CN104263671A - Method for increasing viable count of Lactobacillus brevis for pickles by two-stage dissolved oxygen control strategy - Google Patents
Method for increasing viable count of Lactobacillus brevis for pickles by two-stage dissolved oxygen control strategy Download PDFInfo
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- CN104263671A CN104263671A CN201410352668.2A CN201410352668A CN104263671A CN 104263671 A CN104263671 A CN 104263671A CN 201410352668 A CN201410352668 A CN 201410352668A CN 104263671 A CN104263671 A CN 104263671A
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- dissolved oxygen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a method for high density fermentation to Lactobacillus brevis WJH3 through two-stage dissolved oxygen control. A fermentation strain is Lactobacillus brevis with a preservation number of CCTCC M2014150. At an early cultivation stage (for 6-8 hours), the stirring rotational speed is set to be 280 to 360 r/min, the ventilation capacity is set to 0.7-1.2 vvm, and the dissolved oxygen content is controlled to be 20% to 30%, and at the middle and later periods of logarithmic growth (for 22-23 hours), the ventilation capacity is set to 0.5-1.0 vvm, the stirring rotational speed is set to 50-300 r/min, and the dissolved oxygen content is controlled to be lower than 5%. The method provided by the invention can increase the viable count of Lactobacillus brevis in a fermentation broth by more than 60% markedly, and through the method, a fermentation bacterial agent suitable for industrial production can be prepared.
Description
Technical field
The present invention relates to the high-density cultivation method of a kind of pickles with short lactobacillus, specifically refer to the method adopting two-stage control dissolved oxygen to improve viable count, belong to optimizing fermentation field.
Background technology
Milk-acid bacteria is as a kind of probiotic bacterium, there is regulating intestinal canal microecological balance, eliminate objectionable impurities in body, reduce the nourishing functions such as cholesterol, be subject to the extensive concern of all circles, it is applied to leavened food industry, not only there is raising nutritive value, improve the effect of local flavor, the shelf time of food can also be extended, improve food safety.Pickled vegetable, as the traditional vegetables cold working mode of China, can not destroy under the former nutritious prerequisite of vegetables as far as possible, improves the fresh-keeping rate of vegetables.The leading flora of pickle fermentation is milk-acid bacteria.Artificial inoculation lactobacillus-fermented pickles can not only improve the shortcomings such as the production cycle is long, quality is unstable in traditional natural fermentation, and significantly can reduce kimchi products nitrite, improve the edible safety of kimchi products.At present, milk-acid bacteria has been widely used in cultured milk prod industry as throw type leaven, and the correlative study of the throw type leaven of lactic acid bacteria for kraut is less.Main Bottleneck prepared by milk-acid bacteria throw type leaven is the foundation of high-density culture strategy.
Major part milk-acid bacteria mostly is auxotroph, and its main metabolites is various organic acid, inhibited to own growth.At present the research of the high density fermentation method of milk-acid bacteria is mainly concentrated on to the optimization of fermentation condition, as the exploitation [Ma Honghui of low value culture medium raw material, Kong Baohua, Xia Xiufang etc. lactobacterium casei KLDS1.0381 high-density culturing condition is studied. Northeast Agricultural University's journal 2012, 43 (2): 13-19.], somatomedin probe into [Gu Yuanyi, Ou Jun, Liang Jinzhong. the research of lactobacillus bulgaricus and Promoting Growth Factors of Streptococcus Thermophilus. dairy industry science and technology 2008, 31 (3): 117-120.], circulation yeast culture [Hayakawa K, Sansawa H, Nagamune T, et al.High Density Culture of Lactobacillus casei by a Cross-Flow Culture Method Based on Kinetic Properties of the Microorganism.Journal of fermentation and bioengineering1990, 70 (6): 404-408.] etc.
Short lactobacillus, as the milk-acid bacteria of heterofermentation, plays main contributions effect to the generation of pickle flavor.Short lactobacillus is as the milk-acid bacteria of micro-aerobic, and for its high density fermentation, dissolved oxygen has important impact, and therefore the enrichment of control mode to biomass of dissolved oxygen is most important.At present, be Unareobic fermentation to the high-density cultivation method of milk-acid bacteria, little to the research of the dissolved oxygen level optimization aspect of short lactobacillus, there is not yet bibliographical information.Therefore be necessary that a kind of effective dissolved oxygen control strategy of exploitation improves fermentation level.
Summary of the invention
Technical problem solved by the invention is to provide the high-density cultivation method that a kind of two-stage oxygen dissolving control strategy improves short lactobacillus viable count, improves the utilization ratio of raw material and equipment, shortens the required time that ferments, to significantly improve short lactobacillus viable count.
Described short lactobacillus L.brevis WJH3 is preserved in China typical culture collection center on April 24th, 2014, and deposit number is CCTCC M 2014150, and preservation address is Wuhan, China university.
Described two-stage oxygen dissolving amount control techniques improves the method for short lactobacillus viable count, be fermentation the first stage control dissolved oxygen amount be 20 ~ 30%, at fermentation subordinate phase setting air flow 0.5 ~ 1.0vvm, mixing speed 50 ~ 300r/min, controlling dissolved oxygen amount is less than 5%; Described fermentation time first stage is 6 ~ 8h; The described fermentation subordinate phase time is 22 ~ 23h.
In described method culturing process, pH controls is 6.8, and the ammoniacal liquor being added 20% by stream is realized.
Described fermentative medium formula is (unit g/L): glucose 18-22, yeast powder 27-33, citric acid 1.2-1.9, Trisodium Citrate 15-20, VB
618-22, MgS0
47H
2o 0.3-0.7, MnSO
45H
2o 0.1-0.4, tween-80 0.7-1.2.
Described short lactobacillus high-density cultivation method, comprises the following steps:
(1) seed culture: the short lactobacillus L.brevis WJH3 of freezen protective is inoculated in MRS liquid nutrient medium after 30 DEG C of quiescent culture activation 24h, inoculum size with 3% is transferred in MRS liquid nutrient medium (40mL substratum/250mL triangular flask), 30 DEG C, 200r/min shaking table concussion cultivation 14h.
(2) Fed batch fementation: inoculate according to inoculum size 7% (v/v) in fermented liquid, stream adds ammoniacal liquor control pH6.5 ~ 7.0 of 20%, leavening temperature 28 ~ 31 DEG C, in culturing process, manual stream adds glucose concn 15 ~ 20g/L in the glucose solution maintain base of 400 ~ 600g/L, dissolved oxygen amount is controlled: controlling dissolved oxygen amount in cultivation early stage (6 ~ 8h) is DO20 ~ 30% according to following requirement, to logarithmic growth middle and later periods (22 ~ 23h), setting air flow 0.5 ~ 1.0vvm, mixing speed 50 ~ 300r/min, controls dissolved oxygen amount below 5%.。
Beneficial effect of the present invention: utilize described two-stage oxygen dissolving to control zymotechnique, be cultured to the viable count of logarithmic growth later stage short lactobacillus up to 1.50 × 10
10cFU/mL, not only significantly improves viable count, and shortens fermentation period, has important industrial application value, has certain directive significance to the high density fermentation of other micro-aerobic milk-acid bacteria simultaneously.
Embodiment:
Bacterial classification: short lactobacillus Lactobacillus brevis WJH3, China typical culture collection center preservation, deposit number CCTCC M 2014150.
Vaccination ways: the short lactobacillus L.brevis WJH3 of freezen protective is inoculated in MRS liquid nutrient medium after 30 DEG C of quiescent culture activation 24h, inoculum size with 3% is transferred in MRS liquid nutrient medium (40mL substratum/250mL triangular flask), 30 DEG C, 200r/min shaking table concussion cultivation 14h.
Fermentor tank: 3L NBS automatic fermenter
Fermention medium is (unit g/L): glucose 20, yeast powder 30, citric acid 1.53, Trisodium Citrate 18.58, VB
620, MgSO
47H
2o 0.58, MnSO
45H
2o 0.25, tween-80 1.
The constant dissolved oxygen fermentation of comparative examples 1:3L fermentor tank
The short lactobacillus L.brevis WJH3 of freezen protective is inoculated in MRS liquid nutrient medium after 30 DEG C of quiescent culture activation 24h, inoculum size with 3% is transferred in MRS liquid nutrient medium (40mL substratum/250mL triangular flask), 30 DEG C, 200r/min shaking table concussion cultivation 14h; Seed culture fluid is transferred in the 3L fermentor tank containing 1.1L fermention medium, inoculum size 7% (v/v), auto-feeding 20% ammoniacal liquor control pH6.8, leavening temperature 30 DEG C, in culturing process, manual stream adds glucose concn 15 ~ 20g/L, Ventilation Rate 2.5vvm in the glucose solution maintain base of 500g/L, be associated with dissolved oxygen level DO by mixing speed, control DO30%, carries out fermentation about 14h, and the final viable count of comparative examples 1 is 7.35 × 10
9cFU/mL.
The constant dissolved oxygen fermentation of comparative examples 2:3L fermentor tank
The short lactobacillus L.brevis WJH3 of freezen protective is inoculated in MRS liquid nutrient medium after 30 DEG C of quiescent culture activation 24h, inoculum size with 3% is transferred in MRS liquid nutrient medium (40mL substratum/250mL triangular flask), 30 DEG C, 200r/min shaking table concussion cultivation 14h; Seed culture fluid is transferred in the 3L fermentor tank containing 1.1L fermention medium, inoculum size 7% (v/v), auto-feeding 20% ammoniacal liquor control pH6.8, leavening temperature 30 DEG C, in culturing process, manual stream adds glucose concn 15 ~ 20g/L in the glucose solution maintain base of 500g/L, stuffiness, and mixing speed is set as 50r/min, carry out fermentation about 45.5h, the final viable count of comparative examples 2 is 3.11 × 10
9cFU/mL.
Embodiment 1:3L fermentor tank two-stage oxygen dissolving ferments
Ferment tank adopts two benches culture method, the same comparative examples of all the other conditions.First stage is at logarithmic growth early stage (8h), control dissolved oxygen amount is DO20%, and subordinate phase to logarithmic growth middle and later periods (23h), sets air flow 0.75vvm at yeast culture, mixing speed 250r/min, controls dissolved oxygen amount lower than DO5%.The final viable count of embodiment 1 reaches the highest when 22h is carried out in fermentation, is 1.50 × 10
10cFU/mL.
Embodiment 2:3L fermentor tank two-stage oxygen dissolving ferments
Ferment tank adopts two benches culture method, the same comparative examples of all the other conditions.First stage is at logarithmic growth early stage (6h), control dissolved oxygen amount is DO30%, and subordinate phase to logarithmic growth middle and later periods (22h), sets air flow 0.75vvm at yeast culture, mixing speed 250r/min, controls dissolved oxygen amount lower than DO5%.The final viable count of embodiment 2 reaches the highest when 22h is carried out in fermentation, is 1.21 × 10
10cFU/mL.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (4)
1. a pickles short lactobacillus fermentation process in high density, the mode that described method adopts two-stage oxygen dissolving to control is to L.brevisWJH3) carry out high-density culture, the short lactobacillus of fermentation strain to be deposit number be CCTCC M2014150.
2. method according to claim 1, it is characterized in that, the operation that described two-stage oxygen dissolving amount controls is as follows: inoculate by inoculum size 7% (v/v), stream adds ammoniacal liquor control pH6.5 ~ 7.0 of 20%, leavening temperature 28 ~ 31 DEG C, in culturing process, manual stream adds glucose concn 15 ~ 20g/L in the glucose solution maintain base of 400 ~ 600g/L, dissolved oxygen amount is controlled: controlling dissolved oxygen amount in cultivation early stage (6 ~ 8h) is DO20 ~ 30% according to following requirement, to logarithmic growth middle and later periods (22 ~ 23h), setting air flow 0.5 ~ 1.0vvm, mixing speed 50 ~ 300r/min, dissolved oxygen amount is controlled below 5%.
3., according to the method described in claim 1 to 2, it is characterized in that described fermentative medium formula is for (unit g/L): glucose 18-22, yeast powder 27-33, citric acid 1.2-1.9, Trisodium Citrate 15-20, VB
618-22, MgSO
47H
2o0.3-0.7, MnSO
45H
2o0.1-0.4, tween-80 0.7-1.2.
4. according to the method described in claim 1 to 2, it is characterized in that described ferment-seeded is cultivated is: the short lactobacillus L.brevis WJH3 of freezen protective is inoculated in MRS liquid nutrient medium after 30 DEG C of quiescent culture activation 24h, inoculum size with 3% is transferred in MRS liquid nutrient medium (40mL substratum/250mL triangular flask), 30 DEG C, 200r/min shaking table concussion cultivation 14h.
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Cited By (3)
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|---|---|---|---|---|
| CN104946723A (en) * | 2015-06-05 | 2015-09-30 | 中国检验检疫科学研究院 | General medium for determination of lactobacillus resistance to drugs and use thereof |
| CN106350473A (en) * | 2016-11-30 | 2017-01-25 | 北京市农林科学院 | High-density fermentation culture medium for feed lactobacillus brevis and fermentation method thereof |
| CN109363119A (en) * | 2018-11-21 | 2019-02-22 | 中国农业科学院麻类研究所 | Utilize the method and pickle jar of pickles direct putting type microbial inoculum production pickles |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104946723A (en) * | 2015-06-05 | 2015-09-30 | 中国检验检疫科学研究院 | General medium for determination of lactobacillus resistance to drugs and use thereof |
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| CN106350473A (en) * | 2016-11-30 | 2017-01-25 | 北京市农林科学院 | High-density fermentation culture medium for feed lactobacillus brevis and fermentation method thereof |
| CN106350473B (en) * | 2016-11-30 | 2019-10-11 | 北京市农林科学院 | A kind of high density fermentation culture medium and its fermentation process of feeding Lactobacillus brevis |
| CN109363119A (en) * | 2018-11-21 | 2019-02-22 | 中国农业科学院麻类研究所 | Utilize the method and pickle jar of pickles direct putting type microbial inoculum production pickles |
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Application publication date: 20150107 |