CN104531596A - Improved whey protein culture medium and application thereof - Google Patents

Improved whey protein culture medium and application thereof Download PDF

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CN104531596A
CN104531596A CN201410796931.7A CN201410796931A CN104531596A CN 104531596 A CN104531596 A CN 104531596A CN 201410796931 A CN201410796931 A CN 201410796931A CN 104531596 A CN104531596 A CN 104531596A
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culture medium
viable count
lactobacillus casei
medium
lactalbumin
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CN104531596B (en
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余丽芸
侯喜林
徐超
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Heilongjiang Bayi Agricultural University
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Heilongjiang Bayi Agricultural University
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    • 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

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Abstract

The invention relates to an improved whey protein culture medium comprising the following components in percentage by weight: 4% of whey proteins, 1.5% of yeast powder, 1% of lactose, 0.5% of phosphate and the balance of water, wherein the phosphate comprises the following components in percentage by weight: 0.188% of Na2HPO4 and 0.312% of NaH2PO4. The invention also provides application of the culture medium to culture of recombinant lactobacillus casei. According to the improved whey protein culture medium, a whey protein culture medium for culturing the recombinant lactobacillus casei is further optimized, so that the growth of a strain can be better promoted, the viable count can be increased, and the average measured value of the viable count of the recombinant lactobacillus casei can be up to 7.6*10<9>cfu/mL; and the culture medium can replace an MRS culture medium, so that the production cost is reduced, and the foundation is laid for further preparing an oral solution vaccine for preventing piglet diarrhea.

Description

A kind of lactalbumin medium of improvement and application thereof
Technical field
The invention belongs to microorganism field, relate to a kind of lactalbumin medium and application thereof of improvement.
Background technology
Genetic engineering bacterium utilizes genetic engineering technique, external source goal gene imported host cell and makes it express the recombinant bacterium of desirable proteins.Engineering bacteria fermentation is to obtain a large amount of exogenous gene expression products, having very important relation with the control of fermentation manufacturing technique.
Recombinant lactobacillus casei is a kind of live vector oral vaccine candidate strain, and has the prebiotic function of milk-acid bacteria concurrently, and can play many physiological effects in vivo.Traditional lactobacterium casei amplification culture MRS substratum carries out, bacterial number and expression amount higher, but the price comparison of MRS substratum is expensive, cost is higher, and mouthfeel is not good enough, so sight is placed on cheap by people, nutritious, on the good whey-protein of mouthfeel.Whey-protein from the whey of pasteurize, removes non-protein and makes through filtering-depositing.Whey-protein mouthfeel is good, nutritious, absorption easy to digest.But, find when using simple whey-protein to cultivate recombinant lactobacillus casei, this bacterium fermenting bacteria quantity in lactalbumin powder is low and expression amount is lower, therefore, if the formula of lactalbumin powder can be improved further, obtain cheap, improve lactalbumin powder efficiently, extend shelf time of bacterial classification, the suitability for industrialized production for vaccine lays the foundation and seems particularly important.
Summary of the invention
The object of this invention is to provide a kind of lactalbumin medium of improvement, the price comparison solving MRS substratum used is at present expensive, cost is higher, mouthfeel is not good enough, but simple lactalbumin powder is cultivated recombinant lactobacillus casei as substratum, the problem that the fermenting bacteria quantity of bacterial classification is low and expression amount is lower.
Another object of the present invention is to provide the purposes of the lactalbumin medium of above-mentioned improvement.
The present invention is achieved through the following technical solutions:
One, a lactalbumin medium for improvement, its component and weight percent as follows:
Whey-protein 4%, yeast powder 1.5%, lactose 1%, phosphoric acid salt 0.5%, surplus is water.
Further, described phosphoric acid salt comprises Na 2hPO 4and NaH 2pO 4, wherein, Na 2hPO 4weight percent be 0.188%, NaH 2pO 4weight percent be 0.312%.
The lactalbumin medium of two, above-mentioned improvement is cultivating the application in recombinant lactobacillus casei.
Adopt the positively effect of technique scheme: the present invention has carried out further optimization to the lactalbumin medium cultivating recombinant lactobacillus casei, better can promote growth, improve viable count, make the actual average value of recombinant lactobacillus casei viable count reach 7.6 × 10 9cfu/mL; This substratum can substitute MRS substratum, reducing production cost, laying the foundation for preparing prevention of diarrhea in piglets oral liquid vaccine further.
Accompanying drawing explanation
Fig. 1 is recombinant plasmid PCR qualification result;
In figure, M.DL 2000marker; 1. negative control, 2.TGEV-BC PCR primer, 1349bp;
Fig. 2 is that buffering salt is to recombinant lactobacillus casei growth effect result figure;
Fig. 3 is the pH value variation diagram before and after buffering salt fermentation;
Fig. 4 is that Tryptones is to recombinant lactobacillus casei growth effect result figure;
Fig. 5 is that yeast powder is to recombinant lactobacillus casei growth effect result figure;
Fig. 6 is that SODIUMNITRATE is to recombinant lactobacillus casei growth effect result figure;
Fig. 7 is that ammonium sulfate is to recombinant lactobacillus casei growth effect result figure;
Fig. 8 is that different carbon source is to recombinant lactobacillus casei growth effect result figure;
Fig. 9 is three kinds of substratum comparing result figure;
In figure, 1 common lactalbumin medium, 2 improvement lactalbumin medium, 3MRS substratum.
Embodiment
The source of biomaterial in the present invention:
1, recombinant lactobacillus casei: during this bacterial classification is disclosed in " research that pig infectious gastroenteritis virus S truncated protein is expressed at lactobacterium casei surface display ", author: Tian Bin, China's Preventive Veterinary Medicine report the 35th volume o. 11th, 1008-0589 (2013) 11-0874-04, this author is Heilongjiang Bayi Agricultural Reclamation University student, this bacterial classification is kept in Life Sci-Tech institute of Heilongjiang Bayi Agricultural Reclamation University genetic engineering laboratories at present, and is ensured to provide this biomaterial to the public in Two decades years from the application's day by the applicant of this patent;
2, lactobacterium casei CICC6105 (Lactobacillus casei CICC 6106): purchased from Chinese industrial Culture Collection.
Below in conjunction with embodiment and comparative example, technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
Embodiment 1
A lactalbumin medium for improvement, takes whey-protein 40g, yeast powder 15g, lactose 10g, Na 2hPO 41.88g, NaH 2pO 43.12g, adds distilled water, and configuration final volume is the lactalbumin medium of 1L.
Embodiment 2
A lactalbumin medium for improvement, takes whey-protein 80g, yeast powder 30g, lactose 20g, Na 2hPO 43.76g, NaH 2pO 46.24g, adds distilled water, and configuration final volume is the lactalbumin medium of 2L.
Embodiment 3
The present embodiment is for illustration of the activation of recombinant lactobacillus casei and qualification.
Recombinant lactobacillus casei list bacterium colony on the cultured MRS agar plate of picking, in access 10mL MRS liquid nutrient medium, then 37 DEG C of static gas wave refrigerator 16h are carry out enlarged culturing in the MRS liquid nutrient medium of the inoculum size access 100mL of 2% by weight percentage, 37 DEG C of static gas wave refrigerator 16h, for subsequent use.
Single bacterium colony that random picking MRS substratum grows, be inoculated in the MRS liquid nutrient medium containing paraxin (34ug/mL), 37 DEG C of static gas wave refrigerator spend the night.The method provided according to Daniel J [94] etc. extracts plasmid, and method is as follows:
(1) get the bacterium liquid 2mL of incubated overnight in centrifuge tube, the centrifugal 1min of 12000r/min collects bacterial sediment;
(2) the resuspended precipitation of solution 200uL containing 25% sucrose and 30mg/mL N,O-Diacetylmuramidase is added, after mixing, 37 DEG C of water-bath 20min;
(3) in solution, add the lysate 400uL of the NaOH containing 3%SDS and 0.2N, mix rapidly, room temperature places 7min;
(4) the 3M sodium-acetate 300uL of precooling is being added wherein, 4 DEG C, the centrifugal 15min of 12000r/min;
(5) supernatant liquor is moved on in a new centrifuge tube, after adding the Virahol of 650uL, 4 DEG C, the centrifugal 15min of 12000r/min;
(6) supernatant discarded, adds the washing with alcohol precipitation of 1mL 75%, 4 DEG C, the centrifugal 5min of 12000r/min, repeated washing twice;
(7) topple over supernatant, the TE containing Rnase enzyme adding 30uL wherein suspends and precipitates, and-20 DEG C save backup.
PCR qualification is carried out as pcr template with the plasmid extracted, by Auele Specific Primer (the upstream primer 5 '-CGCGGATCCACTAATAGAACTATAGGCAAC-3 ' of design, downstream primer 5 '-CGCGTCGACTTAAGCACATTGATCAGTGCAATTG-3 ') carry out pcr amplification, the empty bacterium of lactobacterium casei CICC6105 is set simultaneously in contrast.Identify with the agarose gel electrophoresis of 1%, result as shown in Figure 1, the specific band of visible 1349bp, with expection DNA fragmentation in the same size.
Embodiment 4
The present embodiment illustrates that buffering salt is on the impact of viable count.
Different buffering salt is on the impact of lactobacter growth: milk-acid bacteria metabolize sugars in process of growth produces lactic acid, the pH value of nutrient solution is constantly reduced, the breeding of lactic acid bacteria inhibiting.Therefore in the fermention medium of milk-acid bacteria, the different buffering salt of buffering is added on the impact of lactobacter growth: milk-acid bacteria metabolize sugars in process of growth produces lactic acid, the pH value of nutrient solution is constantly reduced, the breeding of lactic acid bacteria inhibiting.Therefore in the fermention medium of milk-acid bacteria, buffering salt is added to alleviate pH value.
By the recombinant bacterium bacterial strain activated, by whey protein concentration 4%, initial pH 6.6, inoculum size 4%, indirectly inoculation, rotating speed 80r/min, be inoculated in 150mL lactalbumin medium, and use phosphate buffered saline buffer, citrate buffer, acetate salt buffer salts solution respectively, the weight percent of addition is 0.5%, sample respectively after the lower 37 DEG C of static gas wave refrigerator 16h of anaerobic condition, be coated with plate count.
Not add any buffering salt basal fermentation medium for blank, result is affected as Fig. 2 to lactobacter growth.Fig. 3 is shown in pH value change before and after often kind of buffering salt fermentation.Analytical results shows, adds the reduction that phosphate buffer salt can better alleviate pH value, and its viable bacteria amount is also the highest.This and other report, the result being realized high-density culture by alleviation pH value mode is consistent.The patience of different milk-acid bacterias to acidic conditions has larger difference, thus maintains certain pH value and can ensure that it grows.
Embodiment 5
The present embodiment illustrates that nitrogenous source is on the impact of viable count.
Use yeast powder, peptone, ammonium sulfate, SODIUMNITRATE as nitrogenous source respectively, add in lactalbumin medium with the inoculum size that weight percent is 1%, sample respectively after the lower 37 DEG C of static gas wave refrigerator 16h of anaerobic condition, be coated with plate count.
(1) Tryptones is on the impact of viable count
The Tryptones that weight percent concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% is shown in Fig. 4 as the impact of nitrogenous source on recombinant bacterium viable count, as we know from the figure, along with the increase of Tryptones concentration, the viable count of recombinant bacterium is also increasing thereupon, but when Tryptones concentration reaches 1%, the quantity of viable count reaches the highest.Then improve whey protein concentration further again, the quantity of viable count does not only raise, and presents downtrending on the contrary, and this illustrates too high concentration and is unfavorable for the growth of recombinant bacterium.Therefore, determine that the optimum concn of Tryptones is weight percentage 1%.
(2) yeast powder is on the impact of recombinant bacterium viable count
The yeast powder that weight percent concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% is shown in Fig. 5 as the impact of nitrogenous source on recombinant bacterium viable count, as we know from the figure, along with the increase of yeast powder concentration, the viable count of recombinant bacterium is also increasing thereupon, but when yeast powder concentration reaches 1.5%, the quantity of viable count reaches the highest.Then improve yeast powder concentration further again, the quantity of viable count does not only raise, and presents downtrending on the contrary, excessive concentration or the too low growth being all unfavorable for genetic engineering bacterium thalline, and therefore, the optimum concn of yeast powder is weight percentage 1.5%.
(3) SODIUMNITRATE is on the impact of recombinant bacterium viable count
The SODIUMNITRATE that weight percent concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% is shown in Fig. 6 as the impact of nitrogenous source on recombinant bacterium viable count, as we know from the figure, along with the increase of ammonium sulfate concentrations, the viable count of recombinant bacterium is also increasing thereupon, but when sodium nitrate concentration reaches 4%, the quantity of viable count reaches the highest.Then improve ammonium sulfate concentrations further again, the quantity of viable count does not only raise, and presents downtrending on the contrary, illustrates that excessive concentration has restraining effect to a certain degree on the contrary.Therefore, the optimum concn of SODIUMNITRATE is weight percentage 2%.
(4) ammonium sulfate is on the impact of recombinant bacterium viable count
The ammonium sulfate that weight percent concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% is shown in Fig. 7 as the impact of nitrogenous source on recombinant bacterium viable count, as we know from the figure, along with the increase of ammonium sulfate concentrations, the viable count of recombinant bacterium is also increasing thereupon, but when ammonium sulfate concentrations reaches 1.5%, the quantity of viable count reaches the highest.Then improve ammonium sulfate concentrations further again, the quantity of viable count does not only raise, and presents downtrending on the contrary, illustrates that excessive concentration has restraining effect to a certain degree on the contrary.Therefore, the optimum concn of ammonium sulfate is weight percentage 1.5%.
Comprehensive the above results, with yeast powder best results, therefore selected yeast powder is as the nitrogenous source of substratum.
Embodiment 6
The present embodiment illustrates that carbon source is on the impact of viable count.
Use glucose, lactose, maltose, sucrose as carbon source respectively, add in lactalbumin medium with the inoculum size that weight percent is 1%, sample respectively after the lower 37 DEG C of static gas wave refrigerator 16h of anaerobic condition, be coated with plate count.
Select lactose, glucose, sucrose, maltose as carbon source, content is 1%, measures and cultivates 16h secondary fermentation liquid viable count, the results are shown in Figure 8.Test-results shows, adds different carbon source and all has a significant effect to viable count.Analyze number of viable, the highest during interpolation lactose, glucose is minimum, illustrates that milk-acid bacteria is different to the meta-bolites of different carbon source; The lactic acid produced during lactose is less, and it is more that Sucrose Metabolism produces acid, is unfavorable for the growth of milk-acid bacteria.
Embodiment 7
The present embodiment illustrates shaking flask amplification culture.
Adopt the processing parameter after optimizing, namely the concentration of lactalbumin medium is 4%, initial pH value is 6.6, thalline inoculum size is 4%, Inclusion of Lactose 1%, yeast powder addition 1.5%, phosphate buffered saline buffer 0.5%, adopt the mode of inoculation indirectly, rotating speed 80r/min shakes cultivation, carrying out repeating to test the actual average value obtaining recombinant bacterium viable count for 10 times is 7.6 × 10 9cfu/mL.
Comparative example
Using simple whey-protein as substratum, with improvement of the present invention after lactalbumin medium, and MRS substratum is simultaneously for cultivating recombinant lactobacillus casei, with the inoculum size access recombinant lactobacillus casei that weight percent is 1%, sample respectively after the lower 37 DEG C of static gas wave refrigerator 16h of anaerobic condition, be coated with plate count.Result is as Fig. 9.
Result shows, and adopt the lactalbumin medium after optimizing to cultivate milk-acid bacteria, viable count improves 1 order of magnitude, and viable count and MRS substratum remain basically stable.Therefore, we can replace MRS substratum with the lactalbumin medium of improvement, reduce production cost.Whey-protein is nutritious, sweet mouthfeel, and therefore gene recombination bacterium and water (or feed) can mix by we, and Direct-fed is to animal, convenient and swift, reduces labour intensity and time.
The present invention has carried out further optimization to the lactalbumin medium cultivating recombinant lactobacillus casei, better can promote growth, improves viable count, makes the actual average value of recombinant lactobacillus casei viable count reach 7.6 × 10 9cfu/mL; This substratum can substitute MRS substratum, reducing production cost, laying the foundation for preparing prevention of diarrhea in piglets oral liquid vaccine further.

Claims (3)

1. improvement a lactalbumin medium, it is characterized in that: its component and weight percent as follows:
Whey-protein 4%, yeast powder 1.5%, lactose 1%, phosphoric acid salt 0.5%, surplus is water.
2. the lactalbumin medium of improvement according to claim 1, is characterized in that: described phosphoric acid salt comprises Na 2hPO 4and NaH 2pO 4, wherein, Na 2hPO 4weight percent be 0.188%, NaH 2pO 4weight percent be 0.312%.
3. the lactalbumin medium of the improvement described in claim 1 or 2 is cultivating the application in recombinant lactobacillus casei.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564082A (en) * 2021-08-09 2021-10-29 中南大学 Fermentation medium and culture condition for increasing yield of lactobacillus crispatus bacteriocin

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CN101225381A (en) * 2007-12-30 2008-07-23 侯喜林 Lactic acid bacillus transformation method

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564082A (en) * 2021-08-09 2021-10-29 中南大学 Fermentation medium and culture condition for increasing yield of lactobacillus crispatus bacteriocin

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