CN103275921B - Method for improving cholate tolerance of lactobacillus - Google Patents
Method for improving cholate tolerance of lactobacillus Download PDFInfo
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- CN103275921B CN103275921B CN201310188667.4A CN201310188667A CN103275921B CN 103275921 B CN103275921 B CN 103275921B CN 201310188667 A CN201310188667 A CN 201310188667A CN 103275921 B CN103275921 B CN 103275921B
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- bacterium lacticum
- lactobacillus
- bacterium
- cholate
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Abstract
The invention discloses a method for improving cholate tolerance of lactobacillus. The method comprises the following steps of: (1) cultivating a lactobacillus seed liquid; (2) inoculating the seed liquid to an MRS (DeMan Rogosa Sharpe) liquid culture medium containing salt in a certain concentration by 3-4%(v/v) inoculum size, and standing and culturing; and (3) centrifugalizing to obtain thallus. According to the method disclosed by the invention, the cholate tolerance of the lactobacillus is improved by cultivating lactobacillus in the saliferous culture medium. The method is good in experimental repeatability and is a novel method for improving the cholate tolerance of the lactobacillus.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of method improving Bacterium lacticum bile tolerance ability.
Background technology
Milk-acid bacteria (Lactic Acid Bacteria, LAB) is one of mankind bacterium of applying the earliest, and for a long time, these bacteriums are always for the process for processing of food and drink.Milk-acid bacteria can improve Nutritive value of food, improves flavour of food products, improves the added value of food, as the beneficial flora of human gastrointestinal tract, has and maintains intestinal flora balance, raising immunity of organisms and promote the multiple prebiotic functions such as absorption of nutrient ingredients.Active and the trophic function of the special physiological of milk-acid bacteria, causes the attention of people just day by day in recent years.Bacterium lacticum (Lactobacillus) is the maximum genus in milk-acid bacteria, has good tolerance, be widely used in fermented vegetables, meat product and milk-product to salt, nitrite.
It is that it plays the prerequisite of prebiotic function that Bacterium lacticum can be survived in vivo.The digestive tube of humans and animals is the natural cover resisting external pathogenic infection body, and this kind of microorganism useful to human body of probiotic lactobacilli equally will in the face of the test of this natural cover.Bacterium lacticum per os take in after enter enteron aisle through stomach, the cholate in small intestine be Bacterium lacticum in vivo, especially in enteron aisle survival and surely grow the huge challenge run into.Cholate can decompose cytolemma as stain remover, finally causes film to cave in and cell injury, and therefore, Bacterium lacticum has stronger cholate tolerance and has very important meaning for its performance prebiotic effect.Bacterium lacticum can run into the salt of high density in the process of fermentation and preserved food, and Bacterium lacticum is by regulating the expression of autogene, and namely salt stress reaction adapts to hypersaline environment.There are some researches show, the stress reaction of Bacterium lacticum can improve spray-dired survival rate (Desmond et al, Environmental adaptation of probioticlactobacilli towards improvement of performance during spray drying.International Dairy Journal, 2001, 11, 801-808), improve its resistance to (Upadrastaet al that is heated, Improving the Stress Tolerance of Probiotic Cultures:RecentTrends and Future Directions.Stress Responses of Lactic Acid Bacteria, 2011, 395-438, Kimetal, Assessment of stress response of the probioticLactobacillus acidophilus.Current microbiology, 2001,43,346-350) etc. stress ability, but the ability whether salt stress reaction can improve Bacterium lacticum tolerance cholate not studies have reported that.
Summary of the invention
The object of this part is some aspects of general introduction embodiments of the invention and briefly introduces some preferred embodiments.May do in the specification digest and denomination of invention of this part and the application a little simplify or omit with avoid making this part, specification digest and denomination of invention object fuzzy, and this simplification or omit and can not be used for limiting the scope of the invention.
In view of the methods of above-mentioned and/or existing raising Bacterium lacticum bile tolerance ability, propose the present invention.
Therefore, one of them object of the present invention is by cultivating in the substratum of saliferous Bacterium lacticum, for the ability improving the tolerance of Bacterium lacticum cholate provides a kind of novel method.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of method improving Bacterium lacticum bile tolerance ability, comprises the following steps: the cultivation of (1) Bacterium lacticum seed liquor; (2) by described seed liquor with 3%-4%(v/v) inoculum size is inoculated in the MRS liquid nutrient medium containing finite concentration salt, quiescent culture; (3) centrifugal, obtain thalline.
As a kind of preferred version of the method for raising Bacterium lacticum bile tolerance ability of the present invention, wherein: the cultivation of Bacterium lacticum seed liquor in described step (1), that Bacterium lacticum is rule on MRS solid plate, 37 DEG C of-42 DEG C of inversions are cultured to and grow single bacterium colony, picking list colony inoculation is in MRS liquid nutrient medium, 37 DEG C-42 DEG C quiescent culture 6h-10h, obtain seed liquor.
As a kind of preferred version of the method for raising Bacterium lacticum bile tolerance ability of the present invention, wherein: in described step (1), Bacterium lacticum comprises one or more in plant lactobacillus, lactobacterium casei, lactobacterium helveticus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacillus reuteri.
As a kind of preferred version of the method for raising Bacterium lacticum bile tolerance ability of the present invention, wherein: in described step (2), Bacterium lacticum is cultivated in the substratum containing finite concentration salt, and wherein salt refers to sodium salt.
As a kind of preferred version of the method for raising Bacterium lacticum bile tolerance ability of the present invention, wherein: in described step (2), Bacterium lacticum is cultivated in the substratum containing finite concentration salt, and wherein finite concentration scope is 2%-10%.
As a kind of preferred version of the method for raising Bacterium lacticum bile tolerance ability of the present invention, wherein: in described step (3), centrifugal speed controls at 4000-6000rpm.
The present invention is by cultivating in the substratum of saliferous Bacterium lacticum, and improve the ability that Bacterium lacticum tolerates cholate, experimental repeatability is good, for the ability improving the tolerance of Bacterium lacticum cholate provides a kind of novel method.
Embodiment
Below in conjunction with specific embodiment, the method for the invention is described in detail.
The following examples are exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
The cultivation of embodiment 1, seed liquor
1, rule on MRS solid plate by lactobacillus plantarum ST-III, 38 DEG C of inversions are cultured to and grow single bacterium colony, choose single colony inoculation to MRS liquid nutrient medium, 38 DEG C of quiescent culture 8h;
2, above-mentioned nutrient solution is inoculated into containing 2%NaCl, 4%NaCl, 6%NaCl, 8%NaCl, 10%NaCl and not containing in the MRS liquid nutrient medium of salt with 3% inoculum size respectively, 38 DEG C of quiescent culture 10h.
Embodiment 2, lactobacillus plantarum ST-III cholate tolerate
1, be collected in containing salt (2%NaCl, 4%NaCl, 6%NaCl, 8%NaCl, 10%NaCl) and not containing the thalline grown in the substratum of salt;
2, with salt solution (the thalline physiological saline cultivated under salt-free conditions) the resuspended thalline with same concentrations when cultivating, cell concentration is regulated to be 10
8-10
9cFU/mL;
3, the above-mentioned bacteria suspension of 1mL is got, 5000rpm, centrifugal 5min;
4, add the resuspended thalline of MRS substratum that 1mL contains 0.6% cholate, hatch 30min.
Thalline viability count after the tolerance of example 3, cholate
1, enumeration is carried out to the thalline tolerated without cholate: the bacterium liquid (namely not containing the bacterium liquid of cholate) not carrying out cholate tolerance is carried out gradient dilution with physiological saline, gets 1mL10 respectively
-6, 10
-7, 10
-8dilution bacterium liquid in sterile petri dish, MRS solid medium pouring plate;
2, enumeration is carried out to the thalline of cholate tolerance: the bacterium liquid (namely containing the bacterium liquid of 0.6% cholate) tolerated by cholate carries out gradient dilution with physiological saline, gets 1mL10 respectively
-4, 10
-5, 10
-6dilution bacterium liquid in sterile petri dish, MRS solid medium pouring plate;
3, after 37 DEG C of Anaerobic culturel 48h, enumeration.
Table 1 lactobacillus plantarum ST-III cholate tolerance survival rate
* :-to represent survival be 0; Different letters represents significant difference (p≤0.05).
As seen from the above table, produce salt stress thalline containing 0.6% cholate substratum in hatch 30min after its survival rate apparently higher than do not produce salt stress thalline, and also improve along with the increase cholate tolerance of salt concn thereupon, when salt concn is 8% and 10% time reach the highest, but the survival rate after the thalline cholate tolerance under these two salt concn are cultivated does not have significant difference, illustrates that lactobacillus plantarum ST-III cholate tolerance has reached the highest when 8%NaCl.
The impact of example 4, microscopic examination cholate tolerance on film integrality
1, the fluorescent dye of cell
(1) get cholate and tolerate forward and backward thalline 50 μ L in aseptic centrifuge tube;
(2) add each 1 μ L of fluorescence dye PI, SYTO9, room temperature lucifuge hatches 15min;
2, microscope observing cell film integrality
(1) the cell suspension 4 μ L getting fluorescent dye, on slide glass, closes surrounding with whiteruss after building cover glass;
(2) the integrity situation of observation of cell film under fluorescent microscope.
The complete type of cytolemma has reacted the physiological status of cell, and the cell membrane integrity of viable cell is good, thalline fluoresced green after fluorescent dyeing, and the thalline of dead cell and cell membrane damage sends out red fluorescence after fluorescent dyeing.It can thus be appreciated that the film integrality of the thalline cultivated in the substratum of saliferous is far away higher than the film integrality of the thalline cultivated in salt-free substratum (contrast).
As can be seen here, by cultivating in the substratum of saliferous Bacterium lacticum, improve the ability that Bacterium lacticum tolerates cholate, experimental repeatability is good, for the ability improving the tolerance of Bacterium lacticum cholate provides a kind of novel method.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (3)
1. improve a method for Bacterium lacticum bile tolerance ability, it is characterized in that:
Comprise the following steps:
(1) cultivation of Bacterium lacticum seed liquor;
(2) by described seed liquor with 3% ~ 4%(v/v) inoculum size is inoculated in the liquid nutrient medium containing finite concentration salt, quiescent culture;
(3) centrifugal, obtain thalline;
In described step (1), Bacterium lacticum is lactobacillus plantarum ST-III;
In described step (2), Bacterium lacticum is cultivated in the substratum containing finite concentration salt, and wherein salt refers to sodium-chlor, and finite concentration scope is 2% ~ 10%.
2. the method for raising Bacterium lacticum bile tolerance ability according to claim 1, it is characterized in that: the cultivation of Bacterium lacticum seed liquor in described step (1), that lactobacillus plantarum ST-III is rule on MRS solid plate, 37 DEG C ~ 42 DEG C inversions are cultured to and grow single bacterium colony, picking list colony inoculation is in MRS liquid nutrient medium, 37 DEG C ~ 42 DEG C quiescent culture 6 h ~ 10 h, obtain seed liquor.
3. the method for raising Bacterium lacticum bile tolerance ability according to claim 1, is characterized in that: in described step (3), centrifugal speed controls at 4000 ~ 6000 rpm.
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| CN104962509B (en) * | 2015-06-30 | 2019-07-09 | 河北工程大学 | A kind of method and its lactobacillus beverage improving lactobacillus acid-fast ability |
| CN104928219B (en) * | 2015-06-30 | 2018-12-18 | 河北工程大学 | A kind of lactobacillus plantarum TH103 and application thereof |
| CN106770250B (en) * | 2016-12-21 | 2020-04-10 | 农业部环境保护科研监测所 | Method for rapidly detecting cadmium resistance of rice |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378591A (en) * | 1999-06-11 | 2002-11-06 | 雀巢制品公司 | Bacterial protection |
| CN1493684A (en) * | 2002-10-30 | 2004-05-05 | ʳƷ��ҵ��չ�о��� | New acid resistant and cholate resistant lactobacillus isolated strain having reducing and assimilating cholesterol ability |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378591A (en) * | 1999-06-11 | 2002-11-06 | 雀巢制品公司 | Bacterial protection |
| CN1493684A (en) * | 2002-10-30 | 2004-05-05 | ʳƷ��ҵ��չ�о��� | New acid resistant and cholate resistant lactobacillus isolated strain having reducing and assimilating cholesterol ability |
Non-Patent Citations (3)
| Title |
|---|
| 交互保护对干酪乳杆菌ATCC 393TM 存活的影响;薛峰等;《微生物学报》;20100404;第50卷(第4期);478-484 * |
| 小米提取物对乳杆菌耐胆盐能力的影响;靳志强;《食品科学》;20081231;第29卷(第8期);414-417 * |
| 氯化钠刺激对保加利亚乳杆菌生长及质膜流动性的影响;刘丽波等;《食品工业科技》;20121231;第33卷(第12期);132-139 * |
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