CN110129217B - Method for improving heat resistance of lactic acid bacteria and application - Google Patents
Method for improving heat resistance of lactic acid bacteria and application Download PDFInfo
- Publication number
- CN110129217B CN110129217B CN201910321964.9A CN201910321964A CN110129217B CN 110129217 B CN110129217 B CN 110129217B CN 201910321964 A CN201910321964 A CN 201910321964A CN 110129217 B CN110129217 B CN 110129217B
- Authority
- CN
- China
- Prior art keywords
- heat resistance
- lactobacillus
- lactic acid
- acid bacteria
- lactobacillus casei
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of lactobacillus processing, and particularly relates to a method for improving heat resistance of lactobacillus and application thereof, wherein the lactobacillus is subjected to acid stress and temperature stress under proper conditions and then added into a thallus protective agent, so that the heat resistance of the lactobacillus can be greatly improved, and compared with an original strain, the heat resistance of the treated lactobacillus is improved by more than 80 times, so that the method is very suitable for large-scale popularization.
Description
Technical Field
The invention belongs to the technical field of lactobacillus processing, and particularly relates to a method for improving heat resistance of lactobacillus and application thereof.
Background
Lactic acid bacteria are gram-positive bacteria capable of producing lactic acid, are the most important industrial microorganisms, and have wide application in food processing. Most of lactic acid bacteria have the probiotic effect, can promote the metabolism of organisms, help digestion and absorption, inhibit exogenous pathogenic bacteria, well adjust the proportion of intestinal flora, and improve the intestinal micro-ecological environment, thereby preventing and treating diseases.
Research results show that Ca with certain concentration is added in the culture process of the lactobacillus2+Can improve the stability of cell membrane, make it difficult to destroy in the thermal environment, protect the cell activity. Physical mutagenesis methods such as ultraviolet mutagenesis and the like are widely applied to the aspect of improving the heat resistance of the lactic acid bacteria, and the bacteria are treated on a gene level to obtain a strain with changed transcription expression. Stress can improve the expression of stress-related proteins of lactic acid bacteria, such as heat shock proteins, and further improve the heat resistance of lactic acid bacteria. The literature reports that the strength of heat resistance of lactic acid bacteria is proportional to the shelf life of the lactic acid bacteria. Therefore, the preparation and storage problems of the lactobacillus powder can be solved to a great extent by improving the heat resistance of the lactobacillus.
The heat shock protein is used as a main protein of lactic acid bacteria for resisting heat stress, can repair and clear protein denaturation caused by heat damage, maintains the stability of a cell membrane to keep the activity of cells, and improves the expression level of the heat shock protein mainly through stress at present. The temperature pre-adaptation is carried out on the Lactobacillus helveticus, and after the Lactobacillus helveticus is treated by water bath for 20min at 52 ℃, the heat resistance is improved by 11 times. The improvement in heat resistance is not significant. The heat resistance of bacteria in alcohol-free beer is analyzed, and the heat resistance of the bacteria in the beer containing 5% (v/v) ethanol is generally higher than that of the alcohol-free beer, wherein the heat resistance of lactic acid bacteria is improved by 4-7 times, and the heat resistance of pathogenic bacteria is improved by 3-17 times.
At present, the research on the microecologics at home and abroad has a great investment. Common preparation methods include drying methods such as spray drying and freeze drying. Drying generally refers to the process of heating the wet material to drive off volatile moisture and obtain a solid product. But the living bacteria number is reduced because the living bacteria are often exposed to the stress environment such as heat, high osmotic pressure and the like in the production and storage processes of the bacteria. The effect of improving the heat resistance by single stress is not strong, and the advantages cannot be obviously reflected in practical application. In the research of the heat resistance of the lactic acid bacteria, not only the protein and the transcription level need to be concerned, but also the heat resistance of the lactic acid bacteria needs to be greatly improved by combining with the external protection effect.
Disclosure of Invention
The invention aims to provide a method for improving the heat resistance of lactic acid bacteria, which effectively improves the heat resistance of lactic acid bacteria, thereby improving the survival rate of the lactic acid bacteria in a drying process and the stability of the lactic acid bacteria during storage.
The invention also aims to provide application of the method for improving the heat resistance of the lactic acid bacteria.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for improving the heat resistance of lactic acid bacteria comprises the following steps:
1) selecting lactobacillus in late stage of logarithmic phase of fermentation, centrifuging, suspending in normal saline, adjusting pH to 3-6, and acid stress for 0.5-1.5 h;
2) centrifugally suspending the strain treated in the step 1) in normal saline, and carrying out water bath at 37-50 ℃ for 0.5-3 h;
3) centrifuging the strains treated in the step 2), and suspending in a strain protective agent;
the strain protective agent comprises the following components: 2.0 to 2.2 percent of glucose; peptone 1.2-1.4%, beef powder 0.9-1.0%; tween-800.1-0.12%; magnesium sulfate 0.02-0.024%; 0.20 to 0.24 percent of ammonium citrate and the balance of water, which are all in percentage by mass.
Of the above-mentioned methods, preferably, the methods of 1) and 2) comprise the steps of:
1) selecting lactobacillus in late stage of logarithmic phase of fermentation, centrifuging, suspending in normal saline, adjusting pH to 3-4, and acid stress for 0.5-1.0 h;
2) centrifugally resuspending the strain treated in the step 1) in normal saline, and carrying out water bath at 40-50 ℃ for 2-3 h;
in the above method, preferably, the strain protecting agent: 2.0% of glucose; peptone 1.2%, and beef powder 1.0%; tween-800.1%; 0.02% of magnesium sulfate; 0.2 percent of ammonium citrate, which are all mass percentages.
In the above method, preferably, the lactic acid bacteria are: lactobacillus johnsonii, lactobacillus acidophilus, lactobacillus bulgaricus, lactobacillus casei;
in the above method, preferably, the lactic acid bacteria are: lactobacillus casei (lactobacillus casei solid state fermentation and drying process study, mytilus edulis, 2013).
The application of the method for improving the heat resistance of the lactic acid bacteria comprises the step of using the lactic acid bacteria prepared by the method of the invention for storage or transportation of the lactic acid bacteria.
Compared with the prior art, the invention has the following advantages:
the method of the invention can improve the heat resistance of the lactic acid bacteria and improve the activity and stability of the lactic acid bacteria during drying and storage. Ensure the effective exertion of the physiological function of the lactobacillus and expand the application range of the lactobacillus.
The invention not only utilizes a stress mode to improve the heat resistance of the lactic acid bacteria, but also provides a strain protective agent suitable for the lactic acid bacteria, so that the final heat resistance is greatly improved.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
a method for improving the heat resistance of lactic acid bacteria comprises the following steps:
the slant culture medium, the seed culture medium and the fermentation culture medium related to the embodiment are MRS;
1) preparing a seed solution: streaking and activating lactic acid bacteria preserved at-80 ℃ on a solid culture medium, selecting a single colony to be inoculated in an MRS liquid culture medium, culturing for 16h at 37 ℃, inoculating the single colony into a seed culture medium in an inoculation amount of 2% (v/v), and culturing for 12h to serve as seed liquid; inoculating the seed solution into fresh MRS medium at an inoculation amount of 2% (v/v), and standing at 37 deg.C for 15 hr at a bacteria concentration of 1.23 × 109CFU/ml. The heat resistance of the initial strain is detected, and the survival rate is 0.62%. (ii) a The lactic acid bacteria are: lactobacillus casei (lactobacillus casei solid state fermentation and drying process study, mytilus edulis, 2013).
2) Centrifuging the lactobacillus liquid obtained in the step 1 at 8000rpm at room temperature for 4min, and washing twice with physiological saline;
3) the cells were resuspended in physiological saline at a concentration of 109CFU/ml, adjusting the pH of the suspension to 4, and detecting the heat resistance of the suspension after 0.5h under acid pressure stress, wherein the survival rate is 2.24%, and the heat resistance is improved by 3.62 times;
4) centrifuging the acid-pressure treated bacteria liquid again at 8000rpm, washing with 50 deg.C preheated physiological saline for 2 times, and resuspending to obtain bacteria with concentration of 109After CFU/ml and water bath treatment at 50 ℃ for 3h, the heat resistance of the strain is detected, the survival rate is 13.20%, and the heat resistance is improved by 21.28 times.
The above-described evaluation method of heat resistance performance: centrifuging the fermented cell liquid, washing with normal saline, removing the influence of the fermented liquid on heat resistance, resuspending with normal saline preheated at 37 deg.C, heating in water bath at 58 deg.C for 10min, immediately freezing for 5min, measuring viable count by dilution spotting method, and calculating survival rate.
5) Centrifuging to obtain the pressure-stressed lactobacillus, and suspending in a preheated thallus protective agent at 37 ℃;
the thallus protective agent is as follows: 2.0% of glucose; peptone 1.2%, and beef powder 1.0%; tween-800.1%; 0.02% of magnesium sulfate; 0.2 percent of ammonium citrate, which are all mass percentages.
6) Adjusting the concentration of the bacterial suspension to 1010CFU/ml, heating in water bath at 58 deg.C for 10min, immediately freezing for 5min, measuring viable count by dilution spotting method, calculating survival rate of 53.44%, and improving heat resistance by 86.20 times.
7) Adding bran powder into the bacterial liquid obtained by the treatment in the step 6) according to the material-water ratio (mass ratio) of 2:1, and uniformly mixing, wherein the bacterial count is 2.43 multiplied by 109CFU/g, the water content is 39%. Placing in a forced air drying oven: the drying temperature was 55 ℃ and the drying time was 0.5 h. After drying, the water content is 5.08%, and the viable count is 7.37 multiplied by 108CFU/g, survival rate after drying is 30.29%.
8) Placing the obtained lactobacillus powder in a glass drier, and preserving at 4 deg.C for 60 days to obtain viable bacteria number of 7.05 × 108CFU/g, the survival rate is 95.65%.
Dilution spotting method: respectively diluting the bacterial suspension 104，105，106，107And (5) fully and uniformly mixing by using a vortex instrument. 10ul of the diluted sample was placed on MRS solid plate and incubated at 37 ℃ for 48h before counting.
Claims (4)
1. A method for improving the heat resistance of lactic acid bacteria comprises the following steps:
1) selecting lactobacillus in late stage of logarithmic phase of fermentation, centrifuging, suspending in normal saline, adjusting pH to 3-4, and acid stress for 0.5-1.0 h;
2) centrifugally resuspending the strain treated in the step 1) in normal saline, and carrying out water bath at 40-50 ℃ for 2-3 h;
3) centrifuging the strains treated in the step 2), and suspending in a strain protective agent;
the strain protective agent comprises the following components: 2.0 to 2.2 percent of glucose; peptone 1.2-1.4%, beef powder 0.9-1.0%; tween-800.1-0.12%; magnesium sulfate 0.02-0.024%; 0.20-0.24% of ammonium citrate and the balance of water, wherein the mass percentages are above;
the lactobacillus is lactobacillus casei (Lactobacillus casei)Lactobacillus casei ） 。
2. The method of claim 1, wherein: the strain protective agent comprises the following components: 2.0% of glucose; peptone 1.2%, and beef powder 1.0%; tween-800.1%; 0.02% of magnesium sulfate; ammonium citrate 0.2%.
3. Use of the method of claim 1 in lactobacillus casei preservation.
4. Use of the method of claim 1 in the transport of lactobacillus casei.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910321964.9A CN110129217B (en) | 2019-04-22 | 2019-04-22 | Method for improving heat resistance of lactic acid bacteria and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910321964.9A CN110129217B (en) | 2019-04-22 | 2019-04-22 | Method for improving heat resistance of lactic acid bacteria and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110129217A CN110129217A (en) | 2019-08-16 |
| CN110129217B true CN110129217B (en) | 2021-07-23 |
Family
ID=67570582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910321964.9A Active CN110129217B (en) | 2019-04-22 | 2019-04-22 | Method for improving heat resistance of lactic acid bacteria and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110129217B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154689A (en) * | 2020-01-20 | 2020-05-15 | 内蒙古农业大学 | Method for improving heat-resistant survival rate of probiotics |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001286278A (en) * | 2001-03-09 | 2001-10-16 | House Foods Corp | Method for preparing heat-resistant lactic bacterium bacillus coagulans and roux containing the same |
| CN101338288A (en) * | 2008-08-27 | 2009-01-07 | 上海交大昂立股份有限公司 | Method for enhancing plant bacterium lacticum stability |
| CN101671642A (en) * | 2009-08-28 | 2010-03-17 | 上海交大昂立股份有限公司 | Method for improving heat resistance and acid resistance of lactobacillus plantarum |
| CN103725639A (en) * | 2013-12-13 | 2014-04-16 | 山东科技大学 | Medium formula for improving heat resistance of strains |
| CN104489097A (en) * | 2014-11-25 | 2015-04-08 | 南通东概念新材料有限公司 | Active lactobacillus or probiotics heat and convective drying protective agent and preparation method thereof |
| WO2015174407A1 (en) * | 2014-05-15 | 2015-11-19 | エバラ食品工業株式会社 | Method for producing lactic acid bacteria medium, method for culturing lactic acid bacteria using same, and lactic acid bacteria powder using lactic acid bacteria obtained by said culture method |
| KR20160117029A (en) * | 2015-03-31 | 2016-10-10 | 영원메이커스 주식회사 | Method for culturing lactic acid bacteria having improved immunoactivity and stability |
| CN107260704A (en) * | 2017-06-08 | 2017-10-20 | 中国农业科学院饲料研究所 | A kind of enterococcus faecalis microcapsules and preparation method thereof |
| CN109303166A (en) * | 2018-10-14 | 2019-02-05 | 浙江大学 | The preparation method of heat resistance lactobacillus micro-capsule |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971798A (en) * | 2019-05-14 | 2019-07-05 | 华中农业大学 | A method of producing high concentration lactic acid |
| CN110628684B (en) * | 2019-10-25 | 2021-05-07 | 华中农业大学 | Lactobacillus enrichment agent and application thereof in high-density fermentation of lactobacillus |
-
2019
- 2019-04-22 CN CN201910321964.9A patent/CN110129217B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001286278A (en) * | 2001-03-09 | 2001-10-16 | House Foods Corp | Method for preparing heat-resistant lactic bacterium bacillus coagulans and roux containing the same |
| CN101338288A (en) * | 2008-08-27 | 2009-01-07 | 上海交大昂立股份有限公司 | Method for enhancing plant bacterium lacticum stability |
| CN101671642A (en) * | 2009-08-28 | 2010-03-17 | 上海交大昂立股份有限公司 | Method for improving heat resistance and acid resistance of lactobacillus plantarum |
| CN103725639A (en) * | 2013-12-13 | 2014-04-16 | 山东科技大学 | Medium formula for improving heat resistance of strains |
| WO2015174407A1 (en) * | 2014-05-15 | 2015-11-19 | エバラ食品工業株式会社 | Method for producing lactic acid bacteria medium, method for culturing lactic acid bacteria using same, and lactic acid bacteria powder using lactic acid bacteria obtained by said culture method |
| CN104489097A (en) * | 2014-11-25 | 2015-04-08 | 南通东概念新材料有限公司 | Active lactobacillus or probiotics heat and convective drying protective agent and preparation method thereof |
| KR20160117029A (en) * | 2015-03-31 | 2016-10-10 | 영원메이커스 주식회사 | Method for culturing lactic acid bacteria having improved immunoactivity and stability |
| CN107260704A (en) * | 2017-06-08 | 2017-10-20 | 中国农业科学院饲料研究所 | A kind of enterococcus faecalis microcapsules and preparation method thereof |
| CN109303166A (en) * | 2018-10-14 | 2019-02-05 | 浙江大学 | The preparation method of heat resistance lactobacillus micro-capsule |
Non-Patent Citations (10)
| Title |
|---|
| Environmental stress responses in Lactobacillus:A review;Maria De Angelis等;《Proteomics》;20040112;第4卷(第1期);106-122 * |
| Functional properties of Lactobacillus plantarum strains: A study in vitro of heat stress influence;Ferrando, V 等;《Food Microbiology》;20151024;第54卷;154-161 * |
| Lactic acid bacteria and proteomics: current knowledge and perspectives;Marie-Christine Champomier-Vergès等;《Journal of Chromatography B》;20020509;第771卷(第1-2期);329-342 * |
| 乳酸菌应激反应及其在生产中的应用;陈乃用;《工业微生物》;20060930;第36卷(第3期);55-60 * |
| 乳酸菌胁迫反应的影响因素及其耐热性的研究;安璟;《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》;20200215(第02期);A006-558 * |
| 喷雾干燥制备益生菌发酵剂的研究;张书猛;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20121015(第10期);B024-332 * |
| 干酪乳杆菌固态发酵及干燥工艺研究;谢贻天;《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》;20140215(第02期);D050-46 * |
| 干酪乳杆菌高密度培养及干燥工艺的研究;胡榴榴;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20150215(第 02 期);B024-407 * |
| 提高喷雾干燥益生菌发酵剂存活率的研究进展;尚一娜等;《中国乳品工业》;20170725;第45卷(第7期);37-41 * |
| 预培养对酸奶冻干发酵剂活力的影响;赵建新等;《食品与机械》;20050825;第22卷(第4期);第7页右栏第2段，第7-8页第1节，第9页左栏第1段-右栏最后1段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110129217A (en) | 2019-08-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110616159B (en) | Preparation method of Kluyveromyces marxianus freeze-dried powder | |
| CN106754472B (en) | One plant of fruits and vegetables fermentation lactobacillus plantarum grx15 and its application | |
| CN109554265B (en) | Sweet fermented-rice low-alcohol beverage and preparation method thereof | |
| CN113444664B (en) | Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof | |
| König et al. | Maintenance of wine-associated microorganisms | |
| Mabbitt et al. | The use of a selective medium for the enumeration of lactobacilli in Cheddar cheese | |
| CN113025521A (en) | Preparation process of lactobacillus bulgaricus powder with high fermentation activity | |
| CN113278554A (en) | Method for improving acid resistance of lactic acid bacteria by using mixed bacteria biological membrane | |
| CN108004177B (en) | Lactobacillus paracasei capable of degrading nitrite and characteristic research thereof | |
| CN115197870A (en) | Preparation method and application of lactobacillus with high antioxidant stress performance | |
| CN113151140B (en) | Method for improving freeze-drying survival rate and storage stability of lactobacillus | |
| CN110129217B (en) | Method for improving heat resistance of lactic acid bacteria and application | |
| CN108018211B (en) | Culture medium for improving freezing tolerance of lactobacillus bulgaricus and application method thereof | |
| CN111154697B (en) | Leuconostoc mesenteroides intestinal membrane subspecies for producing galactosidase and application thereof | |
| CN113475621A (en) | Production method of fermented feed rich in bifidobacterium lactis | |
| CN109749962B (en) | Shanxi mature vinegar dominant local flavor lactobacillus plantarum with strong tolerance and high acid production and application thereof | |
| CN1793325A (en) | Aromatic type direct putting type ferment agent for sour milk | |
| CN113293101B (en) | Inactivation method and application of lactic acid bacteria | |
| US3410755A (en) | Process and media for producing cells of lactic acid bacteria | |
| CN104273467A (en) | Black garlic and preparation method thereof | |
| CN114262680A (en) | Bacterial strain and application thereof | |
| CN114369546A (en) | Pediococcus acidilactici, fermentation microbial inoculum and application thereof, and preparation method of coarse cereal fermented food | |
| CN113005051A (en) | Lactobacillus paracasei and application thereof | |
| CN107043715A (en) | A kind of active probiotic freeze-dried powder and preparation method thereof | |
| CN108004176B (en) | Lactobacillus rhamnosus LRH09, leavening agent prepared from lactobacillus rhamnosus LRH09 and application of leavening agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |