JP7566466B2 - Alcoholic drinks - Google Patents

Description

The present invention relates to an alcoholic beverage. In particular, the alcoholic beverage may contain probiotic bacteria.

The health benefits that can be obtained from probiotics have led to an increased interest in consuming them. Many foods and beverages now contain probiotics. However, most of the foods and beverages are dairy-based products and therefore may not be ideal for people who are lactose intolerant. Therefore, research is ongoing into novel delivery methods for probiotics in new food environments outside the dairy sector.

Malt-based beverages have been shown to exhibit a protective effect on the viability of some Lactobacillus probiotic strains. Alcoholic beverages, especially specialty beers, are becoming increasingly popular among consumers. Beer is a malt-based beverage. However, maintaining probiotic viability in beer remains a major technical challenge, as beer has many inherent antimicrobial barriers that impede the growth and impair the survival of probiotic bacteria. The main antimicrobial compounds in beer are iso-α-acids, which act as ionophores and limit the growth of Gram-positive bacteria through various mechanisms, such as intracellular acidification and inhibition of energy production and redox homeostasis. Probiotic bacteria have not been isolated from beer and do not have mechanisms to overcome the inhibitory effects of acids to grow and survive in beer.

The present invention seeks to address these challenges and/or provide improved beverages containing probiotic bacteria.

Broadly speaking, the present invention relates to alcoholic beverages that contain probiotic bacteria. The alcoholic beverages of the present invention may provide health benefits compared to other alcoholic beverages in that they contain probiotic bacteria. In particular, probiotic bacteria have been shown to provide health benefits such as enhanced gut health and immune system function.

According to a first aspect, the present invention provides an alcoholic beverage comprising probiotic bacteria.

The alcoholic beverage may have a suitable alcohol content. According to a particular embodiment, the alcohol content of the alcoholic beverage may be ≧0.5% by volume. In particular, 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%. Even more particularly, the alcohol content may be 2.0-5.0%.

The alcoholic beverage may be any suitable alcoholic beverage. In particular, the alcoholic beverage is beer. According to a particular embodiment, the alcoholic beverage may further comprise hops. For the purposes of the present invention, reference to hops refers to hops and/or derivatives thereof. The hops may be any suitable hops. For example, the hops may be an isomerized hop extract. The hops may have a suitable bitterness. In particular, the hops may have a bitterness of ≦30 IBU. Even more particularly, the bitterness may be 0-30 IBU, 3-27 IBU, 5-25 IBU, 7.5-20 IBU, 9-18 IBU, 10-16 IBU, 12-15 IBU, 13-14 IBU.

The probiotic bacteria included in the alcoholic beverage may be any suitable probiotic bacteria. For example, the probiotic bacteria may include lactobacilli, bifidobacteria, or a combination thereof. In particular, the lactobacilli may include, but are not limited to, Lactobacillus (L.) paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or a combination thereof.

A suitable amount of probiotic bacteria may be included in the alcoholic beverage. For example, the probiotic bacteria may have a cell count of ≧5.0 log CFU/mL. In particular, the probiotic bacteria included in the alcoholic beverage may have a cell count of 5.0-12.0 log CFU/mL, 5.5-11.5 log CFU/mL, 6.0-11.0 log CFU/mL, 6.5-10.5 log CFU/mL, 7.0-10.0 log CFU/mL, 7.5-9.5 log CFU/mL, 8.0-9.0 log CFU/mL. Even more specifically, the probiotic bacteria included in the alcoholic beverage may have a cell count of about 7.0 log CFU/mL.

The alcoholic beverage may have a suitable pH. For example, the pH of the alcoholic beverage may be between 2 and 6. In particular, the pH may be between 2.5 and 5.5, between 3 and 5, between 3.5 and 4.5, between 3.75 and 4.0. Even more particularly, the pH may be between about 3 and 5.

The alcoholic beverage may have a suitable Brix. For example, the Brix of the alcoholic beverage may be 4-20°Bx. In particular, the Brix may be 5-18°Bx, 6-15°Bx, 7-12°Bx, 8-10°Bx, 9-9.5°Bx. Even more specifically, the Brix may be about 5-15°Bx.

According to a second aspect of the present invention, there is provided a method for forming an alcoholic beverage as described above, comprising the steps of:
- providing wort or must;
- adding probiotic bacteria to the wort or must;
- adding yeast to the wort or must; and
- fermenting the wort or must for a predetermined period of time and at a predetermined temperature to form an alcoholic beverage.

The wort or must may be any wort or must suitable for the purposes of the present invention.

The probiotic bacteria added to the wort or must may be any suitable probiotic bacteria. For example, the probiotic bacteria may be as described above.

The yeast added to the wort or must may be any yeast suitable for the purposes of the present invention. For example, the yeast may be a Saccharomyces yeast, a non-Saccharomyces yeast, or a combination thereof. In certain embodiments, the yeast may be, but is not limited to, Saccharomyces (S.) cerevisiae, S. pasteurianus, Torulaspora (T.) delbrueckii, Lachancea (L.) thermotolerans, Pichia (P.) kluyveri, Metschnikowia (M.) pulcherrima, or a combination thereof.

The method may further include adding hops to the wort or must. The hops added to the wort or must may be any suitable hop described above.

The addition of probiotic bacteria and yeast may be done simultaneously or sequentially.

According to certain embodiments, the addition of the probiotic bacteria and the addition of the yeast may occur simultaneously. Fermentation may then occur under suitable conditions. For example, fermentation may include fermenting the wort or must at a first temperature for a first period of time, and then fermenting the wort or must at a second temperature for a second period of time. The method may further include adding hops at the end of the second period of time.

According to certain embodiments, the addition of the probiotic bacteria and the addition of the yeast may be performed sequentially. In particular, the addition of the yeast may be performed after a third period of time after the addition of the probiotic bacteria. Fermentation may then be performed under suitable conditions. For example, the fermentation may include fermenting the wort or must at a third temperature for a fourth period of time after the addition of the yeast, and then fermenting the wort or must at a fourth temperature for a fifth period of time. The method may further include adding hops at the end of the fifth period of time.

The first period, second period, third period, fourth period and fifth period may be any suitable amount of time. The periods may be selected depending on the probiotic bacteria and yeasts added to the wort or must.

The first temperature, the second temperature, the third temperature, and the fourth temperature may be any temperature suitable for the purposes of the present invention. For example, the first temperature and the third temperature may be the same. For example, the second temperature and the fourth temperature may be the same.

In particular, the first temperature and the third temperature may be the same or different and may be between 20 and 42°C. Even more specifically, the first temperature and the third temperature may be the same or different and may be about 30°C.

In particular, the second temperature and the fourth temperature may be the same or different and may be between 18 and 30°C. Even more specifically, the second temperature and the fourth temperature may be the same or different and may be about 20°C.

According to certain embodiments, the alcoholic beverage formed may be stored at a suitable temperature after fermentation. For example, the alcoholic beverage formed from the method of the present invention may be stored at a temperature of ≦20° C. In particular, the alcoholic beverage may be stored at a temperature of about ≦10° C. Even more particularly, the alcoholic beverage may be stored at a temperature of about 1-5° C.

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

Figure 1 shows a graph of survival of three probiotic bacterial strains during 10 days of fermentation in hopped wort: (●) L. paracasei L26; (X) L. paracasei Lpc-37 and; (◆) L. rhamnosus HN001. ^#Error bars indicate standard deviation (n=3). *Fermentation temperature was 37°C throughout. Figure 2 shows the growth and survival of L. paracasei L26 during co-fermentation and storage at 5°C and 25°C: (○) L. paracasei L26, 5°C; (●) L. paracasei L26, 25°C; (□) co-inoculation of L. paracasei L26 and S. cerevisiae S-04, 5°C and; (■) co-inoculation of L. paracasei L26 and S. cerevisiae S-04, 25°C. ^* Fermentation temperature was 30°C from day 0 to day 2 and 20°C from day 2 to day 10. ^* 27 IBU of isomerized hop extract was added on day 10. Figure 3 shows the growth and survival of L. paracasei L26 during successive fermentations and storage at 5°C and 25°C: (○) L. paracasei L26, 5°C; (●) L. paracasei L26, 25°C; (□) successive inoculations of L. paracasei L26 and S. cerevisiae S-04, 5°C and; (■) successive inoculations of L. paracasei L26 and S. cerevisiae S-04, 25°C. ^* Fermentation temperatures were 30°C from day 0 to day 2 and 20°C from day 2 to day 10. ^S. cerevisiae S-04 was added on day 2. ^# 27 IBU of isomerized hop extract was added on day 10. Figure 4 shows the growth and survival of co-inoculated L. paracasei L26 during storage of beer at 5°C: (□) 0 IBU, 5°C; (X) 7.5 IBU, 5°C; (○) 15 IBU, 5°C; (△) 22.5 IBU, 5°C. ^* Fermentation temperatures were 30°C from day 0 to day 2 and 20°C from day 2 to day 10. ^#Isomerized hop extract was added on day 10. Figure 5 shows the growth and survival of co-inoculated L. paracasei L26 during storage of beer at 25°C: (■) 0 IBU, 25°C; (+) 7.5 IBU, 25°C; (●) 15 IBU, 25°C; (▲) 22.5 IBU, 25°C. ^* Fermentation temperatures were 30°C from days 0 to 2 and 20°C from days 2 to 10. ^#Isomerized hop extract was added on day 10. Figure 6(a) shows the change in pH during fermentation and storage of beer at 5°C: (□) 0 IBU, 5°C; (X) 7.5 IBU, 5°C; (○) 15 IBU, 5°C; (△) 22.5 IBU, 5°C, and Figure 6(b) shows the change in pH during fermentation and storage of beer at 25°C: (■) 0 IBU, 25°C; (+) 7.5 IBU, 25°C; (●) 15 IBU, 25°C; (▲) 22.5 IBU, 25°C. Figure 7(a) shows the change in Brix during fermentation and storage of beer at 5°C: (□) 0 IBU, 5°C; (X) 7.5 IBU, 5°C; (○) 15 IBU, 5°C; (△) 22.5 IBU, 5°C, and Figure 7(b) shows the change in Brix during fermentation and storage of beer at 25°C: (■) 0 IBU, 25°C; (+) 7.5 IBU, 25°C; (●) 15 IBU, 25°C; (▲) 22.5 IBU, 25°C. Figure 8 shows survival of serially inoculated L. paracasei L26 (after prior yeast fermentation) during storage of beer at 5°C and 25°C: (○) 5°C; (△) 25°C. ^#Hopped wort (equivalent to 27 IBU) was fermented with S. cerevisiae S-04 for 10 days prior to addition of L. paracasei L26. FIG. 9 shows the change in pH during fermentation and storage of serially inoculated probiotic beers at 5° C. and 25° C.: (○) 5° C.; (△) 25° C. FIG. 10 shows the change in °Brix during fermentation and storage of serially inoculated probiotic beers at 5° C. and 25° C.: (○) 5° C.; (△) 25° C. Figure 11 shows the growth and survival of the co-inoculated probiotics during fermentation and storage of beer at 5°C: (◆) L. paracasei L26; (■) L. rhamnosus HN001; (▲) L. acidophilus NCFM. ^* Fermentation temperature was 30°C from day 0 to day 2 and 20°C from day 2 to day 10. ^# 7.5 IBU of isomerized hop extract was added on day 10. Figure 12 shows the growth and survival of co-inoculated probiotics during fermentation and storage of beer at 25°C: (◇) L. paracasei L26; (□) L. rhamnosus HN001; (△) L. acidophilus NCFM. ^* Fermentation temperature was 30°C from day 0 to day 2 and 20°C from day 2 to day 10. ^# 27 IBU of isomerized hop extract was added on day 10. Figure 13(a) shows the change in pH during fermentation and storage of beer at 5°C: (◆) L. paracasei L26; (■) L. rhamnosus HN001; (▲) L. acidophilus NCFM, and Figure 13(b) shows the change in pH during fermentation and storage of beer at 25°C: (◇) L. paracasei L26; (□) L. rhamnosus HN001; (△) L. acidophilus NCFM. Figure 14(a) shows the change in °Brix during fermentation and storage of beer at 5°C: (◆) L. paracasei L26; (■) L. rhamnosus HN001; (▲) L. acidophilus NCFM, Figure 14(b) shows the change in °Brix during fermentation and storage of beer at 25°C: (◇) L. paracasei L26; (□) L. rhamnosus HN001; (△) L. acidophilus NCFM. Figure 15 shows the growth of L. paracasei L26 during co-fermentation with non-Saccharomyces yeasts and subsequent storage at 5°C: (■) L. paracasei L26 and T. delbrueckii Prelude; (●) L. paracasei L26 and M. pulcherrima Flavia. Fermentations were carried out at 30°C from days 0 to 2 and at 20°C from days 2 to 12. ^# 7.5 IBU of isomerized hop extract was added on day 12. Figure 16 shows the growth of L. paracasei L26 during co-fermentation with non-Saccharomyces yeasts and subsequent storage at 25°C: (□) L. paracasei L26 and T. delbrueckii Prelude; (◯) L. paracasei L26 and M. pulcherrima Flavia. Fermentations were carried out at 30°C from days 0 to 2 and at 20°C from days 2 to 12. ^# 7.5 IBU of isomerized hop extract was added on day 12. Figure 17(a) shows the pH change of unhopped wort during fermentation and storage at 5°C, and Figure 17(b) shows the pH change of unhopped wort during fermentation and storage at 25°C: (■) L. paracasei L26 and T. delbrueckii Prelude; (●) L. paracasei L26 and M. pulcherrima Flavia. (□); T. delbrueckii Prelude only; (○) M. pulcherrima Flavia only. Figure 18(a) shows the change in °Brix of unhopped wort during fermentation and storage at 5°C, and Figure 18(b) shows the change in °Brix of unhopped wort during fermentation and storage at 25°C: (■) L. paracasei L26 and T. delbrueckii Prelude; (●) L. paracasei L26 and M. pulcherrima Flavia. (□); T. delbrueckii Prelude only; (○) M. pulcherrima Flavia only. Figure 19 shows the growth and survival of L. paracasei L26 during co-inoculation and sequential inoculation with S. cerevisiae W-34/70 during fermentation and storage of beer at 5°C: (●) co-inoculation of L. paracasei L26 and S. cerevisiae W-34/70 at 5°C; (■) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70 at 5°C. ^* Fermentation temperature was 30°C from day 0 to day 1 and 20°C from day 1 to day 22. ^# 7.5 IBU of isomerized hop extract was added on day 22. ^ S. cerevisiae W-34/70 was added on day 1 of fermentation for sequential inoculation. Figure 20 shows the growth and survival of L. paracasei L26 during co-inoculation and sequential inoculation with S. cerevisiae W-34/70 during fermentation and storage of beer at 25°C: (o) co-inoculation of L. paracasei L26 and S. cerevisiae W-34/70 at 25°C; (□) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70 at 25°C. ^* Fermentation temperature was 30°C from day 0 to day 1 and 20°C from day 1 to day 22. ^# 7.5 IBU of isomerized hop extract was added on day 22. ^ S. cerevisiae W-34/70 was added on day 1 of fermentation for sequential inoculation. Figure 21(a) shows the change in pH during fermentation and storage of beer at 5°C: (●) coinoculation of L. paracasei L26 and S. cerevisiae W-34/70, 5°C; (■) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70, 5°C; (▲) S. cerevisiae W-34/70 monoculture, 5°C. Figure 21(b) shows the change in pH during fermentation and storage of beer at 25°C: (◯) coinoculation of L. paracasei L26 and S. cerevisiae W-34/70, 25°C; (□) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70, 25°C; (△) S. cerevisiae W-34/70 monoculture, 25°C. Figure 22(a) shows the change in °Brix during fermentation and storage of beer at 5°C: (●) coinoculation of L. paracasei L26 and S. cerevisiae W-34/70, 5°C; (■) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70, 5°C; (▲) S. cerevisiae W-34/70 monoculture, 5°C, Figure 22(b) shows the change in °Brix during fermentation and storage of beer at 25°C: (◯) coinoculation of L. paracasei L26 and S. cerevisiae W-34/70, 25°C; (□) sequential inoculation of L. paracasei L26 and S. cerevisiae W-34/70, 25°C; (△) S. cerevisiae W-34/70 monoculture, 25°C.

DETAILED DESCRIPTION As explained above, there is a need for non-dairy beverages as a means for delivering probiotic bacteria.

The present invention relates to alcoholic beverages containing probiotic bacteria. Generally, it is difficult to anticipate incorporating probiotic bacteria into alcoholic beverages such as beer due to the presence of antimicrobial compounds that prevent the growth and impair the survival of probiotic bacteria. Thus, the present invention provides a surprising and unexpected alcoholic beverage in which probiotic bacteria are able to grow and survive despite the presence of antimicrobial compounds in the alcoholic beverage.

Probiotic bacteria are known to provide health benefits. For example, it has been shown that certain strains of probiotic bacteria can result in improved gut health and digestion. Thus, an alcoholic beverage according to the present invention may provide health benefits such as improved gut health and enhanced immunity.

According to a first aspect, the present invention provides an alcoholic beverage comprising probiotic bacteria.

For the purposes of this invention, an alcoholic beverage is defined as a beverage that contains alcohol, such as ethanol or ethyl alcohol. In particular, an alcoholic beverage may have an alcohol content of ≥ 0.5% by volume.

The alcoholic beverage may have a suitable alcohol content. According to certain embodiments, the alcohol content of the 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 particularly, the alcohol content may be about 3-4%.

The alcoholic beverage may be any suitable alcoholic beverage. Examples of alcoholic beverages include, but are not limited to, beer, wine, cider, spirits, etc. According to certain aspects, the alcoholic beverage may be beer.

The probiotic bacteria contained in the alcoholic beverage may be any suitable probiotic bacteria. The probiotic bacteria may be any suitable live microorganism that, when provided in an appropriate amount, confers a health benefit to the recipient. For example, the probiotic bacteria may include Lactobacillus, Bifidobacteria, or a combination thereof. In particular, the Lactobacillus may include, but is not limited to, L. paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or a combination thereof. Even more specifically, the Lactobacillus may include Lactobacillus paracasei LAFTI L26, Lactobacillus paracasei Lpc-37, Lactobacillus rhamnosus HN001, Lactobacillus acidophilus NCFM, or a combination thereof.

A suitable amount of probiotic bacteria may be included in the alcoholic beverage. For example, the probiotic bacteria may have a cell count of ≧5.0 log CFU/mL. In particular, the probiotic bacteria included in the alcoholic beverage may have a cell count of 5.0-12.0 log CFU/mL, 5.5-11.5 log CFU/mL, 6.0-11.0 log CFU/mL, 6.5-10.5 log CFU/mL, 7.0-10.0 log CFU/mL, 7.5-9.5 log CFU/mL, 8.0-9.0 log CFU/mL. Even more specifically, the probiotic bacteria included in the alcoholic beverage may have a cell count of about 7.0 log CFU/mL.

According to certain embodiments, the alcoholic beverage may further comprise hops. For purposes of the present invention, hops includes hops and/or derivatives thereof. The hops may be any suitable hops that contain isomerized alpha acids. The hops may be in any suitable form, such as, but not limited to, hop cones, hop pellets, hop resin, hop powder, isomerized hop extract, etc.

The hops may be any suitable hops. For example, the hops may be an isomerized hop extract. The hops may have a suitable bitterness. In particular, the hops may have a bitterness of ≦30 IBU. Even more specifically, the bitterness may be 0-30 IBU, 3-27 IBU, 5-25 IBU, 7.5-20 IBU, 9-18 IBU, 10-16 IBU, 12-15 IBU, 13-14 IBU. IBU refers to International Bitterness Units and is a measure of the concentration of hop compounds in an alcoholic beverage. In particular, IBU measures the parts per million (ppm) of isohumulones in an alcoholic beverage.

The alcoholic beverage may have a suitable pH. For example, the pH of the alcoholic beverage may be between 2 and 6. In particular, the pH may be between 2.5 and 5.5, between 3 and 5, between 3.5 and 4.5, between 3.7 and 4.0. Even more particularly, the pH may be between about 3 and 5.

The alcoholic beverage may have a suitable Brix or its specific gravity equivalent. Brix is a measure of the amount of sugar in an alcoholic beverage. For example, 1°Bx refers to 1g of sucrose in 100g of alcoholic beverage. Thus, the higher the Brix, the higher the alcohol content may be in the alcoholic beverage. The Brix of an alcoholic beverage may be between 4 and 20°Bx. For the purposes of the present invention, reference to the Brix of an alcoholic beverage refers to a measure of the Brix of the wort or must contained in the alcoholic beverage. In particular, the Brix of an alcoholic beverage may be between 5 and 18°Bx, 6 and 15°Bx, 7 and 12°Bx, 8 and 10°Bx, 9 and 9.5°Bx. Even more specifically, the Brix may be about 5 and 15°Bx.

The alcoholic beverage may be stored at a suitable temperature to maintain the probiotic bacteria at an appropriate level. For example, the alcoholic beverage may be stored at a temperature of about ≦20° C. Preferably, the alcoholic beverage may be stored at a temperature of ≦10° C. In particular, the alcoholic beverage may be stored at a temperature of about 1-10° C., 5-8° C., 6-7° C. Even more specifically, the alcoholic beverage may be stored at a temperature of about 1-5° C.

According to a second aspect of the present invention, there is provided a method of forming an alcoholic beverage of the first aspect, comprising the steps of:
- providing wort or must;
- adding probiotic bacteria to the wort or must;
- adding yeast to the wort or must; and
- fermenting the wort or must for a predetermined period of time and at a predetermined temperature to form an alcoholic beverage.

The wort or must may be any wort or must suitable for purposes of the present invention. For purposes of the present invention, wort may include, but is not limited to, barley malt and/or adjuncts thereof, such as wheat, corn, rye, rice, and water (which has been heated, boiled, and cooled). Wort may also include added sugars. For purposes of the present invention, must may include, but is not limited to, fruit juice, such as grape juice (which has been heated, boiled, and cooled). Must may also include added sugars.

The probiotic bacteria added to the wort or must may be any suitable probiotic bacteria. For example, the probiotic bacteria may be as described above. Adding the probiotic bacteria may include adding a suitable amount of the probiotic bacteria to the wort or must. For example, the amount of probiotic bacteria added may be 1-9 log CFU/ml. In particular, the amount of probiotic bacteria added may be about 2-8 log CFU/ml, 3-7 log CFU/ml, 4-6 log CFU/ml, 4.5-5 log CFU/ml. Even more particularly, the amount of probiotic bacteria added may be 5-7 log CFU/ml.

The yeast added to the wort or must may be any yeast suitable for the purposes of the present invention. The yeast may be a Saccharomyces yeast, a non-Saccharomyces yeast, or a combination thereof. According to a particular embodiment, the yeast may be a Saccharomyces yeast. Examples of Saccharomyces yeast include, but are not limited to, S. cerevisiae, S. paradoxus, S. pasteurianus, S. bayanus, and S. cerevisiae var. boulardii. According to another particular embodiment, the yeast may be a non-Saccharomyces yeast. Examples of non-Saccharomyces yeast include, but are not limited to, T. delbrueckii, L. thermotolerans, P. kluyveri, and M. pulcherrima. In particular, adding yeast to the wort or must includes adding S. cerevisiae.

The addition of yeast may include adding a suitable amount of yeast to the wort or must. For example, the amount of yeast added may be between 1 and 9 log CFU/ml. In particular, the amount may be between about 5 and 7 log CFU/mL.

The method may further include adding hops to the wort or must. The hops added to the wort or must may be any suitable hop as described above. Adding hops may include adding a suitable amount of hops to the wort or must. For example, the amount of hops added may be ≦30 IBU. Specifically, the amount of hops added may be 0-30 IBU, 3-27 IBU, 5-25 IBU, 7.5-20 IBU, 9-18 IBU, 10-16 IBU, 12-15 IBU, 13-14 IBU. Even more specifically, the amount of hops added may be about 7.5 IBU.

The addition of probiotic bacteria and yeast may be done simultaneously or sequentially.

According to certain embodiments, the addition of the probiotic bacteria and the addition of the yeast may occur simultaneously. Fermentation may then occur under suitable conditions. For example, the fermentation may include 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 fermentation. In particular, the fermentation may include fermenting the wort or must at a first temperature for a first period of time, and then fermenting the wort or must at a second temperature for a second period of time. The method may further include adding hops at the end of the second period of time.

According to certain embodiments, the addition of the probiotic bacteria and the addition of yeast may occur sequentially. In particular, the addition of yeast may occur after a third period of time after the addition of the probiotic bacteria. Fermentation may then occur under suitable conditions. For example, the fermentation may include 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 fermentation. In particular, the fermentation may include fermenting the wort or must at a third temperature for a fourth period of time, and then fermenting the wort or must at the fourth temperature for a fifth period of time. The method may further include adding hops at the end of the fifth period of time.

The first, second, third, fourth and fifth periods may be any suitable amount of time. The periods may be selected depending on the probiotic bacteria and yeasts added to the wort or must. For example, the first, third and fourth periods may be 1-5 days, preferably 1-2 days. The second and fifth periods may be 8-30 days.

The first temperature, the second temperature, the third temperature, and the fourth temperature may be any temperature suitable for the purposes of the present invention. For example, the first temperature and the third temperature may be the same. For example, the second temperature and the fourth temperature may be the same.

In particular, the first temperature and the third temperature may be the same or different and may be any suitable temperature that provides favorable conditions for the growth of the probiotic bacteria and allows the maximum probiotic bacterial cell number to be reached. For example, the first temperature and the third temperature may be between 25 and 35°C. Even more particularly, the first temperature and the third temperature may be the same or different and may be about 30°C.

In particular, the second temperature and the fourth temperature may be the same or different and may be any suitable temperature to provide for yeast growth. For example, the second temperature and the fourth temperature may be between 13 and 25°C. Even more particularly, the second temperature and the fourth temperature may be the same or different and may be about 20°C.

In one embodiment, the method includes simultaneously adding the probiotic bacteria and adding yeast to the wort. The wort may be hop-free. The probiotic bacteria and yeast are co-inoculated into the wort for a first period of time at a first temperature. For example, the first temperature may be about 25-35°C. In particular, the first temperature may be about 30°C. The first period of time may be about 1-5 days. In particular, the first period of time may be 2 days. Fermentation is then allowed to proceed for a second period of time at a second temperature. The second temperature may be about 13-25°C. In particular, the second temperature may be about 20°C. The second period of time may be about 8-30 days. In particular, the second period of time may be 8, 12 or 21 days. An appropriate concentration of isomerized hop extract is then added to the fermented wort at the end of the second period of time. For example, the hop extract may be a 7.5 IBU hop extract. The formed alcoholic beverage is then stored at an appropriate temperature, such as about 1-5°C.

In another embodiment, the method comprises first adding the probiotic bacteria to the wort and only adding the yeast after a third period of time. The wort may be unhopped. The third period of time may be 1-5 days. In particular, the yeast may be added 1 or 2 days after the addition of the probiotic bacteria to the wort. The probiotic bacteria may be inoculated into the wort for a fourth period of time at a third temperature. The third temperature may be about 25-35°C. In particular, the third temperature may be about 30°C. The fourth period of time may be about 1-5 days. In particular, the fourth period of time may be 2 days. Fermentation is then allowed to proceed for a fifth period of time at a fourth temperature. The fourth temperature may be about 13-25°C. In particular, the fourth temperature may be about 20°C. The fifth period of time may be about 8-30 days. In particular, the fifth period of time may be 8 or 21 days. An appropriate concentration of isomerized hop extract is then added to the fermented wort at the end of the fifth period. For example, the hop extract may be a 7.5 IBU hop extract. The formed alcoholic beverage is then stored at an appropriate temperature, such as about 1-5°C.

According to certain embodiments, the alcoholic beverage formed may be stored at a suitable temperature after fermentation. For example, the alcoholic beverage formed from the method of the present invention may be stored at a temperature of ≦20° C. In particular, the alcoholic beverage may be stored at a temperature of about ≦10° C. Even more particularly, the alcoholic beverage may be stored at a temperature of about 1-5° C.

According to another aspect, there is provided an alcoholic beverage as described above for use in medicine. In particular, the alcoholic beverage of the present invention may be for use in improving the intestinal health and/or digestion of a consumer of the alcoholic beverage.

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

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

All materials used in the examples provided are as follows:

Sweet wort was prepared by reconstituting 12.2% (w/v) dry light malt extract (Thomas Coopers Breweries, South Australia, Australia) in deionized water followed by boiling for 20 min to achieve hot break. Then, 2.0% (w/v) dextrose (Thomas Coopers Breweries, South Australia, Australia) and 0.2% (w/v) Cascade hop pellets (Yakima Chief-Hopunion, Yakima, USA) were mixed into the wort and boiled for an additional 60 min. Then, cold Ice Mountain distilled water (Fraser and Neave, Limited, Malaysia) was added to the original batch weight and the wort was cooled using an ice bath for approximately 60 min to achieve cold break. The cooled wort was then filtered through a double layer of cheesecloth into 250 mL or 500 mL capped glass bottles and pasteurized at 95 °C for 15 min to ensure sterility of the wort, which was confirmed using potato dextrose agar (PDA; Oxoid Ltd., Hampshire, UK). Hops were omitted in the preparation of unhopped wort for use as a preculture and for the simultaneous and sequential fermentation stages.

Example 1 - Antimicrobial effect of iso-α-acids against probiotic bacteria
To clarify the antibacterial effect of hop acids against probiotic bacteria and the difficulties of incorporating probiotic bacteria into hopped beer, hopped wort was fermented with three different probiotic strains, L. paracasei L26, L. paracasei Lpc-37, and L. rhamnosus HN001, and their growth and stability were recorded. In the screening phase, triple fermentations of L. paracasei L26, L. paracasei Lpc-37, and L. rhamnosus HN001 were performed in 500 mL capped glass bottles containing 200 mL of hopped wort. Each glass bottle was inoculated with 1% (v/v) of the respective probiotic preculture and incubated statically at 37°C for 10 days.

Figure 1 shows the survival kinetics of L. paracasei L26, L. paracasei Lpc-37 and L. rhamnosus HN001 in hopped wort throughout the 10-day fermentation period. The cell numbers of all three probiotic strains gradually decreased and were undetectable by day 7. The cell death could be due to the presence of hop iso-α-acids, since the probiotic bacteria were able to reach high viable cell numbers of at least about 8.50 Log CFU/mL in unhopped wort during growth in precultures grown in unhopped wort (data not shown). Thus, the decrease in cell numbers indicated that all three probiotic strains could not survive in hopped wort and therefore were not hop-resistant. Probiotic cell death in hopped wort is not surprising, since these bacteria do not have the complex mechanisms that allow them to successfully adapt to grow and survive in beer.

L. paracasei L26 showed the highest viability, followed by L. paracasei Lpc-37 and L. rhamnosus HN001. L. paracasei L26 still had a detectable cell count of 5.13 Log CFU/mL on day 4, while L. paracasei Lpc-37 and L. rhamnosus HN001 had cell counts of 6.12 and 4.13 Log CFU/mL, respectively, on day 2. Thus, L. paracasei L26 showed a better degree of resistance to hop iso-α-acids before dying out completely. Because L. paracasei L26 showed the greatest viability in hopped wort, L. paracasei L26 was used for further fermentation with yeast, as shown in the following examples.

Example 2 - Probiotic beer fermentation using different inoculation strategies
2.1 Co-inoculation of S. cerevisiae S-04 and L. paracasei L26
During co-fermentation, triple fermentations were performed in 500 mL capped glass bottles containing 400 mL of unhopped wort. L. paracasei L26 and S. cerevisiae S-04 yeasts were co-cultured at approximately 6.72 Log CFU/mL and 5.00 Log CFU/mL, respectively, to favor the growth of L. paracasei L26. Controls contained the same inoculum of L. paracasei L26 and S. cerevisiae S-04 precultures, respectively, in unhopped wort. Fermentation was initially performed statically at 30° C. from day 0 to day 2 to provide favorable conditions for probiotic growth and allow maximum probiotic cell numbers to be reached. Fermentation was then allowed to proceed at 20° C. from day 2 to day 10 to allow for yeast growth to saturate.

After 10 days of fermentation, shelf-life testing was performed on the same samples (shelf-life phase). On day 10, 27 International Bitterness Units (IBU) of isomerized hop extract (Brouwland, Beverlo, Belgium) was added to the unhopped beer at the same hop concentration as in Example 1. To evaluate the effect of refrigerated and ambient storage temperatures on probiotic viability, samples were stored at 5°C and 25°C. The end of shelf-life for each fermentation set was determined when the plate count of L. paracasei L26 fell below 7.0 Log CFU/mL.

The results obtained are shown in Figure 2. Based on Figure 2, during the fermentation period without hop extract, L. paracasei L26 monoculture (in the absence of yeast) was able to achieve a high stationary phase cell count of 9.06 Log CFU/mL on day 10 in the absence of hops. The results therefore reinforce the antibacterial effect of iso-α-acids against L. paracasei L26. In the presence of yeast, L. paracasei L26 still achieved a high stationary phase cell count and was able to maintain this throughout the 10-day fermentation period, recording 8.77 log CFU/mL on day 10. The results show a successful co-fermentation between L. paracasei L26 and S. cerevisiae S-04 during the growth and stationary phases.

As can be seen from Figure 2, after the addition of hop extract on day 10, at 25°C, L. paracasei L26 monoculture cell counts became undetectable within 1 day of storage, while L. paracasei L26 coculture cell counts dropped below 7.0 log CFU/mL by day 13. Thus, the viability-enhancing effect of S. cerevisiae S-04 was not strongly observed at the storage temperature of 25°C. However, the viability-enhancing effect was more pronounced at 5°C. At 5°C, L. paracasei L26 monoculture cell counts dropped below 7.0 log CFU/mL by day 16, while L. paracasei L26 cocultures successfully maintained high viable cell counts throughout storage, only dropping below the 7.0 log CFU/mL threshold (necessary to provide health benefits) by day 32. Thus, the viability-enhancing effect is due to both the refrigerated storage temperature and the presence of yeast.

The characteristics of non-volatile substances of hopped beer at various storage temperatures are shown in Table 1. According to Table 1, lactic acid produced by L. paracasei L26 resulted in low pH values (3.52-3.64) recorded in beers inoculated with both L. paracasei L26 and S. cerevisiae S-04. Therefore, due to the contribution by lactic acid (5.08-5.15 g/L), the resulting beer would have a sour taste.

2.2 Sequential inoculation of yeast and probiotic bacteria The effect of adding S. cerevisiae S-04 yeast on the second day of fermentation on the viability of L. paracasei L26 during the continuous fermentation phase was investigated to determine whether probiotic viability could be improved with delayed addition of yeast.

A similar procedure as for co-inoculation (Example 2.1) was performed for sequential inoculation fermentation. Triplicate fermentations were performed in 500 mL capped glass bottles containing 400 mL of unhopped wort. L. paracasei L26 (1% (v/v) inoculum) was added to each glass bottle. On the second day of fermentation, 1% (v/v) of S. cerevisiae S-04 was added to the same samples. Monocultures of L. paracasei L26 and S. cerevisiae S-04 in the same inoculum served as controls. Fermentation was initially performed statically at 30 °C from day 0 to day 2 to provide favorable conditions for probiotic bacterial growth and allow maximum probiotic bacterial cell numbers to be reached. Fermentation was then allowed to proceed at 20 °C from day 2 to day 10 to allow for yeast growth to be met.

After 10 days of fermentation, shelf-life testing was performed on the same samples. The procedure was similar to that for the co-inoculation experiment outlined in Example 2.1. The same doses of isomerized hop extract, storage temperatures, and end-of-shelf-life conditions were utilized.

The growth kinetics of L. paracasei L26 in hop-free wort during a 10-day continuous fermentation period and its survival kinetics during the subsequent continuous fermentation storage period after the addition of isomerized hop extract are shown in Figure 3.

According to Figure 3, similar growth kinetics were observed as in the co-fermentation. In the absence of yeast, L. paracasei L26 monoculture was able to achieve a high stationary cell count of 8.95 log CFU/mL on day 10 in the absence of hops, reinforcing that unhopped wort without added hops is a suitable growth medium for L. paracasei L26. In the presence of yeast, L. paracasei L26 was able to achieve a similar high viable cell count of 8.99 log CFU/mL on day 10, indicating that yeast does not hinder the growth of L. paracasei L26.

From Figure 3, it can be seen that both the monoculture and continuous culture cell counts of L. paracasei L26 fell below 7.0 log CFU/mL within 1 day of storage at 25°C. Similar to the co-inoculated fermentation, the viability enhancing effect of S. cerevisiae S-04 was more promising at 5°C. At 5°C, the L. paracasei L26 monoculture cell count fell below 7.0 log CFU/mL by day 16, whereas the L. paracasei L26 co-culture cell count only fell below 7.0 log CFU/mL by day 19. It can be seen that the viability enhancing effect can be attributed to both the refrigerated storage temperature and the presence of yeast.

The death of L. paracasei L26 during storage can be attributed to the addition of hop extract, since L. paracasei L26 monocultures stored at 5°C without the addition of isomerized extract on day 10 still had a high viable cell count of 8.88 log CFU/mL on day 22 (data not shown).

The non-volatile characteristics of hopped beer at various storage temperatures are shown in Table 2. From Table 2, it can be seen that the significant amount of lactic acid (8.25-8.39 g/L) produced by L. paracasei L26 resulted in the low pH values (3.30-3.31) recorded in beers inoculated with both L. paracasei L26 and S. cerevisiae S-04. The accumulation of lactic acid will result in a sour taste in the final probiotic beer product.

It can be seen that the synergistic effect of refrigeration and yeast addition on the viability of the probiotic bacteria was weakened compared to using the co-inoculation strategy of Example 2.1.

Example 3 - Improving viability and shelf life of probiotic bacteria
A longer shelf life would reduce losses caused by long cold chain distribution networks and, in addition, mitigate losses resulting from unsold beer that is subject to high alcohol tariffs. Therefore, the effect of hop levels on probiotic bacteria was investigated.

As seen in Example 1, hop iso-α-acids had a negative effect on the viability of the probiotic bacteria. Co-fermentation of probiotic bacteria with yeast was used in this example because L. paracasei L26 showed higher survival during co-inoculation with S. cerevisiae S-04 compared to sequential inoculation (Example 2).

The method followed was similar to that of Example 2.1, but with different levels of hop extract. Triple fermentations of S. cerevisiae S-04 yeast and L. paracasei L26 probiotic were carried out in 1 L capped glass bottles containing 600 mL of unhopped wort. Each glass bottle was inoculated with 0.5% (v/v) yeast preculture and 1% (v/v) probiotic bacterial preculture and incubated statically at 30°C for 2 days followed by 20°C for 8 days.

On day 10, 7.5, 15 and 22.5 International Bitterness Units (IBU) of isomerized hop extract (Brouwland, Beverlo, Belgium) were added to the unhopped beers, respectively. One triplicate set of beers remained unhopped. Samples were stored at 5°C and 25°C for shelf-life phase. The end of shelf-life for each fermentation set was determined when the plate count of L. paracasei L26 fell below 7.0 Log CFU/mL.

Figure 4 shows the growth and survival kinetics of L. paracasei L26 when co-inoculated with S. cerevisiae S-04 in hopped wort at different IBU levels at 5°C. During storage with added hop extract, survival of the probiotic bacteria improved as the hop concentration decreased. This is expected because the reduction of hop iso-α-acids, an inhibitor of bacterial growth, created a less favorable environment for the survival of the probiotic bacteria. At 22.5 and 15 IBU, the beer had a shelf life of 6 and 10 days, respectively. At 7.5 IBU, the probiotic bacteria maintained cell counts above 7.0 log CFU/mL for 1 month, an improvement from the 22-day shelf life in Figure 2. In unhopped beer (0 IBU), the probiotic bacteria maintained cell counts above 8 log CFU/mL for at least 4 months.

Figure 5 shows the growth and survival kinetics of L. paracasei L26 when co-inoculated with S. cerevisiae S-04 in hopped wort at different IBU levels at 25 °C. As expected, probiotic bacterial samples stored at 25 °C had poorer survival rates compared to samples stored under refrigeration. All hopped samples showed probiotic bacterial cell counts below 7.0 log CFU/mL after 3 days of storage, while non-hopped samples maintained probiotic cell counts above 7.0 log CFU/mL for only about 38 days of storage compared to at least 4 months under refrigeration (Figure 4). The results show the importance of cold chain distribution and storage of probiotic beer to maintain the viability of the probiotic bacteria.

Figure 6(a) and Figure 6(b) show the change in pH during fermentation and storage at 5°C and 25°C, respectively. The pH measurements were similar across the different samples. The low pH (around 3.5) achieved across the samples also suggests that lactic acid was produced by the probiotic bacteria, resulting in a sour taste.

Figures 7(a) and 7(b) show the change in °Brix measurements during fermentation and storage at 5°C and 25°C, respectively. °Brix measurements were similar across the different samples, indicating that the differences in hop concentrations did not significantly affect the fermentative capacity and nutrient utilization of the yeast.

Thus, it can be seen that the viability of probiotic bacteria increased with decreasing hop levels and that the antimicrobial effect of hops was more pronounced at 25°C compared to 5°C.

Example 4 - Modification of the fermentation procedure by adding probiotic bacteria after yeast fermentation
Triplicate fermentations were performed in 250 mL capped glass bottles containing 220 mL of hopped wort. The hop concentration was equivalent to the addition of 27 IBU of isomerized hop extract. S. cerevisiae S-04 (0.5% (v/v) inoculum) was added to each glass bottle initially. After 10 days of fermentation at 20° C., 1% (v/v) of L. paracasei L26 was added to the same samples. Shelf-life tests were then performed. The procedure was similar to that of the co-inoculation experiment outlined in Example 2.1. The same storage temperature and end of shelf-life conditions were utilized.

Figure 8 shows the survival of spiked L. paracasei L26 during storage at 5°C and 25°C. After inoculation with probiotic bacteria on day 10 and immediate storage at 5°C and 25°C, L. paracasei L26 showed better survival at 5°C compared to 25°C, with cell counts of 6.67 and 5.27 log CFU/mL on day 42 at 5°C and 25°C, respectively. This further highlights the need for low temperature storage and cold chain distribution for probiotic beer to maintain viability of the probiotic bacteria, regardless of the inoculation strategy used.

Figures 9 and 10 show the pH and °Brix measurements during the fermentation and storage periods. The °Brix values were similar to those of the co-fermented samples in Example 2.1 (Table 1).

From Figure 8, it was observed that L. paracasei L26 showed no growth, but its bacterial count remained unchanged for at least one month during refrigerated storage. This is probably due to the sequestration of hop acids by the yeast, creating a more favorable environment for the survival of the probiotic bacteria. Furthermore, as shown in Figure 9, a higher pH was obtained compared to the co-fermented sample of Example 2.1 (Table 1), 4.6-4.7 vs. 3.5. Higher pH values reduce the efficacy of hop acids, having an overall positive effect on the survival of the probiotic bacteria.

Therefore, based on the results obtained in Figure 8, since the probiotic cell count remains relatively unchanged throughout storage at 5°C, a version of probiotic beer could be created in which a higher loading of probiotic is added after the initial yeast fermentation period (e.g., above the minimum of 7 log CFU/mL, e.g., 8 log CFU/mL). This would also produce a more favorable and acceptable flavor profile, since the acidity of the resulting beer would be reduced, as opposed to allowing the probiotic to participate in the fermentation process with yeast.

Example 5 - Probiotic beer fermentation using different probiotic bacteria and yeast pairs
Examples 2-4 focus on the use of L. paracasei L26 and S. cerevisiae S-04 as a probiotic-yeast pair. Other probiotic bacteria-yeast pairs were also investigated to determine whether other probiotic bacteria and yeast strains could be used to form alternative versions of probiotic beer. These probiotic bacteria-yeast pairs include S. cerevisiae S-04 and other Lactobacillus probiotic bacteria (Example 5.1), L. paracasei L26 and other non-Saccharomyces yeasts (Example 5.2), and L. paracasei L26 and S. cerevisiae W34/70 lager yeast (Example 5.3).

5.1 Fermentation with S. cerevisiae S-04 and other probiotic Lactobacillus species
L. rhamnosus HN001 was used to demonstrate the antibacterial effect of hops (Example 1). Alone, it is susceptible to the antibacterial effect of hops. However, it is unclear whether its viability can be improved in the presence of yeast, similar to L. paracasei L26 (Example 2). Therefore, unhopped wort was fermented with S. cerevisiae S-04 and L. rhamnosus HN001, and 7.5 IBU of hop extract was added at the end of fermentation, followed by storage at 5°C and 25°C. Another probiotic bacterial strain, L. acidophilus NCFM, was also utilized to determine whether its viability could be improved in the presence of yeast.

Triplicate fermentations were performed in 1 L covered glass bottles containing 550 mL of unhopped wort. L. paracasei L26, L. rhamnosus HN001, and L. acidophilus NCFM were used in co-fermentation with S. cerevisiae S-04. 1% (v/v) probiotic bacterial inoculum and 0.5% (v/v) yeast inoculum were added to each glass bottle. Fermentation was performed statically at 30°C for 2 days followed by 20°C for 8 days.

After 10 days of fermentation, 7.5 IBU of isomerized hop extract was added to each sample. Shelf-life testing was performed using a procedure similar to that of the co-inoculation experiment outlined in Example 2.1. The same storage temperatures and end-of-shelf-life conditions were utilized.

Figure 11 shows the growth and survival of L. paracasei L26, L. rhamnosus HN001, and L. acidophilus NCFM when co-inoculated with S. cerevisiae S-04 in hop-free wort followed by addition of 7.5 IBU of hop extract and storage at 5°C. Both L. rhamnosus HN001 and L. acidophilus NCFM were able to coexist with S. cerevisiae S-04 during co-fermentation as indicated by the high cell counts achieved after 10 days of fermentation (8.06 log CFU/mL and 8.49 log CFU/mL, respectively).

From Figure 11, L. rhamnosus HN001 showed the poorest viability during cold storage, with cell counts below 7.0 log CFU/mL after 4 days of refrigeration, while L. paracasei L26 cell counts fell below 7.0 log CFU/mL within 1 month of storage, reinforcing the results previously obtained in Example 3 (Figure 4). L. acidophilus NCFM showed promising cell counts above 7.0 Log CFU/mL for 2 months of cold storage, falling below 7 Log CFU/mL by day 57 of storage. This is a significant improvement from the 1 month shelf life under refrigeration for L. acidophilus NCFM (at the same level of 7.5 IBU for both L. acidophilus NCFM and L. paracasei L26). Thus, L. acidophilus NCFM may also be used in the formulation of probiotic beer. L. acidophilus NCFM has been shown to survive gastrointestinal transit in both healthy and diseased human populations and has a long history of safe human consumption. L. acidophilus NCFM has been shown to induce several health benefits in human trials, including but not limited to reduced incidence of pediatric diarrhea, stabilization of the gut microbiota during antibiotic therapy, and amelioration of small intestinal bacterial overgrowth symptoms. Thus, the incorporation of L. acidophilus NCFM into probiotic beer will provide consumers with a healthier beer alternative option.

Figure 12 shows the growth and survival of L. paracasei L26, L. rhamnosus HN001, and L. acidophilus NCFM when coinoculated with S. cerevisiae S-04 in unhopped wort followed by the addition of 7.5 IBU of hop extract and stored at 25°C. It can be seen that L. rhamnosus HN001 showed the poorest survival, followed by L. paracasei L26 and L. acidophilus NCFM, closely resembling the trend observed at 5°C (Figure 11). The antibacterial effect of hops was also more pronounced at higher temperatures, with cell counts of L. rhamnosus HN001, L. paracasei L26, and L. acidophilus NCFM falling below the baseline of 7.0 log CFU/mL on days 1, 4, and 9 of storage, respectively.

Figures 13(a) and 13(b) show the pH measurements obtained during fermentation with S. cerevisiae S-04 and L. rhamnosus HN001, L. acidophilus NCFM, and L. paracasei L26 and storage at 5°C and 25°C, respectively, and Figures 14(a) and 14(b) show the corresponding °Brix measurements at storage temperatures of 5°C and 25°C. The pH measurements were similar between the samples, but the °Brix values were higher for L. rhamnosus HN001, indicating that less sugar was utilized by this probiotic bacteria and yeast during the fermentation period compared to L. acidophilus NCFM and L. paracasei L26.

5.2 The results of the pre-fermentation example using L. paracasei L26 and other non-Saccharomyces yeasts demonstrate the ability of Saccharomyces cerevisiae S-04 to enhance the survival of Lactobacillus paracasei L26 in hopped beer. With the increasing demand for novel beers, this example investigates the possibility of producing probiotic beer by co-cultivating L. paracasei L26 with non-Saccharomyces yeasts and the survival enhancing effect of these yeasts on probiotic bacteria.

Co-fermentations were performed as described in Example 2.1, using Torulaspora delbrueckii Prelude or Metschnikowia pulcherrima Flavia instead of S. cerevisiae S-04. Fermentations were performed with 300 mL of unhopped wort in 500 mL glass bottles. For co-cultures, unhopped wort was inoculated with 6.69 log CFU/mL L. paracasei L26 and 5.63 log CFU/mL T. delbrueckii Prelude or 5.08 log CFU/mL M. pulcherrima Flavia. Yeast monocultures were also prepared by inoculating unhopped wort with the same yeast inoculum. The inoculated wort was incubated at 30°C under static conditions from day 0 to day 2 to favor the growth of L. paracasei L26. The fermentation temperature was then reduced to 20°C from day 2 to day 12 to accommodate yeast growth.

After 12 days of fermentation, the beer was spiked with isomerized hop extract (Brouwland, Beverlo, Belgium) to a bitterness level of 7.5 IBU. Shelf-life testing of the hopped beer was performed at 5°C and 25°C as described in Example 3.

Figure 15 shows the growth and survival kinetics of L. paracasei L26 during 12 days of fermentation and storage at 5°C when co-cultured with either T. delbrueckii Prelude or M. pulcherrima Flavia in unhopped wort. Growth of L. paracasei L26 in the presence of two non-Saccharomyces yeasts was similar to when this probiotic bacterium was co-cultured with S. cerevisiae S-04 (Example 2.1; Figure 2). After fermentation, probiotic viable cell counts of 8.83-8.97 log CFU/mL in the beer were achieved. This indicated that T. delbrueckii Prelude and M. pulcherrima Flavia did not exert an inhibitory effect on L. paracasei L26 during fermentation.

Upon storage at 5°C, L. paracasei L26 counts in co-cultured beers remained above 7.0 log CFU/mL for at least 8 days during storage at 5°C, then declined to 6.69-6.76 log CFU/mL at day 27. As reported in Example 1, probiotic bacterial cell counts of L. paracasei L26 monocultures in hopped beers were below the minimum therapeutic level (7.0 log CFU/mL) by day 5 of storage. Thus, the presence of non-Saccharomyces yeasts could also improve the survival of probiotic bacteria in beer. Nevertheless, the probiotic viability-enhancing effects of T. delbrueckii Prelude and M. pulcherrima Flavia were not as pronounced as S. cerevisiae S-04. This is because L. paracasei L26 cell counts remained above 7.0 log CFU/mL for one month when co-cultured with the latter yeast (Example 3, Figure 4; Example 5.1, Figure 11).

The change in viable cell count of L. paracasei L26 in hopped beer when co-cultured with either T. delbrueckii Prelude or M. pulcherrima Flavia and subsequently stored at 25°C is shown in Figure 16. When the beer was stored at 25°C, the probiotic viable cell count decreased from 8.83-8.97 log CFU/mL to less than 3 log CFU/mL within 4 days. This was in agreement with previously collected results, where the survival-enhancing effect of yeast on L. paracasei L26 in hopped beer was lowest at room temperature (Figures 2, 3, 5, 8, and 12).

Figures 17(a) and 17(b) show the pH measurements obtained during the 12-day fermentation period and storage period at 5°C and 25°C, respectively, while Figures 18(a) and 18(b) show the corresponding °Brix measurements at storage temperatures of 5°C and 25°C. While the pH measurements were similar between the samples, the °Brix values were higher for L. rhamnosus HN001, indicating that less sugar was utilized by this probiotic bacteria and yeast during the fermentation period compared to L. acidophilus NCFM and L. paracasei L26.

5.3 Fermentation with L. paracasei L26 and other S. cerevisiae W34/70 lager yeasts Previously, S. cerevisiae S-04 ale yeast was used with L. paracasei L26 in co-fermentation and continuous fermentation in unhopped wort (Example 2). During refrigerated storage with added isomerized hop extract, L. paracasei L26 in the co-culture maintained higher viable cell counts of over 7.0 log CFU/mL for 22 days compared to the continuous culture (9 days).

Since the predominant commercial beer is lager beer, this example explores the use of the lager yeast strain Saccharomyces cerevisiae W-34/70 during co-inoculation and sequential inoculation with L. paracasei L26.

The method was similar to that of Example 2.1. Triplicate fermentations were performed in 500 mL capped glass bottles containing 350 mL of unhopped wort. In the co-fermented samples, the unhopped wort was inoculated with L. paracasei L26 (1% (v/v)) and S. cerevisiae W-34/70 yeast (1% (v/v)). In the sequential fermentation samples, the same inoculum of L. paracasei L26 and S. cerevisiae W-34/70 yeast was added to the unhopped wort, but S. cerevisiae W-34/70 was added only on the first day of fermentation. A monoculture of S. cerevisiae W-34/70 served as a control. The fermentation proceeded statically at 30 °C from day 0 to day 1 to provide favorable conditions for the growth of the probiotic bacteria. The temperature was then lowered to 20°C from day 1 to day 22 to allow yeast growth.

After 22 days of fermentation, a 7.5 IBU dose of isomerized hop extract (Brouwland, Beverlo, Belgium) was added to the unhopped beer. The hopped beer was then stored at 5°C and 25°C and the viability of L. paracasei L26 was assessed.

Figures 19 and 20 show the growth and survival of L. paracasei L26 during fermentation and storage with S. cerevisiae W-34/70 at 5°C and 25°C, respectively. It can be seen that L. paracasei L26 was able to coexist with the lager yeast S. cerevisiae W-34/70 during the 22-day fermentation period, maintaining high cell counts of over 8.60 log CFU/mL for both co-inoculated and sequentially inoculated samples.

After addition of 7.5 IBU of isomerized hop extract and subsequent storage, L. paracasei L26 in the co-inoculated and sequentially inoculated samples still maintained cell counts above 8.60 log CFU/mL within 4 days of storage when stored at 5°C. In contrast, L. paracasei L26 cell counts fell below 7.0 log CFU/mL within the same period for all samples stored at 25°C, reinforcing the importance of cold chain distribution and storage of probiotic beer to maintain probiotic viability.

The data in Figure 20 also suggest that the co-inoculation method may be a better technique in extending probiotic viability than sequential inoculation, where no probiotic bacterial cell counts were detected in the sequentially inoculated samples on the fourth day of storage at 25°C, but 6.63 log CFU/mL was still detected for L. paracasei L26 in the co-inoculated samples. These findings reiterate the findings in Example 2, where L. paracasei L26 co-cultured with S. cerevisiae S-04 maintained high viable cell counts of over 7.0 log CFU/mL for 22 days compared to the continuous culture (9 days). Such effects were less pronounced in samples stored at 5°C, with no significant difference in probiotic cell counts between the co-inoculated and sequentially inoculated samples on the 31st day of storage (although the probiotic cell counts from sequential inoculation were slightly lower than those from the co-inoculation method (Figure 19)).

Under refrigeration, the probiotic bacterial cell count fell below the baseline of 7.0 log CFU/mL by day 31 of storage, indicating that S. cerevisiae W34/70 provided a similar degree of probiotic bacterial viability enhancement effect as S. cerevisiae S-04, which had a shelf life of 1 month (Example 3, Figure 4; Example 5.1, Figure 11). Thus, this indicates that a lager-type probiotic beer can be produced.

Figures 21(a) and 21(b) show pH measurements obtained during a 22 day fermentation period and storage at 5°C and 25°C, respectively, while Figures 22(a) and 22(b) show the corresponding °Brix measurements at storage temperatures of 5°C and 25°C. The pH measurements for samples containing L. paracasei L26 were between 3.55 and 3.56, similar to those obtained in probiotic beer fermentations using L. paracasei L26 and S. cerevisiae S-04 (Example 2, Tables 1 and 2).

Figures 22(a) and 22(b) show that the °Brix measurements slowly decreased for all samples, reaching a plateau at the end of the 22 day fermentation period. No further decrease in °Brix values for all samples indicates complete sugar utilization by the yeast, justifying the start of the storage period after 22 days.

The present invention provides the following:
1. Alcoholic beverages containing probiotic bacteria.
2. The beverage according to claim 1, wherein the alcohol content of the beverage is ≧0.5% by volume.
3. The beverage according to claim 1 or 2, further comprising hops.
4. The beverage according to claim 3, wherein the hops or derivatives thereof have a bitterness of ≦30 IBU.
5. The beverage according to any one of 1 to 4 above, wherein the probiotic bacteria comprises Lactobacillus, Bifidobacteria, or a combination thereof.
6. The beverage according to any one of 1 to 5 above, wherein the probiotic bacteria contained in the beverage are lactobacilli selected from L. paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or a combination thereof.
7. The beverage according to any one of 1 to 6 above, wherein the probiotic bacteria has a cell count of ≧5.0 log CFU/mL.
8. The beverage according to any one of 1 to 7 above, having a pH of 2 to 6.
9. The beverage according to any one of 1 to 8 above, having a Brix of 4 to 20°Bx.
10. The beverage according to any one of 1 to 9 above, which is beer.
11. A method for forming an alcoholic beverage according to any one of claims 1 to 10, comprising the steps of:
- providing wort or must;
- adding probiotic bacteria to the wort or must;
- adding yeast to the wort or must; and
- fermenting the wort or must for a predetermined period of time and at a predetermined temperature to form an alcoholic beverage.
12. The method of claim 11, further comprising adding hops or a derivative thereof to the wort or must.
13. The method of claim 11 or 12, wherein the hops have a bitterness of ≦30 IBU.
14. The method according to any one of claims 11 to 13, wherein the probiotic bacteria comprises Lactobacillus, Bifidobacteria, or a combination thereof.
15. The method according to any one of claims 11 to 14, wherein the probiotic bacteria contained in the beverage are lactobacilli selected from L. paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or a combination thereof.
16. The method according to any one of claims 11 to 15, wherein the yeast is a Saccharomyces yeast, a non-Saccharomyces yeast, or a combination thereof.
17. The method according to any one of claims 11 to 16, wherein the yeast is S. cerevisiae, S. pasteurianus, T. delbrueckii, M. pulcherrima, P. kluveri, L. thermotolerans, or a combination thereof.
18. The method according to any one of claims 11 to 17, wherein the addition of the probiotic bacteria and the addition of the yeast are carried out simultaneously.
19. The method of claim 18, wherein fermenting comprises fermenting the wort or must at a first temperature for a first period of time, and then fermenting the wort or must at a second temperature for a second period of time.
20. The method of claim 19, further comprising adding hops or a derivative thereof at the end of the second period of time.
21. The method according to any one of claims 11 to 17, wherein the addition of the probiotic bacteria and the addition of the yeast are carried out successively.
22. The method according to claim 21, wherein the addition of yeast occurs a third period after the addition of the probiotic bacteria.
23. The method of claim 22, wherein fermenting comprises fermenting the wort or must at a third temperature for a fourth period after addition of yeast, and then fermenting the wort or must at the fourth temperature for a fifth period.
24. The method of claim 23, further comprising adding hops at the end of the fifth period.
25. The method of any one of claims 11 to 24, wherein the formed alcoholic beverage is stored at a temperature of ≦20° C.

Claims (13)

1. An alcoholic beverage comprising a probiotic bacteria having a cell count of ≧5.0 log CFU/mL, further comprising hops or a derivative thereof, the hops or derivative being an isomerized hops extract having a bitterness of ≦30 IBU, the alcoholic beverage having a pH of 2 to 6, and wherein the probiotic bacteria cell count of ≧5.0 log CFU/mL is maintained for 4 days or more when stored at 5° C. The beverage of claim 1, wherein the alcohol content of the beverage is ≥ 0.5% by volume. 3. The beverage of claim 1 or 2, wherein the isomerized hop extract has a bitterness of ≦7.5 IBU. The beverage according to any one of claims 1 to 3, wherein the probiotic bacteria include lactobacilli, bifidobacteria, or a combination thereof. The beverage according to any one of claims 1 to 4, wherein the probiotic bacteria contained in the beverage are lactobacilli selected from L. paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or combinations thereof. The beverage according to any one of claims 1 to 5, having a Brix of 4 to 20°Bx. The beverage according to any one of claims 1 to 6, which is beer. A method for forming an alcoholic beverage according to any one of claims 1 to 7, comprising the steps of:
- providing wort or must;
- adding probiotic bacteria to the wort or must, the amount of probiotic bacteria added being between 5 and 7 log CFU/mL ;
- adding yeast to the wort or must, the amount of yeast added being between 5 and 7 log CFU/mL ;
- fermenting the wort or must for a defined period of time and at a defined temperature to form an alcoholic beverage, the fermentation specifically comprising:
Simultaneous addition of probiotic bacteria and yeast;
fermenting the wort or must at 25-35°C for 1-5 days, then fermenting the wort or must at 13-25°C for 8-30 days;
adding an isomerized hop extract to the fermented wort or must at the end of fermentation; and
The resulting alcoholic beverage should be stored at 1-5°C.
Steps including
wherein the isomerized hop extract has a bitterness of ≦30 IBU . 9. The method of claim 8, wherein the isomerized hop extract has a bitterness of ≦7.5 IBU. The method of claim 8 or 9, wherein the probiotic bacteria comprises lactobacilli, bifidobacteria, or a combination thereof. The method according to any one of claims 8 to 10, wherein the probiotic bacteria contained in the beverage are lactobacilli selected from L. paracasei, L. rhamnosus, L. acidophilus, L. casei, L. fermentum, L. plantarum, or a combination thereof. The method according to any one of claims 8 to 11, wherein the yeast is a Saccharomyces yeast, a non-Saccharomyces yeast, or a combination thereof. 13. The method of any one of claims 8 to 12, wherein the yeast is S. cerevisiae, S. pasteurianus, T. delbrueckii, M. pulcherrima, P. kluveri, L. thermotolerans, or a combination thereof.

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