sedimentation in the supplemented coffee brew; this may have undesirable sensory effects. Furthermore, the final pH achieved with 0.06% Optiwhite® (pH 4.29) was significantly higher than that achieved with 0.2% Noblesse® (pH 4.13) (Figure 4). A higher final pH may result in a lower degree of acid stress on the probiotics, thereby potentially preventing a loss of probiotic viability during product storage. Therefore, for practical and sensory reasons, a final supplementation level of 0.06% Optiwhite® was selected in the subsequent examples.

[0073] Example 2 The effect of fermentation of probiotic cultures in monoculture was investigated. Specifically, using a coffee brew formulation containing 0.25% glucose and 0.06% Optiwhite®, this example evaluated the growth and survival of four different probiotic strains during fermentation and storage, with the goal of identifying the probiotic bacterial strain (>7 Log CFU / mL) that could survive the longest during storage of probiotic-fermented coffee brew.

[0074] method Probiotic growth in unsupplemented coffee brews and their supplemented counterparts (0.25% (w / v) glucose, 0.06% (w / v) Optiwhite®) was evaluated over a 24-hour fermentation period. Probiotic survival was then monitored during storage at 4°C and 25°C. To accomplish this, single probiotic bacterial cultures of L. rhamnosus GG (GG), L. acidophilus NCFM (NCFM) (Danisco A / S), L. plantarum 299v (299v) (Probi AB), or L. paracasei Lpc-37 (Lpc37) (Danisco A / S) were first inoculated into 250 mL glass-capped bottles of supplemented (S-) or non-supplemented (N-) coffee brew. The initial inoculum size was standardized to approximately 7 Log CFU / mL. 40 mL or 12 mL aliquots of the inoculated coffee were then dispensed into 50 mL or 15 mL polypropylene centrifuge tubes, respectively. Triplicate batches were then fermented at 30°C for 24 hours before being stored at 4°C and 25°C.

[0075] result L. rhamnosus GG and L. paracasei Lpc-37 exhibited the highest survival rates during storage and were therefore subjected to further analysis of volatile and nonvolatile substances. Analytical time points were 0 h, 24 h, 2 weeks at 25°C, and 10 weeks at 4°C, corresponding to the final shelf-life criteria (7 Log CFU / mL). Analytical measurements included measurements of volatile and nonvolatile substances (sugars, organic acids, amino acids, phenolic compounds, and alkaloids) as well as antioxidant capacity assays for total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC).

[0076] Figure 5 shows the growth and survival of individual probiotic strains in unsupplemented (N-) and supplemented (S-) coffee brews. In unsupplemented coffee, all four probiotic strains showed no growth. In contrast, in supplemented coffee, a significant increase in probiotic biomass was observed, with L. rhamnosus GG (S-GG), L. plantarum 299v (S-299v), L. paracasei Lpc-37 (S-Lpc37), and L. acidophilus NCFM (S-NCFM) reaching stationary-phase cell counts of 7.93, 8.28, 7.67, and 7.58 Log CFU / mL, respectively, after 24 h.

[0077] During storage, probiotic viable cell counts remained above 7 Log CFU / mL for significantly longer periods in the supplemented coffee brews compared with the unsupplemented counterparts. In unsupplemented coffee, viable cell counts for all four probiotic strains fell below the reference point within one week of storage at both temperatures. The exception was L. rhamnosus GG, which maintained cell counts above the reference point for up to two weeks at 4°C. In supplemented coffee brews, viable cell counts for L. rhamnosus GG and L. paracasei Lpc-37 fell below 7 Log CFU / mL within two and ten weeks of storage at 25°C and 4°C, respectively. The shelf life of coffee brews fermented with L. plantarum 299v and L. acidophilus NCFM was four and three weeks, respectively, at both temperatures. These results highlight the need for nutrient supplementation of coffee brews to support both probiotic growth and survival.

[0078] Table 1 shows the changes in nonvolatile components (pH, glucose, lactic acid, alanine, and glutamic acid) during fermentation and storage of coffee brew, and Figure 6 shows the changes in headspace levels of diacetyl, acetoin, and 3-methylbutanoic acid.

[0079] In Table 1, mean values ​​within the same row with different lowercase letters indicate statistically significant differences (P<0.05) between coffees with different supplementation status within the same probiotic strain, while mean values ​​within the same row with different uppercase letters indicate statistically significant differences (P<0.05) between supplemented coffees fermented with L. rhamnosus GG and L. paracasei Lpc-37. Furthermore, the analysis time points were 0 h, 24 h, 2 weeks at 25°C, and 10 weeks at 4°C, corresponding to the final shelf-life criteria (7 Log CFU / mL). Initially, the unsupplemented coffee brew lacked glucose and free amino acids, reinforcing the nutrient deficiencies in the coffee brew.

[0080] [Table 1]

[0081] Supplemented coffee brew was supplied with nutrients in the form of glucose and Optiwhite® (particularly glutamic acid), providing the substrates necessary for probiotic bacterial growth. Glucose, alanine, and glutamic acid were gradually utilized by L. rhamnosus GG and L. paracasei Lpc-37 throughout the fermentation and storage periods. Concomitant production of bacterial metabolites (lactic acid, diacetyl, acetoin, and 3-methylbutanoic acid) was observed as a result of growth. During fermentation, lactic acid production corresponded to a significant decrease in pH, which further decreased during storage, due to the continued utilization of glucose. In addition to lactic acid, bacterial volatile metabolites were also produced (Figure 6), with significant increases in the levels of 3-methylbutanoic acid, diacetyl, and acetoin during fermentation and storage.

[0082] The production of these bacterial metabolites can result in flavor changes and different taste profiles compared to regular coffee brew: for example, lactic acid imparts a sour taste, 3-methylbutanoic acid imparts a cheesy or sweaty flavor (depending on the concentration), while diacetyl and acetoin impart a buttery aroma.

[0083] The levels of chlorogenic acid, alkaloids, and antioxidant capacity of the coffee brew are shown in Table 2. We sought to analyze endogenous alkaloids and phenolic compounds in coffee because they are commonly associated with the therapeutic benefits of coffee consumption. In general, the levels of bioactive components and overall antioxidant capacity of the coffee brew were not affected by nutrient supplementation, probiotic fermentation, or storage. These results suggest that probiotic coffee may retain the inherent therapeutic benefits of coffee. In Table 2, mean values ​​within the same row with different lowercase letters indicate statistically significant differences (P < 0.05) between coffees supplemented with different probiotic strains and different supplementation regimes. Mean values ​​within the same row with different uppercase letters indicate statistically significant differences (P < 0.05) between supplemented coffees fermented with L. rhamnosus GG and L. paracasei Lpc-37.

[0084] [Table 2]

[0085] Example 3 The effects of fermentation of probiotic cultures by monoculture and coculture were investigated.

[0086] L. rhamnosus GG showed excellent growth and survival in coffee brew supplemented with 0.25% glucose and 0.06% Optiwhite®. A 0.8 Log increase in cell biomass was observed, which remained above 7 Log CFU / mL for 10 weeks under refrigeration and 2 weeks at ambient temperature. While a 10-week refrigerated shelf life is reasonable, cold-chain logistics are not only costly but also limit distribution to a wider market, especially in rural areas lacking adequate cold-chain systems. Because products with short ambient shelf lives are not commercially viable, strategies to extend probiotic survival beyond 2 weeks at ambient temperature must be explored.

[0087] Therefore, the potential of yeast used as a co-culture with L. rhamnosus GG was investigated to further extend the shelf life of probiotic-fermented coffee brew. In this example, the survival of L. rhamnosus GG in coffee brew by co-cultivation with the probiotic yeast S. boulardii CNCM-I745 was investigated.

[0088] method Four different fermentation setups were prepared: a monoculture of L. rhamnosus GG (GG), a monoculture of S. boulardii CNCM-I745 (Sb), a mixed culture of probiotic bacteria and yeast (GG+Sb), and a control (blank), which consisted of coffee brew without probiotic inoculation. For the three fermentation setups, 200 mL of coffee brew in 250 mL glass bottles with caps was inoculated; the inoculation volume was standardized to approximately 7 Log CFU / mL for L. rhamnosus GG and approximately 6 Log CFU / mL for S. boulardii CNCM-I745.

[0089] Next, 40 mL or 12 mL aliquots of the inoculated coffee were dispensed into 50 mL or 15 mL polypropylene centrifuge tubes, respectively. The tubes were then held at 30°C for 24 hours during the fermentation period and at 25°C or 4°C during storage. Further analyses (measurements of non-volatile and volatile compounds and antioxidant capacity assays) were performed on unfermented and fermented coffee brews, as well as samples stored at both temperatures for 1 month. All fermentations were performed in triplicate batches.

[0090] result Figure 7 shows the growth of monocultures and mixed cultures of L. rhamnosus GG and S. boulardii CNCM-I745 during fermentation in coffee brew and subsequent storage at 4°C and 25°C. L. rhamnosus GG grew from an initial cell count of 6.9 Log CFU / mL to 7.8 and 7.5 Log CFU / mL in monoculture (GG) and mixed culture (GG+Sb), respectively, after 24 hours of fermentation. During the same period, S. boulardii CNCM-I745 cell counts increased from an initial value of 6.1 Log CFU / mL to 7.1 and 7.2 Log CFU / mL in monoculture (Sb) and mixed culture (GG+Sb), respectively. When probiotic cell counts in monocultures were compared with those in mixed cultures, no significant difference was observed for S. boulardii CNCM-I745, but a significant difference was detected for L. rhamnosus GG. However, it is recognized that growth of L. rhamnosus GG was still satisfactory, as numbers exceeded 7 Log CFU / mL.

[0091] During storage, the viability of L. rhamnosus GG decreased at a much faster rate in monocultures than in mixed cultures. At 4°C, L. rhamnosus GG in monoculture was no longer detectable after 10 weeks, whereas the mixed culture maintained a high biomass of 7 Log CFU / mL after 14 weeks of storage. At 25°C, the cell count of L. rhamnosus GG in monoculture fell below 6 Log CFU / mL after 3 weeks of storage and was no longer detectable after 10 weeks. In contrast, the same probiotic strain in mixed culture recorded an extremely high viability of 6.8 Log CFU / mL after 14 weeks of ambient storage. Thus, the survival of L. rhamnosus GG was significantly enhanced by yeast at ambient and refrigerated temperatures.

[0092] S. boulardii CNCM-I745 proved to be more robust than L. rhamnosus GG during storage. The viable cell counts of the probiotic yeast in both monoculture and mixed culture were maintained above 6 Log CFU / mL during 14 weeks of storage at 4°C and 25°C. Interestingly, significantly lower viable cell counts of the probiotic yeast (approximately 0.5 Log difference) were detected in monoculture compared with mixed culture after 14 weeks of ambient storage. This may indicate that the survival of S. boulardii CNCM-I745 may also be favorably enhanced by L. rhamnosus GG; however, this probiotic survival benefit would likely be demonstrated with extended storage.

[0093] The changes in pH and nonvolatile components during fermentation and storage are shown in Table 3.

[0094] [Table 3]

[0095] In coffee brew containing S. boulardii CNCM-I745, glucose was fully utilized during the fermentation period. In contrast, in the monoculture of L. rhamnosus GG, utilization was slower, with 45% of the original level remaining after fermentation. The consumption of glucose by the probiotic LAB coincided with a significant increase in lactic acid and a corresponding significant decrease in pH. During storage, the pH of the coffee brew fermented with the monoculture of L. rhamnosus GG further decreased, due to the uptake of residual glucose by the probiotic bacteria.

[0096] Lactic acid was also produced by L. rhamnosus GG in the mixed culture, but the yield was significantly lower, resulting in a significantly higher pH compared to the monoculture. This is likely due to competition with the yeast for glucose, limiting the amount of glucose available for lactic acid production by L. rhamnosus GG. During the storage period, no further lactic acid production was observed in the mixed culture because there was no glucose available for lactic acid production.

[0097] In coffee brews containing S. boulardii CNCM-I745, the pH remained relatively constant throughout the 14-week cold storage period. Interestingly, the pH gradually increased during storage at room temperature, an effect that was more pronounced in the mixed-culture coffee brews. In the mixed-culture brews, the pH gradually increased from 4.54, reaching 4.64 after 4 weeks and 4.97 after 14 weeks. The deacidification of the coffee brew by S. boulardii is likely due to the consumption of citric acid by the yeast, which presumably served as an alternative carbon source to support yeast survival during storage.

[0098] Deacidification of coffee brew by S. boulardii CNCM-I745 may alleviate acid stress by either citric acid consumption or limiting lactic acid production, resulting in improved survival of L. rhamnosus GG in the mixed culture compared to the monoculture. Furthermore, it is worth mentioning that prevention of post-acidification by yeasts is considered organoleptically preferable.

[0099] Table 4 shows selected headspace volatile classes detected in coffee brew after fermentation at 30°C. L. rhamnosus GG was primarily responsible for the release of diacetyl and acetoin, whereas S. boulardii CNCM-I745 primarily produced alcohols, esters, and phenolic compounds. Each volatile compound contributes a unique aroma; for example, higher alcohols impart floral notes, and esters impart fruity notes. Therefore, coffee brews fermented with different probiotic strains may result in a variety of flavors.

[0100] [Table 4]

[0101] Figure 8 shows the changes in coffee bioactive components after fermentation and storage. In general, the levels of measured alkaloids (caffeine, trigonelline) and phenolic compounds (chlorogenic acid, caffeic acid) remained unchanged. Although trigonelline and chlorogenic acid levels were detected at significantly higher levels in the mixed culture than in the blank after ambient storage, these changes were small and may not be practically meaningful. Because endogenous bioactive components of coffee are often cited as potent bioactive coffee components that tend to confer physiological effects, it is desirable to preserve these components after fermentation and storage.

[0102] Figure 9 shows the changes in antioxidant capacity of coffee brews after fermentation and storage. TPC assays revealed non-significant changes between coffee brews regardless of time point. DPPH assays consistently showed slightly but significantly higher antioxidant activity in the mixed-culture coffee brew compared to the blank. While significant, the differences were small and may not be practically meaningful. ORAC assays revealed significantly lower Trolox equivalent values ​​in the probiotic coffee brew after ambient storage compared to the blank, indicating a decrease in peroxyl radical scavenging capacity.

[0103] Example 4 The effect of fermentation of probiotic cultures co-cultured with S. boulardii CNCM-I745 was investigated.

[0104] The survival of L. rhamnosus GG was significantly improved when cocultured with S. boulardii CNCM-I745 in Section 3. However, it would be of further interest to investigate whether similar effects would be observed by coculture of S. boulardii CNCM-I745 with other probiotic strains. This is particularly important because various probiotic strains exert different physiological effects upon ingestion. Furthermore, the probiotic survival-enhancing effect of yeast is strain-dependent, and coculture of yeast with probiotic LAB does not necessarily enhance the latter's survival, making it interesting to identify compatible probiotic-yeast combinations.

[0105] Therefore, this example aims to investigate the effect of co-cultivation of S. boulardii CNCM-I745 on the growth and survival of L. plantarum 299v, L. acidophilus NCFM, L. fermentum PCC, and L. gasseri LAC-343.

[0106] method Coffee brew supplemented with 0.25% glucose and 0.06% Optiwhite® was fermented with the probiotic monocultures L. plantarum 299v (299v), L. acidophilus NCFM (NCFM), L. fermentum PCC (PCC) (Chr. Hansen A / S), L. gasseri LAC-343 (LAC343) (Morinaga), and S. boulardii (Sb) (Biocodex), and their cocultures 299vSb, NCFMSb, PCCSb, and LAC343Sb. A blank consisting of unfermented coffee was included as a control. Probiotics were inoculated into 200 mL of coffee brew in 250 mL glass-capped bottles; the inoculum size was standardized to approximately 6.6–7 Log CFU / mL for probiotic LAB and approximately 6 Log CFU / mL for S. boulardii CNCM-I745. Next, 40 mL or 12 mL aliquots of the inoculated coffee were dispensed into 50 mL or 15 mL polypropylene centrifuge tubes, respectively. The tubes were then held at 30°C for 24 hours during fermentation, followed by either 25°C or 4°C for storage. Further analysis (measurement of nonvolatile matter) was performed on the coffee fermented for 24 hours and stored at both temperatures for one month. All fermentations were performed in triplicate batches.

[0107] result Figure 10 shows the growth of mono- and mixed cultures of L. plantarum 299v, L. acidophilus NCFM, L. fermentum PCC, L. gasseri LAC-343, and S. boulardii CNCM-I745 during fermentation in coffee brew and subsequent storage at 4°C and 25°C.

[0108] All probiotics, whether grown alone or in co-culture, were able to grow above 7 Log CFU / mL, demonstrating the compatibility of the probiotic yeast S. boulardii CNCM-I745 with the other four probiotic LAB strains. Furthermore, the excellent growth above the recommended use of 7 Log CFU / mL suggests that the coffee brew formulation (0.25% glucose and 0.06% Optiwhite®) may be applicable to support the growth of other combinations of probiotic yeast and LAB.

[0109] Probiotic yeast was found to be essential for maintaining viable probiotic LAB populations in coffee during storage. Regardless of temperature, all co-cultured probiotic LAB maintained viable populations above 6-7 Log CFU / mL for at least 3 months. In contrast, single LAB probiotic populations were never maintained above 6 Log CFU / mL for more than 3 months, and most remained below 3 Log CFU / mL.

[0110] Interestingly, the viability of S. boulardii CNCM-I745 cultured alone was not significantly different from that when cultured with other probiotic LAB, indicating that the probiotic yeast remained unaffected by the presence of probiotic LAB and therefore highlighting the excellent compatibility of S. boulardii CNCM-I745 with other probiotic LAB strains.

[0111] Figures 11 and 12 show the changes in pH and lactic acid during fermentation and storage for single and mixed coffee fermentations, respectively. In general, the degree of pH decrease for single- and co-fermented coffee brews was similar after 24 hours of fermentation; the exception was L. plantarum 299v, whose monocultures produced significantly lower pH than co-cultures. The decrease in pH during fermentation is the result of lactic acid production by the probiotic LAB. The reduced lactic acid yield and accompanying increase in pH in the co-cultured L. plantarum 299v coffee brew are most likely the result of competition with yeast for nutrients (glucose, Optiwhite®). Therefore, if a less sour coffee brew is desired after fermentation, it is crucial to co-cultivate probiotic LAB with S. boulardii CNCM-I745 while maintaining a viable probiotic population.

[0112] No further changes in pH were observed during 3 months of cold storage, which is attributed to a decrease in probiotic metabolic activity. However, under ambient temperature conditions, a further decrease in pH was observed in monocultures of L. plantarum 299v, L. acidophilus NCFM, and L. gasseri LAC-343, consistent with lactic acid accumulation (Figure 12). The exception was observed in monocultured L. fermentum PCC, which showed no significant decrease in pH and lactic acid production during ambient storage. When using monocultures of probiotic LAB, refrigeration may be preferable under these circumstances to limit excessive pH changes (which could result in an undesirable sour taste).

[0113] In contrast to the probiotic LAB monocultures, no further increase in lactic acid was observed in the cocultures. Therefore, S. boulardii CNCM-I745 can be effectively used to limit excess lactic acid production and pH drop, which can result in an undesirable sour taste, when a cold supply chain is not available.

[0114] Figure 13 shows the changes in coffee bioactive components after fermentation and storage. In general, no significant loss of measured alkaloids (caffeine, trigonelline) and phenolic compounds (chlorogenic acid) was observed. Fermentation and storage of coffee brew did not alter the levels of endogenous coffee bioactive components; this indicates that the inherent health benefits of coffee are preserved.

[0115] While the foregoing description describes exemplary embodiments, those skilled in the art will appreciate that many variations are possible without departing from the invention. The present invention encompasses, for example, the following embodiments: [1] A coffee-based beverage containing a probiotic, wherein the probiotic has a probiotic viable cell count of 6.0 log CFU / mL or greater. [2] The beverage according to [1], wherein the probiotic has a probiotic viable cell count of 7.0 log CFU / mL or more. [3] The beverage according to [1] or [2], wherein the probiotics contained in the beverage have a probiotic viable cell count of 6.0 log CFU / mL or more after storage for 3 months. [4] The beverage according to any one of [1] to [3], wherein the beverage is a fermented beverage. [5] The beverage according to any one of [1] to [4], wherein the probiotics include probiotic bacteria, probiotic yeast, or a combination thereof. [6] The beverage according to any one of [1] to [5], wherein the probiotics include lactic acid bacteria, bifidobacteria, Saccharomyces yeast, non-Saccharomyces yeast, or a combination thereof. [7] The beverage according to [6], wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or a combination thereof. [8] The beverage according to [6], wherein the Saccharomyces yeast is selected from the group consisting of Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof. [9] The beverage of [6], wherein the probiotic comprises Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof.

[10] The beverage according to any one of [1] to [9], further comprising an additive.

[11] The beverage according to

[10] , wherein the additive is selected from a sweetener, a stabilizer, a flavoring, or a combination thereof.

[12] A method for preparing a coffee-based beverage containing probiotics having a viable cell count of 6.0 log CFU / mL or greater, comprising the steps of: - mixing the coffee brew with sugar and an inactivated yeast derivative to form a mixture; - adding probiotics to the mixture to form an inoculated mixture; and - fermenting the inoculated mixture for a predetermined period of time to prepare the beverage; The method comprising:

[13] The method according to

[12] , wherein the beverage has a viable cell count of 7.0 log CFU / mL or more.

[14] The method according to

[12] or

[13] , wherein the sugar is present in the mixture at a concentration of 0.01 to 10% w / v relative to the total volume of the mixture.

[15] The method according to any one of

[12] to

[14] , wherein the inactivated yeast derivative is present in the mixture at a concentration of 0.005 to 5% w / v relative to the total volume of the mixture.

[16] The method according to any one of

[12] to

[15] , wherein the probiotic comprises probiotic bacteria, probiotic yeast, or a combination thereof.

[17] The method according to any one of

[12] to

[16] , wherein the probiotics include lactic acid bacteria, bifidobacteria, Saccharomyces yeast, non-Saccharomyces yeast, or a combination thereof.

[18] The method according to

[17] , wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or a combination thereof.

[19] The method according to

[17] , wherein the Saccharomyces yeast is selected from the group consisting of Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof.

[20] The method of

[17] , wherein the probiotic comprises Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, Bifidobacterium (B.) lactis, Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof.

[21] The method according to any one of

[12] to

[20] , wherein the adding step comprises adding the probiotic to obtain an initial probiotic viable cell count of at least 6 log CFU / mL.

[22] The method according to any one of

[12] to

[21] , wherein the predetermined period is 4 to 100 hours.

[23] The method according to any one of

[12] to

[22] , wherein the fermentation step is carried out at a temperature of 15 to 45°C.

[24] The method according to any one of

[12] to

[23] , further comprising adding an additive to the mixture.

[25] The method according to

[24] , wherein the additive is selected from a sweetener, a stabilizer, a flavoring, or a combination thereof.

Claims

1. 1. A coffee-based beverage comprising a probiotic, the probiotic having a probiotic viable cell count of 6.0 log CFU / mL or greater; The beverage is a fermented beverage, after 3 months of storage, the probiotics contained in the beverage have a probiotic viable cell count of 6.0 log CFU / mL or greater; The probiotic is a probiotic lactic acid bacterium selected from the group comprising Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or a combination thereof, and a probiotic lactic acid bacterium selected from the group comprising Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof.

1. A beverage comprising a probiotic yeast selected from the group comprising: S. cerevisiae, S. cerevisiae, or a combination thereof.

2. 10. The beverage of claim 1, wherein the probiotic has a probiotic viable cell count of 7.0 log CFU / mL or greater.

3. 3. The beverage of claim 1 or 2, wherein the beverage further comprises an additive.

4. 4. The beverage of claim 3, wherein the additive is selected from a sweetener, a stabilizer, a flavoring, or a combination thereof.

5. 1. A method for preparing a coffee-based beverage containing probiotics having a viable cell count of 6.0 log CFU / mL or greater, comprising the steps of: - mixing the coffee brew with sugar and an inactivated yeast derivative to form a mixture; - adding probiotics to the mixture to form an inoculated mixture; and - fermenting the inoculated mixture for a predetermined period of time to prepare a fermented coffee-based probiotic beverage; Including, after 3 months of storage, the probiotics contained in the beverage have a probiotic viable cell count of 6.0 log CFU / mL or greater; The probiotic comprises a probiotic lactic acid bacterium selected from the group comprising Lactobacillus (Lb.) rhamnosus, Lactobacillus (Lb.) paracasei, Lactobacillus (Lb.) plantarum, Lactobacillus (Lb.) acidophilus, Lactobacillus (Lb.) gasseri, Lactobacillus (Lb.) fermentum, or a combination thereof, and a probiotic yeast selected from the group comprising Saccharomyces (S.) boulardii, Saccharomyces (S.) cerevisiae, or a combination thereof; method.

6. 6. The method of claim 5, wherein the beverage has a viable cell count of 7.0 log CFU / mL or greater.

7. 7. The method of claim 5, wherein the sugar is present in the mixture at a concentration of 0.01 to 10% w / v relative to the total volume of the mixture.

8. 8. The method according to any one of claims 5 to 7, wherein the inactivated yeast derivative is present in the mixture at a concentration of 0.005 to 5% w / v relative to the total volume of the mixture.

9. 9. The method of any one of claims 5 to 8, wherein the adding step comprises adding probiotics to obtain an initial probiotic viable count of at least 6 log CFU / mL.

10. 10. The method of any one of claims 5 to 9, wherein the predetermined period of time is between 4 and 100 hours.

11. 11. The method according to any one of claims 5 to 10, wherein the fermenting step is at a temperature of 15 to 45°C.

12. The method of any one of claims 5 to 11, further comprising adding an additive to the mixture.

13. 13. The method of claim 12, wherein the additive is selected from a sweetener, a stabilizer, a flavoring agent, or a combination thereof.

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