CN104774795A - Lactobacillus plantarum fluid nutrient medium and lactobacillus plantarum fermental cultivation method - Google Patents
Lactobacillus plantarum fluid nutrient medium and lactobacillus plantarum fermental cultivation method Download PDFInfo
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Abstract
The invention discloses a lactobacillus plantarum fluid nutrient medium and a lactobacillus plantarum fermental cultivation method. Molasses, corn steep liquor, fish soluble, diammonium hydrogen phosphate and the like serve as raw materials, the molasses replaces MRS glucose, the corn steep liquor replaces beef extract, the fish soluble replaces yeast powder, and inorganic nitrogen sources replaces peptone. Low-cost agricultural by products partially or completely replace glucose and organic nitrogen sources in an MRS nutrient medium, the nutrient medium combination is optimized at the same time, the number of lactobacillus plantarum bodies and the content of organic acid are improved, and the production cost is reduced remarkably.
Description
Technical field
The present invention relates to milk-acid bacteria culture technique field, in particular a kind of fermentation culture method of plant lactobacillus liquid nutrient medium plant lactobacillus.
Background technology
Plant lactobacillus can produce the multiple natural bacteriostatic materials such as organic acid, bacteriocin, hydrogen peroxide, di-acetyl, therefore be applied in biological fodder and both can produce more number of live bacteria of probiotics, natural antibacterial substance can be produced again, while playing feed anticorrosion, feeding animals can better maintain again intestinal flora balance, improve immunity of organism resistance against diseases, promote animal healthy growth more.Because the water-soluble carbon nitrogenous source in solid-state fermentation substrate is fewer, so plant lactobacillus utilizes dregs of beans, Semen Maydis powder effect bad, breeding to be increased rapidly when solid state fermentation also more difficult.
Adopt the special MRS substratum of milk-acid bacteria to produce, lactobacter growth can be made rapid.But, utilize laboratory MRS substratum to carry out the cost of industrialized production also costly, be not suitable for feeding lactobacillus and produce.
Domestic and international research shows, glucose or sucrose is utilized to make carbon source in plant lactobacillus substratum more common, Xue Delin etc. utilize the starting material such as glucose, sucrose, sugar cane molasses, corn steep liquor, the fermentative production feed complex microorganism preparations of highdensity yeast saccharomyces cerevisiae and Lactobacterium acidophilum (CN103173371A, the production of the feed complex microorganism preparations of yeast saccharomyces cerevisiae and Lactobacterium acidophilum).Although bacterium number reaches the high order of magnitude, this technique needs yeast saccharomyces cerevisiae and Lactobacterium acidophilum to separate ferments and centrifugal concentrating is composite again, and technical process is complicated, and cost is not low.
Domesticly utilize fruits and vegetables class to make the patent of carbon source also more as jerusalem artichoke juice, tomato juice substitute glucose, wherein field turbulent waves etc. utilize jerusalem artichoke juice, tomato juice to cultivate lactobacillus bulgaricus, effect is pretty good, and cost reduces 1600 yuan (CN184435 lactobacillus Bulgaria composite enriched mediums).Jia Yingmin utilizes jerusalem artichoke juice to cultivate bifidus bacillus, makes the most height increament of bifidus bacillus reach 10
9cfu/mL (CN1584013, a kind of bifidobacterium fermentation complex medium and preparation method thereof).Korea Spro Jin lactobacillus plantarum ST-III is inoculated in sterilizing tomato juice, and the conventional MRS substratum of the fermentation cultivation effect of plant lactobacillus ST-III in this substratum and laboratory is suitable; But the cost that fermentation obtains the cost of same dose ST-III viable bacteria reduces by 80% (CN102732470A, lactobacillus plantarum ST-III cultural method of a kind of pure natural, low cost and products thereof and application) than MRS substratum.Although utilize fruits and vegetables class starting material to do fermention medium cost have obvious reduction, because jerusalem artichoke and tomato are that people and animals share fruits and vegetables, add a large amount of use as feed and will bring the anxiety of supplying the mankind, price is wayward.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
The object of the present invention is to provide the fermentation culture method of a kind of plant lactobacillus liquid nutrient medium and plant lactobacillus, molasses are adopted to substitute MRS glucose in formula, corn steep liquor substitutes extractum carnis, the molten slurry of fish substitutes yeast powder, inorganic nitrogen-sourced alternative peptone, simplify the raw material little to plant lactobacillus Factors affecting growth, to solve the high cost of plant lactobacillus fermentation culture existence or the problem of culture process flow process complexity in above-mentioned prior art simultaneously.
Technical scheme of the present invention is as follows:
A kind of plant lactobacillus liquid nutrient medium, wherein, its formula is composed as follows:
Molasses 10-50g/L, corn steep liquor 10-50g/L, fish molten slurry 10-50g/L, Secondary ammonium phosphate 5-25g/L, sodium acetate 2-8g/L, magnesium sulfate 0.5-1g/L, manganous sulfate 0.1-0.5g/L, tween 1-3mL/L;
Wherein, the pH value of substratum is about 6.0-6.8.
Described plant lactobacillus liquid nutrient medium, wherein, its formula is composed as follows:
Molasses 40g/L, corn steep liquor 10g/L, fish molten slurry 15g/L, Secondary ammonium phosphate 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween 1mL/L.
Described plant lactobacillus liquid nutrient medium, wherein, the molten slurry fish meal of described fish substitutes.
Described plant lactobacillus liquid nutrient medium, wherein, described corn steep liquor soybean extract substitutes.
A fermentation culture method for plant lactobacillus, wherein, comprises the following steps:
Take plant lactobacillus as fermentation strain, activation treatment in access MRS liquid nutrient medium; By activation after plant lactobacillus, access as arbitrary in Claims 1 to 5 as described in plant lactobacillus liquid nutrient medium in, inoculum size is 4% ~ 10%, carries out fermentation culture 18 ~ 24 hours;
Wherein, the condition of fermentation culture is anaerobism quiescent culture at 30 ~ 35 DEG C.
The fermentation culture method of described plant lactobacillus, wherein, the process of described activation treatment be in MRS liquid nutrient medium 30 DEG C cultivate 24 hours.
Plant lactobacillus liquid nutrient medium provided by the present invention, with molasses, corn steep liquor, the molten slurry of fish, Secondary ammonium phosphate etc. for raw material, molasses substitute MRS glucose, and corn steep liquor substitutes extractum carnis, and the molten slurry of fish substitutes yeast powder, inorganic nitrogen-sourced alternative peptone.Adopt the glucose in the partly or entirely alternative MRS substratum of the agriculture byproduct of low cost and organic nitrogen source, Optimal Medium combination simultaneously, not only increases thalline quantity and the organic acid content of plant lactobacillus, significantly reduces production cost simultaneously.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of experimental group and control group in the embodiment of the present invention 1.
Embodiment
For making object of the present invention, technical scheme and advantage clearly, clearly, below by way of detailed description and for embodiment, the present invention is described in more detail.
The invention provides a kind of plant lactobacillus liquid nutrient medium, with molasses, corn steep liquor, the molten slurry of fish, Secondary ammonium phosphate etc. for raw material, molasses substitute MRS glucose, and corn steep liquor substitutes extractum carnis, and the molten slurry of fish substitutes yeast powder, inorganic nitrogen-sourced alternative peptone.Molasses are sugaring by product, containing a large amount of fermentable sugars (mainly sucrose), are therefore good fermentation raw materials.Corn steep liquor is the by product of W-Gum processed, containing abundant soluble proteins, growth hormone and some precursor substances, is the organic nitrogen source that microorganism growth is very generally applied.The molten slurry main component of fish is total free aminoacids, low molecular peptide, nucleic acid and water-soluble vitamins and trace element, not only supplement the shortcoming of former MRS substratum somatomedin deficiency, also alternative peptone, extractum carnis, yeast extract paste, reduce costs, and there is very strong attractant, for the later stage produces the starting material that solid-state biological fodder provides good.Described plant lactobacillus liquid nutrient medium, both as seed culture based formulas, also as fermentative medium formula, needs to adopt MRS liquid nutrient medium to activate before use.
Particularly, described plant lactobacillus liquid nutrient medium, its formula is composed as follows:
Molasses 10-50g/L, corn steep liquor 10-50g/L, fish molten slurry 10-50g/L, Secondary ammonium phosphate 5-25g/L, sodium acetate 2-8g/L, magnesium sulfate 0.5-1g/L, manganous sulfate 0.1-0.5g/L, tween 80 1-3mL/L.
Wherein, the pH value of substratum is about 6.0-6.8.The molten slurry of fish can adopt fish meal to substitute; Corn steep liquor can adopt soybean extract to substitute.
Further, also provide optimum implementation in the present invention, described plant lactobacillus liquid nutrient medium, its formula is composed as follows:
Molasses 40g/L, corn steep liquor 10g/L, fish molten slurry 15g/L, Secondary ammonium phosphate 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween 80 1mL/L.
Also provide a kind of fermentation culture method of plant lactobacillus in the present invention, adopt plant lactobacillus liquid nutrient medium of the present invention to carry out fermentation culture to plant lactobacillus, its step is as follows:
Be fermentation strain with plant lactobacillus, get slant strains one ring, carry out activation treatment in access MRS liquid nutrient medium, by the plant lactobacillus after activation, access in liquid nutrient medium of the present invention, inoculum size is 4% ~ 10%, carries out fermentation culture 18 ~ 24h.Wherein, the condition of fermentation culture is: anaerobism quiescent culture at 30 ~ 35 DEG C.
The present invention optimizes seed culture medium by changing, domestication culturing plants Bacterium lacticum, make plant lactobacillus can adapt to the environment of later stage fermentation in advance, can grow fast again, make plant lactobacillus viable count in biological fodder, reach some amount level, plant lactobacillus thalline quantity can reach 30 ~ 5,000,000,000 more than cfu/mL, substratum 10 ~ 2,000,000,000 cfu/mL before comparatively optimizing, viable count improves 2 ~ 5 times, and total acid content brings up to more than 1.7 ~ 3% from 1.0-1.5%.And, adopt the glucose in the partly or entirely alternative MRS substratum of the agriculture byproduct of low cost and organic nitrogen source, both with low cost, take full advantage of resource again.Culture medium cost is reduced to about 843 yuan/ton from MRS substratum about 2855 yuan/ton, reduces about 2000 yuan costs.In addition, plant lactobacillus liquid nutrient medium provided by the present invention, is not only applicable to the fermentation culture of plant lactobacillus, may be used for the fermentation culture of Lactobacterium acidophilum yet.
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
1, liquid nutrient medium of the present invention:
Molasses 40g, corn steep liquor 10g, fish molten slurry 15g, Secondary ammonium phosphate 5g, sodium acetate 5g, magnesium sulfate 0.58g, manganous sulfate 0.25g, tween 80 1mL, distilled water 1L, pH value is 6.8.
2, comparative example formula:
(1) seed culture medium: peptone 10g, extractum carnis 10g, yeast powder 5.0g, glucose 5.0g, tween 80 1.0mL, diammonium hydrogen citrate 2.0g, anhydrous sodium acetate 5.0g, K
2hPO
42.0g, MnSO
4h
2o 0.25g, MgSO
47H
2o 0.58g, distilled water 1.0L, pH value 6.8.
(2) MRS solid medium: peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, glucose 20.0g, anhydrous sodium acetate 5.0g, diammonium hydrogen citrate 2.0g, tween 80 1.0mL, K
2hPO
42.0g, MgSO
47H
2o 0.58g, MnSO
4h
2o 0.25g, agar 20.0g, distilled water 1.0L, pH value 6.8.
(3) MRS liquid nutrient medium: peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose 20.0g, tween 80 1.0mL, diammonium hydrogen citrate 2.0g, anhydrous sodium acetate 5.0g, K
2hPO
42.0g, MnSO
4h
2o 0.25g, MgSO
47H
2o 0.58g, distilled water 1.0L, pH value 6.8.
3, collocation method is as follows:
Take substratum respectively to fill a prescription and dissolve, allotment optimum pH.
The culture medium prescription of above-mentioned dissolving is carried out 121 DEG C of sterilizing 20min, containing the independent 115 DEG C of sterilizing 30min of dextrose culture-medium.
4, the cultural method of plant lactobacillus is as follows:
Culture condition: take plant lactobacillus as fermentation strain, being seeded in MRS liquid nutrient medium to turn after activation is seeded in liquid nutrient medium of the present invention again, initial pH value 6.2 ~ 6.8, anaerobism quiescent culture 18 ~ 24h at 30 ~ 35 DEG C, inoculum size (v/v) is 4 ~ 10%.With culture medium prescription culturing plants Bacterium lacticum of the present invention for experimental group, be forwarded under the same conditions and compare group with culturing plants Bacterium lacticum in MRS liquid nutrient medium.
5, the mensuration of growth curve
Get slant strains one ring, in access MRS liquid nutrient medium, cultivate 24h activation for 30 DEG C, by the plant lactobacillus after activation, access respectively in the liquid nutrient medium of the present invention of 50mL and the MRS liquid nutrient medium of 50mL, inoculum size is 4%.Anaerobism quiescent culture 24h at 35 DEG C, every 2h sampling, until fermentation stops.Reference is made, working sample OD600nm value with the liquid nutrient medium not accessing bacterial classification of correspondence.Draw the growth curve of plant lactobacillus in different culture media, growth curve as shown in Figure 1.
6, the mensuration of plant lactobacillus biomass
Get the fermented liquid 2mL of cultivation end in centrifuge tube, centrifugal 8min under 8000r/min rotating speed, remove supernatant liquor, after adding 1mL distilled water, concussion is dissolved into bacteria suspension, after being finally diluted to suitable multiple, spectrophotometer is utilized to record OD600nm value, with the liquid nutrient medium not accessing bacterial classification of correspondence for reference.
After experimental group cultivation 20h, plant lactobacillus OD600nm value is 10.33, and after control group cultivates 18h, plant lactobacillus OD600nm reaches and is up to 9.01.
7, the mensuration of plant lactobacillus viable count
Shake up sampling after cultivation terminates, adopt dull and stereotyped gradient dilution coating method to detect the viable count of the plant lactobacillus in fermented liquid.
Wherein, after experimental group cultivates 20h, plant lactobacillus viable count reaches 4,000,000,000 more than cfu/mL, and after control group cultivates 18h, plant lactobacillus viable count reaches about 1,800,000,000 cfu/mL.
8, the mensuration of total organic acids content
Get 5mL fermented liquid after cultivation terminates in beaker, adopt the method for acid base titration to measure total organic acids content.
Wherein, after experimental group cultivation 20h, total organic acids content is 1.8%, total organic acids content 1.5% after control group cultivation 18h.
9, cost contrast
Experimental group formulation cost and control group are contrasted, comparing result is as shown in table 1 below, and the cost of experimental group is about 843 yuan/ton, and the cost of control group MRS substratum is about 2855 yuan/ton, and experimental group is originally lower about 2000 yuan than contrast composition.
Table 1 two kinds of culture medium prescription cost synopsis
Embodiment 2
Described plant lactobacillus liquid nutrient medium, its formula is composed as follows:
Molasses 20g/L, corn steep liquor 30g/L, fish molten slurry 50g/L, Secondary ammonium phosphate 15g/L, sodium acetate 5g/L, magnesium sulfate 0.85g/L, manganous sulfate 0.5g/L, tween 80 1.5mL/L.
Get slant strains one ring, in access MRS liquid nutrient medium, cultivate 24h activation for 30 DEG C, by the plant lactobacillus after activation, in the liquid nutrient medium of access 50mL the present embodiment, initial pH value 6.2.Anaerobism quiescent culture 20h at 30 DEG C, inoculum size (v/v) is 4%.
Shake up sampling after cultivation terminates, adopt dull and stereotyped gradient dilution coating method to detect the viable count of the plant lactobacillus in fermented liquid, in the present embodiment, plant lactobacillus thalline quantity reaches 3,000,000,000 more than cfu/mL.
Get 5mL fermented liquid after cultivation terminates in beaker, adopt the method for acid base titration to measure total organic acids content, in the present embodiment, total organic acids content is 1.7%.
Embodiment 3
Described plant lactobacillus liquid nutrient medium, its formula is composed as follows:
Molasses 50g/L, corn steep liquor 5g/L, fish molten slurry 15g/L, Secondary ammonium phosphate 5g/L, sodium acetate 3g/L, magnesium sulfate 0.7g/L, manganous sulfate 0.35g/L, tween 80 3mL/L.
Wherein, the pH value of substratum is 6.3.
Get slant strains one ring, in access MRS liquid nutrient medium, cultivate 24h activation for 30 DEG C, by the plant lactobacillus after activation, in the liquid nutrient medium of access 50mL the present embodiment, initial pH value 6.3.Anaerobism quiescent culture 20h at 30 DEG C, inoculum size (v/v) is 4%.
Shake up sampling after cultivation terminates, adopt dull and stereotyped gradient dilution coating method to detect the viable count of the plant lactobacillus in fermented liquid, in the present embodiment, plant lactobacillus thalline quantity reaches 3,700,000,000 more than cfu/mL.
Get 5mL fermented liquid after cultivation terminates in beaker, adopt the method for acid base titration to measure total organic acids content, in the present embodiment, total organic acids content is 2.0%.
Embodiment 4
Described plant lactobacillus liquid nutrient medium, its formula is composed as follows:
Molasses 40g/L, soybean extract 10g/L, fish meal 15g/L, Secondary ammonium phosphate 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween 80 1mL/L.Wherein, the pH value of substratum is 6.8.
Get slant strains one ring, in access MRS liquid nutrient medium, cultivate 24h activation for 30 DEG C, by the plant lactobacillus after activation, in the liquid nutrient medium of access 50mL the present embodiment, initial pH value 6.8.Anaerobism quiescent culture 24h at 30 DEG C, inoculum size (v/v) is 4%.
Shake up sampling after cultivation terminates, adopt dull and stereotyped gradient dilution coating method to detect the viable count of the plant lactobacillus in fermented liquid, in the present embodiment, plant lactobacillus thalline quantity reaches 3,500,000,000 more than cfu/mL.
Get 5mL fermented liquid after cultivation terminates in beaker, adopt the method for acid base titration to measure total organic acids content, in the present embodiment, total organic acids content is 2.0%.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (7)
1. a plant lactobacillus liquid nutrient medium, is characterized in that, its formula is composed as follows:
Molasses 10-50g/L, corn steep liquor 10-50g/L, fish molten slurry 10-50g/L, Secondary ammonium phosphate 5-25g/L, sodium acetate 2-8g/L, magnesium sulfate 0.5-1g/L, manganous sulfate 0.1-0.5g/L, tween 1-3mL/L;
Wherein, the pH value of substratum is about 6.0-6.8.
2. plant lactobacillus liquid nutrient medium according to claim 1, is characterized in that, its formula is composed as follows:
Molasses 40g/L, corn steep liquor 10g/L, fish molten slurry 15g/L, Secondary ammonium phosphate 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween 1mL/L.
3. plant lactobacillus liquid nutrient medium according to claim 1 and 2, is characterized in that, the molten slurry fish meal of described fish substitutes.
4. plant lactobacillus liquid nutrient medium according to claim 3, is characterized in that, described corn steep liquor soybean extract substitutes.
5. plant lactobacillus liquid nutrient medium according to claim 1 and 2, is characterized in that, described corn steep liquor soybean extract substitutes.
6. a fermentation culture method for plant lactobacillus, is characterized in that, comprises the following steps:
Take plant lactobacillus as fermentation strain, activation treatment in access MRS liquid nutrient medium; By activation after plant lactobacillus, access as arbitrary in Claims 1 to 5 as described in plant lactobacillus liquid nutrient medium in, inoculum size is 4% ~ 10%, carries out fermentation culture 18 ~ 24 hours;
Wherein, the condition of fermentation culture is anaerobism quiescent culture at 30 ~ 35 DEG C.
7. the fermentation culture method of plant lactobacillus according to claim 6, is characterized in that, the process of described activation treatment be in MRS liquid nutrient medium 30 DEG C cultivate 24 hours.
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Cited By (2)
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| CN110577918A (en) * | 2019-10-11 | 2019-12-17 | 福建省农业科学院农业工程技术研究所 | Preparation method of economical lactobacillus high-density proliferation garlic juice molasses culture agent |
| CN112779180A (en) * | 2020-12-23 | 2021-05-11 | 中国科学院微生物研究所 | Liquid fermentation medium and application thereof |
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Application publication date: 20150715 |