WO2023163663A2

WO2023163663A2 - A probiotic alcoholic beverage

Info

Publication number: WO2023163663A2
Application number: PCT/SG2023/050116
Authority: WO
Inventors: Shao Quan Liu; Dai CHEN
Original assignee: National University Of Singapore
Priority date: 2022-02-24
Filing date: 2023-02-24
Publication date: 2023-08-31
Also published as: WO2023163663A3

Abstract

A probiotic alcoholic beverage There is provided a probiotic alcoholic beverage comprising microencapsulated probiotics, wherein after storage of the beverage at a predetermined temperature for a predetermined time, the microencapsulated probiotics have a cell count of ≥ 5.0 log CFU/mL, wherein the predetermined temperature is 20-30 °C and the predetermined time is ≥ 7 days. There is also provided a method of forming the probiotic alcoholic beverage.

Description

A probiotic alcoholic beverage

Technical Field

The present invention relates to a probiotic alcoholic beverage.

Background

An alcoholic beverage having probiotics is a non-dairy based alternative delivery method of probiotics. However, such probiotic alcoholic beverages suffer from poor survival rates and low cell counts of probiotics, worsening as more time passes postproduction. Cold chain transport and storage of the beverage is required to slow the rate of probiotics cell death in the alcoholic beverage. However, that is often expensive, difficult to monitor compliance, and inadequate to prevent probiotics cell death, and as a result, conventional probiotic alcoholic beverages often do not have sufficient amount of probiotics to confer any health benefits to the consumer.

There is therefore still a need for an improved probiotic alcoholic beverage.

Summary of the invention

The present invention seeks to address these problems, and/or to provide an improved probiotic alcoholic beverage.

In general terms, the invention relates to a probiotic alcoholic beverage comprising microencapsulated probiotics. The probiotic alcoholic beverage of the present invention may be stored under ambient conditions whilst maintaining an adequate amount of probiotics throughout its shelf life.

According to a first aspect, the present invention provides a probiotic alcoholic beverage comprising microencapsulated probiotics, wherein after storage of the beverage at a predetermined temperature for a predetermined time, the microencapsulated probiotics have a cell count of > 5.0 log CFU/mL, wherein the predetermined temperature is 20-30°C and the predetermined time is > 7 days.

According to a particular aspect, the predetermined time may be > 90 days.

According to a particular aspect, the microencapsulated probiotics may have a cell count of > 6.0 log CFU/mL after the storage of the beverage. According to a particular aspect, the probiotic alcoholic beverage may further comprise a hop or its derivative. The hop or its derivative may be any suitable hop or its derivative and may have a suitable bitterness. In particular, the hop or its derivative may have a bitterness of < 80 IBU.

The microencapsulated probiotics comprised in the probiotic alcoholic beverage may be any suitable microencapsulated probiotics. For example, the microencapsulated probiotics may comprise microencapsulated probiotic yeast, microencapsulated probiotic bacteria, or a combination thereof. In particular, the microencapsulated probiotic yeast may comprise, but is not limited to, Saccharomyces yeast, non- Saccharomyces yeast, or a combination thereof. The microencapsulated probiotic bacteria may comprise, but is not limited to, a microencapsulated spore-forming probiotic bacteria. In particular, the microencapsulated probiotic bacteria may comprise Bacillus.

The microencapsulated probiotics may have any suitable size. In particular, the microencapsulated probiotics may have an average diameter of 5-1000 pm.

The probiotic alcoholic beverage may have a suitable pH after the storage. For example, the pH of the probiotic alcoholic beverage after the storage may be 4-6.

The probiotic alcoholic beverage may have a suitable Brix after the storage. For example, the Brix of the probiotic alcoholic beverage after the storage may be 2-20 °Bx.

According to a second aspect, the present invention provides a method of forming a probiotic alcoholic beverage described above, the method comprising adding microencapsulated probiotics to a fermented alcoholic beverage to form the probiotic alcoholic beverage. The fermented alcoholic beverage may be any suitable fermented alcoholic beverage.

The method may further comprise forming the microencapsulated probiotics prior to the adding the microencapsulated probiotics to the fermented alcoholic beverage. The forming may comprise adding probiotics to a solution to form a probiotics solution, and may further comprise freeze drying the probiotics prior to the adding the probiotics to the solution. The forming may further comprise placing the probiotics solution into a salt solution to form the microencapsulated probiotics.

According to a particular aspect, the method may further comprise centrifuging and/or pasteurizing the fermented alcoholic beverage prior to the adding the microencapsulated probiotics.

According to a particular aspect, the method may further comprise carbonation of the probiotic alcoholic beverage after the adding the microencapsulated probiotics.

The microencapsulated probiotics added to the fermented alcoholic beverage may comprise any suitable probiotics. For example, the probiotics comprised in the microencapsulated probiotics may be as described above.

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 the experimental outline in a process flowchart;

Figure 2 shows particle size distribution of the beads formed of microencapsulated probiotics;

Figure 3 shows survival of (•) microencapsulated S. cerevisiae CNCM I-3856; (▼) microencapsulated S. boulardii CNCM I-745; (O) free S. cerevisiae CNCM I-3856; and (V) free S. boulardii CNCM I-745 during storage at 30 °C; Figure 3A shows the survival in unhopped beer; Figure 3B shows the survival in hopped beer;

Figure 4 shows survival of (■) microencapsulated B. subtilis CU1 and (□) free B. subtilis CU1 ; Figure 4A shows the survival in unhopped beer; Figure 4B shows the survival in hopped beer;

Figure 5 shows survival rate of freeze-dried probiotics (black bar) and microencapsulated probiotics (white stripe bar); Figure 5A shows the survival rate in unhopped beer; Figure 5B shows the survival rate in hopped beer during storage at 30 o/^.

Figure 6 shows changes of °Brix; Figure 6A shows the changes in unhopped beer; Figure 6B shows the changes in hopped beer during storage at 30 °C: ( • ) microencapsulated S. cerevisiae CNCM I-3856, (▼) microencapsulated S. boulardii CNCM I-745, (O) free S. cerevisiae CNCM I-3856 and (V) free S. boulardii CNCM I- 745;

Figure 7 shows changes of °Brix; Figure 7A shows the changes in unhopped beer; Figure 7B shows the changes in hopped beer during storage at 30 °C: ( ■ ) microencapsulated B. subtilis CU1 and (□) free B. subtilis CU1 ;

Figure 8 shows changes of pH; Figure 8A shows the changes in unhopped beer; Figure 8B shows the changes in hopped beer during storage at 30 °C: ( • ) microencapsulated S. cerevisiae CNCM I-3856, (▼) microencapsulated S. boulardii CNCM I-745, (O) free S. cerevisiae CNCM I-3856 and (V) free S. boulardii CNCM I- 745;

Figure 9 shows changes of pH; Figure 9A shows the changes in unhopped beer; Figure 9B shows the changes in hopped beer during storage at 30 °C: ( ■ ) microencapsulated B. subtilis CU1 and (□) free B. subtilis CU1; and

Figure 10 shows sensory profile plot of mean attribute values on a 1 to 4-point scale: solid line - (•) microencapsulated S. cerevisiae CNCM I-3856, (■) microencapsulated S. boulardii CNCM I-745, and (A) microencapsulated B. subtilis CU1 ; dotted line - (O) non-microencapsulated S. cerevisiae CNCM I-3856, (□) non-microencapsulated S. boulardii CNCM I-745, and (A) non-microencapsulated B. subtilis CU1; long dotted line - (O) hopped beer without addition of probiotics.

Detailed Description

As explained above, there is a need for an improved probiotic alcoholic beverage.

In general terms, the present invention provides a probiotic alcoholic beverage comprising probiotics, which is stable when stored in ambient conditions, and a method of forming the same. Generally, it is difficult to expect probiotics incorporated in a beverage to survive without proper cold storage, and the challenge is exacerbated for probiotics incorporated in an alcoholic beverage, in which the presence of antimicrobial compounds would prevent growth and impair survival of the probiotics. As compared to other probiotic alcoholic beverages, the present probiotic alcoholic beverage maintains an adequate amount of probiotics throughout its shelf life under ambient conditions to effectively confer health benefits on the consumer. For example, it has been shown that certain strains of probiotics can bring about improvement in gut health and digestion. Accordingly, the probiotic alcoholic beverage according to the present invention may bring about health benefits such as improvement in gut health and boosting immunity. The colour and taste of the probiotic alcoholic beverage is also maintained throughout its shelf life. The probiotic alcoholic beverage may be stored at ambient temperature without the need for cold chain and/or cold storage, thereby allowing for easy transportation and storage.

According to a first aspect, the present invention provides a probiotic alcoholic beverage comprising microencapsulated probiotics, wherein after storage of the probiotic alcoholic beverage at a predetermined temperature for a predetermined time, the microencapsulated probiotics have a cell count of > 5.0 log CFU/mL, wherein the predetermined temperature is 20-30°C and the predetermined time is > 7 days.

For the purposes of the present invention, references to probiotic alcoholic beverage refers to a beverage which comprises an alcohol comprising microencapsulated probiotics, while references to fermented alcoholic beverage refers to a beverage which comprises an alcohol before any addition of microencapsulated probiotics. Examples of an alcohol include, but is not limited to, ethanol or ethyl alcohol. In particular, the alcoholic beverage may have an alcohol content > 0.5% by volume.

The probiotic alcoholic beverage may have suitable alcohol content. According to a particular aspect, the alcohol content of the 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 4.0-8.0%.

The probiotic alcoholic beverage may comprise any suitable fermented alcoholic beverage. The fermented alcoholic beverage may be fermented by yeast, bacteria, mould, or any combination thereof. Examples of a fermented alcoholic beverage may include, but is not limited to, beer, cider, fermented water, liquor, mead, pulque, rice wine, sparking wine, spirits, wine, and the like. According to a particular aspect, the fermented alcoholic beverage may be beer.

For the purposes of the present invention, microencapsulated probiotics is defined as probiotics that are coated by capsules. The microencapsulated probiotics may have an average size of 5-1000 pm. In particular, the microencapsulated probiotics may have an average size of 100-900 pm, 200-800 pm, 300-700 pm, 400-600 pm. Even more in particular, the microencapsulated probiotics may have an average size of 300-700 pm. For the purposes of the present invention, average size is defined as the mean diameter of > 50% of the microencapsulated probiotics.

The microencapsulated probiotics may have any suitable shape and size. For example, the microencapsulated probiotics may be globular in shape.

The storage of the probiotic alcoholic beverage may be at a predetermined temperature for a predetermined time to maintain the cell count of microencapsulated probiotics at a suitable level over the shelf life period. For example, the predetermined temperature may be about 0-35°C. In particular, the predetermined temperature may be about 5-30°C, 10-25°C, 15-20°C. Even more in particular, the predetermined temperature may be about 20-30°C.

The predetermined time may be > 7 days. In particular, the predetermined time may be about > 30 days, > 60 days, > 90 days, > 120 days. Even more in particular, the predetermined time may be > 90 days. The alcoholic beverage advantageously maintains cell count of microencapsulated probiotics at an effective level to confer health benefits throughout the shelf life period.

The probiotic alcoholic beverage may comprise a suitable initial amount of microencapsulated probiotics before the storage. For example, the initial cell count of microencapsulated probiotics before the storage may be > 6.0 log CFU/mL. In particular, the initial cell count of microencapsulated probiotics before the storage may be > 6.0-8.0 log CFU/mL. A suitable amount of microencapsulated probiotics may be comprised in the probiotic alcoholic beverage after the storage, with an effective amount to confer health benefits. For example, the probiotic bacteria may have a cell count of > 5.0 log CFU/mL. In particular, the probiotic bacteria comprised in the probiotic alcoholic beverage may have a cell count of 5.0-11.0 log CFU/mL, 5.5-10.5 log CFU/mL, 6.0-10.0 log CFU/mL, 6.5-9.5 log CFU/mL, 7.0-9.0 log CFU/mL, 7.5-8.5 log CFU/mL. Even more in particular, the microencapsulated probiotics comprised in the probiotic alcoholic beverage may have a cell count of > 6.0 CFU/mL after the storage.

The probiotic alcoholic beverage may comprise a hop. For the purposes of the present invention, reference to hop refers to a hop and/or its derivatives. The hop may be any suitable hop. For example, the hop may be an isomerised hop extract.

The hop may have a suitable bitterness. For example, the hop may have a bitterness of < 80 IBU. In particular, the bitterness may be 0-80 IBU, 5-75 IBU, 10-70 IBU, 15-65 IBU, 20-60 IBU, 25-55 IBU, 30-50 IBU, 35-45 IBU. Even more in particular, the hop may have a bitterness of 5-30 IBU. IBU refers to International bittering units and is a measure of the concentration of hop compounds in the alcoholic beverage. In particular, the IBU measures the parts per million (ppm) of isohumulone in the probiotic alcoholic beverage.

The microencapsulated probiotics comprised in the probiotic alcoholic beverage may be any suitable microencapsulated probiotic yeast and/or microencapsulated probiotic bacteria. The microencapsulated probiotic yeast and/or microencapsulated probiotic bacteria may comprise any suitable live microorganism which, when provided in an adequate amount, confers a health benefit on the host.

For example, the microencapsulated probiotic yeast may comprise, but is not limited to, Saccharomyces and/or non- Saccharomyces yeast, such as Pichia, Kluyveromyces, Hanseniaspora, Candida, and Zygosaccharomyces. In particular, the microencapsulated probiotic yeast may comprise, but is not limited to, S. boulardi, S. cerevisiae, or a combination thereof.

The microencapsulated probiotic bacteria may comprise microencapsulated sporeforming probiotic bacteria. For the purposes of the present invention, spore-forming probiotic bacteria is defined as a sporulating bacteria which becomes metabolically inert, and resistant to external stress when in the spore form, and which also confers a health benefit on the host when provided in an adequate amount, which is microencapsulated as defined above. The microencapsulated spore-forming probiotic bacteria may comprise Bacillus. In particular, the microencapsulated spore-forming probiotic bacteria may comprise Bacillus subtilis, Bacillus coagulans, Bacillus cereus, Bacillus clausii, Bacillus licheniformis, Bacillus polyfermenticus, Bacillus pumilus, or a combination thereof. Even more in particular, the microencapsulated spore-forming probiotic bacteria may comprise Bacillus subtilis.

The probiotic alcoholic beverage may have a suitable pH. For example, the pH of the probiotic alcoholic beverage may be > 3.8. In particular, the pH may be 4.0-6.0.

The probiotic alcoholic beverage may have a suitable Brix or its specific gravity equivalent. Brix is a measure of the amount of sugars in the alcoholic beverage. For example, 1 °Bx refers to 1 g of sucrose in 100 g of the alcoholic beverage. Accordingly, the higher the Brix, the higher the alcohol content may be in the alcoholic beverage. For example, the Brix of the probiotic alcoholic beverage may be 2-20 °Bx. In particular, the Brix may be 3-19°Bx, 4-18°Bx, 5-17°Bx, 6-16 °Bx, 7-15°Bx, 8-14 °Bx, 9- 13 °Bx, 10-12 °Bx. Even more in particular, the Brix of the probiotic alcoholic beverage may be 5-15 °Bx.

According to a second aspect of the present invention, there is provided a method of forming a probiotic alcoholic beverage of the first aspect, the method comprising adding microencapsulated probiotics to a fermented alcoholic beverage to form the probiotic alcoholic beverage. For the purposes of the present invention, fermented alcoholic beverage is defined as any beverage which comprises an alcohol, which may be fermented by yeast, bacteria, mould, or any combination thereof. The fermented alcoholic beverage may be any suitable fermented alcoholic beverage. Examples of a fermented alcoholic beverage may include, but is not limited to, beer, cider, fermented water, liquor, mead, pulque, rice wine, sparking wine, spirits, wine, and the like. According to a particular aspect, the fermented alcoholic beverage may be beer. The probiotic alcoholic beverage which is formed from the fermented alcoholic beverage thereby comprises microencapsulated probiotics.

According to a particular aspect, the method of forming the probiotic alcoholic beverage may further comprise forming the microencapsulated probiotics prior to the adding the microencapsulated probiotics to the fermented alcoholic beverage. The forming the microencapsulated probiotics may comprise adding probiotics to a solution to form a probiotics solution. The solution may be any solution suitable for forming a coating to encase the probiotics, thereby microencapsulating the probiotics. For example, the solution may be a gelatin solution, an alginate solution, a chitosan solution, a carrageenan solution, a glycerides solution, or a combination thereof. In particular, the solution may be sodium alginate, calcium alginate, or a combination thereof. The solution may be treated to be food-grade, such that ingestion of the formed microencapsulated probiotics does not cause health hazards. For example, the solution may be sterilised.

The forming the microencapsulated probiotics may further comprise freeze drying the probiotics prior to the adding the probiotics to the solution. The freeze drying advantageously prolongs shelf life and improve viability of the probiotics.

The forming the microencapsulated probiotics may further comprise placing the probiotics solution into a salt solution to form the microencapsulated probiotics. The salt solution may be any salt solution suitable for solidifying the probiotics solution to allow the probiotics to be encased within the coating formed from the solution. For example, the salt solution may be calcium chloride.

The method of forming the probiotic alcoholic beverage may further comprise centrifuging and/or pasteurizing the fermented alcoholic beverage prior to the adding the microencapsulated probiotics. The centrifuging may be at any suitable speed at any suitable temperature for any suitable time. For example, the centrifuging may be at 500-10000 rpm, 1000-9000 rpm, 2000-8000 rpm, 3000-7000 rpm, 4000-6000 rpm. In particular, the centrifuging may be at 1000-3000 rpm. The centrifuging may be at a temperature of 4-10 °C, 5-9 °C, 6-8 °C. In particular, the centrifuging may be at a temperature of 4 °C. The centrifuging may be for 5-30 minutes, 10-25 minutes, 15-20 minutes. In particular, the centrifuging may be for 10-20 minutes. The pasteurizing may be at any suitable temperature for any suitable time. For example, the pasteurizing may be at a temperature of 50-90 °C, 55-85 °C, 60-80 °C. In particular, the pasteurizing may be at a temperature of 55-85 °C. The pasteurizing may be for 5-1800 s.

The method of forming the probiotic alcoholic beverage may further comprise carbonation of the probiotic alcoholic beverage after the adding the microencapsulated probiotics. The carbonation may comprise dissolving carbon dioxide into the probiotic alcoholic beverage, thereby giving rise to effervescence and altering the mouthfeel of the probiotic alcoholic beverage. The carbonation may cause the probiotic alcoholic beverage to impart a pleasant and refreshing taste to the consumer.

The microencapsulated probiotics added to the fermented alcoholic beverage may be any suitable microencapsulated probiotics. For example, the microencapsulated probiotics may be as described above. The adding the microencapsulated probiotics may comprise adding a suitable amount of microencapsulated probiotics to the fermented alcoholic beverage. For example, the amount of microencapsulated probiotics added may have a cell count of 6.0-8.0 log CFU/mL.

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

Examples

Materials and methods

The experimental outline is as shown in the process flowchart of Figure 1.

Unhopped wort and hopped wort were separately prepared and inoculated with brewing yeast (wheat beer yeast S. cerevisiae SafAle WB-06). The fermentation vessel was covered with an airlock and was stored under 10-30 °C for 5-30 days of fermentation. The obtained fermented beer was centrifuged and pasteurized at 55-85 °C for 5 seconds to 30 minutes.

Freeze-dried probiotic yeasts (S. cerevisiae CNCM I-3856 and S. boulardii CNCM I- 745) and spore-forming bacteria (B. subtilis CU1 spores) were each mixed with food grade sterilized calcium alginate solution (0.2%-2% w/v, Phoon Huat Pte Ltd, Malaysia) to form the probiotics solutions. The probiotics solutions were filtered through a 70-pm sterilized easy strainer (Greiner Bio-One, Germany).

A microfluidic droplet generation system obtained from Singapore Institute of Manufacturing Technology (SIMtech) was used to perform microencapsulation of the probiotics in a laminar flow cabinet. The probiotics solutions were each injected through a microfluidic disposable chip into sterile CaCh. Based on preliminary testing, droplet dimensions of probiotics solutions were adjustable by setting parameters using a computer. The microencapsulated beads of S. cerevisiae CNCM I-3856, S. boulardii CNCM I-745 and B. subtilis CU1 spores were separated from the CaCl2 solution by filter paper, and then washed with deionised water thrice.

The microencapsulated probiotics were inoculated into 330 mL of unhopped beer and hopped beer, respectively. The S. cerevisiae CNCM I-3856, S. boulardii CNCM I-745, and B. subtilis CU1 (spores) in each of the inoculated beers had minimum cell counts of 6.0 Log CFU/mL. Equal amounts of non-microencapsulated freeze-dried probiotics cultures were also inoculated in unhopped beer and hopped beer as a control group.

The beer samples were immediately aliquoted into 15-mL centrifuge tubes (12 mL per tube) without carbonation, and stored at 30 °C for over 3 months to study the stability and survival of the probiotics. Triplicate samples of each treatment were taken to monitor the probiotic cell counts and physio-chemical parameters.

Morphology

The particle sizes of microcapsules were measured by Laser Diffraction/Scattering Particle Size Distribution Analyzer LA-950 (Horiba, France).

Probiotic cell counts

Probiotic yeast cell count was monitored using spread plating method on potato dextrose agar (PDA) (Oxoid, Basingstoke, Hampshire, England) at 30 °C for 2 days. B. subtilis CU1 cell counts were monitored using pour plating method with Mueller-Hinton Agar (Oxoid, Basingstoke, Hampshire, England) at 37 °C for 24 hours. The microencapsulated probiotic yeasts and B. subtilis CU1 were lysed with 1% w/v sodium citrate, and the cell counts of the probiotic yeasts and B. subtilis CU1 were monitored as aforementioned.

°Brix and pH

The pH and °Brix of the samples were measured with a pH meter (Metrohm, Switzerland) and a refractometer (ATAGO, Tokyo, Japan), respectively. Sensory evaluation

The experiment was repeated, and the beer samples were packaged in glass bottles (330 mL per bottle) for sensory evaluation. After 3 months of storage at 30 °C, the probiotics beer samples were rated by 8 trained panellists using a 4-point scale quantitative descriptive analysis, based on the 5 attributes: appearance, sweetness, aftertaste, body and taste score (Table 1).

Table 1 : 4-point scale quantitative descriptive analysis of beer attributes

Results and discussion

Morphology

The microencapsulated beads of probiotics were globular in shape had similar sizes. Over 70% of microbeads’ diameters were between 300 pm to 700 pm, and the median size was 338 pm. The other 20% of microbeads’ diameters were from 5 pm to 20 pm (Figure 2). The particle morphology of most microencapsulated probiotics had no substantial changes during storage, indicating that the microencapsulated beads will not be easily dissolved in the beers.

Probiotic cell counts

The survival of microencapsulated probiotics in unhopped beer and hopped beer samples during storage up to 120 days at 30 °C is as shown in Figures 3 and 4. In unhopped beer, the cell counts of microencapsulated S. cerevisiae CNCM I-3856 were reduced, from 7.70 Log CFU/mL to 6.28 Log CFU/mL in the first month of storage, and then kept stable at around 6.3 Log CFU/mL in the next 3 months. The microencapsulated S. boulardii CNCM I-745 was more stable and maintained a cell count of more than 6.3 Log CFU/mL in unhopped beer for around 4 months of storage at 30 °C (Figure 3A). Compared with the microencapsulated probiotic yeasts, the cell count of the non-microencapsulated probiotic yeast quickly reduced by about 1 Log CFU/mL from the second month of storage, retaining a cell count of less than 5.7 Log CFU/mL in unhopped beer. This indicates that microencapsulation contributes to the survival of probiotics in unhopped beer during storage at 30 °C, especially from the mid-term stage of storage onwards.

The probiotic yeasts cell counts all maintained more than 6.3 Log CFU/mL in hopped beer with or without microencapsulation treatment, among which the microencapsulated S. boulardii CNCM I-745 was the most stable in hopped beer (Figure 3B). However, microencapsulation treatment had a limited effect on the survival of S. cerevisiae CNCM I-3856 in hopped beer.

Similarly, microencapsulation had more effects on the stability of B. subtilis CU1 (spores) in unhopped beer, than that in hopped beer (Figure 4). In unhopped beer, the cell count of non-microencapsulated B. subtilis CU1 continuously reduced from 7.3 Log CFU/mL to 4.5 Log CFU/mL after nearly 4 months of storage at 30 °C, during which the microencapsulated B. subtilis CU1 only reduced by approximately 0.9 Log CFU/mL, from 6.9 Log CFU/mL to 6.0 Log CFU/mL (Figure 4A). One possible reason could be the germination of non-microencapsulated B. subtilis in unhopped beer in the absence of iso-a-acids, which causes the bacteria to be more susceptible to environmental stress in the absence of its proteinaceous coat. The microencapsulation treatment provided a calcium alginate-coating for B. subtilis.

In hopped beer, the microencapsulated B. subtilis cell count was stable from the midterm stage of storage, while the non-microencapsulated culture slightly decreased over the same period of time. In hopped beer, the B. subtilis cell count retained 6.7 Log CFU/mL and 6.3 Log CFU/mL in the microencapsulated group and the control group, respectively (Figure 4B).

The effects of microencapsulation on the survival rate of probiotics in beer samples are presented in Figure 5. After microencapsulation, the survival rate of S. cerevisiae CNCM I-3856, S. boulardii CNCM I-745, and B. subtilis CU1 in unhopped beer significantly increased from around 3% to 5%, from 4% to 17 % and from 0.1% to 17%, respectively (Figure 5A). This shows that microencapsulation could contribute to the viability of probiotics in unhopped beer. The survival rate of S. boulardii CNCM I-745, and B. subtilis CU1 in hopped beer increased at least 5-fold after microencapsulation, reaching around 45% and 65% respectively after 4 months of storage at 30 °C (Figure 5B). However, the contribution of microencapsulation on the survival of S. cerevisiae CNCM I-3856 in hopped beer was not significant.

°Brix and pH

The °Brix maintained stable in both unhopped beer and hopped beer after adding microencapsulated probiotics, suggesting that the microcapsules protected the probiotic strains from environmental stress during storage at 30 °C (Figures 6 and 7). The addition of non-microencapsulated probiotic yeasts and spore-forming bacteria caused a slight fluctuation in the °Brix of the unhopped and hopped beer during storage.

The effect of microencapsulation on the pH of yeast-based probiotic beer was limited (Figure 8). Both non-microencapsulated and microencapsulated probiotic yeasts did not affect the pH of unhopped beer and hopped beer, except for S. cerevisiae CNCM I- 3856 in hopped beer. Both non-microencapsulated and microencapsulated S. cerevisiae CNCM I-3856 increased the pH of hopped beer by about 0.3 unit (from 4.5 to 4.8) in the later stage of storage, which may be related to the metabolism of acids by released yeasts during storage term. For the beers containing spore-forming probiotic bacteria, the pH of hopped beer added with microencapsulated B. subtilis CU1 was more stable than that in the beer added with non-microencapsulated freeze-dried B. subtilis CU1 (Figure 9).

Sensory evaluation

Sensory evaluation of probiotic beer containing non-microencapsulated probiotics and microencapsulated probiotics are shown in Figure 10. After 3 months of storage, the appearance of beer containing non-microencapsulated probiotics became “too dark”, while the appearance of the non-probiotic beer and the beer containing microencapsulated probiotics were maintained as “refreshing”. This demonstrates that microencapsulation of probiotics effectively prevents beer browning during storage. Microencapsulation may also lower the bitterness of the beer aftertaste. The addition of B. subtilis CU1 (in both non-microencapsulated and microencapsulated forms) slightly increased the sweetness and lightened the body of beer. The addition of microencapsulated S. cerevisiae CNCM I-3856 also lightened the body of beer. Based on the taste score, non-probiotic beer was “satisfactory” after storage, which was the same as the beer added with non-microencapsulated B. subtilis. Addition of non-microencapsulated probiotic yeasts lowered taste scores to “barely satisfactory” after storage. Microencapsulation improved the beer taste scores, among which the beer containing microencapsulated B. subtilis had the highest taste score, as seen in Figure 10. These results suggest that addition of microencapsulated probiotics can maintain, and even improve, sensory quality of probiotic beer during storage at 30 °C.

Thus, the results showed that microencapsulation effectively increased the stability of probiotics in beer. All microencapsulated probiotics maintained over 6 Log CFU/mL cell counts in both hopped and unhopped beer during 4 months of storage at 30 °C. Moreover, addition of microencapsulated probiotics had beneficial effects on the sensory properties of probiotic beer, and effectively prevented the beer from browning during storage. Compared with probiotic yeasts, the stability of B. subtilis in beer was more significantly improved after microencapsulation. Consistently, the taste score of the beer added with microencapsulated B. subtilis was the highest in all beer samples.

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 probiotic alcoholic beverage comprising microencapsulated probiotics, wherein after storage of the probiotic alcoholic beverage at a predetermined temperature for a predetermined time, the microencapsulated probiotics have a cell count of > 5.0 log CFU/mL, wherein the predetermined temperature is 20-30 °C and the predetermined time is > 7 days.

2. The probiotic alcoholic beverage according to claim 1, wherein the predetermined time is > 90 days.

3. The probiotic alcoholic beverage according to claim 1 or 2, wherein the microencapsulated probiotics have a cell count of > 6.0 log CFU/mL after the storage of the probiotic alcoholic beverage.

4. The probiotic alcoholic beverage according to any preceding claim, wherein the probiotic alcoholic beverage comprises a hop or its derivative.

5. The probiotic alcoholic beverage according to claim 4, wherein the hop or its derivative has a bitterness of < 80 IBU.

6. The probiotic alcoholic beverage according to any preceding claim, wherein the microencapsulated probiotics comprises microencapsulated probiotic yeast, microencapsulated probiotic bacteria, or a combination thereof.

7. The probiotic alcoholic beverage according to claim 6, wherein the microencapsulated probiotic yeast comprises Saccharomyces yeast, non- Saccharomyces yeast, or a combination thereof.

8. The probiotic alcoholic beverage according to claim 6 or 7, wherein the microencapsulated probiotic yeast comprises Saccharomyces cerevisiae, Saccharomyces boulardii, or a combination thereof.

9. The probiotic alcoholic beverage according to claim 6, wherein the microencapsulated probiotic bacteria comprises microencapsulated spore-forming probiotic bacteria.

10. The probiotic alcoholic beverage according to claim 9, wherein the microencapsulated spore-forming probiotic bacteria comprises Bacillus.

11. The probiotic alcoholic beverage according to any preceding claim, wherein the microencapsulated probiotics have an average size of 5-1000 pm.

12. The probiotic alcoholic beverage according to any preceding claim, wherein the beverage has a pH of 4-6 after the storage of the probiotic alcoholic beverage.

13. The probiotic alcoholic beverage according to any preceding claim, wherein the probiotic alcoholic beverage has a Brix of 2-20 °Bx after the storage of the probiotic alcoholic beverage.

14. A method of forming a probiotic alcoholic beverage according to any preceding claim, the method comprising adding microencapsulated probiotics to a fermented alcoholic beverage to form the probiotic alcoholic beverage.

15. The method according to claim 14, further comprising forming the microencapsulated probiotics prior to the adding the microencapsulated probiotics to the fermented alcoholic beverage, wherein the forming comprises adding probiotics to a solution to form a probiotics solution.

16. The method according to claim 15, wherein the forming the microencapsulated probiotics further comprises freeze drying the probiotics prior to the adding the probiotics to the solution.

17. The method according to claim 16, wherein the forming the microencapsulated probiotics further comprises placing the probiotics solution into a salt solution to form the microencapsulated probiotics.

18. The method according to any of claims 14-17, further comprising centrifuging and/or pasteurizing the fermented alcoholic beverage prior to the adding the microencapsulated probiotics.

19. The method according to any of claims 14-18, further comprising carbonation of the probiotic alcoholic beverage after the adding the microencapsulated probiotics.

20. The method according to any of claims 14-19, wherein the microencapsulated probiotics comprises microencapsulated probiotic yeast, microencapsulated probiotic bacteria, or a combination thereof.

21. The method according to claim 20, wherein the microencapsulated probiotic yeast comprises Saccharomyces yeast, non- Saccharomyces yeast, or a combination thereof.

22. The method according to claim 20 or 21, wherein the microencapsulated probiotic yeast comprises Saccharomyces cerevisiae, Saccharomyces boulardii, or a combination thereof.

23. The method according to claim 20, wherein the microencapsulated probiotic bacteria comprises microencapsulated spore-forming probiotic bacteria.

24. The method according to claim 23, wherein the microencapsulated spore-forming probiotic bacteria comprises Bacillus.