KR102097458B1 - A method for producing beer - Google Patents

Abstract

The present invention relates to a method for producing a beer through the two-step alcohol fermentation of a fermentation broth prepared by using lactic acid bacteria. According to the present invention, the beer with high preference can be produced, containing functional oligosaccharide without smell of sake.

Description

A method for producing beer

The present invention relates to a beer production method and a beer produced thereby.

Fermentation by lactic acid bacteria is one of the oldest food processing methods that not only increases the value of foods by imparting a variety of flavors and textures to foods, but also creates nutritional and functional ingredients, and increases the shelf life of foods through the synthesis of antibacterial substances. Therefore, when producing a fermented beverage using lactic acid bacteria, it will improve the refreshing acidity and preservation of the product, improve the digestibility of the milk protein, improve the usability of calcium, iron, phosphorus, etc., and enhance palatability. One of the representative fermented foods using lactic acid bacteria is sour beer, and Lamberg and Flander Red Ale in Belgium are among the oldest sour beers. Sour beer using lactic acid bacteria as a starter has recently been re-illuminated because it has high antioxidant and antibacterial activity, improves flavor, and has a prebiotic effect.

Meanwhile, as the import of foreign beer increased, the premium beer market began to grow, and the premium beer market, which was only 3.5% of the total beer market in 2008, increased significantly from 2010, increasing by 5.4% in 2012. Since 2010, since the Tax Law on Small Liquor has been revised, it has been gradually activated, and the number of breweries of homemade beer has been counted as 95 as of 2017, and has grown sharply from 20 billion won in 2016 to 40 billion won in 2017. have. Liquor consumers value the taste and aroma of alcohol, and the preference for low-alcohol alcoholic beverages is increasing. Research and development in which various subsidiary materials are added (for example, sweet potato and rice) are being made in beer production in accordance with the needs of customers. In particular, in the case of beer using rice as a subsidiary material, saccharification efficiency affecting alcohol content may be reduced, and various technology developments are required in the manufacturing process such as a suitable mixing ratio of materials.

Korean Registered Patent No. 0440723

An object of the present invention is to provide a method for producing beer.

Another object of the present invention is to provide a beer containing a functional oligosaccharide.

1. Mixing rice with malt and saccharifying to obtain saccharification solution; And

A method for producing a beer comprising a fermentation step of oligosaccharide production using lactic acid bacteria to which an alginate calcium carbonate bead having a lactic acid strain immobilized thereon is added to the saccharification solution to ferment the saccharification solution to obtain a primary fermentation solution.

2. The method of 1 above, wherein the lactic acid strain is Weissella cibaria or Leuconostoc mesenteroides .

3. The method of 1 above, wherein the primary fermentation broth has a soluble solid content of 15 to 17 brix.

4. In the above 1, after the primary fermentation broth is heated by putting at least one kind of hops, primary fermentation of alcohol to obtain a secondary fermentation broth by adding and fermenting yeast; And

Alcohol fermentation second step of obtaining a tertiary fermentation liquid by fermenting the secondary fermentation liquid in a closed system; further comprising a beer production method.

5. The method of 4 above, wherein the first step of alcohol main fermentation is performed in a condition in which at least a part of carbon dioxide produced by fermentation is discharged.

6. The method of 4, wherein the secondary fermentation broth has a soluble solid content of 9 to 11 brix.

7. The method of 4, wherein the soluble solids content of the tertiary fermentation broth is reduced by 0.5 to 2 Brix compared to the soluble solids content of the secondary fermentation broth.

8. The method of 4, wherein the yeast is 0.005 to 0.05% (w / v) added to the primary fermentation broth.

9. The method according to the above 4, further comprising an alcohol fermentation step of fermenting the tertiary fermentation under refrigerated conditions.

10. Beer prepared by any one of the above 1 to 9.

The present invention relates to a beer production method and a beer produced thereby, the beer produced according to the production method of the present invention has a high functional oligosaccharide content, and a low odor, such as sake. In addition, the beer of the present invention contains mannitol and branched oligosaccharides, and the taste of the beer of the present invention is also good, as in previous studies that mannitol gives a refreshing taste and eggplant oligosaccharides give a light and soft sweet taste. And high demand from consumers.

1 is a schematic diagram of a saccharification solution manufacturing process.
FIG. 2 is a schematic diagram showing the preparation of calcium alginate carbonate (CaCO ₃ -Alginate bead) and the fermentation process of oligosaccharide production using lactic acid bacteria.
Figure 3 is a schematic diagram of the beer production process using oligosaccharide-producing fermentation broth using lactic acid bacteria.
Figure 4 shows the pH change of samples with different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
Figure 5 shows the change in the total number of bacteria and the number of lactic acid bacteria during the fermentation process of lactic acid bacteria in a sample with different types of calcium alginate beads.
Figure 6 shows the change in the soluble solids of the samples of different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
7 shows changes in reducing sugars of samples of different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
Figure 8 shows the change in the amount of sucrose in the samples of different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
Figure 9 shows the change in the amount of maltose of different samples of alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
Figure 10 shows the change in the amount of fructose of the samples having different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
Figure 11 shows the change in the amount of mannitol of the samples having different alginate calcium carbonate beads during the fermentation process of lactic acid bacteria.
12 shows changes in the amount of D-panose and maltosyl-isomaltooligosaccharide (MIMO) during the fermentation process of Control_S.
13 shows changes in the amounts of D-panose and maltosyl-isomaltooligosaccharide (MIMO) during the fermentation process of EWC_S.
14 shows changes in the amount of D-panose and maltosyl-isomaltooligosaccharide (MIMO) during the fermentation process of ELM_S.
15 shows changes in the amount of D-panose and maltosyl-isomaltooligosaccharide (MIMO) during the fermentation process of EWL_S.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing beer, including; a fermentation step of oligosaccharide production using lactic acid bacteria to obtain a primary fermentation broth by mixing saccharified liquid with beads having a lactic acid strain immobilized thereon.

The saccharification solution may be obtained by mixing rice with malt and saccharifying using methods and conditions known in the art.

For example, the saccharification solution may be obtained by saccharification while continuously increasing the temperature for a period of time. For example, the saccharification solution may be obtained by saccharifying while increasing the temperature from 30 ° C to 50 ° C for 30 minutes.

For another example, the saccharification solution may be obtained by saccharifying at a constant temperature for a period of time, and then increasing the temperature to further saccharify at a constant temperature. For example, after saccharifying for 30 minutes at a temperature of 50 ° C, saccharifying for 30 minutes at a temperature of 60 ° C, and then saccharifying for 1 hour at a temperature of 70 ° C.

The rice may be made by mixing 100 to 500 parts by weight of water relative to 100 parts by weight of rice, preferably 100 to 200 parts by weight of water compared to 100 parts by weight of rice, but is not limited thereto.

Malt may be extracted by mixing in a ratio of 1 to 30ml of water per 1g of malt powder, but is not limited thereto. For example, malt may be extracted by mixing at a ratio of 1 to 20, 2 to 18, 4 to 16, 6 to 14 or 8 to 12 ml of water per 1 g of malt powder, but is not limited thereto.

For example, malt may be extracted by mixing 1000 to 3000 ml of water per 200 g of malt powder, but is not limited thereto.

In addition, a filtration process may be performed after the saccharification.

The lactic acid strains are the bacteria to decompose saccharides mainly produce lactic acid as a metabolite, for example, streptococcus (Streptococcus), Lactobacillus nose kusu (Lactococcus), Lactobacillus bacteria (Lactobacillus), flow Pocono stock (Leuconostoc), Pediococcus ( Pediococcus ) and Bifidobacterium ( Bifidobacterium ), may be one or more of the genus Weselella ( Weissella ), but is not limited thereto. Specifically, the lactic acid strain may be one or more of Leuconostoc mesenteroides , Weissella cibaria , but is not limited thereto.

The beads may be alginate calcium carbonate beads, but are not limited thereto.

The diameter of the beads may be 0.1 to 10 mm. It is not limited to this. For example, the diameter of the beads may be 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 5, 5 to 10 mm, and preferably 1 to 2 mm, but is not limited thereto. Does not.

Preparing a colloidal solution by mixing lactic acid strain-immobilized alginate calcium carbonate beads with sodium alginate, calcium carbonate (CaCO ₃ ), and lactic acid strains; And dropping the colloidal liquid into calcium chloride (CaCl ₂ ).

The saccharification solution may be mixed with beads in which one or more lactic acid strains are immobilized. For example, the saccharification solution may be mixed with a first bead immobilized with Leuconostoc mesenteroides strain and a second bead immobilized with Weissella cibaria strain.

The saccharification solution may be fermented at 10 to 55 ° C. by mixing with the beads, but is not limited thereto. For example, the saccharification solution may be fermented under the conditions of 10 to 50, 15 to 45, 20 to 40, and 25 to 35 by mixing with the beads, but is not limited thereto. For another example, the saccharification solution may be fermented under the conditions of 15 to 25, 25 to 35, 35 to 45, and 45 to 55 ° C. by mixing with the beads, but is not limited thereto.

The saccharification solution may be fermented for 5 to 60 hours by mixing with the beads, but is not limited thereto. For example, the saccharification solution may be fermented for 5 to 45, 10 to 40, 15 to 35, 20 to 30 hours by mixing with the beads, but is not limited thereto. For another example, the saccharification solution may be fermented for 6 to 12, 12 to 24, 24 to 36, 36 to 48, 48 to 60 hours by mixing with the beads, but is not limited thereto.

The primary fermentation broth is a product obtained by fermentation of oligosaccharide production using lactic acid bacteria, and may include one or more sugars, enzymes, etc. in addition to oligosaccharides. The sugar may be a monosaccharide, disaccharide, oligosaccharide or polysaccharide, for example, fructose, glucose, sucrose, maltose, panose, isomaltoligosaccharide ( isomaltooligosaccharide) or mannitol (mannitol), but is not limited thereto. The enzyme may be dextransucrase, but is not limited thereto.

The content of soluble solids contained in the primary fermentation broth may be 11 to 20 brix, but is not limited thereto. For example, the soluble solid content contained in the primary fermentation broth may be 11 to 20, 12 to 19, 13 to 18, 14 to 17 or 15 to 16 brix, but is not limited thereto. For another example, the soluble solid content contained in the primary fermentation broth may be 11 to 12, 12 to 14, 15 to 17, 17 to 18 or 18 to 20 brix, but is not limited thereto.

In the case of fermentation using lactic acid bacteria that are not immobilized, the pH of the lactic acid bacteria is lowered due to by-products generated when culturing the lactic acid bacteria, or the growth or activity of the lactic acid bacteria is lowered, or the proper pH of the enzyme contained in the fermentation broth is difficult to maintain. There is a disadvantage that functional materials are difficult to produce. On the other hand, in the case of fermentation using beads in which the lactic acid bacteria are immobilized, there is an advantage that the culture effect of the lactic acid bacteria is high, and the proper pH of the enzyme reaction can be maintained in the fermentation broth. In other words, when beads with immobilized lactic acid bacteria are used, fermentation efficiency is good, and a functional substance generated by an enzyme reaction may be included in the fermentation broth.

In addition, the present invention is a primary fermentation alcohol first step to obtain a secondary fermentation broth by heating and putting at least one type of hop into the primary fermentation broth obtained by the above-described step; And a second step of alcohol main fermentation to obtain a tertiary fermentation liquid by further fermenting the secondary fermentation liquid.

The hop can be used as a raw material for beer as a family of Cannabaceae . In the present specification, the term "hop" may mean the whole plant, or only a flower.

For example, the hops are Mittelfruh, Saaz, Spalt, Tettnang, Mosaic, Amarillo, Apollo, Calypso , Cascade, Centennial, Chinook, Columbus, Galena, Liberty, Vanguard, Admiral, Brewers' Gold , Puggle, Golding, East Kent Golding, Hallertau, Magnum, Polaris, Galaxy, Southern Cross, Green Bullet (Green Bullet), Nelson Sauvin (Nelson Sauvin), Simcoe (Simcoe) and Warrior (Warrior) may be one or more, but is not limited to.

The yeast may be a yeast that can ferment alcohol, which is known in the art, and may be, for example, Saccharomyces .

The yeast may be 0.005 to 0.05% (w / v) added to the primary fermentation broth, preferably 0.005 to 0.02% (w / v), but is not limited thereto.

The first step of alcohol main fermentation may be fermentation under anaerobic conditions.

In the first step of alcohol main fermentation, at least a part of gas generated by fermentation may be discharged. The gas may be carbon dioxide (CO ₂ ), but is not limited thereto.

When the gas generated in the first step of alcohol main fermentation is discharged to the outside, the amount of gas, which is a fermentation product, cannot be increased to a certain amount, and further fermentation may occur until chemical equilibrium is achieved.

The alcohol main fermentation first step may be performed at a known temperature condition, but may preferably be performed at room temperature. The term “room temperature” in the present specification is a general indoor temperature, for example, may be 1 to 35 ° C., but preferably means a temperature within a range of 15 to 25 ° C. For example, the primary fermentation broth may be fermented under the conditions of 5 to 35, 10 to 30, and 15 to 25 ° C, but is not limited thereto.

The main fermentation first step may be to stop fermentation when a portion of the soluble solid content of the primary fermentation broth is reduced.

The primary fermentation first step may be to ferment until the soluble solids content is reduced by 1 to 10 Brix compared to the primary fermentation broth, but is not limited thereto. For example, the primary fermentation first step may be to ferment until the soluble solid content is reduced from 2 to 9. 3 to 8, 4 to 7 or 5 to 6 brix compared to the primary fermentation broth, but is not limited thereto.

The primary fermentation first step may be to ferment the primary fermentation broth to the extent that a secondary fermentation broth having a soluble solid content of 8 to 12, 9 to 11 or 9 to 10 Brix is obtained.

For another example, the fermentation period of the primary fermentation first step may be shorter than a normal alcohol fermentation period for fermentation until the soluble solids content of the primary fermentation liquid is almost consumed. For example, the fermentation period of the primary fermentation first step may be 1 to 9 days, but is not limited thereto. For example, the fermentation period of the primary fermentation first step may be 1 to 9, 2 to 8, 3 to 7 or 4 to 6 days. However, the specific fermentation period may vary depending on the components of the primary fermentation broth, the amount of the primary fermentation broth, the amount of hops, and the amount of yeast.

The secondary fermentation broth may include residual sugars or enzymes remaining after removing some of the sugars contained in the primary fermentation broth after the primary fermentation first step. The residual sugar may be monosaccharide, disaccharide or polysaccharide, for example, fructose, glucose, sucrose, maltose, panose, isomaltooligosaccharide ) Or mannitol, but is not limited thereto. The enzyme may be dextransucrase, but is not limited thereto.

The soluble solid content of the secondary fermentation broth may be 5 to 18 Brix, but is not limited thereto. For example, the soluble solid content of the secondary fermentation broth may be 5 to 15, 6 to 14, 7 to 13, 8 to 12 or 9 to 11 brix, preferably 9 to 11 brix, but is not limited thereto. Does not.

The second step of alcohol main fermentation may be fermentation under anaerobic conditions.

The second step of alcohol main fermentation may be fermentation in a closed system. As used herein, "closed system" refers to a system that does not exchange materials with the outside, and may specifically mean a system in which products or reactants produced by a reaction are difficult to be discharged to the outside.

That is, when the second step of alcohol fermentation is fermentation in a closed system, gas generated by fermentation may not be discharged to the outside. The gas may be carbon dioxide, but is not limited thereto.

If the gas generated in the second step of alcohol main fermentation is not discharged to the outside, the amount of gas, which is a fermentation product, may increase depending on the degree of fermentation. Can be lowered.

On the other hand, if the gas generated in the second step of alcohol main fermentation is not discharged to the outside, the generated gas may be included in the fermentation broth, and the beer produced using the fermentation broth may have a soft taste including carbon dioxide gas.

The alcohol main fermentation second step may be performed at a known temperature condition, but may preferably be performed at room temperature.

For example, the secondary fermentation broth may be fermented at 0 to 45 ° C, but is not limited thereto. Specifically, the secondary fermentation broth may be fermented under the conditions of 5 to 35, 10 to 30, and 15 to 25 ° C, and preferably fermentation under the conditions of 15 to 25 ° C, but is not limited thereto.

The main fermentation second step may be to stop fermentation when some of the soluble solids content of the secondary fermentation broth is reduced.

The main fermentation second step may be to ferment until the soluble solid content decreases by 0.1 to 3 brix compared to the secondary fermentation broth, but is not limited thereto. For example, the second step of the main fermentation may be fermentation until the soluble solid content is reduced by 0.1 to 3, 0.25 to 2.5, and 0.5 to 2 brix compared to the secondary fermentation broth, but is not limited thereto.

The soluble solids content that is reduced in the primary alcohol fermentation second step may be less than the soluble solids content that is reduced in the alcohol fermentation first step. That is, the degree of fermentation in the first step of alcohol fermentation may be greater than the degree of fermentation in the second step of alcohol fermentation.

The main fermentation second step may be to ferment the secondary fermentation broth to the extent that a third fermentation broth having a soluble solid content of 7 to 11 Brix can be obtained. At this time, the 7 to 11 brix may be a content of a functional oligosaccharide. For example, the second step of the main fermentation may be to ferment the secondary fermentation broth to the extent that a third fermentation broth having a soluble solid content of 8 to 11, 8.5 to 10.5 or 9 to 10 Brix can be obtained.

The fermentation period of the main fermentation second step may be 1 to 11 days, but is not limited thereto. For example, the fermentation period of the main fermentation second step may be 3 to 11, 4 to 10, 5 to 9 or 6 to 8 days, but is not limited thereto. However, the specific fermentation period may vary depending on the components of the secondary fermentation broth, the amount of the secondary fermentation broth, the amount of hops, and the amount of yeast.

The tertiary fermentation broth obtained after the second step of main fermentation may be at least partially consumed of the remaining sugars other than the functional oligosaccharide component in the secondary fermentation broth. The residual sugar consumed in the secondary fermentation broth through the main fermentation second step may be monosaccharide, disaccharide or polysaccharide, for example, fructose, glucose, sucrose, maltose , Panose or mannitol, but is not limited thereto.

The soluble solid content of the tertiary fermentation broth may be 1 to 14 brix, but is not limited thereto. For example, the soluble solid content of the tertiary fermentation broth may be 6 to 14, 7 to 13, 8 to 12, 9 to 11 or 8 to 10 brix, but is not limited thereto. For another example, the soluble solid content of the tertiary fermentation broth may be 1 to 4, 5 to 7, 8 to 10, or 11 to 14 brix, but is not limited thereto. Preferably, the soluble solids content of the tertiary fermentation broth may be 8 to 10 Brix.

As described above, according to the beer production method of the present invention, beer produced through two main fermentation stages has less odor, such as liqueur, and consumer preference compared to beer produced through one main fermentation stage. As high as

Furthermore, the present invention provides a method for producing beer further comprising; an alcohol fermentation step of fermenting the tertiary fermentation liquid under refrigerated conditions.

As used herein, the term "refrigeration conditions" may mean a temperature lower than room temperature. For example, the secondary fermentation may be to ferment the tertiary fermentation broth at 0 ° C or higher, 0 to 5, 5 to 10 or 10 to 15 ° C, but is not limited thereto.

The content of soluble solids contained in the product obtained through the above-mentioned fermentation step may be 2 to 7, but is not limited thereto. For example, the content of soluble solids contained in the product obtained through the side-fermentation step may be 2 to 3, 3 to 4, 4 to 5 or 6 to 7 brix, and preferably 6 to 7 brix, It is not limited to this. The content of soluble solids contained in the product obtained through the side-fermentation step may be the content of functional oligosaccharides contained in the product.

In addition to the above-described steps, the beer production method of the present invention may further include steps that may be included in a conventional beer production process.

The present invention can provide beer prepared by the above-described method.

The beer may be rice beer, barley beer, or wheat beer, but is not limited thereto.

The beer may contain functional oligosaccharides. Oligosaccharides are carbohydrates with a degree of polymerization of 2 to 10 monosaccharides and can be used in the food, animal feed, pharmaceutical and cosmetic industries as energy sources, sweeteners, stabilizers or expanders. In addition, oligosaccharides can help the growth and activity of beneficial microorganisms in the intestine and help the beneficial bacteria to settle.

For example, the oligosaccharide may be maltooligosaccharide, isomalttooligosaccharide, galactooligosaccharide, fructooligosaccharide, and specifically, maltosyl-isomaltooligosaccharide. ; MIMO), but is not limited thereto.

Hereinafter, examples will be described in detail to specifically describe the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Experimental material

Reagents used in this experiment are sucrose, glucose, D-mannitol, D-fructose (SAMCHUN Chemicals Co., Ltd, Gyeonggi-do, Korea), maltose (JUNSEI Chemical Co., Ltd, Tokyo, Japan) , And ethyl alcohol (Sigma-Aldrich Co. LLC., MO, USA) were used. As an organic acid standard, oxalic acid, tartaric acid, DL-malic acid, DL-lactic acid, acetic acid, citric acid, succinic acid (Sigma-Aldrich Co. LLC., MO, USA) were used. , isomaltriose (Sigma-Aldrich Co. LLC., MO, USA) and isomaltoteraose (Tokyo Chemical Co. LTD., Tokyo, Japan) were used. As the HPLC organic solvent reagent, potassium sulfate and potassium phosphate monobasic (SAMCHUN Chemicals Co., Ltd, Gyeonggi-do, Korea) were used, and phosphoric acid (Sigma-Aldrich Co. LLC., MO, U.S.A) was used for pH correction.

In addition, a medium (microbial culture medium) for measuring the total number of bacteria (PCA (plate count agar, Difco, USA) was used, the total lactic acid bacteria in Lactobacilli MRS (BD Difco, USA) agar 2% (BD Difco, USA) and A medium containing sodium azide 0.02% (Sigma-Aldrich Co. LLV., MO, USA) was used. The total yeast was measured by YMPG (yeast 0.3%, malt extract 0.3%, peptone 0.5%, glucose 1%) agar 2%, chloramphenicol 0.01%, chlortetracycline hydrochloride 0.01% (Sigma-Aldrich Co. LLC., MO.USA) Was prepared by mixing and used.

Experiment method

1. Preparation of saccharification liquid

For the saccharification solution, 200 g of rice was washed 3 times, soaked in 2 times of water for 2 hours, drained, and water was poured with 1.5 times the weight of rice to make rice. 200 g of malt flour and water were mixed at a ratio of 1:10, and then stirred at 100 rpm for 2 hours at 25 ° C to extract malt and mix with cooked rice. This was saccharified by sequential heating at 45 ° C for 30 minutes, 63 ° C for 80 minutes, and 70 ° C for 30 minutes, and then the activity was stopped at 78 ° C for 10 minutes. The saccharification solution was filtered by centrifugation at 8000 rpm for 15 minutes (HMR-220IV, Hanil Inductiral Co., Korea), followed by addition of 6% (w / v) sucrose, sterilization at autoclave at 121 ° C for 15 minutes, and then 5 It was used by cooling to ℃. The process of manufacturing the saccharification solution as described above is illustrated in FIG. 1.

2. Calcium alginate calcium carbonate beads (CaCO3-Alginate bead) production and oligosaccharide production fermentation using lactic acid bacteria

Sodium alginate 2% (w / v) into distilled water and dissolved sufficiently using a stirrer, and then mixed with CaCO₃ 20%, Weissella cibaria 10% (w / v), and Leuconostoc mesenteroides 10% (w / v) strains. Homogenize until 200 rpm. For the mixed colloidal solution, the mixed colloidal solution was dropped into a 0.1 M CaCl2 solution through a needle (Gauge 18) using a syringe to prepare beads having a diameter of about 1.0-2.0 mm, and allowed to stand for 1 hour. The beads were strengthened and then washed 3 times with sterile water before use. Alginate calcium carbonate beads immobilized with Weissella cibaria (hereinafter, Encapsulated Weissella cibaria ; EWC) and alginate calcium carbonate beads immobilized with Leuconostoc mesenteroides (hereinafter, Encapsulated Leuconostoc mesenteroides ; ELM) were obtained.

400 mL of saccharification solution was mixed with EWC (2%, w / v), ELM (2%, w / v), EWC (1%, w / v) and ELM (1%, w / v). The treated (EWL) and untreated control groups were fermented in a thermostat at 30 ° C. for 24 hours to obtain a primary fermentation broth. Preparation of the CaCO ₃ -Alginate bead as described above and fermentation process of oligosaccharide production using lactic acid bacteria are shown in FIG. 2.

3. Alcohol fermentation

A warrior hop (0.9%, w / v) was added to the lactic acid bacteria fermentation broth (primary fermentation broth) at a temperature of 100 ° C. and heated for 15 minutes, and centennial hop (0.14%, w / v) was added for further heating for 30 minutes. Put centennial hop (0.14%, w / v) again, heat it for 10 minutes, turn off the fire, add simcoe hop (0.14%, w / v), cool to 5 ℃, then French Sasion (mangrove jack's, BGGi NZ Ltd, Newzealand), Safale S-04 (Fermentis, Lesaffre, England) or Safale US-05 (Fermentis, Lesaffre, England) was added 0.014% (w / v) at a condition of 1x10 ⁷ per mL to increase the yeast activity. The yeast was fermented (first step of alcohol main fermentation) while discharging the gas generated in the incubator for 5 days at 20 ° C, and fermented (second stage of alcohol main fermentation) without discharging the gas generated at the same temperature for 7 days. Next, it was stored at 4 ° C for 4 weeks (alcohol fermentation step). The beer production process as described above is illustrated in FIG. 3.

When producing beer, it is common to ferment until all the remaining sugar is consumed for the yield of alcohol, and it usually takes about 10 days to consume all the remaining sugar. As a control, beer produced with a yeast fermentation period of 10 days is used as a control. Comparison was made, and three types of commercial yeast were used to confirm whether the characteristics of beer could be changed according to the type of yeast.

Method of measuring experimental data

1. Soluble solid content

To measure the soluble solid content, 1 mL of the sample was placed in a micro tube and centrifuged using a centrifuge (centrifuge 5415 B, Brinkmann instruments. Inc., Germany) for 10 minutes at 10,000 rpm (xg-force). Subsequently, the supernatant was measured 3 times using a refractometer (refractometer, ATAGO, PR-101, Japan) to obtain the average value and expressed in ° Brix%.

2. pH measurement

The pH change during fermentation was measured by repeating three times using a pH meter (TOA HM -7E, TOA Electrocin Ltd, Japan) by taking a 15 mL stock solution and showing the average value.

3. Quantification of reducing sugar

The reducing sugar was measured by dinitrosalicylic acid (DNS) method. After mixing 100 mL of the diluted sample solution with 1 mL of the diluted sample solution and reacting in boiling water for 5 minutes, the cooled one was added to an Ultra Microplate Reader (EL 808, BIO-Tek Instruments) using a 96 well microplate. INC., USA) was measured at 540 nm and converted using Glucose (Sigma Chemical Co. USA) as a standard.

4. Free sugar measurement

Each sample was diluted with ultrapure water for analysis of free sugar and alcohol, and then filtered using 0.2 μm membrane filterpaper (Whatman, GE, Healthcare Bio-Sciences, Pittsburgh, PA., USA), and the solution was HPLC (2487, Waters Co. , Milford, MA, USA). Detector was analyzed by Waters W410, and column was used Aminex HPX-87c (300mm x 7.8mm, BIO-RAD, Herculse, CA., U.S.A). The temperature of the column oven was set to 85 ° C, the mobile phase was 0.01M potassium sulfate in water, the flow rate was used at 0.6 mL / min, and the sample was injected with 20 μL. Samples were measured three times each, and the average value was used. For the preparation of the standard solution, a special reagent product (Sigma, St. Louis, MO., U.S.A) was used. The concentration of sugar in the samples was measured by using the calibration curve of the standard solution for the HPLC area value of each component, and analysis conditions of the free sugar are shown in Table 1.

parameter Condition Column Aminex HPX-87c
(300 mm x 7.8 mm, BIO-RAD) Detector Waters RI-2414 Flow rate 0.6 mL / min Mobile phase 0.01M potassium sulfate in water Column Oven temp. 85 ℃ Injection Vol. 20 μL

5. Oligosaccharide Measurement

Each sample was diluted with ultrapure water for oligosaccharide analysis, filtered using 0.2 μm membrane filterpaper (Whatman, GE, Healthcare Bio-Sciences, Pittsburgh, PA., USA), and the solution was HPLC (2487, Waters Co., Milford) , MA, USA). Detector was analyzed by Waters W410, column was BP-200 Ag (300 mm x 7.8 mm, BIO-RAD, Herculse, CA., U.S.A). The temperature of the column oven was set to 90 ° C, the mobile phase was DI H2O, the flow rate was used at 0.5 mL / min, and 20 μL of the sample was injected. Samples were measured by repeating each of 3 times, and the average value was used. For the preparation of the standard solution, a special reagent product (Sigma, St. Louis, MO., U.S.A) was used. The concentration of sugar in the samples was measured using the calibration curve of the standard solution for the HPLC area value of each component, and the analysis conditions of the oligosaccharides are shown in Table 2.

parameter Condition Column BP-200 Ag
(300 mm x 7.8 mm, BIO-RAD) Detector Waters RI-2414 Flow rate 0.5 mL / min Mobile phase DI H2O Column Oven temp. 85 ℃ Injection Vol. 20 μL

6. Calcium (Ca) content measurement

The calcium content was pre-treated according to the wet decomposition microwave method of the trace component test method in the food industry. 5 mL of the sample was placed in a microwave digestion tube, 8 mL of nitric acid and 2 mL of hydrogen peroxide were added, and then the microwave digestion system was decomposed at room temperature for 90 minutes at 90 ° C for 10 minutes, decomposed at 150 ° C for 10 minutes, and heated to 190 ° C for 30 minutes. When the temperature is gradually lowered and the temperature reaches 50 ° C, the solution sample is transferred to a 50 mL diaphragm, distilled with water, filtered with filter paper (Whatman No. 2), and filtered to ICP-ASE (Varian Inc., Mulgrave, Victoria, Australia). Ca was quantified

7. Chromaticity measurement

The SRM (standard Reference Method) chromaticity is measured at 430 nm using a spectrophotometer (X-ma 1200V, Human Co., Ltd., Seoul, Korea) with zero point adjusted using distilled water using the mainstream analysis regulation and ASBC method. The chromaticity was calculated by multiplying the value by 10. If the absorbance at 700 nm is less than the absorbance at 430 nm multiplied by 0.039, the beer is considered to be transparent, and the color of the beer is determined from the absorbance at 430 nm.

[Equation 1]

SRM = 10 x D x absorbance (A)

D = dilution factor

8. Bitter taste measurement

To measure the bitterness (Bitterness Unit, BU), put 100 mL of sample in an Erlenmeyer flask according to the mainstream analysis regulations, add 20 mL of octyl alcohol, 0.5 mL of 6N HCl and 20 mL of 2,2,4-trimethylpentane, and then constant at 250 rpm. It was placed in a constant temperature water bath (ESV3025-W, Polyscience, IL, USA) moving at left and right speeds and mixed for 15 minutes. The supernatant was taken and absorbance A was measured at 275 nm using an ultraviolet spectrophotometer (Du730, Beckman coulter, Inc, CA, U.S.A).

[Equation 2]

BU = absorbance (A) x 50

9. Alcohol content

Alcohol content was measured using the distillation method outlined in the liquor analysis textbook of the National Tax Service. Take 100 mL of each assay sample from 15 ° C. to a 100 mL volumetric flask scale, transfer it to a 500 mL Erlenmeyer flask, combine the liquid washed twice with 15 mL distilled water into an Erlenmeyer flask, connect the cooler, and connect 70 mL. The mixture was heated with a direct flame until it became a distillate, and the distillate was transferred to a volumetric flask, and then distilled water was added to fill up to 100 mL, and then the alcohol content was measured using a spirit meter.

10. Proper acidity

The titratable acidity was measured by neutralizing and titrating 0.1 N NaOH solution to pH 8.3 after taking 15 mL of the sample stock solution, and measuring the consumption of 0.1 N NaOH solution consumed by the lactic acid coefficient, and repeated 3 times each The average value was calculated.

[Equation 3]

Optimum acidity (w / v%) = (MxVxF) / S x 100

M = amount of lactic acid with the highest content in the sample in 1 mL of sodium hydroxide sodium hydroxide solution (count): 0.009

V = 0.1N NaOH consumption

F = factor of 0.1N NaOH

S = sample volume mL

11. Total bacteria count and microbial measurement

1 mL of each sample was taken aseptically and aseptically, 9 mL of sterile water was added to 1 mL of the sample taken, and the dilution of the mixture was gradually diluted to make PCA (Plate Count Agar) medium, MRSA medium, YMPGA (Yeast Extract, Malt Extract, Peptone, Glucose, Agar) was plated in 0.1 mL and then incubated for 48 hours in a 30 ° C. incubator to count the colonies and expressed as log CFU (colony forming unit) / mL.

Experiment result

1. Characteristics of beer according to the type of beads used during fermentation using lactic acid bacteria

The present inventors analyzed the samples in the fermentation process using lactic acid bacteria after adding E.W.C and / or E.L.M to the saccharification solution.

Hereinafter, the sample to which E.W.C is added, the sample to which E.W.C_S, E.L.M is added, the sample to which E.L.M_S, E.W.C and E.L.M is added, is expressed as E.W.L_S, and the sample to which no treatment is added is Control_S.

1-1. pH

Figure 4 shows the pH change during the lactic acid bacteria fermentation process of the sample with different alginate calcium carbonate beads. The initial pH of all samples started at 5.98 to 6.04. The rest of the samples except Control_s showed a tendency to rise to pH 6.27 at 6 hours of fermentation, which seems to show a buffering effect due to the neutralizing action of calcium alginate beads.

After 6 hours from the start of fermentation, the optimum pH was achieved by the neutralization of the beads, and the activity of lactic acid bacteria increased. Accordingly, the amount of organic acid produced increased as the fermentation time lapsed by the action of the lactic acid bacteria growing.

Figure 5 shows the change in the total number of bacteria (see (a) of FIG. 5) and the number of lactic acid bacteria (see (b) of FIG. 5) during the fermentation process of lactic acid bacteria in a sample having different alginate calcium carbonate beads.

1-2. Reducing sugar and free sugar content

FIG. 6 shows the soluble solid content of the fermentation broth obtained through oligosaccharide production fermentation using lactic acid bacteria, and as shown in FIG. 6, the soluble solid content was not significantly changed from 15 to 17 brix until 24 hours of fermentation. On the other hand, as shown in FIG. 7, the reducing sugar did not have a significant change in its value until 12 hours after the start of fermentation, and all samples except Control_S increased to 9 to 11 hours after the start of fermentation. This seems to be due to the result that lactic acid bacteria are generated as the fermentation time elapses, and thus the activity of the dextransucrase enzyme, a sugar transfer enzyme, is also increased to hydrolyze sucrose to increase fructose and glucose.

As a result of analyzing the free sugar content using HPLC, all sucrose was consumed within 24 hours of fermentation, and maltose was also consumed by 35 to 40%. It is believed that the sucrose is hydrolyzed and the resulting glucose reacts with maltose to be converted into a receptor product such as isomaltoligosaccharide, and thus, the change in the content of sucrose and maltose tends to decrease. In addition, 1 to 1.24% of mannitol was produced in the remaining samples compared to the control group. Mannitol has a sugar content of 50% compared to sugar alcohol, so it is used as an alternative sweetener because it does not increase blood sugar, and is widely used in foods because of its sweet taste.

Figure 8 shows the results of HPLC analysis of the change in the amount of sucrose in the fermentation process of oligosaccharide production using lactic acid bacteria. Figure 9 shows the HPLC results of analyzing the change in the amount of maltose in the fermentation process of oligosaccharide production using lactic acid bacteria. Figure 10 shows the HPLC results of analyzing the amount of fructose in the fermentation process of oligosaccharide production using lactic acid bacteria. Figure 11 shows the HPLC results of analyzing the change in the amount of mannitol in the oligosaccharide production fermentation process using lactic acid bacteria.

1-3. Functional oligosaccharide content

12 to 15 show the results of changing the detected D-panose and maltosyl-isomaltooligosaccharide (Maltosyl-Isomaltooligosaccharide; MIMO) for each sample (Control_S, E.W.C_S, E.L.M_S, E.W.L_S) by fermentation time. The D-panose of FIGS. 12 to 15 is an α-1,6 oligosaccharide chain linked to the end of α-1,4 bond, and BDP 4 to 7 has a maltose molecule at a reducing end having a polymerization degree of 4 to α- Means 1,6 sugar-linked maltosyl-isomaltooligosaccharide.

As a result of HPLC analysis, it was confirmed that the experimental strains were converted to D-panose, branched maltosyl-isomatooligosaccharides (D.P. 4-7), which are functional oligosaccharides, with mannitol production using sucrose and maltose. In the sample added with lactic acid bacteria, D-panose and BDP4 levels began to increase at 12 hours of fermentation, and E.W.C_S, E.L.M_S, and E.W.L_S showed maximum values of 4.10%, 4.14%, and 3.96%, respectively. BDP4 increased its content to 0.60-1.18% at 18 hours of fermentation and 1.77-2.22% at 24 hours, and the amount of BDP5 to BDP7 having a high degree of polymerization (DP) increased. The final MIMO content after 24 hours of fermentation was 0.89% control, and E.W.C_S, E.L.M_S, and E.W.L_S showed 5.89 to 6.53% content. According to these characteristics, the fermentation broth obtained by using lactic acid bacteria immobilized on calcium alginate can be used to produce a health functional beverage.

According to the above results, it was confirmed that lactic acid bacteria are activated under optimized pH conditions when lactic acid bacteria are immobilized using lactic acid bacteria immobilized on calcium alginate, thereby increasing the activity of sugar transferase, and increasing functional mannitol, functional oligosaccharide content, and the like. On the other hand, the type of lactic acid bacteria did not significantly affect the above properties.

2. Characteristics of beer according to the type of alginate calcium carbonate beads

Hereinafter, a beer sample prepared using a fermentation broth prepared by adding EWC, and a beer sample prepared using a fermentation broth prepared by adding EWC_B and ELM were prepared using a fermentation broth prepared by adding ELM_B, EWC, and ELM. A beer sample prepared using a fermentation broth prepared by adding EWL_B and a non-treated beer sample is expressed as Control_B.

Table 3 below shows the MIMO content and calcium content of the above-mentioned four types of beer samples.

sample MIMO content (%, w / v) Calcium content (mg / 100 g) Control_B 0.89 5.09 E.W.C_B 5.89 84.55 E.L.M_B 6.52 71.38 E.W.L_B 6.53 82.25

As shown in Table 3, compared to the case of Control_B, oligosaccharide production fermentation using lactic acid bacteria showed high MIMO content and calcium content in one beer. It can be seen that the calcium content of E.W.C_B, E.L.M_B and E.W.L_B is not small compared to that of milk having a calcium content of 105.0 mg / 100 g.

Table 4 below shows the color, bitterness, and alcohol content of the four types of beer samples.

sample Beer characteristics SRM
(color) bitterness
(BU) Alcohol content (%, v / v) Control_B 7.26 ± 0.01 39.72 ± 2.22 4.80 E.W.C_B 5.39 ± 0.01 29.18 ± 3.87 2.80 E.L.M_B 5.94 ± 0.01 31.12 ± 1.20 2.90 E.W.L_B 5.18 ± 0.01 32.40 ± 0.77 2.70

The SRM (color) of beer was according to the liquor analysis regulation, and control_B showed a significantly high value. It seems that the fermentation of oligosaccharides using lactic acid bacteria makes the color of beer lighter than that of samples subjected to oligosaccharide production fermentation using lactic acid bacteria. The bitter taste of beer compared with Control_B, the rest of the samples showed a low BU value. This seems to offset the bitter taste due to the high content of sugar components produced by the enzymatic reaction by oligosaccharide production fermentation using lactic acid bacteria. The alcohol content shows lower values for the rest of the samples compared to Control_B.

Table 5 below shows the sensory test results of the four types of beer samples.

sample Sensory characteristics color flavor sweetness bitterness Sour taste Richness aftertaste Overall
Preference Control_B 6.58 ± 1.72 5.53 ± 2.05 3.05 ± 1.48 3.85 ± 1.61 4.18 ± 1.75 4.08 ± 1.69 3.88 ± 2.00 3.73 ± 1.41 E.W.C_B 4.97 ± 1.80 6.08 ± 1.49 5.10 ± 1.69 4.48 ± 1.88 5.13 ± 1.76 5.55 ± 1.08 5.35 ± 1.59 5.98 ± 1.53 E.L.M_B 5.68 ± 1.10 6.20 ± 1.51 4.95 ± 1.57 4.60 ± 1.77 5.38 ± 1.43 5.55 ± 1.08 5.50 ± 1.65 5.90 ± 1.39 E.W.L_B 4.95 ± 1.89 5.63 ± 1.58 4.73 ± 1.68 5.28 ± 1.60 5.13 ± 1.56 5.33 ± 1.58 5.23 ± 1.78 5.65 ± 1.56 G product 5.85 ± 1.10 5.98 ± 1.94 4.68 ± 1.58 5.18 ± 2.47 5.50 ± 1.59 5.63 ± 1.56 5.75 ± 1.56 6.03 ± 1.62 F -value 7.496 1.138 10.707 3.731 4.131 7.994 7.240 16.848

(G product: commercial beer (Green blaze IPA)) As shown in Table 5, Control_B showed lower preference in sweetness, bitterness, sourness, richness, aftertaste, and overall preference than other samples using lactic acid bacteria.

3. Characteristics of beer according to alcohol fermentation method

As described above, the present inventors discharged the gas from the primary fermentation broth produced by oligosaccharide production fermentation using alginate calcium carbonate beads immobilized with Leuconostoc mesenteroides , that is, ELM, at room temperature under anaerobic conditions and alcohol fermentation for 5 days (alcohol main fermentation agent) Step 1) to obtain a second fermentation broth, and the second fermentation broth was obtained by performing additional fermentation (alcohol main fermentation second step) without discharging gas under normal temperature anaerobic conditions, and then refrigerated at 4 ° C. Beer was produced by storage for 4 weeks (alcohol fermentation).

In addition, the present inventors produced a beer by storing the primary fermentation broth obtained after oligosaccharide production fermentation using ELM under anaerobic conditions and fermenting it for 10 days after storage for 4 weeks under refrigerated conditions at 4 ° C for comparison. .

On the other hand, three types of commercial yeast French Sasion (mangrove jack's, BGGi NZ Ltd, Newzealand), Safale S-04 (Fermentis, Lesaffre, England), Safale US- Beer was prepared using 05 (Fermentis, Lesaffre, England) to analyze each characteristic.

Table 6 below shows the soluble solid content, pH, proper acidity, and reducing sugar of the secondary fermentation broth obtained after the first step of alcohol main fermentation using three types of yeast. In addition, it represents the soluble solids content of the tertiary fermentation broth obtained after the second step of alcohol main fermentation.

Experimental method leaven pH Proper acidity (%) 2nd fermentation broth
Soluble solids (Brix˚) 2nd fermentation broth
Reducing sugar (%) Tertiary fermentation broth
Soluble solids (Brix˚) Alcohol primary fermentation first step and
Alcohol fermentation second stage French Saison 3.93 ± 0.01 0.40 ± 0.01 9.8 ± 0.0 2.86 ± 0.06 8.5 ± 0.0 Safale S-04 3.97 ± 0.00 0.34 ± 0.00 10.1 ± 0.0 3.28 ± 0.10 8.9 ± 0.0 Safale US-05 3.76 ± 0.01 0.35 ± 0.02 10.2 ± 0.0 2.64 ± 0.01 9.0 ± 0.0

Table 7 below shows the soluble solid content, pH, appropriate acidity, and reducing sugar of the fermentation broth obtained after alcohol fermentation for 10 days under anaerobic conditions using three types of yeast.

Experimental method leaven pH Proper acidity (%) Soluble solids (Brix˚) of fermentation broth after 10 days fermentation Reducing sugar (%) 10 days effective French Saison 4.00 ± 0.00 0.46 ± 0.02 7.7 ± 0.0 1.00 ± 0.00 Safale S-04 4.03 ± 0.02 0.36 ± 0.02 9.1 ± 0.0 1.74 ± 0.06 Safale US-05 3.83 ± 0.00 0.36 ± 0.00 8.8 ± 0.0 1.57 ± 0.01

As shown in Tables 6 and 7, the secondary fermentation broth obtained in the first step of alcohol main fermentation had a higher soluble solid content than the fermentation broth obtained by discharging gas under anaerobic conditions for 10 days. This is because each sample obtained through the first step of alcohol main fermentation stopped fermentation with a residual sugar remaining compared to the samples obtained after 10 days of fermentation.

On the other hand, the soluble solids content of the tertiary fermentation broth was not significantly different from the soluble solids content of the fermentation broth obtained by discharging gas under anaerobic conditions for 10 days, which is the second step of main fermentation of alcohol in the secondary fermentation broth. Means consumed through fermentation.

On the other hand, there was no difference in pH and titratable acidity, and there was no significant difference in fermentation pattern between the three yeasts.

Table 8 below shows the beer produced using the third fermentation broth obtained through the above-described first alcohol main fermentation step and the second alcohol main fermentation step, and discharged gas under anaerobic conditions, and the beer produced using the fermentation broth obtained after fermentation for 10 days. The palatability also shows the results of sensory testing through irradiation.

Fermentation method leaven flavor Overall preference Alcohol primary fermentation first step and
Alcohol fermentation second stage French Saison Fermented scent without sake The taste is soft and the carbonate is good Safale S-04 Fine scent appears I can feel the fine taste of sake Safale US-05 There is no scent, and a soft scent appears. The taste is similar to that of ordinary beer, and the soft sweetness is felt. 10 days effective French Saison Cheongju Dynasty appears Cheongju tasted, it had a pleasant taste, and fermented odor remained. Safale S-04 Strong alcoholic beverages appear Strong yakju taste, strong bitter taste Safale US-05 Cheongju Dynasty appears Cheongju tastes good

As shown in Table 8, there was an opinion that the samples fermented for 10 days had odors such as scented alcohol (smell of scented alcohol), but after 5 days yeast fermentation (first step of alcoholic fermentation), further fermentation (alcohol) The beer obtained by the second fermentation of the main fermentation) does not show a scent, and has a soft sweet taste, and has a high preference. On the other hand, in the sensory test results, there was no significant difference between the three types of yeast.

That is, it was confirmed that when beer is produced by consuming sugar through yeast fermentation in two stages, there is no odor such as sake, and beer with higher preference can be produced.

Claims (10)

Mixing rice with malt and saccharifying to obtain saccharification solution;
Oligosaccharide production fermentation step using lactic acid bacteria to obtain a primary fermentation broth by adding alginate calcium carbonate beads to which the lactic acid strain is immobilized to the saccharification broth;
After the primary fermentation broth is heated by putting at least one type of hops, alcohol is added and fermented to obtain a secondary fermentation liquor; And
The second fermentation broth fermentation in a closed system alcohol fermentation second step of obtaining a third fermentation broth; beer production method comprising a.
The method of claim 1, wherein the lactic acid strain is Weissella cibaria or Leuconostoc mesenteroides .
The method according to claim 1, wherein the soluble solids content of the primary fermentation broth is 15 to 17 brix.
delete The method according to claim 1, wherein the first step of alcohol main fermentation is a beer production method performed under conditions in which at least a part of carbon dioxide produced by fermentation is discharged.
The method according to claim 1, wherein the soluble solids content of the secondary fermentation broth is 9 to 11 brix.
The method according to claim 1, wherein the soluble solids content of the tertiary fermentation broth is reduced by 0.5 to 2 Brix compared to the soluble solids content of the secondary fermentation broth.
The method of claim 1, wherein the yeast is 0.005 to 0.05% (w / v) added to the primary fermentation broth.
The method of claim 1, further comprising an alcohol fermentation step of fermenting the tertiary fermentation solution under refrigerated conditions.
Beer produced by the method of any one of claims 1 to 3 and 5 to 9.

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