CN102329831A - Method for producing levan by fermenting soy molasses - Google Patents
Method for producing levan by fermenting soy molasses Download PDFInfo
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Abstract
The invention relates to a method for producing levan by fermenting soy molasses, which comprises the following steps: (1) preparation of spore fungus suspension: inoculating Bacillus licheniformis TJZKBA10658 onto a culture medium slope, culturing at the constant temperature of 30 DEG C for 2-4 days, and adding 0.75 wt% normal saline to wash mycelia, thereby obtaining a spore fungus suspension; (2) inoculating the spore fungus suspension into a sterilized liquid seed culture medium to obtain a liquid seed; (3) inoculating the primary liquid seed into a sterilized 5L seed tank, and inoculating 5-12 vol% of the liquid culture medium into the cooled fermentation tank culture medium as a secondary liquid seed; (4) inoculating the secondary liquid seed into the sterilized fermentation tank, and fermenting for 14-30 hours; and (5) after the fermentation finishes, centrifugalizing the fermentation liquid at 6000rpm for 15 minutes to remove the thallus, passing the supernatant through an ultrafiltration membrane, concentrating the trapped liquid, and carrying out powder spraying to obtain the levan of which the yield is 42-138kg. The invention has the advantages of simple technique and obvious effect.
Description
Technical field
The present invention relates to a kind of method of fermentative prodn Polylevulosan, particularly a kind of method of utilizing soy molasses fermentative prodn Polylevulosan.
Background technology
Soy molasses is to produce the sub product that obtains in the soybean protein concentrate process, contains abundant soybean oligosaccharide in the soy molasses, can be used as the raw materials for production of functional soy oligose.Wherein staple content is: total reducing sugar 54.64% (wherein sucrose 32.17 %, cottonseed sugar 4.27%, stachyose 17.54%), total lipoid 18.00%, total steroid glucoside 3.06%, total phospholipids 8.79%, crude protein 8.71%, ash content 8.69%.
Polylevulosan (levan) is a kind of exocellular polysaccharide with extensive use, wraps in the ball-like structure by residue of fructose and forms, and the polysaccharide that the D-fructose that links to each other with β-2,6 glycosidic link constitutes, molecular formula is (C
18H
32O
16)
nMany mikrobes can both produce polymer outside the born of the same parents (EPS) in physical environment, Polylevulosan (levan) is only found wherein a kind of, and the outer polymer majority of these born of the same parents is present in the rapid brook.They can protect mikrobe to avoid dry and toxic erosion infringement, and can store carbon source.Polylevulosan (levan) also can be separated in the cell debris from commodity production.Pharmaceutically, Polylevulosan (levan) can be used as subcutaneous injection and uses SUV, the substitute of antitumor, immunomodulator, anti-inflammatory drug and blood plasma.On food, be used as two fructofuranoses, fructose, oligofructose, also can be used as the substitute of emulsifying agent, encapsulation agent, pigment and flavouring agent and fat.
Levan just came to light before more than 100 years, because its character is different from other polysaccharide, so caused the great interest of people.Just be used in medically at the beginning and consumption seldom because cost is very high, people are devoted to scale operation Polylevulosan (levan), realize maximum economic interests.
Therefore, the method that a kind of technology is simple, obvious results is utilized soy molasses fermentative prodn Polylevulosan (levan) is provided, can effectively reduces cost, improve productive rate.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art, provide a kind of efficient feasible and can reduce Polylevulosan (levan) production cost, reduce investment, be easy to the method for utilizing soy molasses fermentative prodn Polylevulosan of production application.
Following for realizing the embodiment that above-mentioned purpose the present invention adopted:
A kind of method of utilizing soy molasses fermentative prodn Polylevulosan is characterized in that implementation step is following:
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and comprised the 1L of unit: glucose 2g, yeast powder 5g, peptone 10g, agar 15g are settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, liquid seed culture medium is formed and comprised the 1L of unit: sucrose 20-60g, yeast powder 4-8g, peptone 5-10g are settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 5-12% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then; The bottled liquid measure of shaking of 500ml specification is 100ml; Shake on the shaking table that bottle is 120-200rpm in 30-40 ℃, rotating speed and cultivated 24 hours, obtain liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 5-12% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, and leavening temperature 30-40 ℃, dissolved oxygen is controlled at 30-40%, and incubation time 24h is as the secondary liquid seeds;
(4) above-mentioned secondary liquid seeds is used for being seeded to the 500L fermentor tank after the sterilization, soy molasses is diluted 1-3 doubly; Be settled to 300L with zero(ppm) water, pH value 5.0-8.0 cools off fermentor cultivation based on sterilization in 115 ℃, 15 minutes again; Inoculum size according to volume ratio 5-12% inserts liquid seeds in the above-mentioned refrigerative fermentation tank culture medium then, and leavening temperature 30-40 ℃, dissolved oxygen is controlled at 30-40%, fermentation time 14-30 hour;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 6000-8000 dalton ultra-filtration membrane, trapped fluid concentrates and dusts, and the output that obtains Polylevulosan (levan) is 42-138kg.
The invention has the beneficial effects as follows: the present invention utilizes soy molasses fermentative prodn Polylevulosan (levan); Can significantly reduce fermentation costs, common uses sucrose higher as the cost of fermenting raw materials, but utilizes the soy molasses fermentation; Both solved that big soy molasses is deposited difficulty, discharging has a pollution problems; Greatly reduce fermentation costs again, unemployed magazine can all be removed through ultra-filtration membrane in the soy molasses, can effectively improve productive profit.And do not need complex apparatus, treatment process is simple, and fermenting process is simple, is beneficial to the popularization of the Application and Development of Polylevulosan (levan).This method is of very high actual application value, and the market advantage is very obvious, and is suitable for suitability for industrialized production.
Embodiment
Below in conjunction with embodiment, to details are as follows according to embodiment provided by the invention:
Embodiment 1
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and is comprised the 1L of unit: glucose 2g, and yeast powder 5g, peptone 10g, agar 15g is settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis (Bacillus licheniformis) TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, and liquid seed culture medium is formed and comprised the 1L of unit: sucrose 60g, and yeast powder 5g, peptone 10g is settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 10% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then, and the bottled liquid measure of shaking of 500ml specification is 100ml, shakes on the shaking table that bottle is 160rpm in 35 ℃, rotating speed and cultivates 24 hours, obtains liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 10% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 35 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, and incubation time 24h is as the secondary liquid seeds;
(4) above-mentioned secondary liquid seeds is used for being seeded to the 500L fermentor tank after the sterilization, and to 300L, pH value is adjusted to 7.0, and fermentor cultivation is cooled off based on sterilization in 115 ℃, 15 minutes again with 3 times of 100L soy molasses dilutions; Inoculum size according to volume ratio 10% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 35 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, fermentation time 14 hours;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 8000 dalton's ultra-filtration membranes, trapped fluid concentrates and dusts, and obtains Polylevulosan (levan) 42.1kg.
Embodiment 2
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and is comprised the 1L of unit: glucose 2g, and yeast powder 5g, peptone 10g, agar 15g is settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis (Bacillus licheniformis) TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, and liquid seed culture medium is formed and comprised the 1L of unit: sucrose 40g, and yeast powder 4g, peptone 6g is settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 5% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then, and the bottled liquid measure of shaking of 500ml specification is 100ml, shakes on the shaking table that bottle is 160rpm in 40 ℃, rotating speed and cultivates 24 hours, obtains liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 5% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 40 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, and incubation time 24h is as the secondary liquid seeds;
(4) the aforesaid liquid seed is used for being seeded to the 500L fermentor tank after the sterilization, with the 200L soy molasses with 1.5 times of distilled water dilutings to 300L, pH value 6.5, with fermentor cultivation based on sterilization in 115 ℃, 15 minutes cooling again; Inoculum size according to volume ratio 5% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 40 ℃ of leavening temperatures, and dissolved oxygen is controlled at 40%, fermentation time 20 hours;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 6000 dalton's ultra-filtration membranes, trapped fluid concentrates and dusts, and obtains Polylevulosan (levan) 93.5kg.
Embodiment 3
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and is comprised the 1L of unit: glucose 2g, and yeast powder 5g, peptone 10g, agar 15g is settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis (Bacillus licheniformis) TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, and liquid seed culture medium is formed and comprised the 1L of unit: sucrose 60g, and yeast powder 7g, peptone 7g is settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 12% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then, and the bottled liquid measure of shaking of 500ml specification is 100ml, shakes on the shaking table that bottle is 160rpm in 30 ℃, rotating speed and cultivates 24 hours, obtains liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 5% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 30 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, and incubation time 24h is as the secondary liquid seeds;
(4) the aforesaid liquid seed is used for being seeded to the 500L fermentor tank after the sterilization, and the 150L soy molasses is diluted to 300L, pH value 8.0, with fermentor cultivation based on sterilization in 115 ℃, 15 minutes cooling again; Inoculum size according to volume ratio 8% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 30 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, fermentation time 24 hours;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 8000 dalton's ultra-filtration membranes, trapped fluid concentrates and dusts, and obtains Polylevulosan (levan) 72.4g.
Embodiment 4
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and is comprised the 1L of unit: glucose 2g, and yeast powder 5g, peptone 10g, agar 15g is settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis (Bacillus licheniformis) TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, and liquid seed culture medium is formed and comprised the 1L of unit: sucrose 50g, and yeast powder 8g, peptone 5g is settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 10% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then, and the bottled liquid measure of shaking of 500ml specification is 100ml, shakes on the shaking table that bottle is 160rpm in 35 ℃, rotating speed and cultivates 24 hours, obtains liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 5% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 30 ℃ of leavening temperatures, and dissolved oxygen is controlled at 30%, and incubation time 24h is as the secondary liquid seeds;
(4) the aforesaid liquid seed is used for being seeded to the 500L fermentor tank after the sterilization, with the 300L soy molasses as fermention medium, pH value 7.5, with fermentor cultivation based on sterilization in 115 ℃, 15 minutes cooling again; Inoculum size according to volume ratio 12% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, 37 ℃ of leavening temperatures, and dissolved oxygen is controlled at 40%, fermentation time 30 hours;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 6000 dalton's ultra-filtration membranes, trapped fluid concentrates and dusts, and obtains Polylevulosan (levan) 136.9kg.
Above-mentioned detailed description of the method for utilizing soy molasses fermentative prodn Polylevulosan (levan) being carried out with reference to embodiment; Be illustrative rather than determinate; Can be according to institute's limited range several embodiment that give an example out; Therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (1)
1. method of utilizing soy molasses fermentative prodn Polylevulosan is characterized in that implementation step is following:
(1) preparation of pityrosporion ovale suspension: Bacillus licheniformis TJZKBA10658 is inoculated on the medium slant; In 30 ℃ of constant temperature culture 2-4 days; The adding mass concentration is 0.75% saline water washing mycelium, obtains the pityrosporion ovale suspension, and suspension miospore content is about 10
5-10
6Individual, for use; Slant medium is formed and comprised the 1L of unit: glucose 2g, yeast powder 5g, peptone 10g, agar 15g are settled to 1L with zero(ppm) water, pH value 7.0; Wherein used bacterial classification: Bacillus licheniformis TJZKBA10658;
(2) above-mentioned pityrosporion ovale suspension is used for being seeded to the liquid seed culture medium after the sterilization, liquid seed culture medium is formed and comprised the 1L of unit: sucrose 20-60g, yeast powder 4-8g, peptone 5-10g are settled to 1L with zero(ppm) water, pH value 7.0; Liquid seed culture medium is cooled off in sterilization in 121 ℃, 15 minutes again; Inoculum size according to volume percent 5-12% inserts the pityrosporion ovale suspension in the above-mentioned refrigerative liquid seed culture medium then; The bottled liquid measure of shaking of 500ml specification is 100ml; Shake on the shaking table that bottle is 120-200rpm in 30-40 ℃, rotating speed and cultivated 24 hours, obtain liquid seeds;
(3) above-mentioned level liquid seed is used for being seeded to the 5L seeding tank after the sterilization, and substratum is identical with the aforesaid liquid seed culture medium, liquid amount 3L; With fermentor cultivation based on 121 ℃, 15min sterilization cooling again; Inoculum size according to volume ratio 5-12% inserts liquid nutrient medium in the above-mentioned refrigerative fermentation tank culture medium then, and leavening temperature 30-40 ℃, dissolved oxygen is controlled at 30-40%, and incubation time 24h is as the secondary liquid seeds;
(4) above-mentioned secondary liquid seeds is used for being seeded to the 500L fermentor tank after the sterilization, soy molasses is diluted 1-3 doubly; Be settled to 300L with zero(ppm) water, pH value 5.0-8.0 cools off fermentor cultivation based on sterilization in 115 ℃, 15 minutes again; Inoculum size according to volume ratio 5-12% inserts liquid seeds in the above-mentioned refrigerative fermentation tank culture medium then, and leavening temperature 30-40 ℃, dissolved oxygen is controlled at 30-40%, fermentation time 14-30 hour;
(5) after the fermentation ends, 6000 rev/mins of fermented liquids were removed thalline in centrifugal 15 minutes, get supernatant and cross molecular weight 6000-8000 dalton ultra-filtration membrane, trapped fluid concentrates and dusts, and the output that obtains Polylevulosan levan is 42-138kg.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815476A (en) * | 2017-12-26 | 2018-03-20 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
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2011
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815476A (en) * | 2017-12-26 | 2018-03-20 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
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Application publication date: 20120125 |