CN103013890B - Method of culturing lactobacillus and bacillus in mixing way - Google Patents
Method of culturing lactobacillus and bacillus in mixing way Download PDFInfo
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- CN103013890B CN103013890B CN201210581009.7A CN201210581009A CN103013890B CN 103013890 B CN103013890 B CN 103013890B CN 201210581009 A CN201210581009 A CN 201210581009A CN 103013890 B CN103013890 B CN 103013890B
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Abstract
The invention discloses a method of culturing lactobacillus and bacillus in a mixing way, which comprises the following steps of: inoculating lactobacillus plantarum and bacillus subtilis with the mixed inoculation ratio of 1 percent:10 percent by weight in an MRS-Ca culture medium, carrying out vibration culturing for 18h at the temperature of 37 DEG C and 220rpm and harvesting; or inoculating lactobacillus casei and bacillus subtilis with the mixed inoculation ratio of 1.5 percent:10 percent by weight in the MRS-Ca culture medium, carrying out vibration culturing for 24h at the temperature of 37 DEG C and 220rpm and harvesting. The method is particularly suitable for liquid fermentation production of microbial ecological agents and has the remarkable advantages that the lactobacillus and the bacillus are fermented in the mixing way, and the synergistic effect among bacterial strains is fully exerted, thus supplying protection to aerobic metabolism of the lactobacillus, and increasing the equipment utilization ratio.
Description
Technical field
The present invention relates to a kind of lactobacillus and genus bacillus mixed culturing method, relate in particular to the mixed culturing method of a kind of plant lactobacillus or lactobacterium casei and subtilis condition optimizing, belong to biological technical field.
Background technology
Probiotics refers to a kind of important intestinal microflora conditioning agent being added in feed, claim again probiotic agent, there is the intestinal microflora of adjustment balance, promote immunity of organism, improve feed palatability, improve efficiency of feed utilization, strengthen the effects such as animal disease resistant ability, as feed, add the approval that antibiotic substitute has obtained aquaculture.The public associations of united States food and drug administration (FAD) in 1989 and U.S. feed (AAFCO) have announced 44 kinds can Direct-fed and be commonly referred to be safe microorganism and produce bacterial strain as probiotics, comprising 28 kinds of milk-acid bacterias, 2 kinds of sporeformer, 2 Yeasts and 2 kinds of aspergillus.In December, 2008 No. 1126, China Ministry of Agriculture bulletin regulation can Direct-fed animal totally 16 kinds of feed level microbe additives, as Bacillus licheniformis, subtilis, bifidus bacillus, enterococcus faecalis, faecium, lactoenterococcus, Lactobacterium acidophilum, lactobacterium casei, plant lactobacillus, lactobacillus lactis, lactobacillus delbruckii, pediococcus acidilactici, Pediococcus pentosaceus, Rhodopseudomonas palustris, yeast saccharomyces cerevisiae, product Yuan candiyeast.
Probiotics is comprised of multiple-microorganism conventionally, is a kind of mixed live preparation.The probiotics that is applied at present livestock and poultry breeding industry is mainly milk-acid bacteria and genus bacillus.Milk-acid bacteria is that facultative anaerobe, genus bacillus are aerobic bacteria, and both growth characteristics and nutritional requirement are widely different, in traditional industrial fermentation, conventionally adopt single culture, decomposite production technique then, cause thus plant factor low, production cost increases.Research shows, the animal of the sporeformer of feeding, and in its enteron aisle and ight soil, lactic acid bacterium number increases, and illustrates that sporeformer has promoter action to the growth of milk-acid bacteria; Have at present report, some Bacterium lacticum biomass under aerobic conditions improves, and having under the material existence such as protoheme, can activate its catalase, falls Hypoxia Stress, and thalline viability extends.Subtilis is aerobic bacteria, can produce multiple peroxidase, and oxygen is coerced to perfect defense mechanism.Based on this theory, the Optimizing Technical of research and development plant lactobacillus or lactobacterium casei and subtilis mixed culture, for the mixed bacterial fermentation of probiotics provides reference significant.
Summary of the invention
For the deficiency of existing production technique, the object of the present invention is to provide the mixed culturing method of a kind of plant lactobacillus or lactobacterium casei and subtilis condition optimizing.
The method of lactobacillus of the present invention and genus bacillus mixed culture, step is:
(1) get plant lactobacillus or the lactobacillus casei bacterial strain in later stage exponential growth later stage to stationary phase and be inoculated into respectively in MRS substratum, 37 ± 1 ℃ of static cultivations 12 ± 2 hours;
(2) get the bacillus subtilis strain in later stage exponential growth later stage to stationary phase and be inoculated in LB substratum, 37 ± 1 ℃, 220rpm concussion are cultivated 12 ± 2 hours;
(3) bacillus subtilis that plant lactobacillus step (1) being made and step (2) make is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1% ﹕ 10%, 37 ± 1 ℃, 18 ± 1 hours results of 220rpm concussion cultivation; Or the bacillus subtilis that makes of the lactobacterium casei that step (1) is made and step (2) is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1.5% ﹕ 10%, 37 ± 1 ℃, 220rpm concussion are cultivated 24 ± 1 hours results.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture: described plant lactobacillus preferred plant Bacterium lacticum (Lactobacillus plantarum) CGMCC No.6888; The preferred lactobacterium casei of described lactobacterium casei (Lb.casei) ATCC393 (pL251); The preferred B.subtilis ATCC23857 of described subtilis.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described MRS substratum is as follows:
Under 121 ℃ of conditions, sterilizing is 15 minutes.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described LB substratum is: peptone 1g, yeast extract 0.5g, NaCl1g, agar 1.5-2g, water 100ml.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described MRS-Ca substratum is: on MRS substratum, adding whole weight percent concentration is 0.02% CaCl
2.
A kind of plant lactobacillus that is applied to the method for above-mentioned lactobacillus and genus bacillus mixed culture, described bacterial strain called after plant lactobacillus (Lactobacillus plantarum) K2, separated from Xinjiang traditional zymotic Yoghourt, applicant has been preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number is CGMCC No.6888 on November 27th, 2012.
The invention discloses a kind of method of lactobacillus and genus bacillus mixed culture, the liquid fermenting that is particularly suitable for probiotics is produced.The significant advantage of the present invention is: lactobacillus and genus bacillus are carried out to mixed fermentation, give full play to the synergy between bacterial strain, to the aerobic metabolism of milk-acid bacteria, provide protection, improved plant factor.
Accompanying drawing explanation
Shown in Fig. 1, Lb.plantarum CGMCC6888 and B.subtilis ATCC23857, Lb.casei ATCC393(pL215) and B.subtilis ATCC23857 according to 1%+10% and 1.5%+10% inoculative proportion, be inoculated in MRS-Ca substratum respectively, the growth curve that 37 ℃, 220rpm concussion are cultivated, and compare during with independent strain culturing.
Wherein: B.subtilis ATCC23857 and Lb.plantarum CGMCC6888 mixed culture (■), B.subtilis ATCC23857 and Lb.casei ATCC393(pL215) mixed culture (▲), B.subtilis ATCC23857 single culture (●), Lb.plantarum CGMCC6888 single culture (), Lb.casei ATCC393(pL215) growth curve of single culture (△).
Fig. 2 Lb.plantarum CGMCC6888 and B.subtilis ATCC23857, Lb.casei ATCC393(pL215) and B.subtilis ATCC23857 according to the ratio of accompanying drawing 1, be inoculated in MRS-Ca substratum, when 37 ℃, 220rpm concussion are cultivated 12h, 18h and 24h, the viable count of plant lactobacillus, lactobacterium casei and subtilis.
Wherein: (a) Lb.plantarum CGMCC6888 or Lb.caseiATCC393(pL215) viable count; (b) B.subtilis ATCC23857 total count; (c) B.subtilis ATCC23857 gemma number.
Embodiment
Embodiment 1: the optimization of mixed culture inoculation condition
Have basically identical growth velocity when making two kinds of bacteria growings in same system, the growth conditions of seeds before two kinds of bacterium mix, inoculum size and inoculative proportion while mixing are extremely important.The present invention first by seed activation to the logarithmic phase later stage to stationary phase in earlier stage, method is plant lactobacillus (Lactobacillus plantarum) CGMCC No.6888 or lactobacterium casei (Lb.casei) ATCC393(pL215 that (1) gets later stage exponential growth later stage to stationary phase) bacterial strain is inoculated into respectively in MRS substratum, 37 ℃ of static cultivations 12 hours, (2) subtilis (B.subtilis) the ATCC23857 inoculation of getting later stage exponential growth later stage to stationary phase is in LB substratum, and 37 ℃, 220rpm concussion are cultivated 12 hours, then by plant lactobacillus (Lactobacillusplantarum) CGMCC No.6888 or lactobacterium casei (Lb.casei) ATCC393(pL215) with (B.subtilis) ATCC23857 is first by weight 1%+20%, 2%+20%, 1%+10%, 1%+5% inoculum size is mixed in 20mL MRS-Ca substratum, 37 ℃, 18h～24h is cultivated in 220rpm concussion, measure and cultivate the absorbancy (OD600nm) connecing, result determines that best mixing condition (by weight percentage) is: 1% plant lactobacillus (Lactobacillus plantarum) CGMCCNo.6888+10% subtilis (B.subtilis) ATCC23857, or 1.5% lactobacterium casei (Lb.casei) ATCC393(pL215)+10% subtilis (B.subtilis) ATCC23857 biomass is the highest.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described MRS substratum is as follows:
Under 121 ℃ of conditions, sterilizing is 15 minutes.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described LB substratum is: peptone 1g, yeast extract 0.5g, NaCl1g, agar 1.5-2g, water 100ml.
In the method for above-mentioned lactobacillus and genus bacillus mixed culture, the formula of described MRS-Ca substratum is: on MRS medium base, adding whole weight percent concentration is 0.02% CaCl
2.
Above-mentioned plant lactobacillus (Lactobacillus plantarum) CGMCC No.6888, separated from Xinjiang traditional zymotic Yoghourt, applicant is in preservation on November 27 in 2012 to China Committee for Culture Collection of Microorganisms's common micro-organisms center, above-mentioned lactobacterium casei (Lb.casei) ATCC393(pL215) with subtilis (B.subtilis) ATCC23857 all purchased from the biological product preservation of USS center.
Embodiment 2: the determining of the best harvest time of mixed culture
The gemma number of the viable count of Bacterium lacticum, genus bacillus is the standard of weighing probiotics quality.According to the best inoculative proportion of embodiment 1, in weight ratio, by 1% plant lactobacillus (Lb.plantarum) CGMCC No.6888+10% subtilis (B.subtilis) ATCC23857, or 1.5% lactobacterium casei (Lb.casei) ATCC393(pL215)+10% subtilis (B.subtilis) ATCC23857 is inoculated in after 20mL MRS-Ca substratum, 37 ℃, 220rpm shake cultivation, respectively at 12h, 18h, 24h sampling and measuring living preparation of lactobacillus number, genus bacillus total count and gemma number.
Experimental result: suffer oxygen to coerce lighter Lb.casei ATCC393(pL215 during for aerobic metabolism), when incubation time extends to 24h, for its viable count, have no significant effect, and can obtain higher gemma number.During for aerobic metabolism, produce a large amount of H
2o
2plant lactobacillus (Lb.plantarum) CGMCC No.6888, although B.subtilis ATCC23857 can remove H in environment in mixed culture
2o
2(H do not detected
2o
2accumulation), its viable count or because oxygen is to a certain degree coerced starts to occur obvious decline after 18h, so incubation time can not extend again, harvest time is 18h.
Comprehensive above data, for Lb.caseiATCC393(pL215) and the mixed culture of B.subtilis ACTT23857, preferably inoculum size is 1.5%+10%, harvest time is 24h; For the mixed culture of Lb.plantarum CGMCC6888 and B.subtilis ATCC23857, preferably inoculum size is 1%+10%, and harvest time is 18h.
Embodiment 3: preferred lactobacillus and genus bacillus mixed culturing method
(1) get plant lactobacillus (Lb.plantarum) CGMCC6888 or lactobacterium casei (Lb.casei) ATCC393 (pL215) bacterial strain in later stage exponential growth later stage to stationary phase and be inoculated into respectively in MRS substratum, 37 ℃ of static cultivations 12 hours;
(2) subtilis (B.subtilis) the ATCC23857 inoculation of getting later stage exponential growth later stage to stationary phase is in LB substratum, and 37 ℃, 220rpm concussion are cultivated 12 hours;
(3) bacillus subtilis that plant lactobacillus step (1) being made and step (2) make is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1% ﹕ 10%, 37 ℃, 18 hours results of 220rpm concussion cultivation; Or the bacillus subtilis that makes of the lactobacterium casei that step (1) is made and step (2) is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1.5% ﹕ 10%, 37 ℃, 220rpm concussion are cultivated 24 hours results.
In above-described embodiment, the formula of MRS substratum used, LB substratum, MRS-Ca substratum is with embodiment 1.
Claims (2)
1. a method for lactobacillus and genus bacillus mixed culture, step is:
(1) get plant lactobacillus or the lactobacillus casei bacterial strain in later stage exponential growth later stage to stationary phase and be inoculated into respectively in MRS substratum, 37 ± 1 ℃ of static cultivations 12 ± 2 hours;
(2) get the bacillus subtilis strain in later stage exponential growth later stage to stationary phase and be inoculated in LB substratum, 37 ± 1 ℃, 220rpm concussion are cultivated 12 ± 2 hours;
(3) subtilis that plant lactobacillus step (1) being made and step (2) make is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1% ﹕ 10%, 37 ± 1 ℃, 18 ± 1 hours results of 220rpm concussion cultivation; Or the subtilis that makes of the lactobacterium casei that step (1) is made and step (2) is seeded in MRS-Ca substratum by weight the combined inoculation ratio of 1.5% ﹕ 10%, 37 ± 1 ℃, 220rpm concussion are cultivated 24 ± 1 hours results;
Wherein, the formula of above-mentioned MRS-Ca substratum is: on the basis of MRS substratum, adding whole weight percent concentration is 0.02% CaCl
2.
2. the method for lactobacillus and genus bacillus mixed culture as claimed in claim 1, is characterized in that: described plant lactobacillus is plant lactobacillus (Lactobacillus plantarum) CGMCC No.6888; Described lactobacterium casei is lactobacterium casei (Lb.casei) ATCC393; Described subtilis is B.subtilis ATCC23857.
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| CN103392917A (en) * | 2013-07-29 | 2013-11-20 | 湖南圣雅凯生物科技有限公司 | Composite feed additive preparation technology based on gynostemma pentaphylla |
| CN104371958B (en) * | 2014-11-18 | 2017-01-11 | 昆明理工大学 | Method for increasing biomass by virtue of mixed cultivation of enterococcus faecium and bacillus subtilis |
| CN109971675B (en) * | 2019-03-22 | 2019-12-17 | 湖南碧野生物科技有限公司 | Lactobacillus plantarum SCUEC6strain and application thereof |
| CN111575337A (en) * | 2020-06-01 | 2020-08-25 | 汤臣倍健股份有限公司 | Method for detecting lactobacillus number in mixed probiotic product |
| CN112625935A (en) * | 2020-09-25 | 2021-04-09 | 深圳合民生物科技有限公司 | MRS solid culture medium, microbial compound bacterium agent for inhibiting staphylococcus aureus and preparation method thereof |
| CN112940960B (en) * | 2020-12-30 | 2022-02-08 | 北京工商大学 | Bacillus subtilis BS-15, preparation method thereof and application thereof in inducing synthesis of phytolactobacillin EF |
| CN113881600A (en) * | 2021-10-29 | 2022-01-04 | 内蒙古自治区农牧业科学院 | Liquid culture medium for simultaneously culturing two different bacilli and culture method thereof |
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| KR100590913B1 (en) * | 2005-01-24 | 2006-06-19 | 학교법인 계명기독학원 | A method for preparation of chungkukgang with soybean curd residue by using bacillus sp. and lactic acid bacteria |
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