CN109266709A - Nisin production technology - Google Patents

Nisin production technology Download PDF

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Publication number
CN109266709A
CN109266709A CN201710580398.4A CN201710580398A CN109266709A CN 109266709 A CN109266709 A CN 109266709A CN 201710580398 A CN201710580398 A CN 201710580398A CN 109266709 A CN109266709 A CN 109266709A
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CN
China
Prior art keywords
production technology
fermentation
glucose
nisin
obtains
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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CN201710580398.4A
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Chinese (zh)
Inventor
姜均
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SICHUAN ANYI BIOTECHNOLOGY CO Ltd
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SICHUAN ANYI BIOTECHNOLOGY CO Ltd
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Priority to CN201710580398.4A priority Critical patent/CN109266709A/en
Publication of CN109266709A publication Critical patent/CN109266709A/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins

Abstract

The present invention provides a kind of nisin production technology, it is characterised in that: specifically includes step;(1), Lactococcus lactis strain is prepared;(2), it ferments;Lactococcus lactis strain is placed in fermentor, and glucose is added and ferments, obtains fermentation liquid;Concentration of glucose is controlled in fermentation process;(3), it extracts;A: fermentation liquor pretreatment obtains filtrate;B: filtrate concentration obtains concentration filtrate;C: concentration filtrate is saltoutd, and filter residue is obtained by filtration;D: filter residue affords eluent;(4) eluent is spray-dried, and obtains powdery nisin product.The production technology is easy to operate, and obtained nisin good product quality.

Description

Nisin production technology
Technical field
The present invention relates to a kind of production technology of microorganism, in particular to a kind of production technology of nisin.
Background technique
Nisin (Nisin) is also known as nisin or transliteration is that Buddhist nun is pungent, is the one of streptococcus lactis generation Kind peptide material, is made of, molecular weight is about 3500Da 34 amino acid residues.Due to nisin can inhibit it is most of Gram-positive bacterium, and have strong inhibiting effect to the spore of bacillus, therefore answered extensively as food preservative For food service industry.It is hydrolyzed into amino acid quickly under physiological pH condition and α-chymotrypsin protein enzyme effect of human body after edible, Normal flora in human body intestinal canal will not be changed and generate the resistance problem occurred such as other antibiotic, will not more be resisted with other There is cross tolerance in rhzomorph, is a kind of antiseptics for natural food efficient, nontoxic, safe, without side-effects.
Currently, there are many preparation methods of nisin, but technical staff is dedicated to finding a kind of easy to operate and produce The measured preparation method of quality.
Summary of the invention
The present invention provides a kind of nisin production technology, it is therefore an objective to solve prior art problem, provide a kind of easy The preparation method of operation and good product quality.
The present invention solve the problems, such as the technical solution adopted is that:
Nisin production technology, through strain preparation, fermentation, extraction process, it is characterised in that: specifically include Step;
(1), Lactococcus lactis strain is prepared;
(2), it ferments;Glucose is added in fermentor to ferment, obtains fermentation liquid;Concentration of glucose control in fermentation process System is between 3~8g/L, and PH is between 6.0~6.8;
(3), it extracts;
A: fermentation liquor pretreatment-fermentation liquid salt acid for adjusting pH to 2.5~2.7, after be warming up to 80 DEG C~85 DEG C, at this 20min is maintained under part, then reduces temperature to 50 DEG C~60 DEG C, filtrate is obtained by filtration, temperature when control is filtered is 50~60 ℃;
B: concentration-filtrate is concentrated into solid content 30~35%, obtains concentrate;
C: saltout-concentrate is cooled to 30~35 DEG C;It is slowly added to piece alkali, adjusts concentrate pH to 3.2~3.4, is stood 2h-3h;The perlite for accounting for concentrate mass fraction 5 ‰ is added in concentrate after standing, filtering obtains filter residue;
D: elution-purified water dissolves filter residue, adjusts pH to 2.5 or so, stirs 1h, carries out plate-frame filtering, collects clear wash De- liquid, the step carry out at least once;
(4) sodium chloride, which will be added, in spray drying-in eluent that step D is obtained makes filtrate solid content in 3%-4%, It places and is spray-dried in spray dryer afterwards, wherein 200~220 DEG C of inlet air temperature of control, obtains by 80~85 DEG C of leaving air temp Powdery nisin product.
In certain embodiments, in step (2), fermentation initial stage concentration of glucose control is in 10g/L.
In certain embodiments, in step A, fermentation liquid is filtered using ceramic membrane.
In certain embodiments, the elution process of step D carries out 3 times, and the eluent repeatedly afforded is merged.
In certain embodiments, in step C, using plate-frame filtering, and with perlite bed board when filtering.
In certain embodiments, there may be more foams in step B, when concentration, therefore defoaming can be added in right amount Agent.
In certain embodiments, in step (1) strain preparation process, take the separation of glycerol tube strain plate streaking single Bacterium colony, in 30 DEG C of incubator cultures, left and right, the big single colonie of picking are connected to shaking flask culture 16h or so and obtain Lactococcus lactis for 24 hours Strain, glycerol tube preservation control glycerol concentration 15~30%, are put into -20 DEG C of Storage in refrigerator.
In certain embodiments, the seed culture medium (g/L) used in strain preparation process: glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.0~7.2 is adjusted, sterilize 20min at 121 DEG C, and liquid amount is 200mL/500mL;Medium of shaking flask fermentation (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO4 5、NaCl 4、 Tween-80 1 adjusts pH7.5;Sterilize 10min at 115 DEG C.
In certain embodiments, in step (2) fermentation process, the seed culture medium (g/L) that uses: glucose 6, egg White peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.0~7.2 or so are adjusted, sterilize 20min at 121 DEG C.
In certain embodiments, in step (2) fermentation process, strain fermentation uses in fermentor fermentation medium (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1 adjust pH7.5.
Beneficial effects of the present invention: entire production process is easy to operate, and production process is easy to control, obtained nisin Good product quality.
Specific embodiment
The present invention is described in further details below in conjunction with specific embodiment.
Embodiment 1
Nisin production technology specifically includes step;
(1), Lactococcus lactis strain is prepared;
(2), it ferments;Lactococcus lactis strain is placed in fermentor, and glucose is added and ferments, obtains fermentation liquid;Hair Concentration of glucose control is in 3g/L during ferment, and PH is between 6.0, and initial stage concentration of glucose control of fermenting is in 10g/L.
Wherein seed culture medium (g/L) used in Spawn incubation process: glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.0 is adjusted, sterilize 20min at 121 DEG C.
Fermentation medium used in strain fermentation process (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1 adjust pH7.5.
(3), it extracts;
A: fermentation liquor pretreatment-fermentation liquid salt acid for adjusting pH to 2.5~2.7, after be warming up to 80 DEG C~85 DEG C, at this 20min is maintained under part, then reduces temperature to 50 DEG C, ceramic membrane filter obtains filtrate, and temperature when control is filtered is at 50 DEG C.
B: concentration-filtrate is concentrated into solid content 30%, obtains concentrate.There may be more foams when concentration, therefore can It is appropriate that defoaming agent is added.
C: saltout-concentrate is cooled to 35 DEG C;It is slowly added to piece alkali, adjusts concentrate pH to 3.2~3.4, stands 3h;It is quiet The perlite for accounting for concentrate mass fraction 5 ‰ is added in the concentrate postponed, obtains filter residue through plate-frame filtering with perlite bed board.
D: elution-purified water dissolves filter residue, adjusts pH to 2.5 or so, stirs 1h, carries out plate-frame filtering, collects clear wash De- liquid.The elution process of filter residue is carried out 3 times, the clarification eluent obtained after 3 elutions is collected.
(4) sodium chloride, which will be added, in spray drying-in eluent that step D is obtained makes filtrate solid content in 3%-4%, It places and is spray-dried in spray dryer afterwards, wherein 200~220 DEG C of inlet air temperature of control, obtains by 80~85 DEG C of leaving air temp Powdery nisin product.
Embodiment 2
Nisin production technology specifically includes step;
(1), Lactococcus lactis strain is prepared;
(2), it ferments;Lactococcus lactis strain is placed in fermentor, and glucose is added and ferments, obtains fermentation liquid;Hair Concentration of glucose control is between 5g/L during ferment, and PH is 6.5;Fermentation initial stage concentration of glucose is controlled in 10g/L.
Wherein seed culture medium (g/L) used in Spawn incubation process: glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.2 is adjusted, sterilize 20min at 121 DEG C.
Fermentation medium used in strain fermentation process (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1 adjust pH7.5.
(3), it extracts;
A: fermentation liquor pretreatment-fermentation liquid salt acid for adjusting pH to 2.5~2.7, after be warming up to 80 DEG C~85 DEG C, at this 20min is maintained under part, then reduces temperature to 55 DEG C, ceramic membrane filter obtains filtrate, and temperature when control is filtered is at 55 DEG C.
B: concentration-filtrate is concentrated into solid content 30%, obtains concentrate.There may be more foams when concentration, therefore can It is appropriate that defoaming agent is added.
C: saltout-concentrate is cooled to 30 DEG C;It is slowly added to piece alkali, adjusts concentrate pH to 3.2~3.4, stands 2h- 3h;The perlite for accounting for concentrate mass fraction 5 ‰ is added in concentrate after standing, is obtained with perlite bed board through plate-frame filtering Filter residue.
D: elution-purified water dissolves filter residue, adjusts pH to 2.5 or so, stirs 1h, carries out plate-frame filtering, collects clear wash De- liquid.The elution process of filter residue is carried out 3 times, the clear eluent obtained after 3 elutions is collected.
(4) sodium chloride, which will be added, in spray drying-in eluent that step D is obtained makes filtrate solid content in 3%-4%, It places and is spray-dried in spray dryer afterwards, wherein 200~220 DEG C of inlet air temperature of control, obtains by 80~85 DEG C of leaving air temp Powdery nisin product.
Embodiment 3
Nisin production technology specifically includes step;
(1), Lactococcus lactis strain is prepared;
(2), it ferments;Lactococcus lactis strain is placed in fermentor, and glucose is added and ferments, obtains fermentation liquid;Hair Concentration of glucose control is in 8g/L during ferment, and PH is 6.8;Fermentation initial stage concentration of glucose is controlled in 10g/L.
Wherein seed culture medium (g/L) used in Spawn incubation process: glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.0~7.2 or so are adjusted, sterilize 20min at 121 DEG C.
Fermentation medium used in strain fermentation process (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1 adjust pH7.5.
(3), it extracts;
A: fermentation liquor pretreatment-fermentation liquid salt acid for adjusting pH to 2.5~2.7, after be warming up to 80 DEG C~85 DEG C, at this 20min is maintained under part, then reduces temperature to 60 DEG C, ceramic membrane filter obtains filtrate, and temperature when control is filtered is at 60 DEG C.
B: concentration-filtrate is concentrated into solid content 33%, obtains concentrate.There may be more foams when concentration, therefore can It is appropriate that defoaming agent is added.
C: saltout-concentrate is cooled to 33 DEG C;It is slowly added to piece alkali, adjusts concentrate pH to 3.2~3.4, stands 2h- 3h;The perlite for accounting for concentrate mass fraction 5 ‰ is added in concentrate after standing, is obtained with perlite bed board through plate-frame filtering Filter residue.
D: elution-purified water dissolves filter residue, adjusts pH to 2.5 or so, stirs 1h, carries out plate-frame filtering, collects clear wash De- liquid.The elution process of filter residue is carried out 3 times, the clear eluent obtained after 3 elutions is collected.
(4) sodium chloride, which will be added, in spray drying-in eluent that step D is obtained makes filtrate solid content in 3%-4%, It places and is spray-dried in spray dryer afterwards, wherein 200~220 DEG C of inlet air temperature of control, obtains by 80~85 DEG C of leaving air temp Powdery nisin product.

Claims (10)

1. nisin production technology, through strain preparation, fermentation, extraction process, it is characterised in that: specifically include and walk Suddenly;
(1), Lactococcus lactis strain is prepared;
(2), it ferments;Glucose is added in fermentor to ferment, obtains fermentation liquid;Concentration of glucose control exists in fermentation process Between 3~8g/L, PH is between 6.0~6.8;
(3), it extracts;
A: fermentation liquor pretreatment-fermentation liquid salt acid for adjusting pH to 2.5~2.7, after be warming up to 80 DEG C~85 DEG C, under this condition 20min is maintained, then reduces temperature to 50 DEG C~60 DEG C, filtrate is obtained by filtration, temperature when control is filtered is at 50~60 DEG C;
B: concentration-filtrate is concentrated into solid content 30~35%, obtains concentrate;
C: saltout-concentrate is cooled to 30~35 DEG C;It is slowly added to piece alkali, adjusts concentrate pH to 3.2~3.4, stands 2h- 3h;The perlite for accounting for concentrate mass fraction 5 ‰ is added in concentrate after standing, filtering obtains filter residue;
D: elution-purified water dissolves filter residue, adjusts pH to 2.5 or so, stirs 1h, carries out plate-frame filtering, collects clear eluent, The step carries out at least once;
(4) sodium chloride, which will be added, in spray drying-in eluent that step D is obtained makes filtrate solid content in 3%-4%, after put It sets in spray dryer and is spray-dried, wherein 200~220 DEG C of inlet air temperature of control, obtains powdery by 80~85 DEG C of leaving air temp Nisin product.
2. nisin production technology as described in claim 1, it is characterised in that: in step (2), initial stage Portugal of fermenting Grape sugar concentration is controlled in 10g/L.
3. nisin production technology as described in claim 1, it is characterised in that: in step A, using ceramic membrane pair Fermentation liquid is filtered.
4. nisin production technology as described in claim 1, it is characterised in that: the elution process of step D carries out 3 It is secondary, the eluent repeatedly afforded is merged.
5. nisin production technology as described in claim 1, it is characterised in that: in step C, using sheet frame mistake With perlite bed board when filtering, and filtering.
6. nisin production technology as described in claim 1, it is characterised in that: in step B, defoaming agent is added.
7. nisin production technology as described in claim 1, it is characterised in that: in step (1), take glycerol tube bacterium Kind separates single colonie with plate streaking, and in 30 DEG C of incubator cultures, left and right, the big single colonie of picking are connected to shaking flask culture 16h for 24 hours Lactococcus lactis strain is obtained, glycerol tube preservation controls glycerol concentration 15~30%, is put into -20 DEG C of Storage in refrigerator.
8. nisin production technology as described in claim 7, it is characterised in that: cultivating Lactococcus lactis strain is The seed culture medium (g/L) of use: glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, it adjusts PH7.0~7.2, sterilize at 121 DEG C 20min, liquid amount 200mL/500mL;Medium of shaking flask fermentation (g/L): glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1 adjust pH7.5;Sterilize 10min at 115 DEG C.
9. nisin production technology as described in claim 1, it is characterised in that: in step (2), the seed of use Culture medium (g/L): glucose 6, peptone 6, yeast powder 12, K2HPO4 8、NaCl 5、MgSO40.6, pH7.0~7.2 are adjusted, Sterilize 20min at 121 DEG C.
10. nisin production technology as described in claim 1, it is characterised in that: in step (2), fermentation process The fermentation medium (g/L) of middle use: glucose 36, Dried Corn Steep Liquor Powder 2, yeast powder 10, KH2PO45, NaCl 4, Tween-80 1, adjust pH7.5.
CN201710580398.4A 2017-07-17 2017-07-17 Nisin production technology Pending CN109266709A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112159463A (en) * 2020-09-23 2021-01-01 通辽市圣达生物工程有限公司 Preparation method of nisin
CN112225784A (en) * 2020-09-23 2021-01-15 通辽市圣达生物工程有限公司 Method for extracting nisin
CN112971083A (en) * 2021-03-26 2021-06-18 青岛日辰食品股份有限公司 Preparation method of mushroom soup with natural antibacterial function
CN117701362A (en) * 2024-01-31 2024-03-15 通辽市圣达生物工程有限公司 Equipment for extracting nisin from lactococcus lactis fermentation liquor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101113463A (en) * 2007-05-23 2008-01-30 齐齐哈尔安泰生物工程有限公司 Preparation method of lactic acid streptostacin
CN101906153A (en) * 2010-08-16 2010-12-08 张国峰 Extraction process for preparing nisin
CN103798515A (en) * 2012-11-09 2014-05-21 山东福瑞达生物科技有限公司 Method for preparing feed additive by using nisin extraction waste liquid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101113463A (en) * 2007-05-23 2008-01-30 齐齐哈尔安泰生物工程有限公司 Preparation method of lactic acid streptostacin
CN101906153A (en) * 2010-08-16 2010-12-08 张国峰 Extraction process for preparing nisin
CN103798515A (en) * 2012-11-09 2014-05-21 山东福瑞达生物科技有限公司 Method for preparing feed additive by using nisin extraction waste liquid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RONAN GOUGH: "A Simple Method for the Purification of Nisin" *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112159463A (en) * 2020-09-23 2021-01-01 通辽市圣达生物工程有限公司 Preparation method of nisin
CN112225784A (en) * 2020-09-23 2021-01-15 通辽市圣达生物工程有限公司 Method for extracting nisin
CN112971083A (en) * 2021-03-26 2021-06-18 青岛日辰食品股份有限公司 Preparation method of mushroom soup with natural antibacterial function
CN112971083B (en) * 2021-03-26 2023-09-08 青岛日辰食品股份有限公司 Preparation method of mushroom soup with natural antibacterial function
CN117701362A (en) * 2024-01-31 2024-03-15 通辽市圣达生物工程有限公司 Equipment for extracting nisin from lactococcus lactis fermentation liquor
CN117701362B (en) * 2024-01-31 2024-04-23 通辽市圣达生物工程有限公司 Equipment for extracting nisin from lactococcus lactis fermentation liquor

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