CN104762163B - A kind of method preparing biological song - Google Patents

Abstract

The invention discloses a method for preparing biological koji, which belongs to the technical field of brewing. Firstly, the bacterial seed liquid, yeast seed liquid and mold solid seeds are respectively produced, and then the bacterial seed liquid, yeast seed liquid and mold solid seeds are inserted into raw materials in proportion to obtain biological koji. On the basis of the traditional Daqu technology, the present invention combines the purebred cultivation biotechnology with the traditional raw material fermentation technology, which eliminates the process of turning the koji, shortens the production cycle, and saves the three-month storage period of Daqu , has improved the production efficiency of Daqu, and has far-reaching significance for promoting the scientific and technological progress of the liquor industry.

Description

A method for preparing biokoji

technical field

The invention relates to a method for preparing biological koji, which belongs to the technical field of brewing.

Background technique

The traditional fermentation preparation process of Daqu takes 4 months, which consumes a lot of manpower and material resources, and the external bacterial strains change greatly with the seasons. The product quality is affected by many factors and is not easy to control, resulting in unstable product quality of Daqu. In recent years, with the development of brewing technology, in the preparation process of Daqu, from raw material crushing to buckling has been mechanized, while the follow-up process is still at the traditional manual level. This study combines modern biotechnology with traditional koji making technology, and introduces fully enclosed and fully automatic solid fermentation equipment, which reduces unnecessary bacterial contamination, is not limited by seasons, and can be used according to different functional bacteria. The temperature required for the growth stage and each period of enzyme production can be adjusted flexibly to realize the full-line mechanized production of biological koji, opening up a new chapter for the mechanization process of the liquor industry.

Contents of the invention

The purpose of the present invention is to: provide a method for preparing biological koji, improve the traditional production process of Daqu, utilize modern biotechnology, mechanize and automate the production of biological koji, realize continuous controllable production, improve production efficiency, and promote the overall technology of brewing in liquor enterprises Innovation.

The inventive method mainly comprises the following steps:

(1) Prepare bacterial seed liquid: connect Bacillus pumilus, Forestomonas amylovora, and Lactococcus lactis to the seed medium respectively, and carry out activation culture to obtain a mixed bacterial liquid;

(2) Preparation of yeast seed liquid: add a ring of Hansenula anomalies, Pichia petersensis, and Candida stout to the seed medium respectively, and perform activation culture to obtain a mixed bacterial liquid;

(3) Preparation of mold solid-state strains: take the physiological saline of the spores of Mucor circiniferi, insert them into Daqu raw meal for static cultivation, and then inoculate them into the seed koji tank at 32-36°C for 72±8h to obtain mold solid-state strains ;

(4) Preparation of biological koji: wheat and barley are mixed in a mass ratio (8-10): 1, moistened with water to form a wet material with a water content of 47.5±2.5% (w/w), and 1±0.5% (w/w) in the wet material /w) inoculation amount into bacterial seed liquid, 2±0.5% (w/w) inoculum amount into yeast seed liquid, 3±0.5% (w/w) inoculum amount into mold solid strain, Mix well, stack thickness 25±5cm, keep warm at 30±1°C in the initial stage of fermentation, and cultivate for 6-8 days to make the temperature of Quxin top fire reach 60±2°C, and top fire for 3-5 days.

In one embodiment of the present invention, the Bacillus pumilus is Bacillus pumilus CGMCC No 1.1168.

In one embodiment of the present invention, the Forestomonas amylolytica is Silvimonas amylolytica CGMCC No 1.8860.

In one embodiment of the present invention, the Lactococcus lactis is Lactococcus lactis CGMCC No 1.2470.

In one embodiment of the present invention, the Hansenula anomala is Hansenula anomala CGMCC No 2.764.

In one embodiment of the present invention, the Pichia petersonii is Pichia petersonii CGMCC No 2.1473.

In one embodiment of the present invention, the Candida robusta is Candida robusta (Candidarobusta) CGMCC No 2.865.

In one embodiment of the present invention, the Mucor circinelloides is Mucor circinelloides CGMCC No3.2208.

One embodiment of the invention employs the following steps:

(1) Preparation of bacterial seed liquid: connect Bacillus pumilus, Forestomonas amylovora, and Lactococcus lactis to the seed medium for activation and culture, and the concentration of the obtained bacterial liquid is 10 ⁶ -10 ⁷ Individual/mL; the seed medium is preferably beef extract peptone medium; the activation culture conditions are preferably 35-37° C., 100-120 rpm.

(2) Preparation of yeast seed liquid: Inoculate a ring of preserved Hansenula anomalosaccharomyces, Pichia petersensis, and Candida stout into the seed medium for activation culture, and the cell concentration of the obtained bacterial liquid is 10 ⁵ -10 ⁶ cells/mL; the seed medium is preferably wort juice or rice koji juice; the activation culture conditions are preferably 28-30°C, 100-120rpm.

(3) Preparation of mold solid-state strains: take the physiological saline of the spores of Mucor cloverleaf, the spore content is about ^105-106 /mL, take ¹ ±0.5mL and insert it into 30-35g water content to adjust to 45% - 50% Daqu raw material in a 500mL shake flask, 32-36 ° C static culture, the culture time is 72 ± 8 hours; then inoculated into the seed koji tank 32-36 ° C for 72 ± 8 hours to obtain mold solid strains.

(4) Preparation of biological koji: wheat and barley are mixed in a mass ratio (8-10): 1, moistened with water to form a wet material with a water content of 47.5±2.5% (w/w), and 1±0.5% (w/w) in the wet material /w) inoculation amount into bacterial seed liquid, 2±0.5% (w/w) inoculum amount into yeast seed liquid, 3±0.5% (w/w) inoculum amount into mold solid strain, Mix well, stack thickness 25±5cm, keep warm at 30±1°C in the initial stage of fermentation, after 6-8 days of cultivation, make the temperature of the koji heart top fire reach 60±2°C, top fire for 3-5 days, and the entire koji making time is shortened to 15 ± 1 day, and finally get the biological song.

The present invention also provides a method of using the biological koji to brew liquor: the obtained biological koji is crushed and used in a brewery, the amount of koji used is 10%-15% (w/w) of the feeding amount, and the ratio of grain to grain is 1: (4-5), carry out brewing production according to "six points method" process.

The present invention has the following advantages:

1. The combination of modern purebred cultivation biotechnology and traditional raw meal fermentation process eliminates the koji turning process, shortens the production cycle, and saves the three-month storage period of Daqu, improving the production efficiency of biokoji. 2. The production process is simple, easy to operate, mechanized and automated production, saving manpower. 3. This production process avoids the influence of the environment on the product quality, and the influence of human factors is also small. It can be produced all year round, and all the indicators are equal to or better than the traditional Daqu. 4. The biological koji was applied to the cellar, and it was found that although the biological koji had not been stored, the temperature change in the cellar was completely in line with the best state of "slow front, straight in the middle, and slow fall in the back". Skeleton components and evaluation results have also verified that Biokoji has a good application prospect. 5. The application of biological koji not only greatly improves the quality of original wine, but also plays an important role in the domestication and enrichment of functional bacteria in the microecology of winemaking, thereby further improving the quality of original wine.

Description of drawings

Fig. 1 goes into cellar cellar pool temperature curve, biological song 1-3 is respectively the biological song prepared by embodiment 1-3

detailed description

The technical solution of the present invention will be further described below in conjunction with specific examples, and these examples should not be construed as limitations on the technical solution.

Embodiment 1 produces biological song according to the following steps

(1) Bacterial strains: 50mL beef extract peptone liquid medium was placed in a 500mL triangular flask, from three bacterial functional bacteria Bacillus pumilus (CGMCC No 1.1168), Silvimonas amylolytica (CGMCC No 1.8860), Lactococcus lactis (CGMCC No 1.2470) was preserved on a slant and each scraped ring was inserted into beef extract peptone liquid medium, pH value 7.0, cultured on a shaker at 37°C, the shaker speed was 100rpm, and the culture time was 24h; then, Beef extract peptone liquid medium, pH 7.0, cultured in a fermenter at 37°C for 24 hours to obtain bacterial seed solution; microscopic examination showed that the number of bacteria per milliliter remained above 10 ⁷ , the number was large and uniform, and did not contain other miscellaneous bacteria ;

(2) Yeast strains: 50mL wort liquid culture medium was placed in a 500mL Erlenmeyer flask, from three functional yeasts Hansenula anomala (CGMCC No 2.764), Pichiapetersonii (CGMCC No 2.1473) , Candida robusta (CGMCC No 2.865) was preserved on the slant and scraped one ring into the wort liquid medium, cultured on a shaker at 28°C, the shaker speed was 100rpm, and the culture time was 24h; then, using the same method as above The culture medium was adjusted to a pH value of 4.5, and cultured in a fermenter at 28°C for 36 hours to obtain a yeast seed solution; microscopic examination showed that the number of bacteria per milliliter remained above 10 ⁶ , the yeast was relatively robust and uniform in size, and there were no vacuoles in the cells, no Infection with other miscellaneous bacteria;

(3) Mold strains: take a functional mold fungus Mucor circinelloides (Mucor circinelloides) CGMCC No3.2208 slant preservation test tube, wash the spores with sterile saline, and use a hemocytometer to adjust to an order of magnitude of 10 ⁶ /mL , take 1mL and insert it into 30g/500mL Daqu raw material with a water content adjusted to 45%, and culture it statically at 32°C for 72 hours; then, inoculate it into a seed koji tank and culture it statically at 32°C for 72h to obtain solid mold strains ;

(4) Biological koji: Wheat and barley are mixed at a mass ratio of 8:1, moistened with water to form a wet material with a water content of 45.0% (w/w), and bacterial seeds are inserted into the wet material with an inoculation amount of 0.5% (w/w) Liquid, 1.5% (w/w) inoculum amount into yeast seed liquid, 2.5% (w/w) inoculum amount into mold solid strains, stacking thickness 20cm, heat preservation at 29°C in the initial stage of fermentation, after 6 days of cultivation , so that the temperature of the koji heart top fire reaches 58 ℃, and the top fire is 3 days, and the whole koji making time is shortened to 14 days, and finally biological koji 1 is obtained.

Embodiment 2 produces modern biological song according to the following steps

(1) Bacterial strains: 50mL beef extract peptone liquid medium was placed in a 500mL triangular flask, from three bacterial functional bacteria B.pumilus (CGMCC 1.1168), S.amylolytica (CGMCC 1.8860), L.lactis (CGMCC 1.2470 ) into the beef extract-peptone liquid culture medium with a pH value of 7.2, cultivated on a shaker at 37°C, the shaker speed is 100rpm, and the culture time is 24h; then, use beef extract-peptone liquid culture medium, pH The value is 7.2, cultured in a fermenter at 37°C for 24 hours to obtain bacterial seed solution; microscopic examination shows that the number of bacteria per milliliter remains above 10 ⁷ , the number is relatively large, uniform, and does not contain other miscellaneous bacteria;

(2) Yeast strains: 50mL rice koji juice liquid medium was placed in a 500mL Erlenmeyer flask. ) into the rice koji juice liquid culture medium, cultured on a shaker at 29°C, with a shaker rotation speed of 100rpm, and a culture time of 24h; Cultivate in a tank at 29°C for 36 hours to obtain yeast seed liquid; microscopic examination shows that the number of bacteria per milliliter remains above 10 ⁶ , the yeast is relatively robust and uniform in size, there are no vacuoles in the cells, and it does not infect other miscellaneous bacteria;

(3) Mold strains: Take a fungal fungus M.circinelloides (CGMCC 3.2208) slanted preservation test tube, wash the spores with sterile saline, use a hemocytometer to adjust to an order of magnitude of 10 ⁶ /mL, take 1mL and insert Fill 30g/500mL Daqu raw material with water content adjusted to 47.5%, and culture it statically at 34°C for 72 hours; then, inoculate it into a seed koji tank and culture it statically at 34°C for 72 hours to obtain mold solid strains;

(4) Biological koji: Wheat and barley are mixed at a mass ratio of 9:1, moistened with water to form a wet material with a water content of 47.5% (w/w), and bacterial seeds are inserted into the wet material with an inoculation amount of 1% (w/w) Liquid, 2% (w/w) inoculum amount into yeast seed liquid, 3% (w/w) inoculum amount into mold solid strains, stacking thickness 25cm, initial stage of fermentation at 30 ° C, after 7 days of cultivation , so that the temperature of the koji heart reaches 60°C, and the koji is top-fired for 4 days, the whole koji making time is shortened to 15 days, and biological koji 2 is finally obtained.

Embodiment 3 produces modern biological song according to the following steps

(1) Bacterial strains: 50mL beef extract peptone liquid medium was placed in a 500mL triangular flask, from three bacterial functional bacteria B.pumilus (CGMCC 1.1168), S.amylolytica (CGMCC 1.8860), L.lactis (CGMCC 1.2470 ) into the beef extract-peptone liquid culture medium with a pH value of 7.4, cultivated on a shaker at 37°C, the shaker speed is 100rpm, and the culture time is 24h; then, use beef extract-peptone liquid culture medium, pH The value is 7.4, cultured in a fermenter at 37°C for 24 hours to obtain bacterial seed solution; microscopic examination shows that the number of bacteria per milliliter remains above 10 ⁷ , the number is relatively large, uniform, and does not contain other miscellaneous bacteria;

(2) Yeast strains: 50mL wort liquid medium was placed in a 500mL Erlenmeyer flask. Scrape one ring on each preservation slant and put it into the wort liquid culture medium, culture on a shaker at 30°C, the speed of the shaker is 100rpm, and the culture time is 24h; Cultivate at ℃ for 36 hours to obtain yeast seed solution; microscopic examination shows that the number of bacteria per milliliter remains above 10 ⁶ , the yeast is relatively robust and uniform in size, there are no vacuoles in the cells, and no other miscellaneous bacteria are infected;

(3) Mold strains: Take a fungal fungus M.circinelloides (CGMCC 3.2208) slanted preservation test tube, wash the spores with sterile saline, use a hemocytometer to adjust to an order of magnitude of 10 ⁶ /mL, take 1mL and insert Fill it with 30g/500mL Daqu raw meal, adjust the water content to 50%, and culture it statically at 36°C for 72 hours; then, inoculate it into the seed koji tank and culture it statically at 36°C for 72 hours to obtain solid mold strains;

(4) Biological koji: Wheat and barley are mixed at a mass ratio of 10:1, moistened with water to form a wet material with a water content of 50.0% (w/w), and bacterial seeds are inserted into the wet material with an inoculation amount of 1.5% (w/w) Liquid, 2.5% (w/w) inoculum amount into yeast seed liquid, 3.5% (w/w) inoculum amount into mold solid strains, stacking thickness 30cm, heat preservation at 31°C in the initial stage of fermentation, after 8 days of cultivation , so that the temperature of the koji heart top fire reaches 62 ℃, and the top fire is 5 days, the whole koji making time is shortened to 16 days, and finally biological koji 3 is obtained.

The biological koji was tested in the pilot workshop according to the standard of traditional koji, using the "six-point method" process, using sorghum as the raw material for wine making, high-quality barley, wheat, and peas as ingredients, adding biological koji, and using koji The amount is 10%-15% (w/w) of the feeding amount, the ratio of grain to fermented grains is 1: (4-5), solid-state fermentation in a mud cellar, using continuous grain ingredients, mixed steaming and mixed firing, picking wine by quantity and quality, and extracting fermented grains Sprinkle and look at the water in the steamer, spread it to cool, add koji and put it in the cellar. After a 6-month experiment, the results showed that the temperature of the cellar pool using biological koji met the technological requirements of "slow in the front, straight in the middle and slow down after falling" (as shown in Figure 1), and the ethyl hexanoate in the high-grade wine and the first-grade wine was produced. The contents are greater than 300mg/100mL and 200mg/100mL respectively (as shown in Table 2). Score and write comments on color, aroma, taste and style, with 10 points for color, 25 points for aroma, 50 points for taste and 15 points for style. Grade 70 and above. The results of the evaluation showed that the selected wine body had reached the excellent grade and the first-class standard (shown in Table 3), which verified that the modern biokoji has a good application prospect.

Table 1 Comparative analysis of the physical and chemical indicators of biological koji and traditional bag koji

Table 2 Comparison of wines tested in biological koji (mg/100mL)

Table 3 The results of tasting in the wine tasting room

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (6)

1. a kind of method preparing biological song, mainly includes the following steps that：

(1) prepare antibacterial seed liquor：Bacillus pumilus, solution starch forest Zymomonas mobiliss, lactococcus lactis are connect a ring extremely respectively In seed culture medium, carry out activation culture, obtain mixed bacteria liquid；

(2) prepare yeast starter liquid：Hansenula anomala, Pi Texun Pichia sp., Candida valida are connect a ring extremely respectively In seed culture medium, carry out activation culture, obtain mixed bacteria liquid；

(3) prepare mycete solid state bacterial：Take the normal saline of the spore of volume branch Mucor, access Daqu (massive raw stater for alcholic liquor) raw material quiescent culture, then, It is seeded to 32-36 DEG C of culture 72 ± 8h of yeast inoculation tank, obtain mycete solid state bacterial；

(4) prepare biological song：Semen Tritici aestivi and Fructus Hordei Vulgaris are with mass ratio (8-10):1 mixing, adds water and moistens into water content 47.5 ± 2.5% Wet feed in terms of w/w, accesses antibacterial seed liquor by 1 ± 0.5% inoculum concentration in terms of w/w in wet feed, 2 ± 0.5% in terms of w/w Inoculum concentration accesses saccharomycete seed liquor, 3 ± 0.5% inoculum concentrations in terms of w/w access mycete solid state bacterial, mixes, ulking thickness 25 ± 5cm, the fermentation initial stage is incubated 30 ± 1 DEG C, cultivated through 6-8 days, makes bent heart top fire temperature reach 60 ± 2 DEG C, the fiery 3-5 in top My god.

2. method according to claim 1 is it is characterised in that described preparation antibacterial seed liquor：By Bacillus pumilus, solution Starch forest Zymomonas mobiliss, lactococcus lactis connect a ring respectively to seed culture medium, carry out activation culture, the thalline of gained bacterium solution Concentration is 10⁶-10⁷Individual/mL；Described seed culture medium is beef-protein medium；Described activation culture condition is 35-37 ℃、100-120rpm.

3. method according to claim 1 is it is characterised in that described prepare yeast starter liquid：By Hansenula anomala, skin Special inferior Pichia sp., Candida valida connect a ring respectively to seed culture medium, carry out activation culture, the thalline of gained bacterium solution Concentration is 10⁵-10⁶Individual/mL；Described seed culture medium is beerwort or rice-koji juice；Described activation culture condition be 28-30 DEG C, 100-120rpm.

4. method according to claim 1 is it is characterised in that described preparation mycete solid state bacterial：Take the spore of volume branch Mucor The normal saline of son, spore content 10⁵-10⁶Individual/mL, takes 1 ± 0.5mL to access and is adjusted to 45% equipped with 30-35g water content In the 500mL shaking flask of 50% Daqu (massive raw stater for alcholic liquor) raw material, 32-36 DEG C of static gas wave refrigerator, incubation time is 72 ± 8h；Then, it is seeded to yeast inoculation 32-36 DEG C of culture 72 ± 8h of tank, obtains mycete solid state bacterial.

5. the biological song of method preparation according to claim 1.

6. biological bent application in brewed spirit described in claim 5.