CN118632917A - An alcoholic beverage based on probiotic yeast and preparation method thereof - Google Patents

Abstract

An alcoholic beverage based on probiotic yeast and a method for preparing the same. An alcoholic beverage based on probiotic yeast is provided, comprising probiotic yeast, wherein the probiotic yeast has a cell count of ≥5 log CFU/mL after storage at 30°C for 100 days. A method for forming the alcoholic beverage based on probiotic yeast is also provided.

Description

An alcoholic beverage based on probiotic yeast and preparation method thereof

Technical Field

The present invention relates to an alcoholic beverage based on probiotic yeast. Specifically, the alcoholic beverage based on probiotic yeast may contain probiotic yeast.

Background Art

Given the health benefits that probiotics can provide, there is growing interest in the consumption of probiotics, which are now included in many foods and beverages.

Most foods and beverages containing probiotics are stored at low temperatures. However, some dairy-based and plant-based probiotic foods and beverages can be stored at ambient-stable temperatures. However, it is difficult to store alcoholic beverages containing probiotics at ambient conditions while keeping the probiotics alive in the alcoholic beverages.

Summary of the invention

The present invention seeks to address these problems and/or to provide an improved alcoholic beverage comprising probiotic yeast and a method for its preparation, such that the alcoholic beverage is stable when stored under ambient conditions.

According to a first aspect of the present invention, there is provided a method for forming an alcoholic beverage based on probiotic yeast, the method comprising:

- Provide wort or unfermented juice;

- adding a first yeast to the wort or unfermented juice;

- fermenting the wort or unfermented juice for a predetermined period of time and at a predetermined temperature to form alcohol

Beverages; and

- adding a second yeast to the alcoholic beverage to form a probiotic yeast based alcoholic beverage, wherein the second yeast is a probiotic yeast.

The first yeast may be any suitable yeast. For example, the first yeast may be a probiotic yeast or a non-probiotic yeast.

According to a specific aspect, the first yeast may be a probiotic yeast. The first yeast may be the same as the second yeast.

According to another specific aspect, the first yeast may be a non-probiotic yeast. Specifically, the first yeast may include: a non-probiotic Saccharomyces yeast, a non-probiotic non-Saccharomyces yeast, or a combination thereof.

The second yeast may be any suitable probiotic yeast. For example, the second yeast may include: probiotic Saccharomyces yeast, probiotic non-Saccharomyces yeast, or a combination thereof.

The second yeast may be in any suitable form. For example, the second yeast may include freeze-dried probiotic yeast or pre-cultured probiotic yeast.

The first yeast and the second yeast may be added in any suitable amount. For example, the first yeast and the second yeast may each have a cell count of ≥4 log CFU/mL. The method may further include adding the first hops or a derivative thereof to the wort or unfermented juice. The first hops or a derivative thereof added to the wort or unfermented juice may be any suitable hops. The first hops may have a suitable bitterness. Specifically, the first hops or a derivative thereof may have a bitterness of ≤80 IBU.

According to a specific aspect, the method may further comprise removing the precipitate of the first yeast before adding the second yeast. The removing of the precipitate of the first yeast may be performed by any suitable method.

The fermentation of the wort or unfermented juice may be performed under any suitable conditions. For example, the first predetermined fermentation temperature may be any suitable temperature. According to a specific aspect, the first predetermined fermentation temperature may be 10-35°C.

The method may further include adding a second amount of wort or unfermented juice to the alcoholic beverage. Specifically, the adding of the second amount of wort or unfermented juice to the alcoholic beverage is performed simultaneously with the adding of the second amount of yeast, or after the adding of the second amount of yeast.

The method may further include fermenting the second wort or unfermented juice at a second predetermined fermentation time period and a second predetermined fermentation temperature. The second predetermined fermentation temperature may be any suitable temperature. According to a specific aspect, the second predetermined fermentation temperature may be 20-30°C.

According to a second aspect, the present invention provides a probiotic yeast-based alcoholic beverage comprising probiotic yeast, wherein the probiotic yeast has a cell count of ≥ 5 log CFU/mL after storage at 30°C for 100 days.

The probiotic yeast contained in the probiotic yeast-based alcoholic beverage may be any suitable probiotic yeast. For example, the probiotic yeast may include probiotic Saccharomyces yeast, probiotic non-Saccharomyces yeast, or a combination thereof.

According to a specific aspect, the alcoholic beverage based on probiotic yeast may further include hops or a derivative thereof. The hops or a derivative thereof may be any suitable hops. The hops may have a suitable bitterness. Specifically, the hops or a derivative thereof may have a bitterness of ≤80 IBU. The alcoholic beverage based on probiotic yeast may be formed by the method of the first aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the present invention may be fully understood and readily put into practice, exemplary embodiments will now be described by way of non-limiting examples only, with reference to the illustrative drawings. In the drawings:

Figure 1 shows the survival of the probiotic Saccharomyces cerevisiae CNCM I-3856 in beer without hops during storage at 30°C, wherein (●) indicates no sequential inoculation (control group); (▼) indicates sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (FS group); (■) indicates sequential inoculation of primary cultured Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (PS group); Figure 2 shows the changes in the Brix (°Brix) of beer without hops fermented with the probiotic Saccharomyces cerevisiae CNCM I-3856 during storage at 30°C, wherein (●) indicates no sequential inoculation (control group), (▼) indicates sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (FS group), and (■) indicates primary cultured Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (PS group). Sequential inoculation of I-3856 + 5% v/v wort (PS group);

Figure 3 shows the pH changes of hopless beer fermented with probiotic Saccharomyces cerevisiae CNCM I-3856 during storage at 30°C, where (●) indicates no sequential inoculation (control group), (▼) indicates sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (FS group), and (■) indicates sequential inoculation of primary cultured Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (PS group); Figure 4 shows the sensory profile of the average characteristic values of the probiotic hopless beer samples calculated on a scale of 1 to 5 points. (◇) indicates no sequential inoculation (control group); (●) indicates sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (FS group); and (■) indicates sequential inoculation of primary cultured Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort (PS group);

Figure 5 shows the survival of probiotics in beer during storage at 30°C, wherein (△) represents S. boulardii CNCM I-745 in beer without hops; (▼) represents S. boulardii CNCM I-745 in beer with hops; and (●) represents S. cerevisiae CNCM I-3856 in beer with hops;

FIG6 shows the change in the Brix of beer during storage at 30° C., wherein (△) represents Saccharomyces boulardii CNCM I-745 in beer without hops, (▼) represents Saccharomyces boulardii CNCM I-745 in beer with hops, and (●) represents Saccharomyces cerevisiae CNCM I-3856 in beer with hops; FIG7 shows the change in pH of beer during storage at 30° C., wherein (△) represents Saccharomyces boulardii CNCM I-745 in beer without hops, (▼) represents Saccharomyces boulardii CNCM I-745 in beer with hops, and (●) represents Saccharomyces cerevisiae CNCM I-3856 in beer with hops; FIG8 shows the sensory profile of the average characteristic values calculated on a scale of 1 to 5 points for the probiotic beer samples, wherein (△) represents Saccharomyces boulardii CNCM I-745 in beer without hops; (▲) represents Saccharomyces boulardii CNCM I-745 in beer with hops, and (●) represents Saccharomyces cerevisiae CNCM I-3856 in beer with hops. I-3856;

Figure 9 shows the survival of probiotics in beer without hops during storage at 30°C, wherein (●) represents the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort; and (▼) represents the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 10% v/v wort;

Figure 10 shows the change in Brix of hopless beer during storage at 30°C, wherein (●) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort; and (▼) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 10% v/v wort;

Figure 11 shows the pH changes of beer without hops during storage at 30°C, wherein (●) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort; and (▼) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856 + 10% v/v wort;

12 shows a sensory profile of the average characteristic values of probiotic hopless beers calculated on a scale of 1 to 5 points, wherein (▲) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856+5% v/v wort, and (●) indicates the sequential inoculation of Saccharomyces cerevisiae CNCM I-3856+10% v/v wort;

FIG. 13A shows the preference ranking scores of unhopped beers based on probiotic yeast, and FIG. 13B shows the preference ranking scores of hopped beers based on probiotic yeast stored at 30° C. for 120 days; and

14A to 14D show the main volatile aroma compounds of unhopped beer stored at 30° C. for 120 days.

Summary of the invention

As explained above, there is a need for an improved alcoholic beverage containing probiotics that is stable under ambient environmental conditions. In summary, the present invention relates to an alcoholic beverage containing probiotics that can be stored at ambient temperature, in particular an alcoholic beverage containing probiotic yeast, and a method of forming the same. The alcoholic beverage of the present invention may provide advantages in storage and transportation, as a cold chain may not be required to keep the beverage stable, thereby reducing costs and increasing shelf-life.

According to a first aspect of the present invention, there is provided a method for forming an alcoholic beverage based on probiotic yeast, the method comprising:

- Provide wort or unfermented juice;

- adding a first yeast to the wort or unfermented juice;

- fermenting the wort or unfermented juice for a predetermined period of time and at a predetermined temperature to form alcohol

Beverages; and

- adding a second yeast to the alcoholic beverage to form a probiotic yeast based alcoholic beverage, wherein the second yeast is a probiotic yeast.

The probiotic yeast-based alcoholic beverage formed by the method comprises probiotic yeast, wherein the probiotic yeast has a cell count of ≥5 log CFU/mL after storage at 30°C for 100 days.

For the purpose of the present invention, an alcoholic beverage based on probiotic yeast may be defined as a beverage containing alcohol (e.g. ethanol or ethyl alcohol). In particular, the alcohol of the alcoholic beverage may have an alcohol content of ≥ 0.5% (volume percentage).

The alcoholic beverage based on probiotic yeast may have a suitable alcohol content. According to a specific aspect, the alcohol content of the alcoholic beverage based on probiotic yeast may be ≥0.5% (volume percentage). For example, the alcohol content may be 0.5-10%, 1.0-9.0%, 1.5-8.0%, 2.0-7.5%, 2.5-7.0%, 3.0-6.5%, 3.5-6.0%, 4.0-5.5%, 4.5-5.0%. Specifically, the alcohol content may be 2.0-5.0%. Even more specifically, the alcohol content may be about 3-4%.

The alcoholic beverage based on probiotic yeast may be any suitable alcoholic beverage. Examples of alcoholic beverages may include, but are not limited to, beer, wine, sparkling wine, rice wine, cider, spirits, fermented water, liquor, mead, tequila, etc. According to a specific aspect, the alcoholic beverage based on probiotic yeast may be beer.

The wort or unfermented juice may be any wort or unfermented juice suitable for the purpose of the present invention. For the purpose of the present invention, the wort or unfermented juice may include but is not limited to barley, oats, wheat, corn, rye, rice, water or a combination thereof. The wort or unfermented juice may be prepared by mashing, heating, boiling and cooling of malt. The wort or unfermented juice may also include added sugar. The added sugar may be in any suitable form, such as but not limited to fruit juice, pulp or a combination thereof.

The method may further include adding a first batch of hops to the wort or unfermented juice. For the purposes of the present invention, hops include hops and/or derivatives thereof. The hops may be any suitable hops containing isomerized alpha acids. The hops may be in any suitable form, such as but not limited to hop cones, hop pellets, hop resins, hop powders, isomerized hop extracts, and the like.

The first hops may be any suitable hops. For example, the first hops may be an isomerized hop extract. The first hops may have a suitable bitterness. According to a specific aspect, the first hops may have a bitterness of ≤80 IBU. IBU refers to the International Bitterness Unit, and is a unit of measure for the concentration of hop compounds in the alcoholic beverage based on probiotic yeast. Specifically, IBU measures the parts per million (ppm) of isohumulones in the beverage. Specifically, the bitterness may be 0-80 IBU, 5-75 IBU, 10-70 IBU, 15-60 IBU, 20-50 IBU, 25-45 IBU, 30-40 IBU. Even more specifically, the bitterness may be 0-30 IBU.

The first yeast added to the wort or unfermented juice may be any suitable yeast. For example, the first yeast may be a probiotic yeast or a non-probiotic yeast.

According to a specific aspect, the first yeast may be a probiotic yeast. The probiotic yeast may be a probiotic yeast, a probiotic non-yeast yeast, or a combination thereof. Examples of suitable probiotic yeast (S.) yeasts include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces paradoxus, Saccharomyces pasteurianus, Saccharomyces bayanus, or a combination thereof. Specifically, the probiotic yeast may include, but are not limited to, Saccharomyces boulardi CNCM I-745, Saccharomyces cerevisiae CNCM I-3856, Saccharomyces boulardi T1, Saccharomyces boulardi 17, Saccharomyces boulardi CECT 1474, or a combination thereof. Examples of suitable probiotic non-yeast yeasts include, but are not limited to, Pichia (Pichia, P.), Kluyveromyces (Kluyveromyces, K.), Hanseniaspora (H.), Candida (Candida, C.), Zygosaccharomyces (Z.), or a combination thereof.

According to another specific aspect, the first yeast may be a non-probiotic yeast. Any suitable non-probiotic yeast may be used as the first yeast. The non-probiotic yeast may be a non-probiotic Saccharomyces yeast, a non-Saccharomyces yeast, or a combination thereof. Examples of suitable non-probiotic Saccharomyces yeast may include but are not limited to Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces ludwigi, Saccharomyces paradoxica, Saccharomyces pastorianus, or a combination thereof. Examples of suitable non-probiotic non-Saccharomyces yeast include but are not limited to Dekkera bruxellensis, Galactomyces geotrichum, Kazachstania zonata, Kluyveromyces lactis, Lindnera meyerae, Pichia kluyveri, Schizosaccharomyces pombe, Starmera caribae, Torulaspora delbrueckii.

The adding of the first yeast may include adding a suitable amount of yeast to the wort or unfermented juice. For example, the amount of the first yeast may be ≥ 4 log CFU/mL. Specifically, the amount of the first yeast may be 4-9 log CFU/mL, 5-8 log CFU/mL, 6-7 log CFU/mL. Even more specifically, the amount of the first yeast added may be 5-7 log CFU/mL.

The fermentation may be carried out under suitable conditions. For example, the fermentation may include fermenting the wort or unfermented juice at a suitable temperature for a suitable period of time. The temperature may be changed at any time point during the fermentation. Specifically, the fermentation may include fermenting the wort or unfermented juice at a first fermentation predetermined temperature for a first fermentation predetermined period of time.

The first fermentation predetermined time period may be any suitable amount of time. The time period may be selected based on the first yeast added to the wort or unfermented juice. For example, the first fermentation predetermined time period may be ≤30 days. Specifically, the first fermentation predetermined time period may be 5-30 days, 6-28 days, 8-25 days, 10-22 days, 12-20 days, 14-18 days, 15-17 days. Even more specifically, the first fermentation predetermined time period may be 5 to 14 days.

The first fermentation predetermined temperature may be any suitable temperature. The temperature may be selected according to the first yeast added to the wort or unfermented juice. For example, the first fermentation predetermined temperature may be 10-35°C. Specifically, the first fermentation predetermined temperature may be 10-30°C, 12-28°C, 15-25°C, 18-22°C, 20-21°C. Even more specifically, the first fermentation predetermined temperature may be 15-20°C.

The second addition of yeast may be at any suitable time. For example, the second addition of yeast is after the fermentation has ended. Specifically, the fermentation is considered to have ended when the °Bx of the wort or unfermented juice has become stable. Even more specifically, the fermentation may be considered to have ended when the °Bx may be about 2-15 °Bx.

The method may further include removing the precipitate of the first yeast before adding the second yeast. The removing of the precipitate of the first yeast may be performed by any suitable method. For example, the removing may be performed by cold crashing, centrifugation, filtration, or a combination thereof. The removing of the precipitate may include removing yeast and protein particles formed by the fermentation, which, if left in the alcoholic beverage, may make the alcoholic beverage turbid and have a yeast aroma. Specifically, the removing of the precipitate may improve the stability and sensory quality of the alcoholic beverage.

According to a particular aspect, the removal of precipitates may be performed by rapid cooling. The rapid cooling may be performed under suitable conditions, such as at a suitable temperature for a suitable period of time. For example, the temperature of the rapid cooling may be 0-10°C. Specifically, the temperature may be 2-9°C, 3-8°C, 4-7°C, 5-6°C. Even more specifically, the temperature may be 0-4°C. For example, the time period may be 1-10 days. Specifically, the time period may be 2-9 days, 3-8 days, 4-7 days, 5-6 days. Even more specifically, the time period may be 1-2 days.

According to another specific aspect, the removal of precipitate may be performed by centrifugation. The centrifugation may be performed under suitable conditions, such as at a suitable temperature for a suitable period of time. The centrifugation may further include pasteurization. For example, the temperature of the centrifugation and optional pasteurization may be 55-85°C. Specifically, the temperature may be 60-80°C, 65-75°C, 70-73°C. Even more specifically, the temperature may be 60-65°C. For example, the time period may be 1-10 days. Specifically, the time period may be 5s–30min, 30s–25min, 1-20min, 5-15min, 8-10min. Even more specifically, the time period may be 15-20min. Specifically, the centrifugation may be performed until the alcoholic beverage reaches a suitable pasteurization unit. The centrifugation may be performed until the alcoholic beverage reaches a pasteurization unit of 10-500PU.

The yeast added for the second time may include adding a suitable probiotic yeast. For example, the yeast added for the second time may include: probiotic yeast, probiotic non-yeast yeast, or a combination thereof. Examples of suitable probiotic yeast (S.) include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces paradoxica, Saccharomyces pastorianus, Saccharomyces bayanus, or a combination thereof. Specifically, the probiotic yeast may include, but are not limited to, Saccharomyces boulardii CNCM I-745, Saccharomyces cerevisiae CNCM I-3856, Saccharomyces boulardii T1, Saccharomyces boulardii 17, Saccharomyces boulardii CECT 1474, or a combination thereof. Examples of suitable probiotic non-yeast yeast include, but are not limited to, Pichia pastoris (P.), Kluyveromyces (K.), Hansenula spores (H.), Candida (C.), Zygosaccharomyces (Z.), or a combination thereof.

According to a specific aspect, when the first yeast is a probiotic yeast, the second yeast may be the same as the first yeast.

The second yeast may be in any suitable form. For example, the second yeast may include freeze-dried probiotic yeast, pre-cultured probiotic yeast, or a combination thereof.

The adding of the second yeast to the alcoholic beverage may include adding the second yeast in any suitable amount. The second yeast may be added in the same or different amount as the first yeast. For example, the second yeast may be added so that the cell count of the second yeast is ≥ 4 log CFU/mL. Specifically, the amount of the second yeast added may be 4-11 log CFU/mL, 5-10 log CFU/mL, 6-9 log CFU/mL, 7-8 log CFU/mL. Even more specifically, the amount of the second yeast added may be 5-7 log CFU/mL.

The method may further include adding a second wort or unfermented juice to the alcoholic beverage. Specifically, the adding of the second wort or unfermented juice to the alcoholic beverage may be performed simultaneously with the adding of the second yeast, or after the adding of the second yeast. The second wort or unfermented juice may include adding any suitable wort or unfermented juice. For example, the second wort or unfermented juice may be related to the first wort or unfermented juice, as described above.

The wort or unfermented fruit juice may be added in any suitable amount. For example, the amount of wort or unfermented fruit juice added may be 3-20% v/v based on the total volume of the alcoholic beverage. Specifically, the amount of wort or unfermented fruit juice added may be 5-15% v/v, 7-12% v/v, 8-10% v/v. Even more specifically, the amount of wort or unfermented fruit juice added may be 5-10% v/v based on the total volume of the alcoholic beverage.

The method may further include fermenting the second wort or unfermented juice at a second fermentation predetermined time period and a second fermentation predetermined temperature. The second fermentation predetermined time period may be any suitable time period. For example, the second fermentation predetermined time period may be ≤10 days. Specifically, the second fermentation predetermined time period may be 1-10 days, 2-9 days, 3-8 days, 4-7 days, 5-6 days. Even more specifically, the second fermentation predetermined time period may be 2-5 days.

The second predetermined fermentation temperature may be any suitable temperature. According to a specific aspect, the second predetermined fermentation temperature may be ≤35°C. Specifically, the temperature may be 20-30°C.

The method further comprises packaging the alcoholic beverage based on probiotic yeast after the second yeast is added. The packaging may be performed by any suitable method. For example, the packaging may include packaging the alcoholic beverage based on probiotic yeast into a suitable storage container, such as but not limited to bottles, cans, kegs, etc. The packaging may further include carbonating the alcoholic beverage based on probiotic yeast during the packaging. The amount of carbon dioxide added for carbonation may depend on the amount of the second yeast added and/or the second wort or unfermented juice.

According to a specific aspect, at the time of packaging, the alcoholic beverage based on probiotic yeast may not be fully formed. Specifically, the alcoholic beverage based on probiotic yeast may be formed after the packaging and after the second fermentation predetermined time period.

According to one embodiment, the method includes adding a first yeast to wort or unfermented juice, wherein the first yeast is a probiotic yeast. The first yeast may be any suitable probiotic yeast as described above. The wort or unfermented juice may be hopped or hopless. Then, fermentation is allowed to proceed at a predetermined time period and a predetermined temperature of the first fermentation to form an alcoholic beverage. Once the fermentation is complete (indicated by a stable Brix), the precipitate of the first yeast can be removed by, for example, rapid cooling. Then, the alcoholic beverage may be inoculated with a second yeast, wherein the second yeast is a probiotic yeast. Subsequently, the second wort or unfermented juice may be added. The alcoholic beverage with the second yeast may optionally be subjected to carbonation. Then, the alcoholic beverage may be packaged and stored at a temperature of ≤35°C, particularly at a temperature of 20-30°C. Then, a secondary fermentation may be carried out in the packaging to form the alcoholic beverage based on the probiotic yeast. The formed probiotic yeast-containing alcoholic beverage may maintain a probiotic cell count of ≥5 log CFU/mL even after storage at 30°C for 100 days.

According to a second embodiment, the method includes adding a first yeast to wort or unfermented juice, wherein the first yeast is a non-probiotic yeast. The first yeast may be any suitable non-probiotic yeast as described above. The wort or unfermented juice may be hopped or hopless. Then, fermentation is carried out at a predetermined time period and a predetermined temperature of the first fermentation to form an alcoholic beverage. Once the fermentation is complete (indicated by a stable Brix), the precipitate of the first yeast can be removed by, for example, centrifugation and pasteurization. Then, the alcoholic beverage may be inoculated with a second yeast without adding wort or juice, wherein the second yeast is a probiotic yeast to form an alcoholic beverage based on probiotic yeast. The alcoholic beverage with the second yeast may optionally be subjected to carbonation. Then, the alcoholic beverage may be packaged and stored at a temperature of ≤35°C, particularly at a temperature of 20-30°C. No secondary fermentation occurs in the packaging. The formed probiotic yeast-containing alcoholic beverage may maintain a probiotic cell count of ≥ 6 log CFU/mL even after storage at 30°C for 4 months.

According to a third embodiment, the method includes adding a first yeast to wort or unfermented juice, wherein the first yeast is a non-probiotic yeast. The first yeast may be any suitable non-probiotic yeast as described above. The wort or unfermented juice may be hopped or hopless. Then, fermentation is carried out at a predetermined time period and a predetermined temperature of the first fermentation to form an alcoholic beverage. Once the fermentation is complete (indicated by a stable Brix), the precipitate of the first yeast can be removed by, for example, centrifugation and pasteurization. Then, the alcoholic beverage may be inoculated with a second yeast, wherein the second yeast is a probiotic yeast. Subsequently, the second wort or unfermented juice may be added. The alcoholic beverage with the second yeast may optionally be subjected to carbonation. Then, the alcoholic beverage may be packaged and stored at a temperature of ≤35°C, particularly at a temperature of 20-30°C. Then, a secondary fermentation may be carried out in the packaging to form the alcoholic beverage based on the probiotic yeast. The formed probiotic yeast-based alcoholic beverage may still maintain a probiotic cell count of ≥5.5 log CFU/mL after being stored at 30°C for 3 months.

According to a second aspect, a probiotic yeast-based alcoholic beverage is provided, comprising probiotic yeast, wherein the probiotic yeast has a cell count of ≥5 log CFU/mL after storage for 100 days at 30°C. The probiotic yeast-based alcoholic beverage may have a suitable alcohol content. According to a specific aspect, the alcohol content of the probiotic yeast-based alcoholic beverage may be ≥0.5% (volume percentage). For example, the alcohol content may be 0.5-10%, 1.0-9.0%, 1.5-8.0%, 2.0-7.5%, 2.5-7.0%, 3.0-6.5%, 3.5-6.0%, 4.0-5.5%, 4.5-5.0%. Specifically, the alcohol content may be 2.0-5.0%. Even more specifically, the alcohol content may be approximately 4-6%.

The alcoholic beverage based on probiotic yeast may be any suitable alcoholic beverage. Examples of alcoholic beverages may include, but are not limited to, beer, wine, sparkling wine, rice wine, cider, spirits, fermented water, strong wine, mead, tequila, etc. According to a specific aspect, the alcoholic beverage based on probiotic yeast may be beer.

The probiotic yeast contained in the alcoholic beverage based on probiotic yeast may have a cell count of 5-11logCFU/mL, 5.5-10log CFU/mL, 6-9log CFU/mL, 7-8log CFU/mL. Even more specifically, the probiotic yeast contained in the alcoholic beverage based on probiotic yeast may have a cell count of 5-9log CFU/mL. Further, after storage for 100 days at 30°C, the probiotic yeast contained in the alcoholic beverage based on probiotic yeast may have a cell count of 5-7log CFU/mL. Considering that the beverage is not stored under refrigerated conditions, but under ambient conditions, this is relatively high. This is unexpected because it is well known that in order to maintain an appropriate amount of probiotic cell counts, thereby bringing health benefits, beverages (especially alcoholic beverages) must be stored at a refrigerated temperature of about 0-5°C. In the alcoholic beverage according to the second aspect, a high content of probiotic bacteria may be obtained even if the alcoholic beverage is not stored at a refrigerated temperature but at a normal ambient temperature. The alcoholic beverage based on probiotic yeast may be stored at a suitable temperature to maintain the probiotic yeast at a suitable level. For example, the alcoholic beverage based on probiotic yeast may be stored at a temperature of about ≤35°C. Specifically, the alcoholic beverage based on probiotic yeast may be stored at a temperature of about 15-35°C, 20-30°C, 25-28°C. Even more specifically, the alcoholic beverage based on probiotic yeast may be stored at a temperature of about 20-30°C.

The probiotic yeast contained in the probiotic yeast-based alcoholic beverage may be any suitable probiotic yeast. For example, the probiotic yeast may be related to the second yeast, as described above. Specifically, the probiotic yeast may include probiotic Saccharomyces yeast, probiotic non-Saccharomyces yeast, or a combination thereof.

According to a particular aspect, the alcoholic beverage based on probiotic yeast may further include hops or a derivative thereof. The hops or a derivative thereof may be any suitable hop. In particular, the hops or a derivative thereof may be related to the first aspect, as described above. The hops or a derivative thereof may have a suitable bitterness. In particular, the hops or a derivative thereof may have a bitterness of ≤80 IBU. Even more particularly, the hops or a derivative thereof may have a bitterness of 5-30 IBU.

The probiotic yeast-containing alcoholic beverage may be formed by the method of the first aspect.

The alcoholic beverage based on probiotic yeast may have a suitable pH. For example, the pH of the alcoholic beverage based on probiotic yeast may be ≥ 3.8. Specifically, the pH may be 3.8-6.5, 4-6, 5-5.5. Even more specifically, the pH may be about 4-6.

The alcoholic beverage based on probiotic yeast may have a suitable Brix or its specific gravity equivalent. Brix is a unit of measurement for the amount of sugar in the alcoholic beverage based on probiotic yeast. For example, 1°Bx means that 100g of the alcoholic beverage based on probiotic yeast contains 1g of sucrose. Accordingly, the higher the Brix, the higher the alcohol content of the alcoholic beverage based on probiotic yeast may be. The Brix of the alcoholic beverage based on probiotic yeast may be 2-15°Bx. For the purposes of the present invention, the Brix of the alcoholic beverage refers to the measured value of the Brix of the wort or unfermented fruit juice contained in the alcoholic beverage. Specifically, the Brix of the alcoholic beverage based on probiotic yeast may be 2-15°Bx, 3-12°Bx, 4-10°Bx, 5-8°Bx, 6-7°Bx. Even more specifically, the Brix may be about 5-10° Bx.

Having summarized the present invention, the present invention will be more readily understood by reference to the following examples. The following examples are provided by way of illustration and are not intended to limit the present invention.

Example

All materials used in the examples provided are as follows:

Material source Saccharomyces cerevisiae SafAle WB-06 Lesaffre, Lille, France Saccharomyces cerevisiae SafAle S-04 Lesaffre, Lille, France Saccharomyces cerevisiae CNCM I-3856 Lesaffre, Lille, France Saccharomyces cerevisiae CNCM I-745 Biocodex, Gentilly, France

The wort is prepared by mashing the malt, adding 70-90% of drinking water, heating until the mixture boils and boiling for 60 minutes, and then cooling the mixture to room temperature. The wort or unfermented juice may include, but is not limited to, barley, oats, wheat, corn, rye, rice, water, and may also include different forms of sugars (e.g., fruit juice and syrup).

When preparing the hopless wort as used in Examples 1 to 3, no hops or derivatives thereof were added. However, during the boil of the wort mixture for preparing the hopped wort as used in Example 2, hops Cascade and hop extract (IBU 18) were added.

Example 1 - Initial and sequential inoculation of probiotic yeast for fermentation

method

Use the first yeast as probiotic yeast for primary fermentation (main fermentation)

The first probiotic yeast Saccharomyces cerevisiae CNCM I-3856 was inoculated into a sterile flask (containing the pasteurized hop-free wort prepared as above) to achieve a minimum cell count of 5.0 Log CFU/mL. The fermentation vessel was covered with an airlock and stored at 20°C for 7-21 days for the primary fermentation. After the primary fermentation, a portion of the probiotic-free beer was taken as a control group for shelf life testing.

Sequential inoculation of probiotic yeast

After the first probiotic yeast was fermented, most of the probiotic yeast precipitates were removed by rapid cooling (0-4°C, 24 hours). The pasteurized hop-free wort (5% v/v) was added to the clarified beer, and then the second probiotic yeast was inoculated in sequence, specifically freeze-dried saccharomyces cerevisiae CNCM I-3856 (minimum cell count was 5.0Log CFU/mL) (FS group) and primary cultured saccharomyces cerevisiae CNCM I-3856 (minimum cell count was 5.0Log CFU/mL) (PS group). The second probiotic yeast was the same as the first probiotic yeast used.

Packaging and Shelf Life Testing

Beer samples were immediately dispensed into 15 mL centrifuge tubes (12 mL/tube), without carbonation, and stored at 30 ° C for more than 3 months to study stability and yeast survival. Three replicates of each treatment were taken every 5-30 days to monitor probiotic cell counts and physicochemical parameters. Yeast cell counts were monitored for 2 days at 30 ° C using the diffusion plate method on potato dextrose agar (PDA) (Oxoid, Basingstoke, Hampshire, UK). The pH and Brix of the samples were measured using a pH meter (Metrohm, Switzerland) and a refractometer (ATAGO, Tokyo, Japan), respectively.

The experiment was repeated and the beer samples were packaged into glass bottles (330 mL/bottle) for sensory evaluation. After 4 months of storage at 30°C, the beer samples containing probiotic yeast were scored by eight trained panelists using a 5-point scale quantitative descriptive analysis based on five characteristics: sweetness, alcohol content, fruity and off-flavors (1-lowest intensity, 5-highest intensity) and appearance (1-lowest acceptance, 5-highest acceptance).

result

Probiotic Yeast Quantity

Survival of Saccharomyces cerevisiae CNCM I-3856 in beer without hops during storage at 30°C is shown in Figure 1. In the control group, the cell count of Saccharomyces cerevisiae CNCM I-3856 was able to reach 7.62Log CFU/mL after the first fermentation, while the cell count dropped to about 6Log CFU/mL in the first 10 days of storage at 30°C, and remained at about 5Log CFU/mL after 3 months. In the FS and PS groups, the cell count of Saccharomyces cerevisiae CNCM I-3856 dropped to 6.2-6.3Log CFU/mL after rapid cold drop, which recovered to 6.8Log CFU/mL after the second fermentation (mainly in the first 4 days of storage). As shown in Figure 1, the probiotic yeast cell count of the FS group remained stable from the second to the fourth month of storage, greater than 6Log CFU/mL, while the yeast cell count of the PS group dropped to about 5Log CFU/mL in the first two months of storage at 30°C. These results indicate that sequential inoculation of freeze-dried probiotic yeasts is beneficial for the viability of the probiotics during storage at 30°C.

Brix and pH

The Brix of the beer after the primary fermentation was about 7.1, which was stable within the range of 7.1-7.2 of the control group, as shown in Figure 2. In the FS and PS groups, the Brix dropped to 6.7 and 6.5, respectively, in the first four days of storage, and finally dropped to 6.5 and 6.4, respectively, after three months. This indicates that most of the sugars in the added wort were consumed in the first four days of storage. In addition, the sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856 utilized less sugars during the secondary fermentation than the primary cultured yeast.

The initial pH of the beer without hops fermented with Saccharomyces cerevisiae CNCM I-3856 was about 4.7, as shown in Figure 3. During storage for 4 months at 30°C, the pH gradually decreased by about 0.3 units, while the pH of the PS group only decreased by about 0.1 units. This indicates that the sequential inoculation of the primary culture of Saccharomyces cerevisiae CNCM I-3856 contributes to a stable pH.

Sensory evaluation

Sensory evaluation and observation of beer based on probiotic yeast were conducted. The results are shown in Figure 4.

After 4 months of storage at 30°C, the appearance of the probiotic beer in the control group became very turbid and was not accepted by all panelists. The appearance of the probiotic beer in the FS and PS groups was more attractive and had a higher acceptability level. Off-flavors, such as fusel oil and herbal flavors, were clearly detected in the probiotic beer in the control group. Sequential inoculation of probiotic yeast could significantly reduce the intensity of off-flavors (<2), especially in the FS group (1.43). The alcohol intensity score of the beer based on probiotic yeast in the FS group (2.57) was lower than that of other beers (>3.4). All the beers had very similar scores in fruity intensity (2.0-2.3), followed by sweet intensity (2.0-2.8). This result indicates that sequential inoculation of probiotic yeast effectively prevents the development of turbidity and off-flavors in beer during storage.

This result shows that probiotic yeasts are unstable when stored at ambient temperature as primary fermentation cultures. These disadvantages can be mitigated by sequential inoculation of freeze-dried Saccharomyces cerevisiae CNCM I-3856, where the probiotic cell count can be reduced by only 0.8 Log CFU/mL after 3 months of storage at 30°C, remaining greater than 6 Log CFU/mL. The organoleptic quality is also improved.

Example 2 - Sequential inoculation of probiotic yeast without fermentation

method

Use the first yeast as non-probiotic yeast for the first fermentation

Hopless beer and hopped wort were prepared and inoculated with wheat beer yeast, Saccharomyces cerevisiae SafAle WB-06. The beer was centrifuged and pasteurized at 60-65°C for 15-20 minutes to remove yeast sediment and kill most microorganisms in the beer.

Sequential inoculation of probiotic yeast

In addition to Saccharomyces cerevisiae CNCM I-3856, another probiotic yeast Saccharomyces boulardii CNCM I-745 was used in this example, which had not been used in probiotic beer fermentation before. Freeze-dried probiotic yeast Saccharomyces boulardii CNCM I-745 or freeze-dried Saccharomyces cerevisiae CNCM I-3856 were inoculated in pasteurized hopless and/or hopped beer with a minimum added cell count of 6.0 Log CFU/mL, without the addition of wort. The beer samples were packaged and carbonated, and then analyzed in Example 1.

result

Probiotic cell count

The changes in the cell counts of the probiotic yeasts Saccharomyces boulardii CNCM I-745 and Saccharomyces cerevisiae CNCM I-3856 during storage at 30°C in beer without hops and with hops are shown in FIG5 . The cell counts of Saccharomyces boulardii CNCM I-745 in beer without hops and with hops during storage had similar trends, which rapidly decreased by about 0.7 Log CFU/mL in the second month of storage. The cell counts of Saccharomyces boulardii CNCM I-745 in beer without hops remained greater than 5.6 Log CFU/mL during the 4-month storage period, while the probiotic yeast cell counts in beer with hops remained greater than 6.1 Log CFU/mL during the 100-day storage period. The cell counts of Saccharomyces cerevisiae CNCM I-3856 in beer without hops decreased from greater than 7.5 Log CFU/mL to about 6.5 Log CFU/mL in the first month of storage, and then stabilized at 6.3 Log CFU/mL during the next 3 months of storage, as shown in FIG5 . These results showed that the cell counts of different probiotic yeasts had similar trends in different beer samples, mainly decreasing in the early and middle stages of storage at 30 °C.

Brix and pH

There was no significant change in the Brix of the beer with probiotic yeast added during storage at 30°C for 4 months, as can be seen from Figure 6, which indicates that the residual sugar in the beer was not utilized by the probiotic yeast.

The addition of probiotic Saccharomyces cerevisiae CNCM I-3856 slightly increased the pH of the beer from about 4.7 to 4.9 during storage, and the pH of the other probiotic beer samples remained stable at 30°C, as shown in FIG7 .

Sensory evaluation

The sensory evaluation results and appearance of the probiotic beer were evaluated, and the sensory evaluation results are shown in Figure 8. After storage at 30°C for 4 months, the appearance of the probiotic hopless beer containing Saccharomyces boulardii CNCM I-745 remained clear and was accepted by most panelists. This was followed by the probiotic hopped beer with the same probiotic yeast strain. However, the probiotic hopped beer containing Saccharomyces cerevisiae CNCM I-3856 was too turbid and was not accepted by all panelists, which may be related to the higher addition of Saccharomyces cerevisiae CNCM I-3856. Compared with Saccharomyces boulardii CNCM I-745, the probiotic beer with the addition of Saccharomyces cerevisiae CNCM I-3856 had lower alcohol flavor (2.17) and off-flavor (1.33) scores.

All beers had very similar sweetness scores (2.1-2.5), followed by fruitiness (2.0-2.7). This result suggests that the addition of more probiotic yeast can not only increase the production cost of beer, but also affect the appearance of beer during storage. Therefore, a method of reducing the addition of probiotic yeast was explored in Example 3 (see below).

Example 3 - Sequential inoculation of probiotic yeast for secondary fermentation

method

Use the first yeast as non-probiotic yeast for the first fermentation

Beer was prepared without hops and inoculated with wheat beer yeast, Saccharomyces cerevisiae SafAle WB-06. The beer was centrifuged and pasteurized at 60-65°C for 15-20 minutes to remove yeast sediment and kill most microorganisms in the beer.

Sequential fermentation of probiotic yeast

After the first fermentation with the non-probiotic yeast, the centrifuged and pasteurized beer without hops was inoculated with pre-cultured probiotic yeast Saccharomyces cerevisiae CNCM I-3856 (with a minimum cell count of 5.0 Log CFU/mL), and 5% v/v and 10% v/v of hop-free wort were added, respectively. Pure cultures were prepared with freeze-dried Saccharomyces cerevisiae CNCM I-3856, which were expanded at least 100 times. The beer samples were packaged and tested for shelf life using the same method as in Example 1. The probiotic beer samples were scored by eight trained panelists using a 5-point scale quantitative descriptive analysis, as described in Example 1.

In addition, 6 probiotic hopless beer samples and 2 probiotic hopped beer samples from Examples 1 to 3 stored at 30° C. for 120 days were subjected to sensory evaluation by 8 experienced panelists using a preference ranking test (hopless beer: 1-least favorite, 6-most favorite; hopped beer: 1-least favorite, 2-more favorite).

Volatile compound analysis

The effects of probiotic yeast on the volatile aroma compounds of probiotic beer samples were analyzed. The probiotic beer samples included probiotic beer fermented with probiotic yeast for primary fermentation and sequential fermentation (such as Example 1), probiotic beer not fermented with probiotic yeast (such as Example 2) and probiotic beer fermented only with probiotic yeast for sequential fermentation (such as this Example 3).

Before analysis, 5mL beer samples (pH adjusted to 2.5 with 1M HCl) were transferred to screw cap headspace vials. Volatile compounds were extracted by headspace (HS) solid phase microextraction (SPME) and carboxyl/polydimethylsiloxane fiber (85 μm coating, Supelco, Sigma-Aldrich, Barcelona, Spain) and analyzed using gas chromatography (GC)-mass spectrometry (MS)-flame ionization detector (FID). Sample extraction was performed at 60 ° C using SPME automatic sampler (CTC, Combi Pal, Switzerland) under 250rpm stirring for 40 minutes. GC was connected to Agilent 5975C triple axis MS and FID for identification and quantification of volatiles (Chen et al., 2015, International Journal of Food Microbiology, 206: 45-50). These volatiles were identified by comparing their mass spectra with NIS 8.0 and Wiley275MS libraries. Based on its GC-FID peak area, semi-quantitative analysis was performed.

result

Probiotic cell count

The changes of probiotic Saccharomyces cerevisiae CNCM I-3856 in beer without hops during storage at 30°C are shown in Figure 9. Due to secondary fermentation, the cell count of Saccharomyces cerevisiae CNCM I-3856 increased rapidly from about 5.5LogCFU/mL to about 7.0LogCFU/mL in the first 5 days of storage. Then, the probiotic yeast cell count gradually decreased to about 5.5LogCFU/mL after 2 months of storage, and remained stable in the third month of storage, as can be seen from Figure 9. This shows that the addition of 5% v/v and 10% v/v wort has no significant effect on the change of yeast cell count during storage, and therefore 5% v/v wort addition can be used to reduce the production of _CO2 during storage.

Brix and pH

The Brix of beer fermented with non-probiotic yeast is about 6.1, which is much lower than the beer fermented with Saccharomyces cerevisiae CNCM I-3856 (Fig. 2 and Fig. 10). This result shows that the decay rate of probiotic yeast is lower than that of conventional Saccharomyces cerevisiae. After adding 5% v/v wort and 10% v/v wort respectively, the Brix of beer increases to 6.3 and 6.7 respectively, which is consumed to 5.7 after the first 5 days of storage, as shown in Figure 10. This shows that secondary fermentation with Saccharomyces cerevisiae CNCM I-3856 is mainly completed in the first 5 days of bottling. Then, the beer sample without hops is almost stable during storage at 30 ℃. As can be seen from Figure 11, the pH of the beer without hops containing Saccharomyces cerevisiae CNCM I-3856 after storage for 3-4 months at 30 ℃ drops from 4.3 to 4.1.

Sensory evaluation and volatile compound analysis

The sensory evaluation results and appearance of the probiotic hopless beer are shown in Figure 12. The appearance of the probiotic hopless beer remained clear after storage at 30°C for 4 months and was accepted by most panelists. The addition of different levels of wort had no significant effect on the appearance, alcohol flavor, sweetness and off-flavor of the probiotic beer after storage. However, the addition of 10% v/v wort for secondary fermentation to the probiotic beer helped to increase the fruity aroma. This result shows that the sequential inoculation of probiotic yeast and 5%-10% v/v wort for secondary fermentation in the bottle can maintain the appearance of the beer and the beer has similar sensory quality.

The preference ranking scores based on the probiotic yeast-containing beers after 4 months of storage are shown in Figure 13. The samples of probiotic hop-free beers are as follows:

(A) Primary fermentation with probiotic yeast and no sequential inoculation;

(B) Primary fermentation with probiotic yeast followed by sequential inoculation with freeze-dried Saccharomyces cerevisiae CNCM I-3856 + 5% v/v malt extract;

(C) Primary fermentation with probiotic yeast, followed by sequential inoculation with primary culture of Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort;

(D) Primary fermentation with non-probiotic yeast followed by sequential inoculation with freeze-dried Saccharomyces boulardii CNCM I-745 without addition of wort;

(E) primary fermentation with non-probiotic yeast followed by sequential inoculation with pre-cultured Saccharomyces cerevisiae CNCM I-3856 + 5% v/v wort; and

(F) Primary fermentation with non-probiotic yeast followed by sequential inoculation with pre-cultured Saccharomyces cerevisiae CNCM I-3856 + 10% v/v wort.

All panelists dislike probiotic beer sample A the most, which means that carrying out primary fermentation with probiotic yeast is not suitable for the probiotic beer stored at normal ambient temperature (Figure 14 A). After carrying out primary fermentation with probiotics, the sequential inoculation of probiotic yeast can effectively improve the ranking score of probiotic beer (sample B and sample C). Compared with probiotic yeast, the probiotic beer that carries out primary fermentation with conventional saccharomyces cerevisiae (samples D-F) is more liked by panelists. In addition, compared with the probiotic yeast (sample D, Figure 14 B) that is not fermented, the sequential inoculation probiotic yeast is used for secondary fermentation, which may help to improve the sensory quality of beer (sample E & F).

Figures 14A to 14D show the main volatile aroma compounds of different probiotic yeast-based beer samples A to F after storage for 4 months. After the sequential inoculation of probiotic yeast (samples B-F, Figure 14A), the content of ethanol and 2,4-di-tert-butylphenol and other volatiles in the alcohol group (including phenylethanol and isobutanol, Figures 14B and 14C) increased significantly. Phenylethanol (sweet, rose, floral) and isobutanol (fruity, red wine-like aroma) increase the complexity of beer flavor. Compared with beer fermented with probiotic yeast alone, probiotic beer fermented for the first time with conventional brewing yeast (samples D-F) produced a higher amount of main volatile compounds in the alcohol group.

Probiotic yeasts used for primary and sequential fermentations (samples B & C) contributed higher amounts of important acetates such as phenylethyl acetate (floral, rose, sweet) and isoamyl acetate (fruity, banana, sweet) than other inoculation methods (Figure 14C).

The content of ethyl esters (mainly C ₆ -C ₁₀ ethyl esters) in sample A is significantly higher than that in other beer samples ( FIG. 14D ). These ethyl esters can give beer a fruity flavor, and excessive production of ethyl esters can mask other volatiles and cause off-flavors (as shown in the results of FIG. 4 ). This may explain why probiotic beer sample A was the least popular in the sensory evaluation ( FIG. 13A ). This result also shows that probiotic beer that was initially fermented only with probiotic yeast is not suitable for storage at room temperature.

While the foregoing description has described exemplary embodiments, those skilled in the art will appreciate that many variations are possible without departing from the invention.

Claims (21)

1. A method of forming an alcoholic beverage based on probiotic yeast, the method comprising: - Provide wort or unfermented juice; - adding a first yeast to the wort or unfermented juice; - fermenting the wort or unfermented juice for a first fermentation predetermined time period and a first fermentation predetermined temperature to form an alcoholic beverage; and - adding a second yeast to the alcoholic beverage to form a probiotic yeast based alcoholic beverage, wherein the second yeast is a probiotic yeast. 2. The method according to claim 1, characterized in that the first yeast is probiotic yeast. 3 . The method according to claim 1 , wherein the first yeast is the same as the second yeast. 4. The method according to claim 1, characterized in that the first yeast is non-probiotic yeast. 5. The method according to claim 4, characterized in that the first yeast is: non-probiotic Saccharomyces yeast, non-probiotic non-Saccharomyces yeast, or a combination thereof. 6. The method according to any one of the above claims, characterized in that the second yeast is a probiotic Saccharomyces yeast, a probiotic non-Saccharomyces yeast, or a combination thereof. 7. The method according to any one of the preceding claims, wherein the first yeast and the second yeast each have a cell count of ≥ 4 log CFU/mL. 8. The method according to any one of the preceding claims, characterized in that the second yeast comprises freeze-dried probiotic yeast, pre-cultured probiotic yeast, or a combination thereof. 9. The method according to any one of the preceding claims, characterized in that the method further comprises adding a first batch of hops or derivatives thereof to the wort or unfermented juice. 10. The method of claim 9, wherein the first hops have a bitterness of ≤ 80 IBU. 11. The method according to any one of the preceding claims, further comprising removing sediment from the first yeast before adding the second yeast. 12. The method according to any one of the preceding claims, characterized in that the predetermined temperature of the first fermentation is 10-35°C. 13. The method according to any one of the preceding claims, further comprising adding a second amount of wort or unfermented juice to the alcoholic beverage. 14. The method according to claim 13, characterized in that the adding of the second wort or unfermented juice to the alcoholic beverage is performed simultaneously with the adding of the second yeast, or is performed after the adding of the second yeast. 15 . The method according to claim 13 or 14 , characterized in that the method further comprises fermenting the second wort or unfermented juice at a second predetermined fermentation time period and a second predetermined fermentation temperature. 16. The method according to claim 15, characterized in that the second fermentation predetermined temperature is 20-30°C. 17. A probiotic yeast-based alcoholic beverage comprising probiotic yeast, wherein the probiotic yeast has a cell count of ≥ 5 log CFU/mL after storage at 30°C for 100 days. 18. The probiotic yeast-based alcoholic beverage according to claim 17, wherein the probiotic yeast comprises probiotic Saccharomyces yeast, probiotic non-Saccharomyces yeast, or a combination thereof. 19. The alcoholic beverage based on probiotic yeast according to claim 17 or 18, characterized in that the alcoholic beverage based on probiotic yeast further comprises hops or a derivative thereof. 20. The alcoholic beverage based on probiotic yeast according to claim 19, characterized in that the hops or derivatives thereof have a bitterness of ≤80 IBU. 21. An alcoholic beverage based on probiotic yeast according to any one of claims 17 to 20, characterised in that it is formed by the method of claims 1 to 16.