CN105567624A - Synergist for lactococcus lactis subsp. lactis capable of generating nisin through fermentation and use method of synergist - Google Patents

Synergist for lactococcus lactis subsp. lactis capable of generating nisin through fermentation and use method of synergist Download PDF

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CN105567624A
CN105567624A CN201610051680.9A CN201610051680A CN105567624A CN 105567624 A CN105567624 A CN 105567624A CN 201610051680 A CN201610051680 A CN 201610051680A CN 105567624 A CN105567624 A CN 105567624A
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lactococcus lactis
synergistic agent
synergist
nisin
lactic acid
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CN105567624B (en
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汪超
柳念
周梦舟
李冬生
徐宁
胡勇
李玮
李斌
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Hubei University of Technology
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    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

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Abstract

The invention relates to a synergist for lactococcus lactis subsp. lactis capable of generating nisin through fermentation and a use method of the synergist. The synergist is prepared from, by weight, 0.02-1.5 parts of taurine, 0.003-0.006 part of selenium, 0.03-0.8 part of proline, 0.05-1.0 part of lysine and 0.02-0.052 part of inositol. The synergist is added into an M17 culture medium, and then lactic acid bacteria capable of generating the nisin are inoculated for fermentation. All the ingredients of the synergist are easy to obtain, low in purchase cost and simple in use method. Experiments show that the synergist can obviously improve the nisin yield of the lactic acid bacteria, synergia is obvious, therefore, the production cost of the nisin is reduced, the production cycle is shortened, and the production efficiency is improved.

Description

A kind of Lactococcus lactis breast subspecies synergistic agent of the lactic acid producing streptostacin that ferments and using method thereof
Technical field
The invention belongs to technical field of microbial fermentation, be specifically related to a kind of Lactococcus lactis breast subspecies synergistic agent and using method thereof of the lactic acid producing streptostacin that ferments.
Background technology
Milk-acid bacteria is a class can produce the gram positive bacterium of a large amount of lactic acid general designation from fermentability carbohydrate.The milk-acid bacteria found at occurring in nature is at present divided into 43 genus, as Bacillus, Listerial, bud embrace the Pseudomonas such as lactobacillus, lactobacillus, secondary lactobacillus, streptococcus, lactococcus, section's Richter scale Bacillaceae, genus bifidobacterium on systematic bacteriology.Most milk-acid bacteria all can synthesize bacteriocin in metabolic process, and be referred to as lactobacillin, Lactococcus lactis is widely used in dairy industry.The bacterium that Lactococcus lactis can produce have: diplococcin, lactococcin, lacticin and nisin.Wherein that the most outstanding is the bacteriocin Nisin that various LactococcuslactissubsP.Lactis produces.
Nisin is used as food preservatives the earliest, at present, oneself have comprise China, the U.S., Britain more than 50 countries and regions approval Nisin be widely used among food-processing industry as a kind of antiseptics for natural food.Although very wide to the research range of Nisin at present, milk-acid bacteria is easy to reach decline phase in substratum growth, and lactic acid producing streptostacin also reduces accordingly.So at present, the research delaying the decline phase of milk-acid bacteria is imperative, so a lot of research all relates to the optimization of lactic acid bacteria culturing medium at present, postpones the decline phase of milk-acid bacteria, thus strengthen the formation of nisin.Such as patent of invention CN200510071945.3 substratum, its preparation, the method for its preparation method and cultivation nisin.Its preparation method is drop in material-compound tank by yeast proteinoid, soybean protein, add water and stir evenly, drop into bacterial classification again, fill into carbon source, sodium phosphate, magnesium sulfate successively, favourable to the metabolism of Lactococcus lactis, enter logarithmic phase shortening in period, metabolism is vigorous, the consumption of sugar nitrogen is accelerated, and the paracme postpones, and nisin activity greatly improves through estimation of biological potency.Although improve nisin, operate loaded down with trivial details, uncontrollable influence factor increases.
Summary of the invention
The object of the invention is to solve the problems of the technologies described above, there is provided a kind of and significantly can improve lactic acid producing streptostacin ability, delay the decline of Lactococcus lactis breast subspecies, the Lactococcus lactis breast subspecies synergistic agent of the fermented lactic acid producing streptostacin that the turnout of nisin is large.
The using method of the synergistic agent that the present invention also provides a kind of method simple, practical.
Synergistic agent of the present invention is made up of by weight following composition:
Taurine 0.02-1.5, selenium 0.003-0.006, proline(Pro) 0.03-0.8, Methionin 0.05-1.0, inositol 0.02-0.052.
Carry out a step, described synergistic agent is preferably taurine 0.08-1.2, selenium 0.004-0.005, proline(Pro) 0.2-0.5, Methionin 0.07-0.09, inositol 0.03-0.04.
Described Lactococcus lactis breast subspecies are the Lactococcus lactis breast subspecies of lactic acid producing streptostacin of can fermenting, as the M9 Lactococcus lactis of the growth of most of gram-positive microorganism and gemma thereof can be suppressed, especially to streptococcus aureus, Streptococcus hemolyticus, Clostridium botulinum effect is obvious, be widely used in food, the Lactococcus lactis subsp.lactis T0625 screened from Chinese cabbage, it has stronger bacteriostatic activity to streptococcus aureus, and its bacteriostatic activity has genetic stability, strong inhibiting streptococcus acidi lactici L318 is had to vibrio marinopraesens, greatly reduce the loss of aquaculture, particularly preferably preserving number is the Lactococcus lactis milk-globule subspecies (Lactococcuslactissubsp.lactis) of CGMCCNO1.2829, this bacterium has acidproof, high-yield lactic acid streptostacin, the features such as resistance to selenium, be specially adapted to synergistic agent of the present invention, significantly can improve the output of nisin.
The using method of above-mentioned synergistic agent, comprises the following steps:
(1) M17 substratum is prepared;
(2) in described M17 substratum, add described synergistic agent, add-on is described M17 substratum quality 0.12%-3.36%;
(3) carry out cultivation in the M17 substratum obtained to step (2) by lactobacillus inoculum to obtain cultivating bacterium liquid, the inoculum size of described milk-acid bacteria is 1.0%-1.5%
Cultural method in described step (3) adopts shake-flask culture, and control rotating speed is 160-180r/min, and culture temperature is 30-34 DEG C, incubation time 10-14h.
The present invention is based on the Lactococcus lactis breast subspecies that can produce nisin in fermenting process, by adding synergistic agent in its substratum, to promote the production of milk-acid bacteria, improve the turnout of nisin, reduce incubation time, enhance productivity, to reduce production cost.In described synergistic agent, taurine has promotion microorganism generation tropina, for microorganism growth sulphur source is provided, taurine is utilized by Lactococcus lactis breast subspecies and participates in albumen synthesis simultaneously, improve the bacteriostatic action of antibacterial peptide, addition is 0.02-1.5, more being preferably 0.08-1.2 too much can suppress Lactococcus lactis breast subspecies to the enrichment of selenium, crosses that I haven't seen you for ages weakens the antibacterial ability of antibacterial peptide; Selenium has and promotes the Lactococcus lactis breast energy for growth of subspecies and acid resistance, and addition is 0.003-0.006, is more preferably 0.004-0.005, too much can suppresses microbial growth, crosses that I haven't seen you for ages and weaken the generation of nisin element; Proline(Pro) has has strong increment effect to Lactococcus lactis breast subspecies, it contributes to absorbing of Lactococcus lactis breast subspecies as nitrogenous source, producing bacillus subtilis antibacterial peptide is jointly promoted with taurine, addition is 0.03-0.8, more be preferably 0.2-0.5, too much can suppress absorbing of taurine, suppress Lactococcus lactis breast subspecies to produce antibacterial peptide ability, cross that I haven't seen you for ages and be unfavorable for the growth of thalline; Methionin has promotion Lactococcus lactis breast subspecies synthetic protein, Lactococcus lactis breast subspecies synthesizing lactic acid streptostacin is promoted with taurine is collaborative, addition is 0.05-1.0, more be preferably 0.07-0.09, too much can suppress microbial growth, I haven't seen you for ages excessively reduces thalline synthesizing lactic acid streptostacin; Inositol has accelerate growth of microorganism, delays the decline of microorganism, and addition is 0.02-0.052, is more preferably 0.03-0.04, too much can without positive effect, crosses that I haven't seen you for ages causes microorganism to be become feeble and die soon.
When synergistic agent of the present invention is used for Lactococcus lactis breast subspecies fermentation culture, joined in M17 substratum and mixed, its add-on is the 0.12%-3.36% of M17 substratum quality, under this add-on effect, can promote that the breast of the synergy Lactococcus lactis between synergistic agent subspecies have good inrichment to it, promote the growth of Lactococcus lactis breast subspecies self, growth-delaying decline phase; Under the culture condition adding synergistic agent, the nisin that Lactococcus lactis breast subspecies produce increases, and its antibacterial ability is also strengthened greatly.
Beneficial effect:
(1) the Lactococcus lactis breast subspecies that the present invention is directed to the lactic acid producing streptostacin that can ferment develop a kind of synergistic agent, significantly can improve the generation of nisin in Lactococcus lactis breast subspecies culturing process, improve the metabolism of Lactococcus lactis breast subspecies, postpone its growth decline, thus reduce production cost, shortening production cycle, enhance productivity.
(2) each component raw material of synergistic agent of the present invention is easy to get, and purchase cost is low, mix by adding in M17 substratum after simple mixing, and using method is very simple and reliable, controllability good, have wide market application foreground.
(3) same milk-acid bacteria is under same culture conditions, adopt synergistic agent of the present invention compared with the Nostoc commune Vanch process not using synergistic agent, milk-acid bacteria metabolism is more vigorous, the nisin production limits of Lactococcus lactis breast subspecies self can be broken through, nisin output can be improved and reach more than 50%, reduce the production cost of 40%.
Embodiment
Below in conjunction with specific embodiment, the technical scheme in the present invention is clearly and completely described.
Below in conjunction with specific embodiment, the technical scheme in the present invention is clearly and completely described.
One, formulation of synergist embodiment 1-5 example is in table 1.
Table 1 synergistic agent Example formulations (weight ratio)
Taurine Selenium Proline(Pro) Methionin Inositol
Synergistic agent 1 1.5 0.006 0.8 1.0 0.052
Synergistic agent 2 0.02 0.003 0.03 0.05 0.02
Synergistic agent 3 0.10 0.0045 0.3 0.08 0.035
Synergistic agent 4 0.08 0.004 0.2 0.07 0.03
Synergistic agent 5 1.2 0.005 0.5 0.09 0.04
Two, lactobacillus-fermented embodiment 1-5, wherein, the Lactococcus lactis milk-globule subspecies (Lactococcuslactissubsp.lactis) of described Lactococcus lactis breast subspecies to be preserving number be CGMCCNO1.2829.
(1), preparation M17 substratum is placed in triangular flask, and LB culture medium prescription is 1.0g peptone, 0.5g yeast powder, 0.2g ammonium citrate, 2.0g glucose, 0.3g sodium acetate, 0.2g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganous sulfate, 1.0g extractum carnis, 0.1g tween-80,100mL water;
(2), to M17 substratum add synergistic agent, add-on is in table 2;
(3), by the M17 substratum after lactobacillus inoculum to above-mentioned sterilizing, inoculum size, in table 2, carries out shake-flask culture, and rotating speed is 160-180r/min, and culture temperature is 30-34 DEG C, incubation time 10-14h, obtains respectively cultivating bacterium liquid 1-5.
The fermentation embodiment 1-5 of table 2 subtilis
Above-mentioned per-cent in M17 substratum total mass in step (1) for 100%.
Comparative example:
Contrast fermentation embodiment 1: not containing taurine and selenium in the synergistic agent in step (2), all the other obtain contrast cultivate bacterium liquid 1 with fermentation embodiments 1;
Contrast fermentation embodiment 2: not containing proline(Pro), Methionin in the synergistic agent in step (2), all the other are with embodiment 1, obtains contrast and cultivates bacterium liquid 2;
Contrast fermentation embodiment 3: step does not add synergistic agent in (2), and all the other are with embodiment 1, obtains contrast and cultivates bacterium liquid 3.
Method of contrast:
(1), use the HCl of 5mol/L that cultivation bacterium liquid is adjusted to pH2.5, afterwards 90 DEG C of centrifugal 12min of heating 30min, 5400r/min, removing precipitation, collects supernatant liquor for subsequent use;
(2) getting increment is 10 7streptococcus aureus (culture presevation the is numbered CGMCCNO1.8721) nutrient solution of CFU/mL is as indicator suspension, indicator growth medium after sterilizing is poured in the culture dish of diameter 9cm, every ware 15mL, to be cooled solidify after add 0.2mL indicator suspension and smoothen, rear aseptic nipper to be dried places Oxford cup (internal diameter 6.0 ± 0.1mm, high 8.0 ± 0.1mm).Get the supernatant liquor 0.lmL obtained in step (1) after leaving standstill 20min to add in the cup of Oxford.Described Oxford cup is carefully put into incubator, the fermented liquid in cup is fully diffused in substratum, cultivate 48h for 37 DEG C, use vernier caliper measurement antibacterial circle diameter, often group does 3 repetitions, averages.
Cultivation bacterium liquid 1-5 and contrast are cultivated bacterium liquid 1-3 adopts above-mentioned experimental technique to carry out contrast experiment respectively, by cylinder plate method test Lactococcus lactis milk-globule subspecies (preserving number is CGMCCNO1.2829) lactic acid producing streptostacin content, and 10 times are diluted to nutrient solution, use UV spectrophotometry OD value (measurement wavelength is 600nm), the results are shown in Table 3.
Table 3, contrast and experiment.
As shown in Table 3; the growth to Lactococcus lactis breast subspecies of taurine, selenium, proline(Pro), Methionin, inositol has obvious promoter action; and significantly enhance the ability of Lactococcus lactis breast institute lactic acid producing streptostacin, and improve the bacteriostasis of nisin.The invention provides everybody one group of synergistic agent that can strengthen Lactococcus lactis breast subspecies lactic acid producing streptostacin, this group synergistic agent has synergy, and can significantly improve the bacteriostasis of Lactococcus lactis breast subspecies, be a kind of innovation of economic environmental protection.

Claims (5)

1. can ferment the Lactococcus lactis breast subspecies synergistic agent of lactic acid producing streptostacin, and it is characterized in that, described synergistic agent is made up of by weight following composition:
Taurine 0.02-1.5, selenium 0.003-0.006, proline(Pro) 0.03-0.8, Methionin 0.05-1.0, inositol 0.02-0.052.
2. can ferment the Lactococcus lactis breast subspecies synergistic agent of lactic acid producing streptostacin, it is characterized in that,
Taurine 0.08-1.2, selenium 0.004-0.005, proline(Pro) 0.2-0.5, Methionin 0.07-0.09, inositol 0.03-0.04.
3. can ferment the Lactococcus lactis breast subspecies synergistic agent of lactic acid producing streptostacin as claimed in claim 1 or 2, it is characterized in that, described Lactococcus lactis breast subspecies are Lactococcus lactis breast subspecies (Lactococcuslactissubsp.lactis) of lactic acid producing streptostacin of can fermenting, and its preserving number is CGMCCNO1.2829.
4. the using method of the synergistic agent as described in any one of claim 1-3, is characterized in that, comprises the following steps:
(1) M17 substratum is prepared;
(2) in described M17 substratum, add described synergistic agent, add-on is described M17 substratum quality 0.12%-3.36%;
(3) carry out cultivation in the M17 substratum obtained to step (2) by lactobacillus inoculum to obtain cultivating bacterium liquid, the inoculum size of described milk-acid bacteria is the 1.0%-1.5% of described MI7 substratum quality.
5. the using method of synergistic agent as claimed in claim 4, it is characterized in that, the cultural method in described step (3) adopts shake-flask culture, and control rotating speed is 160-180r/min, and culture temperature is 30-34 DEG C, incubation time 10-14h.
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Cited By (8)

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CN106173272A (en) * 2016-09-21 2016-12-07 天津科技大学 A kind of fermentation method prepares the method for livestock and poultry blood protein peptide
CN106727471A (en) * 2017-01-22 2017-05-31 河南师范大学 Inositol as Florfenicol synergist application
CN107173799A (en) * 2017-06-29 2017-09-19 张萌 Extend the preparation method of the shelf life of ferment
CN110016456A (en) * 2018-05-28 2019-07-16 西北工业大学 Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method
CN111621536A (en) * 2020-05-27 2020-09-04 河北圣雪大成制药有限责任公司 Fermentation production process of high-yield nisin
CN114027433A (en) * 2021-11-08 2022-02-11 上海赛瑞益升健康食品有限公司 Slow-release biological preservative for fermentation product
CN114958663A (en) * 2022-05-13 2022-08-30 齐鲁工业大学 Lactococcus lactis subsp lactis A32 strain and derivative product and application thereof
CN116218711A (en) * 2022-12-21 2023-06-06 陕西省微生物研究所 Lactococcus lactis PZ1 and application thereof in preparation of selenium-enriched oligopeptide

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106173272A (en) * 2016-09-21 2016-12-07 天津科技大学 A kind of fermentation method prepares the method for livestock and poultry blood protein peptide
CN106173272B (en) * 2016-09-21 2019-12-13 天津科技大学 Method for preparing livestock and poultry blood protein peptide by fermentation method
CN106727471A (en) * 2017-01-22 2017-05-31 河南师范大学 Inositol as Florfenicol synergist application
CN106727471B (en) * 2017-01-22 2019-12-03 河南师范大学 Application of the inositol as Florfenicol synergist
CN107173799A (en) * 2017-06-29 2017-09-19 张萌 Extend the preparation method of the shelf life of ferment
CN110016456A (en) * 2018-05-28 2019-07-16 西北工业大学 Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method
CN111621536A (en) * 2020-05-27 2020-09-04 河北圣雪大成制药有限责任公司 Fermentation production process of high-yield nisin
CN114027433A (en) * 2021-11-08 2022-02-11 上海赛瑞益升健康食品有限公司 Slow-release biological preservative for fermentation product
CN114958663A (en) * 2022-05-13 2022-08-30 齐鲁工业大学 Lactococcus lactis subsp lactis A32 strain and derivative product and application thereof
CN116218711A (en) * 2022-12-21 2023-06-06 陕西省微生物研究所 Lactococcus lactis PZ1 and application thereof in preparation of selenium-enriched oligopeptide
CN116218711B (en) * 2022-12-21 2024-02-06 陕西省微生物研究所 Lactococcus lactis PZ1 and application thereof in preparation of selenium-enriched oligopeptide

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