CN110373444A - A kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate - Google Patents
A kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate Download PDFInfo
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- CN110373444A CN110373444A CN201910772366.3A CN201910772366A CN110373444A CN 110373444 A CN110373444 A CN 110373444A CN 201910772366 A CN201910772366 A CN 201910772366A CN 110373444 A CN110373444 A CN 110373444A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
Abstract
The invention discloses a kind of methods that compound lactobacillus-fermencucumber eggshell prepares calcium lactate, comprising the following steps: the preparation of egg-shell meal, the production of fermentation medium, the activation of strain and the preparation of calcium lactate.Calcium lactate yield is 40.01 ± 0.035 g/L, purity 92.65% under best technological condition for fermentation.Process conditions optimize, and operability, excellent product performance is conducive to promote and apply.
Description
Technical field
The invention belongs to calcium lactate preparation technical fields, and in particular to a kind of compound lactobacillus-fermencucumber eggshell prepares calcium lactate
Method.
Background technique
China is the first big country of world's egg production and consumption, discarded about 3,000,000 tons of eggshell every year, causes serious ring
Border pollution and the wasting of resources.Eggshell calcium is a kind of ideal diet calcium source, is mentioned from the calcium of egg-shell meal than business calcium carbonate in rat
It is easier to absorb in small intestine.Eggshell calcium can be used for human body and Animal Bone mineralising and the treatment of the calcium deficiency of growth, and as the anti-of toothpaste
Tartar agent.The calcium lactate that food industry is prepared using eggshell calcium is as curing agent, flavoring agent, leavening agent and antibacterial agent.
The calcium of organic acid preparation such as calcium lactate, calcium citrate and calcium gluconate is efficiently prepared as current research hot spot.Cream
Sour calcium is supplement and the perfect additive for strengthening skeletal calcium, is widely used in medicine, food and feed industry.Eggshell is rich in
Calcium carbonate can be used as calcium source and prepare calcium of organic acid, and main method has high-temperature calcination, direct neutralisation and microbial fermentation at present
Method.Energy consumption is high for high-temperature calcination and direct neutralisation, and calcium transformation ratio is lower, and industrial production cost is high.Microbe fermentation method consumption
Energy is low, it is simple to environment generation pollution, preparation process condition to be not easy, and is more suitable for preparing calcium lactate.
Microbial fermentation conversion eggshell calcium is that the common microorganism of calcium lactate is lactic acid bacteria, and lactic acid bacteria can utilize carbohydrate fermentation
Generate lactic acid.Compound lactobacillus can use the Physiology and biochemistry feature of different lactic acid bacterias, and other side provides suitable growth ring each other
Border, and reach the complementation of metabolite.It is had not been reported using the research that 4 kinds of lactic acid bacteria composite fermentation eggshells prepare calcium lactate.
Summary of the invention
The purpose of the present invention is to provide a kind of methods that compound lactobacillus-fermencucumber eggshell prepares calcium lactate, choose lactic acid and produce
Measure 4 kinds of relatively high enterococcus mundtii, lactobacillus bulgaricus, streptococcus thermophilus, Lactobacillus casei lactic acid bacteria composite fermentations
Eggshell biology prepares calcium lactate, and calcium lactate yield is 40.01 ± 0.035 g/L under best technological condition for fermentation, and purity is
92.65%.Process conditions optimize, and operability, excellent product performance is conducive to promote and apply.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate, comprising the following steps:
(1) preparation of egg-shell meal
Eggshell wash with distilled water after, 70 DEG C of 3 h of drying, crush；Distilled water is added, stands 15 min after stirring 10 min, removes
The egg shell membrane for going upper liquid to float；It filters, 70 DEG C of 3 h of drying, crosses 200 meshes；
(2) production of fermentation medium
Formula: 0.5 g of peptone, 0.5 g of tryptone, 1.0 g of yeast extract, 4 mL of salting liquid, 12 g of glucose, eggshell
8 g of powder, 100 mL of distilled water；After each component is sufficiently mixed, in 121 DEG C of 20 min of sterilizing；
The formula of salting liquid: NaHCO3 10.0 2.0 g of g, NaCl, anhydrous CaCl2 0.2 g, K2HPO4 1.0 g、KH2PO4
1.0 g、MgSO4·7H20.48 g of O, distilled water are settled to 1000 mL；
(3) activation of strain
Enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus are inoculated in MRS fluid nutrient medium respectively
In, 37 DEG C of 24 h of culture, 2.0% inoculum concentration passes on 2~3 times, until rejuvenate, low-temperature preservation in 0~4 DEG C of refrigerator；
(4) preparation of calcium lactate
It is sequentially ingressed into enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus in the fermentation medium, controls
Bacterial concentration 1 × 10 processed8 Cfu/mL, 180 r/min anaerobic fermentations.
Enterococcus mundtii in step (4), streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus volume ratio be 1-
2:1-2:1-2:1=2；32-42 DEG C of fermentation temperature, fermentation time 48-80h, fermentation liquid pH value 5.0-9.0, compound bacteria inoculum concentration
8.0-24.0%。
The beneficial effects of the present invention are: lactic acid is prepared by biofermentation conversion for natural green calcium source using eggshell
Calcium, in optimum process condition (37 DEG C of fermentation temperature, 72 h of fermentation time, fermentation liquid pH value 6.5, compound bacteria inoculum concentration 8.0%)
Under, calcium lactate yield is up to 40.01 ± 0.035 g/L, purity 92.65%.Process conditions optimize, operability, properties of product
It is excellent, be conducive to promote and apply.
Detailed description of the invention
Fig. 1 is the antagonistic effect result figure of four kinds of bacterium；
Fig. 2 is lactobacter growth curve graph；
Fig. 3 is influence diagram of the different composite bacterial strain combination to calcium lactate yield；
Fig. 4 is influence diagram of the single factor test to calcium lactate yield；
Fig. 5 is eggshell source lactic acid calcium product and its infrared spectrogram；
Fig. 6 is eggshell calcium lactate X-ray diffractogram.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
A kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate, the specific steps are as follows:
Egg-shell meal preparation
Eggshell after distilled water cleaning dries 3 h in 70 DEG C of Constant Temp. Oven, is subsequently placed into continous way high-performance powder
It is crushed in broken machine.Distilled water immersion is added in egg-shell meal, 15 min is stood after stirring 10 min, removes the eggshell of upper liquid floating
Film.Egg-shell meal is collected spare after 70 DEG C of dry 3 h, excessively 200 meshes after filtering.
Culture medium and actication of culture
Fermentation medium: 0.5 g of peptone, 0.5 g of tryptone, yeast extract 1.0 g, salting liquid (NaHCO310.0 g、
2.0 g of NaCl, anhydrous CaCl20.2 g, K2HPO41.0 g、KH2PO41.0 g、MgSO4·7H2O0.48 g, is settled to 1000
ML) 4 mL, 12 g of glucose, 8 g of egg-shell meal, 100 mL of distilled water.After medium component is sufficiently mixed, in 121 DEG C of sterilizings 20
Min, egg-shell meal individually sterilize, spare after cooling.
Actication of culture: 4 plants of strains (enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus) are respectively
It is inoculated in MRS fluid nutrient medium, in 37 DEG C of 24 h of culture, 2.0% inoculum concentration is passed on 2-3 times, until rejuvenate, 0-4
Low-temperature preservation in DEG C refrigerator.
4 kinds of lactic acid bacterias are combined with each other by different proportion.Each bacterial concentration is uniformly tuned into 1 × 108 Cfu/mL, fermented and cultured
Enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus are sequentially ingressed into base, inoculation volume ratio is 1:1:
1:1,1:2:1:1,1:1:2:1,1:1:1:2,2:1:1:1.180 r/min anaerobic fermentations.When 32-42 DEG C of fermentation temperature, fermentation
Between 48-80h, fermentation liquid pH value 5.0-9.0, compound bacteria inoculum concentration 8.0-24.0%.
The extraction of Lactic Acid from Fermentation Broth calcium
Fermentation liquid contains that tunning calcium lactate, superfluous eggshell calcium, do not ferment residual sugar, pigment, protein etc., need to fermentation liquid into
Row removal of impurities processing.Supernatant is taken after fermentation liquid centrifugation, calcium hydroxide tune pH value is added to 12-13,80 DEG C of 30 min of stirring.It will be thick
The filtering of calcium lactate liquid, pH are adjusted to 4-5,3.0-4.0% Powdered Activated Carbon are added with impurity such as adsorpting pigments, 50-60 DEG C stands 2
H, vacuum filtration filter off active carbon, repeat decoloration until clarification.After collecting the concentration of lactic acid mother liquor of calcium rotary evaporation, sub-cooled knot
Crystalline substance, then crystal is dissolved in distilled water and is recrystallized.Appropriate dehydrated alcohol is added, washs calcium lactate crystal, removes remnants'
Lactic acid and other residues of surface attachment.By calcium lactate product in 80 DEG C of dry 8 h, reaching constant weight can crush, be sieved
Calcium lactate finished product.
Antagonistic effect
As shown in Figure 1, different digital label represents different lactic acid bacterias, 4 kinds of equal well-growns of bacterium on plate, infall is sterile two-by-two
Atrophy or extinction tests are fallen, illustrate do not have antagonism between 4 kinds of bacterium, can be used for mixed bacterium composite fermentation test.
Lactobacter growth curve
It is analyzed by Fig. 2 growth curve it is found that the lag phase of 4 kinds of lactic acid bacterias is shorter, 0~4 h starts slowly to grow after inoculation, should
The breeding of period inner cell is slower, and quantity increases less.Thallus enters logarithmic growth phase after shorter period of delay, 4~10
Growth is rapid in h, exponentially increases, and strain metabolism and breeding are accelerated, and enzyme system is active, is metabolized vigorous, bacterial strain life in culture medium
Long rate reaches maximum.Total bacteria count reaches higher level and is held essentially constant when to 12 h, into stationary phase, thalli growth speed
Rate amplitude of variation tends to be steady, and viable count keeps relative stability, and bacterium colony metabolite, which constantly accumulates, reaches top.To after 20 h
Into decline phase, viable bacteria thalline quantity is gradually slowly reduced, and bacterial activity reduces and tends to stagnate.
According to the growth curve comprehensive descision of 4 kinds of bacterial strains, 12 h activity are higher after strain inoculated, each bacterium solution in 12 h
OD value reaches the larger value, is suitble to follow-on inoculation.Hereafter it keeps relative stability, illustrating bacterium solution, upgrowth situation is most in 12 h
It is good, therefore the stationary phase bacterium solution that 4 kinds of lactic acid bacterias choose 12 h of culture in this test carries out inoculation fermentation.
Different composite bacterial strain combines the influence to calcium lactate yield
Successively it is inoculated with according to the sequence of enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus.It can by Fig. 3
Know, when composite bacteria inoculum concentration ratio is 1:1:2:1, calcium lactate yield highest.The significance difference analysis that test result is carried out
Show inoculation than being 1:1:2:1 and 1:2:2:1,2:1:1:1 without significant difference, there were significant differences with other 12 groups of tests.Have
Research report, the fermentative activity that streptococcus thermophilus and lactobacillus bulgaricus are inoculated with 1:1 are best.The cheese cream of Lactobacillus
Bacillus is the important sources of production of lactic acid, this may be to mix Lactobacillus casei with streptococcus thermophilus and lactobacillus bulgaricus
Play the role of promoting growth mutually to other lactic acid bacterias afterwards, reaches ideal ferment effect.Therefore compound strain inoculum concentration
Than being chosen to be 1:1:2:1.
Single factor experiment
By Fig. 4 (a) it is found that with cultivation temperature raising, calcium lactate yield first increases to be declined afterwards, fermentation temperature to calcium lactate produce
Amount influences significant.Calcium lactate yield reaches maximum value at 37 DEG C, and residual sugar content is minimum, illustrates that compound lactobacillus utilizes the effect of glucose
Fruit is best.When cultivation temperature be lower than 37 DEG C when, temperature is too low, and enzymatic activity is suppressed, enzymatic reaction weaken, cause thalli growth and
Metabolism slows down.But it when the temperature is excessively high, will lead to bacterium protein solidification denaturation, nucleolysis deactivation, therefore after being higher than 37 DEG C
Calcium lactate yield reduces.The suitable fermentation temperature of compound lactobacillus is 37 DEG C.
By Fig. 4 (b) it is found that with fermentation time extension, calcium lactate yield gradually rises, and glucose content gradually drops
It is low.Fermentation time has a significant impact calcium lactate yield and residual sugar content.Between when fermenting more than 60 h after, calcium lactate yield increase
Length is slow and tends to stop.This is because lactic acid bacterium number is continuously increased with the extension of incubation time before 60 h, utilize
The lactic acid content that glucose fermentation generates also gradually increases, the carbon source that glucose ferments as lactobacter growth, generates a large amount of
Calcium lactate.The nutrition contents such as glucose are reduced after 60 h, are slowed down the growth and metabolism of lactic acid bacteria, are led to the production of calcium lactate
Amount is reduced.Fermentation time is advisable with 60-72 h.
By Fig. 4 (c) it is found that with the initial pH of culture medium increase, calcium lactate yield presentation first increase the variation reduced afterwards
Feature.Initial pH has a significant impact calcium lactate yield.It when the ph is lower, has been more than lactic acid due to a large amount of hydrionic presence
Bacterium keeps the ability of pH value dynamic equilibrium intracellular, causes film rouge saturation degree to decline, pH value intracellular also declines therewith, to inhibit
The growth of lactic acid bacteria.Medium pH is controlled at 6.0, and lactobacter growth is preferable, lactic acid production highest, this and lactobacter growth
Optimum pH is consistent.After pH is more than 6.5, pH value raising makes the chemical balance between original culture medium intermediate ion move, give birth to
At precipitating, the effective ingredient of culture medium is reduced, also inhibits the metabolism growth of lactic acid bacteria.Fermentation medium pH value is with 6.0
Preferably.
By Fig. 4 (d) it is found that with inoculum concentration increase, calcium lactate yield gradually decreases.This is because inoculum concentration is excessive
When, thalli growth is too fast, and microorganism enhances the competition of nutriment, and culture solution viscosity increases, and accelerates cell ageing, leads to portion
It is dead to divide thallus, and generates excessive metabolic waste, glucose utilization reduces, and fermentation lacks of staying power, to influence metabolite
Synthesis, produce acid amount declines therewith.Fermentation inoculum concentration is advisable with 8.0%.
By Fig. 4 (e) it is found that low concentration Mn2+With Mg2+Calcium lactate yield can be improved to a certain extent, optium concentration is
0.1 g/L；And high concentration can show certain inhibiting effect.Bivalent metal ion Mn2+、Mg2+It is necessity of activator metal enzyme
Most of enzymes of co-factor, EMP Embden Meyerbof Parnas pathway require Mg2+Participation, Mg2+Content is directly related with the proliferation of normal cell；Mg2+
It is the activator of the Major Enzymes such as glucokinase, phosphoenol enzyme, pyruvic acid enzyme and the center component part of complex enzyme, but makees
It is lower with concentration.Mn2+It is the component part of lactic dehydrogenase and the co-factor of certain phosphotransferases, speed is grown to cell
Degree and biomass concentration have important beneficial effect, can promote lactic acid generation.Guo et al. research discovery Mn2+、Mg2+And Ca2+
It can significantly increase lactobacillus protein enzyme activity, and Co2+、Zn2+、Ni2+The enzymatic activity can then be inhibited with EDTA；With this test
Result of study is consistent, but not significant to the effect for improving calcium lactate yield.
Response surface optimization test
Compound lactobacillus-fermencucumber eggshell prepares calcium lactate optimum process condition are as follows: 37 DEG C of fermentation temperature, 72 h of fermentation time, fermentation
Liquid pH value 6.5, compound bacteria inoculum concentration 8.0% predict that calcium lactate yield is 42.030 g/L with this condition.In order to verify model
The accuracy of prediction, according to the fermentation condition that response surface experiments optimize, 3 parallel tests are verified, with this condition,
Calcium lactate yield differs 4.8% with model predication value higher than 27.28 g/L of the clean report of Chen Wen up to 40.01 ± 0.035 g/L,
It is consistent substantially with theoretical value, illustrates that the parameter obtained using this model optimization is accurate and reliable.Measure prepared calcium lactate purity
It is 92.65%, meets 86.92% that vibration is reported higher than Lee.
Product structure characterization
1. FTIR spectrum
The lactic acid calcium product that extracts from fermentation liquid is as shown in figure 5, eggshell source calcium lactate is white or cream-white crystals powder；With
The infrared spectroscopy of egg-shell meal compares it is found that eggshell calcium is fermented to have been converted into calcium lactate.In 3500-3200 cm-1Occur one
A wider and strong absorption peak, for the bending vibration for the O-H key that dissociates；In the higher calcium lactate sample of concentration, hydroxy chemical produce
The stretching vibration absworption peak of raw association phenomenon, O-H base is formed by slightly towards the displacement of lower wave number direction, and peak point is about in 3350 cm-1
Left and right.In 3000-2800 cm-1Occurs a small and sharp peak in range, this is typical-CH3Base, c h bond stretching vibration
Caused absorption peak.And it should be in 1900-1650 cm-1The absorption peak of the C=O carbonylic stretching vibration occurred in range is due to combining
Ca2+, to lower wave number 1600-1500 cm-1It is deviated in range.In 1350-1450 cm-1The apicule peak of appearance is C-
The bending vibration of H key；Infrared spectroscopy is in 1300-4000 cm-1For functional group region, less than 1300 cm-1Area is rolled into a ball for non-functional, instead
What is reflected is molecule structure change, such as the stretching vibration of C -- C single bond.Pass through the infrared spectrum analysis to product calcium lactate, it was demonstrated that multiple
Closing lactobacillus-fermented eggshell calcium and preparing products therefrom is calcium lactate.
2.X x ray diffraction
It is as shown in Figure 6 that X-ray diffraction measure is carried out to the calcium lactate sample of fermentation preparation.Analysis is it is found that eggshell calcium lactate spreads out
It is sharply clear to penetrate peak, illustrates that calcium lactate crystallization degree is higher.By the X-ray diffracting spectrum of sample and JCPDS standard substance card
Difference angles (2 in 05-0101θ) calcium lactate data comparison carry out Discriminating materials, discovery spread out in calcium lactate main feature
Penetrate peak position (2θ=8.0 °, 22.1 °, 27.4 °, 45.2 ° of equal left and right) equal appearance, characteristic peak positions and standard spectrogram are in same angular
Spending two maps, diffraction maximum occur similar, illustrates that crystal and space structure are similar, verifying tunning again is calcium lactate.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (4)
1. a kind of method that compound lactobacillus-fermencucumber eggshell prepares calcium lactate, it is characterised in that: the following steps are included: egg-shell meal
Preparation, the production of fermentation medium, the activation of strain and calcium lactate preparation.
2. according to the method described in claim 1, it is characterized by: specific step is as follows:
(1) preparation of egg-shell meal
Eggshell wash with distilled water after, 70 DEG C of 3 h of drying, crush；Distilled water is added, stands 15 min after stirring 10 min, removes
The egg shell membrane for going upper liquid to float；It filters, 70 DEG C of 3 h of drying, crosses 200 meshes；
(2) production of fermentation medium
Formula: 0.5 g of peptone, 0.5 g of tryptone, 1.0 g of yeast extract, 4 mL of salting liquid, 12 g of glucose, eggshell
8 g of powder, 100 mL of distilled water；After each component is sufficiently mixed, in 121 DEG C of 20 min of sterilizing；
The formula of salting liquid: NaHCO3 10.0 2.0 g of g, NaCl, anhydrous CaCl2 0.2 g, K2HPO4 1.0 g、KH2PO4 1.0
g、MgSO4·7H20.48 g of O, distilled water are settled to 1000 mL；
(3) activation of strain
Enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus are inoculated in MRS fluid nutrient medium respectively
In, 37 DEG C of 24 h of culture, 2.0% inoculum concentration passes on 2-3 times, until rejuvenate, low-temperature preservation in 0-4 DEG C of refrigerator；
(4) preparation of calcium lactate
It is sequentially ingressed into enterococcus mundtii, streptococcus thermophilus, Lactobacillus casei, lactobacillus bulgaricus in the fermentation medium, controls
Bacterial concentration 1 × 10 processed8 Cfu/mL, 180 r/min anaerobic fermentations.
3. according to the method described in claim 2, it is characterized by: enterococcus mundtii, streptococcus thermophilus, cheese in step (4)
Lactobacillus, lactobacillus bulgaricus volume ratio be 1-2:1-2:1-2:1=2；32-42 DEG C of fermentation temperature, fermentation time 48-
80h, fermentation liquid pH value 5.0-9.0, compound bacteria inoculum concentration 8.0-24.0%.
4. according to the method described in claim 3, it is characterized by: enterococcus mundtii, streptococcus thermophilus, cheese in step (4)
Lactobacillus, lactobacillus bulgaricus volume ratio be 1:1:2:1；37 DEG C of fermentation temperature, 72 h of fermentation time, fermentation liquid pH value
6.5, compound bacteria inoculum concentration 8.0%.
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| WO2021222817A1 (en) * | 2020-05-01 | 2021-11-04 | Packerland Whey Products D/B/A Fermented Nutrition | Compositions and methods for solidified fermented animal feed |
| CN114732092A (en) * | 2022-04-21 | 2022-07-12 | 清远金沣生物药品有限公司 | Active calcium and magnesium microemulsion and preparation method thereof |
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| CN114732092A (en) * | 2022-04-21 | 2022-07-12 | 清远金沣生物药品有限公司 | Active calcium and magnesium microemulsion and preparation method thereof |
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Application publication date: 20191025 |