CN110511885A - A kind of compound nitrogen source and application method being applicable in bacillus coagulans fermentation - Google Patents
A kind of compound nitrogen source and application method being applicable in bacillus coagulans fermentation Download PDFInfo
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Abstract
The invention discloses a kind of compound nitrogen sources and application method for being applicable in bacillus coagulans fermentation, belong to field of microbial fermentation.Compound nitrogen source of the invention is mixed by sesame seed meal protein hydrolysate, maize yellow-powder protein hydrolysate, shrimp shell protein hydrolysate, it is applied in such a way that multi-change speed stream adds in the multistage fermentation of bacillus coagulans, realize efficient, the high-speed fermentation of bacillus coagulans, and the raw material of compound nitrogen source is easy to get, price is low, the fermentation costs for greatly reducing bacillus coagulans are more suitable for the industrialized production of bacillus coagulans.
Description
Technical field
The present invention relates to field of microbial fermentation, and in particular to a kind of to make what bacillus coagulans rapidly and efficiently fermented to answer
Close nitrogen source and its application method.
Background technique
Bacillus coagulans (Bacillus coagulans) are facultative anaerobic bacterias, all may be used in the environment of aerobic and anaerobic
Growth, adapts to the intestinal environment of hypoxemia, has higher tolerance to acid and bile, can ferment lactic acid producing.Therefore, bud is condensed
Spore bacillus food, medical treatment and in terms of, be especially all with a wide range of applications in terms of improving enteron aisle balance.
Bacillus coagulans growth needs a large amount of carbon nitrogen source, and inorganic salts, common medium component includes glucose, ferment
Mother's leaching powder, peptone, sodium chloride etc., such as: patent CN104017751A, using peptone as main nitrogen, with lactobacillus plantarum and
Lactobacillus casei Combined culture, bacterium solution fermentation time 36h, viable count 2.98*109CFU/g；Patent CN109207406A, with egg
White peptone, yeast extract, beef extract are main nitrogen, cultivate 70h, viable count 5*109CFU/mL.Patent CN103160455A, with ferment
Female cream, tryptone are main nitrogen, bacterium solution fermentation time 32h, solid fermentation culture medium semiclosed fermentation 72h, viable count 1*
1010CFU/g.Patent CN101390571A, using fish meal, soy peptone etc. as main nitrogen, bacterium solution fermentation time 36h is living
Bacterium number 1*1010CFU/mL.Patent CN109609432A, with soy peptone, yeast extract is main nitrogen, passes through stream plus grape
The mode of sugar is fermented in 50L fermentor, bacterium solution fermentation time 44h, viable count 4*1010CFU/mL.Patent
CN108085265A, fermentation medium are glucose, maize flour, dregs of beans, sodium chloride, magnesium sulfate, in 0.5m3Fermentor in send out
Ferment 18h, viable count 4*1010CFU/mL.Patent CN104974966A, using peptone, yeast powder as main nitrogen, in 50m3Hair
Ferment 48h in fermentation tank, viable count 1.5 × 1010CFU/mL.It can be seen that there is fermentation training in the fermentation of bacillus coagulans at present
The higher cost of base is supported, fermentation period is long, and viable count is lower, and the industrial fermentation and popularization for constraining bacillus coagulans are answered
With.
Sesame seed meal, is the by-product after sesame oil expression, it is then known as degreasing by the product after the degreasing of extraction method
Sesame seed meal powder, the main component of degreasing sesame seed meal are sesame proteins.Sesame protein is mainly made of alpha-globulin, is accounted for about
80%, sesame protein is dissolved with dilute alkaline soln, then adjusting pH value makes its precipitating, obtains sesame protein isolate；Sesame is separated into egg
White with protease hydrolytic is soluble protein, and soluble protein is concentrated in vacuo (vacuum degree 0.8Mpa, temperature 50oC after), then
Using spray drying, sesame seed meal protein hydrolysate is made.
Maize yellow-powder, also known as corn gluten meal, corn protein powder or zein are wet-grinding technology and relative device production cornstarch
Principal by product contains about 50%~60% protein, 15%~20% starch, 10% moisture, 6%~8% lipid and a small amount of cellulose
With other micro constitutents, but maize yellow-powder itself contains about 40% impurity, so the processing by alpha-amylase and cellulose is gone
It except starch, cellulose impurities, then is solubility by protease hydrolytic through thermal denaturation, the processing of the protein denaturation of sulphite
Albumen, soluble protein are concentrated in vacuo (vacuum degree 0.8Mpa, temperature 50oC after), then using spray drying, corn is made
Bloom protein hydrolysate.
Shrimp shell protein hydrolysate, using cray shell as raw material, use the protein in hydrolysis by novo cray shell for
Soluble protein, soluble protein are concentrated in vacuo (vacuum degree 0.8Mpa, temperature 50oC after), then using spray drying, system
At shrimp shell protein hydrolysate.
Summary of the invention
In view of the deficienciess of the prior art, the present invention propose it is a kind of be applicable in bacillus coagulans fermentation compound nitrogen source and
Application method, the compound nitrogen source are prepared by sesame seed meal protein hydrolysate, maize yellow-powder protein hydrolysate, shrimp shell protein hydrolysate, will
Compound nitrogen source is applied in the fermentation of bacillus coagulans, it can be achieved that the fast and efficiently fermentation of bacillus coagulans, solves existing
There is fermentation period in technology long, the problems such as high production cost.
Inventor passes through a large number of experiments and unremitting effort, is finally obtained a kind of answering for applicable bacillus coagulans fermentation
Nitrogen source is closed, which is mixed by sesame seed meal protein hydrolysate, maize yellow-powder protein hydrolysate, shrimp shell protein hydrolysate, three
Mass ratio be 2:2:1.
Preferably, a kind of compound nitrogen source being applicable in bacillus coagulans fermentation as described above, the compound nitrogen source
Additive amount be the 3-5% for putting tank volume.
A kind of application method for the compound nitrogen source being applicable in bacillus coagulans fermentation, the compound nitrogen source are added using non-uniform flow
Method be applied to bacillus coagulans fermentation.
Preferably, the application method of a kind of compound nitrogen source being applicable in bacillus coagulans fermentation as described above, it is described multiple
Close application method of the nitrogen source in fermentation are as follows: the seed liquor of bacillus coagulans, fermentation are accessed into the fermentor equipped with bottom water
10-18h, fermentation temperature are 45 DEG C -55 DEG C, and stream adds carbon source, nitrogen source and sodium hydroxide solution, specific stream plus side in batches during fermentation
Method are as follows:
Ferment 0-4h, and the flow acceleration of compound nitrogen source is the 0.5-2%, revolving speed 100-150r/min of the volume of stream aggregation per hour；Hair
Ferment 4-10h, the flow acceleration of compound nitrogen source are the 2-15%, revolving speed 150-200r/min of the volume of stream aggregation per hour；Ferment 10-
16h, the flow acceleration of compound nitrogen source are 0.5-2%, the revolving speed 150-200r/min of the volume of stream aggregation per hour, while flowing and adding ferment
Mother's leaching powder solution, flow acceleration are the 8-16% of the stream of stream aggregation per hour plus volume, and dissolved oxygen amount is controlled 30%；Fermentation final stage
16-18 stops stream plus nitrogen source；
Ferment 0-6h, and the flow acceleration of stream plus carbon source is the 2.5-6.25% of the volume of stream aggregation per hour；Ferment 6-10h, stream plus carbon
The flow acceleration in source is the 3.75-6.25% of the volume of stream aggregation per hour；Ferment 10-16h, and the flow acceleration of stream plus carbon source is every
The 3.75-6.25% of hour stream aggregation volume, full fermentation process while stream add the sodium hydroxide solution of 1-2mol/L, and control stream adds
Speed maintains pH in 5.5-6.5.
Preferably, the application method of a kind of compound nitrogen source being applicable in bacillus coagulans fermentation as described above, it is described solidifying
Grafting bud spore bacillus seed liquor prepares culture medium are as follows: glucose 20g/L, yeast extract 2.5g/L, peptone 5g/L, beef extract
5g/L, 12.8g/L sesame seed meal protein hydrolysate powder, 8.5g/L maize yellow-powder protein hydrolysate powder, 3.7g/L shrimp shell protein hydrolysate powder, lemon
Lemon acid hydrogen diammonium 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, pH5.0-
6.0。
Preferably, the application method of a kind of compound nitrogen source being applicable in bacillus coagulans fermentation as described above, the hair
Bottom water in fermentation tank is made of water, yeast powder, dipotassium hydrogen phosphate, epsom salt, manganese sulfate, and pH is adjusted to 5.0-6.0.
Preferably, a kind of application method for the compound nitrogen source for being applicable in bacillus coagulans as described above, flow plus it is compound
Nitrogen concentration is 90-150g/L, yeast extract solution is 20-40g/L, carbon source is 400-500g/L glucose solution.
Compared with prior art, advantages of the present invention are as follows:
1, by the compound nitrogen source application of sesame seed meal protein hydrolysate powder, maize yellow-powder protein hydrolysate powder, shrimp shell protein hydrolysate powder composition
In the fermentation of bacillus coagulans, using non-uniform flow plus method, significantly shortening fermentation period, in 10L fermentation cylinder for fermentation
Period, only 10-18h, coagulated bacillus living number were up to 1.5*1011CFU/mL；In 500L fermentor, ferment 16-18h, coagulates
Knot bacillus viable count is 2.4*1011CFU/mL realizes bacillus coagulans and fast and efficiently ferments.
2, sesame seed meal protein hydrolysate powder, maize yellow-powder protein hydrolysate powder, shrimp shell protein hydrolysate powder price that the present invention uses
It is cheap, the fermentation costs of bacillus coagulans are greatly reduced, and raw material is easy to get, is more suitable for the industrial metaplasia of bacillus coagulans
It produces.
Figure of description
Fig. 1 is that the optimum addition of compound nitrogen source in bacillus coagulans fermentation screens figure.
Specific embodiment
Form is described in further detail above content of the invention again by the following examples, but should not manage this
For solution for the scope of the above subject matter of the present invention is limited to the following embodiments, all technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Embodiment 1: the optimal additive amount of compound nitrogen source prepares bacillus coagulans
(1) bacterial strain
Bacillus coagulans (Bacillus coagulans) IDCC1201, the laboratory Hubei University Of Technology Fermentation Engineering A613 is protected
Hiding.
(2) prepared by raw material
Sesame seed meal: protein content is 39% in raw material, and it is pre- to carry out degreasing to sesame seed meal first with 1% Tween 80 surfactant
Processing, pretreatment time 1h, after room temperature is washed 3 times, centrifuging and taking precipitating carries out proteolysis, is located in advance using hydrolysis by novo
Sesame seed meal after reason, hydrolysis time 4h, 55 DEG C of temperature, material-water ratio 1:10, hydrolysis gained soluble protein is through being concentrated in vacuo (vacuum
Spend 0.8Mpa, temperature 50oC after), then spray drying is used, sesame seed meal protein hydrolysate powder, protein content 82- is made
87%, the total nitrogen recovery of sesame seed meal is 87% or more.
Maize yellow-powder: protein content is 58.9% in raw material, and maize yellow-powder is first smashed it through 60 mesh by airslide disintegrating mill
Sieve, the maize yellow-powder for crushing and being sieved is added and is tuned into certain concentration from water, is first handled with cellulase: temperature 50 C,
PH5.0, enzyme concentration 0.6%, 3.0 h of time, material-water ratio 1:3.It is handled again using alpha-amylase: 65 DEG C of temperature, pH6.5, enzyme concentration
0.8%, sodium sulfite denaturation is being added in 1.0 h of time, material-water ratio 1:4, and temperature 45 C, material-water ratio 1:4,30 min of time add
Enter amount 6%, after adjusting concentration and pH, addition papain hydrolysis, temperature 60 C, pH6.5, material-water ratio 1:6, time 5h,
Hydrolyzate, is then centrifuged by enzyme concentration 3%, and washing collects supernatant, is concentrated in vacuo (vacuum degree 0.8Mpa, temperature 50oC)
Afterwards, then use is spray-dried, maize yellow-powder protein hydrolysate powder, protein content 81-84% is made.Maize yellow-powder total nitrogen
The rate of recovery is 95.1%.
Shrimp shell protein hydrolysate: protein content is 25-30% in shrimp husk as raw material.First shrimp shell is crushed, after broken
Shrimp shell add water and be heated to 50 DEG C, preheat 30min, alkali protease added to be hydrolyzed, temperature 50 C, hydrolysis time 5 hours,
Material-water ratio 1:3, pH8.0 are filtered after enzyme deactivation and are obtained filtrate again, are concentrated in vacuo (vacuum degree 0.8Mpa, temperature 50oC after),
Spray drying is used again, cray shell protein hydrolysate powder, protein content 82-84% is made.The recycling of cray shell total nitrogen
Rate is 66.92%.
(3) prepared by culture medium
Actication of culture fluid nutrient medium contains: glucose 2%, yeast extract 1%, peptone 2%, pH6.0-7.0；
Liquid seed culture medium contains: glucose 20g/L, yeast extract 2.5g/L, peptone 5g/L, beef extract 5g/L,
12.8g/L sesame seed meal protein hydrolysate powder, 8.5g/L maize yellow-powder protein hydrolysate powder, 3.7g/L shrimp shell protein hydrolysate powder, citric acid
Hydrogen diammonium 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, pH6.0-7.0.
(4) the optimal additive amount of compound nitrogen source
Compound nitrogen source presses 1%, 2%, 3%, 4% respectively, 5%, 6%(W/V) ratio be added to 100mL and contain 2%(W/V) glucose
In the 500mL triangular flask of liquid, 1%(V/V is accessed) the bacillus coagulans seed bacterium solution that has activated, pH5.0-6.0 is adjusted,
After 45-55 DEG C of culture 16-18h, coagulated bacillus living number is measured with dilution spread flat band method.The result is shown in Figure 1.It can by Fig. 1
Know, additive amount is in 3-5%(W/V) when, the viable count of bacillus coagulans is higher, and adds 4%(W/V) compound nitrogen source when,
Coagulated bacillus living number highest, can reach 2.14*1010CFU/mL。
Using fed-batch mode fermentation bacillus coagulans on embodiment 2:10L fermentor
(1) bacterial strain: with embodiment 1.
(2) culture medium
Actication of culture Liquid Culture is the same as embodiment 1；
Liquid seed culture medium is the same as embodiment 1；
Secondary seed medium contains: glucose 2%, yeast extract 5%, peptone 1%, beef extract 1%, the hydrolysis of 1.28% sesame seed meal
Albumen powder, 0.85% maize yellow-powder protein hydrolysate powder, 0.37% shrimp shell protein hydrolysate powder, diammonium hydrogen citrate 0.2%, sodium acetate
0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.058%, manganese sulfate 0.025%, pH5.0-6.0；
Bottom water contains in 10L fermentation tank level top fermentation: water 1.5L, yeast powder 15g, dipotassium hydrogen phosphate 1.2g, epsom salt
0.2g, manganese sulfate 0.375g, pH are adjusted to 5.0-6.0.
The preparation of stream plus nitrogen source: the volume of stream plus nitrogen source is 3L, and nitrogen source and 1L including 2L 250g containing compound nitrogen source contain ferment
The nitrogen source of female extract 30g, 115 DEG C of sterilizing 20min.
The preparation of stream plus carbon source: the volume of stream plus carbon source is 1.6L, and composition is glucose 750g, 115 DEG C of sterilizing 20min.
The preparation of stream plus aqueous slkali: the volume of stream plus aqueous slkali is 500mL, and composition is sodium hydroxide, concentration 1.5mol/
L, 115 DEG C of sterilizing 20min.
(3) fermented and cultured
Multistage fed-batch cultivation: by bacillus coagulans seed liquor obtained in secondary seed medium by the 3%(V/ for putting tank volume
V) access is equipped in the 10L automatic fermenter of 1.5L bottom water, and 45-55 DEG C of fermentation temperature, fermentation period 14-18h.Earlier fermentation
0-4h, compound nitrogen source flow acceleration are as follows: 10mL/h -40mL/h, revolving speed 100-150r/min；Ferment middle 4-10h, composite nitrogen
Source stream acceleration are as follows: 40 mL/h -300mL/h, revolving speed 150-200r/min；Ferment later period 10-16h, and composite nitrogen source stream accelerates
Degree are as follows: 40mL/h -100mL/h, while flowing and adding yeast extract: 80 mL/h -160mL/h, revolving speed 150-200r/min, dissolved oxygen
Amount control is 30%；Ferment final stage 16-18h, stops stream plus nitrogen source.
Ferment 0-6h, and carbon source flow acceleration is 40mL/h-100mL/h；Ferment middle 6-10h, carbon source flow acceleration are
60mL/h-100mL/h；Ferment later period 10-16h, and carbon source flow acceleration is 60mL/h-100mL/h.Full fermentation process flows simultaneously to be added
1-2mol/L sodium hydroxide solution controls flow acceleration, maintains pH in 5.0-7.0.
(4) measurement of viable count: dilution spread flat band method is used.
(5) result:
Fermentation time: 18 hours；
Viable count: 1.5*1011CFU/mL。
Using fed-batch mode fermentation bacillus coagulans on embodiment 3:500L fermentor
(1) bacterial strain: with embodiment 1.
(2) culture medium
Actication of culture fluid nutrient medium is the same as embodiment 1；
Liquid seed culture medium is the same as embodiment 1；
Secondary seed medium is the same as embodiment 1；
The preparation of stream plus nitrogen source: the volume of stream plus nitrogen source is 150L, and nitrogen source and 50L including 100L 12kg containing compound nitrogen source contain ferment
The nitrogen source of female extract 2kg, 115 DEG C of sterilizing 20min.
The preparation of stream plus carbon source: the volume of stream plus carbon source is 80L, and composition is glucose 40kg, 115 DEG C of sterilizing 20min.
The preparation of stream plus aqueous slkali: the volume of stream plus aqueous slkali is 300L, and composition is sodium hydroxide, concentration 2mol/L,
115 DEG C of sterilizing 20min.
(3) fermented and cultured
Multistage fed-batch cultivation: by bacillus coagulans obtained in secondary seed medium by the 3%(V/V for putting tank volume) access
In 500L automatic fermenter equipped with 75L bottom water, 45-55 DEG C of fermentation temperature, fermentation period 14-18h.Earlier fermentation 0-4h,
Compound nitrogen source flow acceleration are as follows: 0.5L -2L/h, revolving speed 100-150r/min；Ferment middle 4-10h, compound nitrogen source flow acceleration
Are as follows: 2L-15L/h, revolving speed 150-200r/min；Ferment later period 10-16h, compound nitrogen source flow acceleration are as follows: 2L -5L/h, simultaneously
Stream plus yeast extract: 4L-8L/h, revolving speed 150-200r/min, dissolved oxygen amount are controlled 30%；Ferment final stage 16-18h, stops
Stream plus nitrogen source.
Ferment 0-6h, and carbon source flow acceleration is 2L-5L/h；Ferment middle 6-10h, carbon source flow acceleration are 3L-5L/h；Hair
Ferment later period 10-16h, carbon source flow acceleration are 3L-5L/h.Full fermentation process flows simultaneously plus 2mol/L sodium hydroxide solution, control
Flow acceleration maintains pH in 5.0-7.0.
(4) measurement of viable count: dilution spread flat band method is used.
(5) result:
Fermentation time: 18 hours；
Viable count: 2.4*1011CFU/mL。
Comparative example 1
Implementation method flows the compound nitrogen source added with embodiment 2, but in fermentation process compared with Example 2, only lacks shrimp shell hydrolysis
Albumen measures viable count after fermentation.As a result:
Fermentation time: 44 hours
Viable count: 2.1*108CFU/mL。
Comparative example 2
Implementation method flows the compound nitrogen source added with embodiment 2, but in fermentation process compared with Example 2, only lacks sesame seed meal water
Albumen is solved, measures viable count after fermentation.As a result:
Fermentation time: 36 hours
Viable count: 4.5*108CFU/mL。
Comparative example 3
It is sesame seed meal protein hydrolysate, maize yellow-powder hydrolysis that implementation method, which flows the compound nitrogen source added with embodiment 2, but in fermentation process,
Albumen, shrimp shell protein hydrolysate mix, and the mass ratio of three is 1:1:1, measure viable count after fermentation.As a result:
Fermentation time: 44 hours
Viable count: 3.4*109CFU/mL。
Comparative example 4
It is sesame seed meal protein hydrolysate, maize yellow-powder water that implementation method, which flows the compound nitrogen source added with embodiment 2, but in fermentation process,
Solution albumen, yeast extract mix, and the mass ratio of three is 2:2:1, measure viable count after fermentation.As a result:
Fermentation time: 28 hours
Viable count: 1.2*1010CFU/mL。
Claims (7)
1. a kind of compound nitrogen source for being applicable in bacillus coagulans fermentation, it is characterised in that: the compound nitrogen source hydrolyzes egg by sesame seed meal
White, maize yellow-powder protein hydrolysate, shrimp shell protein hydrolysate mix, and the mass ratio of three is 2:2:1.
2. a kind of compound nitrogen source for being applicable in bacillus coagulans fermentation as described in claim 1, it is characterised in that: described compound
The additive amount of nitrogen source is the 3-5% for putting tank volume.
3. a kind of application method for the compound nitrogen source for being applicable in bacillus coagulans fermentation, it is characterised in that: the compound nitrogen source is adopted
With non-uniform flow plus method be applied in the multistage fermentation of bacillus coagulans.
4. a kind of application method of compound nitrogen source for being applicable in bacillus coagulans fermentation as claimed in claim 3, feature exist
In: application method of the compound nitrogen source in bacillus coagulans fermentation are as follows: condensation is accessed into the fermentor equipped with bottom water
The seed liquor of bacillus, fermentation 10-18h, fermentation temperature are 45 DEG C -55 DEG C, and stream adds carbon source, nitrogen source and hydrogen in batches during fermentation
Sodium hydroxide solution, specific feeding method are as follows:
Ferment 0-4h, and the flow acceleration of compound nitrogen source is the 0.5-2%, revolving speed 100-150r/min of the volume of stream aggregation per hour；Hair
Ferment 4-10h, the flow acceleration of compound nitrogen source are the 2-15%, revolving speed 150-200r/min of the volume of stream aggregation per hour；Ferment 10-
16h, the flow acceleration of compound nitrogen source are 0.5-2%, the revolving speed 150-200r/min of the volume of stream aggregation per hour, while flowing and adding ferment
Mother's leaching powder solution, flow acceleration are the 8-16% of the stream of stream aggregation per hour plus volume, and dissolved oxygen amount is controlled 30%；Fermentation final stage
16-18 stops stream plus nitrogen source；
Ferment 0-6h, and the flow acceleration for flowing carbon source is the 2.5-6.25% of the volume of stream aggregation per hour；Ferment 6-10h, stream plus carbon source
Flow acceleration be per hour stream aggregation volume 3.75-6.25%；Ferment 10-16h, and the flow acceleration of stream plus carbon source is per small
When stream aggregation volume 3.75-6.25%, full fermentation process simultaneously flow plus 1-2mol/L sodium hydroxide solution, control stream accelerate
Degree maintains pH in 5.5-6.5.
5. a kind of application method of compound nitrogen source for being applicable in bacillus coagulans fermentation as claimed in claim 4, feature exist
In: the bacillus coagulans seed liquor prepares culture medium are as follows: glucose 20g/L, yeast extract 2.5g/L, peptone 5g/
L, beef extract 5g/L, 12.8g/L sesame seed meal protein hydrolysate powder, 8.5g/L maize yellow-powder protein hydrolysate powder, the hydrolysis of 3.7g/L shrimp shell
Albumen powder, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/
L, pH5.0-6.0.
6. a kind of application method of compound nitrogen source for being applicable in bacillus coagulans fermentation as claimed in claim 4, feature exist
In: the bottom water of the fermentor is made of water, yeast powder, dipotassium hydrogen phosphate, epsom salt, manganese sulfate, and pH is adjusted to 5.0-
6.0。
7. a kind of application method of compound nitrogen source for being applicable in bacillus coagulans fermentation as claimed in claim 4, feature exist
In: the concentration for flowing the compound nitrogen source added is 90-150g/L, yeast extract solution is 20-40g/L, stream plus carbon source are 400-500g/
L glucose solution.
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