CN111793590B - Brewing microbial inoculum for acidized beer and application thereof - Google Patents
Brewing microbial inoculum for acidized beer and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C11/00—Fermentation processes for beer
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- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
- C12C12/008—Lactic acid bacteria
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention relates to a brewing microbial inoculum for acidizing beer and application thereof. A brewing microbial agent for acidifying beer is prepared by mixing lactobacillus and Brettanomyces (Brettanomyces); the lactobacillus is composed of lactobacillus plantarum and pediococcus acidilactici. The invention also relates to a preparation method of the brewing microbial inoculum for the acidified beer and a method for brewing the acidified beer by using the brewing microbial inoculum. The acidified beer provided by the invention utilizes a pure microbial fermentation technology to inoculate specific microbial lactobacillus and saccharomycetes into wort for fermentation to generate specific aroma and taste, so that the technology is convenient to fix and standardize, and meanwhile, the risk of generating toxic and harmful substances by pollution fermentation of unknown microorganisms can be reduced.
Description
Technical Field
The invention relates to a brewing microbial inoculum for acidizing beer and application thereof, belonging to the technical field of beer brewing.
Background
The acidified beer is a traditional beer originated from Belgium, also called lambic, Lapeck and Lanpeck beer, has no large-scale brewing for the moment in China, and is mostly fermented by utilizing naturally-existing microorganisms in a specific air environment. Different from the common beer brewing process which strictly controls the external microorganisms to enter the wort, the traditional process of the acid beer generally has a special natural inoculation step, namely, a window of a workshop for storing the wort is opened, the environmental microorganisms are naturally inoculated through air convection, and the population of the microorganisms is large and complicated.
At present, no relevant research report of acidified beer exists in China, and more researches are related to influence of biological acidification technology on beer taste. For example, in the application of the biological acidification technology to beer production (Wangzhai et al, beer science and technology, 2010), the biological acidification technology is used for separating non-putrefying beer lactobacillus or excellent lactic acid producing bacteria from the surface of malt, fermenting and producing lactic acid by using first-pass wort as a culture medium, and adjusting the pH values of mash and wort in the saccharification process to meet the requirements of beer brewing without adding acid additionally. The biological acidification technology can not only reduce the production cost, but also improve the beer quality.
In the biological acidification technology, the key point is to reduce the irritant taste caused by the generation of acetic acid in beer by lactic acid bacteria, but the lactic acid bacteria known at present can inhibit the growth of lactic acid due to the bitter taste generated by the isomerization of alpha-acid generated by hops under the condition of hop existence. For example, in "influence of metabolism of lactic acid bacteria and on beer quality under different environmental conditions" (Wangting et al, university of Dalian Industrial science, 2015), reference to beer spoilage bacteria refers to a group of facultative or strict anaerobic microorganisms that are harmful to beer quality and destroy beer flavor and biological stability, among which lactic acid bacteria are the most predominant ones. The hop bitter taste substance which gives the beer bitter taste in the beer has the function of bacteriostasis and can inhibit the growth of various bacteria. Beer spoilage lactic acid bacteria can resist the bacteriostatic action of bitter taste of hops, accompanied by ATP metabolism. Although the beer spoilage lactic acid bacteria can survive and grow under the condition that the lupulus bitter taste exists, the lactic acid generated by the growth of the beer spoilage lactic acid bacteria cannot meet the requirement of the acidified beer, so that the amount of acetic acid generated by yeast cannot be inhibited, and the taste of the acidified beer is influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a brewing microbial inoculum for acidizing beer and application thereof.
The technical scheme of the invention is as follows:
a brewing microbial agent for acidifying beer is prepared by mixing lactic acid bacteria and Brettanomyces Brettanomyces (6-10): 1 viable bacteria number ratio, and the total viable bacteria number is 0.8-1.5 × 109cfu/mL；
The lactobacillus consists of lactobacillus plantarum and pediococcus acidilactici, and the number ratio of viable bacteria of the lactobacillus plantarum to the pediococcus acidilactici is (2-4): 1.
Preferably according to the invention, the lactobacillus plantarum is purchased from lactobacillus plantarum of the encyclopedia bioengineering limited; pediococcus acidilactici and Brett's yeast are available from wyast or whitelab.
The preparation method of the brewing microbial inoculum of the acidified beer comprises the following steps:
(1) activating and expanding the lactobacillus plantarum to prepare lactobacillus plantarum bacterial liquid;
(2) activating and expanding the pediococcus acidilactici to obtain pediococcus acidilactici bacterial liquid;
(3) activating and expanding the brettanomyces to prepare a brettanomyces liquid;
(4) and (3) inoculating a mixed bacterial liquid obtained by mixing the lactobacillus plantarum bacterial liquid obtained in the step (1), the pediococcus acidilactici bacterial liquid obtained in the step (2) and the brettrium yeast bacterial liquid obtained in the step (3) into sterile wort, and performing adaptive amplification culture to obtain the lactobacillus plantarum bacterial liquid when the quantity and the proportion of thalli meet the inoculation requirements.
Preferably, in step (1) or (2), the activation and expansion culture conditions are: culturing at 18-30 ℃ for 18-48 hours in a culture medium of 10-15 DEG P sterile wort, ventilating every 2-4 hours in the culture process, and initially inoculating 0.5-1.5 multiplied by 108cfu/mL。
Preferably, in step (3), the activation and expansion culture conditions are: culturing for 18-48 hours at 18-30 ℃, wherein the culture medium is 10-15 DEG P sterile wort, ventilating is performed every 2-4 hours in the culture process, the ventilation is performed slowly to prevent the foam from overflowing to cause pollution in the container, and the initial inoculation amount is 0.5-1.5 multiplied by 107cfu/mL。
According to the invention, in the step (4), the volume ratio of the lactobacillus plantarum bacterial liquid to the pediococcus acidilactici bacterial liquid to the braytococcus bacterium liquid in the mixed bacterial liquid is (4-6): 2-4): 1;
according to the invention, in the step (4), the volume of the sterile wort is preferably 5-10 times of the volume of the mixed bacteria liquid.
According to the invention, in the step (4), the sterile wort is 10-15 DEG P.
Preferably, in step (4), the conditions for the adaptive scale-up culture are as follows: culturing at 18-30 ℃ for 18-36 hours, and ventilating for 3-5 minutes every 2-4 hours in the culture process.
The application of the brewing microbial inoculum of the acidified beer in preparing the acidified beer.
The application comprises the following steps:
(i) preparing a bacterial solution to be inoculated by carrying out activated culture and expanded culture on a brewing microbial agent of the acidified beer;
(ii) inoculating the microbial inoculum to be inoculated prepared in the step (i) into wort, wherein the inoculation amount is 5-10% of the volume of the wort, the addition amount of the granular hop in the wort is 0-600 mg/L, fermenting at 15-25 ℃ until the sugar degree is 5-6 DEG P, sealing the can, continuously fermenting at the temperature until the total acid is constant, continuously maintaining for 7-10 days in the sealed state, cooling to 0 ℃ and then storing to prepare fermentation liquor;
(iii) and (iii) filling and sterilizing the fermentation liquor prepared in the step (ii) to prepare the acidified beer.
Preferably, in step (i), the activation culturing step is as follows:
inoculating a brewing microbial inoculum of the acidified beer into light-colored wort with the temperature of 10-15 ℃ P, inoculating the inoculation amount of 20-50%, and performing sterile culture at 18-30 ℃ for 18-36 h to obtain the activated microbial inoculum.
Preferably, in step (i), the step of expanding culture is as follows:
transferring the activated and cultured bacterial liquid to 5-20 times of light-colored wort under aseptic conditions, inoculating 20-50%, and culturing at 18-30 ℃ for 18-36 h to obtain the microbial inoculum to be inoculated.
According to the present invention, in the step (ii), the amount of the granular hop in the wort is preferably 200 to 500 mg/L.
Preferably, in step (ii), the wort is obtained by crushing malt, saccharifying, filtering, washing, boiling, whirlpool precipitating, and cooling.
Further preferably, the malt is malt containing not more than 30% unmalted kernels.
Preferably, the wort comprises the following ingredients in parts by weight:
6 parts of malt, 2 parts of wheat kernel, 0.014 part of home-made granular hop and 42 parts of water.
The germinated malt can be combined in various proportions according to the requirements on beer color, and common types of the germinated malt include Belson malt, Aier malt, burnt malt, dark malt and the like.
More preferably, the sugar degree of the wort is 12-16 DEG P.
Preferably, in the step (ii), the total acid content is 4.5-5.5 g/100 mL.
Preferably according to the invention, in step (ii), the wort has a bittering profile of no more than 25 IBU.
Preferably, in step (iii), the filling step further comprises a solid-liquid separation step; further preferably, the solid-liquid separation is filtration or centrifugation.
Preferably, step (iii) of mixing the juice or/and blending with other types of beer and/or supplementing CO is further included before the filling step2The step (2). The steps can be completed in the conventional beer production field, belongs to the conventional technology, and is used for blending and supplementing CO2The selection of the product is mainly determined by market factors such as the sales mode, the sales channel, the product type demand and the like of the product.
Description of the principles
The acidified beer of the invention is brewed by adopting a microorganism mixed fermentation mode, namely required microorganisms are simultaneously added into wort for fermentation, and the fermentation modes of various microorganisms have different functions in the fermentation process, lactobacillus plantarum can produce lactic acid by homotype fermentation, 2 parts of lactic acid can be produced when 1 part of glucose is consumed, and 1 part of lactic acid +1 part of CO is produced when 1 part of glucose is consumed by pediococcus2Can ensure the higher content of lactic acid in the fermentation liquor, and the lactic acid is mainly contained in the beer than the common beerSome organic acids, i.e., acetic acid, have a soft mouthfeel, and lactic acid bacteria of the genus Pediococcus have a higher tolerance to hops although they have a lower efficiency in metabolizing lactic acid. Hops contain a series of compounds that inhibit the growth of gram-positive bacteria. The key material of these substances is iso-alpha-acids, which are generated by isomerisation of alpha-acids in hops during wort boiling, so that the bitter taste of the acid beer described in this application is a conventional level of bitter taste to ensure that no inhibition of lactic acid bacteria growth is formed. The inhibitory effect is due to the ability of the iso-alpha-acids to bind H in the liquid+Ions, H dispersed across the surface of the bacterial cell membrane+Concentrations which impair enzyme activity and influence nutrient transport, ultimately acting as a cell stop, lower levels of iso-alpha-acids which are responsible for H+Has limited binding activity and cannot inhibit the growth of the corresponding microorganisms.
Advantageous effects
The acidified beer provided by the invention utilizes a pure microbial fermentation technology to inoculate specific microbial lactobacillus and saccharomycetes into wort for fermentation to generate specific aroma and taste, so that the technology is convenient to fix and standardize, and meanwhile, the risk of generating toxic and harmful substances by pollution fermentation of unknown microorganisms can be reduced.
Drawings
FIG. 1 is a graph showing the total acid content change in the fermentation process of example 4 and general beer;
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
Source of raw materials
Malt, wheat kernel and granular hop are all conventional raw materials for brewing beer and are common commercial products; the brewing water is tap water.
Detection method
The lactic acid bacteria counting is carried out by adopting a bacteria counting plate, the viable bacteria counting can adopt a flat plate coating culture counting method, and the specific steps refer to GB4789.35 food safety national standard food microbiology inspection lactic acid bacteria inspection; counting the yeasts by a blood cell counting plate;
the detection methods of items such as total acid, pH value, alcohol content and the like of beer fermentation liquor refer to GB/T4928; the flavor substances are detected by a gas chromatograph.
Microbial origin
Lactobacillus plantarum was purchased from lactobacillus plantarum of interference bioengineering limited;
pediococcus acidilactici and Bretts yeast were purchased from wyesast.
Example 1
A preparation method of a brewing microbial inoculum for acidizing beer comprises the following steps:
(1) activating and expanding the lactobacillus plantarum to prepare lactobacillus plantarum bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 25 deg.C for 48 hr in 12 ° P sterile wort, ventilating every 4 hr during culture, and inoculating with 1.0 × 108cfu/mL；
(2) Activating and expanding the pediococcus acidilactici to obtain pediococcus acidilactici bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 25 deg.C for 48 hr in 12 ° P sterile wort, ventilating every 4 hr during culture, and inoculating with 1.0 × 108cfu/mL；
(3) Activating and expanding the brettanomyces to prepare a brettanomyces liquid;
the activation and expansion culture conditions are as follows: culturing at 25 deg.C for 48 hr in 12 ° P sterile wort, ventilating every 4 hr during culture, and inoculating with 1.0 × 107cfu/mL；
(4) Inoculating a mixed bacterium solution of the lactobacillus plantarum bacterium solution prepared in the step (1), the pediococcus acidilactici bacterium solution prepared in the step (2) and the brerett yeast solution prepared in the step (3) into sterile wort, performing adaptive amplification culture at 25 ℃ for 48 hours, wherein a culture medium is 12-DEG P sterile wort, intermittently ventilating every 4 hours for 3 minutes in the culture process, preventing the pollution in a container caused by foam overflow, and obtaining the lactobacillus plantarum bacterium liquid by refrigeration and preservation when the quantity and proportion of bacteria meet the inoculation requirement;
in the mixed bacterial liquid, the volume ratio of lactobacillus plantarum bacterial liquid to pediococcus acidilactici bacterial liquid to brettanomyces bacteria liquid is 6:3: 1; the volume of the sterile wort is 10 times of the volume of the mixed bacteria liquid;
detecting the brewing microbial inoculum of the acidified beer, wherein the viable count ratio of the lactobacillus to the Brettanomyces is 9:1, and the total viable count is 1.0 × 109cfu/mL; the lactobacillus consists of lactobacillus plantarum and pediococcus acidilactici, and the number ratio of viable bacteria of the lactobacillus plantarum to the pediococcus acidilactici is 2: 1.
Example 2
A preparation method of a brewing microbial inoculum for acidizing beer comprises the following steps:
(1) activating and expanding the lactobacillus plantarum to prepare lactobacillus plantarum bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 18 deg.C for 48 hr in 10 ° P sterile wort, ventilating every 2 hr during culture, and inoculating with 1.5 × 108cfu/mL；
(2) Activating and expanding the pediococcus acidilactici to obtain pediococcus acidilactici bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 18 deg.C for 48 hr in 10 ° P sterile wort, ventilating every 2 hr during culture, and inoculating with 1.5 × 108cfu/mL；
(3) Activating and expanding the brettanomyces to prepare a brettanomyces liquid;
the activation and expansion culture conditions are as follows: culturing at 18 deg.C for 48 hr in 10 ° P sterile wort, ventilating every 2 hr during culture, and inoculating with 1.5 × 107cfu/mL；
(4) Inoculating a mixed bacterium solution of the lactobacillus plantarum bacterium solution prepared in the step (1), the pediococcus acidilactici bacterium solution prepared in the step (2) and the brerett yeast solution prepared in the step (3) into sterile wort, performing adaptive amplification culture at 18 ℃ for 36 hours, wherein a culture medium is the sterile wort with 13-DEG P, intermittently ventilating for 4 minutes every 2 hours in the culture process, preventing the pollution in a container caused by foam overflow, and obtaining the lactobacillus plantarum bacterium solution by refrigeration and preservation when the quantity and proportion of bacteria meet the inoculation requirement;
in the mixed bacterial liquid, the volume ratio of lactobacillus plantarum bacterial liquid to pediococcus acidilactici bacterial liquid to brettanomyces bacteria liquid is 4:2: 1; the volume of the sterile wort is 6 times of the volume of the mixed bacteria liquid;
detecting the brewing microbial inoculum of the acidified beer, wherein the viable count ratio of the lactobacillus to the Brettanomyces is 6:1, and the total viable count is 1.2 × 109cfu/mL; the lactobacillus consists of lactobacillus plantarum and pediococcus acidilactici, and the number ratio of viable bacteria of the lactobacillus plantarum to the pediococcus acidilactici is 2: 1.
Example 3
A preparation method of a brewing microbial inoculum for acidizing beer comprises the following steps:
(1) activating and expanding the lactobacillus plantarum to prepare lactobacillus plantarum bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 30 deg.C for 18 hr in sterile wort at 15 ° P in a medium with ventilation every 4 hr, and inoculating with an initial inoculum of 0.5 × 108cfu/mL；
(2) Activating and expanding the pediococcus acidilactici to obtain pediococcus acidilactici bacterial liquid;
the activation and expansion culture conditions are as follows: culturing at 25 deg.C for 48 hr in 12 ° P sterile wort, ventilating every 4 hr during culture, and inoculating with 1.0 × 108cfu/mL；
(3) Activating and expanding the brettanomyces to prepare a brettanomyces liquid;
the activation and expansion culture conditions are as follows: culturing at 25 deg.C for 48 hr in 12 ° P sterile wort, ventilating every 4 hr during culture, and inoculating with 1.0 × 107cfu/mL；
(4) Inoculating a mixed bacterium solution of the lactobacillus plantarum bacterium solution prepared in the step (1), the pediococcus acidilactici bacterium solution prepared in the step (2) and the brerett yeast solution prepared in the step (3) into sterile wort, performing adaptive amplification culture at 30 ℃ for 40 hours, wherein a culture medium is the sterile wort with 15 DEG P, intermittently ventilating for 3 minutes every 3 hours in the culture process, preventing the pollution in a container caused by foam overflow, and obtaining the lactobacillus plantarum bacterium liquid by refrigeration and preservation when the quantity and proportion of bacteria meet the inoculation requirement;
in the mixed bacterial liquid, the volume ratio of lactobacillus plantarum bacterial liquid to pediococcus acidilactici bacterial liquid to brettanomyces bacteria liquid is 5:3: 1; the volume of the sterile wort is 8 times of the volume of the mixed bacteria liquid;
detecting the brewing microbial inoculum of the acidified beer, wherein the viable count ratio of the lactobacillus to the Brettanomyces is 8:1, and the total viable count is 1.5 multiplied by 109cfu/mL; the lactobacillus consists of lactobacillus plantarum and pediococcus acidilactici, and the number ratio of viable bacteria of the lactobacillus plantarum to the pediococcus acidilactici is 5: 3.
Example 4
A method for preparing acidified beer by using the brewing microbial inoculum for acidified beer described in example 1, comprising the following steps:
(a) bacterial liquid activation: inoculating the preserved brewing microbial inoculum into 10mL of pre-sterilized wort, and culturing at 28 ℃ for 24h, and fully shaking every 3h to obtain activated bacterial liquid;
expanding and culturing bacterial liquid: transferring the activated bacterium liquid into 170mL of wort under aseptic conditions, and culturing at 25 ℃ for 48 h; then, continuously inoculating the cultured bacterial liquid into 1600mL of wort, culturing at 22 ℃ for 48h, taking the cultured bacterial liquid as a seed liquid, inoculating the seed liquid into 30L of corresponding acid beer wort for fermentation, wherein the wort used for expanding culture is 12-DEG P light-color hop-free wort, and preparing the bacterial liquid to be inoculated;
(b) preparing wort: pulverizing fructus Hordei Germinatus, saccharifying, filtering, washing distiller's grains, boiling, vortex precipitating, and cooling to obtain wort;
wherein the ingredients for preparing the wort are as follows: 30L of water for saccharification, 12L of water for washing lees, 6Kg of Belson malt, 2Kg of wheat kernel and 14g of home-made granular hop;
the saccharification process comprises blanking at 37 ℃ and keeping for 10 minutes, then heating to 50 ℃ and keeping for 15 minutes, keeping for 60 minutes at 65 ℃, keeping for 10 minutes at 72 ℃, filtering wort at 78 ℃, washing grains with 12L of pure water at 78 ℃ and collecting the wort; boiling the wort for 70 minutes after washing the lees, and adding hops when the wort is boiled for 10 minutes; after boiling, carrying out vortex precipitation on the wort for 20 minutes, collecting the supernatant of the wort, cooling the wort to 18 ℃, transferring the wort into a clean fermentation tank, and obtaining 30L of wort with the concentration of 14.5 DEG P through the step;
(c) inoculating 1.78L of the bacterial liquid to be inoculated prepared in the step (a) into the wort prepared in the step (b), and fermenting at 18 +/-0.3 ℃; immediately introducing sterile air to the fermentation substrate in the tank for 5min after inoculation, and ventilating again for 5min after fermentation for 18h to promote uniform mixing and rapid initiation of bacterial liquid in the fermentation liquid; sealing the tank when the sugar degree is 5.8P, continuously maintaining the temperature for fermentation, sealing the tank for 13 days until the total acid is constant and reaches 4.8g/100mL, maintaining the fermentation liquor at 18 ℃ for 7 days, cooling to 0 ℃ for post-storage to obtain mature fermentation liquor;
(d) directly filling the fermentation liquor prepared in the step (c) into a glass bottle without filtering, and sterilizing in a water bath at 62 ℃ for 8 minutes to prepare the non-blended acidified beer.
Through detection, the alcohol content is 4.83g/100g, the total acid content is 4.8g/100mL, the diacetyl content is 0.07mg/L, the ethyl acetate content is 35mg/L, the isoamyl acetate content is 7.5mg/L, and the bitter taste quality is 15 IBU;
during the main fermentation period, the highest cell concentration of each bacterium is as follows, and the highest cell concentration of the Brettt yeast is 6.2 multiplied by 107cfu/mL, Lactobacillus plantarum 4.2X 108cfu/mL, Pediococcus acidilactici 1.4X 108cfu/mL。
From the example 4, it can be seen that the total acid in the fermentation of the present invention is constant, and generally reaches about 4.5-5.5 g/100mL when the fermentation is performed for 3 weeks, while the acidity of the general beer (i.e. the beer fermented by the single saccharomyces cerevisiae) is constant within 2 weeks, and the total acid is generally less than or equal to 2.2g/100mL, and the results are shown in fig. 1.
Comparative example 1
The method as described in example 1, except that the lactic acid bacteria in the mixed starter culture used were replaced with the lactobacillus bulgaricus and lactobacillus casei in a direct vat set commonly used for dairy fermentation, with the amount of bacteria unchanged; to prepare the contrast agent 1.
Preparation of acidified beer was carried out by using comparative inoculum 1 according to the method of example 4.
Through detection, the alcohol content is 4.91g/100g, the total acid content is 3.6g/100mL, the diacetyl content is 0.16mg/L, the ethyl acetate content is 32mg/L, the isoamyl acetate content is 7.0mg/L, and the bitter taste quality is 15 IBU;
during the main fermentation period, the highest cell concentration of each bacterium is as follows, and the highest cell concentration of the Brettt yeast is 6.5 multiplied by 107cfu/mL, total number of cells of direct-fed lactic acid bacteria 2.3X 108cfu/mL。
Diacetyl is a typical flavor substance normally existing in milk, but is an off-flavor for beer, the content of diacetyl is generally controlled to be lower than 0.1mg/L, and a typical flavor defect phenomenon is caused when the diacetyl content is higher; the diacetyl content of comparative example 1 is significantly higher than that of the conventional requirement, and the fact that the replacement of different lactic acid bacteria has a significant effect on the index in the fermentation environment is proved. The flavor substances such as ethyl acetate, isoamyl acetate and the like are mainly related to the metabolism of yeast and are slightly influenced.
Comparative example 2
The method as described in example 1, except that brettanomyces was replaced with aleyrodids fermented with general beer, and the amount of bacteria was unchanged; to prepare a contrast agent 2.
Preparation of acidified beer was carried out by using comparative inoculum 2 in accordance with the method of example 4.
Through detection, the alcohol content is 5.15g/100g, the total acid content is 3.2g/100mL, the diacetyl content is 0.16mg/L, the ethyl acetate content is 10mg/L, the isoamyl acetate content is 0.2mg/L, and the bitter taste quality is 15 IBU;
during the main fermentation period, the highest cell concentration of each bacterium is as below, and the concentration of the Aier type saccharomyces cerevisiae is 7.8 multiplied by 107cfu/mL, Lactobacillus plantarum 3.0X 108cfu/mL, Pediococcus acidilactici 1.0X 108cfu/mL；
In the fermentation process, the blood sugar reduction speed of the fermentation liquor is obviously improved, lactic acid bacteria are influenced by the rapid growth of yeast, the acid production is insufficient, the total acid is 3.2g/100mL, the alcohol content is 5.15g/100g, and the content difference of ethyl acetate and isoamyl acetate presenting fruit aroma in the flavor substances after the fermentation is finished is obviously lower than that in example 1 and is respectively 10mg/L and 0.2 mg/L. The overall harmony of flavor and taste is poor.
Comparative example 3
The process as described in example 4, except that hops were not added during the wort preparation.
In the comparative example, on the 3 rd day and the 4 th day of fermentation, the sterile wind is respectively supplemented and introduced for 1 time due to the lower blood sugar reducing speed, the total fermentation time of reducing the sugar degree of wort from 14.5 degrees P to 5 degrees P is 25 days, and the example 4 is 19 days;
the detection proves that the alcohol content is 4.76g/100g, the total acid content is 3.2g/100mL, the diacetyl content is 0.13mg/L, the ethyl acetate content is 16mg/L, and the isoamyl acetate content is 3.8 mg/L.
During the main fermentation period, the highest cell concentration of each bacterium is as follows, and the highest cell concentration of the Brettt yeast is 3.8 multiplied by 107cfu/mL, Lactobacillus plantarum 2.8X 108cfu/mL, Pediococcus acidilactici 0.7X 108cfu/mL。
In the fermentation process, the blood sugar reducing speed of the fermentation liquor is obviously reduced, hops are not added in the wort boiling process, and cannot be fully separated out by means of the combination and precipitation of polyphenols in the hops and coagulable protein in the wort, so that physical wrapping effect is formed on microorganisms in the fermentation process, the microorganisms cannot be fully contacted with nutrient substances to be metabolized, the consumption speed of the nutrient substances, the microbial quantity and the fermentation speed in the fermentation process are reduced, the formation of various metabolites is further influenced, the flavor of beer is poor, and the taste harmony of the beer is poor because of no bitter taste given by hops. Meanwhile, under the condition, the inoculated microorganisms cannot form the advantages of flora as soon as possible, and the mixed bacteria are easily polluted to cause the fermentation liquor to deteriorate.
Analysis of results
As can be seen from the results of the example 1 and the comparative examples 1-2, in the initial stage of fermentation, the selected strain is a common food fermentation microorganism, so that the propagation and the initiation speed are high, glucose, fructose and maltose in a fermentation substrate are rapidly utilized to enter an acid production stage to accumulate a large amount of lactic acid, the selected yeast is a wild yeast, the fermentation speed is slow compared with that of common saccharomyces cerevisiae, but the utilization rate of a substrate is high, and maltotriose can also be utilized, so that the fermentation is generally carried out until the fermentation is carried outAfter 3-5 days and the substrate concentration is consumed to be 0.5-2.5 DEG P, the accumulation of lactic acid begins to slow down, and the brettanomyces begins to metabolize to generate alcohol and CO2And accumulate abundant aroma substances such as mellow aroma, ester aroma and phenolic aroma, and contain 4-vinyl guaiacol, ethyl acetate, phenethyl alcohol and other aroma substances. This phenomenon can only be produced after the specific bacteria described in example 1 are collocated, and at the same time under the specific hop concentration conditions.
In the early stage of inoculation, the nutrient conditions of a fermentation substrate (namely wort) are favorable for the growth of various microorganisms and are difficult to generate mutual inhibition, but as the dissolved oxygen in the culture medium is gradually consumed in the early stage of fermentation, the growth of each microorganism enters different stages and is possibly limited by the stress of adverse factors or metabolism, the propagation speed of the Eleutherococcus barus is high under the condition of sufficient dissolved oxygen, the quantity of the Eleutherococcus is rapidly increased, and the lactic acid bacteria also gradually start to propagate and metabolize to generate acid along with the oxygen consumption in the microenvironment. Compared with the brettanomyces, the common alexandrium type yeast has poorer tolerance to acid stress, so that although a larger number of cells are accumulated in the early stage of fermentation, the aroma production capability is weakened under the influence of the acid stress in the later stage, the flavor type of the alexandrium type yeast is generally not full-bodied fruit flavor but relatively flat ester flavor, and the flavor effect on enhancing the acid beer is weaker; the direct-vat lactic acid bacteria have strong acid-producing ability in milk base material, weak acid-producing ability in wheat juice, and high diacetyl-producing ability, while the general Aier yeast has limited activity of reducing and degrading diacetyl with high concentration, which results in insufficient total acid and flavor of fermentation liquor, and high diacetyl.
In addition, under the action of multiple strains, the ethyl acetate content of the acid beer prepared in the embodiment 1 reaches 20-35 mg/L, fruit aroma is guaranteed, meanwhile, the isoamyl acetate content is between that of common beer and wheat beer, the phenethyl alcohol content reaches 70mg/L, high lactic acid content is combined, the acid beer is endowed with specific taste and aroma from the two aspects of taste and aroma, the taste acidity is high, but the acid beer is mainly characterized by combining lactic acid with a small amount of acetic acid, and unlike a composite strain fermented beverage containing acetic acid bacteria, the acetic acid is used as a main acid, the acetic acid has strong and pungent flavor, and meanwhile, the acetic acid is used as a volatile acid to easily cover other flavors of the beer.
Claims (18)
1. The preparation method of the acidified beer is characterized by comprising the following steps:
(i) preparing a bacterial solution to be inoculated by carrying out activated culture and expanded culture on a brewing microbial agent of the acidified beer;
(ii) inoculating the microbial inoculum to be inoculated prepared in the step (i) into wort, wherein the inoculation amount is 5-10% of the volume of the wort, the addition amount of the granular hop in the wort is 200-500 mg/L, fermenting at 15-25 ℃ until the sugar degree is 5-6 DEG P, sealing the can, continuously fermenting at the temperature until the total acid is constant, continuously maintaining for 7-10 days in the sealed state, cooling to 0 ℃ and then storing to prepare fermentation liquor;
the sugar degree of the wort is 12-16 degrees P;
(iii) (iii) filling and sterilizing the fermentation liquor prepared in the step (ii) to prepare acidified beer;
in the step (i), the brewing microbial inoculum for acidifying the beer is formed by mixing lactic acid bacteria and Brettanomyces (Brettanomyces), the viable bacteria number ratio of the lactic acid bacteria to the Brettanomyces is (6-10): 1, and the total viable bacteria number is 0.8-1.5 multiplied by 109cfu/mL；
The lactobacillus consists of lactobacillus plantarum and pediococcus acidilactici, and the number ratio of viable bacteria of the lactobacillus plantarum to the pediococcus acidilactici is (2-4): 1.
2. The method according to claim 1, wherein the brewer's microbial inoculum for acidified beer in step (i) is prepared by the steps of:
(1) activating and expanding the lactobacillus plantarum to prepare lactobacillus plantarum bacterial liquid;
(2) activating and expanding the pediococcus acidilactici to obtain pediococcus acidilactici bacterial liquid;
(3) activating and expanding the brettanomyces to prepare a brettanomyces liquid;
(4) and (3) inoculating a mixed bacterial liquid obtained by mixing the lactobacillus plantarum bacterial liquid obtained in the step (1), the pediococcus acidilactici bacterial liquid obtained in the step (2) and the brettrium yeast bacterial liquid obtained in the step (3) into sterile wort, and performing adaptive amplification culture to obtain the lactobacillus plantarum bacterial liquid when the quantity and the proportion of thalli meet the inoculation requirements.
3. The method according to claim 2, wherein in the step (1) or (2), the activation and expansion culture conditions are: culturing at 18-30 ℃ for 18-48 hours in a culture medium of 10-15 DEG P sterile wort, ventilating every 2-4 hours in the culture process, and initially inoculating 0.5-1.5 multiplied by 108cfu/mL。
4. The method according to claim 2, wherein in the step (3), the activation and expansion culture conditions are: culturing at 18-30 ℃ for 18-48 hours in a culture medium of 10-15 DEG P sterile wort, ventilating every 2-4 hours in the culture process, and initially inoculating 0.5-1.5 multiplied by 107cfu/mL。
5. The method according to claim 2, wherein in the step (4), the volume ratio of the mixed bacterial liquid to the lactobacillus plantarum bacterial liquid, the pediococcus acidilactici bacterial liquid and the brettanomyces albuminosus bacterial liquid is (4-6): (2-4): 1.
6. The method according to claim 2, wherein in the step (4), the volume of the sterile wort is 5 to 10 times that of the mixed bacterial liquid.
7. The method according to claim 2, wherein in the step (4), the sterile wort is 10 to 15 ° P.
8. The method according to claim 2, wherein in the step (4), the conditions for the adaptive scale-up culture are: culturing at 18-30 ℃ for 18-36 hours, and ventilating for 3-5 minutes every 2-4 hours in the culture process.
9. The method according to claim 1, wherein in the step (i), the activation culturing step is as follows:
inoculating a brewing microbial inoculum of the acidified beer into light-colored wort with the temperature of 10-15 ℃ P, inoculating the inoculation amount of 20-50%, and performing sterile culture at 18-30 ℃ for 18-36 h to obtain the activated microbial inoculum.
10. The method according to claim 1, wherein in the step (i), the step of expanding culture is as follows:
transferring the activated and cultured bacterial liquid to 5-20 times of light-colored wort under aseptic conditions, inoculating 20-50%, and culturing at 18-30 ℃ for 18-36 h to obtain the microbial inoculum to be inoculated.
11. The method according to claim 1, wherein in the step (ii), the wort is prepared by pulverizing malt, saccharifying, filtering, washing, boiling, whirlpool precipitating, and cooling.
12. The method of claim 11, wherein the malt is malt containing no more than 30% unmalted wheat kernels.
13. The method for preparing the wort according to claim 12, wherein the wort is prepared from the following ingredients in parts by weight:
6 parts of malt, 2 parts of wheat kernel, 0.014 part of home-made granular hop and 42 parts of water.
14. The method of claim 1, wherein in step (ii), the total acid is 4.5-5.5 g/100 mL.
15. The process of claim 1, wherein in step (ii), the wort has a bittering quality of no more than 25 IBU.
16. The method of claim 1, wherein step (iii) further comprises a step of solid-liquid separation before the filling step.
17. The method of claim 16, wherein the solid-liquid separation is filtration or centrifugation.
18. The process according to claim 1, wherein step (iii) of filling further comprises a step of blending the juice or/and other types of beer and/or CO supplementation2The step (2).
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| CN112920918B (en) * | 2021-03-23 | 2022-05-03 | 青岛啤酒股份有限公司 | Acid beer and preparation method thereof |
| CN113004992B (en) * | 2021-03-23 | 2022-08-12 | 青岛啤酒股份有限公司 | Lanbeck acid beer and preparation method thereof |
| CN113061498B (en) * | 2021-03-23 | 2022-11-08 | 青岛啤酒股份有限公司 | Light sour beer and preparation method thereof |
| CN112760273A (en) * | 2021-03-24 | 2021-05-07 | 中国农业大学 | Complex microbial inoculant and food prepared from same |
| CN113736592A (en) * | 2021-04-26 | 2021-12-03 | 蜀汉益德(成都)精酿啤酒有限公司 | Lactic acid beer containing lactic acid bacteria and capable of relieving piquancy and removing greasy and preparation method thereof |
| CN113201527B (en) * | 2021-04-30 | 2022-09-20 | 齐鲁工业大学 | Hop picric acid yeast microcapsule and preparation method and application thereof |
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