CN101597585A - A kind of substratum of suitable industrial production of bifidobacteria and compound method thereof - Google Patents
A kind of substratum of suitable industrial production of bifidobacteria and compound method thereof Download PDFInfo
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Abstract
The invention discloses a kind of substratum and compound method thereof of suitable industrial production of bifidobacteria.Described substratum is grouped into by following one-tenth: Dried Corn Steep Liquor Powder (protein content 40%), 55～63g/L; Glucose, 5～10g/L; Tween-80,0～1g/L; Initial pH 6.5～7.2.Bifidus longum bb BBMN68 and BB12 be well-grown in this substratum, and the maximum growth amount reaches 10
9Cfu/ml is better than classical bifidus bacillus substratum MRS and NPNL.This culture medium cost cheap (compare NPNL substratum price and descend 95%, compare MRS substratum price and descend 80%), making processes is simple, is fit to the commercial scale production of bifidus bacillus.
Description
Technical field
The present invention relates to substratum of a kind of bifidus bacillus and preparation method thereof, particularly a kind of suitable industrialized bifidus bacillus substratum.。
Background technology
Bifidus bacillus (Bifidobacterium) is that people and animals wait one of important physical bacterium in the homoiothermy animal intestinal, it is one of important indicator bacterium of HUMAN HEALTH, its proportion in intestinal microflora directly affects host's healthy state, constituted the field planting resistance that stops the foreign bacteria invasion with other normal microflora, has certain biological antagonist effect, the bifidus bacillus body contains a large amount of digestive ferments simultaneously, the enzyme that can digest dependence itself in some bodies is the nutritive substance that can not digest, promote the absorption of endotrophic material, bifidus bacillus and surface tissue composition thereof can be used as biological response agent oral administration or non-stomach and intestine approach strengthens host's immune surveillance function, can strengthen various cytokines and production of antibodies, improve NK and macrophage activity, improve the defense function of part or whole body, performance is regulated from steady, anti-infective, anti-tumour effect.Bifidus bacillus is many at present is added in the dairy products as probiotic bacterium, dominates in functional foodstuff market American-European, Japan and other countries, and 379 kinds of probiotic products listings were only just arranged in 2005.
At present, the substratum that bifidus bacillus is commonly used has MRS, NPNL, TPY, PTYG etc.TPY and PTYG substratum surge capability a little less than, be not suitable for extensive use; MRS and NPNL nutritive ingredient are abundant, and surge capability is strong, but at present MRS and NPNL substratum there are the following problems: price higher (13 yuan/L of NPNL, 3 yuan/L of MRS; Phosphoric acid salt easily causes lactobacillus to become not anti-freeze fine bar-shaped by the anti-stub that freezes in the buffer system; More inorganic salt composition has potential harmful effect to human body and animal health; The L-cysteine hydrochloride produces problems such as unhappy smell and limits it in industrial application through high-temperature sterilization.For screening the bifidus bacillus substratum, the researchist that reduces cost is mostly on the basis of biochemical medium, to add a certain proportion of crude substance with bifid growth factor, as tomato juice, jerusalem artichoke juice, Radix Dauci Sativae juice etc., also reached the purpose that improves viable count, but this type of substratum preparation steps is more, complex operation is not suitable for being used for industrialization.
Summary of the invention
For overcoming above-mentioned deficiency of the prior art, the invention provides a kind of is the bifidus bacillus substratum of main raw material with the Dried Corn Steep Liquor Powder.The characteristics that it has cost economy and is easy to prepare.
The screening of 1 bifidus bacillus template substratum
For the growth characteristics to bifidus bacillus have a preliminary understanding, the bifidus longum bb of having selected better performances such as acidproof bile tolerance is an experimental strain, has measured the growth curve of bifidus longum bb under four kinds of different substratum.Adopt 25mL anaerobism screw-cap test tube, NPNL, MRS, TPY, PTYG substratum, inoculum size leaves standstill cultivation for 1%, 37 ℃, and viable count is measured in sampling in per three hours, the speed of growth and the number of viable of contrast bacterial strain in different substratum.Experimental result as shown in Figure 1.
By the SAS statistical study as can be known, viable count was compared with other substratum significant difference is all arranged after the cultivation 12h of bifidus longum bb in the NPNL substratum entered stationary phase, and therefore selecting NPNL is that template is carried out the screening of industrialization substratum.
The NPNL medium component is: peptone 10g, and Tryptones 5g, soy peptone 3g, yeast soak powder 10g, glucose 10g, beef liver powder 15g, cysteine hydrochloride 0.5g, tween-80 1ml, Zulkovsky starch 0.5g, extractum carnis 3g, solution A 10ml, solution B 5ml.(wherein, A liquid: K2HPO425g, KH2PO4 25g, distilled water 250ml; B liquid: MgSO47H2O5g, FeSO47H2O 0.5g, NaCl 0.5g, MnSO4H2O 0.337g, distilled water 250ml) pH7.2.
The factor of 2 restriction NPNL substratum industrial applications
NPNL substratum moiety comprise carbon source (glucose), nitrogenous source (peptone, Tryptones, soy peptone, yeast soak powder, beef liver powder), buffer system (K2HPO4, KH2PO4), antioxidant (L-cysteine hydrochloride), trace element (MgSO47H2O, FeSO47H2O, NaCl, MnSO4H2O).
Different types of nutrient substance proportion difference is bigger in the NPNL substratum, as accompanying drawing 2.The nitrogenous source cost accounts for 93% of overall cost, and carbon source accounts for 2.5%, and antioxidant accounts for 4.0%, and buffer system, trace element, emulsifying agent account for 0.5% altogether.By accompanying drawing 2 as can be known, the higher NLNL substratum industrial applications that influenced of nitrogenous source cost.In addition, phosphate buffered saline buffer is to thalline, the more inorganic salt composition harmful effect to humans and animals health, and the bad flavor that the sterilization of L-cysteine hydrochloride produces all is factors of restriction NPNL substratum industrial applications.
3 industrialization substratum are selected
Carry out the screening of industrial culture medium at the shortcoming of NPNL, under the prerequisite that does not reduce bifidus bacillus biological fermentation amount, template NPNL substratum is suitably revised.
3.1 determining of nitrogenous source
The cost of nitrogenous source accounts for more than 90% of total cost among the NPNL, and therefore reducing culture medium cost mainly is to reduce the cost of nitrogenous source.
Select for use nitrogenous sources such as comparatively common and lower-cost skim-milk, whey-protein, corn steep liquor to study, other nutritive ingredients and consumption all add according to composition and consumption in the template NPNL substratum.The addition of nitrogenous sources such as skim-milk, whey-protein, Dried Corn Steep Liquor Powder is a gross protein value (25g/L) in the template NPNL substratum, measures the viable count of bifidus longum bb in various substratum, the result as shown in Figure 3:
Be at protein content under the condition of 25g/L, bifidus longum bb enters stationary phase at 9h, viable count in Dried Corn Steep Liquor Powder has significant difference and template substratum NPNL not to have significant difference than the viable count in skim-milk and the whey-protein, therefore selects for use Dried Corn Steep Liquor Powder as nitrogenous source.
Under all constant situation of other conditions such as carbon source, antioxidant, emulsifying agent, select the substratum of different Dried Corn Steep Liquor Powder protein contents for use, 1% inoculation, 12h carries out viable count and measures, and the result is as shown in Figure 4.
By the SAS statistical study as can be known, bifidus longum bb protein content be in the substratum of 22g/L and 25g/L the viable bacteria number average than the substratum of other content in viable count significant difference is arranged, the two does not have significant difference, therefore with corn oar dry powder during as culture media nitrogen source, the protein content of preferred culture medium is 22～25g/L.
The corn oar dry powder that can select different protein contents is as nitrogenous source, and regulates the addition of corn oar dry powder according to required culture medium protein content.The protein content of Dried Corn Steep Liquor Powder is preferably 40 weight %, and its addition can be 55～63g/L.
3.2 determining of buffer system
Having more solid substance in the Dried Corn Steep Liquor Powder substratum, is a kind of good buffer system.Imagination can replace phosphate buffer and play shock absorption, and verifies.On the medium base of 3.1 screenings, add and do not add phosphate buffer respectively, 1% inoculation, when reaching stationary phase, 12h takes a sample respectively, measure viable count, the substratum viable count logarithmic value of adding phosphate buffer is 9.61 ± 0.03, the substratum viable count logarithmic value of not adding phosphate buffer is 9.65 ± 0.05, and both do not have significant difference as can be known through the SAS statistical study.
By the result as can be known, add or do not add phosphate buffer and the pH of corn steep liquor substratum and viable count are changed have no significant effect, can play the buffered effect.Therefore can reject phosphate buffered saline buffer in the template NPNL substratum, to avoid its influence to thalline freeze-drying survival rate.
3.3 determining of inorganic salt solution
There are some researches show, in the substratum that contains the corn steep liquor composition, add or do not add external inorganic salt composition lactobacillus rhamnosus lactic acid producing ability is not had significant difference, show that the inorganic salt content in the corn steep liquor can satisfy the demand of milk-acid bacteria to mineral salt, can omit the inorganic salt solution in the NPNL substratum, and whether carry out experimental verification influential to the growth of bifidus bacillus.On the medium base of 3.2 screenings, add and do not add the inorganic salt solution among the NPNL respectively, 1% inoculation, it is the sampling and measuring viable count that 12h reaches stationary phase, the substratum viable count logarithmic value of adding inorganic salt solution among the NPNL is 9.73 ± 0.03, and the substratum viable count that does not add inorganic salt solution among the NPNL is 9.60 ± 0.07.
Both do not have significant difference as can be known through the SAS statistical study, and promptly the inorganic salt composition in the corn steep liquor can satisfy the growth of bifidus bacillus, therefore needn't add the inorganic salt solution among the NPNL in selected corn steep liquor substratum.
3.4 determining of antioxidant
There are some researches show, xitix also has as a kind of antioxidant to be eliminated oxygen and not to have unpleasant odor to produce in membrane filtration degerming and bifidus bacillus culturing process to the effect of coercing of bifidus bacillus and its, therefore can consider to replace the L-cysteine hydrochloride, select for use xitix to add in 3.3 substratum that screen as antioxidant, 1% inoculation, sampling and measuring viable count during 12h, the result as shown in Figure 5:
Wherein, any antioxidant is not added in 0 expression.The result shows, the xitix that adds different amounts does not have the significance influence to the growth of bifidus bacillus in the corn steep liquor substratum, therefore can not add any antioxidant. trace it to its cause may for, the Gelucystine that contains have an appointment 0.39% (dry matter content) in the corn steep liquor, and Gelucystine can transform mutually with halfcystine, can play the chemical stress effect of oxygen to thalline of removing in the process of bifidobacterium fermentation.
The present invention is with the nitrogenous source of corn oar as the bifidus bacillus substratum.Have following advantage:
Contain rich soluble albumen, amino acid, VITAMIN, inorganic salt etc. in the corn steep liquor, contain about 70% solid matter, protein content is about 40%, and aminoacids content is about 20%, and its trophic structure is well suited for the needs of lactobacter growth breeding.
Its wide material sources, and the spray-dried Dried Corn Steep Liquor Powder that makes, accumulating is convenient, adapts to the needs that large-scale industrialization is produced.
In recent years W-Gum production increase is very fast, and the outlet of corn steep liquor has also become problem, the W-Gum factory producing region of relatively concentrating particularly, and a lot of little factories all directly discharge soak water of maize, the waste resource, and cause the pollution of environment.The present invention has opened up the use range of corn steep liquor.
It is with low cost, has reduced the composition of substratum significantly, compares NPNL substratum price and descends 95%, compares MRS substratum price and descends 80%.
Its making processes is simple, is fit to the commercial scale production of bifidus bacillus.
Description of drawings
Accompanying drawing 1: the growth curve of bifidus bacillus in four kinds of bifid substratum.
The cost of a nutritive ingredient in the accompanying drawing 2:NPNL substratum.
Accompanying drawing 3: the result of three kinds of nitrogenous source culture medium culturing bifidus bacilluss of replacement.
Accompanying drawing 4: the result of different Dried Corn Steep Liquor Powder protein concn culture medium culturing bifidus bacilluss.
Accompanying drawing 5: add the contrast of cultivating bifidus bacillus in the Dried Corn Steep Liquor Powder substratum of different antioxidants.
Embodiment
Embodiment 1:
(1) preparation corn steep liquor: after the Dried Corn Steep Liquor Powder of weighing, supply water to 55～63g/L, filtered through gauze, standby;
(2) add glucose, 10～20g/L; Tween-80,0～1g/L;
(3) regulate pH value to 6.5～7.2;
(4) 110 ℃～121 ℃, 10～15min sterilization is standby.
Embodiment 2:
(1) preparation corn steep liquor: select refining Dried Corn Steep Liquor Powder, after weighing, supply water, regulate pH value to 7.2 to 63g/L through spraying drying processing, 8 layers of filtered through gauze, standby;
(2) add glucose, 10g/L;
(3) regulate pH value to 6.5;
(4) packing, 110 ℃～121 ℃, 10～15min sterilization is standby.
Embodiment 3:
(1) preparation corn steep liquor: select refining Dried Corn Steep Liquor Powder, after weighing, supply water, regulate pH value to 7.2 to 58g/L through spraying drying processing, 8 layers of filtered through gauze, standby;
(2) add glucose, 15g/L; Tween-80,0.5g/L;
(3) regulate pH value to 7.0;
(4) packing, 110 ℃～121 ℃, 10～15min sterilization is standby.
Embodiment 4:
(1) preparation corn steep liquor: select refining Dried Corn Steep Liquor Powder, after weighing, supply water, regulate pH value to 7.2 to 55g/L through spraying drying processing, 8 layers of filtered through gauze, standby;
(2) add glucose, 20g/L; Tween-80,1g/L;
(3) regulate pH value to 7.2;
(4) packing, 110 ℃～121 ℃, 10～15min sterilization is standby.
Claims (6)
1. the substratum of a suitable industrial production of bifidobacteria is characterized in that being grouped into by following one-tenth:
Be that to make culture medium protein matter content be 22～25g/L to nitrogenous source with the Dried Corn Steep Liquor Powder; Glucose, 10～20g/L; Tween-80,0～1g/L; Initial pH 6.5～7.2, surplus is a water.
2. substratum as claimed in claim 1 is characterized in that wherein the protein wt content of Dried Corn Steep Liquor Powder is 40%.
3. according to claims 1 or 2 described suitable industrial production of bifidobacteria substratum, it is characterized in that described Dried Corn Steep Liquor Powder is the refining Dried Corn Steep Liquor Powder through spraying drying processing.
4. according to claims 1 or 2 described suitable industrial production of bifidobacteria substratum, it is characterized in that described glucose is analytical pure glucose.
5. according to claims 1 or 2 described suitable industrial production of bifidobacteria substratum, it is characterized in that described tween-80 is the analytical pure tween-80.
6. according to the preparation method of the described suitable industrial production of bifidobacteria substratum of claims 1-5, it is characterized in that may further comprise the steps:
(1) preparation corn steep liquor: after the Dried Corn Steep Liquor Powder of weighing, supply water to normality, after the filtered through gauze, standby;
(2) glucose and/or the tween-80 of interpolation specified amount in the corn steep liquor of step (1);
(3) regulate pH value to 6.5～7.2;
(4) 110 ℃～121 ℃, sterilization 10～15min promptly.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760635A (en) * | 2017-11-30 | 2018-03-06 | 成都欧林生物科技股份有限公司 | Beneficial to the bacterium culture medium of anti-microbiological contamination |
| CN112592860A (en) * | 2020-12-23 | 2021-04-02 | 深圳科兴药业有限公司 | Bifidobacterium infantis fermentation medium without animal-derived nitrogen source and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1225551C (en) * | 2002-07-16 | 2005-11-02 | 李盛学 | Liquid-solid combined fermentation process of probiotics by using bifidobacteria as main component |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760635A (en) * | 2017-11-30 | 2018-03-06 | 成都欧林生物科技股份有限公司 | Beneficial to the bacterium culture medium of anti-microbiological contamination |
| CN112592860A (en) * | 2020-12-23 | 2021-04-02 | 深圳科兴药业有限公司 | Bifidobacterium infantis fermentation medium without animal-derived nitrogen source and application thereof |
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