CN103255083B - Fermentation method for lactobacillus with high cell density - Google Patents
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- 230000004151 fermentation Effects 0.000 title claims abstract description 67
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- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 9
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims abstract description 6
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 64
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 62
- 239000000835 fiber Substances 0.000 claims description 55
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- 230000001954 sterilising effect Effects 0.000 claims description 9
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
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- 229920004935 Trevira® Polymers 0.000 claims description 4
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- 244000082204 Phyllostachys viridis Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 239000002657 fibrous material Substances 0.000 abstract 2
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- 240000001046 Lactobacillus acidophilus Species 0.000 abstract 1
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- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 abstract 1
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to the technical field of biological products, and discloses a fermentation method for lactobacillus with high cell density. The fermentation method comprises the steps of constructing a fibrous bed reactor, preparing a culture medium, inoculating, culturing by fermenting, detecting microbial content, collecting fermentation liquor and the like. According to the fermentation method, fiber materials are successfully arranged inside the reactor as carriers of immobilized lactobacillus cells, and the number of the lactobacillus cells can be substantially enhanced in fermentation of lactobacillus with the fibrous bed and under specific fermentation conditions. The biological reactor can be separately used or be taken as a seed fermentation tank to be combined with a common reactor for carrying out sequence fermentation in batches or continuous fermentation, and has the characteristics of being high in production efficiency and stable in batch production, shortening a fermentation period, ensuring high cell density and the like; fiber materials used by the method are cheap; the device is simple and convenient to operate, the production cost is low, and the fermentation method can be well applied to industrialized production of cells of lactobacillus such as lactobacillus acidophilus, lactobacillus bulgaricus and bifidobacterium.
Description
Technical field
The present invention relates to biological product technical field, more specifically, relate to a kind of method for fermenting lactic acid bacteria of high-cell density.
Technical background
Milk-acid bacteria is a class probiotic bacterium common in animal intestinal, and it can improve microecological balance in animal intestinal, suppress effect such as growth, promoting digestion, raising body immunity of pathogenic bacteria.Current is also a feast for the eyes using it as the product of effective constituent, therefore also have stimulated the rise of lactobacillus-fermented industry.But the milk-acid bacteria that current traditional zymotic technique is produced, there is bacteria concentration low, fermentation time is long, complex process, the shortcomings such as survival rate is low, make the milk-acid bacteria high expensive fermenting out, also make the cost of lactic acid bacteria class product remain high always, become the bottleneck problem of lactobacillus-fermented industry.
Summary of the invention
The technical problem to be solved in the present invention is for the technical deficiency in lactobacillus-fermented, provides a kind of method for fermenting lactic acid bacteria of high-cell density.
Object of the present invention is achieved by the following technical programs:
A kind of method for fermenting lactic acid bacteria of high-cell density is provided, comprises the following steps:
S1. fibre bed reactor is built;
S2. substratum is prepared: join in fibre bed reactor by the lactic acid bacteria culturing medium prepared, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. the lactobacillus solution in substratum after access activation, inoculum size is 5 ~ 10% of substratum cumulative volume;
S4. regulate temperature to be 30 ~ 40 DEG C, adjust ph is 6.0 ~ 6.5, carries out fermentation culture, and timing sampling detects bacterium number;
S5. when bacterium number reaches 10
10fermentation is stopped during more than cfu/ml.
Wherein, S1. described fibre bed reactor mainly comprises reactor body, reactor body is provided with opening for feed, discharge port, thief hatch, heating system, pH electrode, temperature electrode, whipping appts is established in reactor, and the filamentary material be fixed in reactor, the Controlling System of control temperature and pH value can also be comprised.
Described filamentary material, as the carrier of fixing lactic acid bacteria cell, can increase substantially lactic-acid bacteria cells quantity when liquid fermenting.For milk-acid bacteria, the present invention adopt vegetable fibre or synthon one or both as fiber bed material in reactor of the present invention, preferably, described vegetable fibre comprises: cotton fibre, bamboo fibers, stalk fibre.
Preferably, described synthon comprise: trevira, tynex and polyacrylonitrile fibre.
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L.
The present invention carries out sequence batch (fermentation as being used alone, and from the discharge port of reactor, fermented liquid can all be discharged collection and carry out downstream processing, prepares next batch fermentation simultaneously; Continous way fermentation is carried out as being used alone, while discharge fermented liquid while add fresh culture from the opening for feed stream of reactor from the discharge port of reactor, making it ensureing that the prerequisite of bacterium number is issued to running balance, continuing fermentation, the fermented liquid ejected being carried out downstream processing simultaneously.
Described bio-reactor can be used alone, also can as seeding tank and common biological fermentation tank conbined usage; As in parallel in fibre bed reactor of the present invention is produced as seeding tank and conventional milk-acid bacteria the common fermentation tank fermented, during fermentation ends, close recycle pump, cut off mutual closed tube, all release or partly release the bacterium liquid producing fermentor tank and make downstream processing; Dose new fermention medium more afterwards, UNICOM's fibre bed seeding tank, ON cycle, carries out second batch fermentation.
Described bio-reactor is preferably provided with automatic control pH, the Controlling System system of temperature realizes automatic control fermentation.
One or more in the preferred Lactobacterium acidophilum of milk-acid bacteria of the present invention, lactobacillus bulgaricus, bifidus bacillus, faecium or thermophilus streptococcus, when adopting multi-cultur es, the ratio of mixing is not strict with.
Adopt traditional method for fermenting lactic acid bacteria, bacterium Particle density is low, and fermentation time is long, complex process, and survival rate is low.
Although fibre bed reactor has been reported, but all can only be applied to fermentative production acetic acid or propionic acid, have no and be applied to lactobacillus-fermented, chief reason is: milk-acid bacteria easily degenerates and inactivation, and the lactic acid produced once run up to certain concentration will the growth and breeding of feedback inhibition milk-acid bacteria somatic cells, thus making somatic cells quantity be difficult to improve, these technical barriers do not find effective and reasonable method to solve.
High quality, high efficiency probiotics are produced and are be unable to do without high performance fermentor tank support.Fibre bed reactor technology is successfully applied to lactobacillus-fermented by the present invention, mainly improves from these aspects and optimizes:
The first, the present invention is by continuous analysis and summary, and provide specific fiber bed material for milk-acid bacteria, mainly comprise vegetable fibre or synthon, vegetable fibre comprises cotton fibre, bamboo fibers, stalk fibre three kinds.Synthon comprise trevira, tynex and polyacrylonitrile fibre three kinds;
Second, optimize medium component, prior art adopts degreasing reconstituted milk as nutrient media components usually, the present invention adopts hydrolyzing lactoalbumin and corn juice as substratum main ingredient, and the rational proportion accurately limited between each component, under new fibre bed and substratum conjugation condition, for milk-acid bacteria provides suitable yeasting;
3rd, present invention optimizes technological condition for fermentation, selecting 30 ~ 40 DEG C to be leavening temperature, selecting pH value to be 6.0-6.5 is fermentation pH value, under above-mentioned condition provided by the invention, somatic cells output increased 5-10 is left and right doubly, and for lactic acid bacteria class product provides the somatic cells of high yield, product has that viable count is many, stability is high, prepare the features such as simple and easy to use, overcome the deficiency in current China suitability for industrialized production, promote the quality product of lactobacillus product.
Filamentary material is successfully installed on inside reactor as the carrier of fixing lactic acid bacteria cell by the present invention, and sums up and obtain, under fibre bed and specific fermentation condition, increasing substantially lactic-acid bacteria cells quantity when realizing lactobacillus-fermented.Bio-reactor of the present invention can be used alone, also can further fibre bed reactor be carried out batch sequence and ferments as use in parallel with common response device of seed fermentation tank or continuously ferment, there is production efficiency high, batch administration measure, shorten fermentation period, cell density high, the filamentary material used is cheap, device is simple, easy to operate, production cost is low, Lactobacterium acidophilum can be advantageously applied to, lactobacillus bulgaricus, the suitability for industrialized production of the lactic-acid bacteria cellss such as bifidus bacillus, the development promoting China's fermentation lactic acid bacteria product industry is had great importance.
Accompanying drawing explanation
The fibre bed reactor structural representation of Fig. 1 milk-acid bacteria of the present invention;
Fig. 2 embodiment of the present invention 4 fermentation system structural representation.
Embodiment
The present invention is further described below in conjunction with specific embodiment.The test method used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc. if no special instructions, are the reagent that can obtain from routine business approach and material.
Embodiment 1
S1. fibre bed reactor is built; As shown in Figure 1.Accompanying drawing 1 provides a kind of structural representation of fibre bed reactor, comprise reactor body 3, reactor body 3 outside is provided with heating unit 9, opening for feed 1 is provided with in reactor body 3 upper end, thief hatch 2 and soda acid feeding-system 6, reactor body 3 lower end arranges discharge port 11, reactor body 3 internal fixtion is provided with filamentary material 10, whipping appts 4 is set in reactor body 3, pH electrode 7, temperature electrode 8, soda acid feeding-system 6, pH electrode 7 is connected with Controlling System 5 with temperature electrode 8, can in real time by pH and Temperature Feedback to Controlling System 5, Automated condtrol is realized by soda acid feeding-system 6 and heating system 9.
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L and is mixed with substratum and joins in fibre bed reactor, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. in substratum, access the Lactobacterium acidophilum seed liquor after the activation of MRS liquid nutrient medium, inoculum size is 5 ~ 10% of substratum cumulative volume;
S4. regulate temperature to be 35 DEG C, adjust ph is 6.0, carries out fermentation culture, and timing sampling detects bacterium number;
S5. when bacterium number reaches 10
10fermentation is stopped during more than cfu/ml.From discharge port, fermented liquid is all discharged collection and carry out downstream processing, prepare next batch fermentation simultaneously.Also continous way fermentation can be carried out as being used alone reactor, while discharge fermented liquid limit from discharge port 11 to add fresh culture from opening for feed 1 stream, make it ensure that the prerequisite of bacterium number is issued to running balance, continuing fermentation, the fermented liquid simultaneously ejected carries out downstream processing.
The fiber bed material that the present embodiment adopts is cotton fibre.The rear bacterium number that fermented can reach 8.5 × 10
10cfu/ml, and by fermentor tank can high stable continue production thalline, do not need renewed vaccination.And after adopting identical bacterial classification, leavening temperature and pH value and same medium conventional liq fermentation same time, bacterium number only has 3.4 × 10
9cfu/ml.
Embodiment 2
S1. fibre bed reactor is built; With embodiment 1;
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L and is mixed with substratum and joins in fibre bed reactor, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. in substratum, access the lactobacillus bulgaricus after the activation of MRS liquid nutrient medium and thermophilus streptococcus seed liquor, inoculum size is respectively 3% and 3% of substratum cumulative volume;
S4. regulate temperature to be 37 DEG C, adjust ph is 6.5, carries out fermentation culture, and timing sampling detects bacterium number;
S5. when bacterium number reaches 10
10fermentation is stopped during more than cfu/ml.From discharge port, fermented liquid is all discharged collection and carry out downstream processing, prepare next batch fermentation simultaneously.Also continous way fermentation can be carried out as being used alone reactor, while discharge fermented liquid limit from discharge port 11 to add fresh culture from opening for feed 1 stream, make it ensure that the prerequisite of bacterium number is issued to running balance, continuing fermentation, the fermented liquid simultaneously ejected carries out downstream processing.
The fiber bed material that the present embodiment adopts is bamboo fibers.The rear bacterium number that fermented can reach 2.4 × 10
10cfu/ml, and by fermentor tank can high stable continue production thalline, do not need renewed vaccination.And after adopting identical bacterial classification, leavening temperature and pH value and same medium conventional liq fermentation same time, bacterium number only has 5.4 × 10
9cfu/ml.
Embodiment 3
S1. fibre bed reactor is built; With embodiment 1;
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L and is mixed with substratum and joins in fibre bed reactor, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. in substratum, access the Lactobacterium acidophilum after the activation of MRS liquid nutrient medium, lactobacillus bulgaricus, thermophilus streptococcus seed liquor, inoculum size is respectively 2%, 2% and 2% of substratum cumulative volume;
S4. regulate temperature to be 37 DEG C, adjust ph is 6.4, carries out fermentation culture, and timing sampling detects bacterium number;
S5. when bacterium number reaches 10
10fermentation is stopped during more than cfu/ml.From discharge port, fermented liquid is all discharged collection and carry out downstream processing, prepare next batch fermentation simultaneously.
Also continous way fermentation can be carried out as being used alone reactor, while discharge fermented liquid limit from discharge port 11 to add fresh culture from opening for feed 1 stream, make it ensure that the prerequisite of bacterium number is issued to running balance, continuing fermentation, the fermented liquid simultaneously ejected carries out downstream processing.
The fiber bed material that the present embodiment adopts is trevira, and the rear three kinds of bacterium total counts that fermented can reach 6.2 × 10
10cfu/ml, and by fermentor tank can high stable continue production thalline, do not need renewed vaccination.And after adopting identical bacterial classification, leavening temperature and pH value and same medium conventional liq fermentation same time, three kinds of bacterium total counts only have 2.9 × 10
9cfu/ml.
Embodiment 4
S1. build fibre bed reactor as seeding tank, structure is with embodiment 1; Using the fermentor tank 13 of routine as production fermentor tank, the seed fermentation tank of fiber biological reactor is assembled sterilizing with production fermentor tank and circulation device 12 and uses for extensive continous way fermentative production.Circulation device 12 maintains the circulation of nutrient solution between fibre bed bio-reactor and common fermentation tank (13).System after assembling as shown in Figure 2.
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L and is mixed with substratum and joins in fibre bed reactor, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. bifidus bacillus is inoculated in seed fermentation tank after 37 DEG C of activation 21h, opens peristaltic pump and start Cyclic culture, after cultivating 21h, when bacterium number reaches 10
10stop fermentation during more than cfu/ml, close recycle pump 12, cut off mutual closed tube, whole releasing or part are released the bacterium liquid producing fermentor tank and are made downstream processing, supplement new substratum circulating fermentation, seeding tank is constant, reaches the production of continuously fermenting of high cell density fermentation;
The fiber bed material that the present embodiment adopts is tynex.The rear bacterium number that fermented can reach 2.4 × 10
10cfu/ml, and by fermentor tank can high stable continue production thalline, do not need renewed vaccination.And after adopting identical bacterial classification, leavening temperature and pH value and same medium conventional liq fermentation same time, bacterium number only has 5.4 × 10
9cfu/ml.
Embodiment 5
S1. the assembling of fermentation system is with embodiment 4.
Substratum described in S2 is: hydrolyzing lactoalbumin (g/L) (food grade) of 3%, the glucose (g/L) of 2 ~ 2.5%, the bitter salt (g/L) of 0.5 ~ 1%, the corn steep liquor (ml/L) of 10%, distilled water is settled to 1L and is mixed with substratum and joins in fibre bed reactor, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. in substratum, access the bifidus bacillus seed liquid after the activation of BS substratum, inoculum size is 5 ~ 10% of substratum cumulative volume;
S4. fermentation culture: in Controlling System 5, temperature and pH value are set to 37 DEG C, initial pH value is 6.2, carries out automatic control fermentation, and timing sampling detects bacterium number;
S5. continue to expand fermentation: after cultivating 12h, when bio-reactor bacterium number reaches 10
10during more than cfu/ml, open the valve with common fermentation tank (having the fresh culture after sterilizing in common fermentation tank), start recycle pump simultaneously, continue fermentation;
S6. collect: after cultivating the 24h time, when in common fermentation tank, bacterium number reaches 10
10stop fermentation during more than cfu/ml, from discharge port, fermented liquid is all discharged collection and carry out downstream processing, prepare next batch fermentation simultaneously.Close recycle pump, cut off mutual closed tube, all release or partly release the bacterium liquid producing fermentor tank and make downstream processing; Dose new fermention medium more afterwards, UNICOM's fibre bed seeding tank, ON cycle, carries out second batch fermentation.
The fiber bed material that the present embodiment adopts is bamboo fibers.The rear bacterium number that fermented can reach 2.4 × 10
10cfu/ml, and by fermentor tank can high stable continue production thalline, do not need renewed vaccination.And after adopting identical bacterial classification, leavening temperature and pH value and same medium conventional liq fermentation same time, bacterium number only has 5.4 × 10
9cfu/ml.
Claims (3)
1. a method for fermenting lactic acid bacteria for high-cell density, is characterized in that, comprises the following steps:
S1. fibre bed reactor is built;
S2. substratum is prepared: join in fibre bed reactor by the lactic acid bacteria culturing medium prepared, for subsequent use after 121 DEG C of sterilizing 15 ~ 20min;
S3. in substratum access activation after lactobacillus solution, inoculum size be substratum cumulative volume 5 ~
10%；
S4. regulate temperature to be 30 ~ 40 DEG C, adjust ph is 6.0 ~ 6.5, carries out fermentation culture, and timing sampling detects bacterium number;
S5. when bacterium number reaches 10
10fermentation is stopped during more than cfu/ml;
Wherein, fibre bed reactor described in S1 mainly comprises reactor body, reactor body is provided with opening for feed, discharge port, thief hatch, heating system, pH electrode, temperature electrode, in reactor, establishes whipping appts, and be fixed on the filamentary material in reactor;
Described filamentary material is one or both of vegetable fibre or synthon;
Described vegetable fibre is one or both in cotton fibre, bamboo fibers;
Described synthon are one or both in trevira, tynex;
Substratum described in S2 is: according to g/L meter, food grade hydrolyzing lactoalbumin, the glucose of 2 ~ 2.5%, the bitter salt of 0.5 ~ 1% of 3%, according to mL/L meter, and the corn steep liquor of 10%, distilled water is settled to 1L;
Described milk-acid bacteria is one or more in Lactobacterium acidophilum, lactobacillus bulgaricus, bifidus bacillus or thermophilus streptococcus.
2. according to the method for fermenting lactic acid bacteria of high-cell density described in claim 1, it is characterized in that, described bio-reactor is provided with Controlling System.
3. the method for fermenting lactic acid bacteria of high-cell density according to claim 1, is characterized in that, S1 also comprise using fibre bed reactor as seed fermentation tank and produce fermentor tank, circulation device assembling obtains continous way fermentative production system; Described circulation device maintains fibre bed bio-reactor and produces the circulation of nutrient solution between fermentor tank.
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| CN103555776B (en) * | 2013-10-31 | 2016-01-20 | 南京工业大学 | Method for producing D-lactic acid by repeated batch fermentation by utilizing surface immobilization technology |
| CN103555777B (en) * | 2013-11-15 | 2015-07-01 | 厦门大学 | Method for producing lactic acid by using immobilized cells for converting glycerin |
| CN103642854B (en) * | 2013-12-03 | 2015-07-15 | 南京工业大学 | Method for producing succinic acid by immobilized corynebacterium glutamicum and repeated batch fermentation |
| CN109943509A (en) * | 2019-04-12 | 2019-06-28 | 沈阳药科大学 | A method of utilizing full simple substance culture medium High Density Cultivation lactic acid bacteria |
| CN112869171A (en) * | 2021-01-29 | 2021-06-01 | 陕西科技大学 | Probiotic dietary fiber powder and preparation method thereof |
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