CN101497892A - Natural yoghourt essence bottom flavoring and microbial fermentation production method thereof - Google Patents
Natural yoghourt essence bottom flavoring and microbial fermentation production method thereof Download PDFInfo
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Abstract
The invention relates to a natural yoghurt flavor basic charge and a microbiological fermentation method thereof. The method mainly comprises the following steps: lactic acid bacteria freeze starters are prepared, yoghurt fermentation medium is prepared, and the natural yoghurt flavor basic charge is prepared; fermentation treatment is carried out on the yoghurt fermentation medium which takes fresh milk, full cream milk, or nonfat dried milk powder as main basic charge by using the various lactic acid bacteria freeze starters, centrifuging for primary solid-liquid separation; and clear liquid, i.e. the natural yoghurt flavor basic charge, is obtained by refinedly filtering supernatant. The natural yoghurt flavor basic charge produced by the method of microbiological fermentation contains various important milk flavor fragrant ingredients such as acids, alcohols, ketones, lactones and sulfur-containing compounds and can be made into the yoghurt flavor by proper modification, which can be widely applied to various beverages, dairy foods, ice creams, bakeries, candies, and the like. Compared with common formulated yoghurt flavors, the fermented yoghurt flavor has not only good flavor but also fermented taste.
Description
Technical field
The present invention relates to a kind of essence bed material and production method thereof, particularly relate to and a kind ofly utilize the natural yoghourt essence bottom flavoring that biotechnology produces and produce used microbe fermentation method.
Background technology
Milk flavor fragrance is that people are familiar with and one of the fragrance liked the most, because flavour ingredient wrecks unavoidably in the diary processing process, the milk of milk-product need be replenished, so dairy products perfuming technology is paid close attention to by people always.On the other hand, milk essence is an Applications in Food Industry essence the most widely, can be applicable to various beverages, dairy products, ice cream, bakery, candy etc., has vast market prospect.The fermentation sense with sour milk essence is the most important especially in the milk essence.The preparation method of sour milk essence mainly is synthetic perfume material allotment (allotment type sour milk essence) and passes through microbe fermentation method or enzymolysis process preparation (fermented type sour milk essence) two big class methods at present.Shortcomings such as natural sense is poor owing to existing for the fragrance of allotment type essence, fragrance remaining time is lacked, fragrance is mellow and full inadequately and plentiful make the preparation of natural fermented type sour milk essence more and more become the emphasis that people pay close attention to.
Along with improving constantly of dairy products perfuming technology, people combine microbial fermentation technology and zymolysis technique and traditional meat flavor formulating technology, develop high-quality natural milk-taste essence.Utilize the milk essence bed material of microbe fermentation method and enzymolysis process preparation, not only fragrance is natural, soft, true to nature; And flavour ingredient is abundant, has both comprised the original aroma component of milk-product, has increased the fragrance matter with fermentation sense again, and aroma strength can improve 100～300 times, this be artificial blending technology be difficult to up to and.The natural milk-taste essence bed material is through after the suitable modification, and it is satisfactory for result to compose perfume, and strong persistent fragrance and careful lubricated mouthfeel both were provided, and it is big to have remedied the compound essence pungency again, not soft defective.
Enzyme process prepares milk essence, is to be raw material with cream (or rare cream), utilizes zymolysis technique that cream is hydrolyzed under certain condition, produces the compound with milk feature, with 100～300 times of flavourings.Yet research and production about fermented type sour milk essence bed material do not appear in the newspapers so far as yet.
Summary of the invention
Shortcoming such as the present invention is directed to that above-mentioned artificial allotment type sour milk essence fragrance exists that natural sense is poor, fragrance remaining time is short, fragrance is mellow and full inadequately and plentiful provides a kind of microbial fermentation production method of natural yoghourt essence bottom flavoring and the natural yoghourt essence bottom flavoring that is prepared by this method.
To achieve these goals, the present invention has adopted following technical scheme:
A kind of microbial fermentation production method of producing natural yoghourt essence bottom flavoring of the present invention, its microbial fermentation production method sequence of steps mainly comprises the agent of preparation milk-acid bacteria frozen fermented, preparation sour milk fermention medium and preparation natural yoghourt essence bottom flavoring, and its concrete steps order is as follows: the agent of preparation milk-acid bacteria frozen fermented:
(1) select for use following arbitrary lactic acid bacteria culturers to cultivate the agent of preparation milk-acid bacteria frozen fermented as follows respectively: thermophilus streptococcus, (Streptococcus thermophilus) CGMCC1.2471 or lactobacillus delbruockii subspecies bulgaricus, (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 or Lactobacterium acidophilum, (Lactobacillus acidophilus) CGMCC1.1854 or lactococcus lactis subsp, (Lactococcus lactis subsp.cremoris) CGMCC1.9;
(2) the common cultivation of milk-acid bacteria: above-mentioned milk-acid bacteria is inoculated in respectively on the MRS solid medium that contains 15～20g/L agar, cultivated 16～48 hours under 25 ℃～45 ℃ conditions; Above-mentioned MRS solid medium, concrete prescription is: peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 15～20g/L; Regulating pH is 6.2～6.4, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) bacterium that increases of milk-acid bacteria is cultivated: the lactic bacterium strains that step (2) is cultivated is encircling in the liquid enrichment medium of 30～100ml with inoculation articulating 1～2 under the aseptic condition respectively, under 30 ℃～45 ℃ conditions, shaking culture is 4～16 hours on shaking table, make various streptococcus acidi lactici fermented solutions, wherein viable count reaches 10
9～10
10CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is: skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(4) collect thalline: respectively at 0 ℃～20 ℃, rotating speed is 5000～10000 rev/mins with above-mentioned various streptococcus acidi lactici fermented solutions, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 5～10 minutes;
(5) add the thalline lyophilized vaccine: under aseptic condition, will above-mentioned various milk-acid bacterias precipitate thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is: skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(6) precooling treatment before the thalline freeze-drying: the various milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carry out 6～16 hours precooling treatment under-15 ℃～-18 ℃ conditions;
(7) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the various milk-acid bacteria thalline of precooling treatment are carried out vacuum lyophilization respectively, make the agent of milk-acid bacteria frozen fermented, and store for future use under-18 ℃～-20 ℃ conditions;
(8) preparation sour milk fermention medium: comprise fresh milk or whole milk powder or skim-milk, glucose, whey powder, diammonium hydrogen citrate and water, concrete prescription is: fresh milk or whole milk powder or skim-milk 100～900g/L, glucose 5～50g/L, whey powder 10～90g/L, diammonium hydrogen citrate 5～50g/L;
(9) homogeneous: after the Yoghourt fermentation substratum for preparing stirred, carry out homogenization treatment under the room temperature, rotating speed is 3000～6000 rev/mins, and the time is 5～20 minutes;
(10) sterilization: with the fermention medium heat sterilization behind the homogeneous, temperature is 85 ℃～105 ℃, and sterilization time is 5～30 minutes;
(11) cooling: the fermention medium after will sterilizing is cooled to 30 ℃～45 ℃;
(12) inoculation: will cultivate two kinds of making or two or more milk-acid bacteria frozen fermented agent is mixed and made into working stock culture by above-mentioned steps (1)-(7), and be that 0.05～80g/L is inoculated in the Yoghourt fermentation substratum by above-mentioned steps (8)-(11) preparation by total addition level with it;
Carry out the preparation of natural yoghourt essence bottom flavoring then:
(13) fermentation: the substratum of step (12) inoculation various lactobacillus frozen fermented agent is carried out fermentative processing, and leavening temperature is 37 ℃～45 ℃, and fermentation time is 4～24 hours; Acidity after the control fermentation is 70～130 ° of T;
(14) preliminary solid-liquid separation: the Yoghourt fermentation thing that step (13) is obtained under 3000～4000 rev/mins condition centrifugal 5～8 minutes, carry out preliminary solid-liquid separation;
(15) the refining filtration: the Yoghourt fermentation thing supernatant liquor that step (14) obtains is made with extra care filtration, obtain transparent clarifying natural yoghourt essence bottom flavoring;
(16) sample detection:, carry out the qualitative detection of milk aroma component with GC/MS with the transparent clarifying liquid sample that step (15) obtains.
Liquid enrichment medium described in the above-mentioned steps (3), concrete prescription is: skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions.
Preferred 0 ℃～10 ℃ of collection thalline temperature described in the above-mentioned steps (4), preferred 8000～10000 rev/mins of rotating speed, preferred 5～8 minutes of time.
Thalline lyophilized vaccine described in the above-mentioned steps (5), concrete prescription is: skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L.
Yoghourt fermentation substratum described in the above-mentioned steps (8), concrete formula optimization is: skim-milk 100～200g/L, glucose 30～50g/L, whey powder 20～60g/L, diammonium hydrogen citrate 5～30g/L.
Preferred 4500～6000 rev/mins of homogenization treatment rotating speed described in the above-mentioned steps (9), preferred 5～10 minutes of homogenization treatment time.
Preferred 100 ℃～105 ℃ of fermention medium sterilising temp described in the above-mentioned steps (10), preferred 5～15 minutes of sterilization time.
The working stock culture that various lactobacillus frozen fermented agent described in the above-mentioned steps (12) is mixed and made into, the inoculation total addition level is preferably 1～50g/L.
Be combined as two kinds or two or more combinations in the various lactobacillus frozen fermented agent described in the above-mentioned steps (12).
The working stock culture that various lactobacillus frozen fermented agent described in the above-mentioned steps (12) is mixed and made into, wherein the mass percent ratio of coccus and bacillus is 2:1 or 1:1.
Preferred 40 ℃～44 ℃ of leavening temperature described in the above-mentioned steps (13), preferred 10～22 hours of fermentation time.
Preferred 90～120 ° of T of acidity control after the fermentation described in the above-mentioned steps (13).
Refining filter method described in the above-mentioned steps (15) comprises: supercentrifugal process, rotating speed are 8000～10000 rev/mins, and centrifugation time is 5～10 minutes; The filter press method; The polypropylene screen filtration method.
The polypropylene screen filtration method is preferably used in refining filtration described in the above-mentioned steps (15).
The GC/MS qualitative detection analytical procedure that relates in the above-mentioned steps (16), gas-chromatography adopts Agilent 6890 type instruments, capillary gas chromatographic column is Agilent HP-5 post (30m * 0.25mm * 0.25 μ m), injector temperature is 250 ℃, splitting ratio is 50:1, and carrier gas is high-purity helium, and flow velocity is 0.8ml/min, detector is FID, and detected temperatures is 300 ℃.
The GC/MS method for qualitative analysis that relates in the above-mentioned steps (16), mass spectrum adopts Varian Saturn series 2000 type instruments, electron-bombardment (EI) ion source, ionization voltage is 70eV, ion source temperature is 180 ℃, interface temperature is 280 ℃, and the quality of scanning scope is 29～38amu, and the solvent delay time is 2.5 minutes.
A kind of natural yoghourt essence bottom flavoring that the present invention is obtained by method for preparing, this natural yoghourt essence bottom flavoring is transparent clarified liq, mainly comprises following composition: butanols, acetoin, butyric acid, furfuryl alcohol, caproic acid, furanone, Tetramethylpyrazine, n-nonanoic acid, capric acid, vanillin food grade,1000.000000ine mesh, styracin, 2-butylene two dibutyl phthalates, laurostearic acid, δ-Shi Erneizhi, TETRADECONIC ACID, palmitic acid.Wherein the relative proportion of various compositions is: butanols 0.12～0.34, acetoin 0.09～0.44, butyric acid 0.06～1.57, furfuryl alcohol 0.08～0.97, caproic acid 0.02～5.00, furanone 0.02～0.96, Tetramethylpyrazine 0.08～4.47, n-nonanoic acid 0.38～5.48, capric acid 0.97～6.06, vanillin food grade,1000.000000ine mesh 0.16～0.98, styracin 0.03～0.36,2-butylene two dibutyl phthalates 0.84～5.15, laurostearic acid 0.05～1.33, δ-Shi Erneizhi 0.23～2.55, TETRADECONIC ACID 0.06～0.69, palmitic acid 0.32～5.35.
The beneficial effect that the present invention possesses is:
Frozen fermented agent through the preparation various lactobacillus, pass through microbial fermentation technology, utilizing the agent of various lactobacillus frozen fermented is that the fermention medium of main substrate carries out fermentative processing to fresh milk or whole milk powder or skim-milk, and obtaining transparent clarifying liquid product after the centrifugal and refining filtration is a kind of natural yoghourt essence bottom flavoring.Analyze through the GC/MS qualitative detection, show multiple important milk aroma components such as containing acids, alcohols, ketone, lactone, sulfocompound in the product.This product had both had the basic fragrance and the mouthfeel feature of natural sour milk; And because the prolongation of fermentation time makes that the fragrance of sour milk is stronger, fragrance remaining time is long, fermentation sense taste also increases.
The present invention has prepared the milk-acid bacteria frozen fermented agent that makes things convenient for industrialization to use, and with reference to the Yoghourt fermentation principle, utilizes comparatively simple production technique, has obtained that fragrance is pure, strong, mellow and full, fragrance remaining time is long; Compose fragrant satisfactory for result; And the careful lubricated natural yoghourt essence bottom flavoring of mouthfeel, this very is fit to and helps suitability for industrialized production.By controlling different reaction conditionss, adopting different modifying method, can obtain the diversified sour milk essence of different-style in conjunction with meat flavor formulating technology.
The upper strata clear product that utilizes fermented yogurt is as bed material, make that the milk essence product category after modifying is diversified more, not only can be deployed into the oil-in-water type milk essence, also can be deployed into liquid soluble perfume, thereby widen its range of application to a greater degree.
Embodiment
Further specify embodiments of the invention below.
Embodiment one
The preparation of milk-acid bacteria frozen fermented agent:
1. the preparation of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented agent:
(1) the common cultivation of milk-acid bacteria: CGMCC1.2471 is inoculated on the MRS solid medium that contains 15g/L agar with thermophilus streptococcus (Streptococcus thermophilus), and 45 ℃ of temperature condition were cultivated 16 hours down;
Above-mentioned MRS solid medium, concrete prescription is:
Peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 15g/L; Regulating pH is 6.2, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(2) bacterium that increases of milk-acid bacteria is cultivated: with the bacterial strain of above-mentioned cultivation, encircle in the liquid enrichment medium of 30ml with inoculation articulating 1 under aseptic condition, under 45 ℃ of temperature, shaking culture is 4 hours on shaking table, makes streptococcus acidi lactici fermented solution, and wherein viable count reaches 10
9CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is:
Skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) collect thalline: at 0 ℃, rotating speed is 5000 rev/mins with above-mentioned streptococcus acidi lactici fermented solution, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 10 minutes;
(4) add the thalline lyophilized vaccine: under aseptic condition, with above-mentioned precipitation thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is:
Skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(5) precooling treatment before the thalline freeze-drying: the milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carries out 6 hours precooling treatment under-15 ℃ of conditions;
(6) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the milk-acid bacteria thalline of precooling treatment is carried out vacuum lyophilization, make the frozen fermented agent, and store for future use under-18 ℃ of conditions.
2. the preparation of lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 frozen fermented agent:
(1) the common cultivation of milk-acid bacteria: CGMCC1.2161 is inoculated on the MRS solid medium that contains 20g/L agar with lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckiisubsp.bulgaricus), and 37 ℃ of temperature condition were cultivated 20 hours down;
Above-mentioned MRS solid medium, concrete prescription is:
Peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 20g/L; Regulating pH is 6.4, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(2) bacterium that increases of milk-acid bacteria is cultivated: with the bacterial strain of above-mentioned cultivation, encircle in the liquid enrichment medium of 100ml with inoculation articulating 2 under aseptic condition, under 37 ℃ of temperature, shaking culture is 8 hours on shaking table, makes streptococcus acidi lactici fermented solution, and wherein viable count reaches 10
10CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is:
Skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) collect thalline: at 4 ℃, rotating speed is 8000 rev/mins with above-mentioned streptococcus acidi lactici fermented solution, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 8 minutes;
(4) add the thalline lyophilized vaccine: under aseptic condition, with above-mentioned precipitation thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is:
Skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(5) precooling treatment before the thalline freeze-drying: the milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carries out 12 hours precooling treatment under-18 ℃ of conditions;
(6) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the milk-acid bacteria thalline of precooling treatment is carried out vacuum lyophilization, make the frozen fermented agent, and store for future use under-20 ℃ of conditions.
3. the preparation of Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented agent:
(1) the common cultivation of milk-acid bacteria: CGMCC1.1854 is inoculated on the MRS solid medium that contains 20g/L agar with Lactobacterium acidophilum (Lactobacillus acidophilus), and 30 ℃ of temperature condition were cultivated 36 hours down;
Above-mentioned MRS solid medium, concrete prescription is:
Peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 20g/L; Regulating pH is 6.2, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(2) bacterium that increases of milk-acid bacteria is cultivated: with the bacterial strain of above-mentioned cultivation, encircle in the liquid enrichment medium of 50ml with inoculation articulating 2 under aseptic condition, under 30 ℃ of temperature, shaking culture is 16 hours on shaking table, makes streptococcus acidi lactici fermented solution, and wherein viable count reaches 10
10CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is:
Skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) collect thalline: at 20 ℃, rotating speed is 10000 rev/mins with above-mentioned streptococcus acidi lactici fermented solution, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 5 minutes;
(4) add the thalline lyophilized vaccine: under aseptic condition, with above-mentioned precipitation thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is:
Skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(5) precooling treatment before the thalline freeze-drying: the milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carries out 16 hours precooling treatment under-15 ℃ of conditions;
(6) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the milk-acid bacteria thalline of precooling treatment is carried out vacuum lyophilization, make the frozen fermented agent, and store for future use under-18 ℃ of conditions.
4. the preparation of lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) CGMCC1.9 frozen fermented agent:
(1) the common cultivation of milk-acid bacteria: CGMCC1.9 is inoculated on the MRS solid medium that contains 20g/L agar with lactococcus lactis subsp (Lactococcus lactis subsp.cremoris), and 25 ℃ of temperature condition were cultivated 48 hours down;
Above-mentioned MRS solid medium, concrete prescription is:
Peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 20g/L; Regulating pH is 6.2, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(2) bacterium that increases of milk-acid bacteria is cultivated: with the bacterial strain of above-mentioned cultivation, encircle in the liquid enrichment medium of 100ml with inoculation articulating 2 under aseptic condition, under 25 ℃ of temperature, shaking culture is 16 hours on shaking table, makes streptococcus acidi lactici fermented solution, and wherein viable count reaches 10
9CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is:
Skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) collect thalline: at 20 ℃, rotating speed is 10000 rev/mins with above-mentioned streptococcus acidi lactici fermented solution, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 5 minutes;
(4) add the thalline lyophilized vaccine: under aseptic condition, with above-mentioned precipitation thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is:
Skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(5) precooling treatment before the thalline freeze-drying: the milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carries out 10 hours precooling treatment under-15 ℃ of conditions;
(6) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the milk-acid bacteria thalline of precooling treatment is carried out vacuum lyophilization, make the frozen fermented agent, and store for future use under-18 ℃ of conditions.
The preparation of natural yoghourt essence bottom flavoring:
The preparation fermention medium, concrete prescription is: fresh milk 900g/L, glucose 20g/L, whey powder 10g/L, diammonium hydrogen citrate 20g/L;
The fermention medium for preparing, back 3000 rev/mins of homogeneous 20 minutes stir, with 105 ℃ of sterilising treatment of fermention medium 5 minutes, be cooled to 41 ℃, thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented agent and the agent of lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckiisubsp.bulgaricus) CGMCC1.2161 frozen fermented are mixed and made into working stock culture according to mass ratio 2:1, with the addition adding fermention medium of this working stock culture according to 0.05g/L, 37 ℃ of ferment at constant temperature 24 hours, acidity after the control fermentation is 70 ° of T, tunning low-speed centrifugal 8 minutes under 3000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, use 8000 rev/mins of centrifugal 10 minutes refinement treatment of supercentrifuge, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is soft naturally, has the full aroma of fresh milk simultaneously, and has the fermented flavour mouthfeel.
The GC/MS qualitative analysis of milk aroma component in the natural yoghourt essence bottom flavoring:
Get natural yoghourt essence bottom flavoring 100g, adopt the extraction of distillation extraction instrument, the extraction liquid vacuum concentration is to 1ml, and sample size is 0.8 μ l, the GC/MS qualitative analysis.The main milk aroma component that obtains thus is as shown in the table:
| Sequence number | Retention time (min) | Relative proportion (%) | Compound | Molecular formula |
| 1 | 3.256～3.538 | 0.12～0.34 | Butanols | C 4H 10O |
| 2 | 3.832～3.854 | 0.09～0.44 | Acetoin | C 4H 8O 2 |
| 3 | 4.716～5.076 | 0.06～1.57 | Butyric acid | C 4H 8O 2 |
| 4 | 6.347～6.581 | 0.08～0.97 | Furfuryl alcohol | C 5H 6O 2 |
| 5 | 11.263～12.485 | 0.02～5.00 | Caproic acid | C 6H 12O 2 |
| 6 | 15.757～15.819 | 0.02～0.96 | Furanone | C 6H 8O 3 |
| 7 | 16.395～17.080 | 0.08～4.47 | Tetramethylpyrazine | C 8H 12N 2 |
| 8 | 29.648～29.901 | 0.38～5.48 | N-nonanoic acid | C 9H 18O 2 |
| 9 | 35.349～35.607 | 0.97～6.06 | Capric acid | C 10H 20O 2 |
| 10 | 36.784～36.898 | 0.16～0.98 | Vanillin food grade,1000.000000ine mesh | C 8H 8O 3 |
| 11 | 36.738～37.491 | 0.03～0.36 | Styracin | C 9H 8O 2 |
| 12 | 44.352～44.648 | 0.84～5.15 | 2-butylene two dibutyl phthalates | C 12H 20O 4 |
| 13 | 46.438～46.636 | 0.05～1.33 | Laurostearic acid | C 12H 24O 2 |
| 14 | 53.791～54.194 | 0.23～2.55 | δ-Shi Erneizhi | C 12H 22O 2 |
| 15 | 58.140～58.264 | 0.06～0.69 | TETRADECONIC ACID | C 14H 28O 2 |
| 16 | 63.404～64.008 | 0.32～5.35 | Palmitic acid | C 16H 32O 2 |
Embodiment two
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: fresh milk 850g/L, glucose 5g/L, whey powder 45g/L, diammonium hydrogen citrate 50g/L;
The fermention medium for preparing, back 4000 rev/mins of homogeneous 20 minutes stir, with 100 ℃ of sterilising treatment of fermention medium 10 minutes, be cooled to 30 ℃, thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented agent and the agent of lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckiisubsp.bulgaricus) CGMCC1.2161 frozen fermented are mixed and made into working stock culture according to mass ratio 1:1, with the addition adding fermention medium of this working stock culture according to 0.5g/L, 37 ℃ of ferment at constant temperature 20 hours, acidity after the control fermentation is 100 ° of T, tunning low-speed centrifugal 8 minutes under 3000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, use 10000 rev/mins of centrifugal 5 minutes refinement treatment of supercentrifuge, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is soft naturally, strong lasting, and has the pure mouthfeel of fermented flavour.
Embodiment three
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: whole milk powder 150g/L, glucose 50g/L, whey powder 10g/L, diammonium hydrogen citrate 20g/L;
The fermention medium for preparing, back 6000 rev/mins of homogeneous 10 minutes stir, with 100 ℃ of sterilising treatment of fermention medium 10 minutes, be cooled to 45 ℃, with the agent of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented, lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 frozen fermented agent and the agent of Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented are mixed and made into working stock culture according to mass ratio 5:4:1, with the addition adding fermention medium of this working stock culture according to 2g/L, 42 ℃ of ferment at constant temperature 16 hours, acidity after the control fermentation is 130 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, through the refining filtration of polypropylene screen, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is true to nature naturally, and fragrance remaining time is long, and has the pure mouthfeel of fermented flavour.
Embodiment four
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: whole milk powder 200g/L, glucose 50g/L, whey powder 90g/L, diammonium hydrogen citrate 5g/L;
The fermention medium for preparing, back 6000 rev/mins of homogeneous 10 minutes stir, with 85 ℃ of sterilising treatment of fermention medium 30 minutes, be cooled to 43 ℃, with the agent of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented, lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 frozen fermented agent and the agent of lactococcus lactis subsp (Lactococcus lactissubsp.cremoris) CGMCC1.9 frozen fermented are mixed and made into working stock culture according to mass ratio 5:4:1, with the addition adding fermention medium of this working stock culture according to 30g/L, 45 ℃ of ferment at constant temperature 10 hours, acidity after the control fermentation is 100 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, through the refining filtration of polypropylene screen, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is soft naturally, strong lasting, and has the pure mouthfeel of fermented flavour.
Embodiment five
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: skim-milk 200g/L, glucose 15g/L, whey powder 20g/L, diammonium hydrogen citrate 30g/L;
The fermention medium for preparing, back 5000 rev/mins of homogeneous 15 minutes stir, with 105 ℃ of sterilising treatment of fermention medium 5 minutes, be cooled to 42 ℃, with the agent of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented, lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 frozen fermented agent, Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented agent and lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) CGMCC1.9 frozen fermented agent is mixed and made into working stock culture according to mass ratio 10:3:1:1, with the addition adding fermention medium of this working stock culture according to 50g/L, 45 ℃ of ferment at constant temperature 4 hours, acidity after the control fermentation is 90 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, carry out the refining filtration of filter press, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is pure, strong, mellow and full, and it is effective to compose perfume, and has the pure mouthfeel of fermented flavour.
Embodiment six
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: skim-milk 200g/L, glucose 10g/L, whey powder 90g/L, diammonium hydrogen citrate 5g/L;
The fermention medium for preparing, back 5000 rev/mins of homogeneous 15 minutes stir, with 105 ℃ of sterilising treatment of fermention medium 10 minutes, be cooled to 42 ℃, with the agent of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented, Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented agent and lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) CGMCC1.9 frozen fermented agent is mixed and made into working stock culture according to mass ratio 5:3:2, with the addition adding fermention medium of this working stock culture according to 80g/L, 43 ℃ of ferment at constant temperature 10 hours, acidity after the control fermentation is 100 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, 8000 rev/mins of high speed centrifugations 10 minutes, refinement treatment, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is soft naturally, strong lasting, and has the pure mouthfeel of fermented flavour.
Embodiment seven
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: whole milk powder 100g/L, glucose 20g/L, whey powder 10g/L, diammonium hydrogen citrate 5g/L;
The fermention medium for preparing, back 6000 rev/mins of homogeneous 10 minutes stir, with 100 ℃ of sterilising treatment of fermention medium 15 minutes, be cooled to 45 ℃, thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented agent and the agent of Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented are mixed and made into working stock culture according to mass ratio 2:1, with the addition adding fermention medium of this working stock culture according to 10g/L, 37 ℃ of ferment at constant temperature 16 hours, acidity after the control fermentation is 80 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, the filter press refinement treatment, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is pure, strong lasting, and has the pure mouthfeel of fermented flavour.
Embodiment eight
Identical with embodiment one, different is in the present embodiment, the preparation fermention medium, and concrete prescription is: skim-milk 500g/L, glucose 50g/L, whey powder 90g/L, diammonium hydrogen citrate 5g/L;
The fermention medium for preparing, back 5000 rev/mins of homogeneous 10 minutes stir, with 90 ℃ of sterilising treatment of fermention medium 25 minutes, be cooled to 42 ℃, with the agent of thermophilus streptococcus (Streptococcus thermophilus) CGMCC1.2471 frozen fermented, lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 frozen fermented agent, Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC1.1854 frozen fermented agent and lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) CGMCC1.9 frozen fermented agent is mixed and made into working stock culture according to mass ratio 14:3:1:3, with the addition adding fermention medium of this working stock culture according to 50g/L, 43 ℃ of ferment at constant temperature 10 hours, acidity after the control fermentation is 100 ° of T, tunning low-speed centrifugal 5 minutes under 4000 rev/mins condition, carry out preliminary solid-liquid separation, get supernatant liquid, the filter press refinement treatment, obtaining transparent clarifying supernatant liquid is natural yoghourt essence bottom flavoring.This product acid milk fragrance is soft naturally, strong lasting, and has the pure mouthfeel of fermented flavour.
Claims (9)
1. the microbial fermentation production method of a natural yoghourt essence bottom flavoring, its microbial fermentation production method sequence of steps mainly comprises the agent of preparation milk-acid bacteria frozen fermented, preparation sour milk fermention medium and preparation natural yoghourt essence bottom flavoring, and its concrete steps order is as follows: the agent of preparation milk-acid bacteria frozen fermented:
(1) select for use following arbitrary lactic acid bacteria culturers to cultivate the agent of preparation milk-acid bacteria frozen fermented as follows respectively: thermophilus streptococcus, (Streptococcus thermophilus) CGMCC1.2471 or lactobacillus delbruockii subspecies bulgaricus, (Lactobacillus delbrueckii subsp.bulgaricus) CGMCC1.2161 or Lactobacterium acidophilum, (Lactobacillus acidophilus) CGMCC1.1854 or lactococcus lactis subsp, (Lactococcus lactis subsp.cremoris) CGMCC1.9;
(2) the common cultivation of milk-acid bacteria: above-mentioned milk-acid bacteria is inoculated in respectively on the MRS solid medium that contains 15～20g/L agar, cultivated 16～48 hours under 25 ℃～45 ℃ conditions; Above-mentioned MRS solid medium, concrete prescription is: peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, agar powder 15～20g/L; Regulating pH is 6.2～6.4, adds the tween 80 of 1ml/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(3) bacterium that increases of milk-acid bacteria is cultivated: the lactic bacterium strains that step (2) is cultivated is encircling in the liquid enrichment medium of 30～100ml with inoculation articulating 1～2 under the aseptic condition respectively, under 30 ℃～45 ℃ conditions, shaking culture is 4～16 hours on shaking table, make various streptococcus acidi lactici fermented solutions, wherein viable count reaches 10
9～10
10CFU/ml;
Above-mentioned liquid enrichment medium, concrete prescription is: skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO35g/L; Sterilization is 20 minutes under 115 ℃ of conditions;
(4) collect thalline: respectively at 0 ℃～20 ℃, rotating speed is 5000～10000 rev/mins with above-mentioned various streptococcus acidi lactici fermented solutions, and the time is to carry out centrifugal treating under the condition, the collecting precipitation thalline in 5～10 minutes;
(5) add the thalline lyophilized vaccine: under aseptic condition, will above-mentioned various milk-acid bacterias precipitate thalline respectively with thalline lyophilized vaccine uniform mixing;
Above-mentioned thalline lyophilized vaccine, concrete prescription is: skim-milk 100g/L, glycerine 10g/L, trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L;
(6) precooling treatment before the thalline freeze-drying: the various milk-acid bacteria precipitation thalline that will be mixed with lyophilized vaccine carry out 6～16 hours precooling treatment under-15 ℃～-18 ℃ conditions;
(7) vacuum lyophilization prepares the agent of milk-acid bacteria frozen fermented: the various milk-acid bacteria thalline of precooling treatment are carried out vacuum lyophilization respectively, make the agent of milk-acid bacteria frozen fermented, and store for future use under-18 ℃～-20 ℃ conditions;
(8) preparation sour milk fermention medium: comprise fresh milk or whole milk powder or skim-milk, glucose, whey powder, diammonium hydrogen citrate and water, concrete prescription is: fresh milk or whole milk powder or skim-milk 100～900g/L, glucose 5～50g/L, whey powder 10～90g/L, diammonium hydrogen citrate 5～50g/L;
(9) homogeneous: after the Yoghourt fermentation substratum for preparing stirred, carry out homogenization treatment under the room temperature, rotating speed is 3000～6000 rev/mins, and the time is 5～20 minutes;
(10) sterilization: with the fermention medium heat sterilization behind the homogeneous, temperature is 85 ℃～105 ℃, and sterilization time is 5～30 minutes;
(11) cooling: the fermention medium after will sterilizing is cooled to 30 ℃～45 ℃;
(12) inoculation: will cultivate two kinds of making or two or more milk-acid bacteria frozen fermented agent is mixed and made into working stock culture by above-mentioned steps (1)-(7), and be that 0.05～80g/L is inoculated in the Yoghourt fermentation substratum by above-mentioned steps (8)-(11) preparation by total addition level with it;
Carry out the preparation of natural yoghourt essence bottom flavoring then:
(13) fermentation: the substratum of step (12) inoculation various lactobacillus frozen fermented agent is carried out fermentative processing, and leavening temperature is 37 ℃～45 ℃, and fermentation time is 4～24 hours; Acidity after the control fermentation is 70～130 ° of T;
(14) preliminary solid-liquid separation: the Yoghourt fermentation thing that step (13) is obtained under 3000～4000 rev/mins condition centrifugal 5～8 minutes, carry out preliminary solid-liquid separation;
(15) the refining filtration: the Yoghourt fermentation thing supernatant liquor that step (14) obtains is made with extra care filtration, obtain transparent clarifying natural yoghourt essence bottom flavoring;
(16) sample detection:, carry out the qualitative detection of milk aroma component with GC/MS with the transparent clarifying liquid sample that step (15) obtains.
2. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1, it is characterized in that the liquid enrichment medium described in the step (3), concrete prescription is: skim-milk 80g/L, whey powder 30g/L, tomato juice 10g/L, yeast extract 5g/L, lactose 10g/L, CaCO
35g/L; Sterilization is 20 minutes under 115 ℃ of conditions.
3. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1 is characterized in that the thalline lyophilized vaccine described in the step (5), and concrete prescription is: skim-milk 100g/L; glycerine 10g/L; trehalose 20g/L, L-glutamic acid 30g/L, tween 80 5ml/L.
4. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1 is characterized in that the working stock culture that the various lactobacillus frozen fermented agent described in the step (12) is mixed and made into, and total addition level is 0.05～80g/L.
5. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1 is characterized in that two kinds or the two or more combinations of being combined as in the various lactobacillus frozen fermented agent described in the step (12).
6. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1, it is characterized in that the working stock culture that the various lactobacillus frozen fermented agent described in the step (12) is mixed and made into, wherein the mass percent ratio of coccus and bacillus is 2:1 or 1:1.
7. the microbial fermentation production method of a kind of natural yoghourt essence bottom flavoring according to claim 1, it is characterized in that the refining filter method described in the step (15) comprises: supercentrifugal process, rotating speed is 8000～10000 rev/mins, and centrifugation time is 5～10 minutes; The filter press method; The polypropylene screen filtration method.
8. the method with claim 1 prepares a kind of natural yoghourt essence bottom flavoring, it is characterized in that natural yoghourt essence bottom flavoring is transparent clarified liq, mainly comprises following composition: butanols, acetoin, butyric acid, furfuryl alcohol, caproic acid, furanone, Tetramethylpyrazine, n-nonanoic acid, capric acid, vanillin food grade,1000.000000ine mesh, styracin, 2-butylene two dibutyl phthalates, laurostearic acid, δ-Shi Erneizhi, TETRADECONIC ACID, palmitic acid.
9. a kind of natural yoghourt essence bottom flavoring according to claim 8 is characterized in that the relative proportion of various compositions in the described natural yoghourt essence bottom flavoring is: butanols 0.12～0.34, acetoin 0.09～0.44, butyric acid 0.06～1.57, furfuryl alcohol 0.08～0.97, caproic acid 0.02～5.00, furanone 0.02～0.96, Tetramethylpyrazine 0.08～4.47, n-nonanoic acid 0.38～5.48, capric acid 0.97～6.06, vanillin food grade,1000.000000ine mesh 0.16～0.98, styracin 0.03～0.36,2-butylene two dibutyl phthalates 0.84～5.15, laurostearic acid 0.05～1.33, δ-Shi Erneizhi 0.23～2.55, TETRADECONIC ACID 0.06～0.69, palmitic acid 0.32～5.35.
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