CN114231515B - Method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria - Google Patents
Method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 115
- 241000894006 Bacteria Species 0.000 title claims abstract description 65
- 241000186660 Lactobacillus Species 0.000 title claims abstract description 38
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 38
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 38
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 38
- 102000004317 Lyases Human genes 0.000 title claims abstract description 28
- 108090000856 Lyases Proteins 0.000 title claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 97
- 102000004190 Enzymes Human genes 0.000 claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 claims abstract description 50
- 238000012258 culturing Methods 0.000 claims abstract description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 28
- 241000556533 uncultured marine bacterium Species 0.000 claims description 15
- 239000004310 lactic acid Substances 0.000 claims description 14
- 235000014655 lactic acid Nutrition 0.000 claims description 14
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 9
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- 235000019319 peptone Nutrition 0.000 claims description 9
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 8
- 235000010413 sodium alginate Nutrition 0.000 claims description 8
- 239000000661 sodium alginate Substances 0.000 claims description 8
- 229940005550 sodium alginate Drugs 0.000 claims description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
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- 235000015097 nutrients Nutrition 0.000 description 2
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- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02003—Poly(beta-D-mannuronate) lyase (4.2.2.3)
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- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02011—Poly(alpha-L-guluronate) lyase (4.2.2.11), i.e. alginase II
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Abstract
The invention provides a method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, which comprises the steps of preparing a fermentation culture solution of the XS1412C strain by pre-culturing the marine bacteria XS1412C strain, inoculating the XS1412C strain into the fermentation culture solution, pre-culturing the halophilic lactobacillus, inoculating the halophilic lactobacillus into the fermentation culture solution of the XS1412C strain for symbiotic fermentation culture to obtain a symbiotic fermentation culture solution, and finally centrifuging the symbiotic fermentation culture solution to obtain an enzyme solution containing the algin lyase.
Description
Technical Field
The invention belongs to the technical field of marine organisms, and relates to a method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria.
Background
Algins are sodium salts of alginic acid, sodium alginate, also known as sodium alginate, a class of water-soluble acidic polysaccharide substances widely found in various brown algae. The algin has wide application value, is an important component of the cell wall of brown algae, has the characteristics of large molecular mass, high polymerization degree, high viscosity, high fibrosis and the like, and therefore, the absorption and utilization degree of human bodies is poor, and the application range of the algin is limited to a great extent. The algin is decomposed into absorbable and utilizable active ingredients by a scientific method, for example, the algin is decomposed into micromolecular substances by an enzymatic reaction, and the algin oligosaccharide is the main product of the enzymolysis of the algin, has biological activities such as immunoregulation, bacteriostasis, antiviral capability and the like, is widely applied to the fields of foods, medicines, environmental protection and chemical industry, and has high application value. The algin lyase is used as a tool enzyme for producing the algin oligosaccharide, has high reaction efficiency, mild reaction conditions and strong controllability, and is convenient for directionally preparing the algin oligosaccharide, so that the research of the algin lyase has profound theoretical significance and application prospect.
In order to improve the enzyme activity, in terms of utilizing molecular biology, the most common is to carry out excessive heterologous expression on the enzyme, but the method is complex, has certain difficulty in operation and high cost, and is not suitable for daily production and application of factories. In recent years, research has been reported that microorganisms can ferment to produce algin lyase, such as bacillus, but most microorganisms have low activity to ferment to produce algin lyase and long fermentation period, and cannot be applied to actual industrial production.
Lactic acid bacteria are important components of probiotics, have outstanding probiotics characteristics and fermentation activity, and can be widely applied to the production of foods, such as dairy products, meats, vegetables, baked products and the like. Numerous studies have shown that lactic acid bacteria not only improve the organoleptic properties of food, produce organic acids, lower the pH of food, promote the growth of beneficial bacteria, even affect the expression of enzymes, but also are beneficial to human health. In the past few decades, a great deal of research on functional properties of lactic acid bacteria, regulation of host metabolism, interaction with microbiome in a host, and other health effects has been conducted by using in vitro tests, animal models, etc., and a great deal of test results show that lactic acid bacteria can regulate immune functions, reduce cholesterol, promote digestion, prevent cancer, etc., and bring health benefits to the diet of lactic acid bacteria. Therefore, the use of lactic acid bacteria to promote the production of algin lyase has good research prospect and application value.
In summary, the existing methods for producing algin lyase by microorganism have the technical problems of low enzyme yield, long fermentation period, complex production process and high industrialization cost, and need to be further improved.
Disclosure of Invention
The application aims to provide a method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, which aims to solve the technical problems of low enzyme yield, long fermentation period, complex production process and high industrialization cost of the existing method for producing algin lyase by microorganisms.
The embodiment of the application provides a method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, which comprises the following steps:
step (1): pre-culturing marine bacteria: inoculating marine bacteria into a culture solution for preculture;
Step (2): preparation of marine bacteria fermentation culture solution: inoculating the marine bacteria cultured in the step (1) into a fermentation culture solution for culturing to prepare a marine bacteria fermentation culture solution;
step (3): pre-culturing halophilic lactobacillus: inoculating halophilic lactobacillus into MRS liquid culture medium for preculture;
Step (4): halophilic lactobacillus-marine bacterium symbiotic fermentation culture: inoculating the halophilic lactobacillus cultured in the step (3) into the marine bacterium fermentation culture solution prepared in the step (2), and performing halophilic lactobacillus-marine bacterium symbiotic fermentation culture to prepare a symbiotic fermentation culture solution;
Step (5): and (3) centrifugal collection: and (3) centrifuging the symbiotic fermentation culture solution prepared in the step (4), and collecting supernatant to obtain enzyme solution containing algin lyase.
Preferably, the marine bacterium in step (1) is strain XS 1412C.
Preferably, the culture solution in the step (1) comprises the following components: sodium alginate 2%, monopotassium phosphate 0.1%, ammonium chloride 0.2%, peptone 5%, and the treatment conditions are as follows: adding filtered seawater for dissolving.
Preferably, the culture conditions of the marine bacteria in step (1) are: the culture temperature is 26 ℃ and the culture time is 7 hours.
Preferably, the fermentation broth in step (2) comprises the following components: 5% of peptone, 5% of glucose, 2% of sodium alginate, 0.1% of monopotassium phosphate, 0.4% of ammonium chloride and 0.38% of magnesium sulfate.
Preferably, the inoculation amount of the marine bacteria in the step (2) is 1-5%, and the culture conditions of the marine bacteria fermentation culture solution are as follows: the temperature is 25-35 ℃ and the time is 30-42 h.
Preferably, the components of the MRS liquid medium in step (3) are: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 0.4g of dipotassium hydrogen phosphate, 5g of sodium acetate, 0.58g of magnesium sulfate, 20g of calcium carbonate, 0.29g of manganese sulfate, 1g of tween-80 and 2g of diammonium citrate, wherein the treatment conditions are as follows: pH 6.0, water to 1000mL, and autoclaving at 121℃for 20min.
Preferably, the culture conditions of the halophilic lactic acid bacteria in step (3) are: the inoculation amount is 2%, the culture temperature is 28 ℃, and the culture time is 36h.
Preferably, the inoculum size of the halophilic lactobacillus in the step (4) is 1-5%, and the conditions of the halophilic lactobacillus-marine bacterium symbiotic fermentation culture are as follows: the culture temperature is 25-35 ℃ and the culture time is 30-42 h.
Preferably, the centrifugation conditions in step (5) are: the temperature is 4 ℃, the rotating speed is 8000r/min, and the centrifugation is carried out for 20min.
The invention provides a method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, which comprises the steps of preparing a marine bacterial fermentation culture solution by culturing marine bacteria in advance, inoculating the marine bacteria into the fermentation culture solution, then inoculating the halophilic lactobacillus into the marine bacterial fermentation culture solution for symbiotic fermentation culture by culturing the halophilic lactobacillus in advance, obtaining a symbiotic fermentation culture solution, and finally centrifuging the symbiotic fermentation culture solution to obtain an enzyme solution containing the algin lyase, wherein a marine bacterial XS1412C strain is preserved in China center for type culture collection (address: wuhan university, china) with a preservation number of CCTCC NO: m2015149, which is classified as Corynebacterium sp.XS1412C, was deposited for a period of 2015, 3, 23 days. Compared with the prior art, the invention has the beneficial effects that: the strain XS1412C is a coryneform bacterium screened from abalone intestinal tracts, can secrete algin lyase, and the halophilic lactobacillus itself has the common probiotics of lactobacillus, can ferment to generate beneficial substances, has the coordination function with other strains, promotes the generation of enzyme, and can promote the growth of the strain XS1412C and the metabolite thereof and improve the enzyme production capacity thereof by the co-fermentation culture of the halophilic lactobacillus and the strain XS1412C in the same growth environment due to the common utilization of resources.
Drawings
FIG. 1 is a graph showing the change in enzyme activity of a strain in a fermentation broth of marine bacterial XS1412C strain at different culture temperatures in an example of the present application;
FIG. 2 shows growth curves of strains of marine bacteria XS1412C in fermentation broths of strain at different temperatures in examples of the present application;
FIG. 3 is a graph showing the change in enzyme activity of a strain in a fermentation broth of marine bacterium XS1412C strain at different culture times in the examples of the present application;
FIG. 4 shows the growth curves of strains of marine bacteria XS1412C in fermentation broth for different culture times in the examples of the present application;
FIG. 5 is a graph showing the change in enzyme activity of the strain in the fermentation broth of marine bacterium XS1412C strain at different inoculum sizes in the example of the application;
FIG. 6 is a graph showing the growth of strains of marine bacterium XS1412C in fermentation broth at various inoculum sizes in the example of the application;
FIG. 7 is a graph showing the change of enzyme activity of strains in symbiotic fermentation culture solutions at different culture temperatures in the examples of the present application;
FIG. 8 shows growth curves of strains in symbiotic fermentation broths at different temperatures in examples of the present application;
FIG. 9 is a graph showing the change of enzyme activity of strains in symbiotic fermentation culture solutions at different culture times in the examples of the present application;
FIG. 10 shows growth curves of strains in symbiotic fermentation broths at different cultivation times in examples of the present application;
FIG. 11 is a graph showing the change of enzyme activity of strains in symbiotic fermentation culture solutions under different inoculum sizes in the examples of the application;
FIG. 12 is a graph showing the growth of strains in symbiotic fermentation broths at different inoculum sizes in examples of the application;
FIG. 13 is a graph showing the change in enzyme activity of a strain in fermentation broth of marine bacterium XS1412C strain (without halophilic lactic acid bacteria) under optimal culture conditions and a growth curve of the strain in the examples of the present application;
FIG. 14 is a graph showing the change in enzyme activity of the strain in the symbiotic fermentation broth (inoculated with halophilic lactic acid bacteria) and the growth curve of the strain under the optimal culture conditions in the examples of the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
Example 1
(1) Marine bacterial xs1412C strain preculture: single colonies of the marine bacterial xs1412C strain were inoculated into the culture broth, and pre-cultured with shaking at 26 ℃ for 7 hours to obtain a high-activity marine bacterial xs1412C strain.
The culture solution comprises the following components: sodium alginate 2%, monopotassium phosphate 0.1%, ammonium chloride 0.2%, peptone 5%, and the treatment conditions are as follows: adding filtered seawater for dissolving.
(2) Preparation of marine bacterium XS1412C strain fermentation culture solution: inoculating the marine bacterial strain XS1412C cultured in the step (1) into a fermentation broth according to an inoculum size of 3%, and fermenting and culturing for 36 hours at 30 ℃ to prepare the marine bacterial strain XS1412C fermentation broth.
The components of the fermentation culture solution are as follows: 5% of peptone, 5% of glucose, 2% of sodium alginate, 0.1% of monopotassium phosphate, 0.4% of ammonium chloride and 0.38% of magnesium sulfate.
(3) Pre-culturing halophilic lactobacillus: the halophilic lactobacillus is inoculated into MRS liquid culture medium according to the inoculation amount of 2 percent, and is pre-cultured for 36 hours at the temperature of 28 ℃ to obtain the halophilic lactobacillus with high activity.
The MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 0.4g of dipotassium hydrogen phosphate, 5g of sodium acetate, 0.58g of magnesium sulfate, 20g of calcium carbonate, 0.29g of manganese sulfate, 1g of tween-80 and 2g of diammonium citrate, wherein the treatment conditions are as follows: pH 6.0, water to 1000mL, and autoclaving at 121℃for 20min.
(4) Symbiotic fermentation culture of halophilic lactobacillus-marine bacterium xs1412C strain: inoculating the halophilic lactobacillus cultured in the step (3) into the marine bacterium XS1412C strain fermentation culture solution prepared in the step (2) according to the inoculation amount of 3%, performing halophilic lactobacillus-marine bacterium XS1412C strain symbiotic fermentation culture, performing symbiotic fermentation culture for 36 hours at 30 ℃, and preparing symbiotic fermentation culture solution to produce algin lyase.
(5) And (3) centrifugal collection: centrifuging the symbiotic fermentation culture solution prepared in the step (4) at the temperature of 4 ℃ at the rotating speed of 8000r/min for 20min, and collecting supernatant, namely enzyme solution containing algin lyase.
After the test is finished, the enzyme activity, the biomass and the growth curve of the strain are measured according to the following method:
(1) Determination of bacterial strain enzyme activity:
The enzyme activity of the strain is measured by adopting a 3, 5-dinitrosalicylic acid (DNS) method, and the enzyme activity is measured by sampling every 6 hours. Definition unit of enzyme Activity (U/mL): the amount of enzyme required to release 1 μg of reducing sugar per minute is one enzyme activity unit (U).
The enzyme activity calculation formula:
Wherein: m is the amount of reducing sugar (mg) (from the standard curve according to the OD value); n is dilution multiple (enzyme solution is fermentation culture solution after centrifugation); t is the reaction time (20 min); v is the enzyme liquid volume (0.2 mL).
(2) Strain biomass and growth curve determination thereof
Continuously culturing the marine bacteria XS1412C strain fermentation culture solution in the step (2) or the symbiotic fermentation culture solution in the step (4), taking 200 mu L of fermentation culture solution every 6 hours, measuring the absorbance value (OD 600) of the fermentation culture solution at 600nm, and recording biomass.
And drawing by taking time as an abscissa and respectively taking enzyme activity and OD600 as an ordinate, namely an enzyme activity change graph and a strain growth graph.
Example 2
The present embodiment differs from example 1 in that: the culture temperature of the fermentation culture solution of the marine bacteria XS1412C strain in the step (2) is 25 ℃, and other steps are the same.
Example 3
The present embodiment differs from example 1 in that: the culture temperature of the fermentation culture solution of the marine bacteria XS1412C strain in the step (2) is 35 ℃, and other steps are the same.
Referring to fig. 1 and 2, the change of the enzyme activity of the strain in the fermentation broth of the marine bacterial XS1412C strain at different culture temperatures and the growth curve of the strain are shown, respectively, and as shown in examples 1,2 and 3 above, the strain grows slowly and has lower enzyme activity when the temperature is too low, and the strain grows faster in a short time when the temperature is too high, but the continuous higher temperature inhibits the growth of the strain and affects the enzyme production thereof, so the culture temperature of the fermentation broth of the marine bacterial XS1412C strain is preferably 30 ℃.
Example 4
The present embodiment differs from example 1 in that: the culture time of the fermentation culture solution of the marine bacterium XS1412C strain in the step (2) is 30 hours, and other steps are the same.
Example 5
The present embodiment differs from example 1 in that: the culture time of the fermentation culture solution of the marine bacterium XS1412C strain in the step (2) is 42 hours, and other steps are the same.
Referring to fig. 3 and 4, the change of the enzyme activity of the strain in the fermentation broth of the marine bacterial XS1412C strain and the growth curve of the strain are shown in the above examples 1, 4 and 5, respectively, wherein the strain is not in the optimal state for growth and enzyme production when the fermentation time is short, and the growth of the strain is slow due to the consumption of nutrients and the enzyme activity is reduced when the fermentation time is too long, so that the culture time of the fermentation broth of the marine bacterial XS1412C strain is preferably 36h.
Example 6
The present embodiment differs from example 1 in that: the inoculum size of the marine bacterium XS1412C strain in the step (2) was 1%, and the other steps were the same.
Example 7
The present embodiment differs from example 1 in that: the inoculum size of the marine bacterium XS1412C strain in the step (2) was 5%, and the other steps were the same.
Referring to FIGS. 5 and 6, which are respectively graphs of change in enzyme activity of the strain in fermentation broth of marine bacterium XS1412C strain and growth curves of the strain, according to examples 1, 6 and 7 above, when the inoculum size is too low, the enzyme productivity is low due to the limited number of strains, and when the inoculum size is too high, the strain activity is affected due to the limitation of nutrients, thereby affecting the enzyme production, so that the inoculum size of XS1412C strain in fermentation broth of marine bacterium XS1412C strain is preferably 3%.
In summary, the optimal culture conditions for the fermentation broth of marine bacteria xs1412C strain in step (2) are: the inoculation amount of the marine bacteria XS1412C strain is 3%, the culture temperature is 30 ℃ and the culture time is 36h.
Example 8
The present embodiment differs from example 1 in that: the culture temperature of the co-fermentation culture solution in the step (4) is 25 ℃, and other steps are the same.
Example 9
The present embodiment differs from example 1 in that: the culture temperature of the co-fermentation culture solution in the step (4) is 35 ℃, and other steps are the same.
Referring to FIGS. 7 and 8, the enzyme activity change pattern of the strain in the symbiotic fermentation broth and the growth curve of the strain at different culture temperatures are shown, respectively, and as can be seen from examples 1, 8 and 9 above, the culture temperature of the symbiotic fermentation broth is preferably 30 ℃.
Example 10
The present embodiment differs from example 1 in that: the culture time of the co-fermentation culture solution in the step (4) is 30 hours, and other steps are the same.
Example 11
The present embodiment differs from example 1 in that: the culture time of the co-fermentation culture solution in the step (4) is 42 hours, and other steps are the same.
Referring to fig. 9 and 10, the enzyme activity change pattern of the strain in the symbiotic fermentation broth and the growth curve of the strain at different cultivation times are shown, respectively, and as can be seen from examples 1, 10 and 11 above, the cultivation time of the symbiotic fermentation broth is preferably 36h.
Example 12
The present embodiment differs from example 1 in that: the inoculum size of the halophilic lactobacillus in the step (4) is 1%, and other steps are the same.
Example 13
The present embodiment differs from example 1 in that: the inoculum size of the halophilic lactobacillus in the step (4) is 5%, and the other steps are the same.
Referring to fig. 11 and 12, the enzyme activity change pattern of the strain in the symbiotic fermentation broth and the growth curve of the strain at different inoculum sizes are shown, respectively, and as can be seen from examples 1, 12 and 13 above, the inoculum size of halophilic lactic acid bacteria in the symbiotic fermentation broth is preferably 3%.
In summary, the optimal culture conditions for the co-fermentation broth in step (4) are: the inoculum size of the halophilic lactobacillus is 3 percent, the culture temperature is 30 ℃, and the culture time is 36 hours.
Referring to fig. 13 and 14, the enzyme activity change patterns and strain growth curves of the strain in the marine bacterial XS1412C strain fermentation broth (without inoculating halophilic lactobacillus) and the symbiotic fermentation broth (with inoculating halophilic lactobacillus) under the optimal culture conditions are respectively shown, so that the enzyme activity of the marine bacterial XS1412C strain fermentation broth (without inoculating halophilic lactobacillus) is 88.54U/mL, the OD600 value is 1.72 at most, the enzyme activity of the symbiotic fermentation broth (with inoculating halophilic lactobacillus) is 192.32U/mL, and the OD600 value is 2.33 at most, so that the growth of marine bacteria can be remarkably promoted after the fermentation culture of the halophilic lactobacillus and the marine bacterial XS1412C strain, and the enzyme production capacity of the marine bacteria can be remarkably improved.
According to the method for producing the algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, provided by the invention, the marine bacterial XS1412C strain is cultivated in advance, the marine bacterial XS1412C strain is inoculated into the fermentation culture solution to prepare the marine bacterial XS1412C strain fermentation culture solution, then the halophilic lactobacillus is cultivated in advance, the halophilic lactobacillus is inoculated into the marine bacterial XS1412C strain fermentation culture solution to carry out symbiotic fermentation culture, the symbiotic fermentation culture solution is obtained, and finally the enzyme solution containing the algin lyase is obtained by centrifuging the symbiotic fermentation culture solution.
The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application, and are intended to be included in the scope of the present application.
Claims (5)
1. A method for producing algin lyase by co-fermentation of halophilic lactobacillus and marine bacteria, which is characterized by comprising the following steps:
(1) Pre-culturing marine bacteria: inoculating marine bacteria into a culture solution for preculture;
the marine bacteria is an XS1412C strain;
the culture solution comprises the following components: sodium alginate 2%, monopotassium phosphate 0.1%, ammonium chloride 0.2%, peptone 5%, and the treatment conditions are as follows: adding filtered seawater for dissolution;
(2) Preparation of marine bacteria fermentation culture solution: inoculating the marine bacteria cultured in the step (1) into a fermentation culture solution for culturing to prepare a marine bacteria fermentation culture solution;
the components of the fermentation culture solution are as follows: 5% of peptone, 5% of glucose, 2% of sodium alginate, 0.1% of monopotassium phosphate, 0.4% of ammonium chloride and 0.38% of magnesium sulfate;
the inoculation amount of the marine bacteria is 1-5%, and the culture conditions of the marine bacteria fermentation culture solution are as follows: the temperature is 25-35 ℃ and the time is 30-42 h;
(3) Pre-culturing halophilic lactobacillus: inoculating halophilic lactobacillus into MRS liquid culture medium for preculture;
(4) Halophilic lactobacillus-marine bacterium symbiotic fermentation culture: inoculating the halophilic lactobacillus cultured in the step (3) into the marine bacterium fermentation culture solution prepared in the step (2), and performing halophilic lactobacillus-marine bacterium symbiotic fermentation culture to prepare a symbiotic fermentation culture solution;
the inoculation amount of the halophilic lactobacillus is 1-5%, and the conditions of the halophilic lactobacillus-marine bacterium symbiotic fermentation culture are as follows: the culture temperature is 25-35 ℃ and the culture time is 30-42 h;
(5) And (3) centrifugal collection: and (3) centrifuging the symbiotic fermentation culture solution prepared in the step (4), and collecting supernatant to obtain enzyme solution containing algin lyase.
2. The method for producing algin lyase by co-fermentation of halophilic lactic acid bacteria and marine bacteria according to claim 1, wherein the culture conditions of the marine bacteria in the step (1) are as follows: the culture temperature is 26 ℃ and the culture time is 7 hours.
3. The method for co-fermenting halophilic lactobacillus-marine bacteria to produce algin lyase according to claim 1, wherein the composition of the MRS liquid medium in the step (3) is: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 0.4g of dipotassium hydrogen phosphate, 5g of sodium acetate, 0.58g of magnesium sulfate, 20g of calcium carbonate, 0.29g of manganese sulfate, 1g of tween-80 and 2g of diammonium citrate, wherein the treatment conditions are as follows: pH 6.0, water to 1000mL, and autoclaving at 121℃for 20min.
4. The method for producing algin lyase by co-fermentation of halophilic lactic acid bacteria and marine bacteria according to claim 1, wherein the culture conditions of the halophilic lactic acid bacteria in the step (3) are as follows: the inoculation amount is 2%, the culture temperature is 28 ℃, and the culture time is 36h.
5. The method for producing algin lyase by co-fermentation of halophilic lactic acid bacteria and marine bacteria according to claim 1, wherein the centrifugation conditions in the step (5) are as follows: the temperature is 4 ℃, the rotating speed is 8000r/min, and the centrifugation is carried out for 20min.
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| CN103695341A (en) * | 2013-12-16 | 2014-04-02 | 江南大学 | Alginate lyase secreted from marine bacterium and preparation method thereof |
| CN104195080A (en) * | 2014-08-23 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | Bacillus sp capable of producing alginate lyase and application thereof |
| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN111961619A (en) * | 2020-08-19 | 2020-11-20 | 天津科技大学 | Vibrio maritima capable of producing alginate lyase with good thermal stability and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695341A (en) * | 2013-12-16 | 2014-04-02 | 江南大学 | Alginate lyase secreted from marine bacterium and preparation method thereof |
| CN104195080A (en) * | 2014-08-23 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | Bacillus sp capable of producing alginate lyase and application thereof |
| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN111961619A (en) * | 2020-08-19 | 2020-11-20 | 天津科技大学 | Vibrio maritima capable of producing alginate lyase with good thermal stability and application |
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