CN103005240B - Preparation method and application of compound leavening agent - Google Patents
Preparation method and application of compound leavening agent Download PDFInfo
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- CN103005240B CN103005240B CN201210585827.4A CN201210585827A CN103005240B CN 103005240 B CN103005240 B CN 103005240B CN 201210585827 A CN201210585827 A CN 201210585827A CN 103005240 B CN103005240 B CN 103005240B
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Abstract
The invention relates to a preparation method and application of a compound leavening agent. The preparation method comprises the steps of: separating lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus, saccharomyces cerevisiae, candida rugosa, candida tropicalis and fermented pichia pastoris, as a leavening agent, from naturally fermented panada to produce a strain so as to prepare freeze-dried strain powder; weighing 1-2 parts of lactobacillus plantarum, 1-2 parts of lactobacillus bulgaricus, 1-2 parts of streptococcus thermophilus, 3-5 parts of saccharomyces cerevisiae, 3-5 parts of candida rugosa, 3-5 parts of candida tropicalis and 3-5 parts of fermented pichia pastoris based on parts by weight, and uniformly mixing to obtain the compound leavening agent. When the compound leavening agent is applied to the cooked wheaten food, bacterial contamination can be effectively prevented, the sanitation of the cooked wheaten food is guaranteed, the quality of the cooked wheaten food is improved, the production cycle is shortened, a production process is controllable, and the preparation method of the compound leavening agent lays a foundation for industrial production.
Description
Technical field
The invention belongs to food processing field, particularly a kind of preparation method of composite ferment and application.
Background technology
Thin pancake is one of Chinese traditional food, is five cereals to be added to water mill become batter, by fermentation, pours griddle into, shakeouts to poker to form with thin pancake bamboo rake.The thin pancake unique flavor of making by fermentation, mouthfeel chewiness, is liked deeply.
At present, still adopt the traditional zymotic mode of spontaneous fermentation in thin pancake production process, the production cycle is long, and in sweat, is subject to the impact of external environment, and activity and the ratio of bacterial classification easily change, and causes that sweat is wayward, food quality is unstable.Adopt traditional zymotic method, food hygiene situation does not reach the requirement of modern food enterprise, makes this traditional food with distinct cultural features only have individual workship to produce, the long-term stage in original backwardness, there is hidden danger in food security, urgently realizes its industrialization and produce.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method and application of composite ferment, use this leavening to make thin pancake and can ensure the health of food, the quality of raising food, can shorten the thin pancake production cycle again, realize thin pancake production process controlled, for thin pancake making realizes suitability for industrialized production, lay the foundation.
Technical solution of the present invention is:
A preparation method for composite ferment, its concrete steps are as follows:
From the batter of spontaneous fermentation, isolate Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus, S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris and produce bacterial classification as leavening, each produces starter culture viable count>=10
9individual/ml, produces bacterial classification by leavening and makes freeze-dried vaccine powder; According to parts by weight meter, get 1 part～2 parts, Lactobacillus plantarum freeze-dried vaccine powder, 1 part～2 parts, lactobacillus bulgaricus freeze-dried vaccine powder, 1 part～2 parts, streptococcus thermophilus freeze-dried vaccine powder, 3 parts～5 parts, S. cervisiae freeze-dried vaccine powder, 3 parts～5 parts, fold candidiasis freeze-dried vaccine powder, 3 parts～5 parts, Candida tropicalis freeze-dried vaccine powder, 3 parts～5 parts, fermentation Pichia pastoris freeze-dried vaccine powder, mixes, and obtains composite ferment.
Described batter is at least one in the ripe powder of brown rice, corn flour, millet powder, soy meal, pearling cone meal, wheat flour, coixlacrymajobi powder, acorn nut powder, sorghum flour, black rice flour, buckwheat, naked oats flour, sweet potato powder, milled glutinous broomcorn millet powder, mung bean flour, adds after water and grinds and form.
The application of a kind of composite ferment in preparing fermented pasta.
Described wheaten food is thin pancake.
The application of above-mentioned composite ferment in preparing thin pancake, its concrete steps are as follows:
Composite ferment is joined in batter, mix and stir, described composite ferment and the mass ratio of batter are 1:100～1:1000, add NaHCO
3control batter pH value 6.0～7.0, at 20 ℃～35 ℃, fermentation 2h～12h.
Described batter is at least one in the ripe powder of brown rice, corn flour, millet powder, soy meal, pearling cone meal, wheat flour, coixlacrymajobi powder, acorn nut powder, sorghum flour, black rice flour, buckwheat, naked oats flour, sweet potato powder, milled glutinous broomcorn millet powder, mung bean flour, adds after water and grinds and form.
Described composite ferment and the mass ratio of batter are 1:100～1:500, and fermentation time is 2h～4h.
Beneficial effect of the present invention:
Choose isolated bacterial classification from the batter of spontaneous fermentation, the control of composite and addition two aspects by bacterial classification, makes beneficial microbe become dominant bacteria, avoids the growth of miscellaneous bacteria; Dominant bacteria carries out growth metabolism, promotes in fermented pasta the flavor substances such as alcohols, aldehydes, ketone, acids, ester class, terpene to produce, and makes its taste abundanter, has improved the quality of fermented pasta; Adopt multiple saccharomycete to compare and can produce more flavor substance with single culture leavening with lactic acid bacteria composite ferment, wheaten food local flavor and the traditional zymotic wheaten food of making are more approaching; The wheaten food that uses this composite ferment to make can effectively prevent living contaminants, ensures the health of wheaten food, the quality that improves wheaten food, shortening production cycle, and production process is controlled, for realizing suitability for industrialized production, lays the foundation.
The specific embodiment
Embodiment 1
1, the purifying of single bacterial classification in fermentation batter
The ripe powder of 50g brown rice, 20g corn flour, 20g millet powder, 10g soy meal are mixed, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 30 ℃, fermentation 6h, obtains spontaneous fermentation batter; Adopt sterile working to get spontaneous fermentation paste and carry out gradient dilution to 10
-1~ 10
-6, proceed to respectively PDA plating medium and MRA plating medium, at 30 ℃, cultivate 48h; The doubtful bacterium colony streak inoculation of saccharomycete of picking plate SC diameter >0.5mm is in PDA plating medium, in picking plate, the doubtful colony inoculation of lactic acid bacteria of molten calcium circle >0.5mm is in MRS plating medium, respectively at 30 ℃, cultivate 48h, with PDA plating medium and MRS plating medium plate streaking, purified for 4 generations respectively again, microscopic examination is observed after its form, be transferred to 4 ℃ of storages in slant medium, obtain single bacterial classification for bacterial screening.
2. saccharomycetic screening
Primary dcreening operation: the doubtful saccharomycete bacterial classification after purifying is had access in the test tube of YPD fluid nutrient medium of Du Shi tubule, at 30 ℃, cultivate 12h, every 4h observes the aerogenesis situation in Du Shi tubule, filters out the fastest 30 maximum strain bacterial strains of gas production;
Secondary screening (multiple sieve): by the bacterial strain access YPD fluid nutrient medium of primary dcreening operation, at 30 ℃, cultivate 12h, getting 6mL access is equipped with in the triangular flask of 200mL YPD fluid nutrient medium, at 30 ℃, cultivate 12h, every 4h record is weightless situation once, the soluble solid content of zymotic fluid, content of reducing sugar, pH value and alcoholic strength are analyzed, select fermentation strain 20 strains that soluble solid content is high, content of reducing sugar is low, pH value is high, alcoholic strength is high, be defined as the bacterial strain of fermentation character the best;
Three level screen: by the bacterial strain access YPD fluid nutrient medium of multiple sieve, cultivate 12h for 30 ℃, every 2h weighs once, with the YPD fluid nutrient medium of not inoculating, does blank, and in OD value and the pH value of 600nm wavelength place mensuration zymotic fluid, filter out the bacterial strain that fertility is the strongest, acid resistance is good.
3, the screening of lactic acid bacteria
Primary dcreening operation: the doubtful lactic acid bacteria culturers after purifying is carried out to Gram's staining, the acid of glucose product, aerogenesis experiment and catalase experiment;
Secondary screening (multiple sieve): by the bacterial classification access MRS fluid nutrient medium of primary dcreening operation, cultivate 12h at 30 ℃, every 4h gets sample one time, measures the pH value of zymotic fluid, and the bacterial strain that acid is the fastest is produced in screening;
Three level screen: by the lactic acid bacteria strains access MRS fluid nutrient medium sifting out again, at 30 ℃, cultivate 12h, every 4h gets sample one time, with the MRS fluid nutrient medium of not inoculating, does blank, at 600nm wavelength place, measure zymotic fluid OD value, filter out the bacterial strain that fertility is the strongest.
4, the evaluation of bacterial classification
The yeast strain that separation, purifying, screening are drawn, by the D1/D2 sequence analysis of 26S rDNA, draws S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris; The lactic acid bacteria that separation, purifying, screening are drawn, by 16S rDNA sequence analysis, draws Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus.
5, the preparation of composite leavening
5.1, zymogenic preparation
The preparation of a, mother culture
Measure respectively YPD fluid nutrient medium 200mL in 4 500mL triangular flasks and MRS culture medium 200mL in 3 500mL triangular flasks, 121 ℃ of sterilizing 20min, be cooled to 30 ℃, saccharomycete is accessed to YPD fluid nutrient medium, in lactic acid bacteria access MRS culture medium, by 10% inoculation of culture volume, at 30 ℃, shaking table is cultivated 24h, as mother culture;
The preparation that b, saccharomycete are produced leavening
Configure 0.5% peptone, 1.5% yeast extract, 0.4% sodium chloride, 1% glucose, 2% agar, pH value is 7.0, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by saccharomycete mother culture in 2% volume inoculation step a, at 30 ℃, shaking table is cultivated 36h, detects the saccharomycete of producing leavening, and each produces saccharomycete viable count>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
The preparation that c, lactic acid bacteria produce leavening
Prepare 5 ° of Be brewer's wort 1L, 0.5% yeast extract, 0.6% calcium carbonate, 1.5% agar, nature pH value, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by lactic acid bacteria mother culture in 2% volume inoculation step a, 30 ℃ of shaking tables are cultivated 36h, detect and produce starter lactic acid bacteria, and each produces viable count of lactobacillus>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL.
5.2, the preparation of powder freeze-drying lactobacillus
Ripe production leavening is poured in glass ampoule under aseptic condition, and liquid level, lower than 1cm, is added a cover after bottle stopper, puts into-30 ℃ of refrigerator-freezer quick-frozens, after freezing, glass ampoule is used to pallet splendid attire, puts into freeze dryer freeze drying 24h, obtains freeze-dried vaccine powder.
5.3, zymogenic composite
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 1g, lactobacillus bulgaricus freeze-dried vaccine powder 2g, streptococcus thermophilus freeze-dried vaccine powder 1g, S. cervisiae freeze-dried vaccine powder 5g, fold candidiasis freeze-dried vaccine powder 3g, Candida tropicalis freeze-dried vaccine powder 5g, fermentation Pichia pastoris freeze-dried vaccine powder 3g; After mixing, vacuum packaging obtains composite ferment.
6, the application of composite ferment in preparing thin pancake, its concrete steps are as follows:
The ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, milled glutinous broomcorn millet powder 10g are mixed, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 0.3g composite ferment, add NaHCO
3control batter pH value 6.0～7.0 at 20 ℃, fermentation 12h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
Embodiment 2
By the ripe powder 50g of brown rice, corn flour 10g, wheat flour 20g, millet powder 20g, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 30 ℃, fermentation 6h, obtains spontaneous fermentation batter; According to the method for embodiment 1, from the batter of spontaneous fermentation, isolate Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus, S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris and produce bacterial classification as leavening, each produces starter culture viable count>=10
9individual/ml, produces bacterial classification to leavening and carries out quick-frozen, freeze drying, obtains freeze-dried vaccine powder.
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 2g, lactobacillus bulgaricus freeze-dried vaccine powder 1g, streptococcus thermophilus freeze-dried vaccine powder 2g, S. cervisiae freeze-dried vaccine powder 3g, fold candidiasis freeze-dried vaccine powder 5g, Candida tropicalis freeze-dried vaccine powder 3g, fermentation Pichia pastoris freeze-dried vaccine powder 5g; After mixing, vacuum packaging obtains composite ferment.
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: by the ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 1g composite ferment, add NaHCO
3control batter pH value 6.0～7.0, at 20 ℃, fermentation 8h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
Embodiment 3
By the ripe powder 50g of brown rice, corn flour 10g, Chinese sorghum ground rice 10g, millet powder 10g, soy meal 10g, buckwheat 10g, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 35 ℃, fermentation 6h, obtains spontaneous fermentation batter; According to the method for embodiment 1, from the batter of spontaneous fermentation, isolate Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus, S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris and produce bacterial classification as leavening, each produces starter culture viable count>=10
9individual/ml, produces bacterial classification to leavening and carries out quick-frozen, freeze drying, obtains freeze-dried vaccine powder.
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 1.5g, lactobacillus bulgaricus freeze-dried vaccine powder 1.5g, streptococcus thermophilus freeze-dried vaccine powder 1.5g, S. cervisiae freeze-dried vaccine powder 4g, fold candidiasis freeze-dried vaccine powder 4g, Candida tropicalis freeze-dried vaccine powder 4g, fermentation Pichia pastoris freeze-dried vaccine powder 4g; After mixing, vacuum packaging obtains composite ferment.
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: by the ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 3g composite ferment, add NaHCO
3control batter pH value 6.0～7.0, at 30 ℃, fermentation 3h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
Embodiment 4
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: the ripe powder 50g of brown rice, corn flour 10g, sweet potato powder 10g, millet powder 10g, soy meal 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to the composite ferment in 1.5g embodiment 1, add NaHCO
3control batter pH value 6.0～7.0, at 20 ℃, fermentation 4h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
Embodiment 5
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: the ripe powder 50g of brown rice, buckwheat 10g, oatmeal 10g, sorghum flour 10g, mung bean flour 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to the composite ferment in 3g embodiment 1, add NaHCO
3control batter pH value 6.0～7.0, at 25 ℃, fermentation 2h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
Claims (3)
1. a preparation method for composite ferment, is characterized in that:
1.1, the purifying of single bacterial classification in fermentation batter
The ripe powder of 50g brown rice, 20g corn flour, 20g millet powder, 10g soy meal are mixed, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 30 ℃, fermentation 6h, obtains spontaneous fermentation batter; Adopt sterile working to get spontaneous fermentation paste and carry out gradient dilution to 10
-1~ 10
-6, proceed to respectively PDA plating medium and MRA plating medium, at 30 ℃, cultivate 48h; The doubtful bacterium colony streak inoculation of saccharomycete of picking plate SC diameter >0.5mm is in PDA plating medium, in picking plate, the doubtful colony inoculation of lactic acid bacteria of molten calcium circle >0.5mm is in MRS plating medium, respectively at 30 ℃, cultivate 48h, with PDA plating medium and MRS plating medium plate streaking, purified for 4 generations respectively again, microscopic examination is observed after its form, be transferred to 4 ℃ of storages in slant medium, obtain single bacterial classification for bacterial screening;
1.2. saccharomycetic screening
Primary dcreening operation: the doubtful saccharomycete bacterial classification after purifying is had access in the test tube of YPD fluid nutrient medium of Du Shi tubule, at 30 ℃, cultivate 12h, every 4h observes the aerogenesis situation in Du Shi tubule, filters out the fastest 30 maximum strain bacterial strains of gas production;
Secondary screening: by the bacterial strain access YPD fluid nutrient medium of primary dcreening operation, at 30 ℃, cultivate 12h, get 6mL access and be equipped with in the triangular flask of 200mL YPD fluid nutrient medium, at 30 ℃, cultivate 12h, every 4h record is weightless situation once, the soluble solid content of zymotic fluid, content of reducing sugar, pH value and alcoholic strength are analyzed, selected fermentation strain 20 strains that soluble solid content is high, content of reducing sugar is low, pH value is high, alcoholic strength is high, be defined as the bacterial strain of fermentation character the best;
Three level screen: by the bacterial strain access YPD fluid nutrient medium after secondary screening, cultivate 12h for 30 ℃, every 2h weighs once, with the YPD fluid nutrient medium of not inoculating, do blank, and in OD value and the pH value of 600nm wavelength place mensuration zymotic fluid, filter out the bacterial strain that fertility is the strongest, acid resistance is good;
1.3, the screening of lactic acid bacteria
Primary dcreening operation: the doubtful lactic acid bacteria culturers after purifying is carried out to Gram's staining, the acid of glucose product, aerogenesis experiment and catalase experiment;
Secondary screening: by the bacterial classification access MRS fluid nutrient medium of primary dcreening operation, cultivate 12h at 30 ℃, every 4h gets sample one time, measures the pH value of zymotic fluid, and the bacterial strain that acid is the fastest is produced in screening;
Three level screen: by the lactic acid bacteria strains access MRS fluid nutrient medium after secondary screening, at 30 ℃, cultivate 12h, every 4h gets sample one time, with the MRS fluid nutrient medium of not inoculating, does blank, at 600nm wavelength place, measure zymotic fluid OD value, filter out the bacterial strain that fertility is the strongest;
1.4, the evaluation of bacterial classification
The yeast strain that separation, purifying, screening are drawn, by the D1/D2 sequence analysis of 26S rDNA, draws S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris; The lactic acid bacteria that separation, purifying, screening are drawn, by 16S rDNA sequence analysis, draws Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus;
1.5, the preparation of composite leavening
1.5.1, zymogenic preparation
The preparation of a, mother culture
Measure respectively YPD fluid nutrient medium 200mL in 4 500mL triangular flasks and MRS culture medium 200mL in 3 500mL triangular flasks, 121 ℃ of sterilizing 20min, be cooled to 30 ℃, saccharomycete is accessed to YPD fluid nutrient medium, in lactic acid bacteria access MRS culture medium, by 10% inoculation of culture volume, at 30 ℃, shaking table is cultivated 24h, as mother culture;
The preparation that b, saccharomycete are produced leavening
Configure 0.5% peptone, 1.5% yeast extract, 0.4% sodium chloride, 1% glucose, 2% agar, pH value is 7.0, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by saccharomycete mother culture in 2% volume inoculation step a, at 30 ℃, shaking table is cultivated 36h, detects the saccharomycete of producing leavening, and each produces saccharomycete viable count>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
The preparation that c, lactic acid bacteria produce leavening
Prepare 5 ° of Be brewer's wort 1L, 0.5% yeast extract, 0.6% calcium carbonate, 1.5% agar, nature pH value, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by lactic acid bacteria mother culture in 2% volume inoculation step a, 30 ℃ of shaking tables are cultivated 36h, detect and produce starter lactic acid bacteria, and each produces viable count of lactobacillus>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
1.5.2, the preparation of powder freeze-drying lactobacillus
Ripe production leavening is poured in glass ampoule under aseptic condition, and liquid level, lower than 1cm, is added a cover after bottle stopper, puts into-30 ℃ of refrigerator-freezer quick-frozens, after freezing, glass ampoule is used to pallet splendid attire, puts into freeze dryer freeze drying 24h, obtains freeze-dried vaccine powder;
1.5.3, zymogenic composite
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 1g, lactobacillus bulgaricus freeze-dried vaccine powder 2g, streptococcus thermophilus freeze-dried vaccine powder 1g, S. cervisiae freeze-dried vaccine powder 5g, fold candidiasis freeze-dried vaccine powder 3g, Candida tropicalis freeze-dried vaccine powder 5g, fermentation Pichia pastoris freeze-dried vaccine powder 3g; After mixing, vacuum packaging obtains composite ferment;
1.6, the application of composite ferment in preparing thin pancake, its concrete steps are as follows:
The ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, milled glutinous broomcorn millet powder 10g are mixed, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 0.3g composite ferment, add NaHCO
3control batter pH value 6.0～7.0 at 20 ℃, fermentation 12h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
2. a preparation method for composite ferment, is characterized in that:
1.1, the purifying of single bacterial classification in fermentation batter
By the ripe powder 50g of brown rice, corn flour 10g, wheat flour 20g, millet powder 20g, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 30 ℃, fermentation 6h, obtains spontaneous fermentation batter; Adopt sterile working to get spontaneous fermentation paste and carry out gradient dilution to 10
-1~ 10
-6, proceed to respectively PDA plating medium and MRA plating medium, at 30 ℃, cultivate 48h; The doubtful bacterium colony streak inoculation of saccharomycete of picking plate SC diameter >0.5mm is in PDA plating medium, in picking plate, the doubtful colony inoculation of lactic acid bacteria of molten calcium circle >0.5mm is in MRS plating medium, respectively at 30 ℃, cultivate 48h, with PDA plating medium and MRS plating medium plate streaking, purified for 4 generations respectively again, microscopic examination is observed after its form, be transferred to 4 ℃ of storages in slant medium, obtain single bacterial classification for bacterial screening;
1.2. saccharomycetic screening
Primary dcreening operation: the doubtful saccharomycete bacterial classification after purifying is had access in the test tube of YPD fluid nutrient medium of Du Shi tubule, at 30 ℃, cultivate 12h, every 4h observes the aerogenesis situation in Du Shi tubule, filters out the fastest 30 maximum strain bacterial strains of gas production;
Secondary screening: by the bacterial strain access YPD fluid nutrient medium of primary dcreening operation, at 30 ℃, cultivate 12h, get 6mL access and be equipped with in the triangular flask of 200mL YPD fluid nutrient medium, at 30 ℃, cultivate 12h, every 4h record is weightless situation once, the soluble solid content of zymotic fluid, content of reducing sugar, pH value and alcoholic strength are analyzed, selected fermentation strain 20 strains that soluble solid content is high, content of reducing sugar is low, pH value is high, alcoholic strength is high, be defined as the bacterial strain of fermentation character the best;
Three level screen: by the bacterial strain access YPD fluid nutrient medium after secondary screening, cultivate 12h for 30 ℃, every 2h weighs once, with the YPD fluid nutrient medium of not inoculating, do blank, and in OD value and the pH value of 600nm wavelength place mensuration zymotic fluid, filter out the bacterial strain that fertility is the strongest, acid resistance is good;
1.3, the screening of lactic acid bacteria
Primary dcreening operation: the doubtful lactic acid bacteria culturers after purifying is carried out to Gram's staining, the acid of glucose product, aerogenesis experiment and catalase experiment;
Secondary screening: by the bacterial classification access MRS fluid nutrient medium of primary dcreening operation, cultivate 12h at 30 ℃, every 4h gets sample one time, measures the pH value of zymotic fluid, and the bacterial strain that acid is the fastest is produced in screening;
Three level screen: by the lactic acid bacteria strains access MRS fluid nutrient medium after secondary screening, at 30 ℃, cultivate 12h, every 4h gets sample one time, with the MRS fluid nutrient medium of not inoculating, does blank, at 600nm wavelength place, measure zymotic fluid OD value, filter out the bacterial strain that fertility is the strongest;
1.4, the evaluation of bacterial classification
The yeast strain that separation, purifying, screening are drawn, by the D1/D2 sequence analysis of 26S rDNA, draws S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris; The lactic acid bacteria that separation, purifying, screening are drawn, by 16S rDNA sequence analysis, draws Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus;
1.5, the preparation of composite leavening
1.5.1, zymogenic preparation
The preparation of a, mother culture
Measure respectively YPD fluid nutrient medium 200mL in 4 500mL triangular flasks and MRS culture medium 200mL in 3 500mL triangular flasks, 121 ℃ of sterilizing 20min, be cooled to 30 ℃, saccharomycete is accessed to YPD fluid nutrient medium, in lactic acid bacteria access MRS culture medium, by 10% inoculation of culture volume, at 30 ℃, shaking table is cultivated 24h, as mother culture;
The preparation that b, saccharomycete are produced leavening
Configure 0.5% peptone, 1.5% yeast extract, 0.4% sodium chloride, 1% glucose, 2% agar, pH value is 7.0, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by saccharomycete mother culture in 2% volume inoculation step a, at 30 ℃, shaking table is cultivated 36h, detects the saccharomycete of producing leavening, and each produces saccharomycete viable count>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
The preparation that c, lactic acid bacteria produce leavening
Prepare 5 ° of Be brewer's wort 1L, 0.5% yeast extract, 0.6% calcium carbonate, 1.5% agar, nature pH value, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by lactic acid bacteria mother culture in 2% volume inoculation step a, 30 ℃ of shaking tables are cultivated 36h, detect and produce starter lactic acid bacteria, and each produces viable count of lactobacillus>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
1.5.2, the preparation of powder freeze-drying lactobacillus
Ripe production leavening is poured in glass ampoule under aseptic condition, and liquid level, lower than 1cm, is added a cover after bottle stopper, puts into-30 ℃ of refrigerator-freezer quick-frozens, after freezing, glass ampoule is used to pallet splendid attire, puts into freeze dryer freeze drying 24h, obtains freeze-dried vaccine powder;
1.5.3, zymogenic composite
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 2g, lactobacillus bulgaricus freeze-dried vaccine powder 1g, streptococcus thermophilus freeze-dried vaccine powder 2g, S. cervisiae freeze-dried vaccine powder 3g, fold candidiasis freeze-dried vaccine powder 5g, Candida tropicalis freeze-dried vaccine powder 3g, fermentation Pichia pastoris freeze-dried vaccine powder 5g; After mixing, vacuum packaging obtains composite ferment;
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: by the ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 1g composite ferment, add NaHCO
3control batter pH value 6.0～7.0, at 20 ℃, fermentation 8h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
3. a preparation method for composite ferment, is characterized in that:
1.1, the purifying of single bacterial classification in fermentation batter
By the ripe powder 50g of brown rice, corn flour 10g, Chinese sorghum ground rice 10g, millet powder 10g, soy meal 10g, buckwheat 10g, add 20 ℃ of water 200g and grind into batter, add 10g leaven, at 35 ℃, fermentation 6h, obtains spontaneous fermentation batter; Adopt sterile working to get spontaneous fermentation paste and carry out gradient dilution to 10
-1~ 10
-6, proceed to respectively PDA plating medium and MRA plating medium, at 30 ℃, cultivate 48h; The doubtful bacterium colony streak inoculation of saccharomycete of picking plate SC diameter >0.5mm is in PDA plating medium, in picking plate, the doubtful colony inoculation of lactic acid bacteria of molten calcium circle >0.5mm is in MRS plating medium, respectively at 30 ℃, cultivate 48h, with PDA plating medium and MRS plating medium plate streaking, purified for 4 generations respectively again, microscopic examination is observed after its form, be transferred to 4 ℃ of storages in slant medium, obtain single bacterial classification for bacterial screening;
1.2. saccharomycetic screening
Primary dcreening operation: the doubtful saccharomycete bacterial classification after purifying is had access in the test tube of YPD fluid nutrient medium of Du Shi tubule, at 30 ℃, cultivate 12h, every 4h observes the aerogenesis situation in Du Shi tubule, filters out the fastest 30 maximum strain bacterial strains of gas production;
Secondary screening: by the bacterial strain access YPD fluid nutrient medium of primary dcreening operation, at 30 ℃, cultivate 12h, get 6mL access and be equipped with in the triangular flask of 200mL YPD fluid nutrient medium, at 30 ℃, cultivate 12h, every 4h record is weightless situation once, the soluble solid content of zymotic fluid, content of reducing sugar, pH value and alcoholic strength are analyzed, selected fermentation strain 20 strains that soluble solid content is high, content of reducing sugar is low, pH value is high, alcoholic strength is high, be defined as the bacterial strain of fermentation character the best;
Three level screen: by the bacterial strain access YPD fluid nutrient medium after secondary screening, cultivate 12h for 30 ℃, every 2h weighs once, with the YPD fluid nutrient medium of not inoculating, do blank, and in OD value and the pH value of 600nm wavelength place mensuration zymotic fluid, filter out the bacterial strain that fertility is the strongest, acid resistance is good;
1.3, the screening of lactic acid bacteria
Primary dcreening operation: the doubtful lactic acid bacteria culturers after purifying is carried out to Gram's staining, the acid of glucose product, aerogenesis experiment and catalase experiment;
Secondary screening: by the bacterial classification access MRS fluid nutrient medium of primary dcreening operation, cultivate 12h at 30 ℃, every 4h gets sample one time, measures the pH value of zymotic fluid, and the bacterial strain that acid is the fastest is produced in screening;
Three level screen: by the lactic acid bacteria strains access MRS fluid nutrient medium after secondary screening, at 30 ℃, cultivate 12h, every 4h gets sample one time, with the MRS fluid nutrient medium of not inoculating, does blank, at 600nm wavelength place, measure zymotic fluid OD value, filter out the bacterial strain that fertility is the strongest;
1.4, the evaluation of bacterial classification
The yeast strain that separation, purifying, screening are drawn, by the D1/D2 sequence analysis of 26S rDNA, draws S. cervisiae, fold candidiasis, Candida tropicalis, fermentation Pichia pastoris; The lactic acid bacteria that separation, purifying, screening are drawn, by 16S rDNA sequence analysis, draws Lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus;
1.5, the preparation of composite leavening
1.5.1, zymogenic preparation
The preparation of a, mother culture
Measure respectively YPD fluid nutrient medium 200mL in 4 500mL triangular flasks and MRS culture medium 200mL in 3 500mL triangular flasks, 121 ℃ of sterilizing 20min, be cooled to 30 ℃, saccharomycete is accessed to YPD fluid nutrient medium, in lactic acid bacteria access MRS culture medium, by 10% inoculation of culture volume, at 30 ℃, shaking table is cultivated 24h, as mother culture;
The preparation that b, saccharomycete are produced leavening
Configure 0.5% peptone, 1.5% yeast extract, 0.4% sodium chloride, 1% glucose, 2% agar, pH value is 7.0, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by saccharomycete mother culture in 2% volume inoculation step a, at 30 ℃, shaking table is cultivated 36h, detects the saccharomycete of producing leavening, and each produces saccharomycete viable count>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
The preparation that c, lactic acid bacteria produce leavening
Prepare 5 ° of Be brewer's wort 1L, 0.5% yeast extract, 0.6% calcium carbonate, 1.5% agar, nature pH value, at 121 ℃, sterilizing 20min, is cooled to 30 ℃, respectively by lactic acid bacteria mother culture in 2% volume inoculation step a, 30 ℃ of shaking tables are cultivated 36h, detect and produce starter lactic acid bacteria, and each produces viable count of lactobacillus>=10 of leavening
9individual/mL, is considered as fermenting-ripening, if fermentation viable count≤10
9individual/mL, continues to cultivate, until reach 10
9individual/mL;
1.5.2, the preparation of powder freeze-drying lactobacillus
Ripe production leavening is poured in glass ampoule under aseptic condition, and liquid level, lower than 1cm, is added a cover after bottle stopper, puts into-30 ℃ of refrigerator-freezer quick-frozens, after freezing, glass ampoule is used to pallet splendid attire, puts into freeze dryer freeze drying 24h, obtains freeze-dried vaccine powder;
1.5.3, zymogenic composite
According to parts by weight meter, get Lactobacillus plantarum freeze-dried vaccine powder 1.5g, lactobacillus bulgaricus freeze-dried vaccine powder 1.5g, streptococcus thermophilus freeze-dried vaccine powder 1.5g, S. cervisiae freeze-dried vaccine powder 4g, fold candidiasis freeze-dried vaccine powder 4g, Candida tropicalis freeze-dried vaccine powder 4g, fermentation Pichia pastoris freeze-dried vaccine powder 4g; After mixing, vacuum packaging obtains composite ferment;
The application of composite ferment in preparing thin pancake, its concrete steps are as follows: by the ripe powder 50g of brown rice, corn flour 10g, wheat flour 10g, millet powder 10g, soy meal 10g, coixlacrymajobi powder 10g, mix, add the water 200g mixing defibrination of 20 ℃, obtain batter; Batter is added to 3g composite ferment, add NaHCO
3control batter pH value 6.0～7.0, at 30 ℃, fermentation 3h, is then used thin pancake spoon spoon to ladle out and burns the thin pancake of rolling uniformly stand flakiness shape on hot griddle, and baking 1.5min is lower with shovel shovel.
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