CN113234597B - Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof - Google Patents

Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof Download PDF

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CN113234597B
CN113234597B CN202110594646.7A CN202110594646A CN113234597B CN 113234597 B CN113234597 B CN 113234597B CN 202110594646 A CN202110594646 A CN 202110594646A CN 113234597 B CN113234597 B CN 113234597B
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崔树茂
高欣伟
唐鑫
毛丙永
赵建新
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Jiangnan University
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Abstract

The invention discloses a culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof, belonging to the technical field of microorganisms and the technical field of fermentation and freeze-drying. The invention provides a method for fermenting bifidobacteria at high density by adding compatible solute into a culture medium, thereby improving the freeze-drying survival rate of the bifidobacteria, inoculating the bifidobacteria into the culture medium containing the compatible solute for high-density culture to a stable period, and being capable of obviously improving the freeze-drying stress resistance of the bifidobacteria, so that the freeze-drying survival rate of the bifidobacteria is improved by 3.02 times (under the action of the same protective agent); the culture method can obviously improve the freeze-drying survival rate of the bifidobacteria and improve the freeze-drying survival rate of the bifidobacteria from 10-20% to 40-60%.

Description

Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof
Technical Field
The invention relates to a culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof, belonging to the technical field of microorganisms and the technical field of fermentation and freeze-drying.
Background
Bifidobacterium (Bifidobacterium longum) is a gram-positive and catalase-negative rod-shaped bacterium existing in human gastrointestinal tract, and comprises 32 species such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium bifidum. Bifidobacteria are a microaerotolerant anaerobic bacterium believed to be located primarily in the gastrointestinal tract of infants. When grown in a solid medium in an anaerobic environment, bifidobacteria typically form white, smooth, convex colonies. Although bifidobacteria are present in relatively small amounts in the adult gastrointestinal tract, they are thought to be part of the gut microbiota, which is widely distributed in the human gut and whose production of lactic acid is thought to prevent the growth of pathogenic microorganisms. Researches show that the bifidobacterium has the beneficial effects of improving intestinal microbiota, regulating intestinal permeability, improving intestinal immunity, relieving Irritable Bowel Syndrome (IBS), resisting intestinal inflammation and the like. In addition, bifidobacteria are non-pathogenic and are often added to food products.
With the increasingly intensive study and wide application of bifidobacteria on the health promotion effect, researchers are required to adopt a proper storage method. The industrial spread of bifidobacteria as probiotics depends to a large extent on preservation techniques, which require the viability and viability of long-term stable cultures. Therefore, the bifidobacterium storage method requires two goals: both long-term storage of the pure culture and ready application of the pure culture. At present, freeze-drying preservation is the first choice long-term preservation method for bacteria culture for decades and is also a long-term preservation method for bifidobacteria because of low maintenance cost and easy transplantation of freeze-dried cultures. There are many microbial resource centers that rely on freeze-drying to preserve various cells for future propagation.
The bifidobacterium is stressed by low temperature, mechanical damage, osmotic pressure damage and the like in the freeze drying process, so that the conditions of cell membrane permeability change, protein denaturation inactivation, pH dynamic balance damage and the like are caused, and the viable count of the bifidobacterium strain is obviously reduced. Factors affecting the freeze-drying stress resistance of bifidobacteria include the selection of freeze-drying protective agents and the freeze-drying stress resistance of the bifidobacteria themselves. At present, although much research focuses on optimizing the freeze-drying protective agent of the strain, the freeze-drying stress resistance of the strain is required to be improved by controlling the fermentation conditions.
Disclosure of Invention
In order to solve the problems that the method for improving the freeze-drying stress resistance of the strain in the prior art is single, the effect is not obvious, the freeze-drying stress resistance of the bifidobacterium is improved by controlling the fermentation condition and the like, the invention is based on the following principle: high osmotic stress reduces the growth rate, survival rate and metabolic activity of lactic acid bacteria. Theoretically, cells need to maintain a higher intracellular osmotic pressure than the osmotic pressure of the culture medium to produce cellular engorgement, which is the driving force for cell elongation and growth. Lactic acid bacteria can accumulate compatible substances during hypertonic stress to maintain the turgor pressure required for cell growth. Compatible solutes are primarily small organic molecules that are polar, soluble, uncharged under physiological conditions, and can accumulate in high concentrations (1mol/kg) in the cell without affecting their function and protein folding. The hypertonic condition induces the cells to accumulate compatible solutes in the cells, effectively relieves the ice crystal damage and osmotic pressure damage in the cells and maintains the structures of cell membranes and proteins in the freeze drying process, thereby improving the freeze-drying survival rate of the bifidobacteria. The invention provides a culture medium which is added with compatible solute in an initial culture medium and is used for high-density culture to a stable period, the bifidobacteria are in a relative hypertonic condition in the middle and later periods of high-density fermentation, and the bifidobacteria accumulate the compatible solute in cells under high deep stress, thereby improving the freeze-drying survival rate of the bifidobacteria.
The invention also provides a preparation method of the freeze-dried bifidobacterium powder with improved freeze-drying survival rate, which comprises the following steps:
(1) inoculating bifidobacterium into a fermentation medium containing compatible solutes for fermentation, wherein the compatible solutes comprise one or more of amino acid, quaternary ammonium salt, sugar and sugar alcohol; after fermentation, centrifugally collecting bacteria to obtain thalli;
(2) and (2) washing the thalli prepared in the step (1) by adopting a solution with osmotic pressure of 550-1100 mOsm/kg, mixing the thalli with a protective agent, and freeze-drying to prepare bifidobacterium powder.
In one embodiment of the present invention, the bifidobacterium is a seed liquid of activated bifidobacterium.
In one embodiment of the present invention, the solution having an osmotic pressure of 550 to 1100mOsm/kg is a solution in a state close to the osmotic pressure of Bifidobacterium, and the NaCl solution has a concentration of 16.5 to 33 g/L.
In one embodiment of the invention, the compatible solutes comprise: one or more of glutamic acid, arginine, glycine, proline, cysteine, glycine betaine, trehalose, lactose, stachyose, arabinose, sorbitol, mannitol, inositol.
In one embodiment of the invention, the compatible solute is one or more of glycine, proline, cysteine.
In one embodiment of the invention, the bifidobacterium comprises: bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium animalis.
In one embodiment of the invention, the end of fermentation refers to the cultivation to stationary phase.
In one embodiment of the present invention, in the step (2), when the Bifidobacterium is Bifidobacterium adolescentis, the osmotic pressure is adjusted to 750 to 950mOsm/kg (NaCl solution concentration: 22.5 to 28.5 g/L); when the bifidobacterium is bifidobacterium bifidum, the osmotic pressure is adjusted to 550-850 mOsm/kg (the concentration of NaCl solution is 16.5-25.5 g/L); when the Bifidobacterium is Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium animalis, the osmotic pressure is adjusted to 900-1000 mOsm/kg (NaCl solution concentration: 27-30 g/L).
In one embodiment of the invention, the bifidobacterium longum includes, but is not limited to: bifidobacterium longum subsp. infantus CCFM 687, Bifidobacterium longum subsp. longum CCFM1102, Bifidobacterium longum subsp. longum CCFM 1077.
In one embodiment of the invention, the bifidobacterium breve includes, but is not limited to: the Bifidobacterium breve CCFM1026, the Bifidobacterium breve CCFM1067 and the Bifidobacterium breve CCFM 1078.
In one embodiment of the present invention, said bifidobacterium adolescentis includes, but is not limited to: bifidobacterium adolescents CCFM 1108, Bifidobacterium adolescents CCFM 1062, and Bifidobacterium adolescents CCFM 1061.
In one embodiment of the invention, said bifidobacterium bifidum includes, but is not limited to: bifidobacterium duplex CCFM1063, Bifidobacterium duplex CGMCC NO.13632, Bifidobacterium duplex HNJ6, Bifidobacterium duplex CCFM 1150.
In one embodiment of the invention, the bifidobacterium animalis includes, but is not limited to: bifidobacterium animalis CCFM1148, Bifidobacterium animalis BB 12.
In one embodiment of the invention, the fermentation medium comprises: 1-50 g/L of nitrogen source, 1-100 g/L of carbon source, 0.1-30 g/L of buffer salt, 0.01-0.30 g/L of magnesium sulfate heptahydrate, 0.01-0.1 g/L of manganese sulfate monohydrate, 800.1-2 mL/L of tween-800, 0.1-2 g/L of cysteine and 10-30g/L of compatible solute.
In one embodiment of the present invention, the carbon source comprises: glucose, lactose, sucrose, oligosaccharide or a plurality of saccharides.
In one embodiment of the present invention, the nitrogen source comprises: yeast extract, beef extract, fish peptone, soybean peptone, tryptone, and whey protein powder.
In one embodiment of the invention, the buffer salt is: one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate or sodium phosphate.
In one embodiment of the present invention, in step (1), a culture solution of bifidobacterium is inoculated into a fermentation medium containing a compatible solute for fermentation; the preparation method of the bifidobacterium culture solution comprises the following steps: selecting a bifidobacterium single colony, inoculating the bifidobacterium single colony in an MRS liquid culture medium, and performing activated culture for 18-24 hours under an anaerobic condition at 37 ℃ to obtain an activated solution; inoculating the activated liquid to an MRS liquid culture medium in an inoculation amount of 1-5% (v/v), and continuing activated culture for 18-24 h to obtain a bifidobacterium seed liquid; and (3) inoculating the prepared seed solution into a triangular flask filled with an MRS liquid culture medium according to the inoculation amount of 1-5% (v/v), and performing anaerobic culture at 37 ℃ for 24 hours to obtain a culture solution.
In one embodiment of the invention, the prepared bifidobacterium is cultured and inoculated into a fermentation medium containing compatible solute in an inoculation amount of 1-5% (v/v), the culture temperature is 30-40 ℃, the pH is 5.0-6.0, and the bifidobacterium is cultured for 14-24 hours to obtain a fermentation liquor; and (4) centrifugally collecting the prepared fermentation liquor to prepare thalli.
In one embodiment of the present invention, the cell and the protective agent are mixed in a ratio of 1: 1.
In one embodiment of the invention, the prepared bifidobacterium seed solution is inoculated into a fermentation medium containing compatible solutes in an inoculation amount of 1-5% (v/v), the culture temperature is 37 ℃, the pH is 5.0-7.0, and the fermentation solution is obtained after 20 hours of culture.
In one embodiment of the invention, the protectant is a 20% sorbitol solution, trehalose or sucrose.
In one embodiment of the invention, the fermentation medium is: glucose 60g/L, nitrogen source 24g/L, MgSO 4 ·7H 2 O0.35g/L, Tween-801 mL, cysteine 1g/L and compatible solute 20 g/L.
In one embodiment of the invention, the compatible solute is proline.
In one embodiment of the invention, the fermentation medium is: 10-50 g/L of nitrogen source, 20-100 g/L of anhydrous glucose, 0.1-30 g/L of buffer salt, 0.10-0.30 g/L of magnesium sulfate heptahydrate, 0.01-0.1 g/L of manganese sulfate monohydrate, 0.5-2 g/L of tween-800.5.
In one embodiment of the invention, the fermentation medium is: 24g/L of yeast extract powder, 60g/L of anhydrous glucose, 0.35g/L of magnesium sulfate heptahydrate, 801 mL/L of tween-801, 1g/L of cysteine and 20g/L of proline.
The invention also provides a method for improving the freeze-drying survival rate of bifidobacterium, which is characterized by comprising the following steps: inoculating bifidobacterium into a fermentation medium containing compatible solutes for fermentation, wherein the compatible solutes comprise one or more of amino acid, quaternary ammonium salt, sugar and sugar alcohol; after fermentation, centrifugally collecting bacteria to obtain thalli; and washing the thalli by adopting a solution with the osmotic pressure of 550-1100 mOsm/kg, mixing the thalli with a protective agent, and freeze-drying.
In one embodiment of the invention, the compatible solutes comprise: one or more of glutamic acid, arginine, glycine, proline, cysteine, glycine betaine, trehalose, lactose, stachyose, arabinose, sorbitol, mannitol, inositol.
In one embodiment of the present invention, the cell and the protective agent are mixed in a ratio of 1: 1.
In one embodiment of the invention, the protective agent is trehalose, sucrose or sorbitol.
In one embodiment of the invention, the fermentation medium comprises: 1-50 g/L of nitrogen source, 1-100 g/L of carbon source, 0.1-30 g/L of buffer salt, 0.01-0.30 g/L of magnesium sulfate heptahydrate, 0.01-0.1 g/L of manganese sulfate monohydrate, 800.1-2 mL/L of tween-800, 0.1-2 g/L of cysteine and 10-30g/L of compatible solute.
The invention also provides the bifidobacterium freeze-dried powder prepared by the method.
Advantageous effects
(1) The invention provides a method for high-density fermentation of bifidobacteria by adding compatible solutes into a culture medium, thereby improving the freeze-drying and freeze-drying survival rate of the bifidobacteria. Bifidobacteria were inoculated into media containing compatible solutes for constant pH culture to stationary phase, ensuring that the final fermentation broth was stopped due to hypertonic stress (rich in carbon and nitrogen sources and appropriate temperature and pH). Centrifuging to collect and wash the bacteria twice, mixing with a freeze-drying protective agent, and freeze-drying. Compared with the blank group, the freeze-drying survival rate of the bifidobacterium in the experimental group is obviously improved.
(2) The invention provides a culture strategy capable of improving the freeze-drying survival rate of bifidobacteria by adding proline, which is characterized in that a bifidobacteria seed solution is inoculated into a culture medium with 10-30g/L of proline to carry out anaerobic constant-temperature (37 ℃) and constant-pH (5.0) culture, and the culture is fermented until bacteria are harvested in a stable period. The culture strategy can obviously improve the freeze-drying survival rate of the bifidobacteria, and can obviously improve the freeze-drying survival rate of the bifidobacteria and improve the freeze-drying survival rate of the bifidobacteria from 10-20% to 40-60%.
Detailed Description
The invention is further illustrated with reference to specific examples. The information on the strains referred to in the following examples is as follows:
said Bifidobacterium longum subsp. infantis CCFM 687 is disclosed in patent application text with publication number CN 110693919A; the Bifidobacterium longum subsp. longum CCFM1102 is described in patent application text with publication number CN 111073834A; said Bifidobacterium longum subsp. longum CCFM 1077 is described in the patent application text with publication number CN 111073828A.
The Bifidobacterium breve CCFM1026 is described in patent text with publication number CN 109576185B; the Bifidobacterium breve CCFM1067 is described in the patent application with publication number CN 110616167A; the Bifidobacterium breve CCFM1078 is described in the patent application publication No. CN 112111424A;
the Bifidobacterium adolescentis CCFM 1108 is described in patent application publication No. CN 111534453A; the Bifidobacterium adolescentis CCFM 1062 is described in the patent application with publication number CN 110432332A; the Bifidobacterium adolescentis CCFM1061 is described in patent document with publication number CN 110331118B;
the Bifidobacterium bifidum CCFM1063 is described in the patent application publication No. CN 110331119A; the Bifidobacterium bifidum CGMCC NO.13632 is described in the patent text with the publication number CN 106834187B; the Bifidobacterium bifidum HNJ6 is described in patent application publication No. CN 107629988A; the Bifidobacterium bifidum CCFM1150 is described in the patent application with publication number CN 112694992A;
the Bifidobacterium animalis CCFM1148 is described in the patent document with the publication number CN 112159778A; the Bifidobacterium animalis BB12 is described in the patent publication CN 107893044A.
Angel yeast FM803, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween 80, cysteine hydrochloride, peptone, beef extract, glucose, sodium acetate, diammonium hydrogen citrate, agar powder, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, agar powder, glutamic acid, arginine, glycine, proline, glycine betaine, trehalose, lactose, stachyose, arabinose, sorbitol, mannitol, inositol, which are mentioned in the examples below, were purchased from Shanghai Chuisashi technologies, Ltd.
The media involved in the following examples are as follows:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 0.05g/L of O, 801 mL/L of Tween, 15g/L of agar powder and 0.5g/L of cysteine hydrochloride.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O0.05 g/L, Tween 801 mL/L and cysteine hydrochloride 0.5 g/L.
The detection methods referred to in the following examples are as follows:
and (3) measuring the viable count of the lactic acid bacteria:
the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
And (3) determining the freeze-drying survival rate of the lactic acid bacteria:
the freeze-drying survival rate of the bifidobacteria was calculated according to the following formula:
Figure BDA0003090682390000061
example 1: fermentation and freeze-drying process of bifidobacterium
The method comprises the following specific steps:
(1) preparation of bifidobacterium seed liquid:
marking the preserved bifidobacteria on an MRS solid culture medium, and culturing at the constant temperature of 37 ℃ for 36h to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at a constant temperature of 37 ℃ for 24 hours to obtain a seed solution;
(2) inoculating the prepared seed solution into a triangular flask filled with an MRS liquid culture medium in an inoculation amount of 5% (v/v), and performing anaerobic culture at 37 ℃ for 24h to obtain a culture solution;
(3) preparing fermentation medium containing compatible solute
Yeast extract powder FM 80324 g/L, anhydrous glucose 60g/L, Tween 801 mL/L, cysteine hydrochloride 1g/L, MgSO 4 ·7H 2 0.35g/L of O and 10-30g/L of compatible solute; placing the fermentation medium in 5L fermentation tank, sterilizing at 115 deg.C for 15min, introducing nitrogen, maintaining pressure of 0.1Mpa, and cooling;
(4) inoculating the culture solution prepared in step (2) into the fermentation medium of step (3) according to the inoculation amount of 5% (v/v), culturing at constant temperature of 37 ℃ at pH 5.0, sampling every 2h, and determining OD 600 To stationary phase, at which time OD 600 15-20, and obtaining fermentation liquor after fermentation is finished; centrifuging the fermentation liquor, and collecting thalli;
(5) after the cells were washed twice with a solution (NaCl solution) having an osmotic pressure of 550 to 1100mOsm/kg, the cells were mixed with sorbitol as a protective agent in a ratio of 1:1 is resuspended in a sorbitol solution with the concentration of 200g/L to obtain a resuspension solution; when the Bifidobacterium is Bifidobacterium adolescentis, the osmotic pressure is adjusted to 750-950 mOsm/kg (the concentration of NaCl solution is 22.5-28.5 g/L);
when the Bifidobacterium is Bifidobacterium bifidum, the osmotic pressure is adjusted to 550-850 mOsm/kg (the concentration of NaCl solution is 16.5-25.5 g/L);
when the Bifidobacterium is Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium animalis, the osmotic pressure is adjusted to 900-1000 mOsm/kg (NaCl solution concentration: 27-30 g/L).
(6) And (3) freeze-drying the heavy suspension, wherein the freeze-drying process comprises the following steps: placing the heavy suspension in an environment with the temperature of 50 ℃ below zero for 4 hours; primary drying at-30 deg.C under 200 μ bar for 30 hr; drying for the second time at 25 deg.C under 0 μ bar for 20 hr; preparing bifidobacterium powder.
Example 2: fermentation and freeze-drying of Bifidobacterium longum subsp
The method comprises the following specific steps:
the first scheme is as follows: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to 20g/L of anhydrous glucose to prepare the Bifidobacterium longum subsp.
Scheme II: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L glutamic acid, so as to prepare the Bifidobacterium longum subsp.
The third scheme is as follows: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L arginine, so as to prepare the Bifidobacterium longum subsp.
And the scheme is as follows: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L glycine, so as to prepare the Bifidobacterium longum subsp.
And a fifth scheme: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to 20g/L proline, so as to prepare the Bifidobacterium longum subsp.
Scheme six: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to be glycine betaine of 20g/L to prepare the Bifidobacterium longum subsp.
The scheme is seven: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L of trehalose to prepare the Bifidobacterium longum subsp.
And the eighth scheme is as follows: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L lactose to prepare the Bifidobacterium longum subsp.
The scheme is nine: the specific implementation manner is the same as that of example 1, except that stachyose with compatible solute adjusted to 20g/L in step (3) is prepared to obtain Bifidobacterium longum subsp.
And a scheme ten: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to 20g/L of arabinose, so as to prepare the Bifidobacterium longum subsp.
Scheme eleven: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L of sorbitol to prepare Bifidobacterium longum subsp.
Scheme twelve: the specific implementation manner is the same as that of example 1, except that the compatible solute in the step (3) is adjusted to 20g/L mannitol to prepare the Bifidobacterium longum subsp.
Scheme thirteen: the specific implementation manner is the same as example 1, except that the compatible solute in step (3) is adjusted to 20g/L inositol to prepare Bifidobacterium longum subsp.
A fourteen scheme: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to 20g/L raffinose to prepare Bifidobacterium longum subsp.
A fifteenth scheme: the specific implementation manner is the same as that in example 1, except that the compatible solute in the step (3) is adjusted to 20g/L erythritol, so that Bifidobacterium longum subsp. The results are shown in table 1:
table 1: bifidobacterium longum subsp. infantis CCFM 687 lyophilized results were fermented using the method of this example
Figure BDA0003090682390000081
Figure BDA0003090682390000091
The results show that the glucose content of the fermentation liquor is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the effect of adding proline is most obvious, and the number of live bacteria after freeze-drying is 2.17 +/-0.3 multiplied by 10 11 The CFU/g is increased to 10.36 +/-0.33 multiplied by 10 11 The freeze-drying survival rate of CFU/g is improved from 22.18 +/-0.26% to 98.88 +/-0.17%, and the freeze-drying survival rate is improved by about 4.68 times.
Example 3: fermentation and freeze-drying of Bifidobacterium longum subsp
On the basis of example 2, the Bifidobacterium longum subsp. infarnatum CCFM 687 of example 2 was replaced by Bifidobacterium longum subsp. longum CCFM1102, and the experimental results are shown in table 2.
Table 2: bifidobacterium longum subsp. longum CCFM1102 Freeze-drying results were fermented using the method of this example
Figure BDA0003090682390000092
Figure BDA0003090682390000101
The results show that the glucose content of the fermentation liquor is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with a blank group, the number of viable bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, and the improvement effect of arabinose addition is smaller; the effect of adding proline is most obvious, and the number of viable bacteria after freeze-drying is 2.42 +/-0.47 multiplied by 10 11 CFU/g is increased to 10.28 +/-0.35X 10 11 CFU/g, the freeze-drying survival rate is improved from 26.6 +/-0.41% to 99.43 +/-0.37%, and the freeze-drying survival rate is improved by about 3.89 times.
Example 4: fermentation and lyophilization of Bifidobacterium longum subsp
On the basis of example 2, the Bifidobacterium longum subsp. infarnatum CCFM 687 of example 2 was replaced by Bifidobacterium longum subsp. longum CCFM 1077, and the experimental results are shown in table 3.
TABLE 3 Bifidobacterium longum subsp. longum CCFM 1077 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000102
The results show that the glucose content of the fermentation broth is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compareThe number of live bacteria and the freeze-drying survival rate of the experiment group after freeze-drying are obviously improved in the blank group, wherein the number of live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of live bacteria after freeze-drying is 3.09 +/-0.31 multiplied by 10 11 The CFU/g is improved to 9.71 +/-0.18 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 31.39 +/-0.19% to 97.93 +/-0.06%, and the freeze-drying survival rate is improved by about 3.12 times.
Example 5: fermentation and freeze-drying of Bifidobacterium breve CCFM1026
On the basis of example 2, the Bifidobacterium longum subsp. infarnationis CCFM 687 of example 2 was replaced with Bifidobacterium breve CCFM1026, and the results are shown in Table 4.
TABLE 4 Bifidobacterium breve CCFM1026 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000111
The results show that the glucose content of the fermentation broth is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 3.78 +/-0.3 multiplied by 10 11 The CFU/g is improved to 9.12 +/-0.44 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 41.47 +/-0.3% to 91.21 +/-0.33%, and the freeze-drying survival rate is improved by about 2.20 times.
Example 6: fermentation and freeze-drying of Bifidobacterium breve CCFM1067
On the basis of example 2, the Bifidobacterium longum subsp. infarnatis CCFM 687 of example 2 was replaced by Bifidobacterium breve CCFM1067, and the experimental results are shown in table 5.
TABLE 5 Bifidobacterium breve CCFM1067 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000121
The results show that the glucose content of the fermentation broth is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.84 +/-0.23 multiplied by 10 11 The CFU/g is improved to 9.68 +/-0.28 multiplied by 10 11 The freeze-drying survival rate is improved from 30.67 +/-0.49% to 97.77 +/-0.28% by CFU/g, and the freeze-drying survival rate is improved by about 3.19 times.
Example 7: fermentation and freeze-drying of Bifidobacterium breve CCFM1078
On the basis of example 2, the Bifidobacterium longum subsp. infarnatis CCFM 687 of example 2 was replaced by Bifidobacterium breve CCFM1078, and the experimental results are shown in table 6.
TABLE 6 Bifidobacterium breve CCFM1078 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000122
Figure BDA0003090682390000131
The results show that the glucose content of the fermentation liquor is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 3.88 +/-0.22 multiplied by 10 11 The CFU/g is improved to 9.69 +/-0.33 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 39.4 +/-0.24% to 97.41 +/-0.15%, and the freeze-drying survival rate is improved by about 2.90 times.
Example 8: fermentation and lyophilization of Bifidobacterium adolescentis CCFM 1108
Based on example 2, the Bifidobacterium longum subsp. inside infection CCFM 687 of example 2 was replaced with Bifidobacterium adolescents CCFM 1108, and the osmotic pressure was adjusted to 750 to 950mOsm/kg (NaCl solution concentration: 22.5 to 28.5g/L), and the results of the experiment are shown in Table 7.
TABLE 7 Bifidobacterium adolescentis CCFM 1108 Freeze-drying results of fermentation using the method of this example
Figure BDA0003090682390000132
Figure BDA0003090682390000141
The results show that the glucose content of the fermentation liquor is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.71 +/-0.19 multiplied by 10 11 The CFU/g is improved to 9.59 +/-0.45 multiplied by 10 11 The freeze-drying survival rate is improved from 27.84 +/-0.37% to 96.87 +/-0.61% by CFU/g, and the freeze-drying survival rate is improved by about 3.48 times.
Example 9: fermentation and freeze-drying of Bifidobacterium adolescentis CCFM 1062
In addition to example 2, the Bifidobacterium longum subsp. multifacies CCFM 687 of example 2 was replaced with Bifidobacterium adolescentis CCFM 1062, and the osmotic pressure was adjusted to 750 to 950mOsm/kg (NaCl solution concentration: 22.5 to 28.5g/L), and the results of the experiments are shown in Table 8.
Table 8: bifidobacterium adolescentis CCFM 1062 fermentation and freeze-drying results by the method of this example
Figure BDA0003090682390000142
Figure BDA0003090682390000151
The results show that the glucose content of the fermentation liquor is more than or equal to 18.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 3.41 +/-0.24 multiplied by 10 11 CFU/g is increased to 10.65 +/-0.45 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 37.27 +/-0.07% to 99.52 +/-0.27%, and the freeze-drying survival rate is improved by about 2.85 times.
Example 10: fermentation and freeze-drying of Bifidobacterium adolescentis CCFM1061
In addition to example 2, the Bifidobacterium longum subsp. multifacies CCFM 687 of example 2 was replaced with Bifidobacterium adolescentis CCFM1061, and the osmotic pressure was adjusted to 750 to 950mOsm/kg (NaCl solution concentration: 22.5 to 28.5g/L), and the results of the experiments are shown in Table 9.
TABLE 9 Bifidobacterium adolescentis CCFM1061 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000152
The results show that the glucose content of the fermentation liquor is more than or equal to 18.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.81 +/-0.29 multiplied by 10 11 CFU/g is increased to 9.91 +/-0.37 multiplied by 10 11 CFU/g，The freeze-drying survival rate is improved from 31.07 +/-0.56% to 99.22 +/-0.33%, and the freeze-drying survival rate is improved by about 3.19 times.
Example 11: fermentation and freeze-drying of Bifidobacterium bifidum CCFM1063
Based on example 2, the Bifidobacterium longum subsp. infantis CCFM 687 of example 2 was replaced with Bifidobacterium bifidum CCFM1063, and the osmotic pressure was adjusted to 550 to 850mOsm/kg (NaCl solution concentration: 16.5 to 25.5g/L), and the results of the experiment are shown in Table 10.
TABLE 10 Bifidobacterium bifidum CCFM1063 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000161
The results show that the glucose content of the fermentation broth is more than or equal to 18.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the freeze-dried viable count and the freeze-dried survival rate of the experimental group are both obviously improved, wherein the freeze-dried viable count and the freeze-dried survival rate of the experimental group are highest after proline is added, and the freeze-dried viable count is 1.76 +/-0.34 multiplied by 10 11 The CFU/g is improved to 9.19 +/-0.44 multiplied by 10 11 The freeze-drying survival rate is improved from 20.7 +/-0.4% to 92.63 +/-0.44% by CFU/g, and the freeze-drying survival rate is improved by about 4.47 times.
Example 12: fermentation and freeze-drying of Bifidobacterium bifidum CGMCC NO.13632
In addition to example 2, the Bifidobacterium longum subsp. infarnata CCFM 687 of example 2 was replaced with Bifidobacterium bifidum CGMCC NO.13632, and the osmotic pressure was adjusted to 550 to 850mOsm/kg (NaCl solution concentration: 16.5 to 25.5g/L), and the results of the experiments are shown in Table 11.
TABLE 11 Bifidobacterium bifidum CGMCC NO.13632 fermentation and freeze-drying results by the method of this example
Figure BDA0003090682390000171
The results show that the glucose content of the fermentation liquor is more than or equal to 18.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.14 +/-0.24 multiplied by 10 11 The CFU/g is improved to 9.82 +/-0.31 multiplied by 10 11 The freeze-drying survival rate of CFU/g is improved from 23.05 +/-0.27% to 98.34 +/-0.38%, and the freeze-drying survival rate is improved by about 4.27 times.
Example 13: fermentation and lyophilization of Bifidobacterium bifidum HNJ6
In addition to example 2, the Bifidobacterium longum subsp. infarnata CCFM 687 of example 2 was replaced with Bifidobacterium bifidum HNJ6, and the osmotic pressure was adjusted to 550 to 850mOsm/kg (NaCl solution concentration: 16.5 to 25.5g/L), and the results of the experiments are shown in Table 12.
TABLE 12 Bifidobacterium bifidum HNJ6 Freeze-drying results were fermented by the method of this example
Figure BDA0003090682390000181
The results show that the glucose content of the fermentation liquor is more than or equal to 18.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.71 +/-0.35 multiplied by 10 11 The CFU/g is improved to 9.81 +/-0.36 multiplied by 10 11 The freeze-drying survival rate of the CFU/g is improved from 27.87 +/-0.58% to 98.46 +/-0.58%, and the freeze-drying survival rate is improved by about 3.53 times.
Example 14: fermentation and freeze-drying of Bifidobacterium bifidum CCFM1150
In addition to example 2, the Bifidobacterium longum subsp. infarnatum CCFM 687 of example 2 was replaced with Bifidobacterium bifidum CCFM1150, and the osmotic pressure was adjusted to 550 to 850mOsm/kg (NaCl solution concentration: 16.5 to 25.5g/L), and the results of the experiments are shown in Table 13.
TABLE 13 Bifidobacterium bifidum CCFM1150 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000191
The results show that the glucose content of the fermentation liquor is more than or equal to 19.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 2.6 +/-0.42 multiplied by 10 11 The CFU/g is increased to 10.77 +/-0.18 multiplied by 10 11 The freeze-drying survival rate is improved from 30.35 +/-0.16% to 99.03 +/-0.42% by CFU/g, and the freeze-drying survival rate is improved by about 3.56 times.
Example 15: fermentation and freeze-drying of Bifidobacterium animalis CCFM1148
On the basis of example 2, the Bifidobacterium longum subsp. infarnatis CCFM 687 of example 2 was replaced by Bifidobacterium animalis CCFM1148, and the experimental results are shown in table 14.
TABLE 14 Bifidobacterium animalis CCFM1148 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000192
Figure BDA0003090682390000201
The results show that the glucose content of the fermentation liquor is more than or equal to 16.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; fermentation broth after fermentationThe osmotic pressure is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 3.58 +/-0.23 multiplied by 10 11 The CFU/g is improved to 9.72 +/-0.41 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 39.07 +/-0.5 percent to 97.7 +/-0.36 percent, and the freeze-drying survival rate is improved by about 2.50 times.
Example 16: fermentation and lyophilization of Bifidobacterium animalis BB12
On the basis of example 2, the Bacillus longum subsp. infarnatis CCFM 687 of example 2 was replaced with Bacillus animalis BB12, and the results are shown in Table 15.
TABLE 15 Bifidobacterium animalis BB12 fermentation and lyophilization results using the method of this example
Figure BDA0003090682390000202
Figure BDA0003090682390000211
The results show that the glucose content of the fermentation liquor is more than or equal to 17.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; compared with the blank group, the number of the live bacteria and the freeze-drying survival rate of the experimental group after freeze-drying are both obviously improved, wherein the number of the live bacteria and the freeze-drying survival rate are the highest after freeze-drying by adding proline, and the number of the live bacteria after freeze-drying is 3.15 +/-0.2 multiplied by 10 11 The CFU/g is improved to 9.87 +/-0.37 multiplied by 10 11 CFU/g, the freeze-drying survival rate is improved from 33.85 +/-0.41% to 99.05 +/-0.52%, and the freeze-drying survival rate is improved by about 2.92 times.
Example 17: fermented and freeze-dried bifidobacterium added with different amounts of proline
In this example, referring to the results of examples 2 to 7, proline fermentation was selected to increase the freeze-drying survival rate of bifidobacteria, as follows.
On the basis of example 2, the proline concentrations were set at 10g/L, 20g/L, and 30g/L, and controls were set: adjusting the compatible solute to be anhydrous glucose with the concentration of 30 g/L; then, lyophilized Bifidobacterium (Bifidobacterium longum subsp. infantis CCFM 687, Bifidobacterium longum subsp. longum CCFM1102, Bifidobacterium longum subsp. longum CCFM 1077, Bifidobacterium breve CCFM1026, Bifidobacterium breve CCFM1067, Bifidobacterium breve FM1078, Bifidobacterium adolescent CCFM 1108, Bifidobacterium adolescent CCFM 1062, Bifidobacterium adolescent CCFM1061, Bifidobacterium adolescent CCFM1063, Bifidobacterium bism CGMCC NO.13632, Bifidobacterium J.illusm J. 6, Bifidobacterium brevicum CCFM1063, Bifidobacterium brevicum CGMCC NO.13632, Bifidobacterium brevicum J. 6, Bifidobacterium brevicum CCFM1150, Bifidobacterium brevicum CCFM 12, Bifidobacterium brevicum CCFM 30-11416, and the optimum addition amounts of the solutes are shown in the Table.
TABLE 16 fermentation and lyophilization results of Bifidobacterium longum subsp. infarninatis CCFM 687 by the method of this example
Figure BDA0003090682390000212
TABLE 17 Bifidobacterium longum subsp. longum CCFM1102 Freeze-drying results of fermentation using the method of this example
Figure BDA0003090682390000221
TABLE 18 Bifidobacterium longum subsp. longum CCFM 1077 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000222
TABLE 19 Bifidobacterium breve CCFM1026 fermentation lyophilization results using the procedure of this example
Figure BDA0003090682390000223
TABLE 20 Bifidobacterium breve CCFM1067 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000224
TABLE 21 Bifidobacterium breve CCFM1078 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000225
TABLE 22 Bifidobacterium adolescentis CCFM 1108 Freeze-drying results of fermentation using the method of this example
Figure BDA0003090682390000231
TABLE 23 Bifidobacterium adolescentis CCFM 1062 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000232
TABLE 24 Bifidobacterium adolescent CCFM1061 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000233
TABLE 25 fermentation and lyophilization results of Bifidobacterium bifidum CCFM1063 by the method of this example
Figure BDA0003090682390000234
TABLE 26 Bifidobacterium bifidum CGMCC NO.13632 fermentation and freeze-drying results of the method of this example
Figure BDA0003090682390000235
Figure BDA0003090682390000241
TABLE 27 Bifidobacterium bifidum HNJ6 Freeze-drying results were fermented by the method of this example
Figure BDA0003090682390000242
TABLE 28 Bifidobacterium bifidum CCFM1150 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000243
TABLE 29 Bifidobacterium animalis CCFM1148 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000244
TABLE 30 Bifidobacterium animalis BB12 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000245
Results in tables 16-30 show that the glucose content of the fermentation broth is more than or equal to 8.00g/L after the culture is finished, which indicates that the glucose content in the fermentation medium of the invention is sufficient; after fermentation, the osmotic pressure of fermentation liquor is close to the completely inhibited osmotic pressure; when the addition amount of proline is between 0 and 30g/L, the freeze-drying survival rate of the bifidobacterium and the number of live bacteria after freeze-drying are both obviously improved along with the increase of the addition amount of the proline, and the maximum survival rate is reached when the addition amount of the proline is between 20 and 30 g/L.
The highest osmotic pressure resistance of the bifidobacterium is lower, and the excessive addition of proline can improve the freeze-drying survival rate of the strain but also cause the increase of the osmotic pressure to a certain extent, so that the yield of the bifidobacterium fermentation broth bacterial sludge is reduced, and 20g/L is selected for subsequent experiments.
Example 18: adding proline fermented bifidobacterium to collect bacteria under different osmotic pressures and freeze-drying
Referring to the results of example 8, the fermentation with addition of 20g/L proline was selected to improve the freeze-dried survival rate of Bifidobacterium.
In order to explore the conditions for increasing the freeze-drying survival rate of bifidobacterium by adding proline, a blank group (group A) is adopted on the basis of the embodiment 2;
the concentration of proline is set to be 20g/L, the content of glucose added in a fermentation medium is changed, anhydrous glucose of 20g/L and 60g/L is respectively added to be set as a group B and a group C, freeze-dried bifidobacteria (bifidobacterium longum CCFM1102, bifidobacterium longum CCFM1109, bifidobacterium breve CCFM1067, bifidobacterium breve CCFM1078, bifidobacterium adolescentis CCFM1061 and bifidobacterium bifidum CCFM116) are respectively prepared to be harvested in a stationary phase, and conditions for improving the freeze-drying survival rate of the bifidobacterium by adding compatible solutes are researched.
TABLE 31 Bifidobacterium longum subsp. infarninatis CCFM 687 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000251
TABLE 32 Bifidobacterium longum subsp. longum CCFM1102 Freeze-drying results of fermentation using the method of this example
Figure BDA0003090682390000252
TABLE 33 Bifidobacterium longum subsp. longum CCFM 1077 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000253
TABLE 34 Bifidobacterium breve CCFM1026 fermentation lyophilization results Using the procedure of this example
Figure BDA0003090682390000254
Figure BDA0003090682390000261
TABLE 35 Bifidobacterium breve CCFM1067 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000262
TABLE 36 Bifidobacterium breve CCFM1078 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000263
TABLE 37 Bifidobacterium adolescentis CCFM 1108 Freeze-drying results of fermentation using the method of this example
Figure BDA0003090682390000264
TABLE 38 Bifidobacterium adolescentis CCFM 1062 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000265
TABLE 39 Bifidobacterium adolescent CCFM1061 Freeze-drying results were fermented by the method of this example
Figure BDA0003090682390000266
TABLE 40 Bifidobacterium bifidum CCFM1063 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000271
TABLE 41 Bifidobacterium bifidum CGMCC NO.13632 fermentation and freeze-drying results of the method of this example
Figure BDA0003090682390000272
TABLE 42 Bifidobacterium bifidum HNJ6 Freeze-drying results were fermented by the method of this example
Figure BDA0003090682390000273
TABLE 43 Bifidobacterium bifidum CCFM1150 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000274
TABLE 44 Bifidobacterium animalis CCFM1148 fermentation freeze-drying results using the method of this example
Figure BDA0003090682390000275
TABLE 45 Bifidobacterium animalis BB12 fermentation lyophilization results using the method of this example
Figure BDA0003090682390000276
Figure BDA0003090682390000281
Results from tables 31-45 show that comparing group a and group B, it is known that adding proline to ferment to low osmotic pressure to harvest bacteria does not significantly improve the freeze-drying survival rate of bifidobacteria; comparing group B and group C, it can be seen that the freeze-drying survival rate of bifidobacteria and the number of viable bacteria after freeze-drying can be significantly improved only when the osmotic pressure is nearly completely inhibited under the synergistic effect of proline addition and hypertonic stress.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (3)

1. A preparation method of bifidobacterium lyophilized powder is characterized by comprising the following steps:
(1) inoculating bifidobacterium into a fermentation culture medium containing compatible solute proline for fermentation; after fermentation, centrifugally collecting bacteria to obtain thalli;
the fermentation medium is as follows: 24g/L yeast extract powder, 60g/L anhydrous glucose, 801 mL/L tween and 1g/L, MgSO g/L cysteine hydrochloride 4 ·7H 2 0.35g/L of proline, 20-30g/L of O, wherein the bifidobacteria are bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis;
(2) washing the thalli prepared in the step (1) by adopting a solution with an osmotic pressure value of 550-1100 mOsm/kg, and then mixing the thalli with a protective agent sorbitol according to a ratio of 1:1, and freeze-drying to obtain the bifidobacterium powder.
2. A method for improving freeze-drying survival rate of bifidobacteria, which is characterized by comprising the following steps: inoculating bifidobacterium into a fermentation culture medium containing compatible solute proline for fermentation, and centrifugally collecting bacteria after the fermentation is finished to obtain thalli; washing the thalli by using a solution with osmotic pressure of 550-1100 mOsm/kg, and mixing the thalli with a protective agent sorbitol according to the ratio of 1:1, and freeze-drying;
the fermentation medium comprises: 24g/L yeast extract powder, 60g/L anhydrous glucose, 801 mL/L tween and 1g/L, MgSO g/L cysteine hydrochloride 4 ·7H 2 0.35g/L of O, 20-30g/L of proline, wherein the bifidobacteria are bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis.
3. A lyophilized powder of Bifidobacterium prepared by the method of claim 1.
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