CN107937320B - Lactobacillus plantarum, lactobacillus plantarum freeze-dried powder and preparation method thereof - Google Patents
Lactobacillus plantarum, lactobacillus plantarum freeze-dried powder and preparation method thereof Download PDFInfo
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Abstract
The application provides a strain of Lactobacillus plantarum (Lactobacillus plantarum LP4), and the microorganism preservation number of the strain is CGMCC No. 14533. The application also provides a method for preparing the lactobacillus plantarum LP4 freeze-dried powder and lactobacillus plantarum LP4 freeze-dried powder prepared by the method, the method comprises the steps of coating the lactobacillus plantarum LP4 before freeze-drying the lactobacillus plantarum LP4, combining a plurality of protective agents, and particularly, carrying out secondary coating after carrying out primary coating on the surface of the lactobacillus plantarum LP4, so as to make up for the defect of incomplete coating possibly existing in the primary coating, forming an at least completely compact protective layer on the surface of the lactobacillus plantarum LP4, forming a microcapsule structure with the lactobacillus plantarum LP4, effectively isolating the lactobacillus plantarum LP4 from the external environment, and forming a stable internal environment inside the microcapsule, so that the lactobacillus plantarum LP4 can keep activity at normal temperature.
Description
Technical Field
The application relates to the technical field of biology, in particular to lactobacillus plantarum, lactobacillus plantarum freeze-dried powder and a preparation method thereof.
Background
Compared with lactobacillus cryogenic preparation, lactobacillus freeze-dried powder is widely used due to the advantages of convenient storage and use and the like. Stability indexes such as viable count, survival rate and activity of the thallus in the lactobacillus freeze-dried powder have important influence on whether the lactobacillus freeze-dried powder can be normally used.
Generally, the freeze-drying method of the lactic acid bacteria is to rapidly freeze the lactic acid bacteria thallus containing liquid phase components in a low-temperature environment and then obtain the lactic acid bacteria freeze-dried powder according to programmed temperature rise. During storage, the lactobacillus thallus in the lactobacillus freeze-dried powder can be damaged due to contact with water, oxygen and free radicals in the environment, and further the thallus is inactivated. Due to the double loss of the freeze-drying process and the storage period, the number of live bacteria in the lactic acid bacteria freeze-dried powder is low, the survival rate is low, the activity of the bacteria is insufficient and the like in actual use.
Compared with the method of directly freeze-drying, the method can not solve the problem of the loss of the live bacteria in the storage stage, the prepared freeze-dried lactic acid bacteria powder still needs to be preserved at low temperature, and the activity of the lactic acid bacteria is maintained by a method of enabling the lactic acid bacteria to be dormant. Under the condition of no low-temperature preservation equipment, the lactobacillus freeze-dried powder is placed in a normal-temperature environment, so that the survival rate of live bacteria in the lactobacillus freeze-dried powder is rapidly reduced, the failure of the lactobacillus freeze-dried powder is shown, the effect of the lactobacillus freeze-dried powder is further influenced, and great inconvenience is brought to the use of the lactobacillus freeze-dried powder.
Disclosure of Invention
One of the purposes of the application is to provide a lactobacillus plantarum strain, and the other purpose of the application is to provide a method for preparing the lactobacillus plantarum into freeze-dried powder, so as to solve the problems that the number of viable bacteria in the lactobacillus freeze-dried powder prepared by the existing freeze-drying method is low, the bacteria are easy to inactivate during normal-temperature storage, and the number of viable bacteria cannot be effectively ensured within the shelf life.
In order to achieve the purpose, the application is realized by the following technical scheme:
lactobacillus plantarum LP4(Lactobacillus plantarum LP4) was selected by the following method:
the Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) is a probiotic with potential probiotic properties isolated from traditional kimchi; the strain is preserved in China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 14533; the classification is named as: lactobacillus plantarum LP 4; preservation time: 8 month and 18 days 2017; and (4) storage address: western road No.1 institute 3, institute of microbiology, china academy of sciences, north chen, chaoyang, china.
The isolated Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) has the following biological properties: the strains are gram-positive bacteria, are straight rods, and are single, sometimes paired or chained. The diameter of a bacterial colony on the MRS solid culture medium is 1-3mm, and the bacterial colony is milky white, convex, circular and smooth in surface. The optimal growth temperature is 30-37 ℃, the anaerobic or facultative anaerobic is carried out, the optimal pH is about 6.5, and the strain grows turbid in an MRS liquid culture medium.
The application provides a method for preparing the Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) freeze-dried powder, which comprises the following steps:
step 1-1, mixing the wet thalli with an antioxidant and stirring;
step 1-2, mixing the system prepared in the step 1-1 with polyhydric alcohol, and stirring;
step 1-3, mixing the system prepared in the step 1-2 with alkaline polysaccharide or a water dispersion system thereof, and stirring;
step 1-4, mixing the system prepared in the step 1-3 with non-alkaline polysaccharide or a water dispersion system thereof, and stirring;
step 1-5, mixing the system prepared in the step 1-4 with protein and micromolecular sugar, and stirring;
step 1-6, mixing the system prepared in the step 1-5 with alkaline polysaccharide or a water dispersion system thereof, and stirring;
and 1-7, freezing and drying the system prepared in the step 1-6.
The method provided by the application comprises the steps of coating the lactobacillus plantarum LP4 before freeze-drying the lactobacillus plantarum LP4, using a plurality of protective agents in combination, wherein the protective agents comprise antioxidants, polyols, basic polysaccharides, non-basic polysaccharides, proteins, small molecular sugars and the like, particularly, after the surface of the lactobacillus plantarum LP4 is coated for the first time, performing secondary coating, so as to make up for the defect that the coating is incomplete when the coating is incomplete for the first time, forming an at least completely compact protective layer on the surface of the lactobacillus plantarum LP4, forming a microcapsule structure with the lactobacillus plantarum LP4, effectively isolating the lactobacillus plantarum LP4 from the external environment, and forming a stable internal environment inside the microcapsule, so that the lactobacillus plantarum LP4 can keep vitality under normal temperature conditions.
By adopting the method provided by the application, the outer surface of the thallus in the prepared lactobacillus plantarum LP4 freeze-dried powder is coated with two layers of coatings, so that the coatings and the thallus form a microcapsule structure, and the microcapsule has an internal environment which is oxygen-insulated, water-insulated, air-insulated and relatively stable, thereby reducing the freezing impact of the freeze-drying process on the lactobacillus plantarum LP4 thallus and reducing the thallus loss in the freeze-drying process. Moreover, the prepared lactobacillus plantarum LP4 freeze-dried powder keeps a microcapsule structure, and the contact between the bacteria and water, oxygen, free radicals and the like in the environment is isolated, so that the activity of the bacteria can be maintained for a long time even if the lactobacillus plantarum LP4 freeze-dried powder is stored at normal temperature. Meanwhile, the inventor finds that the lactobacillus plantarum LP4 freeze-dried powder prepared by the method provided by the application can be used, and the coating substances coated on the surfaces of the bacteria can be easily removed from the surfaces of the bacteria, so that the bacteria are exposed and play a role.
The method provided by the application is different from a common vacuum freeze-drying scheme, the live bacteria content in the newly prepared freeze-dried powder is pursued on one side, the bacteria are doubly coated before freeze-drying to form microcapsules, so that the bacteria are isolated and protected, the problems that the live bacteria number of the lactobacillus plantarum LP4 freeze-dried powder is volatile and can not be effectively guaranteed in the shelf life in the normal-temperature storage process are solved, and the method is convenient for development and application of the lactobacillus plantarum LP4 freeze-dried powder in products in different fields.
The method for preparing the lactobacillus plantarum LP4 freeze-dried powder can also be applied to preparing other lactobacillus freeze-dried powder.
The application also provides the lactobacillus plantarum LP4 freeze-dried powder prepared according to the method, the viable bacteria content in the lactobacillus plantarum LP4 freeze-dried powder is 6015 hundred million CFU/g or more, and the viable bacteria survival rate after 6 months of storage at 25 ℃ is 65% or more.
The surface of the thallus in the lactobacillus plantarum LP4 freeze-dried powder is coated with a compact coating, and the coating thallus forms a micro-capsule structure, so that the thallus can be effectively isolated from the outside, the activity of the thallus can be maintained by the lactobacillus plantarum LP4 freeze-dried powder at normal temperature, low-temperature storage is not needed, the storage condition of the lactobacillus plantarum LP4 freeze-dried powder is mild, the lactobacillus plantarum LP4 freeze-dried powder is more convenient to store, and the shelf life of the lactobacillus plantarum LP4 freeze-dried powder is longer.
Drawings
FIG. 1 is a graph showing the results of survival (%) of viable bacteria in 6 months of Lactobacillus plantarum LP4 lyophilized powder prepared in examples 1-4;
FIG. 2 is a graph showing the results of viable bacteria survival (%) of Lactobacillus plantarum LP4 lyophilized powder prepared in examples 5-9 for 6 months;
FIG. 3 is a graph showing the results of survival (%) of viable bacteria in 6 months on lyophilized powder of Lactobacillus plantarum LP4 prepared in examples 10-13;
FIG. 4 is a graph showing the results of survival (%) of viable bacteria in 6 months of lyophilized powder of Lactobacillus plantarum LP4 prepared in examples 14 to 18;
FIG. 5 is a graph showing the results of viable cell survival (%) at 6 months of Lactobacillus plantarum LP4 lyophilized powder prepared in comparative examples 1-2 and example 19.
Detailed Description
The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention.
The present invention is described in detail below.
The application provides a Lactobacillus plantarum strain LP4(Lactobacillus plantarum LP4), wherein the Lactobacillus plantarum strain LP4(Lactobacillus plantarum LP4) is a probiotic with potential probiotic properties, and is separated from traditional pickle; the strain is preserved in China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 14533; the classification is named as: lactobacillus plantarum LP 4; preservation time: 8 month and 18 days 2017; and (4) storage address: western road No.1 institute 3, institute of microbiology, china academy of sciences, north chen, chaoyang, china.
The Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) can be used for fermented foods, such as fermented milk, soybean milk, pickled vegetables and the like, can also be used for preparing healthy foods, such as solid beverages, capsules and the like, can also be used for preparing animal microecological preparations, such as feed additives, feed leavening agents and the like, and can also be used for preparing agricultural microecological preparations, such as microbial fertilizers, organic fertilizer leavening agents and the like.
The application also provides a method for preparing lactobacillus plantarum LP4 freeze-dried powder, which comprises the following steps:
step 1-1, mixing the wet thallus with an antioxidant and stirring.
In the step 1-1 of the application, the wet thallus is Lactobacillus plantarum LP4 thallus, wherein the preservation number of the Lactobacillus plantarum LP4 is CGMCC No. 14533.
The inventor finds that the antioxidant added into wet thalli can remove free radicals, effectively protect thalli, prevent oxidative damage in a drying process, reduce damage of cell membranes of the thalli and improve the storage stability of lactobacillus plantarum LP4 freeze-dried powder.
Further, the antioxidant is selected from one or more of glutathione, tea polyphenol and vitamin E. The inventor finds that the antioxidants such as glutathione, tea polyphenol and vitamin E can rapidly remove oxygen free radicals on cell membranes of thalli and reduce oxidative damage of thalli.
In one embodiment, the weight ratio of the wet cells to the antioxidant is 1kg (10 to 100) g of the weight of the wet cells to the weight of the antioxidant, wherein the weight of the wet cells is based on the total weight of the wet cells. The inventor finds that the antioxidant with the weight part can be uniformly dispersed in the thallus of lactobacillus plantarum LP4 to form an antioxidant core, and oxygen free radicals on the thallus can be rapidly removed to reduce the oxidative damage of the thallus.
The number of viable bacteria in the wet thallus is more than 6500 hundred million CFU/g, and in an realizable mode, the wet thallus can be prepared according to a method comprising the following steps:
step 2-1, inoculating Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) frozen and preserved at-80 ℃ in 5ml of MRS liquid medium, and carrying out anaerobic culture at 37 ℃ for 24 hours, thus carrying out subculture for 2 times to obtain activated Lactobacillus plantarum LP4 strain.
Step 2-2, inoculating the strain activated in step 2-1 into a fermentation culture medium sterilized at 121 ℃ for 20min, and inoculating the live bacteria of Lactobacillus plantarum LP4Metering, total inoculum size is 1 × 107CFU/mL, fermenting at 37 ℃ under a constant-temperature anaerobic condition, automatically controlling the pH value of a fed-batch ammonia water maintaining system to be 5.9 in the fermentation process, stopping acid production, stopping fermentation when ammonia water is not fed-batch to obtain a lactobacillus plantarum high-density culture solution, wherein the viable count of lactobacillus plantarum LP4 in the high-density culture solution is more than 300 hundred million CFU/mL, and the fermentation culture medium is prepared from the following components in parts by weight: 100kg of cane sugar, 10kg of yeast powder, 10kg of soybean peptone, 5kg of casein peptone, 5kg of tryptone, 1.0kg of citric acid, 20kg of sodium citrate and MgSO 24·7H2O 1.5kg，MnSO4·5H20.08kg of O, 801.0 kg of Tween and 1000L of distilled water.
And 2-3, cooling the high-density culture solution prepared in the step 2-2 to 4 ℃ at the temperature of 4-8 ℃, centrifugally concentrating and extracting wet thalli by 14000Xg, mixing and stirring for 30min, and then centrifugally treating to remove residual ions and pigments in an original culture medium system, wherein the weight ratio of the wet thalli to the sterile physiological saline is 1:2, and the treated wet thalli is high in purity and white in color.
In step 1-1 of the present application, the temperature of the system is maintained at 2 ℃ to 8 ℃, preferably 4 ℃. The inventor finds that the metabolic activity of the lactobacillus plantarum LP4 is reduced at the temperature, and the lactobacillus plantarum LP4 is in a dormant state and is easy to maintain the activity.
In step 1-1, the mixing may be any method for uniformly mixing the two substances, such as emulsification using an emulsifier, emulsification using a homogenizer, mechanical stirring, and the like, and preferably mechanical stirring for 5 min.
And step 1-2, mixing the system prepared in the step 1-1 with polyhydric alcohol, and stirring.
The present inventors have found that a polyol can scavenge free radicals and prevent oxidative damage to cells during drying.
In step 1-2 of the present application, the polyhydric alcohol is selected from one or more of glycerol, mannitol and sorbitol, preferably glycerol and a combination of mannitol and sorbitol, such as a combination of glycerol, mannitol and sorbitol in a ratio of 2:1: 1.
In an achievable mode, the weight ratio of the system obtained in the step 1-1 to the polyhydric alcohol is 1kg (100-800) g, such as 1kg to 400g, of the weight of the system obtained in the step 1-1, based on the weight of the wet bacterial cells obtained in the step 1-1. The inventor finds that the added polyol can be dispersed around the cells of the lactobacillus plantarum LP4 thallus to form an anti-freezing protective layer by adding the polyol according to the weight ratio, so that the physical damage of ice crystals to cell membranes is reduced, and the drying survival rate in the freezing process is improved by more than 20 percent.
And 1-3, mixing the system prepared in the step 1-2 with the alkaline polysaccharide or the water dispersion system thereof, and stirring.
The inventors found that basic polysaccharides can form a microcapsule structure by charge interaction with acidic cells, protect cells, and immobilize low-temperature microcapsules formed on the surface of Lactobacillus plantarum LP4 cells.
In steps 1-3 of the present application, the basic polysaccharide is a polysaccharide that exhibits basicity in aqueous dispersion, such as chitosan.
Optionally, the aqueous dispersion of the basic polysaccharide is a chitosan solution having a concentration of 1 wt% to 8 wt%, preferably 2.5 wt%.
In the present application, the concentration refers to the weight part of the solute per unit volume of the dispersion, wherein 1L is taken as 1 unit volume and 1g is taken as 1 weight part of the solute. For example, a 2.5 wt% chitosan solution refers to a chitosan dispersion having a weight of 25g per liter of chitosan dispersion.
The inventor finds that chitosan with the concentration can generate condensation reaction between the surface of lactobacillus plantarum LP4 and lactobacillus plantarum LP4 in wet cells, so that a compact gel layer is formed on the surface of lactobacillus plantarum LP4, the gel water holding capacity is good, and the sterile liquid of the cells coated with the gel layer is precipitated, so that the coating effect is good.
In the step 1-3, the weight ratio of the system prepared in the step 1-2 to the alkaline polysaccharide or the aqueous dispersion thereof is 1kg (12.5-50) g, preferably 1kg to 25g, based on the weight of the wet bacterial cells in the step 1-1, and the weight of the aqueous dispersion of the alkaline polysaccharide is based on the weight of the alkaline polysaccharide in the step 1-1. The present inventors have found that the addition of the basic polysaccharide at the above weight ratio allows the formation of a coating layer on the surface of Lactobacillus plantarum LP4 in wet cells.
And 1-4, mixing the system prepared in the step 1-3 with non-alkaline polysaccharide or a water dispersion system thereof, and stirring.
The present inventors have found that non-basic polysaccharides can undergo charge aggregation reaction with basic polysaccharides, and gel polymers are continuously formed on the surface of existing microcapsules, thereby more tightly protecting the cells of Lactobacillus plantarum LP 4.
The non-alkaline polysaccharide is a polysaccharide with a neutral or acidic water dispersion system, is selected from one or more of cellulose, maltodextrin, sodium alginate, gelatin, pectin, xanthan gum, pullulan, soybean polysaccharide, beta-glucan, tara gum, guar gum, fenugreek gum, locust bean gum, algin gum and arabic gum, is preferably a composition consisting of gelatin, maltodextrin, sodium alginate and arabic gum, and is a composition of gelatin, maltodextrin, sodium alginate and arabic gum in weight.
In the step 1-4, the concentration of the non-alkaline aqueous polysaccharide dispersion is 3 wt% to 8 wt%, preferably, the non-alkaline aqueous polysaccharide dispersion is a non-alkaline polysaccharide composition solution with a concentration of 5 wt%, and the non-alkaline polysaccharide composition is a composition of gelatin, maltodextrin, sodium alginate and arabic gum in equal weight. The inventor finds that the non-basic polysaccharide with the concentration can generate charge condensation reaction with the basic polysaccharide to form a gel polymer with a compact structure, and the bacterial cells of the lactobacillus plantarum LP4 are isolated from the outside.
In step 1-4, the weight ratio of the system obtained in step 1-3 to the non-basic polysaccharide or its aqueous dispersion is 1kg (25-100) g, preferably 1kg to 50g, based on the weight of the wet bacterial cells in step 1-1, and the weight of the non-basic polysaccharide aqueous dispersion is based on the weight of the non-basic polysaccharide in step 1-1. The inventor finds that the non-alkaline polysaccharide with the concentration can generate equivalent charge condensation reaction with alkaline sugar to form gel polymer with compact structure, and the water content of the prepared freeze-dried bacterial powder is less than or equal to 3%; and the addition of excessive non-alkaline polysaccharide can cause the system viscosity to rise, the freeze-dried bacterial powder is difficult to remove water, the water content is more than or equal to 5 percent, and the activity of the bacterial strain is not easy to maintain.
And 1-5, mixing the system prepared in the step 1-4 with protein and micromolecular sugar, and stirring.
The inventor finds that the small molecular sugar can permeate into the cell, the protein can be dispersed in the intercellular space, and the small molecular sugar and the protein cooperate to protect the thallus and reduce the damage of the ice crystal to the cell structure of the lactobacillus plantarum LP4 during the freezing process.
The protein is selected from one or more of skimmed milk powder, soybean protein powder and desalted whey powder, preferably a composition consisting of skimmed milk powder and desalted whey powder, such as the equal weight composition of skimmed milk powder and desalted whey powder.
The small molecular sugar is selected from one or more of glucose, sucrose, lactose, maltose, trehalose, fructose, fructo-oligosaccharide and galacto-oligosaccharide, preferably trehalose, lactose, a composition consisting of sucrose and fructose, such as trehalose, lactose, a composition consisting of sucrose and fructose in equal weight. The inventor finds that after the small molecular sugar is added, biological structures such as cell membranes, proteins and the like of the lactobacillus plantarum LP4 can be effectively protected, and the capacity of the thallus to resist the action of external adverse environment stress is improved.
In step 1-5, the weight ratio of the protein to the small molecule sugar is (0.5-1.2): (0.5-1.2), preferably (0.5-1.0): 0.8-1.0), such as 1.0:1.0, by weight of the protein, based on the total weight of the small molecule sugar composition, if the protein is a composition, and/or the small molecule sugar is also a composition, wherein the weight of the protein is based on the total weight of the protein composition, and the weight of the small molecule sugar is based on the total weight of the small molecule sugar composition.
In step 1-5, the weight ratio of the system prepared in step 1-4 to the protein and the small molecular sugar is the weight of the system prepared in step 1-4, the weight of the protein to the weight of the small molecular sugar is (1-3): (0.5-1.2): (0.5-1.2), preferably (1-1.5): (0.5-1.0): 0.8-1.0), such as 1.0:1.0:1.0, wherein the weight of the system prepared in step 1-4 is the weight of the wet bacterial cells in step 1-1, and if the protein is a composition and/or the small molecular sugar is a composition, the weight of the protein is the total weight of the protein composition and the weight of the small molecular sugar is the total weight of the small molecular sugar composition. The inventor finds that the addition of the protein and the micromolecular sugar according to the weight ratio can slow down the formation speed of the crystallization ice crystals in the freeze drying process, reduce the number and the granularity of the ice crystals, reduce the damage of the ice crystals to the cell structure of the lactobacillus plantarum LP4 in the freezing process and improve the drying survival rate in the freezing process by more than 30 percent.
And 1-6, mixing the system prepared in the step 1-5 with the alkaline polysaccharide or the water dispersion system thereof, and stirring.
The inventor finds that the alkaline polysaccharide can perform charge condensation reaction with the thalli and the non-alkaline polysaccharide in the steps 1-1 and 1-4 to form gel, multilayer and multiple embedding protection is performed on the thalli of the lactobacillus plantarum LP4, and the influence of adverse environments such as air, temperature and the like on the activity of the strain is completely isolated
The basic polysaccharide used in steps 1-6 may be the same as or different from the basic polysaccharide used in steps 1-3. For example, chitosan or an aqueous chitosan dispersion may be used in steps 1-6.
Optionally, the concentration of the aqueous dispersion of the basic polysaccharide is 1 wt% to 5 wt% of the aqueous dispersion, and preferably, the concentration of the basic polysaccharide or the aqueous dispersion thereof is 2.5 wt% of chitosan solution. The inventors found that 2.5 wt% chitosan acted equally as the non-basic polysaccharide in steps 1 to 4 to form a gel, resulting in a low-temperature multiple-embedding treatment of the cells of Lactobacillus plantarum LP 4.
In the step 1-6, the weight ratio of the system prepared in the step 1-5 to the alkaline polysaccharide or the aqueous dispersion thereof is 1kg (12.5-50) g, preferably 1kg to 25g, based on the weight of the wet bacterial cells in the step 1-1, and the weight of the alkaline polysaccharide aqueous dispersion is based on the weight of the alkaline polysaccharide in the system prepared in the step 1-5. The present inventors have found that the addition of the alkaline polysaccharide solution in the above weight ratio can act on the non-alkaline polysaccharide in steps 1 to 4 in an equivalent amount to form a gel, thereby forming a low-temperature multiple-embedding treatment of the cells of Lactobacillus plantarum LP 4.
The steps 1-1 to 1-6 of the application are all carried out at the temperature of 2-8 ℃, so that the lactobacillus plantarum LP4 thalli is in a dormant state to keep the activity of the thalli.
And 1-7, freezing and drying the system prepared in the step 1-6.
In the steps 1-7, the method for freeze drying can be any method for freeze drying the lactobacillus in the prior art, for example, the system prepared in the steps 1-6 is quickly frozen to-50 ℃ by liquid nitrogen, and then is dried according to the programmed temperature rise under the environment with the vacuum degree of less than or equal to 30pa, the temperature gradient of the programmed temperature rise can be-50 ℃ to-15 ℃ to-5 ℃ to-15 ℃ to-30 ℃, and the lactobacillus plantarum LP4 freeze-dried powder is prepared after 48 hours.
The activity of the lactobacillus freeze-dried powder is closely related to the oxygen content in the preparation process environment, and in short, the lower the oxygen content in the preparation process environment, the higher the activity of the prepared lactobacillus freeze-dried powder is. The present inventors have found that oxygen in the production process environment can interact with the cell membrane of the strain to impair DNA synthesis of the strain and can generate free radicals before freeze-drying of the strain. Therefore, the application selects that the steps 1-1 to 1-7 are performed under anaerobic conditions, such as under the protection of nitrogen or carbon dioxide, so as to reduce the generation of free radicals, thereby improving the number of viable bacteria in the lactobacillus plantarum LP4 freeze-dried powder and the survival rate of the viable bacteria.
The interaction and the mixing sequence proportion between the wet lactobacillus plantarum LP4 thalli and different substances have great influence on the storage stability of the lactobacillus plantarum LP4 freeze-dried powder. Alkaline polysaccharide, such as chitosan, preferentially undergoes condensation reaction with gelatin, sodium alginate, gum arabic and the like, and the polysaccharide substances are added simultaneously, so that the lactobacillus plantarum LP4 thallus, the antioxidant and the alkaline polysaccharide cannot be condensed to form a microcapsule, a complete embedding structure cannot be formed, and the later freeze drying and freeze-dried powder storage are not facilitated.
The application also provides a freeze-dried powder of lactobacillus plantarum LP4 prepared according to the method, wherein the viable bacteria content of the freeze-dried powder is 6015 hundred million CFU/g or more, and the viable bacteria survival rate is 65% or more after the freeze-dried powder is stored for 6 months at 25 ℃.
The lactobacillus plantarum LP4 freeze-dried powder provided by the invention is completely coated, the activity of the strain is not required to be ensured by making the strain dormant in a low-temperature environment, the activity can be kept in the process of normal-temperature storage in food, feed and medical health products, and the viable count can be effectively ensured in the shelf life.
Examples
The lactobacillus plantarum LP4 used in the examples of the present application has the accession number: CGMCC No. 14533.
The MRS liquid medium used in this application was purchased from: guangdong Huanji microbial science and technology Limited, for microbial detection;
the physiological saline used in the application is self-made.
Preparation of Lactobacillus plantarum culture solution
a. Activating strain, inoculating Lactobacillus plantarum LP4(Lactobacillus plantarum LP4) frozen and preserved at-80 deg.C into 5ml MRS liquid culture medium, anaerobically culturing at 37 deg.C for 24 hr, and subculturing for 2 times to obtain activated strain.
b. Inoculating and fermenting: inoculating the seed culture solution into a fermentation culture medium sterilized at 121 ℃ for 20min, fermenting under a constant-temperature anaerobic condition at 37 ℃, automatically controlling the fed-batch ammonia water to keep pH5.9 for fermentation until acid production stops, and stopping fermentation when the ammonia water is not fed-batch to obtain the Lactobacillus plantarum high-density culture solution, wherein the viable count of Lactobacillus plantarum LP4 in the culture solution is 300 hundred million CFU/mL.
Fermentation ofThe culture medium was prepared from the following components: 100kg of cane sugar, 10kg of yeast powder, 10kg of soybean peptone, 5kg of casein peptone, 5kg of tryptone, 1.0kg of citric acid, 20kg of sodium citrate and MgSO 24·7H2O 1.5kg，MnSO4·5H20.08kg of O, 801.0 kg of Tween and 1000L of distilled water.
c. Carrying out centrifugal concentration on Lactobacillus plantarum LP4 fermentation liquor cooled to 4 ℃ by 14000g to extract wet thalli, mixing and stirring the extracted wet thalli and sterile PBS buffer solution (the weight of the wet thalli is 1:2) for 30min, carrying out centrifugal treatment, removing metal ions and extracting residual culture medium for later use, wherein the temperature of the liquid material in the operation process is controlled to be 4-8 ℃.
Examples 1-4 selection of Small molecule sugar dosage in cell protectant
Taking 1kg of the wet thallus prepared in the step one, adding 1kg of protein (equal weight composition of skim milk powder and desalted whey powder), adding different amounts of micromolecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose), freeze-drying for 48 hours under the same condition to prepare lactobacillus plantarum LP4 freeze-dried powder, storing the prepared lactobacillus plantarum LP4 freeze-dried powder for 6 months at 25 ℃, and determining the survival rate of viable bacteria, wherein the specific contents are shown in the following table 1:
TABLE 1
| Sample (I) | Small molecule sugar addition amount (g) | Survival rate of viable bacteria at 6 months (%) (25 ℃ C.) |
| Example 1 | 0 | 0.050 |
| Example 2 | 500 | 6.130 |
| Example 3 | 1000 | 11.025 |
| Example 4 | 1500 | 9.052 |
Comparative analysis shows that the addition of micromolecular sugar (trehalose, lactose, sucrose, fructose and other compositions in equal weight) in the vacuum freeze-drying process of the lactobacillus plantarum LP4 can effectively protect the thallus, reduce the freeze damage and improve the storage stability of the freeze-dried powder. Through optimization, the addition proportion of 1kg of wet thallus to 1kg of micromolecular sugar is more appropriate, and the specific effect is shown in figure 1.
In the examples described below, the amount of small-molecular sugar added was 1kg/kg based on 1kg of wet cells.
Examples 5-9 Effect of polyols on Lactobacillus plantarum LP4 lyophilized powder
Taking 1kg of the wet bacterial cells prepared in the step (I), adding 1kg of protein (equal weight composition of skim milk powder and desalted whey powder) and 1000g of micromolecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose), adding polyols with different proportions (weight of glycerol: weight of mannitol: weight of sorbitol: 2:1: 1) on the basis of the protein and the micromolecular sugar, carrying out vacuum freeze-drying for 48 hours under the same conditions to prepare lactobacillus plantarum LP4 freeze-dried powder, storing for 6 months at 25 ℃, and determining the survival rate of viable bacteria, wherein the survival rate is specifically shown in the following table 2:
TABLE 2
| Sample (I) | Polyol addition amount (g) | Survival rate of viable bacteria at 6 months (%) (25 ℃ C.) |
| Example 5 | 100 | 10.835 |
| Example 6 | 200 | 11.319 |
| Example 7 | 400 | 15.741 |
| Example 8 | 600 | 15.562 |
| Example 9 | 800 | 12.376 |
Comparative analysis shows that the addition of the polyhydric alcohol (the weight of glycerol: the weight of mannitol: the weight of sorbitol: the composition is 2:1: 1) can effectively protect the thallus, reduce the freezing damage and improve the storage stability of the lactobacillus plantarum LP4 freeze-dried powder in the vacuum freeze-drying process. Through optimization, the addition proportion is that 0.4kg of polyhydric alcohol is added into 1kg of wet thalli, and the specific effect is shown in figure 2.
In the examples described below, the amount of the polyol added was 0.4kg/kg based on 1kg of wet cells.
Example 10-13 Effect of polysaccharide dosage on Lactobacillus plantarum LP4 lyophilized powder
Taking 1kg of the wet bacterial cells prepared in the step (I), adding 1kg of protein (equal weight composition of skim milk powder and desalted whey powder), 1000g of micromolecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose), 400g of polyalcohol (weight of glycerol: weight composition of mannitol: weight of sorbitol: 2:1: 1), adding polysaccharides in different proportions on the basis of the protein, specifically, adding chitosan for the first time after adding polyalcohol, then adding non-alkaline polysaccharide composition (equal weight composition of gelatin, maltodextrin, sodium alginate and Arabic gum), then adding chitosan for the second time, performing vacuum freeze-drying for 48h under the same condition, preparing into lactobacillus plantarum LP4 freeze-dried powder, storing for 6 months at 25 ℃, and determining the survival rate of viable bacteria, which is specifically shown in the following table 3:
TABLE 3
Comparative analysis shows that during the vacuum freeze drying process of lactobacillus plantarum LP4, polysaccharide substances are sequentially added according to the sequence of alkaline polysaccharide-non-alkaline polysaccharide, so that the thalli of the lactobacillus plantarum LP4 can be effectively protected, the freezing damage is reduced, and the storage stability of the lactobacillus plantarum LP4 freeze-dried powder is improved. Through optimization, the adding proportion is based on 1kg of wet thallus, 25g of alkaline polysaccharide and 50g of non-alkaline polysaccharide are added for the first time, 25g of alkaline polysaccharide is added for the second time, and the specific effect is shown in figure 3.
In the examples described below, the amount of the first basic polysaccharide added was 25g/kg, the amount of the non-basic polysaccharide added was 50g/kg, and the amount of the second basic polysaccharide added was 25g/kg, based on 1kg of wet cells, and in the examples described below, the total amount of polysaccharide added was 0.10 kg.
Examples 14-18 Effect of antioxidant addition on Lactobacillus plantarum LP4 lyophilized powder
Taking 1kg of the wet bacterial cells prepared in the step (a), adding 1000g of protein (equal weight composition of skim milk powder and desalted whey powder, small molecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose), 400g of polyalcohol (weight of glycerol: weight of mannitol: weight of sorbitol: 2:1:1 composition), and 0.1kg of total added polysaccharide, adding antioxidants (weight of glutathione: weight of tea polyphenol: weight of vitamin E: 1:1:2 composition) in different proportions into the wet bacterial cells, performing vacuum freeze-drying for 48h under the same conditions, preparing lactobacillus plantarum LP4 freeze-dried bacterial powder, storing the powder at 25 ℃ for 6 months, and measuring the survival rate of viable bacteria, wherein the survival rate is specifically shown in the following table 4:
TABLE 4
| Sample (I) | Antioxidant addition amount (g) | Survival rate of viable bacteria at 6 months (%) (25 ℃ C.) |
| Example 14 | 10 | 30.015 |
| Example 15 | 20 | 35.438 |
| Example 16 | 40 | 40.713 |
| Example 17 | 80 | 39.802 |
| Example 18 | 100 | 38.985 |
Comparative analysis shows that the addition of an antioxidant (a composition in which the weight of glutathione is the weight of tea polyphenol and the weight of vitamin E is 1:1: 2) in the vacuum freeze-drying process of lactobacillus plantarum LP4 can effectively protect the thallus, reduce the damage of thallus cell membranes and improve the storage stability of the freeze-dried powder. Through optimization, the addition proportion is that 0.04kg of antioxidant is added into 1kg of wet thalli, and the specific effect is shown in figure 4.
In the examples described below, the antioxidant was added in an amount of 0.04kg/kg based on 1kg of wet cells.
Example 19 preparation of Lactobacillus plantarum LP4 lyophilized powder by Low-temperature multiple microencapsulation Process
Step 1: adding 10g of antioxidant (glutathione: tea polyphenol: vitamin E: 1:1: 2) into 1kg of wet thallus prepared in step one, stirring for 5min, adding 400g of polyalcohol (glycerol: mannitol: sorbitol: 2:1: 1) and stirring for 5 min;
step 2: adding the system prepared in the step 1 into 1000mL of 2.5 wt% chitosan solution, stirring for 15min, and then mixing and dispersing by a homogenizer to obtain uniform liquid;
and step 3: adding 1000mL of 5 wt% non-alkaline polysaccharide (gelatin, maltodextrin, sodium alginate, Arabic gum and other compositions in weight) solution into the uniform liquid prepared in the step 2, stirring for 15min, and then mixing and dispersing by a homogenizer to obtain uniform liquid;
and 4, step 4: adding 1000g of protein (equal weight composition of skim milk powder and desalted whey powder) and 1000g of micromolecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose) into the uniform liquid prepared in the step 3, and uniformly stirring and mixing;
and 5: and (4) adding the system prepared in the step (4) into 1000mL of 2.5 wt% chitosan solution, stirring for 15min, and then mixing and dispersing into uniform liquid through a homogenizer.
The temperature of the materials and the environment is controlled to be 2-8 ℃ in the above operation process.
And (3) carrying out vacuum freeze drying on the mixed solution for 48h to prepare lactobacillus plantarum LP4 freeze-dried powder, wherein the viable bacteria content in the lactobacillus plantarum LP4 freeze-dried powder is more than 6015 hundred million CFU/g, and the viable bacteria survival rate is 67.149% after the lactobacillus plantarum LP4 freeze-dried powder is stored for 6 months at 25 ℃.
Comparative example
Comparative example 1 preparation of Lactobacillus plantarum LP4 lyophilized powder by one-time mixing at room temperature
Wet cells prepared from 10g of an antioxidant (glutathione, weight: tea polyphenol, weight: vitamin E, composition: 1:2), 400g of a polyol (glycerol, mannitol, sorbitol, composition: 2:1: 1), 50g of a non-basic polysaccharide (gelatin, maltodextrin, sodium alginate, gum arabic, etc.), 1000g of a protein (skim milk powder, desalted milk powder, etc.), 1000g of a small molecular sugar (trehalose, lactose, sucrose, fructose, etc.), and 1kg of a saccharide were stirred for 30min, mixed and dispersed by a homogenizer to form a uniform liquid.
And (3) carrying out vacuum freeze-drying on the prepared system for 48h to prepare lactobacillus plantarum PL4 freeze-dried powder, wherein the viable bacteria survival rate of the lactobacillus plantarum PL4 freeze-dried powder is 41.529% after the lactobacillus plantarum PL4 freeze-dried powder is stored for 6 months at 25 ℃.
Comparative example 2 preparation of Lactobacillus plantarum LP4 lyophilized powder by fixation of microcapsules at Low temperature
Step 1: adding 10g of antioxidant (glutathione: tea polyphenol: vitamin E: 2:1: 1) into 1kg of wet thallus prepared in the step (I), stirring for 5min, adding 400g of polyalcohol (glycerol: mannitol: sorbitol: 2:1: 1) and stirring for 5 min;
step 2: adding the uniformly mixed liquid obtained in the step 1 into 1000mL of 2.5 wt% chitosan solution, stirring for 15min, and then mixing and dispersing into uniform liquid through a homogenizer;
and step 3: mixing the uniform liquid obtained in the step 2 with 1000mL of 5 wt% non-alkaline polysaccharide (equal weight composition of gelatin, maltodextrin, sodium alginate and Arabic gum) solution, stirring for 15min, and mixing and dispersing by a homogenizer to obtain uniform liquid;
and 4, step 4: and (3) uniformly stirring and mixing the uniform liquid obtained in the step (3) with 1000g of protein (equal weight composition of the skim milk powder and the desalted whey powder) and 1000g of small molecular sugar (equal weight composition of trehalose, lactose, sucrose and fructose).
The temperature of the materials and the environment is controlled to be 2-8 ℃ in the above operation process.
The mixed solution is subjected to vacuum freeze drying for 48 hours to prepare freeze-dried bacterial powder, and the survival rate of live bacteria is 52.372 percent after the freeze-dried bacterial powder is stored for 6 months at 25 ℃.
Comparative analysis shows that in the vacuum freeze-drying process of lactobacillus plantarum PL4, the thalli are treated by the microencapsulation process without the microcapsule technology (comparative example 1) and only once coated (comparative example 2) and the thalli treated by the secondary coating microencapsulation process provided by the application (example 19), so that the adverse environment of the thalli to the outside, such as stress action of temperature and oxygen, can be effectively reduced, cell damage is reduced, and the storage stability of lactobacillus plantarum PL4 freeze-dried powder is improved. Through optimization, the low-temperature multi-microcapsule coating process is suitable for processing and preparing lactobacillus plantarum PL4 freeze-dried powder with high storage stability, and the specific effect is shown in figure 5.
The lactobacillus plantarum LP4 freeze-dried powder prepared by adopting a low-temperature multi-microcapsule coating process avoids the defects that the lactobacillus freeze-dried powder is volatile and alive in the normal-temperature storage process, the viable count cannot be effectively ensured in the shelf life, and the effect is influenced, can be widely applied to food, feed and medical products, stabilizes the product quality and exerts the health effect.
The present application has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.
Sequence listing
<110> Jinhua Galaxy Biotech Co., Ltd
<120> lactobacillus plantarum, lactobacillus plantarum freeze-dried powder and preparation method thereof
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atccattgta gcacgtgtgt agcccaggtc ataaggggca tgatgatttg acgtcatccc 300
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gayaaccatg caccacmtgt atccatgtcc cygragggar sgtstaatmt yttagatttg 480
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tctacgcatt tcaccgctac acatggagtt ccactgtcct cttctgcact caagtttccc 840
agtttccgat gcacttcytc ggtkgagccg aaggctttca catcaractt aaaaaaccgc 900
ctgcgctcgc tttacgccca ataaatccgg acaacgcttg scwcctacrt attaccgcgs 960
ctgctggcac gtarttagcc gwggctttct ggttaaatac cgtcaatacc tgaacagtta 1020
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Claims (2)
1. A method for preparing lactobacillus plantarum LP4 freeze-dried powder, which comprises the following steps:
step 1-1, mixing and stirring wet thalli and an antioxidant, wherein the wet thalli is the thalli of lactobacillus plantarum LP4, the microbial preservation number of the wet thalli is CGMCC No.14533, and the antioxidant is a composition of glutathione, tea polyphenol and vitamin E in a weight ratio of 1:1: 2;
step 1-2, mixing the system prepared in the step 1-1 with polyhydric alcohol, and stirring, wherein the polyhydric alcohol is a composition of glycerol, mannitol and sorbitol in a weight ratio of 2:1: 1;
step 1-3, mixing the system prepared in the step 1-2 with an alkaline polysaccharide water dispersion system, and stirring, wherein the alkaline polysaccharide water dispersion system is a chitosan water dispersion system with the weight percentage of 3-8 percent;
step 1-4, mixing the system prepared in the step 1-3 with a non-alkaline polysaccharide water dispersion system, and stirring, wherein the non-alkaline polysaccharide is a composition of maltodextrin, sodium alginate, gelatin and Arabic gum in equal weight, and the concentration of the non-alkaline polysaccharide water dispersion system is 3-8 wt%;
step 1-5, mixing the system prepared in the step 1-4 with protein and micromolecular sugar, and stirring, wherein the protein is a composition of skim milk powder and desalted whey powder in equal weight, and the micromolecular sugar is a composition of sucrose, lactose, trehalose and fructose in equal weight;
step 1-6, mixing the system prepared in the step 1-5 with an alkaline polysaccharide water dispersion system, and stirring;
step 1-7, freezing and drying the system prepared in the step 1-6;
wherein, the steps 1-1 to 1-7 are all carried out under the anaerobic condition at the temperature of 2-8 ℃.
2. The lactobacillus plantarum LP4 freeze-dried powder prepared according to the method of claim 1, wherein the viable bacteria content in the lactobacillus plantarum LP4 freeze-dried powder is 6015 hundred million CFU/g or more, and the viable bacteria survival rate after 6 months of storage at 25 ℃ is 65% or more.
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| CN108841757A (en) * | 2018-07-13 | 2018-11-20 | 甘肃省科学院生物研究所 | A kind of lactic acid bacteria culture collection process |
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| CN110387330B (en) * | 2019-07-17 | 2022-10-14 | 上海理工大学 | Freeze-drying method for improving survival rate of lactobacillus plantarum by using composite protective agent |
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| CN113604381A (en) * | 2021-07-16 | 2021-11-05 | 上海商学院 | Acetic acid bacteria freeze-dried powder and preparation method thereof |
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