CN103898016B - One plant height lactic acid-producing bacteria and fermentation eggshell thereof prepare the method for calcium lactate - Google Patents
One plant height lactic acid-producing bacteria and fermentation eggshell thereof prepare the method for calcium lactate Download PDFInfo
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Abstract
The invention discloses a plant height lactic acid-producing bacteria, be enterococcus mundtii (Enterococcus mundtii) HNMD-25, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC NO.8699.The invention also discloses described high-yield lactic acid bacterium and prepare application in calcium lactate and a kind of method utilizing eggshell to prepare calcium lactate in fermentation calcium source, calcium lactate preparation method provided by the invention, eggshell utilization ratio is high, reaches 77.98%, and every 1kg eggshell can prepare the five water lactic acid calcium of about 2.5kg; Inversion rate of glucose is high, reaches as high as 79.3%, is better than the inversion rate of glucose of the conventional bacterial classification of industry; Calcium lactate purity is high, and tunning is through refining, and calcium lactate purity can reach more than 99%.
Description
Technical field
The invention belongs to microorganism field, the milk-acid bacteria and the fermentation eggshell thereof that are specifically related to a plant height lactic acid producing prepare the method for calcium lactate.
Background technology
Faecalis (Enterococcus) is the gram-positive cocci of the interior extensively distribution of nature and humans and animals body, enterococcus mundtii (Enterococcus mundtii) is a kind of faecalis being reported as bacteriocinogeny more, can kill or suppress the growth of some pathogenic bacterias, wherein Enterococcus mundtii ST4SA has been listed in the probiotic bacterium bacterial classification of domestic preservation, the report of enterococcus mundtii relating to antibiotic-producing and antiviral peptide is very common, report enterococcus mundtii Enterococcus mundtii ST4SA and plant lactobacillus Lactobacillusplantarum423 is had to be that matrix is as probiotic bacterium with powdered milk formula.Faecalis has the ability of lactic acid producing, but there is no the report utilizing faecalis fermentation for lactic acid at present.
In recent years, the comprehensive utilizating research of eggshell is deepening continuously.The calcium of eggshell is changed into the importance that the organic calcium easily absorbed is eggshell comprehensive utilization.Wherein calcium lactate has the advantages such as calcium contents is high, solubleness is comparatively large, specific absorption is high, security is high, reasonable price because of it and receives much concern.In current research, eggshell is prepared calcium lactate and is mainly adopted chemical process, and studying more is calcination method, and calcination method power consumption is large, and cost is high, and calcining produces a large amount of carbonic acid gas and dust, causes pollution to environment.Hydrothermal method and direct neutralisation avoid the environmental pollution that high-temperature calcination eggshell causes, but these two kinds of methods need make a big purchase lactic acid in large quantities, and cost is higher.Microbial method prepare calcium lactate be one can decreasing pollution and increase the effective way that reduces costs of economic benefit, also by the method for fermentable, eggshell is not directly prepared into the bibliographical information of calcium lactate at present.
Summary of the invention
First object of the present invention is to provide a plant height lactic acid-producing bacteria.
High-yield lactic acid bacterium provided by the present invention is enterococcus mundtii (Enterococcus mundtii), called after HNMD-25, deliver the China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation being positioned at Institute of Microorganism, Academia Sinica on January 9th, 2014, deposit number is: CGMCC NO.8699.
High-yield lactic acid bacterium provided by the present invention is separated in discarded eggshell soil sample buried for a long time.
High-yield lactic acid bacterium preparation method provided by the present invention is:
(1) sample.Sample picks up from the soil of the long-term buried eggshell in egg processing place.
(2) primary dcreening operation is separated.MRS substratum is adopted to be substrate, and carry out Anaerobic culturel, MRS substratum to be rule repeatedly separation and purification, observe colonial morphology, choose the milky of needle point size, the moistening and glossiness bacterium colony of tool, observe thalli morphology with sediments microscope inspection further, filter out Gram-positive micrococcus X strain and proceed line separation, obtain pure culture respectively.
(3) multiple sieve.Point sample on MC substratum, selects lactic acid producing and forms the bacterial strain of molten calcium circle.
(4) acquisition of mutagenesis and high-yield lactic acid bacterial strain.By ultraviolet (UV) and nitrosoguanidine (NTG) mutagenesis and with the molten calcium circle in MC substratum for standard, obtain Lactic Acid High-yield Strains: the bacterium liquid collected by centrifugation thalline after 18-24h will be cultivated and add sterilized water several times afterwards with sterilized water washing and make bacteria suspension, NTG is added with 0.3mg/mL, oscillation treatment 40min on shaking table, repeatedly centrifuge washing thalline, and make bacteria suspension, get 0.1mL bacterium liquid and coat MRS solid plate and after Anaerobic culturel 18-24h, flat board is placed in mutagenesis under 20W ultraviolet lamp, irradiation distance is 30cm, 1.5min, thalline point sample on MC flat board is got after continuing Anaerobic culturel 18-24h, choose the bacterium colony that molten calcium circle is greater than contrast, go down to posterity, obtain stable pure culture bacterial strain, by its called after Enterococcus mundtii HNMD-25.
(5) identify.Physiology and biochemistry Property Identification.Sugar fermentating test shows, and bacterial strain can utilize sucrose, wood sugar, lactose, maltose, seminose, raffinose, saligenin, Vitamin C2, sorbitol fermentation to produce acid, can not utilize cellobiose, can partly utilize melizitose and melibiose; Litmus milk test, acetyl methyl carbinol V-P test, methyl red (M.R.) test, citrate test are shown as the positive, catalase test, Starch Hydrolysis is tested, casein hydrolysis experiment, gelatin liquification test, anaerobism nitrate aerogenesis is tested, and hydrogen sulfide produces test, and each biochemical property such as indole test all shows feminine gender; Still can grow at the test of resistance to temperature, soda acid, salt is presented at 43 DEG C, all can grow under less salt and slant acidity environment, but in the higher salt concentrations of 10% and the comparatively sour environment not regrowth of pH4.3.
Bacterial strain 16S rDNA Sequence Identification.CTAB method is adopted to extract thalline STb gene and carry out 16S pcr amplification, glue order-checking is cut after carrying out agarose gel electrophoresis purifying, sequence and GenBank amplifying nucleic acid data (http://www.ncbi.nlm.nih.gov/blast) are carried out blast comparison, 99% is reached with the homology of a strain enterococcus mundtii ATCC43186, in conjunction with Physiology and biochemistry character result and the outstanding Bacteria Identification handbook of uncle, common bacteria system identification handbook and the judgement such as lactic-acid-bacterium isolation identification and experimental technique, the final bacterial strain confirming that separation obtains is enterococcus mundtii (Enterococcus mundtii).
(6) lactic acid producing ability.Adopt the fermentation of shaking table liquid culture, the lactic acid content in fermented liquid can reach 60.375-72.064g/L, and inversion rate of glucose (glucose be fermented accounts for the ratio of glucose total amount) is 66.4-79.3%.
Another object of the present invention, is to provide described high-yield lactic acid bacterium and prepares application in calcium lactate in fermentation calcium source, especially prepares the application in calcium lactate at fermentation eggshell.
Invention further provides a kind of method utilizing eggshell to prepare calcium lactate, the method is to the milk-acid bacteria according to claim 1 of the inoculation of medium containing glucose and egg-shell meal, ferments, then refining spearation calcium lactate.
Preferably, in described substratum, the weight ratio of egg-shell meal and glucose is 0.2-1.5: 1.
Best, in described substratum, the weight ratio of egg-shell meal and glucose is 0.6: 1.
Preferred further, described substratum is grouped into by the one-tenth of following weight proportioning:
Peptone 0.5%, Tryptones 0.5%, yeast extract 1%, glucose 10%, egg-shell meal 6%, CaCl
20.008%, MgSO
40.192%, K
2hPO
40.04%, KH
2pO
40.04%, NaHCO
30.4%, NaCl0.08%, distilled water is surplus.
Preferably, every 100g inoculation of medium 10
5-10
8the milk-acid bacteria of cfu.
Preferably, be 32-37 DEG C in temperature, shaking table speed is the condition bottom fermentation of 150-200r/min, and fermentation time is 60-84h.
The invention has the beneficial effects as follows:
1) be separated the milk-acid bacteria of acquisition in the present invention, its spontaneous fermentation lactic acid producing performance is better than current many common bacterial strains; And bacterial strain is easily cultivated, reproduction speed is fast, and patience is strong.
2) calcium lactate preparation method provided by the invention, eggshell utilization ratio is high, reaches 77.98%, and every 1kg eggshell can prepare the five water lactic acid calcium of about 2.5kg; Inversion rate of glucose is high, reaches as high as 79.3%, is better than the inversion rate of glucose of the conventional bacterial classification of industry; Calcium lactate purity is high, and tunning is through refining, and calcium lactate purity can reach more than 99%.
3) compared with neutralisation, the present invention does not need to buy lactic acid, can save cost, also overcomes calcination method simultaneously and prepares the environmental pollution of eggshell source calcium lactate and destroy the bioactive problem of eggshell.
Accompanying drawing explanation
Fig. 1 is M-25 bacterial strain microscopy picture under 100 times of oily mirrors that primary dcreening operation obtains.
Fig. 2 is the M-25 bacterial strain molten calcium circle that point sample is formed in MC substratum after multiple sieve.
Fig. 3 be in embodiment 2 different time of ultraviolet irradiation to the influence curve figure of molten calcium circle size.
Fig. 4 be in embodiment 2 the different nitrosoguanidine treatment time to the influence curve figure of molten calcium circle size.
Fig. 5 is the HNMD-25 bacterial strain 16S rDNA agarose gel electrophoresis figure obtained after mutagenesis.
Fig. 6 is the 16S rDNA sequence of HNMD-25 bacterial strain.
Fig. 7 is fermentation time to the influence curve figure of calcium lactate output and residual glucose.
Fig. 8 is fermentation time on the impact of inversion rate of glucose and egg-shell meal transformation efficiency.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
The separation of embodiment 1 bacterial strain
Isolation medium is MRS substratum, and substratum is purchased from Hai Bo bio tech ltd, Qingdao, and composition contains: peptone 10.0g, beef powder 8.0g, yeast powder 4.0g, glucose 20.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, tween 80 1.0g, magnesium sulfate 0.2g, manganous sulfate 0.04g, agar 14g, distilled water 1000mL, pH value 6.5 ± 0.2(25 DEG C).Take this product 66.2g during use, heating for dissolving in 1000mL distilled water, 121 DEG C of autoclaving 15min, for subsequent use.
Milk-acid bacteria sieves checking substratum MC substratum again, purchased from Qingdao Hai Bo biotechnology company, composition contains: soy peptone 5.0g, beef extract powder 3.0g, yeast leaching powder 3.0g, glucose 20.0g, lactose 20.0g, calcium carbonate 10.0g, agar 15.0g, toluylene red 0.05g, distilled water 1000mL, pH6.0(25 DEG C).Take this product 80.0g during use, heated and boiled is dissolved in 1000mL distilled water, and 121 DEG C of autoclaving 15min are for subsequent use.
1.1 bacterial strain initial gross separation purifying
Get the every increment product of buried eggshell pedotheque respectively and be about 25g, add the normal saline dilution of 225mL, sample after dilution does spread plate and line two kinds of mode process respectively respectively on MRS substratum, Anaerobic culturel 48h at 37 DEG C, observes the color etc. of colony shape, size, edge, surface, projecting shape, transparency, bacterium colony and substratum.
The single bacterium colony of the different typical case of bacterium picking growing way previous step cultivated repeatedly is rule separation and Culture further on MRS substratum, until colonial morphology is consistent, observes colonial morphology further.
Bacterium colonies different for growing way in isolation medium is got single bacterium colony smear and dry on slide glass fixing after, adopt gram staining method dyeing, and oily mirror microscopy is observed under microscope, checking thalli morphology and gram character.Fig. 1 is the microscopy result being separated the wild type strain M-25 obtained.
1.2 bacterial strains sieve again
To rule isolated bacterial classification inoculating needle point sample on MC agar plate, every ware point sample 5 points, Anaerobic culturel 48h at 37 DEG C, observes thalline color, and observe whether molten calcium form transparent ring, whether checking bacterial classification has molten calcium characteristic.Fig. 2 is the wild type strain M-25 molten calcium circle that point sample is formed in MC substratum after multiple sieve.
The mutagenesis of embodiment 2 bacterial strain and qualification
2.1 study ultraviolet mutagenesis and nitrosoguanidine mutagenesis respectively on the impact of lactic acid acid producing ability:
2.1.1 ultraviolet (UV) mutagenesis
Get thalline inclined-plane, add sterilized water 10mL and make suspension, getting 0.1mL respectively coats on 8 flat boards, at 37 DEG C, Anaerobic culturel 24h is placed on mutagenesis under 20W ultraviolet lamp, irradiation distance is 30cm, irradiate 0.5min, 1.0min, 1.5min, 2.0min, 2.5min, 3min, 3.5min respectively, with the plate without uv irradiating for contrast (CK), then with 37 DEG C of Anaerobic culturel 24h.By culture point sample in MC solid plate, Anaerobic culturel 42h at 37 DEG C, with the diameter of the molten calcium circle of vernier caliper measurement, surveys 3 molten calcium circles, averages, the molten calcium circle size of more different mutation time.
Result: different irradiation time molten calcium circle size result is as Fig. 3, and during irradiation 1.5min, molten calcium circle is maximum.
2.1.2 nitrosoguanidine (NTG) mutagenesis
At being taken at 37 DEG C, 180r/min cultivates the bacterium liquid 30mL of 36h, the centrifugal 15min of 6000r/min, and collect thalline, the stroke-physiological saline solution with 8% washs 3 times, then adds appropriate sterilized water in thalline, and vibration, being mixed with concentration is 10
8the thallus suspension liquid of cfu/ml.NTG is added in the triangular flask of bacteria suspension, make its concentration be 0.3mg/ml, not add the thallus suspension liquid of NTG for contrast (CK).On shaking table triangular flask being placed in 180r/min at 37 DEG C after oscillation treatment different time (20min, 30min, 40min, 50min, 60min, 70min), with stroke-physiological saline solution repeatedly centrifuge washing thalline, get bacterial sediment, add stroke-physiological saline solution to shake up, suitable dilution, get after 0.1ml bacterium liquid coats the dull and stereotyped upper 37 DEG C of Anaerobic culturel 48h of MRS solid medium, with inoculating needle point sample on primary dcreening operation MC solid plate, Anaerobic culturel 42h at being placed in 37 DEG C, the molten calcium circle size of more different action time.
Result: the nitrosoguanidine different treatment time, molten calcium circle size result was as Fig. 4, during process 40min, molten calcium loop diameter is maximum.
2.1.3NTG and UV complex mutation
By the bacteria suspension after NTG mutagenic treatment different time (20min, 30min, 40min, 50min, 60min) repeatedly centrifuge washing, get after 0.1ml bacterium liquid coats the dull and stereotyped upper 37 DEG C of Anaerobic culturel 24h of MRS solid medium, under being placed in ultraviolet lamp, different time (0.5min, 1.0min, 1.5min, 2.0min, 2.5min) irradiates, again by the thalline of combination of two mutagenesis point sample on primary dcreening operation MC solid medium, Anaerobic culturel 42h at being placed in 37 DEG C, more molten calcium circle size.Accessed respectively by bacterial strain in the 250mL triangular flask having filled 100mL PYG substratum simultaneously and carry out shaking table cultivation, 37 DEG C, 180r/min cultivates 48h, measures bacterial strain lactic acid producing content.
Result: the while of complex mutation employing nitrosoguanidine process 40min, during uv irradiating 1.5min, molten calcium loop diameter is maximum, and shaking flask lactic acid content is the highest simultaneously, and final bacterial classification adopts complex mutation bacterium, is HNMD-25 by bacterium numbering.
The biochemical property qualification of 2.2 mutagenic strains
2.2.1 sugar-fermenting character
The carbohydrate such as the sugar of mensuration or alcohols add in fermention medium by mass volume ratio 0.5% by experiment, be sub-packed in the test tube containing 10mL sugar-fermenting substratum, sugar-fermenting pipe composition is: extractum carnis 5.0g, peptone 5.0g, yeast extract 5.0g, tween 80 0.5mL, agar 1.5g, 1.6% purpurum bromocresolis spirituous solution 1.4mL, distilled water 1000mL, 121 DEG C of autoclaving 15min.To use in the carbohydrate medium after the identified bacterial strain percutaneous puncture-inoculation to sterilizing of inoculating needle picking 160r/min shaking culture 24h at 37 DEG C respectively.The fermentation test of following sugar has been carried out in this test: sucrose, wood sugar, lactose, maltose, seminose, cellobiose, melibiose, melizitose raffinose, saligenin, Vitamin C2, sorbyl alcohol.Result: be negative to cellobiose nonfermented, presents Partial fermentation to melizitose and melibiose, is all fermented into the positive to all the other sugar.
2.2.2 all the other biochemical properties and resistance test
Verify other Physiology and biochemistry character and acid and alkali-resistance salt tolerant and resistance to humid test.According to the outstanding systematic bacteriology handbook of uncle, the method for common bacteria system identification handbook and Ling Daiwen etc. (1999), mainly carries out following biochemical property qualification: catalase test, Starch Hydrolysis is tested, litmus milk is tested, casein hydrolysis experiment, gelatin liquification test, acetyl methyl carbinol V-P tests, methyl red (M.R.) is tested, citrate test, and anaerobism nitrate aerogenesis is tested, hydrogen sulfide produces test, indole test.Result is as follows:
Table 1 bacterial strain biochemical property result
Note :+represent that biochemical reaction is positive ,-represent that biochemical reaction is negative.
2.2.3 resistance test
The bacterial strain of separation is verified respectively acid and alkali-resistance heatproof and salt tolerance test, operates as follows:
Configuration MRS substratum, inoculates with plate streak, then Anaerobic culturel at 25 DEG C, 37 DEG C, 43 DEG C, 57 DEG C respectively, observes the growing state of bacterial classification after cultivating 48h.
Configuration saltiness is respectively the MRS substratum of 2%, 5%, 7%, 10%, inoculates, then Anaerobic culturel at 37 DEG C with plate streak, observes the growing state of bacterial classification after cultivating 48h.
Configuration MRS substratum, adjusts PH to 4.3,5.7,6.8,8.7 with NaOH solution and HCl solution, inoculates, then Anaerobic culturel at 37 DEG C with plate streak, observe the growing state of bacterial classification after cultivating 48h.
Result is as follows:
Table 2 bacterial strain tolerance test result
Note :+represent that biochemical reaction is positive ,-represent that biochemical reaction is negative.
The molecular biology identification of 2.3 mutagenic strains
The 16S rDNA of bacterial strain increases and sequencing analysis
2.3.1 total DNA extraction
Method for extracting adopts CTAB method, and with reference to the method for (2005) such as Ao Baisi, concrete operations are as follows:
(1) a ring bacterium is connect in 10mL PYG substratum, 37 DEG C, Anaerobic culturel 24h.
(2) 1.5mL bacterium liquid is got in 1.5mL Eppendorf centrifuge tube, the centrifugal 5min of 8000r/min, supernatant discarded.
After adding the washing of 1.5ml TE buffer by centrifugation, with 567uL TE buffer solution thalline, repeatedly blow and beat with pipettor and make it resuspended.
(3) Proteinase K of SDS and 3uL20mg/mL of 30uL10% is added, mixing, incubation 1h in 37 DEG C of dry-type thermostats.
(4) add the NaCl solution that 100uL5mol/L is aseptic, fully mix, then add 80uLCTAB/NaCl, mixing, 65 DEG C of incubation 10min in dry-type thermostat.
(5) add isopyknic chloroform/primary isoamyl alcohol (24:1) (0.8mL), mixing, the centrifugal 5min of 12000r/min, proceeds to supernatant liquor in a new pipe.
(6) add isopyknic phenol in the new pipe of supernatant liquor: chloroform: primary isoamyl alcohol (25: 24: 1) (0.8mL), mixing, the centrifugal 5min of 12000r/min, proceeds to supernatant liquor in a new pipe.
(7) add the Virahol of 0.48mL times of volume, mixing is until DNA precipitates gently, the centrifugal 15min of 12000r/min, collects DNA precipitation, washs DNA precipitation, vacuum-drying 0.5h with the centrifugal 5min of 75% ethanol (1mL) 12000r/min.
(8) precipitation is heavily dissolved in the TE damping fluid of 100uL, saves backup in-20 DEG C.
2.3.2 the amplification of bacterial 16 S rDNA and product purification
The primer of bacterial 16 S rDNA pcr amplification adopts a pair universal primer, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Forward primer Eubac27F is 5 '-AGAGTTTGATCCTGGCTCAG-3 '; Reverse primer Eubac1492R is 5 '-GGTTACCTTGTTACGACTT-3 '.Polishing to the 20uL system that 2*Taq PCRMasterMix and primer (each 1uL of forward and reverse primer), template 4uL added water after mixing carries out PCR, PCR response procedures in table 3.
Table 3PCR response procedures
Product is placed in-20 DEG C save backup.
With the DNA that 0.8% agarose gel electrophoresis qualification is extracted, with Gold View staining agent (5uL/100uL) dyeing, every hole point sample 5uL, mark point sample 5uL, 110V, electrophoresis 1 hour.Electrophoresis is placed in gel imaging system and observes, and as shown in Figure 5, (what sample need introduce each swimming lane is respectively, and electrophoresis result how), shows PCR success to result.Cut band clearly again, reclaim test kit requirement to specifications with sepharose and reclaim product, be placed in-20 DEG C of preservations.
2.3.316S rDNA order-checking and sequence alignment
By the product sample presentation order-checking of PCR rear electrophoresis purifying of preserving, checking order is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The splice program of ContigExpress is utilized the forward and reverse sequence recorded to be carried out splicing and revising base mismatch, then blast program is utilized, the sequence of known bacterial classification in the 16S rDNA sequence of spliced bacterial strain and GenBank database is compared (http://www.ncbi.nlm.nih.gov/blast), finds the known bacterial classification the highest with goal gene sequence homology.Result: find that the similarity level of the 16S rDNA of isolated strains and enterococcus mundtii is the highest, reach more than 99%, in conjunction with morphology and the physiologic character of bacterial strain, determines that the bacterial strain be separated is enterococcus mundtii (Enterococcusmundtii).The 16S rDNA sequence of bacterial strain is shown in Fig. 6.
The research of embodiment 3 bacterial strain lactic acid producing ability and fermentation time
1. the acid producing ability of bacterial strain
Fermentation medium components is by following configuration: peptone 0.5g, Tryptones 0.5g, yeast extract 1.0g, glucose 10g, CaCl
20.008%, MgSO
40.192%, K
2hPO
40.04%, KH
2pO
40.04%, NaHCO
30.4%, NaCl0.08%, distilled water 100mL.115 DEG C of sterilizing 20min.Neutralizing agent calcium carbonate 6g, separately sterilizing.Inoculum size with 2% accesses the milk-acid bacteria logarithmic phase seed liquor that the present invention is separated, and produces the most frequently used bacterial classification of lactic acid with current industrial fermentation simultaneously---and lactobacillus bulgaricus is contrast.72h is cultivated in 35 DEG C of 180r/min shaking tables.30min at being placed in 85 DEG C, kills thalline, filters.Get the fermented liquid 1.0mL after filtration, add distilled water 50mL, then add NaOH solution 4.0mL and the fluorexon indicator 20mg of lmol/L, titration adopts the EDTANa of 0.05mol/L
2solution, is titrated to when back-light observation yellow-green fluorescence changes into orange and stops, and the volume (mL) of the EDTANa2 that record consumes, is calculated as follows lactic acid content:
W(g/L)=K×9.008×V
In formula: W=lactic acid content (g/L)
The volume (mL) of the EDTANa2 consumed during V=titration
K=[V+W-(Wl-W2)]/V
Wherein Wl represents: fermentation weight just; W2 represents: the weight after fermentation ends; W is: inoculum size; V is: initial liquid amount.
Result: the lactic acid production of strain fermentation glucose of the present invention is 60.375-72.064g/L, and inversion rate of glucose is 66.4-79.3%, and the lactic acid production of lactobacillus bulgaricus is 54.247-63.056g/L, the transformation efficiency of glucose is 57.5-69.4%.
2. the fermentation time of bacterial strain
Design the different fermentations time (36h, 48h, 60h, 72h, 84h) respectively, the rear high temperature that fermented kills thalline, filters, and measures the calcium lactate content in fermented liquid.
Result: shown in calcium lactate output and residual sugar content and glucose and egg-shell meal transformation efficiency following Fig. 7, Fig. 8.Fermentation has reached 76.741g/L completely to substantially fermenting during 60h, and glucose now transformation efficiency is 69.69%, and ferment to 84h and be increased to 82.196g/L, inversion rate of glucose reaches 74.65%.Therefore fermentation time selects 60-84h to be advisable.
Embodiment 4
The high-yield lactic acid bacteria strain fermentation eggshell utilizing deposit number to be CGMCC NO.8699 prepares a method for calcium lactate, comprises the following steps:
1) eggshell treatment: by dry after the washing of Fresh Egg shell, pulverize with pulverizer, cross 200 mesh sieves.
2) ferment: to inoculation of medium lactic acid bacterial liquid, be 34 DEG C in temperature, shaking table speed is the condition bottom fermentation 72h of 180r/min.
Described substratum is grouped into by the one-tenth of following weight proportioning:
Peptone 0.5%, Tryptones 0.5%, yeast extract 1%, glucose 10%, egg-shell meal 6%, CaCl
20.008%, MgSO
40.192%, K
2hPO
40.04%, KH
2pO
40.04%, NaHCO
30.4%, NaCl0.08%, distilled water is surplus.
10 are contained in every 100g substratum after inoculation
7the milk-acid bacteria of cfu.
3) filtration and removal of impurities: the rear high temperature that fermented kills thalline, filters; Filtrate adjustment pH to 10, reacts 15min, and adds decolorizing with activated carbon with 10g/L, while hot suction filtration, retain filtrate at 94 DEG C.
4) crystallization: after being concentrated into about 2/5 volume at filtrate being placed in 80 DEG C, is placed in 5 DEG C and goes out white crystal down to crystallization, suction filtration; White crystal after filtering heavily is dissolved in the distilled water of original fermented solution volume, again concentrates in 80 DEG C, concentrated be placed on 5 DEG C at cool recrystallization, suction filtration, repeat above recrystallization process 2 times, to remove the impurity such as nutrient solution in crystal.
5) finished product: the white crystal after crystallization is dried at 70 DEG C, namely obtains calcium lactate finished product.Purity is 99.25%, and egg-shell meal utilization ratio is 77.98%, and every 1kg eggshell can prepare about 2.5kg five water lactic acid calcium.
Claims (9)
1. a plant height lactic acid-producing bacteria, be enterococcus mundtii (Enterococcus mundtii) HNMD-25, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCCNO.8699.
2. high-yield lactic acid bacterium according to claim 1 prepares the application in calcium lactate in fermentation calcium source.
3. application according to claim 2, is characterized in that: described calcium source is eggshell.
4. utilize eggshell to prepare a method for calcium lactate, it is characterized in that: to the high-yield lactic acid bacterium according to claim 1 of the inoculation of medium containing glucose and egg-shell meal, ferment, then refining spearation calcium lactate.
5. the method utilizing eggshell to prepare calcium lactate according to claim 4, is characterized in that: in described substratum, the weight ratio of egg-shell meal and glucose is 0.2-1.5: 1.
6. the method utilizing eggshell to prepare calcium lactate according to claim 5, is characterized in that: in described substratum, the weight ratio of egg-shell meal and glucose is 0.6: 1.
7. the method utilizing eggshell to prepare calcium lactate according to claim 6, is characterized in that: described substratum is grouped into by the one-tenth of following weight proportioning:
Peptone 0.5%, Tryptones 0.5%, yeast extract 1%, glucose 10%, egg-shell meal 6%, CaCl
20.008%, MgSO
40.192%, K
2hPO
40.04%, KH
2pO
40.04%, NaHCO
30.4%, NaCl0.08%, distilled water is surplus.
8. prepare the method for calcium lactate according to the eggshell that utilizes of claim 4-7 described in any one, it is characterized in that: every 100g inoculation of medium 10
5-10
8the high-yield lactic acid bacterium of cfu.
9. prepare the method for calcium lactate according to the eggshell that utilizes of claim 4-7 described in any one, it is characterized in that: be 32-37 DEG C in temperature, shaking table speed is the condition bottom fermentation of 150-200r/min, and fermentation time is 60-84h.
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| CN105475429A (en) * | 2015-11-20 | 2016-04-13 | 广西壮族自治区农业科学院农产品加工研究所 | Dietary fiber sugar-free biscuits prepared by utilization of fruit processed waste residue enzyme method |
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| CN110373444A (en) * | 2019-08-21 | 2019-10-25 | 福建农林大学 | A kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate |
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