CN104130956A - Lactobacillus amylolyticus L6 and application thereof in fermentation of yellow serofluid - Google Patents
Lactobacillus amylolyticus L6 and application thereof in fermentation of yellow serofluid Download PDFInfo
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- CN104130956A CN104130956A CN201410277246.3A CN201410277246A CN104130956A CN 104130956 A CN104130956 A CN 104130956A CN 201410277246 A CN201410277246 A CN 201410277246A CN 104130956 A CN104130956 A CN 104130956A
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Abstract
The invention discloses lactobacillus amylolyticus L6 and an application thereof in fermentation of a yellow serofluid. The lactobacillus amylolyticus L6 has the preservation number of CGMCC NO.9090, and the 16S rDNA sequence is shown in SEQ.ID.NO.1. When the lactobacillus amylolyticus L6 is applied in fermentation of the bean curd yellow serofluid, firstly, the yellow serofluid is sterilized at the temperature of 115-121 DEG C; based on the yellow serofluid volume, 3%-6% of a lactobacillus amylolyticus L6 seed liquid is introduced; heat preservation acidification is carried out for 24-48 h at the temperature of 35-47 DEG C, the acidity value of the yellow serofluid can reach 60 DEG T-110 DEG T, and thus a novel bean curd curdling agent is obtained. The problem of clutter growth of microorganisms in a natural fermentation pulp slurry is solved, and the quality safety of sour pulp bean curd is improved. The lactobacillus amylolyticus L6 directly utilizes oligosaccharides, starch and the like in the yellow serofluid for growth production of acids, can reach the acidity required for the curdling agent without supplement of other carbon sources and nutrients, and reduces the production cost.
Description
Technical field
The present invention relates to milk-acid bacteria and bean product manufacture field, be specifically related to strain solution starch milk-acid bacteria (Lactobacillus amylolyticus) L6 and in the application of preparing in bean curd coagulant.
Background technology
Yellow seriflux is the yellow draining producing in bean curd making processes, contains a large amount of water miscible carbohydrates as the oligose such as stachyose, raffinose, and the nutritive substance such as isoflavones, protein and VITAMIN, is applicable to very much microbial growth.Directly discharge not only can cause the waste of resource, and meeting serious environment pollution and soil, therefore realizes the concern that the comprehensive utilization of yellow seriflux has been caused to domestic and international scientific worker.
In China's traditional bean curd production process, gypsum and bittern are as peptizer life-time service.Use bittern to select the bean curd local flavor of starching splendid, but retentiveness is poor, is difficult for storage period long; Use gypsum good as the bean curd retentiveness of selecting slurry agent, but finished product is slightly with bitter taste, lacks soybean fragrance.Recent study is found, the yellow seriflux producing in the bean product course of processing, the wintercherry water generating through spontaneous fermentation in the process of storage, it is a kind of good bean curd coagulant, use in part bean product manufacturer, the micro-Huang of bean curd color and luster, the mouthfeel made with wintercherry water are fresh and tender, without the bitter taste of chemical point slurry agent, belong to pure natural food.But the wintercherry water using is at present spontaneous fermentation, involved microorganism is except producing sour milk-acid bacteria, also contain other bacterium and yeast etc. of part, wherein also likely pollute the miscellaneous bacteria such as bacillus cereus, staphylococcus, the edible safety of bean curd and goods thereof is formed to harm.
In the time using commercial lactic acid bacterium, as Lactobacterium acidophilum, yellow seriflux is carried out to the agent of pure-blood ferment preparation point slurry, although can obtain safe wintercherry water, but the element that needs to supplement the nutrients just can reach suitable acidity as sucrose, increased production cost, the milk-acid bacteria that therefore finds suitable fermenting yellow serofluid has important practical usage.
Summary of the invention
The object of the invention is to for problems of the prior art, provide a strains of lactic acid bacteria to separate starch milk bacillus (Lactobacillus amylolyticus) L6.
Another object of the present invention is to provide the one application of above-mentioned milk-acid bacteria Lactobacillus amylolyticus L6 in fermentation bean curd yellow pulp water using.
The present invention is achieved by the following technical programs:
One strain solution starch milk bacillus (Lactobacillus amylolyticus) L6, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and postcode is 100101; Preserving number is CGMCC NO.9090, and preservation date is on April 25th, 2014.
In the present invention, separate starch milk bacillus (Lactobacillus amylolyticus) L6 and screen acquisition by dilution-plate method.The wintercherry water that uses the spontaneous fermentation of aseptic sampling bottle spot sampling to form, low temperature is taken back; Use immediately sterilized water to be diluted to respectively 10
-3, 10
-4, 10
-5doubly, coat on the MRS solid medium of improvement; Then put into the constant incubator of 37 DEG C, cultivate 48h; The doubtful bacterium colony of picking, carries out plate streaking separation, so repeats 4～5 times, until obtain pure single bacterium colony.By single bacterium colony percutaneous puncture-inoculation of purifying in MRS solid medium, in 4 DEG C of Refrigerator stores.Wherein MRS liquid culture medium (g/L) formula is: extractum carnis 10g, and peptone 10g, yeast soaks powder 5g, glucose 20g, tween-80 1mL, K
2hPO
43H
2o2g, NaAc3H
2o5g, Triammonium citrate 2g, MgSO
47H
2o0.58g, MnSO
4h
2o0.25g, adds distilled water to 1L, adjusts pH to 6.4 ± 0.2 (adding 2% or 0.7% agar on the basis of MRS solid medium liquid medium within), 121 DEG C of sterilizing 15min.
In the present invention, the characteristic of biological activity of Bacterium lacticum L6 is as follows: bacterium colony projection on MRS solid medium flat board, and rounded, diameter is generally 1-3mm, and canescence is opaque, moisteningly smoothly (sees Fig. 1 a); Thalline is Gram-positive sporeless bacterium, is thin rod-short, and paired or heap shape is arranged and (seen Fig. 1 b); Glucose homofermentation, growth wide temperature range (30-47 DEG C can normal growth, see Fig. 2), fast growth; Conventional Physiology and biochemistry experimental result (in table 1) shows that bacterial strain L6 is Gram-positive, catalase feminine gender, can utilizes starch, homo-fermentative sporeless bacterium (table 1).Surveyed 16S rDNA sequence (as SEQ.ID.NO1) is compared by BLAST, identified that it is to separate starch milk bacillus (Lactobacillus amylolyticus) (seeing Fig. 3).
The invention provides and separate the application of starch milk bacillus (Lactobacillus amylolyticus) L6 in fermentation bean curd yellow pulp water using.
This application method comprises the steps:
(1) by yellow seriflux 115-121 DEG C of sterilizing;
(2) press yellow seriflux volumeter, the seed liquor of the solution starch milk bacillus L6 of access 3%-6%;
(3) at 35-47 DEG C of insulation acidifying 24-48h, the acidity value of yellow seriflux is reached to 60 ° more than T.
Preferably, the time of described sterilizing is 15-30 minute.
With respect to prior art, the advantage that the present invention has and beneficial effect:
(1) Lactobacillus amylolyticus L6 seed liquor is linked in the yellow seriflux of sterilizing, carry out pure-blood ferment, obtain a kind of novel bean curd coagulation agent, solved the mixed and disorderly problem of microorganism growth in natural fermented sourdough pulp-water, improved the quality and safety of wintercherry bean curd.
(2) this bacterial strain can directly utilize the growths such as oligomeric sugar and starch in yellow seriflux to produce acid, can reach and starch the required acidity of agent without supplementary other carbon source and nutrient substance, has reduced production cost.
(3) this bacterial strain acid production speed is fast, and culture condition is simple, is easy to suitability for industrialized production, has good development prospect.
Brief description of the drawings
Fig. 1 a is the colonial morphology figure of Lactobacillus amylolyticus L6 bacterial strain of the present invention;
Fig. 1 b is the thalli morphology figure of Lactobacillus amylolyticus L6 bacterial strain of the present invention;
Fig. 2 is the adaptability curve synoptic diagram of Lactobacillus amylolyticus L6 bacterial strain of the present invention to temperature;
The systematic evolution tree that Fig. 3 does according to 16S rDNA sequence for Lactobacillus amylolyticus L6 bacterial strain of the present invention.
Embodiment
For understanding better the present invention, below in conjunction with drawings and Examples, the present invention is further illustrated, but embodiment does not form the restriction to the claimed scope of the present invention.
In embodiment, it should be noted that:
(1) " lactic-acid-bacterium classification qualification and test method " (the 1999 year version) of Physiology and biochemistry qualification substratum with reference to Ling Daiwen chief editor observed and commonly used to gramstaining, shape.
(2) to the adaptive detection of temperature in accordance with the following methods:
Get 100mL triangular flask, add 50mL MRS liquid culture medium, 121 DEG C of sterilizing 15min, cooling rear inoculation Lactobacillus amylolyticus L6 bacterial strain, 37 DEG C of overnight incubation, for subsequent use as seed culture fluid.In the MRS liquid culture medium triangular flask of sterilizing, add 1mL inoculum (fermentation inoculum size is 2% (V/V)), respectively at 22 DEG C, 27 DEG C, 32 DEG C, 37 DEG C, 42 DEG C, 47 DEG C, constant temperature culture under 52 DEG C of equitemperatures, arranges 3 groups of parallel fermentations, after 18h, survey its OD value at 600nm place with ultraviolet-visible spectrophotometer.
(3) MRS liquid culture medium (for the cultivation of milk-acid bacteria):
Extractum carnis 10g, peptone 10g, yeast soaks powder 5g, glucose 20g, tween-80 1mL, K
2hPO
43H
2o 2g, NaAc3H
2o 5g, Triammonium citrate 2g, MgSO
47H
2o 0.58g, MnSO
4h
2o 0.25g; Distilled water 1L, pH6.4 ± 0.2 (on the basis of MRS solid medium liquid medium within, adding the agar of 0.7-2%), 121 DEG C of sterilizing 15min.
(4) contrast is Lactobacillus acidophilus20272 with Lactobacterium acidophilum, is purchased from Chinese industrial microbial strains preservation administrative center.
(5) acid test is measured with reference to the method for GB541334-2010.Concrete operations are: sample is stirred, accurately take 10g sample, add 20mL without CO
2distilled water, mix, add 0.5mL phenolphthalein indicator, is titrated to solution with 0.1N NaOH standardized solution and is blush, and nondiscoloration is terminal in 30s, the calculating of acidity is according to formula below:
X＝(C×V×100)/(m×0.1)
The acidity of X---sample, ° T of unit;
The volumetric molar concentration of C---standard solution of sodium hydroxide, the mol/L of unit;
V---consume the volume of standard solution of sodium hydroxide, Unit/mL;
The quality of m---sample, the g of unit.
Embodiment 1 bacterial strain screening
(1) use aseptic sampling bottle, the wintercherry water that spot sampling spontaneous fermentation forms, low temperature is taken back.Use immediately sterilized water to be diluted to respectively 10
-3, 10
-4, 10
-5doubly, coat on the MRS solid medium of improvement.Then put into the constant incubator of 37 DEG C, cultivate 48h.The doubtful bacterium colony of picking, carries out plate streaking separation, so repeats 4～5 times, until obtain pure single bacterium colony.Single bacterium colony of purifying is inserted to the test tube center of containing MRS solid medium with inoculating needle, in 4 DEG C of refrigerators, preserve.
(2) from the wintercherry water of spontaneous fermentation, isolate bacterial strain L6, its bacterium colony on MRS substratum is rounded, diameter is generally 1-3mm, canescence, opaque shape, and it is moistening that smooth (Fig. 1 is a); Carry out gramstaining and cell shape and observe, the thalline of bacterial strain L6 is rod-short, and paired or heap is raw, and (Fig. 1 b).Conventional Physiology and biochemistry experimental result (in table 1, the physio-biochemical characteristics table of Lactobacillus amylolyticus L6 bacterial strain) shows that bacterial strain L6 is Gram-positive, catalase feminine gender, homo-fermentative sporeless bacterium.Glucose homofermentation, growth wide temperature range (30-47 DEG C can normal growth, see Fig. 2), fast growth;
Table 1
Embodiment 2 identification of strains
By obtained strains L6 activation culture, send professional feeler mechanism's order-checking, obtain 16S rDNA sequence (seeing SEQ.ID.NO1), result is compared on the gene pool of NCBI, find out the reference culture KC456629.1 that bacterium relationship is close therewith (Lactobacillus amylolyticus strain TUST015), FR683095.1 (Lactobacillus amylolyticus) and KC456630.1 (Lactobacillus amylolyticus strain TUST016), the partial sequence of the 16S rDNA of bacterial strain L6 and standard bacterium are carried out to similarity analysis, L6 exceedes 96% with solution starch milk bacillus Lactobacillus amylolyticus strain TUST015 sequence homology, should be same.Use Clustal X and PHYLIP Software on Drawing system fertility tree (seeing Fig. 3), then in conjunction with bacterium colony, thalli morphology, physiological and biochemical property is accredited as bacterial strain L6 to separate starch milk bacillus (Lactobacillus amylolyticus).
This solution starch milk bacillus (Lactobacillus amylolyticus) L6, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preserving number is CGMCC NO.9090, and preservation date is on April 25th, 2014.
Embodiment 3Lactobacillus amylolyticus L6 bacterial strain is in the effect of 35 DEG C of fermenting yellow serofluids
(1) get 100mL triangular flask, add 50mL MRS liquid culture medium, 121 DEG C of sterilizing 15min, inoculation Lactobacillus amylolyticus L6 bacterial strain, 37 DEG C of overnight incubation, for subsequent use as seed culture fluid.
(2) bean curd yellow pulp water using is sub-packed in 1.8 × 20cm test tube, makes liquid height reach 18cm, with rubber jam-pack plug, as substratum.121 DEG C of sterilizing 15min access Lactobacillus amylolyticus L6 seed culture fluid, constant temperature culture 24h and 48h at 35 DEG C respectively by the addition of 6% (V/V) in gnotobasis.
(3) after testing, when fermentation 24h, the acidity value of wintercherry water is 52 ° of T, and when fermentation 48h, acidity value is the wintercherry of 61～65 ° of T.
Utilizing lactobacillus-fermented yellow plasm of soybean to prepare bean curd coagulant is mainly to utilize carbohydrate production organic acid wherein to realize through milk-acid bacteria, but because the carbohydrate in yellow plasm of soybean mostly is oligomeric sugar and starch, and be not suitable for all milk-acid bacterias, only can utilize the milk-acid bacteria of oligomeric sugar and starch could in bean curd yellow pulp water using, grow product acid well; Therefore can utilize the variation of acidity value to embody the advantage of bacterial strain of the present invention.
Embodiment 4Lactobacillus amylolyticus L6 bacterial strain is in the effect of 42 DEG C of fermenting yellow serofluids
(1) get 100mL triangular flask, add 50mL MRS liquid culture medium, 121 DEG C of sterilizing 15min, inoculation Lactobacillus amylolyticus L6 bacterial strain, 37 DEG C of overnight incubation, for subsequent use as seed culture fluid.
(2) bean curd yellow pulp water using is sub-packed in 1.8 × 20cm test tube, makes liquid height reach 18cm, with rubber jam-pack plug, as substratum.121 DEG C of sterilizing 15min access Lactobacillus amylolyticus L6 bacterial strain seed culture fluid, constant temperature culture 24h, 48h at 42 DEG C by the addition of 5% (V/V) in gnotobasis.
(3) after testing, when fermentation 24h, acidity value is 96 ° of T, and when fermentation 48h, acidity value reaches 110 ° of T.
Embodiment 5Lactobacillus amylolyticus L6 bacterial strain is in the effect of 47 DEG C of fermenting yellow serofluids
(1) get 100mL triangular flask, add 50mL MRS liquid culture medium, 121 DEG C of sterilizing 15min, inoculation Lactobacillus amylolyticus L6 bacterial strain, 37 DEG C of overnight incubation, for subsequent use as seed culture fluid.
(2) bean curd yellow pulp water using is sub-packed in 1.8 × 20cm test tube, makes liquid height reach 18cm, with rubber jam-pack plug, as substratum.121 DEG C of sterilizing 15min access Lactobacillus amylolyticus L6 bacterial strain seed culture fluid, constant temperature culture 24h, 48h at 47 DEG C by the addition of 5% (V/V) in gnotobasis.
(3) after testing, when fermentation 24h, acidity value is 78 ° of T, and when fermentation 48h, acidity value reaches 84 ° of T.
The effect of embodiment 6 (comparative example) Lactobacillus acidophilus20272 fermenting yellow serofluid
Prior art 1 (Du Xin, Li Li. the anti-oxidant research of lactobacillus-fermented yellow seriflux. China brewages, 2013 (007): 24-27) and prior art 2 (Du Xin, Li Li, Liu Dongmei. the impact [J] of carbon source and bacteriostatic activity anti-oxidant on probiotics fermention yellow seriflux. modern food science and technology .2014,30 (2): 129-133) all use Lactobacillus acidophilus20272 strain fermentation yellow plasm of soybean, and use Lactobacillus acidophilus20272 strain fermentation yellow plasm of soybean to prepare bean curd coagulant to have obtained good effect.Therefore with this bacterial strain as a comparison, concrete grammar comprises the steps.
(1) get 100mL triangular flask, add 50mL MRS liquid culture medium, 121 DEG C of sterilizing 15min, inoculation Lactobacillus acidophilus20272 bacterial strain, 37 DEG C of overnight incubation, for subsequent use as seed culture fluid.
(2) bean curd yellow pulp water using is sub-packed in 1.8 × 20cm test tube, makes liquid height reach 18cm, with rubber jam-pack plug, as substratum.121 DEG C of sterilizing 15min access Lactobacillus acidophilus20272 seed culture fluid, constant temperature culture 24h, 48h under the condition of 35 DEG C, 42 DEG C, 47 DEG C respectively by the addition of 5% (V/V) in gnotobasis.
(3) after testing, when 35 DEG C of fermentation 24h, acidity value is 48 ° of T, and when fermentation 48h, acidity value is 53 ° of T; 42 DEG C of fermentation 24h, acidity value is 57 ° of T, when fermentation 48h, acidity value is 58 ° of T.47 DEG C of fermentation 24h, acidity value is 38 ° of T, when fermentation 48h, acidity value is 43 ° of T.
Comparative example's demonstration, under differing temps, the sour ability of preparing bean curd coagulant of Lactobacillus amylolyticus L6 strain fermentation yellow plasm of soybean product is all obviously better than Lactobacillus acidophilus20272.
Claims (5)
1. separate a starch milk bacillus L6, it is characterized in that, the preserving number of this solution starch milk bacillus (Lactobacillus amylolyticus) L6 is CGMCC NO.9090.
2. solution starch milk bacillus L6 according to claim 1, is characterized in that, the 16S rDNA sequence of separating starch milk bacillus (Lactobacillus amylolyticus) L6 is SEQ.ID.NO1.
3. described in claim 1, separate the one application of starch milk bacillus L6 in fermentation bean curd yellow pulp water using.
4. the application of solution starch milk bacillus L6 according to claim 3 in fermentation bean curd yellow pulp water using, is characterized in that, this application method comprises the steps:
(1) by yellow seriflux 115-121 DEG C of sterilizing;
(2) press yellow seriflux volumeter, the seed liquor of the solution starch milk bacillus L6 of access 3%-6%;
(3) at 35-47 DEG C of insulation acidifying 24-48h, the acidity value of yellow seriflux can reach 60 ° of T-110 ° of T.
5. the application of solution starch milk bacillus L6 according to claim 3 in fermentation bean curd yellow pulp water using, is characterized in that, the time of described sterilizing is 15-30 minute.
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| CN112136904A (en) * | 2020-09-29 | 2020-12-29 | 广东广中皇食品有限公司 | Acid water and preparation method thereof |
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| CN102258085A (en) * | 2010-05-28 | 2011-11-30 | 河南农业大学 | Method for preparing bean curd coagulant with yellow serofluid fermented with lactic acid bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102258085A (en) * | 2010-05-28 | 2011-11-30 | 河南农业大学 | Method for preparing bean curd coagulant with yellow serofluid fermented with lactic acid bacteria |
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| CN112136904A (en) * | 2020-09-29 | 2020-12-29 | 广东广中皇食品有限公司 | Acid water and preparation method thereof |
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