CN104996931A - A method of producing flavor fermented soya beans - Google Patents
A method of producing flavor fermented soya beans Download PDFInfo
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- CN104996931A CN104996931A CN201510411823.8A CN201510411823A CN104996931A CN 104996931 A CN104996931 A CN 104996931A CN 201510411823 A CN201510411823 A CN 201510411823A CN 104996931 A CN104996931 A CN 104996931A
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- fermented soya
- soya bean
- subtilis
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- soya beans
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- 244000068988 Glycine max Species 0.000 title claims abstract description 118
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 118
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- 238000000034 method Methods 0.000 title description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 49
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- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 13
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 11
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 8
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 8
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- 239000000126 substance Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Abstract
The present invention discloses a method for producing flavor fermented soya beans by respectively mixing Bacillus subtilis (B. subtilis) and Lactobacillus plantarum with soya beans to ferment the soya beans by steps and followed by combining low-temperature fermentation to produce good flavor fermented soya beans; compared with B. subtilis pure fermentation produced natto, the produced fermented soya beans have a better sensory quality, a significantly improved flavor, and significantly increased concentrations of two important flavor compounds, 2,3-butanedione and 1,2-butanediol; and the fermented soya beans have rich nutrient substances, and also maximize the retain of the number of live bacteria of the B. subtilis and the Lactobacillus plantarum at the same time.
Description
Technical field
The present invention relates to a kind of preparation method of excellent flavor fermented soya bean, belong to using microbe technical field.
Background technology
Fermented soya bean be a kind of with black soya bean, soya bean for primary raw material, the one tradition seasoned bean products utilizing fermentable to make.Traditional zymotic fermented soya bean have very high nutritive value and health-care efficacy, except containing except the nutritional labelings such as rich in protein, vitamin, amino acid, also containing physiologically active ingredients such as protease, amylase, fibrinolysin, active peptide, isoflavones, there is coordinating intestines and stomach, drive wind, dissipating cold, prevention of osteoporosis, antitumor, thrombolysis and improve physiological hygiene function (the Shan Jin Xiao etc. such as immunity of organisms, China brewages. 2007,3:1-4).
The production of fermented soya bean is divided into koji and two stages of after fermentation, different by the major microorganisms kind participated in during Koji fermentation, can be divided into the large class of bacteria type, aspergillus type, head mold type, Mucor type and arteries and veins born of the same parents bacterial type 5 (Jia Dongxu etc., Food Science. 2009,30:217-219).Bacterial fermented douchi in China with cloud, the natto that expensive, Shandong one is with making among the people for representative, if its microbial host bacillus subtilis (
bacillus subtilis) and a small amount of lactic acid bacteria (Lactic acid bacteria, LAB) and micrococcus luteus.
b. subtilisas a kind of multifunctional safety, nontoxic, harmless bacterial classification, food and medical and health industry are widely used in.
b. subtilismultiple enzyme can be produced in growth and breeding process, as protease, amylase, dextranase, mannonase phytase etc., the macromolecular substances such as the protein in decomposing soybean and starch, fermented soya bean are produced can the emplastic of wire drawing, has antibacterial, thrombus dissolving and the function such as anticancer.In addition,
b. subtilisthe materials such as the polypeptide, amino acid and the monose that produce in metabolism soybean process provide required nutritional factors for LAB, promote the breeding of LAB raised growth.
Bacterial fermented douchi the most well-known be in the world Japan natto, it take soybean as raw material, through purebred bacillus subtilis (
b. subtilis) fermentation forms, its distinguishing feature on local flavor is that local hobby property is strong, has ammonia taste in various degree, be not suitable for most of Chinese residents taste (Sun Sen, Shandong Light Ind College master thesis. 2008).And China's traditional zymotic fermented soya bean have excellent in color characteristic, main cause is the synergy of multiple-microorganism in fermented soya bean sweat, and wherein LAB is that excellent flavor has made outstanding contributions.LAB is Gram-positive, without spore, can not move, form is shaft-like or spherical, can be referred to as by the fermentable carbohydrates bacterioid that produces acid, mainly comprise lactobacillus (
lactobacillus), lactococcus (
lactococcus), enterococcus spp (
enterococcus), Bifidobacterium etc. (
bifidobacterium).LAB can not only produce the pH value that organic acid reduces product by the carbohydrate in metabolism raw material, also can produce the antibacterial substance such as hydrogen peroxide, bacteriocin, suppress the growth of pathogen and spoilage organisms etc., extend the shelf life of product, improve security.
In addition, the function that LAB gives fermented food one important is: during the fermentation, in the especially after fermentation stage, can produce the multiple flavor substances such as acid, alcohol, aldehyde and ketone, improve by purebred
b. subtilisthe natto peculiar taste that fermentation causes " local flavor is more single, and local hobby property is strong, is not suitable for most of Chinese residents taste ".In order to make fermented soya bean have prebiotic and delicious dual-use function concurrently, will
b. subtilisseemingly one is rationally and effectively select with LAB mixed culture fermentation and after fermentation.Mixed culture fermentation comprises synchronized mixes fermentation and substep mixed culture fermentation, due to the biocidal property feature of LAB, is first used by soybean
b. subtilisafter fermentation a period of time, add the step fermentation pattern that LAB carries out mixed culture fermentation, while making fermented soya bean have abundant nutrition material, the reservation of energy maximum
b. subtilisviable count, on this basis, can make the local flavor of fermented soya bean significantly improve through the low temperature after fermentation stage.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, a kind of preparation method of soya beans with distinctive flavour is provided; The inventive method utilize bacillus subtilis (
bacillus subtilis) and Lactobacillus plantarum (
lactobacillus plantarum) substep mixed culture fermentation, and the fermented soya bean with excellent flavor are made in conjunction with low temperature after fermentation; With the local flavor that the fermented soya bean that the method makes have clear improvement relative to the natto tool of purebred fermentation of bacillus subtilis, and some main aroma compound concentration significantly improve.
Soybean is soaked in water by the inventive method, autoclaving, and after cooling, inoculation bacillus subtilis bacteria suspension mixes in cooled soybean, and fermented soya bean semi-finished product are made in fermentation; Then by Lactobacillus plantarum bacterial suspension inoculation in fermented soya bean semi-finished product, continue fermentation obtain accurate finished product fermented soya bean; Finally accurate finished product fermented soya bean are placed in 4 ~ 8 DEG C of refrigerators or freezer after fermentation 24 ~ 48 h, obtain finished product soya beans with distinctive flavour.
The concrete operations of the inventive method are as follows:
1,
b. subtiliswith Lactobacillus plantarum (
lb. plantarum) preparation of seed liquor
By what preserve
b. subtilistake out from-80 DEG C after thawing, get 75 μ L and be seeded in 5 mL bactericidal nurishing broth bouillons, 200 rpm, 40 DEG C of shaken cultivation 16 h; Get 1.5 mL and cultivated 16 h's
b. subtilisbe seeded in 100 mL nutrient broth mediums, 200 rpm 40 DEG C shaken cultivation 16 h again, obtaining cell concentration is 10
8the seed liquor of cfu/mL; Take out from-80 DEG C of refrigerators
lb. plantarum20 μ L preservative fluids, after thawing, are seeded in 5 mL sterilizing MRS broth bouillons, quiescent culture 12 h in 30 DEG C of constant incubators by preservative fluid; By 4 ‰ (v/v) inoculum concentration, will cultivate 12 h's
lb. plantarumbe seeded in 100 mL sterilizing MRS broth bouillons, 30 DEG C of quiescent culture 15 h again, obtaining cell concentration is 10
10the seed liquor of cfu/mL.
2, the preparation of fermentation soybean
Get 120 grams of soya beans, by 1
:3 ~ 1:5(m/v) ratio, at room temperature use distilled water immersion 12 ~ 16 h, after being drained by water, put into vessel, with gauze by mouth close be placed on 115 ~ 121 DEG C of boiling 40 min in high-pressure sterilizing pot, well-done soybean is cooled to 30 DEG C for subsequent use.
3,
b. subtiliswith
lb. plantarumsubstep mixed culture fermentation
Will
b. subtilisseed liquor is sub-packed in sterile centrifugation tube, under room temperature, after centrifugal 10 min of 7000 rpm, and abandoning supernatant, the thalline obtained 0.7% physiological saline wash-out 2 times, and then with the resuspended thalline of physiological saline, making concentration is 10
6~ 10
8the physiological saline bacteria suspension of cfu/mL, get this bacterial suspension inoculation of 5 mL in well-done soybean, sterilizing meal spoon stirs gently, after bacterium liquid is mixed with soybean, is placed in 37 ~ 40 DEG C of constant incubator fermentation 15 ~ 20 h, makes fermented soya bean semi-finished product.
Will
lb. plantarumin seed liquor packing sterile centrifugation tube, adopt with
b. subtilisthe centrifugal condition that seed liquor is identical and elution program, obtaining concentration is 10
8~ 10
10the physiological saline bacteria suspension of cfu/mL, accesses this bacteria suspension of 5 mL
b. subtilisin the fermented soya bean semi-finished product of fermentation, 30 ~ 37 DEG C of constant incubator relaying supervention ferment 16 ~ 24 h, obtain accurate finished product fermented soya bean.
4, low temperature after fermentation
The accurate finished product fermented soya bean that previous step obtains are placed in 4 ~ 8 DEG C of refrigerators or freezer, after after fermentation 24 ~ 48 h, obtain finished product fermented soya bean.
5, the separation andpreconcentration of sensory evaluation and main flavor
Select the academics and students of 12 (6 male 6 female) Majors of Foods as valuation officer, with reference to table 1 index, sensory evaluation is carried out to finished product fermented soya bean; Using the sensory evaluation score value of the average mark sum of each assessment item in sensory evaluation test paper (table 1) as fermented soya bean, in order to judge the quality of fermented soya bean organoleptic quality.
With the good fermented soya bean of organoleptic quality for experiment material, utilize headspace solid-phase microextraction (Head space-Solid phase microextraction, HS-SPME) technology extracts the volatile materials in these fermented soya bean, and adopt gas chromatography-mass spectrum (Gas chromatography-mass spectrometry, GC-MS) technology to carry out identifying and analyzing.Meanwhile, with
b. subtilisthe natto that pure-blood ferment makes is contrast, and the kind of comparative analysis compound and concentration difference obtain the important compound of contribution fermented soya bean excellent flavor.
6, in finished product fermented soya bean
b. subtiliswith
lb. plantarumviable count measures
Take 1.0 g finished product fermented soya bean, join in 5 mL sterile salines, vibration mixing, 4 DEG C are soaked 6 h.After 0.7% 10 times, physiological saline gradient dilution, get the suitable dilution bacteria suspension of 20 μ L and coat (the selection cultivation of nutrient broth agar flat board respectively
b. subtilis) and (the selection cultivation of MRS agar plate
lb. plantarum), be inverted cultivation 24 h, choose the agar plate of clump count between 30 ~ 300 and carry out colony counting, and then be converted into viable count in every gram of fermented soya bean for 37 DEG C.
Compared with prior art, the present invention has following beneficial effect:
1, fermented soya bean have good organoleptic quality, especially relative to
b. subtilisthe natto that pure-blood ferment makes, local flavor significantly improves; Two kinds important, and aroma compound---the concentration of-biacetyl and 2,3-butanediol significantly improves.
2, while fermented soya bean have abundant nutrition material, the reservation of energy maximum
b. subtiliswith
lb. plantarumviable count.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but they are not limitation of the invention.The reagent used in embodiment and method, if no special instructions, all adopt conventional reagent and use conventional method.
Embodiment 1: the preparation method of this soya beans with distinctive flavour, particular content is as follows:
(1)
b. subtiliswith
lb. plantarumthe preparation of seed liquor
By what preserve
b. subtilistake out from-80 DEG C after thawing, get 75 μ L and be seeded in 5 mL bactericidal nurishing broth bouillons, 200 rpm, 40 DEG C of shaken cultivation 16 h.Get 1.5 mL and cultivated 16 h's
b. subtilisbe seeded in 100 mL nutrient broth mediums, 200 rpm 40 DEG C shaken cultivation 16 h again, obtaining cell concentration is 10
8the seed liquor of cfu/mL; Take out from-80 DEG C of refrigerators
lb. plantarum20 μ L preservative fluids, after thawing, are seeded in 5 mL sterilizing MRS broth bouillons, quiescent culture 12 h in 30 DEG C of constant incubators by preservative fluid.By 4 ‰ (v/v) inoculum concentration, get 400 μ L and cultivated 12 h's
lb. plantarumbe seeded in 100 mL sterilizing MRS broth bouillons, 30 DEG C of quiescent culture 15 h again, obtaining cell concentration is 10
9the seed liquor of cfu/mL.
(2) preparation of fermentation soybean
Get 120 g soya beans, by 1
:ratio 3(m/v), at room temperature uses distilled water immersion 16 h, puts into vessel after being drained by water, with gauze by mouth close be placed on 121 DEG C of boiling 40 min in high-pressure sterilizing pot, well-done soybean is cooled to 30 DEG C for subsequent use.
(3)
b. subtiliswith
lb. plantarumsubstep mixed culture fermentation
Step (1) is obtained
b. subtilisseed liquor is sub-packed in sterile centrifugation tube, under room temperature, after centrifugal 10 min of 7000 rpm, and abandoning supernatant, the thalline obtained 0.7% physiological saline wash-out 2 times, and then with the resuspended thalline of physiological saline, making concentration is 10
6the physiological saline bacteria suspension of cfu/mL, get in the soybean that this bacterial suspension inoculation of 5 mL obtains in step (2), sterilizing meal spoon stirs gently, after bacterium liquid is mixed with soybean, is placed in 40 DEG C of constant incubators and ferments 20 h, make fermented soya bean semi-finished product.
Step (1) is obtained
lb. plantarumin seed liquor packing sterile centrifugation tube, adopt with
b. subtilisthe centrifugal condition that seed liquor is identical and elution program, obtaining concentration is 10
10the physiological saline bacteria suspension of cfu/mL, accesses this bacteria suspension of 5 mL
b. subtilisin the fermented soya bean semi-finished product of fermentation, 30 DEG C of constant incubator relaying supervention ferment 24 h, obtain accurate finished product fermented soya bean.
(4) low temperature after fermentation
Accurate finished product fermented soya bean step (3) obtained are placed in 4 DEG C of refrigerators, after after fermentation 48 h, obtain finished product fermented soya bean.
The separation andpreconcentration of the Salt black bean sensory evaluation of the present embodiment finished product and main flavor is as follows:
The fermented soya bean made with embodiment 1, for experiment material, select the academics and students of 12 (6 male 6 female) Majors of Foods as valuation officer, carry out sensory evaluation with reference to table 1 index.Using the sensory evaluation score value of the average mark sum of each assessment item in sensory evaluation test paper (table 1) as fermented soya bean, in order to judge the quality of fermented soya bean organoleptic quality.
In addition, while making fermented soya bean by embodiment 1, with
b. subtilispure-blood ferment makes natto and carries out parallel test.The general comment of Analyses Methods for Sensory Evaluation Results display natto is subsequently divided into 25.9, much smaller than the fermented soya bean (table 1) that embodiment 1 makes, illustrates that these fermented soya bean have higher organoleptic quality.
Table 1: fermented soya bean Analyses Methods for Sensory Evaluation Results
。
The fermented soya bean made with embodiment 1 are for experiment material, HS-SPME technology is utilized to extract volatile materials in these fermented soya bean, and adopt GC-MS technology to carry out identifying and analyzing, obtain 12 acids, 7 ester classes, 5 ketones, 5 Pyrazine, 3 alcohols, 2 phenols, totally 34 compounds (table 2).In addition, with
b. subtilisthe natto that pure-blood ferment makes finds to have biacetyl and the 2,3-butanediol of higher concentration with the fermented soya bean of embodiment 1 making for contrast, comparative analysis, illustrates that they are important compound of contribution fermented soya bean excellent flavor.
Table 2: the main volatile compound of the excellent flavor fermented soya bean that the present embodiment obtains and concentration thereof
a"/" numeric representation below
b. subtilisthe compound concentration that when pure-blood ferment makes natto, there were significant differences.
The fermented soya bean made with embodiment 1 are for experiment material, and measure viable count in finished product fermented soya bean, result shows
b. subtilisviable count is (6.40 ± 1.41) × 10
9cfu/ gram,
lb. plantarumviable count is (6.63 ± 1.24) × 10
10cfu/ gram.
Embodiment 2: the preparation method of this soya beans with distinctive flavour, particular content is as follows:
(1)
b. subtiliswith
lb. plantarumthe preparation process of seed liquor is with embodiment 1 step (1);
(2) preparation of fermentation soybean
Get 120 g soya beans, by 1
:ratio 4(m/v), at room temperature uses distilled water immersion 14 h, puts into vessel after being drained by water, with gauze by mouth close be placed on 115 DEG C of boiling 40 min in high-pressure sterilizing pot, well-done soybean is cooled to 30 DEG C for subsequent use.
(3)
b. subtiliswith
lb. plantarumsubstep mixed culture fermentation
Step (1) is obtained
b. subtilisseed liquor is sub-packed in sterile centrifugation tube, under room temperature, after centrifugal 10 min of 7000 rpm, and abandoning supernatant, the thalline obtained 0.7% physiological saline wash-out 2 times, and then with the resuspended thalline of physiological saline, making concentration is 10
7the physiological saline bacteria suspension of cfu/mL, get in the soybean that this bacterial suspension inoculation of 5 mL obtains in step (2), sterilizing meal spoon stirs gently, after bacterium liquid is mixed with soybean, is placed in 37 DEG C of constant incubators and ferments 20 h, make fermented soya bean semi-finished product.
Step (1) is obtained
lb. plantarumin seed liquor packing sterile centrifugation tube, adopt with
b. subtilisthe centrifugal condition that seed liquor is identical and elution program, obtaining concentration is 10
8the physiological saline bacteria suspension of cfu/mL, accesses this bacteria suspension of 5 mL
b. subtilisin the fermented soya bean semi-finished product of fermentation, 37 DEG C of constant incubator relaying supervention ferment 16h, obtain accurate finished product fermented soya bean.
(4) low temperature after fermentation
Accurate finished product fermented soya bean step (3) obtained are placed in 5 DEG C of refrigerators, after after fermentation 35 h, obtain finished product fermented soya bean.
Carry out the sensory evaluation of finished product Salt black bean with reference to embodiment 1, result is 28.8, higher than with
b. subtilispure-blood ferment makes 24.7 of natto.
Measure the viable count in the finished product fermented soya bean of embodiment 2 making, in every gram of fermented soya bean,
b. subtiliswith
lb. plantarumviable count is respectively (7.67 ± 0.53) × 10
8cfu and (8.98 ± 0.18) × 10
9cfu.
Embodiment 3: the preparation method of this soya beans with distinctive flavour, particular content is as follows:
(1)
b. subtiliswith
lb. plantarumthe preparation process of seed liquor is with embodiment 1 step (1);
(2) preparation of fermentation soybean
Get 120 g soya beans, by 1
:ratio 5(m/v), at room temperature uses distilled water immersion 12 h, puts into vessel after being drained by water, with gauze by mouth close be placed on 119 DEG C of boiling 40 min in high-pressure sterilizing pot, well-done soybean is cooled to 30 DEG C for subsequent use.
(3)
b. subtiliswith
lb. plantarumsubstep mixed culture fermentation
Step (1) is obtained
b. subtilisseed liquor is sub-packed in sterile centrifugation tube, under room temperature, after centrifugal 10 min of 7000 rpm, and abandoning supernatant, the thalline obtained 0.7% physiological saline wash-out 2 times, and then with the resuspended thalline of physiological saline, making concentration is 10
8the physiological saline bacteria suspension of cfu/mL, get in the soybean that this bacterial suspension inoculation of 5 mL obtains in step (2), sterilizing meal spoon stirs gently, after bacterium liquid is mixed with soybean, is placed in 38 DEG C of constant incubators and ferments 15 h, make fermented soya bean semi-finished product.
Step (1) is obtained
lb. plantarumin seed liquor packing sterile centrifugation tube, adopt with
b. subtilisthe centrifugal condition that seed liquor is identical and elution program, obtaining concentration is 10
9the physiological saline bacteria suspension of cfu/mL, accesses this bacteria suspension of 5 mL
b. subtilisin the fermented soya bean semi-finished product of fermentation, 35 DEG C of constant incubator relaying supervention ferment 20h, obtain accurate finished product fermented soya bean.
(4) low temperature after fermentation
Accurate finished product fermented soya bean step (3) obtained are placed in 8 DEG C of refrigerators, after after fermentation 24 h, obtain finished product fermented soya bean.
Carry out the sensory evaluation of finished product Salt black bean with reference to embodiment 1, result is 29.1, higher than with
b. subtilispure-blood ferment makes 25.5 of natto.
Measure the viable count in the finished product fermented soya bean of embodiment 3 making, in every gram of fermented soya bean,
b. subtiliswith
lb. plantarumviable count is respectively (6.33 ± 1.41) × 10
9cfu and (6.54 ± 1.24) × 10
10cfu.
Claims (4)
1. a preparation method for soya beans with distinctive flavour, is characterized in that: be soaked in water by soybean, autoclaving, and after cooling, inoculation bacillus subtilis bacteria suspension mixes in cooled soybean, and fermented soya bean semi-finished product are made in fermentation; Then by Lactobacillus plantarum bacterial suspension inoculation in fermented soya bean semi-finished product, continue fermentation obtain accurate finished product fermented soya bean; Finally accurate finished product fermented soya bean are placed in 4 ~ 8 DEG C of refrigerators or freezer after fermentation 24 ~ 48 h, obtain finished product soya beans with distinctive flavour.
2. the preparation method of soya beans with distinctive flavour according to claim 1, is characterized in that: the ratio of soybean and water 1:3 ~ 1:5 by volume mixes, and soak time is 12 ~ 16 h, and autoclaving condition is 115 ~ 121 DEG C of boiling 30 ~ 40 min.
3. the preparation method of soya beans with distinctive flavour according to claim 1 and 2, is characterized in that: bacillus subtilis bacteria suspension and Lactobacillus plantarum bacteria suspension are the bacteria suspension obtained with the physiological saline that mass percent concentration is 0.7%, and inoculum density is respectively 10
6~ 10
8cfu/mL and 10
8~ 10
10cfu/mL.
4. the preparation method of soya beans with distinctive flavour according to claim 3, is characterized in that: at 37 ~ 40 DEG C of bottom fermentation 15 ~ 20 h after inoculation bacillus subtilis, at 30 ~ 37 DEG C of bottom fermentation 16 ~ 24 h after inoculation Lactobacillus plantarum.
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| CN110192564A (en) * | 2019-07-05 | 2019-09-03 | 嘉兴杰赛生物科技有限公司 | A kind of generation meal cake of high heat low carbohydrate content and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106418210A (en) * | 2016-06-30 | 2017-02-22 | 山东凤凰生物有限公司 | Flavor lobster sauce having thrombolysis and pressure reduction function, and preparation method thereof |
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| CN108813368A (en) * | 2018-04-28 | 2018-11-16 | 贵州省铜仁市永进食品有限公司 | A kind of flavor black bean sauce and preparation method thereof |
| CN109820127A (en) * | 2019-04-10 | 2019-05-31 | 泸州市龙马潭区天富调味品厂 | A kind of natto steam sterilization case |
| CN110192564A (en) * | 2019-07-05 | 2019-09-03 | 嘉兴杰赛生物科技有限公司 | A kind of generation meal cake of high heat low carbohydrate content and preparation method thereof |
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