CN115595286A - Lactobacillus plantarum microbial inoculum and preparation method and application thereof - Google Patents
Lactobacillus plantarum microbial inoculum and preparation method and application thereof Download PDFInfo
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- CN115595286A CN115595286A CN202211383833.1A CN202211383833A CN115595286A CN 115595286 A CN115595286 A CN 115595286A CN 202211383833 A CN202211383833 A CN 202211383833A CN 115595286 A CN115595286 A CN 115595286A
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- lactobacillus plantarum
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- microbial inoculum
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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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- A—HUMAN NECESSITIES
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/45—Addition of, or treatment with, microorganisms
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- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
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- A23L2/382—Other non-alcoholic beverages fermented
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method of a lactobacillus plantarum microbial inoculum and a novel protective agent in freeze dryingThe process has the effect of protecting plant lactobacillus. The preparation method comprises the following steps: lactobacillus plantarum LP115 was inoculated into MRS broth and activated at 37 ℃ for a period of time as seed liquid. Inoculating the seed liquid to another MRS liquid culture medium for amplification culture, and growing to the earlier stage of the stationary phase. Taking out and subpackaging the bacteria liquid into a centrifuge tube, centrifuging, pouring out supernate, washing with sterile normal saline, collecting bacteria mud, adding a certain proportion of a novel freeze-drying protective agent, and uniformly mixing in a vortex manner. Then placing the mixture at the low temperature of minus 80 ℃ for quick prefreezing, and freezing and drying the mixture to obtain the lactobacillus plantarum microbial inoculum. The unit viable count of the lactobacillus plantarum microbial inoculum prepared by the invention is 2.5 multiplied by 10 11 CFU/g, the freeze-drying survival rate is as high as 85%.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus plantarum microbial inoculum as well as a preparation method and application thereof.
Background
Lactobacillus plantarum is a common lactobacillus, belongs to anaerobic or facultative anaerobic gram-positive bacilli, and is widely applied to fermented vegetables, fruit juice, meat products and fermented dairy products. According to the regulations of the Food and Agricultural Organization (FAO) and the world health trade organization (WHO) of the United nations, the number of the live bacteria of the lactobacillus plantarum planted in the gastrointestinal tract of the human body is more than 1 x 10 6 CFU/mL can exert its probiotic function, such as regulating the balance of intestinal flora and enhancing immunity. Therefore, it becomes important to prepare lactobacillus plantarum inocula with high activity from the source, which has become one of the hot spots for research on lactobacillus in recent years.
The vacuum freeze-drying technique is a process of freezing an aqueous solution containing cells, and performing sublimation drying and deicing at a low temperature. Compared with other drying technologies, the technology can maintain the cell viability and the stability of the lactic acid bacteria at a higher level, and provides convenience for the transportation and storage of products, so that the technology is favored. However, certain damage to the cells, such as the formation of large-particle ice crystals, osmotic stress, mechanical damage, solute effects, and denaturation of DNA and proteins, is inevitable during the process, and therefore, in the freeze-drying technology, an anti-freezing protective agent is frequently added to reduce the damage and improve the survival rate of the cells to the maximum extent.
At present, carbohydrate, protein and amino acid are mainly used as common freeze-drying protective agents, and trehalose, skim milk, sucrose, glucose and sodium glutamate are used as protective agents for most researches. In particular, carbohydrates are considered to be the best protective agents compared to the other two classes because of their glassy capacity to allow cells to form low molecular interactions between spaces or surfaces of molecular arrangements. More particularly, the carbohydrate has a polyhydroxy structure, hydrogen bonds can be formed around cells, and natural structures of biological macromolecules such as phospholipid and protein are stabilized instead of water molecules, so that the activity of the cells is maintained. According to the report, the macromolecular carbohydrate has the advantages of higher glass transition temperature and disintegration temperature and providing a physical barrier, and the micromolecular carbohydrate can enter the inside of cells to inhibit the formation of ice crystals, so that a novel protective agent formed by compounding macromolecules and micromolecules is needed to be researched, a better protection effect on microorganisms can be provided, the cell activity is effectively maintained, and the freeze-drying survival rate of the microbial agent is improved.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a lactobacillus plantarum microbial inoculum taking a trehalose solution and a cellulase hydrolysate as a freeze-drying protective agent, and a preparation method and application thereof. According to the lactobacillus plantarum microbial inoculum provided by the invention, micromolecule and macromolecule saccharides are adopted as freeze-drying protective agents to be mixed in the preparation process, the disadvantage of a single protective agent is overcome, the micromolecule and macromolecule saccharides complement each other in the whole protection system, and the respective effects are exerted for preparing the high-activity lactobacillus plantarum microbial inoculum together, so that the prepared lactobacillus plantarum microbial inoculum has higher freeze-drying survival rate, the freeze-dried cells can be maintained in a lower water content range, and the subsequent storage of the microbial inoculum is facilitated.
The technical scheme of the invention is as follows:
a preparation method of a lactobacillus plantarum microbial inoculum comprises the following steps:
(1) Activating strains: inoculating lactobacillus plantarum LP115 to an MRS liquid culture medium, and activating for a period of time to serve as seed liquid;
(2) And (3) expanding culture: inoculating the seed liquid obtained in the step (1) to another MRS liquid culture medium for amplification culture, and growing to the early stage of a stable period;
(3) Collecting bacterial sludge: taking out the bacterial liquid after the enlarged culture in the step (2), subpackaging the bacterial liquid into a centrifuge tube, centrifuging, pouring out supernatant, and washing with sterile normal saline to obtain bacterial sludge;
(4) Adding a freeze-drying protective agent: adding a freeze-drying protective agent into the bacterial sludge obtained in the step (3), and uniformly mixing in a vortex manner to obtain a uniformly mixed solution;
(5) And (3) freeze drying: and (4) pre-freezing the mixed solution obtained in the step (4), and then freezing and drying to obtain the lactobacillus plantarum microbial inoculum.
Further, in the step (4), the freeze-drying protective agent is composed of a trehalose solution and a cellulose hydrolysate according to the weight ratio of (6-10) to (0-4), and the mass fraction of the trehalose solution is 8-12%.
Furthermore, in the step (4), the freeze-drying protective agent is composed of a trehalose solution and a cellulose hydrolysate according to a weight ratio of 8: 2, and the mass fraction of the trehalose solution is 10%.
In the preparation process of the lactobacillus plantarum microbial inoculum provided by the invention, a composite freeze-drying protective agent consisting of trehalose and a cellulase hydrolysate according to a specific weight ratio is creatively added by the inventor of the application, and the combination of the trehalose and the cellulase hydrolysate is found to generate an obvious synergistic effect, so that some soluble oligosaccharides generated in the hydrolysis process of the trehalose and the cellulase can replace water molecules in a freeze-drying stage by virtue of polyhydroxy groups contained in the soluble oligosaccharides, the natural structure of original hydration state of cellular biomacromolecules is maintained, a vitrified matrix formed around cells by macromolecular fibrous substances is combined to resist the damage of the external environment, and the combined effect of the trehalose and the cellulase is similar to a physical barrier to resist the damage of the external environment to the cells, so that the cells bear less activity loss.
Further, the freeze-drying protective agent is used after being sterilized for 17min at the temperature of 121 ℃ and under the pressure of 0.1MPa and cooled.
Further, the weight ratio of the bacterial sludge to the freeze-drying protective agent in the step (4) is 1: 10.
Further, the preparation method of the cellulase hydrolysate in the lyoprotectant of step (4) is as follows:
dissolving cellulose in acetic acid-sodium acetate buffer solution with the pH value of 4.8 to prepare cellulose suspension with the concentration of 0.02g/mL, stirring the cellulose suspension for 12 to 24 hours by a magnetic stirrer, then adding 1.75U/mL of cellulose solution, uniformly mixing the cellulose suspension and the cellulose solution, hydrolyzing the mixture for 0.5 to 2.5 hours at the temperature of 50 ℃ and at the speed of 150rpm, and after the reaction time is over, immersing the mixed solution into boiling water to heat for 5 minutes to inactivate enzyme, thereby stopping enzymatic reaction. And cooling to room temperature, and adjusting the pH of the enzymolysis solution to 6.7-6.9 by using a NaOH solution with the mass concentration of 4% to obtain a cellulase hydrolysate.
Further, the volume ratio of the cellulose suspension to the cellulase solution is 4: 1.
Further, the cellulose is derived from grapefruit peel, and the preparation method of the cellulose is as follows:
drying the grapefruit peel at 50 deg.C for 24h, pulverizing, and sieving with 40 mesh sieve; performing alkali treatment by using a NaOH solution with the mass concentration of 15%, wherein the material-liquid ratio is 1: 30, washing the mixture to be neutral by using distilled water and 95% ethanol, and drying the mixture for 12 hours at 50 ℃; using 6% NaClO by mass fraction 2 The solution is subjected to bleaching treatment, and the NaClO is obtained 2 Adjusting the pH of the solution to 3.8-4.0 by adopting a hydrochloric acid solution, and then washing the solution to be neutral by using distilled water; freeze drying at-70 deg.C and 0Pa for 48 hr to obtain cellulose.
Further, the inoculation amount of the lactobacillus plantarum LP115 in the step (1) is 3 x 10 7 CUF/mL, the activation time is 10h, and the activation temperature is 37 ℃; the inoculation amount of the seed liquid in the step (2) is 4.4% of the mass of the MRS liquid culture medium, and the culture time is 9.5h.
Further, the centrifugation conditions in the step (3) are as follows: centrifuging at 14136 Xg and 4 deg.C for 10min; after centrifugation, the supernatant was decanted off and washed 2 times with sterile physiological saline at a mass concentration of 0.85%.
Further, the freeze-drying conditions in the step (5) are as follows: the temperature of the cold trap is-70 ℃, the vacuum degree is 0Pa, and the freeze drying is carried out for 48 hours.
The invention also provides a lactobacillus plantarum microbial inoculum prepared by the preparation method.
The lactobacillus plantarum microbial inoculum prepared by the method can be applied to food.
The lactobacillus plantarum LP115 is activated, enlarged and cultured, bacterial sludge is centrifugally collected, a certain proportion of novel freeze-drying protective agent is added to obtain a mixture of the lactobacillus plantarum LP115 and the novel protective agent, the mixture is pre-frozen at the temperature of minus 80 ℃, and then the mixture is freeze-dried to obtain the lactobacillus plantarum microbial inoculum.
Compared with the prior art, the lactobacillus plantarum microbial inoculum provided by the invention has the following advantages:
(1) The freeze-drying survival rate of the lactobacillus plantarum microbial inoculum prepared by the invention reaches 88.86%. Compared with the traditional single trehalose protective agent, the novel freeze-drying protective agent consisting of trehalose solution and cellulose enzymolysis products in a mass ratio of 8: 2 improves the freeze-drying survival rate by 30.23%, and remarkably enhances the protection effect. And the raw materials are cheap and easy to obtain, so that the method has strong production and popularization values.
(2) Scanning electron microscope analysis shows that the lactobacillus plantarum microbial inoculum prepared by the method is short rod-shaped, is compactly distributed, is greatly gathered on a structure with a rough and multi-fold surface, is beneficial to maintaining cell activity, and has relatively better protection effect.
(3) The low-field nuclear magnetic resonance hydrogen spectrum analysis shows that the lactobacillus plantarum microbial inoculum prepared by the method mainly contains bound water and water which is difficult to flow, wherein the bound water content is more than 85%, and the water content is maintained in a lower water content range, so that the subsequent storage of the microbial inoculum is facilitated.
Drawings
FIG. 1 is a graph showing growth curves before and after Lactobacillus plantarum activation in MRS medium.
FIG. 2 is a schematic diagram showing the survival rate of Lactobacillus plantarum under the influence of different compounding ratios of the novel protective agent.
FIG. 3 is a schematic representation of the survival rate of Lactobacillus plantarum under the influence of different enzymatic hydrolysis times for cellulose.
FIG. 4 is a schematic representation of a Scanning Electron Microscope (SEM) of Lactobacillus plantarum under the influence of different protectant compositions.
FIG. 5 is a schematic representation of the T2 relaxation profile of Lactobacillus plantarum under the influence of different protectant compositions.
FIG. 6 is a schematic representation of the fluorescence spectra of Lactobacillus plantarum under the influence of different protectant compositions.
In FIGS. 4 to 6: a represents a 10% trehalose solution; b represents a product of hydrolysis of 10% trehalose solution and cellulose for 0.5 h; c represents a 10% trehalose solution and a product of cellulase hydrolysis for 1 h; d represents a 10% trehalose solution and a product of cellulase hydrolysis for 1.5 h; e represents a 10% trehalose solution and a product of cellulase hydrolysis for 2h; f represents the product of hydrolysis with 10% trehalose solution and cellulase for 2.5 h.
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but the invention is within the protection scope of the invention.
In the following examples, reagents not specifically described were conventional reagents and were commercially available from conventional reagent production and distribution companies.
Example 1 preparation method of Lactobacillus plantarum microbial inoculum
The preparation method of the lactobacillus plantarum microbial inoculum comprises the following steps:
(1) Activating strains: inoculating Lactobacillus plantarum LP115 to MRS liquid culture medium in an inoculation amount of 3 × 10 7 Activating CUF/mL at 37 deg.C for 10 hr to obtain seed solution;
(2) And (3) amplification culture: inoculating the seed solution obtained in the step (1) to another MRS liquid culture medium for amplification culture, wherein the inoculation amount of the seed solution is 4.4% of the mass of the MRS liquid culture medium, and the seed solution grows for 9.5h to the early stage of a stabilization period;
(3) Collecting bacterial sludge: taking out the bacterial liquid after the enlarged culture in the step (2), subpackaging the bacterial liquid into centrifuge tubes, centrifuging the centrifugal tubes for 10min at the temperature of 14136 Xg and 4 ℃, pouring out supernate, and washing the supernate for 2 times by using sterile normal saline with the mass concentration of 0.85 percent to obtain bacterial sludge;
(4) Adding a freeze-drying protective agent: adding a freeze-drying protective agent into the bacterial sludge obtained in the step (3), wherein the weight ratio of the bacterial sludge to the freeze-drying protective agent is 1: 10, and uniformly mixing by vortex to obtain a mixed solution;
(5) And (3) freeze drying: quickly pre-freezing the mixed solution obtained in the step (4) at the temperature of minus 80 ℃, and freeze-drying to obtain a lactobacillus plantarum microbial inoculum; the conditions of freeze drying are as follows: the temperature of the cold trap is-70 ℃, the vacuum degree is 0Pa, and the freeze drying is carried out for 48 hours.
The MRS liquid culture medium formula (in parts by weight) is as follows: 10 parts of bacteriological peptone, 20 parts of anhydrous glucose, 5 parts of beef extract, 4 parts of yeast extract powder, 1 part of tween-80, 5 parts of anhydrous sodium acetate, 2 parts of triammonium citrate, 2 parts of dipotassium phosphate heptahydrate, 0.2 part of magnesium sulfate heptahydrate, 0.05 part of manganese sulfate tetrahydrate and 1000 parts of distilled water.
The freeze-drying protective agent in the step (4) is composed of trehalose solution and cellulose hydrolysate according to the weight ratio of 8: 2, and the mass fraction of the trehalose solution is 10%. The freeze-drying protective agent is sterilized for 17min at the temperature of 121 ℃ and under the pressure of 0.1MPa and then is cooled for use.
The preparation method of the cellulase hydrolysate comprises the following steps:
dissolving cellulose in acetic acid-sodium acetate buffer solution with the pH value of 4.8 to prepare cellulose suspension with the concentration of 0.02g/mL, stirring for 20 hours by a magnetic stirrer, then adding 1.75U/mL cellulase solution, wherein the volume ratio of the cellulose suspension to the cellulase solution is 4: 1, uniformly mixing, hydrolyzing for 2 hours at the temperature of 50 ℃ and at the speed of 150rpm, and after the reaction time is over, immersing the mixed solution into boiling water, heating for 5min, inactivating enzyme and further stopping enzymatic reaction. And cooling to room temperature, and adjusting the pH of the enzymolysis solution to 6.7-6.9 by using a NaOH solution with the mass concentration of 4% to obtain a cellulase hydrolysate.
The cellulose is derived from the grapefruit skin, and the preparation method of the cellulose comprises the following steps:
drying the grapefruit peel at 50 ℃ for 24h, then crushing at high speed, and sieving by a 40-mesh sieve; performing alkali treatment by using a NaOH solution with the mass concentration of 15%, wherein the material-liquid ratio is 1: 30, washing the mixture to be neutral by using distilled water and 95% ethanol, and drying the mixture for 12 hours at 50 ℃; using 6% NaClO by mass fraction 2 The solution is subjected to bleaching treatment, and the NaClO is 2 Adjusting the pH of the solution to 3.8-4.0 by adopting a hydrochloric acid solution, and then washing the solution to be neutral by using distilled water; freeze drying at-70 deg.C and 0Pa for 48 hr to obtain cellulose.
Test example I analysis of growth curves of Lactobacillus plantarum in MRS Medium before and after activation
(1) Determination and analysis of growth curve before lactobacillus plantarum activation
Lactobacillus plantarum LP115 was prepared at 3X 10 7 Inoculating the CUF/mL inoculum size into a liquid MRS culture medium, culturing in a water-proof incubator at 37 ℃, and respectively sampling for fermentation for 0, 2, 4, 6, 8, 10, 12 and 24 hours to determine the viable count. And (3) drawing a growth curve by taking the fermentation time as an abscissa and the common logarithm of the viable count of the lactobacillus plantarum as an ordinate, and determining an activation end point.
(2) Determination and analysis of growth curve after lactobacillus plantarum activation
Lactobacillus plantarum LP115 was prepared at 3X 10 7 Inoculating the CUF/mL inoculum size to a liquid MRS culture medium, and culturing to an activation end point to obtain a seed solution. Inoculating the seed liquid into another liquid MRS culture medium with an inoculation amount of 4.4% (w/w), performing amplification culture at 37 ℃, and sampling at 0, 2, 4, 6, 8, 10, 12 and 24h of fermentation respectively to determine the viable count. And (3) drawing a growth curve by taking the fermentation time as an abscissa and the common logarithm of the viable count of the lactobacillus plantarum as an ordinate, and determining the harvest period of the bacterial sludge.
(3) The method for measuring the viable count of the lactobacillus refers to the test of the lactobacillus of GB 4789.35-2016 national food safety standard.
(4) And (3) test results: as can be seen from fig. 1, the growth of lactobacillus plantarum in the liquid MRS medium before activation is: 0-2 h in the lag phase, the viable count change of the lactobacillus plantarum is small, and the growth is slow; 2-12 h, in logarithmic phase, the viable count of the lactobacillus plantarum is rapidly increased and shows exponential growth trend; 12-24 h, in the stable period, the viable count of the lactobacillus plantarum basically keeps unchanged, and the growth reaches dynamic balance. Therefore, the late logarithmic phase of lactobacillus plantarum growth for 10h was chosen as the activation endpoint. The growth conditions of the activated lactobacillus plantarum in a liquid MRS culture medium are as follows: the delay period does not exist, the lactobacillus plantarum viable count is rapidly increased after the logarithmic phase is directly started for 0h and the logarithmic phase is ended for 8 h; and 8-24 h, and in a stable period, the viable count of the lactobacillus plantarum is basically kept unchanged. Cells in stationary phase were reported to be the best choice for the freeze-drying process, so the first 9.5h in stationary phase was chosen as harvest phase for the bacterial sludge.
Test example two, optimized analysis of compounding ratio of lactobacillus plantarum freeze-drying protective agent and cellulose enzymolysis time
(1) Optimization of compounding ratio of freeze-drying protective agent
In the process of preparing the lactobacillus plantarum microbial inoculum, a freeze-drying protective agent with the mass 10 times that of bacterial mud is added according to the mass of the bacterial mud. Wherein, 10 times of 10% trehalose solution is added into the control group, protective agents are respectively added into the experimental group according to the proportion of 9: 1, 8: 2, 7: 3 and 6: 4 (m/m) of the 10% trehalose solution and the cellulase hydrolysate (enzymolysis for 2 h), which are respectively marked as 10: 0, 9: l, 8: 2, 7: 3 and 6: 4, and other operation steps are not changed. And then, measuring the viable count of the freeze-dried sample, calculating the freeze-drying survival rate, and further optimizing the compounding ratio of the protective agent.
(2) Optimization of cellulase hydrolysis time
In the process of preparing the lactobacillus plantarum microbial inoculum, according to the mass of bacterial sludge, an experimental group keeps the compounding ratio of trehalose solution to cellulose enzymolysis product to be 8: 2, the cellulose enzymolysis time is changed, 5 gradients of 0.5, 1, 1.5, 2 and 2.5h are respectively set, a control group is kept unchanged, 10% trehalose solution with the mass being 10 times that of the bacterial sludge is still added, and other operation steps are unchanged. And then, carrying out viable count determination on the freeze-dried sample, calculating the freeze-drying survival rate, and screening out the optimal cellulose enzymolysis time.
The calculation formula of the freeze-drying survival rate is as follows:
(3) And (3) test results: (1) The optimized experimental data of the compounding ratio of the lyoprotectant are shown in table 1 and fig. 2, and (2) the optimized experimental data of the cellulase enzymolysis time are shown in table 2 and fig. 3; in fig. 2 and 3, significant difference analysis was performed at a p <0.05 level, and a, b, c indicate that there is a significant difference in the data (Duncan' step).
TABLE 1
| Different proportions of protective agent | Freeze-drying survival rate (%) |
| 10∶0 | 62.17 |
| 9∶l | 75.78 |
| 8∶2 | 85.37 |
| 7∶3 | 61.17 |
| 6∶4 | 54.83 |
TABLE 2
| Time of enzymolysis (h) | Freeze-drying survival rate (%) |
| 0 | 58.63 |
| 0.5 | 75.35 |
| 1 | 77.65 |
| 1.5 | 81.94 |
| 2 | 88.86 |
| 2.5 | 59.61 |
As can be seen from Table 1 and FIG. 2, when the compounding ratio of the 10% trehalose solution to the cellulase hydrolysate is 8: 2 (m/m), the freeze-drying survival rate of the Lactobacillus plantarum microbial inoculum prepared by the novel freeze-drying protective agent reaches 85.37%, which is 23.2% higher than that of the conventional single trehalose protective agent. As can be seen from Table 2 and FIG. 3, when the enzymolysis time of the cellulase is 2 hours, the freeze-drying survival rate of the lactobacillus plantarum microbial inoculum prepared by the novel freeze-drying protective agent consisting of the corresponding product reaches 88.86%, which is 30.23% higher than that of the conventional single trehalose protective agent, and the survival rate of the microbial inoculum is remarkably improved.
Test example three, micro-morphology and T of Lactobacillus plantarum microbial inoculum 2 Relaxation map analysis
(1) Micro-topography analysis
After the lactobacillus plantarum is freeze-dried by different freeze-drying protective agents, a scanning electron microscope is adopted to observe and analyze the microscopic morphology, and a scanning electron microscope picture reveals the morphological characteristics of the lactobacillus plantarum microbial inoculum and visually reflects the distribution state of the lactobacillus plantarum in different protective agent compositions. Wherein, a represents 10% trehalose solution; b represents a product of hydrolysis of 10% trehalose solution and cellulose for 0.5 h; c represents a 10% trehalose solution and a product of cellulase hydrolysis for 1 h; d represents a 10% trehalose solution and a product of cellulase hydrolysis for 1.5 h; e represents a 10% trehalose solution and a product of cellulase hydrolysis for 2h; f represents the product of hydrolysis with 10% trehalose solution and cellulase for 2.5 h.
As can be seen from FIG. 4, the shape of Lactobacillus plantarum shown in FIGS. a-f is short rod-like, and the length is kept around 2 μm, with similar morphology. However, the surface structure of the graph a is relatively smooth, and the lactobacillus plantarum is embedded in the surface of the trehalose substance, is wrapped in the trehalose in a large amount and is not easy to find. The graphs b-f show similar structures, rough and multi-fold surfaces, large effective inhabitation area and compact cell arrangement. In addition, the lactobacillus plantarum is mostly adhered to the concave part of the sheet-shaped structure of the fiber substrate, exposes the complete cell form, is compactly distributed, is favorable for rehydration of strains and recovery of cell activity.
(2)T 2 Relaxation map analysis
And (3) determining the water distribution condition of the lactobacillus plantarum added with different protective agents after freeze-drying by using a nuclear magnetic resonance imager. Weighing about 0.7g of lactobacillus plantarum powder in a transparent test tube with a plug, selecting a magnet probe of 40mm, and performing water model correction and sample parameter determination under a Q-FID sequence, wherein the parameters are as follows: SF =20mhz, sw =200khz, rfd =0.002ms, p1=9.52 μ s, RG1=20db, drg1=3, prg =2, ns =8, td =60002, tw =6000ms, p2=19.04 μ s, then TE =0.150ms, nech =2000 was set under Q-CPMG sequence, measurement of sample was performed, and inversion was performed to obtain T =0.150ms, tach =2000 2 Relaxation spectrum (as shown in figure 5). The moisture distribution status of lactobacillus plantarum with different protectant compositions after lyophilization is shown in table 3.
TABLE 3
Note: t is 2i ，A 2i And W 2i (i =1,2,3) means respectively T of FIG. 5 after lyophilization of Lactobacillus plantarum 2 Three peaks in the relaxation spectrum, peak areas and occupied proportions thereof.
As can be seen from FIG. 5 and Table 3, T of all samples 21 Ratio W of peak area 21 At the maximum, the water content of the freeze-dried lactobacillus plantarum exists mainly in a state of strongly bound water, and represents the lipid and protein of the lactobacillus plantarumWater with closely bound macromolecules such as lipids. Taking into account weakly bound water T 22 Is occupied ratio W of 22 It can be known that the content of the bound water of the lactobacillus plantarum after freeze-drying exceeds 85%, which indicates that most of the free water in the bacterial powder is removed and is maintained in a lower water content range, and is beneficial to maintaining good cell viability in the subsequent storage process. But does not easily flow water T 23 Compared with T 21 And T 22 The relaxation time is longest, which indicates that the part of water is relatively loosely combined with the lactobacillus plantarum and has larger variation and fluctuation in the later storage process.
Experimental example four fluorescence Spectroscopy analysis of Lactobacillus plantarum microbial inoculum
0.1g of freeze-dried lactobacillus plantarum microbial inoculum is fully dissolved in 9.9mL of physiological saline, and the solution is diluted in a gradient manner (the dilution times of different samples are kept the same) until the OD600nm is about 0.2. 3mL of bacterial liquid is taken, 0.3mL of 1mg/mL Fluorescein Diacetate (FDA) dye solution is added and mixed evenly, then 0.3mL of 200 mu g/mL Propidium Iodide (PI) dye solution is added and mixed evenly by vortex, and the mixture is kept stand for 10 to 15min at 4 ℃ in a dark place. Each sample absorbs 150 mu L of liquid and is dripped to an enzyme label plate, the excitation wavelength is set to be 480nm, the step length is 2nm, and fluorescence spectrum scanning is carried out within the range of the emission wavelength of 500-670 nm.
The FDA dye liquor preparation method comprises the following steps: 50mg FDA was dissolved in 10mL acetone to prepare a mother liquor with a final concentration of 5mg/mL, and stored at 4 ℃ in the dark for further use.
The PI dye solution preparation method comprises the following steps: 10mg of PI is dissolved in 10mL of ultrapure water to prepare a PI mother solution with the final concentration of 1mg/mL, and the PI mother solution is stored at 4 ℃ in a dark place for later use.
The characteristics of the integrity of the cell membranes of lactobacillus plantarum agents consisting of different protective agents are shown in table 4.
TABLE 4
As can be seen from FIG. 6 and Table 4, the fluorescence spectrum curves of the control group a and the experimental groups b-f show similar trend, and two distinct peaks are present near 520nm and 630nm, and the peak heights thereof respectively reflect the degree of normal and damaged cell membranes of Lactobacillus plantarum. The ratio of the peak heights of the control group a and the experimental groups b-f around 520nm and 630nm was calculated to be 0.92,1.40,1.45,1.73,2.21 and 0.83, respectively. The larger the ratio, the less the lactobacillus plantarum cell membrane is damaged and the higher the integrity. Therefore, compared with the control group a, the integrity of the cell membranes of the other b-e lactobacillus plantarum is higher and gradually increased except for the experimental group f, and the integrity of the cell membranes of the lactobacillus plantarum microbial inoculum prepared by taking the product of cellulose enzymolysis for 2 hours and the trehalose solution as the novel protective agent is highest.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a lactobacillus plantarum microbial inoculum is characterized by comprising the following steps:
(1) Activating strains: inoculating lactobacillus plantarum to an MRS liquid culture medium, and activating for a period of time to serve as seed liquid;
(2) And (3) expanding culture: inoculating the seed liquid obtained in the step (1) to another MRS liquid culture medium for amplification culture, and growing to the early stage of a stable period;
(3) Collecting bacterial sludge: taking out the bacterial liquid after the enlarged culture in the step (2), subpackaging the bacterial liquid into a centrifuge tube, centrifuging, pouring out supernatant, and washing with sterile normal saline to obtain bacterial sludge;
(4) Adding a freeze-drying protective agent: adding a freeze-drying protective agent into the bacterial sludge obtained in the step (3), and uniformly mixing in a vortex manner to obtain a mixed solution;
(5) And (3) freeze drying: and (4) pre-freezing the mixed solution obtained in the step (4), and then freezing and drying to obtain the lactobacillus plantarum microbial inoculum.
2. The preparation method of the lactobacillus plantarum microbial inoculum according to claim 1, wherein in the step (4), the freeze-drying protective agent is composed of a trehalose solution and a cellulase hydrolysate according to a weight ratio of (6-10) to (0-4), and the mass fraction of the trehalose solution is 8-12%; the freeze-drying protective agent is used after being sterilized for 17min at the temperature of 121 ℃ and under the pressure of 0.1MPa and cooled.
3. The method for preparing lactobacillus plantarum microbial inoculum according to claim 2, wherein in the step (4), the freeze-drying protective agent is trehalose solution and cellulase hydrolysate in a weight ratio of 8: 2; the weight ratio of the bacterial sludge to the freeze-drying protective agent in the step (4) is 1: 10.
4. The method for preparing a lactobacillus plantarum microbial inoculum according to claim 2, wherein the method for preparing the cellulase hydrolysate in the lyoprotectant of step (4) is as follows:
dissolving cellulose in acetic acid-sodium acetate buffer solution with the pH value of 4.8 to prepare cellulose suspension with the concentration of 0.02g/mL, stirring for 12-24 h, then adding 1.75U/mL cellulase solution, uniformly mixing, hydrolyzing for 0.5-2.5 h at 50 ℃ and 150rpm, soaking the mixed solution into boiling water to heat for 5min after the reaction time is over, inactivating enzyme and further stopping enzymatic reaction, cooling to room temperature, and regulating the pH value of enzymatic hydrolysate to 6.7-6.9 by using NaOH solution with the mass concentration of 4% to obtain a cellulase hydrolysate.
5. The method for preparing a Lactobacillus plantarum microbial inoculum according to claim 4, wherein the volume ratio of the cellulose suspension to the cellulase solution is 4: 1.
6. The method for preparing a lactobacillus plantarum microbial inoculum according to claim 4, wherein the cellulose is derived from grapefruit peel, and the method for preparing the cellulose is as follows:
drying the grapefruit peel at 50 deg.C for 24h, pulverizing, and sieving with 40 mesh sieve; performing alkali treatment with 15% NaOH solution at a material-liquid ratio of 1: 30, distilled water and 95% ethanolWashing with alcohol to neutral, and drying at 50 deg.C for 12 hr; using 6% NaClO by mass fraction 2 The solution is subjected to bleaching treatment, and the NaClO is obtained 2 Adjusting the pH of the solution to 3.8-4.0 by adopting a hydrochloric acid solution, and then washing the solution to be neutral by using distilled water; freeze drying at-70 deg.C and 0Pa for 48 hr to obtain cellulose.
7. A method for preparing Lactobacillus plantarum microbial inoculum according to claim 1, wherein the inoculation amount of Lactobacillus plantarum LP115 in step (1) is 3 x 10 7 CUF/mL, the activation time is 10h, and the activation temperature is 37 ℃; the inoculation amount of the seed liquid in the step (2) is 4.4% of the mass of the MRS liquid culture medium, and the culture time is 9.5h.
8. The method for preparing a lactobacillus plantarum microbial inoculum according to claim 1, wherein the centrifugation conditions in the step (3) are as follows: centrifuging at 14136 Xg and 4 deg.C for 10min; after centrifugation, the supernatant is poured off and washed for 2 times with sterile normal saline with the mass concentration of 0.85%;
in the step (5), the pre-freezing temperature is-80 ℃, and the freeze-drying conditions are as follows: the temperature of the cold trap is-70 ℃, the vacuum degree is 0Pa, and the freeze drying is carried out for 48 hours.
9. A Lactobacillus plantarum microbial inoculum prepared according to the preparation method of any one of claims 1-8.
10. Use of the lactobacillus plantarum microbial inoculum according to claim 9 in food.
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