CN114806972B - Method for preparing high-activity lactobacillus starter by using natural eutectic solvent - Google Patents
Method for preparing high-activity lactobacillus starter by using natural eutectic solvent Download PDFInfo
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- CN114806972B CN114806972B CN202210641024.XA CN202210641024A CN114806972B CN 114806972 B CN114806972 B CN 114806972B CN 202210641024 A CN202210641024 A CN 202210641024A CN 114806972 B CN114806972 B CN 114806972B
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- 241000186660 Lactobacillus Species 0.000 title claims abstract description 30
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 30
- 230000005496 eutectics Effects 0.000 title claims abstract description 17
- 239000002904 solvent Substances 0.000 title claims abstract description 17
- 230000000694 effects Effects 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 12
- 239000007858 starting material Substances 0.000 title claims abstract description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000001694 spray drying Methods 0.000 claims abstract description 14
- 229960003237 betaine Drugs 0.000 claims abstract description 12
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 10
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 235000014655 lactic acid Nutrition 0.000 claims description 5
- 239000004310 lactic acid Substances 0.000 claims description 5
- 229920002774 Maltodextrin Polymers 0.000 claims description 4
- 239000005913 Maltodextrin Substances 0.000 claims description 4
- 229940035034 maltodextrin Drugs 0.000 claims description 4
- 238000002390 rotary evaporation Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000011159 matrix material Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000010802 sludge Substances 0.000 claims description 3
- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 230000006641 stabilisation Effects 0.000 claims description 2
- 238000011105 stabilization Methods 0.000 claims description 2
- 239000003223 protective agent Substances 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 8
- 230000004083 survival effect Effects 0.000 abstract description 8
- 238000004108 freeze drying Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 description 3
- 238000001035 drying Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- 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
-
- 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
- C12N1/02—Separating microorganisms from their culture media
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- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (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)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a method for preparing a high-activity lactobacillus starter by using a natural eutectic solvent, belonging to the technical field of lactobacillus preparations. The invention discloses a method for preparing a high-activity lactobacillus starter by using a natural eutectic solvent, which uses a novel glycerol-betaine natural eutectic solvent of lactobacillus as a protective agent, and improves the spray drying survival rate of the lactobacillus by more than 3 times after being treated by using the protective agent relative to glycerol and betaine as the protective agent.
Description
Technical Field
The invention relates to the technical field of lactobacillus preparations, in particular to a method for preparing a high-activity lactobacillus starter by using a natural eutectic solvent.
Background
Lactic acid bacteria are an important component in the food fermentation industry and the functional food industry. At present, lactobacillus preparation is mainly prepared by a freeze drying technology, and has the defects of higher survival rate, incapability of continuous production, high equipment cost, high energy consumption, long production period and the like; becomes an important factor for limiting the development of the lactobacillus dry preparation. The spray drying technology has the advantages of low cost, high efficiency, continuity and the like, the production cost is only 18 percent of that of the freeze drying technology, and the preparation method is hopeful to replace freeze drying to produce the lactobacillus preparation. However, the cell damage caused by the contact of the thallus with hot air in the spray drying process can lead to massive death and great reduction of the activity of the lactobacillus after drying.
At present, how to reduce the death and activity reduction of lactobacillus caused by drying is a main technical difficulty in preparing high-activity lactobacillus ferment, lactobacillus powder and probiotic powder, and although the addition of protective agents such as glycerol, betaine and the like can improve the survival rate of lactobacillus after freeze drying and spray drying, the effect of the two cannot meet the production requirements, so that the existing protective agents are treated, and the improvement of the protection efficiency of the existing protective agents on lactobacillus spray drying is a main problem to be solved by the technology.
The natural eutectic solvent (NADES) has high biocompatibility, biodegradability and safety, and the preparation process is simple and the cost is low. The NADES has higher glass transition temperature and polarity, can be adsorbed on the cell surface through hydrogen bond action, is arranged and enters cells, and reduces the damage of cell structures and enzyme inactivation of thalli during the dry preservation period. The protective agents such as glycerol and betaine are commonly used in lactobacillus freeze drying and spray drying, but are prepared into natural eutectic solvents for use, and have not been reported at present.
Accordingly, it is a urgent need to solve the problem of the skilled person to provide a method for preparing a high-activity lactic acid bacteria starter using a natural eutectic solvent.
Disclosure of Invention
In view of the above, the invention provides a method for preparing a high-activity lactobacillus starter by using a natural eutectic solvent, which is mainly used for preparing probiotic bacteria powder, lactobacillus preparation and lactobacillus powder production.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preparing high-activity lactobacillus starter by using natural eutectic solvent (NADES) comprises the following specific steps:
(1) Culturing lactobacillus in culture medium to logarithmic phase and pre-stabilization phase;
(2) Centrifugally collecting thalli, and flushing the thalli for 2 times by using normal saline or 0.01M phosphate buffer solution; centrifuging again, and collecting thalli to obtain bacterial sludge;
(3) The molar ratio is 1:2, rotary evaporating betaine and glycerol in a rotary evaporation bottle at 75-85 ℃ for 1-4 hours until the betaine and the glycerol are clear and transparent, and preparing the NADES;
(4) 30% (w/v) maltodextrin is used as a dry matrix, 1-15% (w/v) NADES is added, and the mixture is fully mixed with bacterial mud according to the proportion of 2-10: mixing the materials according to the mass ratio of 1, and performing spray drying to prepare the bacterial powder.
Further, the centrifugation in the step (2) is performed at 4 ℃ and 6000r/min for 10min.
Compared with the prior art, the invention discloses a method for preparing the high-activity lactobacillus starter by using the natural eutectic solvent, wherein the novel glycerol-betaine natural eutectic solvent of the lactobacillus is used as a protective agent, and compared with glycerol and betaine which are used as protective agents, the spray drying survival rate of the lactobacillus after being treated by using the protective agent is improved by more than 3 times.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The protective effect on lactobacillus delbrueckii subsp 1.1 (l.bulgaricus sp 1.1) was investigated using natural eutectic solvents (NADES) as a lactic acid bacteria protective agent. The molar ratio is 1:2, the betaine and the glycerol are subjected to rotary evaporation in a rotary evaporation bottle at 80 ℃ for 2 hours until the betaine and the glycerol are clear and transparent, and the NADES is prepared for standby. After two generations of activation, L.Bulgaricus sp1.1 was inoculated into MRS at an inoculum size of 2% (v/v), and after 24 hours of culture, the cells were collected by centrifugation at 6000r/min at 4℃for 10 minutes. Washing bacteria twice with physiological saline, centrifuging again at 4deg.C and 6000r/min for 10min, and collecting bacterial sludge.
Preparing L.bulgaricus sp1.1 bacterial powder by adopting a laboratory-grade spray drying device: to be used for30% (w/v) maltodextrin of the feed liquid is used as a dry matrix, 5% (w/v) NADES is added into an experimental group, and after being fully and uniformly mixed, the mixture is mixed with bacterial mud according to the following formula of 5: mixing the materials according to the mass ratio of 1; control group 1 (Con 1) without any substance added, control group 2 (Con 2) with equal mass of NADES starting material added but without spin-steaming. The spray drying conditions were as follows: 30% (w/v) of maltodextrin in the feed liquid, and air inlet quantity: 380m 3 And/h, the inlet/outlet air temperature is 120/70 ℃, and the atomization pressure is 0.9m 3 And/h, adjusting the feeding rate to enable the temperature of the inlet air and the outlet air to meet a preset value. The results of survival and moisture content after spray drying are shown in table 1.
Table 1 spray drying survival and moisture content under different treatment conditions
The survival rate of the lactic acid bacteria after spray drying after adding NADES is improved from 5.18% to 20.09% under the condition of not changing other conditions, and the survival rate is improved by more than 3 times.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. A method for preparing a high-activity lactobacillus starter by using a natural eutectic solvent is characterized by comprising the following specific steps:
(1) Culturing lactobacillus in a culture medium to the end of logarithmic phase and the earlier stage of stabilization, wherein the lactobacillus is Lactobacillus delbrueckii subspecies bulgaricus;
(2) Centrifugally collecting thalli, and flushing the thalli for 2 times by using normal saline or 0.01M phosphate buffer solution; centrifuging again, and collecting thalli to obtain bacterial sludge;
(3) The molar ratio is 1:2, rotary evaporating betaine and glycerol in a rotary evaporation bottle at 75-85 ℃ for 1-4 hours until the betaine and the glycerol are clear and transparent, so as to prepare a natural eutectic solvent;
(4) 30% w/v maltodextrin is used as a dry matrix, 5-15% w/v natural eutectic solvent is added, and after being fully and uniformly mixed, the mixture is mixed with bacterial mud according to the proportion of 5-10: mixing the materials according to the mass ratio of 1, and performing spray drying to prepare the bacterial powder.
2. The method for preparing a high activity lactic acid bacteria starter according to claim 1, wherein the centrifugation in the step (2) is performed at 4 ℃ and 6000r/min for 10min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210641024.XA CN114806972B (en) | 2022-06-07 | 2022-06-07 | Method for preparing high-activity lactobacillus starter by using natural eutectic solvent |
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| CN202210641024.XA CN114806972B (en) | 2022-06-07 | 2022-06-07 | Method for preparing high-activity lactobacillus starter by using natural eutectic solvent |
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