CN110760404A - Bacillus mixed bran koji and preparation process and application thereof - Google Patents
Bacillus mixed bran koji and preparation process and application thereof Download PDFInfo
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- CN110760404A CN110760404A CN201911177646.6A CN201911177646A CN110760404A CN 110760404 A CN110760404 A CN 110760404A CN 201911177646 A CN201911177646 A CN 201911177646A CN 110760404 A CN110760404 A CN 110760404A
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 98
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 35
- 238000002156 mixing Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 24
- 239000001888 Peptone Substances 0.000 claims abstract description 23
- 108010080698 Peptones Proteins 0.000 claims abstract description 23
- 235000015278 beef Nutrition 0.000 claims abstract description 23
- 235000019319 peptone Nutrition 0.000 claims abstract description 23
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 238000009630 liquid culture Methods 0.000 claims abstract description 10
- 230000003321 amplification Effects 0.000 claims abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 235000011844 whole wheat flour Nutrition 0.000 claims description 22
- 238000001816 cooling Methods 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 235000014101 wine Nutrition 0.000 claims description 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 15
- 244000063299 Bacillus subtilis Species 0.000 claims description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 241000194108 Bacillus licheniformis Species 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 8
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 8
- 241000194107 Bacillus megaterium Species 0.000 claims description 8
- 241000194103 Bacillus pumilus Species 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 241000304886 Bacilli Species 0.000 abstract 4
- 239000000969 carrier Substances 0.000 abstract 1
- 238000010025 steaming Methods 0.000 description 19
- 235000013339 cereals Nutrition 0.000 description 17
- 239000000796 flavoring agent Substances 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 230000008569 process Effects 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 11
- 240000006394 Sorghum bicolor Species 0.000 description 9
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 239000004576 sand Substances 0.000 description 8
- 244000052616 bacterial pathogen Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000009835 boiling Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 235000019634 flavors Nutrition 0.000 description 5
- 235000015067 sauces Nutrition 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 238000003892 spreading Methods 0.000 description 5
- 230000007480 spreading Effects 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- -1 Nitrogen-containing compound Chemical class 0.000 description 2
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 238000013124 brewing process Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015096 spirit Nutrition 0.000 description 2
- IWTBVKIGCDZRPL-LURJTMIESA-N 3-Methylbutanol Natural products CC[C@H](C)CCO IWTBVKIGCDZRPL-LURJTMIESA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical class O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003216 pyrazines Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000020097 white wine Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H6/00—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages
- C12H6/02—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages by distillation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses bacillus mixed bran koji and a preparation process and application thereof. The method comprises the steps of firstly culturing various bacilli in a beef extract peptone liquid culture medium, then inoculating the cultured bacilli in primary mouldy bran carriers respectively, and culturing at constant temperature to obtain primary single-strain mouldy bran containing different bacilli respectively. And then, carrying out amplification culture on the obtained primary single-strain mouldy bran respectively to obtain secondary single-strain mouldy bran. And mixing and inoculating a plurality of secondary single-strain bran koji into the secondary bran koji carrier, and culturing at constant temperature to obtain the mixed strain bran koji containing a plurality of bacillus. Because the bran koji prepared by the invention contains various bacilli, the bran koji is more beneficial to improving aroma and reducing higher alcohol in liquor brewing. The whole preparation process is simple, and the bacillus is fused in the mouldy bran, so that the bacillus can be stored and used conveniently.
Description
Technical Field
The invention relates to the technical field of wine brewing, and particularly relates to bacillus mixed bran koji and a preparation process and application thereof.
Background
The bacillus is one of the bacteria with a large number in the yeast, has the capability of hydrolyzing protein and starch, and can metabolize various flavor components in the solid state fermentation of the white spirit. Recent researches show that bacillus subtilis produces enzyme and metabolite in the fermentation process, has rich functional effects, proves the research on functional microorganisms in the white wine production process and the production application thereof, and gives full evidence to the research results in the fields of vinegar brewing and sauce making. A large number of research results show that bacillus licheniformis (Bacillus licheniformis) capable of producing Maotai-flavor substances exists in the micro-ecological environment and the brewing process of Maotai-flavor liquor production in a large number, plays an important role in the brewing process of Maotai-flavor liquor, and is a dominant bacterium in the microbial population for brewing Maotai-flavor liquor.
Currently, the use of bacillus in brewing presents several problems: (1) there is no complete process for using bacillus in liquor brewing; (2) many of the used bacillus are utilized in a single bacterium form, and the aroma of the white spirit is still to be improved; (3) the brewing product prepared by the bacillus is not beneficial to long-term storage; (4) the applied method is not suitable for the production of white spirit.
Disclosure of Invention
In order to solve the application problem of the bacillus in wine brewing, the invention aims to develop the bacillus mixed bran koji which can be stored in a large amount, has simple process flow and is suitable for liquor enterprises, and the aroma components of the liquor are improved. The technical scheme for solving the technical problems is as follows:
a preparation process of bacillus mixed bran koji comprises the following steps:
(1) respectively inoculating bacterial suspensions of various bacillus into beef extract peptone liquid culture media for constant-temperature culture;
(2) uniformly mixing bran, whole wheat flour and water, sterilizing and cooling to obtain a primary bran koji carrier; respectively adding the bacillus obtained in the step (1) into sterile physiological saline, respectively adding the obtained bacterial suspensions into the primary mouldy bran carrier, uniformly mixing, and culturing at constant temperature to respectively obtain primary single-bacterium mouldy bran corresponding to each bacillus;
(3) respectively carrying out amplification culture on the primary single-strain bran koji obtained in the step (2) to obtain secondary single-strain bran koji;
(4) uniformly mixing bran, whole wheat flour and water, sterilizing and cooling to obtain a secondary bran koji carrier; and (4) mixing and inoculating the multiple secondary single-strain bran koji prepared in the step (3) into the secondary bran koji carrier, uniformly mixing, culturing at constant temperature, and drying to obtain the bacillus mixed-strain bran koji.
The invention firstly cultures various bacilli in beef extract peptone liquid culture medium, then mixes the bacilli obtained by culture with the first-level bran koji carrier and cultures them at constant temperature, and then first-level single-strain bran koji containing different bacilli is obtained. And then, carrying out amplification culture on the obtained primary single-strain mouldy bran respectively to obtain secondary single-strain mouldy bran. And then mixing various secondary single-strain bran koji, inoculating the mixture into the secondary bran koji carrier of the invention, and culturing at constant temperature to obtain the mixed-strain bran koji containing various bacilli.
The bran koji prepared by the invention contains various bacilli, so that the bran koji is more beneficial to fragrance improvement in liquor brewing. The whole preparation process is simple, and the bacillus is fused in the mouldy bran, so that the bacillus can be stored and used conveniently.
Further, in a preferred embodiment of the present invention, in the step (1), the plurality of bacillus comprises: bacillus subtilis, bacillus pumilus, bacillus licheniformis, bacillus amyloliquefaciens, bacillus belgii and bacillus megaterium; the culture conditions were: culturing for 14-18h at 36-38 ℃ under a shaking table with the speed of 130-.
According to the invention, Bacillus belgii, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus pumilus are selected as mixed bacteria components, and the three bacteria can inhibit the quantity of yeasts in the middle fermentation stage, so that the content of high-grade alcohol generated by the yeasts is reduced, and the effect of reducing the high-grade alcohol in the white spirit is achieved; in addition, bacillus licheniformis and bacillus megatherium are added, and can generate pyrazine compounds, so that the flavor of the white spirit paste is increased; moreover, the bacilli selected by the invention are beneficial to producing acid, thereby improving the quality of white spirit.
Preferably, the culture conditions are that the culture is carried out for 18 hours at 36 ℃ under a shaking table at 160 r/min; or culturing for 14h at 38 deg.C under 130r/min shaking table; alternatively, the cells were cultured for 16 hours at 37 ℃ under a shaker at 150 r/min.
Further, in a preferred embodiment of the present invention, in the step (1), the beef extract peptone liquid medium is prepared by the following method:
according to the weight portion, 2-6 portions of beef extract, 6-18 portions of peptone, 3.5-10 portions of NaCl, 15-20 portions of agar and 1000 portions of distilled water are mixed, and then sterilized for 15-25min under the conditions of pH 7.2-7.4 and temperature of 110-.
Preferably, the beef extract peptone liquid medium is prepared by the following method: according to parts by weight, 2 parts of beef extract, 6 parts of peptone, 3.5 parts of NaCl, 15 parts of agar and 1000 parts of distilled water are mixed, and then the mixture is sterilized for 15min under the conditions of pH 7.2 and 110 ℃.
Preferably, the beef extract peptone liquid medium is prepared by the following method: according to parts by weight, 6 parts of beef extract, 18 parts of peptone, 10 parts of NaCl, 20 parts of agar and 1000 parts of distilled water are mixed, and then the mixture is sterilized for 25min under the conditions of pH7.4 and 130 ℃.
Preferably, the beef extract peptone liquid medium is prepared by the following method: taking 3 parts of beef extract, 10 parts of peptone, 5 parts of NaCl, 16 parts of agar and 1000 parts of distilled water by weight, mixing, and sterilizing at the temperature of 121 ℃ for 20min at the pH value of 7.3.
Further, in a preferred embodiment of the present invention, in the step (2), the total number of bacteria of each bacillus is 107-108piece/mL, the inoculation amount is (4mL-5mL)/100 g; the weight ratio of the bran, the whole wheat flour and the water is (8-12) 1: (12-15), the culture conditions are as follows: culturing at 36-38 deg.C for 1.5-2.5 days.
Preferably, in step (2), the total number of bacteria per Bacillus is 5X 107seed/mL, the inoculation amount is 4.5mL/100 g; alternatively, the total number of bacteria per Bacillus is 108seed/mL, the inoculation amount is 5mL/100 g; alternatively, the total number of bacteria per Bacillus is 107piece/mL, the inoculum size was 4mL/100 g.
Preferably, in step (2), the weight ratio of bran, whole wheat flour and water is 8: 1: 12 or 12: 1: 15 or 10: 1: 14.
preferably, the cultivation temperature is 37 ℃.
Further, in a preferred embodiment of the present invention, in the step (3), the 5-to 15-fold expansion culture is performed under the following conditions: culturing at 36-38 deg.C for 55-65 h.
Further, in the preferred embodiment of the present invention, in the step (4), the weight ratio of the bran, the whole wheat flour and the water is (11-15): (1.1-1.5): (19-23); the total inoculation amount of the second-level single-strain bran koji is 0.5-0.6%; the culture conditions were: culturing at 36-38 deg.C for 6-8 days; the drying temperature is 35-40 ℃.
Preferably, in the step (4), the weight ratio of the bran, the whole wheat flour and the water is 11: 1.1: 19 or 15: 1.5: 23 or 13: 1.3: 20. it should be noted that the mixing ratio of the plurality of secondary single-strain bran koji may be selected according to the actual situation, and the present invention is not particularly limited thereto.
The culture conditions of each culture step are optimally designed to obtain the optimal culture environment suitable for the corresponding step, including limitation on the component proportion of the culture medium, culture temperature, culture time and the like, so that the strain quantity and the protease activity obtained in each culture step are good, and the preservation is facilitated.
The bacillus mixed bran koji prepared by the preparation process.
Before warehousing, the bacillus mixed bran koji disclosed by the invention is prepared by the following steps: water content 4-8%, starch content 8-12%, reducing sugar 35-45g/Kg, acidity 2-4%, protease activity 50U/g, bacillus number 3 x 106-3*107. Under the condition of no influence of aspergillis, the quality guarantee period of the bacillus mixed bran koji can reach 2 years.
The application of the bacillus mixed bran koji in wine brewing.
Further, in a preferred embodiment of the present invention, the above-mentioned bacillus mixed bran koji is used for brewing Xiaoqu liquor, and the mass ratio of the bacillus mixed bran koji to the Xiaoqu liquor is (2-3): 2, the total yeast consumption is 0.5-0.7%.
The specific process of the application of the bacillus mixed bran koji in brewing the Xiaoqu liquor is as follows:
s1: soaking the sorghum in boiling water for 6-8 hours to increase the weight by 45% -50%.
S2: steaming for 15-20min, and then suffocating the grain for 2-2.5 h at 70 ℃.
S3: steaming for 65-70min to increase weight by 120-130%.
S4: spreading the steamed sorghum for cooling, adding bacillus mixed bran koji and Xiaoqu, and keeping the mixture at the temperature of 45-50 ℃ for 2h, wherein the mass ratio of the bacillus mixed bran koji to the Xiaoqu is (2-3): 2, the total yeast consumption is 0.5-0.7%.
S5: stacking culture (time 20-24 h, 25 ℃ before stacking, 33-35 ℃ after stacking, yeast number 10)6Sugar/g, 4% -5%) of sugar.
S6: fermenting in a jar (the temperature in the jar is 24-25 ℃, the fermentation time is 8-12 days, and the temperature is 'front slow, middle slow and back slow').
S7: distilling (gas detection is carried out in a retort, fermented grains are even and loose, cannot be too slow, the firepower is even, and the alcohol content of the picked wine is more than 52%).
Further, in the preferred embodiment of the present invention, the above-mentioned bacillus mixed bran koji is used in brewing Daqu liquor, and the amount of the bacillus mixed bran koji is 0.3-0.5%.
The specific process of the application of the bacillus mixed bran koji in the brewing of the Daqu liquor comprises the following steps:
the invention is suitable for the production of Daqu liquor with accumulation process, the invention adopts the production process of sauce liquor, two rounds of adding sand and brown sand do not add bran koji, and the 3 rd to 8 th rounds add bran koji.
Taking out the fermented grains after the previous round of liquor steaming, adding water, cooling, then adding tail liquor with the total amount of 3 percent, high-temperature Daqu and bran koji, stacking, fermenting in a cellar, and taking out the fermented grains and steaming the liquor.
The amount of the medium-temperature Daqu used for brewing Luzhou-flavor liquor is 12-20%, the amount of the high-temperature Daqu used for brewing Maotai-flavor liquor is 40-50%, and the amount of the low-temperature Daqu used for brewing fen-flavor liquor is about 10-18%. (although the use amount of the Daqu is large, the amount of microorganisms contained in the Daqu is not more than that of the bran koji, and the types of the strains contained in the Daqu are more, so that the number of the single strains is not high).
The invention has the following beneficial effects:
the bacillus mixed bran koji is easy to prepare, easy to store and convenient to use, can be used for producing various white spirits, can select multiple bacillus strains to prepare the bran koji, and has the functions of increasing aroma and reducing higher alcohol for brewing the white spirits.
Detailed Description
The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The bacillus species employed in the following examples of the invention include: bacillus subtilis, Bacillus belgii, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus megaterium. In other embodiments of the invention, there may be other combinations of Bacillus, for example, at least two of Bacillus subtilis, Bacillus belgii, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus megaterium may be selected to produce bran koji.
Example 1:
the preparation process of the bacillus mixed bran koji comprises the following steps:
(1) respectively inoculating bacterial suspensions of bacillus subtilis, bacillus belgii, bacillus pumilus, bacillus licheniformis, bacillus amyloliquefaciens and bacillus megaterium into a beef extract peptone liquid culture medium for constant-temperature culture, wherein the culture conditions are as follows: culturing at 37 deg.C under 150r/min shaking table for 16 h.
The beef extract peptone liquid culture medium is prepared by the following method:
according to parts by weight, 2 parts of beef extract, 8 parts of peptone, 3.5 parts of NaCl, 15 parts of agar and 1000 parts of distilled water are mixed, and then the mixture is sterilized for 15min under the conditions of pH 7.2 and 110 ℃.
(2) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is 8: 1: 12, sterilizing and cooling to obtain a primary bran koji carrier; respectively adding the bacillus obtained in the step (1) into sterile physiological saline, then respectively adding into the primary mouldy bran carrier, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 2 days to obtain primary single strain bran koji corresponding to each Bacillus.
(3) Respectively carrying out 10-time amplification culture on the primary single-strain bran koji obtained in the step (2), wherein the culture conditions are as follows: culturing at 37 deg.C for 60 hr to obtain secondary single-strain bran koji.
(4) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is 11: 1.1: 19, sterilizing and cooling to obtain a secondary bran koji carrier; and (3) proportionally mixing the secondary single-strain mouldy bran prepared in the step (3), inoculating the mixture into the secondary mouldy bran carrier according to the total inoculation amount of 0.5%, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 7 days; drying at 35 deg.C to obtain Bacillus mixed bran koji.
Example 2:
the preparation process of the bacillus mixed bran koji comprises the following steps:
(1) respectively inoculating bacterial suspensions of bacillus subtilis, bacillus belgii, bacillus pumilus, bacillus licheniformis, bacillus amyloliquefaciens and bacillus megaterium into a beef extract peptone liquid culture medium for constant-temperature culture, wherein the culture conditions are as follows: culturing at 37 deg.C under 150r/min shaking table for 16 h.
The beef extract peptone liquid culture medium is prepared by the following method:
according to parts by weight, 6 parts of beef extract, 18 parts of peptone, 10 parts of NaCl, 20 parts of agar and 1000 parts of distilled water are mixed, and then the mixture is sterilized for 25min under the conditions of pH7.4 and 130 ℃.
(2) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is 12: 1: 15, sterilizing and cooling to obtain a primary bran koji carrier; respectively adding the bacillus obtained in the step (1) into sterile physiological saline, then respectively adding into the primary mouldy bran carrier, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 2 days to obtain primary single strain bran koji corresponding to each Bacillus.
(3) Respectively carrying out 10-time amplification culture on the primary single-strain bran koji obtained in the step (2), wherein the culture conditions are as follows: culturing at 37 deg.C for 60 hr to obtain secondary single-strain bran koji.
(4) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is (15: 1.5): 23, sterilizing and cooling to obtain a secondary bran koji carrier; and (3) proportionally mixing the secondary single-strain mouldy bran prepared in the step (3), inoculating the mixture into the secondary mouldy bran carrier according to the total inoculation amount of 0.6%, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 7 days; drying at 40 deg.C to obtain Bacillus subtilis mixed bran koji.
Example 3:
the preparation process of the bacillus mixed bran koji comprises the following steps:
(1) respectively inoculating bacterial suspensions of bacillus subtilis, bacillus belgii, bacillus pumilus, bacillus licheniformis, bacillus amyloliquefaciens and bacillus megaterium into a beef extract peptone liquid culture medium for constant-temperature culture, wherein the culture conditions are as follows: culturing at 37 deg.C under 150r/min shaking table for 16 h.
The beef extract peptone liquid culture medium is prepared by the following method:
according to parts by weight, 6 parts of beef extract, 18 parts of peptone, 10 parts of NaCl, 20 parts of agar and 1000 parts of distilled water are mixed, and then the mixture is sterilized for 25min under the conditions of pH7.4 and 130 ℃.
(2) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is 10: 1: 14, sterilizing and cooling to obtain a primary bran koji carrier; respectively adding the bacillus obtained in the step (1) into sterile physiological saline, then respectively adding into the primary mouldy bran carrier, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 2 days to obtain primary single strain bran koji corresponding to each Bacillus.
(3) Respectively carrying out 10-time amplification culture on the primary single-strain bran koji obtained in the step (2), wherein the culture conditions are as follows: culturing at 37 deg.C for 60 hr to obtain secondary single-strain bran koji.
(4) Uniformly mixing bran, whole wheat flour and water, wherein the weight ratio of the bran to the whole wheat flour to the water is 12: 1.3: 20, sterilizing and cooling to obtain a secondary bran koji carrier; and (3) proportionally mixing the secondary single-strain mouldy bran prepared in the step (3), inoculating the mixture into the secondary mouldy bran carrier according to the total inoculation amount of 0.55%, uniformly mixing, and culturing at constant temperature, wherein the culture conditions are as follows: culturing at 37 deg.C for 7 days; drying at 37 deg.C to obtain Bacillus subtilis mixed bran koji.
Example 4:
application of bacillus mixed bran koji in brewing Xiaoqu liquor.
The specific process of the application of the bacillus mixed bran koji in brewing the Xiaoqu liquor is as follows:
s1: soaking sorghum in boiling water for 6 hr to increase weight by 45%.
S2: steaming for 15min, and then stewing the grain at 70 deg.C for 2.5 h.
S3: steaming for 65min to increase weight by 120%.
S4: spreading the steamed sorghum for cooling, adding bacillus mixed bran koji and Xiaoqu, and keeping the mixture at 45 ℃ for 2 hours, wherein the mass ratio of the bacillus mixed bran koji to the Xiaoqu is 1:1, and the total yeast consumption is 0.5%.
S5: stacking culture (time 20h, 25 ℃ before stacking, 33 ℃ after stacking, yeast number 10)6Piece/g, sugar 4%).
S6: fermenting in a jar (the temperature in the jar is 24 ℃, the fermentation time is 12 days, and the temperature is 'front slow, middle slow and back slow').
S7: distilling (gas detection is carried out in a retort, fermented grains are even and loose, cannot be too slow, the firepower is even, and the alcohol content of the picked wine is more than 52%).
Example 5:
application of bacillus mixed bran koji in brewing Xiaoqu liquor.
The specific process of the application of the bacillus mixed bran koji in brewing the Xiaoqu liquor is as follows:
s1: soaking sorghum in boiling water for 8 hr to increase weight by 50%.
S2: steaming for 20min, and then steaming the grains at 70 deg.C for 2.5 h.
S3: steaming for 70min to increase weight by 130%.
S4: spreading and cooling the steamed sorghum, adding bacillus mixed bran koji and Xiaoqu, and keeping the mixture at 50 ℃ for 2 hours, wherein the mass ratio of the bacillus mixed bran koji to the Xiaoqu is 3:2, the total yeast consumption is 0.7 percent.
S5: stacking culture (time 24h, 25 ℃ before stacking, 35 ℃ after stacking, yeast number 10)6Sugar/g, 5%) of sugar.
S6: fermenting in a jar (the temperature in the jar is 25 ℃, the fermentation time is 8 days, and the temperature is 'front slow, middle slow and back slow').
S7: distilling (gas detection is carried out in a retort, fermented grains are even and loose, cannot be too slow, the firepower is even, and the alcohol content of the picked wine is more than 52%).
Example 6:
application of bacillus mixed bran koji in brewing Xiaoqu liquor.
The specific process of the application of the bacillus mixed bran koji in brewing the Xiaoqu liquor is as follows:
s1: soaking sorghum in boiling water for 7 hr to increase weight by 48%.
S2: steaming for 18min, and then stewing the grain at 70 deg.C for 2.2 h.
S3: steaming for 68min to increase weight by 125%.
S4: spreading and cooling the steamed sorghum, adding bacillus mixed bran koji and Xiaoqu, and keeping the mixture at 48 ℃ for 2 hours, wherein the mass ratio of the bacillus mixed bran koji to the Xiaoqu is 1.2: 1, the total yeast consumption is 0.6 percent.
S5: stacking culture (time 22h, 25 ℃ before stacking, 34 ℃ after stacking, yeast number 10)6Sugar/g, 4.5% sugar).
S6: fermenting in a jar (the temperature in the jar is 24.5 ℃, the fermentation time is 10 days, and the temperature is 'front slow, middle slow and back slow').
S7: distilling (gas detection is carried out in a retort, fermented grains are even and loose, cannot be too slow, the firepower is even, and the alcohol content of the picked wine is more than 52%).
Example 7:
the specific process of the application of the bacillus mixed bran koji in the brewing of the Daqu liquor comprises the following steps:
the invention is suitable for the production of Daqu liquor with accumulation process, the invention adopts the production process of sauce liquor, two rounds of adding sand and brown sand do not add bran koji, and the 3 rd to 8 th rounds add bran koji.
Taking out the fermented grains after the previous round of liquor steaming, adding water, cooling, then adding tail liquor with the total amount of 3 percent, high-temperature Daqu and bran koji, stacking, fermenting in a cellar, and taking out the fermented grains and steaming the liquor. The dosage of the bacillus mixed bran koji is 0.3 percent.
Example 8:
the specific process of the application of the bacillus mixed bran koji in the brewing of the Daqu liquor comprises the following steps:
the invention is suitable for the production of Daqu liquor with accumulation process, the invention adopts the production process of sauce liquor, two rounds of adding sand and brown sand do not add bran koji, and the 3 rd to 8 th rounds add bran koji.
Taking out the fermented grains after the previous round of liquor steaming, adding water, cooling, then adding tail liquor with the total amount of 3 percent, high-temperature Daqu and bran koji, stacking, fermenting in a cellar, and taking out the fermented grains and steaming the liquor. The dosage of the bacillus mixed bran koji is 0.4%.
Example 9:
the specific process of the application of the bacillus mixed bran koji in the brewing of the Daqu liquor comprises the following steps:
the invention is suitable for the production of Daqu liquor with accumulation process, the invention adopts the production process of sauce liquor, two rounds of adding sand and brown sand do not add bran koji, and the 3 rd to 8 th rounds add bran koji.
Taking out the fermented grains after the previous round of liquor steaming, adding water, cooling, then adding tail liquor with the total amount of 3 percent, high-temperature Daqu and bran koji, stacking, fermenting in a cellar, and taking out the fermented grains and steaming the liquor. The dosage of the bacillus mixed bran koji is 0.5 percent.
Test example 1:
in this test example, the effect of the Bacillus subtilis strain bran koji in wine brewing according to the present invention will be described by taking the brewing of Xiaoqu wine as an example.
The index parameters of the bacillus mixed bran koji of the experimental groups 1-3 are shown in the table 1.
TABLE 1
| Experimental group 1 | Experimental group 2 | Experimental group 3 | |
| Number of bacteria (number) | 1.3*109 | 2.5*109 | 1.7*109 |
| Protease activity U | 51 | 47 | 54 |
| Starch content | 11.32％ | 9.13％ | 10.46％ |
The application of bacillus mixed bran koji in Xiaoqu comprises the following process conditions: soaking sorghum in boiling water for 7 hours, increasing the weight by 47 percent, steaming for 17min for the first time, sealing grains for 2.5h, steaming again for 65min, increasing the weight by about 125 percent, stacking and cultivating bacteria for 20h-24h, 25 ℃ before stacking, 33 ℃ to 35 ℃ after stacking, 106 yeast numbers/g, 4 percent to 5 percent of sugar, spreading for cooling for 2h, 0.6 percent of mixed yeast and bran koji: adding Xiaoqu (3: 2), fermenting in a jar for 7 days, and distilling. Calculating wine yield, detecting flavor components in wine, and directly performing fermentation production with 6% Xiaoqu in control group. The detection method refers to: GB/T10345-2007 method for analyzing white spirit, and the detection results are shown in Table 2.
TABLE 2
Experimental groups 1, 2, and 3 were prepared by brewing the bacillus subtilis strain bran koji prepared in examples 1, 2, and 3, respectively, according to the above-described process.
Test example 2:
the flavor analysis of the third liquor taking of the bacillus mixed bran koji in the brewing of the Maotai-flavor liquor according to the embodiment of the invention is shown in the table 3. The control group was directly produced by fermentation using the same process without adding bran having the same weight as the bran koji.
TABLE 3
| 7 experiments | 8 experiments | 9 experiments | Control group | |
| Number of flavors | 78 | 73 | 75 | 75 |
| Esters (a) | 31 | 30 | 32 | 31 |
| Concentration (mg/L) | 3679.3 | 3893.4 | 3745.4 | 3765.4 |
| Alcohols (a) | 7 | 7 | 7 | 7 |
| Concentration (mg/L) | 724.6 | 712.2 | 721.3 | 833.6 |
| Aromatic (a) | 15 | 14 | 13 | 14 |
| Concentration (mg/L) | 157.8 | 158.5 | 168.3 | 185.4 |
| Nitrogen-containing compound(s) | /4 | 4 | 4 | 4 |
| Concentration (mg/L) | 34.4 | 32.3 | 37.4 | 11.2 |
| Aldehyde ketone (a) | 8 | 8 | 7 | 8 |
| Concentration (mg/L) | 78.9 | 73.4 | 75.6 | 84.3 |
| Acids (a) | 6 | 6 | 6 | 6 |
| Concentration (mg/L) | 139.4 | 138.8 | 128.5 | 134.3 |
| Others (a) | 11 | 8 | 10 | 9 |
| Concentration (mg/L) | 343.2 | 359.4 | 329.1 | 338.3 |
The wine yield of the four experiments is respectively as follows: 54.6 percent, 53.6 percent, 54.2 percent and 54.4 percent, the liquor yield is basically not different, the components of the liquor taken for the third time of each experiment are detected, and the main differences can be seen from the above that the main differences are aromatic (the furfural compounds have certain content differences of 113.4, 114.3, 123.6 and 148.9 respectively), nitrogen-containing compounds (pyrazine) and alcohols (the main differences are 3-methyl butanol: 593.2, 589.4, 578.9 and 734.3).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A preparation process of bacillus mixed bran koji is characterized by comprising the following steps:
(1) respectively inoculating bacterial suspensions of various bacillus into beef extract peptone liquid culture media for constant-temperature culture;
(2) uniformly mixing bran, whole wheat flour and water, sterilizing and cooling to obtain a primary bran koji carrier; respectively adding the bacillus obtained in the step (1) into sterile physiological saline, respectively adding the obtained bacterial suspensions into the primary mouldy bran carrier, uniformly mixing, and culturing at constant temperature to respectively obtain primary single-bacterium mouldy bran corresponding to each bacillus;
(3) respectively carrying out amplification culture on the primary single-strain bran koji obtained in the step (2) to obtain secondary single-strain bran koji;
(4) uniformly mixing bran, whole wheat flour and water, sterilizing and cooling to obtain a secondary bran koji carrier; and (4) mixing and inoculating the multiple secondary single-strain bran koji prepared in the step (3) into the secondary bran koji carrier, uniformly mixing, culturing at constant temperature, and drying to obtain the bacillus mixed-strain bran koji.
2. The process for preparing bacillus mixed bran koji as claimed in claim 1, wherein the plurality of bacillus comprises: bacillus subtilis, bacillus pumilus, bacillus licheniformis, bacillus amyloliquefaciens, bacillus belgii and bacillus megaterium; the culture conditions were: culturing for 14-18h at 36-38 ℃ under a shaking table with the speed of 130-.
3. The process for preparing bacillus mixed bran koji as claimed in claim 2, wherein in the step (1), the beef extract peptone liquid medium is prepared by the following method:
according to the weight portion, 2-6 portions of beef extract, 6-18 portions of peptone, 3.5-10 portions of NaCl, 15-20 portions of agar and 1000 portions of distilled water are mixed, and then sterilized for 15-25min under the conditions of pH 7.2-7.4 and temperature of 110-.
4. The process for preparing bacillus mixed bran koji as claimed in claim 1, wherein the total number of bacillus of each kind in step (2) is 107-108piece/mL, the inoculation amount is (4mL-5mL)/100 g; the weight ratio of the bran, the whole wheat flour and the water is (8-12) 1: (12-15), the culture conditions are as follows: culturing at 36-38 deg.C for 1.5-2.5 days.
5. The process for preparing the bacillus mixed bran koji as claimed in claim 4, wherein in the step (3), the 5-15 times of expansion culture is performed under the following culture conditions: culturing at 36-38 deg.C for 55-65 h.
6. The process for preparing bacillus mixed bran koji as claimed in any of claims 1 to 5, wherein in the step (4), the weight ratio of bran, whole wheat flour and water is (11-15): 1.1-1.5): (19-23); the total inoculation amount of the second-level single-strain bran koji is 0.5-0.6%; the culture conditions were: culturing at 36-38 deg.C for 6-8 days; the drying temperature is 35-40 ℃.
7. The bacillus mixed bran koji prepared by the preparation process according to any one of claims 1 to 6.
8. The use of the bacillus subtilis mixed bran koji of claim 7 in wine brewing.
9. The use of claim 8, wherein the use of the bacillus mixed bran koji for brewing the Xiaoqu wine is characterized in that the mass ratio of the bacillus mixed bran koji to the Xiaoqu wine is (2-3): 2, the total yeast consumption is 0.5-0.7%.
10. The use of claim 8, wherein the amount of the bacillus mixed bran koji is 0.3-0.5% in brewing the Daqu wine.
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