CN109868246B - Method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri - Google Patents
Method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri Download PDFInfo
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Abstract
The invention discloses a freeze-drying protection method of rana japonica oil enzymolysis liquid on lactobacillus reuteri. The invention comprises the following steps: firstly, carrying out enzymolysis on wood frog oil by using alkaline protease to prepare wood frog oil enzymolysis liquid, preparing a freeze-drying protective agent according to the proportion of 20-30% of the wood frog oil enzymolysis liquid, 15-25% of trehalose and 3-5% of ascorbic acid, then adding lactobacillus reuteri thalli obtained by culture and centrifugation into the prepared and sterilized freeze-drying protective agent, and mixing uniformly, wherein the volume mass ratio of the thalli to the protective agent is 1: 1-10, and finally, carrying out vacuum freeze drying on the uniformly mixed bacterial suspension. The survival rate of the freeze-dried powder of the lactobacillus reuteri is over 60 percent by using the freeze-drying protective agent.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri.
Background
Lactic acid bacteria, an important probiotic, have been widely used in the industries of food, medicine, cultivation, and the like in recent years. The lactobacillus reuteri selected in the patent belongs to space mutation strains, and research shows that the strain has poor freezing resistance and stability in a storage process, but has the effects of protecting gastric mucosa, regulating intestinal flora, resisting inflammation and resisting allergy. At present, the product is applied to health-care food for infants and adults, functional solid beverage and nutritional food.
The wood frog oil is a traditional health food, contains rich protein, alpha-linolenic acid, linoleic acid, oleic acid and other components, and has the effects of resisting fatigue, resisting aging and regulating endocrine. After dissolved in water, the wood frog oil has poor viscosity and fluidity, and is not easy to eat and absorb. The preparation method of the oviductus ranae peptide by hydrolyzing oviductus ranae by an enzymolysis method improves the dissolubility and functionality of oviductus ranae.
At present, the lactobacillus powder is mainly prepared by a vacuum freeze-drying method, the activity of enzyme and the death rate of thalli can be reduced by the vacuum freeze-drying technology, and the moisture in the thalli is fully removed, so that the transportation and the storage of the lactobacillus powder are facilitated. However, it is shown by some studies that ice crystals formed during the freeze-drying process can destroy the cell membrane and phospholipid molecular layer of the thallus cells, the substances in the membrane flow out, and the osmotic pressure inside and outside the membrane is unbalanced, which finally leads to thallus death. The spatial configuration of protein and DNA can be damaged by the change of pressure and temperature in the vacuum freeze drying process, so that the physiological activity of the thalli is reduced after the thalli is rehydrated, and the thalli even die.
The freeze-drying protective agent of the strain can reduce the damage of the freeze-drying process to the thalli, weaken the damage of ice crystals to the thalli by combining with phospholipid bilayers or membrane proteins, and inhibit the formation of the ice crystals by converting the phase transition temperature of components such as membrane lipid and the like. The freeze-drying protective agent suitable for probiotics at present mainly comprises proteins, saccharides, alcohols, amino acids, ascorbic acid and the like, but documents using wood frog egg protein hydrolysate and fungal extract as protective agents cannot be searched at present. The freeze-drying protective agent uses the wood frog egg protein hydrolysate and the hericium erinaceus extract as the freeze-drying protective agent of the lactobacillus reuteri, has a good protective effect on lactobacillus reuteri space mutation strains, and simultaneously improves the stability of the lactobacillus reuteri space mutation strains in the storage process.
Disclosure of Invention
The invention provides a freeze-drying protection method of rana japonica oil enzymolysis liquid on lactobacillus reuteri. The invention can effectively improve the survival rate and the biological activity of the freeze-dried lactobacillus reuteri.
The invention provides a method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri, which mainly comprises the following components: forest frog oil enzymolysis liquid, trehalose and ascorbic acid.
In one embodiment of the present invention, the lactobacillus reuteri lyoprotectant comprises the following components in parts by weight: 20-30% of oviductus ranae enzymolysis liquid, 15-25% of trehalose and 3-5% of ascorbic acid.
The lactobacillus reuteri freeze-drying protective agent preferably comprises the following components: 25% of oviductus ranae enzymolysis liquid, 20% of trehalose and 3% of ascorbic acid.
The second aspect of the invention provides a preparation method of freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri, which comprises the following steps: preparing the oviductus ranae enzymolysis liquid, and weighing the components to prepare the freeze-drying protective agent.
According to the following steps: 20, adding distilled water into the selected rana japonica oil, soaking and swelling for a certain time, homogenizing, adding 0.5 percent of alkaline protease, carrying out enzymolysis for a certain time, inactivating enzyme at 85 ℃ for 20 minutes, carrying out centrifugal separation at 3000r/min20 minutes, adsorbing supernatant liquid by activated carbon (treating by using 2 percent of activated carbon at 40 ℃ for 1 hour), filtering and cooling to obtain the rana japonica oil enzymolysis liquid.
And (3) fully dissolving the weighed oviductus ranae enzymatic hydrolysate, trehalose and ascorbic acid in a phosphate buffer solution with the pH value of 6.5, and then sterilizing by using a microfiltration membrane for later use.
In one embodiment of the present invention, in the step of the lyoprotectant preparation method, the phosphate buffer at pH 6.5 is 4 ℃.
In one embodiment of the present invention, in the step of the lyoprotectant preparation method, the pore size of the microfiltration membrane is 0.2 μm.
In a third aspect, the invention provides a lactobacillus reuteri freeze-drying protective agent as defined in claim 1, and an application of a preparation method of the lactobacillus reuteri freeze-drying protective agent as defined in claim 3 in preparation of lactobacillus reuteri freeze-dried powder.
In one embodiment of the present invention, the preparation of the lactobacillus reuteri lyophilized powder comprises the following steps: activating and culturing lactobacillus reuteri, centrifuging to obtain bacterial sludge, and mixing the bacterial sludge with a freeze-drying protective agent 1: adding a freeze-drying protective agent in a weight-volume ratio of 1-10, uniformly mixing, and carrying out vacuum freeze-drying to obtain the lactobacillus reuteri freeze-dried powder.
Preferably, the mass-volume ratio of the bacterial cells to the protective agent is 1: 8.
the present invention is described in detail below with reference to specific examples, it should be noted that the present invention is exemplary, and it will be apparent to those skilled in the art that the details and modifications of the technical solution of the present invention and the modifications or substitutions may be made without departing from the principle and scope of the present invention, and these modifications and substitutions are also considered to be within the scope of the present invention. Reagents and equipment not specifically described in the present invention are commercially available.
The invention provides a method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri, which mainly comprises the following components:
preparing a protective agent according to the volume number of 8 times of the mass of the bacterial sludge, wherein the wood frog oil enzymolysis liquid is 20-30%, the trehalose is 15-25%, and the ascorbic acid is 3-5%.
The invention provides a freeze-drying protection method of oviductus ranae enzymolysis liquid for lactobacillus reuteri, which mainly comprises the following steps:
according to the following steps: 20, adding distilled water into the selected rana japonica oil, soaking and swelling for a certain time, homogenizing, adding 0.5 percent of alkaline protease, carrying out enzymolysis for a certain time, inactivating enzyme at 85 ℃ for 20 minutes, carrying out centrifugal separation at 3000r/min20 minutes, adsorbing supernatant liquid by activated carbon (treating by using 2 percent of activated carbon at 40 ℃ for 1 hour), filtering and cooling to obtain the rana japonica oil enzymolysis liquid.
And (3) fully dissolving the weighed oviductus ranae enzymatic hydrolysate, trehalose and ascorbic acid in a phosphate buffer solution with the pH value of 6.5, and then sterilizing by using a microfiltration membrane for later use.
And (3) uniformly mixing the centrifuged bacterial sludge with a freeze-drying protective agent according to the mass-volume ratio of 1:8, and preparing the freeze-dried powder of the lactobacillus reuteri.
Detailed Description
Example 1
1. Culture of Lactobacillus reuteri
(1) Frozen Lactobacillus reuteri (Lactobacillus reuteri F-9-35, a laboratory-derived space mutant strain) was taken out from a refrigerator at-80 deg.C, inoculated in 5ml of MRS medium at a ratio of 2%, and cultured in a thermostat at 37 deg.C for 12 h.
(2) The activated Lactobacillus reuteri F-9-35 plate is streaked and cultured in a thermostat at 37 ℃ for 20 h.
(3) The single colony after streaking is selected and inoculated in 5ml of MRS medium and cultured in a 37 ℃ incubator for 12 h.
(4) Inoculating the bacterial liquid in the step (3) into 100ml of MRS medium according to the proportion of 3%, and culturing for 10h at 37 ℃ in an incubator.
(5) Inoculating the bacterial liquid in the step (4) into 900ml of MRS medium according to the proportion of 3 percent, and culturing for 10 hours at 37 ℃ in an incubator.
2. Preparation of lactobacillus reuteri protective agent
Dissolving trehalose 15g, ascorbic acid 3g, and oviductus Ranae hydrolysate 20ml in phosphate buffer solution with pH of 6.5 at 4 deg.C 100ml, and sterilizing with 0.2 μm microfiltration membrane.
3. Mixing thallus with protectant
(1) And (3) uniformly distributing 900ml of the bacterial liquid in the step (1) in 6 centrifugal bottles, and adjusting parameters: the rotation speed was 6000g, the time was 10min and the temperature was 4 ℃.
(2) Pouring out the culture medium after centrifugation, adding a protective agent containing 15% trehalose, 3% ascorbic acid and 20% oviductus Ranae enzymolysis liquid according to the volume number of 8 times of the thallus mass, mixing uniformly to obtain a bacterial suspension, sucking a certain amount of bacterial liquid, performing gradient dilution by using normal saline, and selecting 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates0。
3. Vacuum freeze drying
Sucking 1ml of bacterial suspension into a freeze-drying bottle with the thickness of about 0.8cm, pre-freezing the freeze-drying bottle at-80 ℃ for 2h, then putting the freeze-drying bottle into a freeze-drying machine for vacuum freeze-drying for 12h, adding physiological saline with the same volume as that before freeze-drying into the freeze-dried bacterial powder for redissolution, sucking a certain amount of bacterial liquid, and using the physiological saline for carrying outGradient dilution, selection 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates1。
4. Freeze-drying results
Survival rate of freeze drying0/N1(N0And N1All units are cfu/ml)
Number of bacteria before lyophilization N0＝1.71*1010Number of lyophilized bacteria N1＝8.8*109The survival rate of lactobacillus reuteri is 51%.
Example 2
1. Culture of Lactobacillus reuteri
(1) Frozen Lactobacillus reuteri (Lactobacillus reuteri F-9-35, a laboratory-derived space mutant strain) was taken out from a refrigerator at-80 deg.C, inoculated in 5ml of MRS medium at a ratio of 2%, and cultured in a thermostat at 37 deg.C for 12 h.
(2) The activated Lactobacillus reuteri F-9-35 plate is streaked and cultured in a thermostat at 37 ℃ for 20 h.
(3) The single colony after streaking is selected and inoculated in 5ml of MRS medium and cultured in a 37 ℃ incubator for 12 h.
(4) Inoculating the bacterial liquid in the step (3) into 100ml of MRS medium according to the proportion of 3%, and culturing for 10h at 37 ℃ in an incubator.
(5) Inoculating the bacterial liquid in the step (4) into 900ml of MRS medium according to the proportion of 3 percent, and culturing for 10 hours at 37 ℃ in an incubator.
2. Preparation of lactobacillus reuteri compound protective agent
Dissolving 20g trehalose, 3g ascorbic acid, and 25ml oviductus Ranae enzymolysis solution in 100ml phosphate buffer solution with pH of 6.5 at 4 deg.C, and filtering with 0.2 μm microfiltration membrane for sterilization.
3. Mixing thallus with protectant
(1) And (3) uniformly distributing 900ml of the bacterial liquid in the step (1) in 6 centrifugal bottles, and adjusting parameters: the rotation speed was 6000g, the time was 10min and the temperature was 4 ℃.
(2) Pouring out the culture medium after centrifugation, and taking the culture medium as 8 times of the thallus massAdding protectant containing 20% trehalose, 3% ascorbic acid and 25% oviductus Ranae enzymolysis solution, mixing well to obtain bacterial suspension, sucking a certain amount of bacterial liquid, performing gradient dilution with normal saline, and selecting 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates0。
3. Vacuum freeze drying
Sucking 1ml of bacterial suspension into a freeze-drying bottle with the thickness of about 0.8cm, pre-freezing the freeze-drying bottle at-80 ℃ for 2h, then putting the freeze-drying bottle into a freeze-drying machine for vacuum freeze-drying for 12h, adding physiological saline with the same volume as that before freeze-drying into the freeze-dried bacterial powder for redissolving, sucking a certain amount of bacterial liquid, performing gradient dilution by using the physiological saline, and selecting 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates1。
4. Freeze-drying results
Survival rate of freeze drying0/N1(N0And N1All units are cfu/ml)
Number of bacteria before lyophilization N0＝1.9*1010Number of lyophilized bacteria N1＝1.2*1010The survival rate of lactobacillus reuteri is 63.2%.
Example 3
1. Culture of Lactobacillus reuteri
(1) Frozen Lactobacillus reuteri (Lactobacillus reuteri F-9-35, a laboratory-derived space mutant strain) was taken out from a refrigerator at-80 deg.C, inoculated in 5ml of MRS medium at a ratio of 2%, and cultured in a thermostat at 37 deg.C for 12 h.
(2) The activated Lactobacillus reuteri F-9-35 plate is streaked and cultured in a thermostat at 37 ℃ for 20 h.
(3) The single colony after streaking is selected and inoculated in 5ml of MRS medium and cultured in a 37 ℃ incubator for 12 h.
(4) Inoculating the bacterial liquid in the step (3) into 100ml of MRS medium according to the proportion of 3%, and culturing for 10h at 37 ℃ in an incubator.
(5) Inoculating the bacterial liquid in the step (4) into 900ml of MRS medium according to the proportion of 3 percent, and culturing for 10 hours at 37 ℃ in an incubator.
2. Preparation of lactobacillus reuteri compound protective agent
Dissolving trehalose 25g, ascorbic acid 4g, and oviductus Ranae hydrolysate 20ml in phosphate buffer solution with pH of 6.5 at 4 deg.C 100ml, and sterilizing with 0.2 μm microfiltration membrane.
3. Mixing thallus with protectant
(1) And (3) uniformly distributing 900ml of the bacterial liquid in the step (1) in 6 centrifugal bottles, and adjusting parameters: the rotation speed was 6000g, the time was 10min and the temperature was 4 ℃.
(2) Pouring out the culture medium after centrifugation, adding protective agent containing 25% trehalose, 4% ascorbic acid and 20% oviductus Ranae enzymolysis solution according to volume number 8 times of thallus mass, mixing well to obtain bacterial suspension, sucking a certain amount of bacterial solution, performing gradient dilution with normal saline, selecting 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates0。
3. Vacuum freeze drying
Sucking 1ml of bacterial suspension into a freeze-drying bottle with the thickness of about 0.8cm, pre-freezing the freeze-drying bottle at-80 ℃ for 2h, then putting the freeze-drying bottle into a freeze-drying machine for vacuum freeze-drying for 12h, adding physiological saline with the same volume as that before freeze-drying into the freeze-dried bacterial powder for redissolving, sucking a certain amount of bacterial liquid, performing gradient dilution by using the physiological saline, and selecting 10-6、10-7、10-8The number of bacteria was counted by streaking three dilution gradient coated plates, and the number of bacteria counted as the initial number of bacteria N was counted for each gradient of three plates1。
4. Freeze-drying results
Survival rate of freeze drying0/N1(N0And N1All units are cfu/ml)
Number of bacteria before lyophilization N0＝1.9*1010Number of lyophilized bacteria N1＝1.2*1010The survival rate of lactobacillus reuteri is 63.2%.
Claims (5)
1. A method for freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri is characterized by comprising the following components: the wood frog oil enzymolysis liquid comprises 1: 20, adding distilled water into the wood frog oil, soaking and swelling for a certain time, homogenizing, adding 0.5 percent of alkaline protease, performing enzymolysis for a certain time, inactivating enzyme at 85 ℃ for 20 minutes, performing centrifugal separation at 3000r/min20 minutes, performing adsorption treatment on supernatant for 1 hour by 2 percent of activated carbon at 40 ℃, filtering and cooling to obtain the wood frog oil.
2. The method for freeze-drying protection of lactobacillus reuteri by the rana japonica oil enzymolysis liquid as claimed in claim 1, which comprises the following steps: 20-30% of oviductus ranae enzymolysis liquid, 15-25% of trehalose and 3-5% of ascorbic acid.
3. A preparation method of freeze-drying protection of rana japonica oil enzymolysis liquid on lactobacillus reuteri is characterized by comprising the following steps:
(1) mixing the raw materials in a ratio of 1: 20, adding distilled water into the oviductus ranae, soaking and swelling for a certain time, homogenizing, adding 0.5 percent of alkaline protease, performing enzymolysis for a certain time, inactivating the enzyme at 85 ℃ for 20 minutes, performing centrifugal separation at 3000r/min20 minutes, performing adsorption treatment on supernatant at 40 ℃ for 2 percent of activated carbon for 1 hour, filtering and cooling to obtain oviductus ranae enzymatic hydrolysate;
(2) preparing a freeze-drying protective agent according to a proportion, fully dissolving weighed oviductus ranae enzymolysis liquid, trehalose and ascorbic acid in a phosphate buffer solution with the pH =6.5, and then using a microfiltration membrane for sterilization for later use, wherein the pore diameter of the microfiltration membrane is 0.2 mu m.
4. The use of the method for protecting lactobacillus reuteri from the oviductus ranae enzymatic hydrolysate as claimed in claim 1 or the method for preparing lactobacillus reuteri freeze-drying protection from the oviductus ranae enzymatic hydrolysate as claimed in claim 3 in the preparation of lactobacillus reuteri freeze-dried powder.
5. The use according to claim 4, comprising the steps of: activating and culturing lactobacillus reuteri, centrifuging to obtain bacterial sludge, and mixing the bacterial sludge with a freeze-drying protective agent 1: adding a freeze-drying protective agent in a weight-volume ratio of 1-10, uniformly mixing, and carrying out vacuum freeze-drying to obtain the lactobacillus reuteri freeze-dried powder.
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