CN114806883A - Method for protecting and recovering live bacteria in fermentation liquor - Google Patents
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Abstract
The invention provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following steps: mixing the fermentation liquor with whey protein concentrate WPC-34, konjac glucomannan and calcium salt, reacting, and centrifuging to recover the strain. The protection and recovery method provided by the invention can effectively protect the viable bacteria in the fermentation liquor, reduce the death of the viable bacteria after centrifugal separation, improve the survival rate of the strains and simultaneously improve the yield of the strains.
Description
Technical Field
The invention belongs to the technical field of microbial post-treatment, particularly relates to a method for protecting and recovering viable bacteria in fermentation liquor, and particularly relates to a method for protecting and recovering viable bacteria in fermentation liquor with high yield.
Background
Lactococcus is widely used in food industry, such as cheese, yoghurt and probiotic solid beverage, and accounts for more than half of the production of lactic acid bacteria. At present, the lactococcus is mainly cultured by a liquid fermentation method, the key link of the fermentation process is not only culture medium optimization, but also the fermentation liquid centrifugation effect is a key technology of the production process. However, in the course of lactococcus fermentation, metabolites such as organic acids, small peptides and polysaccharides are liable to form sol substances in the fermentation liquid, and the recovery and separation of bacterial cells affecting high-density fermentation are present, so that the yield of lactococcus is reduced, and the production efficiency and cost are seriously affected. Therefore, solving this problem can bring a great economic benefit. The existing separation techniques of the somatic cells mainly comprise two types, namely centrifugation and ultrafiltration. The centrifugal separation of the bacterial cells has the advantages of simple operation, convenient equipment cleaning and difficult pollution, and is adopted by most researches and producers, but the yield of the colloidal substances generated by lactococcus is difficult to improve in the actual production, and the centrifugal mechanical action and the damage of acid or oxygen can cause the death of partial bacteria and reduce the survival rate of the viable bacteria cells; the ultrafiltration centrifugation has high centrifugation effect, but is difficult to be applied to actual production due to high material cost and unsatisfactory stability. In conclusion, a method for protecting and rapidly recovering viable bacteria cells in lactococcus fermentation liquor is urgently needed to be explored.
CN108251342A discloses a method for recovering viable bacteria of lactobacillus in lactobacillus fermentation liquor by rapid flocculation and sedimentation, which comprises adjusting the pH value of the lactobacillus fermentation liquor to a proper range, adding a natural safe nontoxic flocculant and coagulant solution, fully stirring to combine the flocculant with cells in the fermentation liquor and flocculate into clusters, standing for a period of time, removing the supernatant, and recovering the lower-layer bacterial-rich floccule sediment solution. By the flocculation deposition method, the lactobacillus fermentation liquor can be concentrated to the original volume of 1/5-1/16, and the maximum recovery rate of bacteria can reach 99%. According to the method, the lactobacillus cells in the fermentation liquor are flocculated into clusters and quickly deposited by adding the flocculating agent into the fermentation liquor, and the thalli can be quickly recovered from the fermentation liquor. However, the method of directly adding the flocculating agent can cause the death of live bacteria, so that the survival rate of the strains is reduced.
The existing separation technology of the somatic cells has the conditions of low survival rate of strains and poor yield. Therefore, how to provide a method for rapidly recovering viable bacteria cells in lactococcus fermentation liquor with high strain survival rate becomes a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for protecting and recovering viable bacteria in fermentation liquor, in particular to a method for protecting and recovering viable bacteria in fermentation liquor with high yield. The protection and recovery method provided by the invention can effectively protect the viable bacteria in the fermentation liquor, reduce the death of the viable bacteria after centrifugal separation, improve the survival rate of the strains and simultaneously improve the yield of the strains.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following steps:
mixing the fermentation liquor with whey protein concentrate WPC-34, Konjac Glucomannan (KGM) and calcium salt, reacting, and centrifuging to recover strain.
According to the method, the whey protein concentrate WPC-34, the konjac glucomannan and the calcium salt are compounded and cooperate, so that the ionic bond, the hydrogen bond and the hydrophobic interaction of the whey protein concentrate WPC-34, the konjac glucomannan and the calcium salt can be fully utilized, a stable system of the WPC-34 protein, the konjac glucomannan and cells is formed in fermentation liquor, the guarantee is provided for the subsequent improvement of the centrifugal survival rate and the cell activity, and the survival rate and the yield of strains after centrifugation are improved.
The viable bacteria include cocci, further lactococcus and streptococcus bacteria, including lactococcus cremoris, lactococcus lactis or streptococcus thermophilus.
In the reaction system, the concentration of whey protein concentrate WPC-34 is 0.1-5.5 mg/L, the concentration of konjac glucomannan is 0.01-0.3 mg/L, wherein the concentration of the whey protein concentrate WPC-34 can be 0.1 mg/L, 0.5 mg/L, 1 mg/L, 1.5 mg/L, 2 mg/L, 2.5 mg/L, 3 mg/L, 3.5 mg/L, 4 mg/L, 4.5 mg/L, 5 mg/L or 5.5 mg/L, etc., the concentration of the konjac glucomannan can be 0.01 mg/L, 0.05 mg/L, 0.1 mg/L, 0.15 mg/L, 0.2 mg/L, 0.25 mg/L or 0.3 mg/L, etc., but not limited to, the above-listed numerical values, and other numerical values not listed in the above numerical range are also applicable.
Preferably, the whey protein concentrate WPC-34 is mixed with the fermentation liquor in the form of a solution, and the mass fraction of the whey protein concentrate WPC-34 in the solution is 2-10%.
Preferably, the volume ratio of the fermentation liquor to the solution of the whey protein concentrate WPC-34 is (20-40): 1.
Preferably, the konjac glucomannan is mixed with the fermentation liquor in the form of solution, and the mass fraction of the konjac glucomannan in the solution is 0.2-1.4%.
Preferably, the volume ratio of the fermentation liquor to the konjac glucomannan solution is (50-100): 1.
Preferably, the feed-liquid ratio of the calcium salt to the fermentation liquid is 0.4-1.2 mg/mL.
Wherein, the mass fraction of the solution of the whey protein concentrate WPC-34 can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc., the volume ratio of the fermentation liquid to the solution of the whey protein concentrate WPC-34 can be 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, 32:1, 34:1, 36:1, 38:1 or 40:1, etc., the mass fraction of the solution of the konjac glucomannan can be 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2% or 1.4%, etc., the volume ratio of the fermentation liquid to the solution of the konjac glucomannan can be 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, etc., the ratio of the fermentation liquid to the fermentation liquid can be 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, etc, 0.9 mg/mL, 1 mg/mL, 1.1 mg/mL, 1.2 mg/mL, etc., but is not limited to the above-mentioned values, and other values not listed in the above-mentioned numerical ranges are also applicable.
Preferably, the temperature of the reaction is 38-45 ℃.
Preferably, the reaction time is 10-20 min.
Preferably, the pH of the reaction is between 5.5 and 7.0.
Preferably, the reaction is carried out with stirring, the rate of stirring being from 10 to 100 rpm.
The reaction temperature may be 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃, the reaction time may be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, the reaction pH may be 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, 6.7 or 7.0, the stirring rate may be 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm or 100 rpm, but the above-mentioned values are not limited thereto, and other values not listed in the above-mentioned value range are also applicable.
The reaction parameters can effectively form a stable system of WPC-34 protein, konjac glucomannan and cells, and the survival rate and the yield of the strains are improved.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for protecting and recovering live bacteria in fermentation liquor, which adopts the compounding of whey protein concentrate WPC-34, konjac glucomannan and calcium salt, has a synergistic effect, can fully utilize the ionic bond, hydrogen bond and hydrophobic interaction of the whey protein concentrate WPC-34, konjac glucomannan and cells to form a stable system of WPC-34 protein, konjac glucomannan and cells in the fermentation liquor, provides guarantee for the subsequent improvement of the centrifugal survival rate and cell activity, and improves the survival rate and yield of strains after centrifugation; and a stable system of WPC-34 protein, konjac glucomannan and cells can be effectively formed by controlling reaction parameters, so that the survival rate and the yield of the strains are improved.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following examples, skim milk powder was purchased from Henan natural in New Zealand;
casein was purchased from Jiangsu Yukun Biotech limited;
mannan was purchased from north Heibei Runshou Biotech limited;
chitosan was purchased from clone biotechnology limited, hebei;
konjac glucomannan was purchased from Shandong Lu lemon Biotech Ltd.
Streptococcus thermophilus (S.thermophilus) to which the following statements relateStreptococcus thermophilus) The ST81 bacterial strain has preservation unit of China general microbiological culture Collection center, preservation time of 2018, 5 months and 11 days, preservation number of CGMCC 15752, and address of No. 3 Siro No. 1 of Beijing.
The formula of the culture medium is as follows: 20 g/L of peptone, 5 g/L of yeast extract, 40 g/L of glucose, 2 g/L of diammonium citrate, 1 g/L of Tween, 0.58 g/L of magnesium sulfate, 0.19 g/L of manganese sulfate and 2 g/L of dipotassium phosphate.
Activating the seed liquid: inoculating streptococcus thermophilus by using a culture medium according to the inoculation amount of 5%, and culturing at the constant temperature of 37 ℃ for 8 hours to prepare first-level seeds; then according to the same method, the first-class seeds are used as initial strains to prepare a second-class seed liquid.
Preparing fermentation liquor: adjusting the pH of the sterilized culture medium to 6.8 by using ammonia water, inoculating a secondary seed solution according to the proportion of 5% of the culture medium, culturing at the constant temperature of 37 ℃, controlling the pH to 5.4, stirring at the rotating speed of 50 rpm, intermittently introducing sterile air, culturing without maintaining pressure, and discharging the culture medium when the residual amount of glucose is less than 0.5%.
Preparation of whey protein concentrate WPC-34 aqueous solution: weighing whey protein concentrate WPC-34, adding into deionized water, stirring for 2 hr to dissolve completely, and placing in refrigerator at 4 deg.C overnight to hydrate completely.
Preparation of Konjac Glucomannan (KGM) sol: weighing Konjac Glucomannan (KGM), adding into deionized water at 40 ℃, stirring at a high speed of 1000 rpm until the Konjac Glucomannan (KGM) is completely dissolved, and placing in a refrigerator at 4 ℃ for later use.
CaCl 2 After irradiation, the mixture is added into a fermentation tank according to a calculated proportion for use.
Example 1
The embodiment provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following specific steps:
concocting 2L fermentation broth with ammonia water, maintaining pH at 5.5 and fermentation tank temperature at 38 deg.C, adding 50 mL 2% whey protein concentrate WPC-34, stirring at 100 rpm to mix WPC-34 with lactobacillus, and mixingThen 20 mL of 0.2% Konjac Glucomannan (KGM) was added, and 8 g CaCl was added 2 The reaction was carried out by stirring slowly at 10 rpm. After 10 min, taking the mixture out of the tank, centrifuging by using a tubular centrifuge, setting the frequency to be 50 Hz, and the rotating speed of a separating cylinder to be: 20000 r/min, separation factor: 16700 R.C.F. And centrifuging and recovering to obtain live bacteria.
Example 2
The embodiment provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following specific steps:
concocting 1L fermentation broth with ammonia water, maintaining pH at 6.0, fermenting at 38 deg.C, adding 50 mL whey protein concentrate WPC-34 with concentration of 4%, stirring at 140 rpm to mix WPC-34 and lactobacillus, adding 20 mL 0.6% Konjac Glucomannan (KGM), adding 4 g Ca (NO), and stirring 3 ) 2 The reaction was carried out by stirring slowly at 30 rpm. After 20 min, taking the mixture out of the tank, centrifuging by using a tubular centrifuge, setting the frequency to be 50 Hz, and the rotating speed of a separating cylinder to be: 20000 r/min, separation factor: 16700 R.C.F. And centrifuging and recovering to obtain live bacteria.
Example 3
The embodiment provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following specific steps:
adjusting pH of 1L fermentation broth with ammonia water, maintaining the fermentation broth at 40 deg.C, adding 50 mL whey protein concentrate WPC-34 with concentration of 6%, stirring at 160 rpm to mix WPC-34 and lactobacillus, adding 20 mL 0.8% Konjac Glucomannan (KGM), adding 6 g CaCl 2 The reaction was carried out by stirring slowly at 50 rpm. Taking the mixture out of the tank after 15 min, centrifuging by using a tubular centrifuge, setting the frequency to be 50 Hz, and the rotating speed of a separating cylinder to be: 20000 r/min, separation factor: 16700 R.C.F. And centrifuging and recovering to obtain live bacteria.
Example 4
The embodiment provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following specific steps:
concocting 1.5L fermentation broth with ammonia water, maintaining pH at 6.5, fermenting at 40 deg.C, adding 50 mL whey protein concentrate WPC-34 with concentration of 8%, stirring at 200 rpm to mix WPC-34 and lactobacillus, adding 20 mL 1.2% Konjac Glucomannan (KGM),12 g of CaCl are added 2 The reaction was carried out by stirring slowly at 50 rpm. And (4) taking the material out of the tank after 15 min, centrifuging by using a tubular centrifuge, setting the frequency to be 50 Hz, and setting the rotating speed of a separating cylinder to be as follows: 20000 r/min, separation factor: 16700 R.C.F. And centrifuging and recovering to obtain live bacteria.
Example 5
The embodiment provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the following specific steps:
concocting 1L fermentation broth with ammonia water, maintaining pH at 7.0, fermenting at 45 deg.C, adding 50 mL 10% whey protein concentrate WPC-34, stirring at 200 rpm to mix WPC-34 and lactobacillus, adding 20 mL 1.4% Konjac Glucomannan (KGM), adding 12 g CaCl 2 The reaction was carried out by stirring slowly at 50 rpm. Taking the mixture out of the tank after 15 min, centrifuging by using a tubular centrifuge, setting the frequency to be 50 Hz, and the rotating speed of a separating cylinder to be: 20000 r/min, separation factor: 16700 R.C.F. And centrifuging and recovering to obtain live bacteria.
Comparative example 1
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the specific steps of removing whey protein concentrate WPC-34, konjac glucomannan and CaCl 2 Otherwise, the rest was the same as example 4.
Comparative example 2
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, and the method is consistent with the method in example 4 except that the temperature of a fermentation tank is 37 ℃.
Comparative example 3
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, and the method is consistent with the example 4 except that the temperature of a fermentation tank is 46 ℃.
Comparative example 4
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, and the steps are the same as those in example 4 except that the pH is kept to be 5.3.
Comparative example 5
The comparative example provides a method for protecting and recovering viable bacteria in fermentation liquor, and the steps are the same as those in example 4 except that the pH is kept at 7.2.
Comparative example 6
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the specific steps of removing WPC-34 without whey protein concentrate, and distributing a reduced part of WPC-34 to CaCl 2 Otherwise, the rest was the same as example 4.
Comparative example 7
This comparative example provides a method for protecting and recovering viable bacteria in a fermentation broth, which is the same as example 4 except that no KGM is contained in the specific steps.
Comparative example 8
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, which specifically comprises the steps of removing CaCl 2 The reduced fraction was distributed over the whey protein concentrate WPC-34, the remainder being in accordance with example 4.
Comparative example 9
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, and the concrete steps do not contain KGM and CaCl 2 The reduced fraction was distributed over the whey protein concentrate WPC-34, the remainder being in accordance with example 4.
Comparative example 10
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the specific steps of removing WPC-34 and CaCl which do not contain whey protein concentrate 2 Otherwise, the rest was the same as example 4.
Comparative example 11
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, which comprises the specific steps of removing KGM and whey protein concentrate WPC-34, and distributing a reduced part to CaCl 2 Otherwise, the rest was the same as example 4.
Comparative example 12
The comparative example provides a method for protecting and recovering live bacteria in fermentation broth, and the specific steps are the same as those in example 4 except that the same amount of skimmed milk powder is used for replacing the whey protein concentrate WPC-34.
Comparative example 13
This comparative example provides a method for protecting and recovering viable bacteria in a fermentation broth, which is the same as that of example 4 except that the whey protein concentrate WPC-34 is replaced with casein of the same amount in the specific steps.
Comparative example 14
The comparative example provides a method for protecting and recovering live bacteria in fermentation liquor, and the specific steps are the same as those in example 4 except that the whey protein concentrate WPC-34 is replaced by the same amount of sodium alginate.
Comparative example 15
The comparative example provides a method for protecting and recovering viable bacteria in fermentation broth, and the specific steps are the same as those in example 4 except that KGM is replaced by equal amount of hydroxypropyl distarch phosphate.
Comparative example 16
This comparative example provides a method for protecting and recovering viable bacteria in a fermentation broth, which is the same as example 4 except that KGM is replaced with mannan in an equivalent amount in the specific steps.
Comparative example 17
This comparative example provides a method for protecting and recovering viable bacteria in a fermentation broth, which is the same as example 4 except that KGM is replaced with chitosan in the same amount in the specific steps.
And (3) effect testing:
the parameters of examples 1-5 and comparative examples 1-17 were tested with reference to the testing method for lactic acid bacteria of GB 4789.35-2016 food safety national standard food microbiology, and the initial fermentation broth of example 4 was directly centrifuged as in example 4, wherein:
total viable count of fermentation liquor: sucking 1 mL of fermentation liquor, adding the fermentation liquor into 9 mL of physiological saline, uniformly mixing in a vortex manner, taking 1 mL of bacterial liquid to 9 mL of physiological saline for gradient dilution, sucking 1 mL, adopting a pouring method, selecting 3 gradients, enabling each gradient to be parallel to 3, putting a culture dish into a 30 ℃ constant-temperature incubator, culturing for 48 hours, taking out and counting, and calculating to obtain the total viable count of the fermentation liquor;
and (3) detecting the total bacteria number of the supernatant after centrifugation: sucking 1 mL of centrifuged supernatant, adding the centrifuged supernatant into 9 mL of physiological saline, uniformly mixing by vortex, taking 1 mL of bacterial liquid to 9 mL of physiological saline for gradient dilution, sucking 1 mL of the physiological saline, selecting 3 gradients by adopting a pouring method, enabling 3 gradients to be parallel, putting a culture dish in a 30 ℃ constant-temperature incubator for culturing for 48 hours, taking out the culture dish for counting, and calculating to obtain the total bacterial count of the centrifuged supernatant;
and (3) detecting the total number of settled bacteria after centrifugation: sucking 1 g of centrifuged bacterial sludge, adding the centrifuged bacterial sludge into 9 mL of physiological saline, uniformly mixing in a vortex manner, taking 1 mL of bacterial liquid into 9 mL of physiological saline for gradient dilution, sucking 1 mL of the bacterial liquid, selecting 3 gradients by adopting a pouring method, enabling 3 gradients to be parallel to each other, putting a culture dish into a 30-DEG C constant-temperature incubator for culture for 48 hours, taking out and counting, and calculating to obtain the total bacterial count of the centrifuged sediment.
And calculating the centrifugal loss rate and the centrifugal yield.
Centrifugal loss rate (%) = total number of supernatant bacteria/total number of fermentation broth viable bacteria x 100% after centrifugation;
centrifugal yield (%) = total number of settled bacteria after centrifugation/total number of viable bacteria in fermentation liquor x 100%;
flow cytometry analysis of the survival rate of sedimented bacteria after centrifugation: the pellet after centrifugation was washed twice with sterile cold PBS (0.01 mol/L, pH 7.0, 0.9% NaCl). The cells were then resuspended and diluted to 1X 10 with sterile cold PBS 7 CFU/mL. The diluted suspension was first incubated with 10. mu.L of 1.496 mmol/L Propidium Iodide (PI) solution at 40 ℃ for 20 min in the dark. Then 10. mu.L of 0.460 mmol/L cFDA solution was added to the mixture and incubated at 40 ℃ for 10 minutes in the absence of light before using a FACStort flow cytometer. Fluorescence emission spectra (excitation 470 nm, emission 490-670 nm, BW 5 nm) were recorded and analyzed for viable count after centrifugation, total count of bacteria after centrifugation using Cell Quest 3.0 software.
The centrifugal survival rate (%) = the number of live bacteria after centrifugation/the total number of bacteria after centrifugation × 100%.
The results are as follows:
the results show that the protection and recovery method provided by the invention effectively improves the survival rate and yield of the strains; comparing example 4 with comparative examples 2 to 5, it can be found that the reaction can be significantly promoted by controlling the fermentation temperature and pH value, and the survival rate of the strain and the centrifugal yield can be improved; comparing example 4 with comparative examples 1 and 6-11, it can be found that the invention remarkably improves the survival rate of strains and the centrifugal yield by adding the whey protein concentrate WPC-34, KGM and calcium salt and utilizing the compounding and synergistic effect of the three; comparing example 4 with comparative examples 12-17, it can be seen that the specific raw materials adopted in the present invention can significantly improve the survival rate of the strains and the centrifugal yield compared to other raw materials; comparing the example 4 with the fermentation liquor group, the invention can find that the survival rate of strains and the centrifugal yield can be obviously improved compared with the untreated condition by adding the whey protein concentrate WPC-34, KGM and calcium salt and utilizing the compounding and the synergistic effect of the three.
The applicant states that the present invention is described in the above examples as a method for protecting and recovering viable bacteria in a fermentation broth according to the present invention, but the present invention is not limited to the above examples, which means that the present invention is not limited to the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (9)
1. A method for protecting and recovering live bacteria in fermentation liquor is characterized by comprising the following steps:
mixing the fermentation liquor with whey protein concentrate WPC-34, konjac glucomannan and calcium salt for reaction, and centrifuging to recover strains;
in the reaction system, the concentration of the whey protein concentrate WPC-34 is 0.1-5.5 mg/L, and the concentration of the konjac glucomannan is 0.01-0.3 mg/L.
2. The method for protecting and recovering viable bacteria in fermentation broth according to claim 1, wherein the whey protein concentrate WPC-34 is mixed with the fermentation broth in the form of solution, and the mass fraction of the whey protein concentrate WPC-34 in the solution is 2-10%;
the volume ratio of the fermentation liquor to the solution of the whey protein concentrate WPC-34 is (20-40): 1.
3. The method for protecting and recovering viable bacteria in a fermentation broth according to claim 1, wherein the konjac glucomannan is mixed with the fermentation broth in the form of a solution, and the mass fraction of the konjac glucomannan in the solution is 0.2-1.4%.
4. The method for protecting and recovering viable bacteria in fermentation broth according to claim 3, wherein the volume ratio of the fermentation broth to the solution of konjac glucomannan is (50-100): 1.
5. The method for protecting and recovering viable bacteria in fermentation broth according to claim 1, wherein the feed-to-liquid ratio of the calcium salt to the fermentation broth is 0.4-1.2 mg/mL.
6. The method for protecting and recovering viable bacteria in a fermentation broth according to claim 1, wherein the reaction temperature is 38-45 ℃.
7. The method for protecting and recovering viable bacteria in a fermentation broth according to claim 1, wherein the reaction time is 10-20 min.
8. The method for protecting and recovering viable bacteria in a fermentation broth according to claim 1, wherein the pH of the reaction is 5.5-7.0.
9. The method for protecting and recovering viable bacteria in a fermentation broth according to claim 1, wherein the reaction is carried out under stirring at a rate of 10-100 rpm.
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