WO2023163661A2

WO2023163661A2 - A yeast-based probiotic alcoholic beverage and a method of preparing the same

Info

Publication number: WO2023163661A2
Application number: PCT/SG2023/050114
Authority: WO
Inventors: Shao Quan Liu; Dai CHEN; Mingzhan TOH
Original assignee: National University Of Singapore
Priority date: 2022-02-24
Filing date: 2023-02-24
Publication date: 2023-08-31
Also published as: WO2023163661A3

Abstract

A yeast-based probiotic alcoholic beverage and a method of preparing the same There is provided a yeast-based probiotic alcoholic beverage comprising probiotic yeast wherein the probiotic yeast has a cell count of ≥log CFU/mL after 100 days of storage at 30C. There is also provided a method of forming the yeast-based probiotic alcoholic beverage.

Description

A yeast-based probiotic alcoholic beverage and a method of preparing the same

Technical Field

The present invention relates to a yeast-based probiotic alcoholic beverage. In particular, the yeast-based probiotic alcoholic beverage may comprise probiotic yeast.

Background

There has been an increase in interest in the consumption of probiotics in view of the health benefits one can derive from them. At present, many food and beverages comprise probiotics.

Most of the food and beverages which comprise probiotics are stored at a cool temperature. There are some dairy-based and plant-based probiotic food and beverages which are ambient-stable, however, it has not been possible to store alcoholic beverages comprising probiotics under ambient conditions while maintaining survival of the probiotics in the alcoholic beverage.

Summary of the invention

The present invention seeks to address these problems, and/or provides an improved alcoholic beverage comprising probiotic yeast and a method of preparing the same 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 of forming a yeast-based probiotic alcoholic beverage, the method comprising: providing a wort or must; adding a first yeast to the wort or must; fermenting the wort or must at a pre-determined period of time and at a pre-determined temperature to form an alcoholic beverage; and adding a second yeast to the alcoholic beverage to form a yeast-based probiotic 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 particular aspect, the first yeast may be a probiotic yeast. The first yeast may be the same as the second yeast.

According to another particular aspect, the first yeast may be a non-probiotic yeast. In particular, the first yeast may comprise: non-probiotic Saccharomyces yeast, non- probiotic non- Saccharomyces yeast, or a combination thereof.

The second yeast may be any suitable probiotic yeast. For example, the second yeast may comprise: 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 comprise 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 comprise adding a first hop or its derivative to the wort or must. The first hop or its derivative added to the wort or must may be any suitable hop. The first hop may have a suitable bitterness. In particular, the first hop or its derivative may have a bitterness of < 80 IBU.

According to a particular aspect, the method may further comprise removing sediments of the first yeast prior to the adding a second yeast. The removing sediments of the first yeast may be by any suitable method.

The fermenting the wort or must may be carried out under any suitable conditions. For example, the first pre-determined temperature may be any suitable temperature. According to a particular aspect, the first pre-determined temperature may be 10-35°C.

The method may further comprise adding a second wort or must to the alcoholic beverage. In particular, the adding a second wort or must to the alcoholic is carried out simultaneously with or following the adding a second yeast.

The method may further comprise fermenting the second wort or must at a second predetermined period of time and at a second pre-determined temperature. The second pre- determined temperature may be any suitable temperature. According to a particular aspect, the second pre-determined temperature may be 20-30°C.

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

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

According to a particular aspect, the yeast-based probiotic alcoholic beverage may further comprise a hop or derivative thereof. The hop or derivative thereof may be any suitable hop. The hop may have a suitable bitterness. In particular, the hop or derivative thereof may have a bitterness of < 80 IBU.

The yeast-based probiotic alcoholic beverage may be formed from the method of the first aspect.

Brief Description of the Drawings

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments, the description being with reference to the accompanying illustrative drawings. In the drawings:

Figure 1 shows survival of probiotic S. cerevisiae CNCM I-3856 in unhopped beer during storage at 30°C, in which (•) represents no sequential inoculation (control group); (▼) represents sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 +5% v/v wort (FS group); (■) represents sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 +5% v/v wort (PS group);

Figure 2 shows changes in °Brix of unhopped beer fermented with probiotic S. cerevisiae CNCM I-3856 during storage at 30°C in which (•) represents no sequential inoculation (control group), (▼) represents sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 +5% v/v wort (FS group), and (■) represents sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 +5% v/v wort (PS group); Figure 3 shows changes in pH of unhopped beer fermented with probiotic S. cerevisiae CNCM I-3856 during storage at 30°C in which (•) represents no sequential inoculation (control group), (▼) represents sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 +5% v/v wort (FS group), and (■) represents sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 +5% v/v wort (PS group);

Figure 4 shows the sensory profile plot of mean attribute values on a 1 to 5-point scale of probiotic unhopped beer samples. (O) represents no sequential inoculation (control group); (•) represents sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 +5% v/v wort (FS group); and (■) represents sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 +5% v/v wort (PS group);

Figure 5 shows survival of probiotics in beer during storage at 30°C in which (A) represents S. boulardii CNCM I-745 in unhopped beer; (▼) represents S. boulardii CNCM I-745 in hopped beer; and (•) represents S. cerevisiae CNCM I-3856 in hopped beer;

Figure 6 shows changes of °Brix of beer during storage at 30°C in which (A) represents S. boulardii CNCM I-745 in unhopped beer, (▼) represents S. boulardii CNCM I-745 in hopped beer and (•) represents S. cerevisiae CNCM I-3856 in hopped beer;

Figure 7 shows changes of pH of beer during storage at 30°C in which (A) represents S. boulardii CNCM I-745 in unhopped beer, (▼) represents S. boulardii CNCM I-745 in hopped beer and (•) represents S. cerevisiae CNCM I-3856 in hopped beer;

Figure 8 shows sensory profile plot of mean attribute values on a 1 to 5-point scale of probiotic beer samples in which (A) represents S. boulardii CNCM I-745 in unhopped beer; (A) represents S. boulardii CNCM I-745 in hopped beer; and (•) represents S. cerevisiae CNCM I-3856 in hopped beer;

Figure 9 shows survival of probiotics in unhopped beer during storage at 30°C in which (•) represents sequential inoculation of S. cerevisiae CNCM I-3856 +5% v/v wort; and (▼) represents sequential inoculation of S. cerevisiae CNCM I-3856 +10% v/v wort; Figure 10 shows changes of °Brix of unhopped beer during storage at 30°C in which (•) represents sequential inoculation of S. cerevisiae CNCM I-3856 +5% v/v wort; and (▼) represents sequential inoculation of S. cerevisiae CNCM I-3856 +10% v/v wort;

Figure 11 shows changes of pH of unhopped beer during storage at 30°C in which (•) represents sequential inoculation of S. cerevisiae CNCM I-3856 +5% v/v wort; and (▼) represents sequential inoculation of S. cerevisiae CNCM I-3856 +10% v/v wort;

Figure 12 shows sensory profile plot of mean attribute values of probiotic unhopped beer on a 1 to 5-point scale in which (A) represents sequential inoculation of S. cerevisiae CNCM I-3856 +5% v/v wort and (•) represents sequential inoculation of S. cerevisiae CNCM I-3856 +10% v/v wort;

Figure 13A shows preference ranking scores of yeast-based probiotic unhopped beer and Figure 13B shows preference ranking scores of yeast-based probiotic hopped beer stored for 120 days at 30°C; and

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

Detailed Description

As explained above, there is a need for and improved alcoholic beverage comprising probiotics which is stable under ambient conditions.

In general terms, the invention relates to an ambient-stable probiotic alcoholic beverage, particularly an alcoholic beverage comprising probiotic yeast, as well as a method of forming the same. The alcoholic beverage of the present invention may provide advantages for storage and transportation since a cold chain may not be required to keep the beverage stable, thereby reducing cost and improving shelf-life.

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

For the purposes of the present invention, a yeast-based probiotic alcoholic beverage may be defined as a beverage which comprises an alcohol, such as ethanol or ethyl alcohol. In particular, the alcoholic beverage may have an alcohol content > 0.5% by volume.

The yeast-based probiotic alcoholic beverage may have a suitable alcohol content. According to a particular aspect, the alcohol content of the yeast-based probiotic alcoholic beverage may be > 0.5% by volume. 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%. In particular, the alcohol content may be 2.0-5.0%. Even more in particular, the alcohol content may be about 3-4%.

The yeast-based probiotic alcoholic beverage may be any suitable alcoholic beverage. Examples of an alcoholic beverage may include, but is not limited to, beer, wine, sparkling wine, rice wine, cider, spirits, fermented water, liquor, mead, pulque, and the like. According to a particular aspect, the yeast-based probiotic alcoholic beverage may be beer.

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

The method may further comprise adding a first hop to the wort or must. For the purposes of the present invention, a hop comprises a hop and/or its derivatives. The hop may be any suitable hop comprising isomerised alpha acids. The hop may be in any suitable form such as, but not limited to, hop cones, hop pellets, hop resins, hop powder, isomerised hop extracts and the like.

The first hop may be any suitable hop. For example, the first hop may be an isomerised hop extract. The first hop may have a suitable bitterness. According to a particular aspect, the first hop may have a bitterness of < 80 IBU. IBU refers to International bittering units and is a measure of the concentration of hop compounds in the yeastbased probiotic alcoholic beverage. In particular, the IBU measures the parts per million (ppm) of isohumulone in the beverage. In particular, 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 in particular, the bitterness may be 0-30 IBU.

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

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

According to another particular 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 comprise, but is not limited to, S. bayanusr, S. boulardii, S. cerevisiae, S. ludwigi, S. paradoxus, S. pasteurianus or a combination thereof. Examples of suitable non-probiotic non- Saccharomyces yeast comprise, but is not limited to, Dekkera bruxellensis, Galactomyces geotrichum, Kazachstania zonata, Kluyveromyces lactis, Lindnera meyerae, Pichia kluyveri, Schizosaccharomyces pombe, Starmera caribae, Torulaspora delbrueckii. The adding a first yeast may comprise adding a suitable amount of yeast to the wort or must. For example, the amount of first yeast added may be > 4 log CFU/mL. In particular, the amount of first yeast added may be 4-9 CFU/mL, 5-8 CFU/mL, 6-7 CFU/mL. Even more in particular, the amount of first yeast added may be 5-7 CFU/mL.

The fermenting may be carried out under suitable conditions. For example, the fermenting may comprise fermenting the wort or must at a suitable temperature for a suitable period of time. The temperature may be changed at any point during the fermenting. In particular, the fermenting may comprise fermenting the wort or must at a first pre-determined temperature for a first pre-determined period of time.

The first pre-determined period of time may be any suitable amount of time. The period of time may be selected depending on the first yeast added to the wort or must. For example, the first pre-determined period of time may be < 30 days. In particular, the first pre-determined period of time 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 in particular, the first pre-determined period of time may be 5-14 days.

The first pre-determined temperature may be any suitable temperature. The temperature may be selected depending on the first yeast added to the wort or must. For example, the first pre-determined temperature may be 10-35°C. In particular, the first predetermined temperature may be 10-30°C, 12-28°C, 15-25°C, 18-22°C, 20-21°C. Even more in particular, the first pre-determined temperature may be 15-20°C.

The adding a second yeast may be at any suitable time. For example, the adding a second yeast is after the fermenting has ended. In particular, the fermenting is considered to have ended when the °Bx of wort or must has become stable. Even more in particular, the fermenting may be considered to have ended when the °Bx may be about 2-15 °Bx.

The method may further comprise removing sediments of the first yeast prior to the adding a second yeast. The removing sediments of the first yeast may be by any suitable method. For example, the removing may be by cold crashing, centrifugation, filtration, or a combination thereof. The removing sediments may comprise removing yeast and protein particles formed from the fermentation, which if left in the alcoholic beverage, may impart turbidity and a yeasty aroma to the alcoholic beverage. In particular, the removing sediments may improve the stability and sensory quality of the alcoholic beverage.

According to a particular aspect, the removing sediments may be by cold crashing. The cold crashing may be under suitable conditions such as at a suitable temperature for a suitable period of time. For example, the temperature for cold crashing may be 0-10°C. In particular, the temperature may be 2-9°C, 3-8°C, 4-7°C, 5-6°C. Even more in particular, the temperature may be 0-4°C. For example, the period of time may be 1-10 days. In particular, the period of time may be 2-9 days, 3-8 days, 4-7 days, 5-6 days. Even more in particular, the period of time may be 1-2 days.

According to another particular aspect, the removing sediments may be by centrifugation. The centrifugation may be under suitable conditions such as at a suitable temperature for a suitable period of time. The centrifugation may further comprise pasteurization. For example, the temperature for centrifugation and optionally pasteurization may be 55-85°C. In particular, the temperature may be 60-80°C, 65-75°C, 70-73°C. Even more in particular, the temperature may be 60-65°C. For example, the period of time may be 1-10 days. In particular, the period of time may be 5 s - 30 min, 30 s - 25 min, 1-20 min, 5-15 min, 8-10 min. Even more in particular, the period of time may be 15-20 min. In particular, the centrifugation may be carried out until the alcoholic beverage reaches a suitable pasteurization unit. The centrifugation may be carried out until the alcoholic beverage reaches a pasteurization unit of 10-500 Pll.

The adding a second yeast may comprise adding a suitable probiotic yeast. For example, the second yeast may comprise: probiotic Saccharomyces yeast, probiotic non- Saccharomyces yeast, or a combination thereof. Examples of suitable probiotic Saccharomyces (S.) yeast comprise, but is not limited to, S. cerevisiae, S. boulardii, S. paradoxus, S. pasteurianus, S. bayanus or a combination thereof. In particular, the probiotic Saccharomyces yeast may comprise, but not limited to, S. boulardii CNCM I- 745, S. cerevisiae CNCM I-3856, S. boulardii T1 , S. boulardii 17, S. boulardii C ECT 1474, or a combination thereof. Examples of suitable probiotic non- Saccharomyces yeast comprise, but is not limited to, Pichia (P.), Kluyveromyces (K.), Hanseniaspora (H.), Candida (C.), Zygosaccharomyces (Z.), or a combination thereof. According to a particular aspect, the second yeast may be the same as the first yeast when the first yeast is a probiotic yeast.

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

The adding a second yeast to the alcoholic beverage may comprise 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 such that the cell count of the second yeast is > 4 log CFU/mL. In particular, the amount of second yeast added may be 4-11 CFU/mL, 5-10 CFU/mL, 6-9 CFU/mL, 7-8 CFU/mL. Even more in particular, the amount of second yeast added may be 5-7 CFU/mL.

The method may further comprise adding a second wort or must to the alcoholic beverage. In particular, the adding a second wort or must to the alcoholic may be carried out simultaneously with or following the adding a second yeast. The second wort or must may comprise adding any suitable wort or must. For example, the second wort or must may be as described above in relation to the first wort or must.

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

The method may further comprise fermenting the second wort or must at a second predetermined period of time and at a second pre-determined temperature. The second predetermined period of time may be any suitable period of time. For example, the second pre-determined period of time may be < 10 days. In particular, the second pre-determined period of time may be 1-10 days, 2-9 days, 3-8 days, 4-7 days, 5-6 days. Even more in particular, the second pre-determined period of time may be 2-5 days.

The second pre-determined temperature may be any suitable temperature. According to a particular aspect, the second pre-determined temperature may be < 35°C. In particular, the temperature may be 20-30°C. The method may further comprise packaging the yeast-based probiotic alcoholic beverage following the adding a second yeast. The packaging may be by any suitable method. For example, the packaging may comprise packaging the yeast-based probiotic alcoholic beverage into suitable storage containers, such as, but not limited to, bottles, cans, kegs, and the like. The packaging may further comprise carbonating the yeastbased probiotic alcoholic beverage during the packaging. The amount of carbon dioxide added for carbonating may depend on the amount of second yeast and/or second wart or must added.

According to a particular aspect, at the time of packaging, the yeast-based probiotic alcoholic beverage may not have fully formed. In particular, the yeast-based probiotic alcoholic beverage may be formed following the packaging and after the second predetermined period of time.

According to one embodiment, the method comprises adding a first yeast to a wort or must, wherein the first yeast is a probiotic yeast. The first yeast may be any suitable probiotic yeast as described above. The wort or must may be hopped or unhopped. Fermentation is then allowed to proceed for a first pre-determined period of time and at a first pre-determined temperature to form an alcoholic beverage. Once the fermentation is completed, indicated by a stable °Brix, sediments of the first yeast may be removed, for example, by cold crashing. The alcoholic beverage may then be inoculated with a second yeast, wherein the second yeast is a probiotic yeast. A second wort or must may be sequentially added. The alcoholic beverage with the second yeast may be optionally subjected to carbonation. The alcoholic beverage may then be packaged and stored at a temperature of < 35°C, particularly at a temperature of 20-30°C. Secondary fermentation may then occur in the packaging, thereby forming the yeast-based probiotic alcoholic beverage. The formed yeast-based probiotic alcoholic beverage may maintain a probiotic cell count of > 5 CFU/mL, even after 100 days of storage at 30°C.

According to a second embodiment, the method comprises adding a first yeast to a wort or must, 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 must may be hopped or unhopped. Fermentation is then allowed to proceed for a first pre-determined period of time and at a first pre-determined temperature to form an alcoholic beverage. Once the fermentation is completed, indicated by a stable °Brix, sediments of the first yeast may be removed, for example, by centrifugation and pasteurisation. The alcoholic beverage may then be inoculated with a second yeast without wort or must addition, wherein the second yeast is a probiotic yeast to form a yeast-based probiotic alcoholic beverage. The alcoholic beverage with the second yeast may be optionally subjected to carbonation. The alcoholic beverage may then 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 yeast-based probiotic alcoholic beverage may maintain a probiotic cell count of > 6 CFU/mL, even after 4 months of storage at 30°C.

According to a third embodiment, the method comprises adding a first yeast to a wort or must, 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 must may be hopped or unhopped. Fermentation is then allowed to proceed for a first pre-determined period of time and at a first pre-determined temperature to form an alcoholic beverage. Once the fermentation is completed, indicated by a stable °Brix, sediments of the first yeast may be removed, for example, by centrifugation and pasteurisation. The alcoholic beverage may then be inoculated with a second yeast, wherein the second yeast is a probiotic yeast. A second wort or must may be sequentially added. The alcoholic beverage with the second yeast may be optionally subjected to carbonation. The alcoholic beverage may then be packaged and stored at a temperature of < 35°C, particularly at a temperature of 20- 30°C. Secondary fermentation may then occur in the packaging, thereby forming the yeast-based probiotic alcoholic beverage. The formed yeast-based probiotic alcoholic beverage may maintain a probiotic cell count of > 5.5 CFU/mL, after 3 months of storage at 30°C.

According to a second aspect, there is provided a yeast-based probiotic alcoholic beverage comprising probiotic yeast, wherein the probiotic yeast has a cell count of > 5 log CFU/mL after 100 days of storage at 30°C.

The yeast-based probiotic alcoholic beverage may have a suitable alcohol content. According to a particular aspect, the alcohol content of the yeast-based probiotic alcoholic beverage may be > 0.5% by volume. 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%. In particular, the alcohol content may be 2.0-5.0%. Even more in particular, the alcohol content may be about 4-6%. The yeast-based probiotic alcoholic beverage may be any suitable alcoholic beverage. Examples of an alcoholic beverage may include, but is not limited to, beer, wine, sparkling wine, rice wine, cider, spirits, fermented water, liquor, mead, pulque, and the like. According to a particular aspect, the yeast-based probiotic alcoholic beverage may be beer.

The probiotic yeast comprised in the yeast-based probiotic alcoholic beverage may have a cell count of 5-11 CFLI/mL, 5.5-10 CFLI/mL, 6-9 CFLI/mL, 7-8 CFLI/mL. Even more in particular, the probiotic yeast comprised in the yeast-based probiotic alcoholic beverage may have a cell count of 5-9 CFU/mL. Further, after 100 days of storage at 30°C, the probiotic yeast comprised in the yeast-based probiotic alcoholic beverage may have a cell count of 5-7 CFU/mL. This is relatively high given that the beverage is not stored under cold storage, but rather under ambient conditions. It is unexpected as it has been known that beverages, particularly alcoholic beverages, must be stored under refrigerated temperature of about 0-5°C in order to maintain a probiotic cell count of a suitable amount so as to confer health benefits. In the alcoholic beverage according to the second aspect, a high probiotic cell count may be obtained even without the alcoholic beverage being stored under refrigerated temperatures, but instead under ambient temperature storage.

The yeast-based probiotic alcoholic beverage may be stored at a suitable temperature so as to maintain the probiotic yeast at a suitable level. For example, the yeast-based probiotic alcoholic beverage may be stored at a temperature of about < 35°C. In particular, the yeast-based probiotic alcoholic beverage may be stored at a temperature of about 15-35°C, 20-30°C, 25-28°C. Even more in particular, the yeast-based probiotic alcoholic beverage may be stored at a temperature of about 20-30°C.

The probiotic yeast comprised in the yeast-based probiotic alcoholic beverage may be any suitable probiotic yeast. For example, the probiotic yeast may be as described above in relation to the second yeast. In particular, the probiotic yeast may comprise probiotic Saccharomyces yeast, probiotic non- Saccharomyces yeast, or a combination thereof.

According to a particular aspect, the yeast-based probiotic alcoholic beverage may further comprise a hop or derivative thereof. The hop or derivative thereof may be any suitable hop. In particular, the hop or derivative may be as described above in relation to the first aspect. The hop or its derivative may have a suitable bitterness. In particular, the hop or derivative thereof may have a bitterness of < 80 IBU. Even more in particular, the hop or derivative may have a bitterness of 5-30 IBU.

The yeast-based probiotic alcoholic beverage may have a suitable pH. For example, the pH of the yeast-based probiotic alcoholic beverage may be > 3.8. In particular, the pH may be 3.8-6.5, 4-6, 5-5.5. Even more in particular, the pH may be about 4-6.

The yeast-based probiotic alcoholic beverage may have a suitable Brix or its specific gravity equivalent. Brix is a measure of the amount of sugars in the yeast-based probiotic alcoholic beverage. For example, 1 °Bx refers to 1 g of sucrose in 100 g of the yeastbased probiotic alcoholic beverage. Accordingly, the higher the Brix, the higher the alcohol content may be in the yeast-based probiotic alcoholic beverage. The Brix of the yeast-based probiotic alcoholic beverage may be 2-15 °Bx. For the purposes of the present invention, reference to Brix of the alcoholic beverage refers to the measure of the Brix of the wort or must comprised in the alcoholic beverage. In particular, the Brix of the yeast-based probiotic alcoholic beverage may be 2-15 °Bx, 3-12 °Bx, 4-10 °Bx, 5-8 °Bx, 6-7 °Bx. Even more in particular, the Brix may be about 5-10 °Bx.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting.

EXAMPLE

All materials used in the examples provided are as follows:

A wort was prepared by mashing of malt, adding 70-90% drinking water, heating until the mixture was boiling and boiled for 60 minutes, and subsequently cooling the mixture to room temperature. The wort or must may include, but is not limited to barley, oats, wheat, corn, rye, rice, water and may also include sugars in different forms (such as fruit juice, and fruit puree).

No hop or its derivatives was added in the preparation of the unhopped worts as used in Examples 1 to 3. However, hop Cascade and hop extracts (IBU 18) was added during the boiling of the wort mixture prepared for the hopped wort as used in Example 2.

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

Method

Primary fermentation with a first probiotic yeast

A first probiotic yeast S. cerevisiae CNCM I-3856 was inoculated into a sterilised flask, containing pasteurized unhopped wort as prepared above to achieve minimum cell counts of 5.0 Log CFU/mL. The fermentation vessel was covered with an airlock and was stored under 20°C for 7-21 days for fermentation to occur. After primary fermentation, part of unhopped beer was taken along with the probiotic yeast for shelflife testing as a control group.

Sequential inoculation of a probiotic yeast

After primary fermentation with the first probiotic yeast, probiotic yeast sediments were mostly removed by cold crashing (0-4°C for 24 hours). Pasteurized unhopped wort (5 % v/v) was added to the clarified beer, and then was sequentially inoculated with a second probiotic yeast, particularly freeze-dried S. cerevisiae CNCM I-3856 (minimum cell counts of 5.0 Log CFU/mL) for the FS group and the primary-cultured S. cerevisiae CNCM I-3856 (minimum cell counts of 5.0 Log CFU/mL) for the PS group, respectively. The second probiotic yeast was the same as the first probiotic yeast used.

Packaging and shelf-life testing

The beer samples were immediately aliquoted into 15-mL centrifuge tubes (12 mL/ tube) without carbonation, and stored at 30°C for over 3 months to study the stability and survival of the yeasts. Triplicate beer samples of each treatment were taken every 5-30 days to monitor the probiotic cell counts and physio-chemical parameters. Yeast cell count was monitored using spread plating method on potato dextrose agar (PDA) (Oxoid, Basingstoke, Hampshire, England) at 30°C for 2 days. The pH and °Brix of the samples were measured with a pH meter (Metrohm, Switzerland) and a refractometer (ATAGO, Tokyo, Japan), respectively.

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

Results

Probiotic yeast population

Survival of S. cerevisiae CNCM I-3856 in unhopped beer during storage at 30°C is shown in Figure 1. In the control group, the cell counts of S. cerevisiae CNCM I-3856 were able to attain 7.62 Log CFU/mL after primary fermentation, while the cell counts dropped to approximately 6 Log CFU/mL in the first 10 days of storage at 30°C, remaining at around 5 Log CFU/mL after 3 months. In the FS and PS groups, the cell counts of S. cerevisiae CNCM I-3856 fell to 6.2-6.3 Log CFU/mL after cold crashing, which were back to 6.8 Log CFU/mL after secondary fermentation mainly in the first 4 days of storage. The probiotic yeast cell counts in FS group kept stable from the second to fourth months of storage with over 6 Log CFU/mL, while the yeast cell counts in PS group dropped to around 5 Log CFU/mL in the first two months of storage at 30°C, as shown in Figure 1. These results suggested that the sequential inoculation of freeze-dried probiotic yeast is beneficial to the viability of probiotics during storage at 30°C.

°Brix and pH

The °Brix of beer after primary fermentation was around 7.1 , which was stable within 7.1- 7.2 in control group, as shown in Figure 2. In the FS and PS groups, the °Brix decreased to 6.7 and 6.5 in the first 4 days of storage, finally falling to 6.5 and 6.4 after 3 months, respectively. This indicated that most sugars in the added wort were consumed in the first 4 days of storage. In addition, the sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 utilized less sugar than primary-cultured yeast during sensory fermentation.

The original pH of the unhopped beer fermented with S. cerevisiae CNCM I-3856 was approximately 4.7, as shown in Figure 3. The pH declined gradually by around 0.3 units during 4 months of storage at 30°C, while the pH only reduced by around 0.1 unit in the PS group. This indicated that the sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 contributed to the stable pH.

Sensory evaluation

Sensory evaluation and observations on appearance of the yeast-based probiotic beer were carried out. The results are as shown in Figure 4.

After 4 months of storage at 30°C, the appearance of probiotic beer in the control group became too cloudy, and was not accepted by all panelists. The appearance of probiotic beer in FS and PS groups was more appealing with higher acceptability levels. The off- flavour of probiotic beer in the control group was obviously detected, such as fusel oil and herbal-like flavour. The sequential inoculation of probiotic yeast could significantly lower the intensity of off-flavour (< 2), especially in the FS group (1.43). The alcoholic intensity ratings for the yeast-based probiotic beer in the FS group was much lower (2.57) than other beers (> 3.4). All beers had very similar ratings for fruity intensity (2.0-2.3), followed by sweetness intensity (2.0-2.8). This result demonstrated that sequential inoculation of probiotic yeast effectively prevented beer cloudiness and off-flavour production during storage.

This result demonstrated that the probiotic yeasts, as primary fermentation culture, were not stable under ambient temperature storage. These drawbacks could be alleviated by sequential inoculating of freeze-dried S. cerevisiae CNCM I-3856, by which the probiotic cell count could only decrease 0.8 Log CFU/mL after 3 months of storage at 30°C, remaining at over 6 Log CFU/mL. The sensory quality was also improved.

Example 2 -Sequential inoculation of probiotic yeast without fermentation

Method Primary fermentation with a first non-probiotic yeast

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

Sequential inoculation of a probiotic yeast

Besides S. cerevisiae CNCM I-3856, another probiotic yeast S. boulardii CNCM I-745 was used in this example, which was not evaluated as a brewing culture for probiotic beer before. Pasteurized unhopped and/or hopped beer was inoculated with freeze-dried probiotic yeast S. boulardii CNCM I-745 or freeze-dried S. cerevisiae CNCM I-3856 with minimum cell counts of 6.0 Log CFU/mL, without wort addition. The beer samples were packaged with carbonation, and then analysed as was also done in Example 1.

Results

Probiotic cell count

The changes of cell count of probiotic yeast S. boulardii CNCM I-745 and S. cerevisiae CNCM I-3856 in unhopped and hopped beer during storage at 30°C is shown in Figure 5. The cell count of S. boulardii CNCM I-745 in unhopped and hopped beer had a similar trend during storage, which rapidly decreased by around 0.7 Log CFU/mL in the second month of storage. The S. boulardii CNCM I-745 cell count in unhopped beer kept over 5.6 Log CFU/mL in the 4 months of storage, while the probiotic yeast cell count was maintained over 6.1 Log CFU/mL in hopped beer till 100 days of storage. The S. cerevisiae CNCM I-3856 cell count in unhopped beer declined from over 7.5 Log CFU/mL to around 6.5 Log CFU/mL in the first month of storage, and then was stable with 6.3 Log CFU/mL for the next 3 months of storage as shown in Figure 5. These results showed that the cell counts of different probiotic yeasts had similar trends in different beer samples, which dropped mainly in the early and mid-stage of storage at 30°C.

°Brix and pH The °Brix of beer added with probiotic yeast had no substantial changes during 4 months of storage at 30°C, as can be seen from Figure 6, indicating the residual sugars in beer were not utilized by probiotic yeasts.

The addition of probiotic S. cerevisiae CNCM I-3856 slightly increased the pH of beer from about 4.7 to 4.9 during storage, and pH of other probiotic beer samples kept stable at 30°C, as seen in Figure 7.

Sensory evaluation

Sensory evaluation results and appearance of probiotic beer were evaluated and the sensory evaluation results are as shown in Figure 8.

The appearance of probiotic unhopped beer containing S. boulardii CNCM I-745 remained clear and was acceptable to most panelists after 4 months of storage at 30°C, followed by the probiotic hopped beer added with the same probiotic yeast strain. However, the probiotic hopped beer containing S. cerevisiae CNCM I-3856 was too turbid and was unacceptable to all panelists, which could be related to the higher amount of S. cerevisiae CNCM I-3856 addition. Compared with S. boulardii CNCM I-745, the probiotic beer added with S. cerevisiae CNCM I-3856 had lower ratings for alcoholic flavour (2.17) and off-flavour (1.33).

All beers had very similar ratings for sweetness (2.1-2.5), followed by fruity flavour (2.0- 2.7). This result demonstrated that addition of more probiotic yeast not only could increase the cost of beer production, but also affected the appearance of beer during storage. Thus, a method to lower the addition of probiotic yeast was explored in Example 3 (see below).

Example 3 -Sequential inoculation of probiotic yeast with secondary fermentation

Method

Primary fermentation with a first non-probiotic yeast

Unhopped beer was prepared and inoculated with wheat beer yeast S. cerevisiae SafAle WB-06. The beer was centrifuged and pasteurized at 60-65°C for 15-20 minutes, to remove yeast sediments and kill most microorganisms in beer. Sequential fermentation with a probiotic yeast

After primary fermentation with the first non-probiotic yeast, centrifuged and pasteurized unhopped beer was inoculated with pre-cultured probiotic yeast S. cerevisiae CNCM I- 3856 with minimum cell counts of 5.0 Log CFU/mL, along with 5% v/v and 10% v/v of unhopped wort, respectively. Probiotic pre-cultured yeast was prepared from pure culture of freeze-dried S. cerevisiae CNCM I-3856, with at least 100 times of growth. The beer samples were packaged and tested for shelf-life with the same methods as was done in Example 1. The probiotic beer samples were rated by eight trained panelists using a 5- point scale quantitative descriptive analysis as described in Example 1.

Additionally, the six probiotic unhopped beer samples and two probiotic hopped beer samples from Examples 1 to 3 were sensorially evaluated by eight experienced panelists using a preference ranking test (unhopped beer: 1 - least preferred, 6 - most preferred; hopped beer: 1 -least preferred, 2-more preferred) on the 120^th day of storage at 30°C.

Volatile compound analysis

The effects of probiotic yeasts on the volatile aroma compounds of probiotic beer samples were analyzed. The probiotic beer samples included the probiotic beer primary and sequential fermented with probiotic yeasts (as in Example 1), the probiotic beer without being fermented with probiotic yeasts (as in Example 2) and the probiotic beer only sequential fermented with probiotic yeasts (as in the present Example 3).

A 5-mL beer sample (pH adjusted to 2.5 with 1 M HCI) was transferred into a screwcapped headspace vial before analysis. Volatile compounds were extracted by headspace (HS) solid-phase microextraction (SPME) with a carboxen/polydimethylsiloxane fibre (85 pm coating, Supelco, Sigma-Aldrich, Barcelona, Spain) and analysed using gas chromatography (GC)-mass spectrometer (MS)-flame ionisation detector (FID). Sample extraction was performed using a SPME autosampler (CTC, Combi Pal, Switzerland) at 60°C for 40 min under 250 rpm agitation. The GC was coupled to the Agilent 5975C triple-axis MS and FID for identification and quantification of the volatiles (Chen et al., 2015, International Journal of Food Microbiology, 206:45- 50). The volatiles were identified by comparing their mass spectra with those from NIST 8.0 and Wiley 275 MS libraries. Semi-quantification was carried out based on their GC- FID peak areas. Results

Probiotic cell counts

The changes of probiotic S. cerevisiae CNCM I-3856 in unhopped beer during storage at 30°C is shown in Figure 9. Because of secondary fermentation, the cell counts of S. cerevisiae CNCM I-3856 rapidly increased from around 5.5 Log CFU/mL to around 7.0 Log CFU/mL during the first 5 days of storage. Then, probiotic yeast cell counts declined gradually to around 5.5 Log CFU/mL after 2 months of storage, and remained stable in the third month of storage as can be seen in Figure 9. This indicated that 5% v/v or 10% v/v of wort addition had no significant difference on changes of yeast cell counts during storage, and thus 5% v/v of wort addition could be used to lower the production of CO2 during storage.

°Brix and pH

The °Brix of beer fermented with non-probiotic yeast was around 6.1 , which was much lower than that in the beer fermented with S. cerevisiae CNCM I-3856 (Figures 2 & 10). This result demonstrated that the attenuation rates of probiotic yeasts was lower than conventional brewing yeasts. The °Brix of beer increased to 6.3 and 6.7 after 5% v/v wort and 10% v/v wort addition, respectively, which was consumed to 5.7 after the first 5 days of storage as can be seen from Figure 10. This indicated that the secondary fermentation with probiotic yeasts S. cerevisiae CNCM I-3856 was mainly completed in the first 5 days of bottle storage. The °Brix of unhopped beer samples was then almost stable during storage at 30°C.

The pH of both unhopped beer containing S. cerevisiae CNCM I-3856 slightly declined from 4.3 to 4.1 during the 3-4 months of storage at 30°C as can be seen from Figure 11 .

Sensory evaluation and volatile compound analysis

Sensory evaluation results and appearance of probiotic unhopped beer are presented in Figure 12. The appearance of probiotic unhopped beer kept clear after 4 months of storage at 30°C, and was accepted by most panelists. Different levels of wort additions did not significantly affect the appearance, alcoholic flavour, sweetness and off-flavour of the probiotic beer after storage. However, the probiotic beer added with 10% v/v wort for secondary fermentation contributed to higher in density of fruity flavour. This result presented that sequential inoculation of probiotic yeast and 5%-10% v/v wort for secondary fermentation in bottle could maintain the appearance of beer, and the beer had a similar sensory quality.

Preference ranking score of yeast-based probiotic beer after 4 months of storage is presented in Figure 13.

Samples of the probiotic unhopped beer were as follows:

(A) primary fermentation with probiotic yeasts and no sequential inoculation;

(B) primary fermentation with probiotic yeasts, followed by sequential inoculation of freeze-dried S. cerevisiae CNCM I-3856 +5% v/v wort;

(C) primary fermentation with probiotic yeasts, followed by sequential inoculation of primary-cultured S. cerevisiae CNCM I-3856 +5% v/v wort;

(D) primary fermentation with non-probiotic yeasts, followed by sequential inoculation of freeze-dried S. boulardii CNCM I-745 without wort addition;

(E) primary fermentation with non-probiotic yeasts, followed by sequential inoculation of pre-cultured S. cerevisiae CNCM I-3856 +5% v/v wort; and

(F) primary fermentation with non-probiotic yeasts, followed by sequential inoculation of pre-cultured S. cerevisiae CNCM I-3856 +10% v/v wort.

The probiotic beer Sample A was least preferred by all panelists, which implied that the primary fermentation with probiotic yeasts was not suitable for probiotic beer under ambient temperature storage (Figure 14A). After primary fermentation with probiotic yeasts, sequential inoculation of probiotic yeasts could effectively increase the ranking score of probiotic beer (Sample B and Sample C). Compared with probiotic yeast, the probiotic beers primarily fermented with a conventional brewing yeast (Sample D-F) were more preferred by panelists. Additionally, the sequential inoculation of probiotic yeast for secondary fermentation could contribute to the sensory quality of beer (Sample E&F), compared with the probiotic yeast without fermentation (Sample D, Figure 14B). Main volatile aroma compounds of the different yeast-based probiotic beer samples A to F described above after 4 months of storage are presented in Figures 14A to 14D. The ethanol content significantly increased after sequential inoculation of probiotic yeasts (Samples B-F, Figure 14A), as well as 2,4-di-tert-butylphenol and other volatiles in the alcohols group (including phenethyl alcohol and isobutyl alcohol, Figures 14B and 14C). The phenethyl alcohol (sweet, rose, floral) and isobutyl alcohol (fruity, wine-like) improved the complexity of the beer flavour. Compared with solely probiotic yeast fermented beers, the probiotic beers primarily fermented with a conventional brewing yeast (Samples D-F) produced higher amounts of the main volatile compounds in the alcohols group.

The probiotic yeast used for both primary and sequential fermentations (Sample B & C) contributed to higher amounts of important acetate esters, such as phenethyl acetate (floral, rose, sweet) and isoamyl acetate (fruity, banana, sweet) than other inoculation methods (Figure 14C).

The amounts of ethyl esters (mainly Ce-C ethyl esters) in Sample A were significantly higher than that in other beer samples (Figure 14D). These ethyl esters could impart fruity flavour to beer, while the over-production of ethyl esters could mask other volatiles and cause off-flavour (as the result in Figure 4). This could explain the reason that the probiotic beer Sample A was least preferred in sensory evaluation (Figure 13A). This result also indicated that probiotic yeasts was not suitable for only primary fermentation of probiotic beer under ambient temperature storage.

Whilst the foregoing description has described exemplary embodiments, it will be understood by those skilled in the technology concerned that many variations may be made without departing from the present invention.

Claims

1 . A method of forming a yeast-based probiotic alcoholic beverage, the method comprising: providing a wort or must; adding a first yeast to the wort or must; fermenting the wort or must at a first pre-determined period of time and at a first pre-determined temperature to form an alcoholic beverage; and adding a second yeast to the alcoholic beverage to form a yeast-based probiotic alcoholic beverage, wherein the second yeast is a probiotic yeast.

2. The method according to claim 1 , wherein the first yeast is a probiotic yeast.

3. The method according to claim 1 or 2, wherein the first yeast is the same as the second yeast.

4. The method according to claim 1 , wherein the first yeast is a non-probiotic yeast.

5. The method according to claim 4, wherein the first yeast is: non-probiotic

Saccharomyces yeast, non-probiotic non- Saccharomyces yeast, or a combination thereof.

6. The method according to any preceding claim, wherein the second yeast is probiotic Saccharomyces yeast, probiotic non- Saccharomyces yeast, or a combination thereof.

7. The method according to any preceding claim, wherein each of the first yeast and the second yeast has a cell count of > 4 log CFU/mL.

8. The method according to any preceding claim, wherein the second yeast comprises freeze-dried probiotic yeast, pre-cultured probiotic yeast, or a combination thereof.

9. The method according to any preceding claim, wherein the method further comprises adding a first hop or its derivative to the wort or must.

10. The method according to claim 9, wherein the first hop has a bitterness of < 80 IBU.

11. The method according to any preceding claim, wherein the method further comprises removing sediments of the first yeast prior to the adding a second yeast.

12. The method according to any preceding claim, wherein the first pre-determined temperature is 10-35°C.

13. The method according to any preceding claim, wherein the method further comprises adding a second wort or must to the alcoholic beverage.

14. The method according to claim 13, wherein the adding a second wort or must to the alcoholic is carried out simultaneously with or following the adding a second yeast.

15. The method according to claim 13 or 14, wherein the method further comprises fermenting the second wort or must at a second pre-determined period of time and at a second pre-determined temperature.

16. The method according to claim 15, wherein the second pre-determined temperature is 20-30°C.

17. A yeast-based probiotic alcoholic beverage comprising probiotic yeast, wherein the probiotic yeast has a cell count of > 5 log CFU/mL after 100 days of storage at 30°C.

18. The yeast-based probiotic alcoholic beverage according to claim 17, wherein the probiotic yeast comprises probiotic Saccharomyces yeast, probiotic non- Saccharomyces yeast, or a combination thereof.

19. The yeast-based probiotic alcoholic beverage according to claim 17 or 18, wherein the yeast-based probiotic alcoholic beverage further comprises a hop or derivative thereof.

20. The yeast-based probiotic alcoholic beverage according to claim 19, wherein the hop or derivative thereof has a bitterness of < 80 IBU.

21. The yeast-based probiotic alcoholic beverage according to any of claims 17 to

20, wherein the yeast-based probiotic alcoholic beverage is formed from the method of claims 1 to 16.