CN111235073A - Lactobacillus plantarum producing inulinase and application thereof - Google Patents
Lactobacillus plantarum producing inulinase and application thereof Download PDFInfo
- Publication number
- CN111235073A CN111235073A CN202010237731.3A CN202010237731A CN111235073A CN 111235073 A CN111235073 A CN 111235073A CN 202010237731 A CN202010237731 A CN 202010237731A CN 111235073 A CN111235073 A CN 111235073A
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- CN
- China
- Prior art keywords
- lactobacillus
- acid
- plantarum3
- inulase
- inulin
- Prior art date
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Abstract
The invention discloses a Lactobacillus plantarum3-2 producing acid inulinase and application thereof, wherein the Lactobacillus plantarum is preserved in China Center for Type Culture Collection (CCTCC) in 11 and 25 months in 2019 with the preservation number of CCTCC NO: m2019972. The Lactobacillus plantarum3-2 is screened from Guangxi Zhuang traditional food raw rice flour, grows well in a fermentation culture medium taking inulin as a carbon source and beef extract as a nitrogen source, has strong acid resistance, has a survival rate of 96% after 1h under the condition of pH4.5, and has the capacity of degrading inulin and levan substances and preparing high fructose syrup. The invention reports lactobacillus plantarum producing acid inulase for the first time, and provides a new strain and inulase for industrial degradation of inulin and fructan substances and preparation of high fructose syrup.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to the field of development of functional lactic acid bacteria and production and development of products thereof. In particular to Lactobacillus plantarum3-2 for producing acid inulase and application thereof.
Background
Inulin is a natural fructan mixture, is formed by connecting fructose furanose through β -2, 1-glycosidic bond, and is linked with glucose at the reduction end, the polymerization degree is generally 2-60, the inulin is widely distributed in dicotyledonous plants such as Compositae and Campanulaceae and monocotyledonous plants such as Liliaceae and Gramineae, especially inulin in Jerusalem artichoke and chicory accounts for more than 90% of the dry weight of carbohydrate, is rich and excellent fructosyl renewable resource, can be used for producing bioethanol, high fructose syrup, fructo oligosaccharide, acetone-butanol, succinic acid, mannitol and the like (ursowood,wangchenghua2013,11(3):40-45.) inulinase is an enzyme capable of specifically cutting β -2, 1-glycosidic bond in fructan such as inulin, and can be divided into two major classes of exoinulinase and endo-inulinase according to different cutting modes, endo-inulinase is a fructoside bond randomly acting in inulin, the product is mainly fructo-oligosaccharide, exoinulinase (EC 3.2.1.80) cuts off fructosyl one by one from the glycosidic bond at the non-reducing end of the inulin chain to form a fermentable carbon source and D-fructoseHas important application prospect and economic value.
There are many microorganisms that can produce exoinulinase in nature, such as bacteria, fungi and yeasts, and most of the current researches are aspergillus (Aspegillius sp.), Penicillium (Penicillium sp.) and Kluyveromyces (Kluyveromyces sp.), but the researches on the acid inulinase from food-grade microorganisms are rarely reported. In 2002, Paludan-Muller et al reported for the first time a Strain of Lactobacillus pentosus producing weakly acidic inulinase, which is also the only food-derived acid inulinase from lactic acid bacteria (Christin agarose-Muller, Lone Gram, feed P.ray.purification and characterization of an extracellular fructan b-fractional from a Lactobacillus pentosus Strain Isolated from fermented Fish.systematics and Applied Microbiology,2002,25(1): 13-20.).
The raw rice flour is a traditional fermented food with the characteristics of Guangxi Zhuang nationality, and contains abundant lactic acid bacteria resources. The method screens the Guangxi characteristic raw rice flour source lactic acid bacteria library constructed in the early stage of the laboratory at a high throughput, screens out novel acid-producing inulase lactic acid bacteria, and applies the novel acid-producing inulase lactic acid bacteria to the field of foods, particularly to the production and preparation of acid inulase, and the degradation of fructan substances, such as inulin, to produce high fructose syrup and the like.
Disclosure of Invention
An object of the invention is to provide a new lactobacillus plantarum3-2 producing acid inulinase and application thereof. The strain has the capability of producing acid inulase by fermentation and degrading fructan substances to prepare products such as fructose and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
lactobacillus plantarum3-2, the sequence of which is as follows:
and is preserved in China center for type culture Collection in 2019, 11 months and 25 days, with the preservation number of CCTCC NO: m2019972, address: eight-way 299 in Wuchang area, Wuhan university Collection, postcode: 430072.
the lactobacillus plantarum CCTCC NO: m2019972, which is obtained by separating from traditional raw rice-pressed rice noodles of Guangxi Nanning Zhuang nationality from the early stage of the laboratory.
The lactobacillus plantarum CCTCC NO: m2019972, good growth on MRS solid medium, milky colony, smooth surface, neat edge, and opaque.
The lactobacillus plantarum CCTCC NO: m2019972, adopting the 27F/1492R of the bacterial universal primer 16S rDNA to carry out PCR amplification and sequencing on the genome DNA, obtaining the gene sequence of 1476bp, as shown in SEQ ID No. 1. BLAST analysis showed that it was 99.59% similar to Lactobacillus plantarum JL-1.
The use of the above-mentioned strains for the production of inulinase is also within the scope of the present invention.
Another object of the present invention is the use of Lactobacillus plantarum3-2 for the production of inulinase.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. the application of Lactobacillus plantarum3-2 for producing the acid-producing inulase in producing the inulase comprises the following steps:
(1) seed culture
Lactobacillus plantarum3-2 is streaked to an MRS solid culture medium, and is subjected to static culture at the temperature of 30 ℃ until single colonies appear.
The formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization at the pH of 6.6-6.8 and the temperature of 121 ℃ for 15 min;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
glucose stock solution with final concentration of 2% is added at the time of use.
(2) Fermentation culture
And (4) selecting a single colony which normally grows in the solid culture medium, streaking the single colony into the solid fermentation culture medium, and carrying out static culture at the temperature of 30 ℃ until the single colony appears.
The solid fermentation medium comprises the following components:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min.
And (3) selecting a single colony which normally grows on the solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at 30 ℃ to obtain fermentation liquor.
(3) Preparation of acid inulinase
Sucking 10mL of fermentation liquid, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, and keeping precipitate. Adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate for resuspending the precipitate, then adding 200 mu L of lysozyme of 100mg/ml, standing for 2h at 30 ℃, then adding 0.12g of quartz sand, then grinding for 240s at-30 ℃ in a high-throughput tissue grinder, after cell breaking, centrifuging for 30min at 4 ℃ and 13000r/min, and taking supernatant, namely the acid inulase.
In order to achieve the above objects, another technical means adopted by the present invention is,
2. the application of Lactobacillus plantarum3-2 for producing the acid-producing inulase in producing the inulase comprises the following steps:
(1) seed culture
Streaking Lactobacillus plantarum3-2 into an MRS solid culture medium, and performing static culture at the temperature of 30 ℃ until a single colony appears; the formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
when in use, the inulin stock solution with the final concentration of 2% is added;
(2) fermentation culture
Streaking a single colony in an MRS solid culture medium into a solid fermentation culture medium, and performing static culture at the temperature of 30 ℃ until the single colony appears; selecting a single colony which normally grows on a solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at the temperature of 30 ℃ to obtain a fermentation liquid; the solid fermentation medium comprises the following components:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1mL of tween-801, 1000mL of distilled water, 6.6-6.8 of pH, and autoclaving at 121 ℃ for 15 min;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
adding inulin solution with final concentration of 2% when in use;
(3) preparation of acid inulinase
Absorbing 10mL of fermentation liquor, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, keeping precipitate, adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate, suspending the precipitate, adding 200 mu L of 100mg/mL lysozyme, standing for 2h at 30 ℃, adding 0.12g of quartz sand, grinding for 240s at-30 ℃ in a high-throughput tissue grinder, centrifuging for 30min at 4 ℃ and 13000r/min after cell breaking, and taking supernatant, namely the acid inulase.
The optimal reaction temperature of the acid inulase prepared by the method is 35 ℃, the activity of the acid inulase can be kept more than 50% at 20-40 ℃ for 30min, the optimal pH value is 4.5, the activity of the acid inulase can be kept more than 65% at pH value of 3.5-8, and the pH tolerance range is wide.
Wherein, the enzyme activity of inulinase is defined as follows: under the conditions of optimal temperature and optimal pH, the enzyme amount required for generating 1 mu mol of reducing sugar per minute is 1 enzyme activity unit (U).
It is another object of the present invention to provide the use of a fermentation broth of Lactobacillus plantarum3-2 for the degradation of fructan-like substances, preferably inulin-like substances, including the use in inulin fructose and inulin high fructose syrups.
The application of the acid inulase produced by Lactobacillus plantarum3-2 fermentation in degrading fructan substances, preferably inulin substances, including the application in inulin fructose and inulin high fructose syrup, is within the protection scope of the invention.
The invention has the beneficial effects that:
(1) the lactobacillus plantarum for producing the acid-producing inulase is provided, the strain takes inulin as a carbon source, beef extract as a nitrogen source and the like to form a fermentation culture medium, the fermentation culture medium is kept stand and fermented at the temperature of 30 ℃ at the pH value of 6.6-6.8 to produce the acid-producing inulase, and fermentation liquor and enzyme have both inulase activity and invertase activity.
(2) The plant lactobacillus acidic inulinase has the optimum pH value of 4.5 and higher stability in a wide range of pH 3.5-pH 8.0, and is particularly suitable for hydrolyzing fructan substances under acidic conditions, such as inulin and other industrial applications.
Drawings
FIG. 1 shows the Lactobacillus plantarum CCTCC NO: colony morphology map of M2019972
FIG. 2 is a graph showing the change of the enzyme activity of acid inulinase with temperature in example 3
FIG. 3 is a graph showing the residual enzyme activity of acid inulinase in example 3 as a function of treatment temperature
FIG. 4 is a graph showing the change of the enzyme activity of acid inulinase with pH in example 4
FIG. 5 is a graph showing the pH-dependent change in residual enzyme activity of acid inulinase in example 4
Detailed Description
The present invention will be described in further detail with reference to specific examples.
EXAMPLE 1 cultivation and characterization of the Strain of inulinase acidogenic Lactobacillus plantarum3-2
(1) Culture of acid inulinase-producing Lactobacillus plantarum3-2 Strain
Taking out 3-2 lactobacillus plantarum screened from traditional raw pressed rice flour of Guangxi Zhuang nationality at the earlier stage in a ultralow-temperature refrigerator at minus 80 ℃, inoculating the lactobacillus plantarum in an MRS solid culture medium (10 g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium hydrogen phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 801 mL of Tween-801, 20g of agar, 1000mL of distilled water, 6.6-6.8 of pH, autoclaving at 121 ℃ for 15min by using an inoculating loop streak, adding 2% of glucose when in use, and culturing at 30 ℃ until a single colony appears; a single colony is picked by an inoculating loop and inoculated into a solid fermentation culture medium (10 g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 801 mL of tween, 20g of agar, 1000mL of distilled water, pH 6.6-6.8, autoclaving at 121 ℃ for 15min, adding 2% inulin when in use) with inulin as a unique carbon source by streaking, and culturing at 30 ℃ until the single colony appears.
(2) Identification of acid inulase producing Lactobacillus plantarum3-2 Strain
As shown in figure 1, the bacterial colony characteristics of the Lactobacillus plantarum3-2 strain in an MRS culture medium taking inulin as a unique carbon source belong to typical Lactobacillus plantarum, and the bacterial colony is milky white, smooth and fine in surface, slightly convex, neat in edge and opaque.
The lactobacillus plantarum3-2 genome DNA is used as a template, and the 16S rRNA gene sequence is amplified by universal primers 27F and 1492R to carry out the molecular identification of the strain. The specific sequences of the primers 27F and 1492R are respectively SEQ ID No.2 and SEQ ID No.3 of the sequence table. A PCR reaction system was constructed according to Table 1, and amplification was carried out by means of a PCR thermal cycler, the amplification procedure being: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, 30 cycles, and final extension at 72 deg.C for 2 min. After the PCR product was purified according to the procedure of the DNA product purification kit, forward and reverse sequencing was performed by the Kyowa Dagene technology Co., Ltd. (Guangzhou department of sequencing). Sequencing results show that the 16s rRNA gene of the lactobacillus plantarum3-2 consists of 1476bp, and the specific sequence is shown as SEQ ID No. 1.
TABLE 1 PCR reaction System
Lactobacillus plantarum3-2, which is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 11 and 25 months, with the preservation number of CCTCC NO: m2019972, address: eight-way 299 in Wuchang area, Wuhan university Collection, postcode: 430072.
example 2
The application of Lactobacillus plantarum3-2 for producing the acid-producing inulase in producing the inulase comprises the following steps:
(1) seed culture
Lactobacillus plantarum3-2 is streaked to an MRS solid culture medium, and is subjected to static culture at the temperature of 30 ℃ until single colonies appear.
The formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization at the pH of 6.6-6.8 and the temperature of 121 ℃ for 15 min;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
glucose stock solution with final concentration of 2% is added at the time of use.
(2) Fermentation culture
And (4) selecting a single colony which normally grows in the solid culture medium, streaking the single colony into the solid fermentation culture medium, and carrying out static culture at the temperature of 30 ℃ until the single colony appears.
The solid fermentation medium comprises the following components:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min.
And (3) selecting a single colony which normally grows on the solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at 30 ℃ to obtain fermentation liquor.
(3) Preparation of acid inulinase
Sucking 10mL of fermentation liquid, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, and keeping precipitate. Adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate for resuspending the precipitate, then adding 200 mu L of lysozyme of 100mg/ml, standing for 2h at 30 ℃, then adding 0.12g of quartz sand, then grinding for 240s at-30 ℃ in a high-throughput tissue grinder, after cell breaking, centrifuging for 30min at 4 ℃ and 13000r/min, and taking supernatant, namely the acid inulase.
(4) Determination of the enzyme Activity of inulinase
The enzyme activity determination method comprises the following steps: adding 100 μ L acid inulase into 200 μ L0.5% inulin solution prepared with pH 5.0 and 0.2M acetic acid buffer solution and 200 μ L0.5% sucrose solution prepared with pH 5.0 and 0.2M acetic acid buffer solution, reacting in 30 deg.C water bath for 30min, immediately boiling water bath for 5min to terminate the reaction, adding 300 μ L DNS into the reaction solution, boiling water bath for 7min, rapidly cooling, and measuring the amount of reducing sugar generated at 540nm to obtain enzyme solution inactivated by heating in boiling water bath as blank control. Definition of enzyme activity: under the conditions of optimal temperature and optimal pH, the enzyme amount required for generating 1 mu mol of reducing sugar per minute is 1 enzyme activity unit (U). One inulase activity (I) unit is defined as the amount of enzyme required to catalyze the conversion of inulin to 1. mu. mol fructose per minute and one invertase activity (S) unit is defined as the amount of enzyme required to catalyze the conversion of sucrose to 1. mu. mol fructose per minute. The protein content was measured by using a BCA protein quantification kit (Shiji Beijing kang, Inc.) using BSA (bovine serum albumin) as a standard protein.
As shown in Table 2, the specific activity of acid inulase produced by fermentation of Lactobacillus plantarum3-2 was 0.07U/mg, and the I/S value was 0.23, indicating that it is an exoinulase.
TABLE 2 acid inulinase enzyme activity, invertase activity, total protein content and specific enzyme activity results
Example 3
The application of Lactobacillus plantarum3-2 for producing the acid-producing inulase in producing the inulase comprises the following steps:
(1) seed culture
Streaking Lactobacillus plantarum3-2 into an MRS solid culture medium, and performing static culture at the temperature of 30 ℃ until a single colony appears; the formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
the inulin stock solution with final concentration of 2% is added for application.
(2) Fermentation culture
Streaking a single colony in an MRS solid culture medium into a solid fermentation culture medium, and performing static culture at the temperature of 30 ℃ until the single colony appears; selecting a single colony which normally grows on a solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at the temperature of 30 ℃ to obtain a fermentation liquid; the solid fermentation medium comprises the following components:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1mL of tween-801, 1000mL of distilled water, 6.6-6.8 of pH, and autoclaving at 121 ℃ for 15 min;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
inulin solution with final concentration of 2% is added at the time of use.
(3) Preparation of acid inulinase
Absorbing 10mL of fermentation liquor, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, keeping precipitate, adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate, suspending the precipitate, adding 200 mu L of 100mg/mL lysozyme, standing for 2h at 30 ℃, adding 0.12g of quartz sand, grinding for 240s at-30 ℃ in a high-throughput tissue grinder, centrifuging for 30min at 4 ℃ and 13000r/min after cell breaking, and taking supernatant, namely the acid inulase.
Example 4
Optimum reaction temperature and temperature stability of Lactobacillus plantarum3-2 acid inulinase
The preparation of acid inulinase was performed as in example 2, and the optimum reaction temperature was determined by adding 200. mu.L of 0.5% inulin solution prepared with pH 5.0, 0.2M acetate buffer to 100. mu.L acid inulinase, reacting at 20 ℃,25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃ for 30min, immediately boiling in a water bath for 5min to terminate the reaction, measuring the enzyme activity by DNS method, and calculating the relative enzyme activity of each measurement group with the highest enzyme activity of the measurement results as 100%. 3 parallel samples were set at each temperature.
The temperature tolerance determination method of acid inulase is that 100 mu L of acid inulase is kept at the temperature ranging from 20 ℃ to 80 ℃ for 30min, then 200 mu L of 0.5% inulin solution prepared by using acetic acid buffer solution with pH 5.0 and 0.2M is added to react at the temperature of 30 ℃ for 30min, finally, the DNS method is used for determining the amount of generated reducing sugar, and the residual enzyme activity under different temperature treatment conditions is calculated by taking the highest enzyme activity of the determination result as 100%. 3 parallel samples were set at each temperature.
The results are shown in FIG. 2, the optimum reaction temperature of Lactobacillus plantarum3-2 inulase is 35 ℃, the temperature is basically unchanged within the range of 30-40 ℃, and the enzyme activity is sharply reduced when the temperature exceeds 40 ℃. As shown in figure 3, the activity of the acid inulase can be maintained at more than 60% below 40 ℃, but the enzyme activity is rapidly reduced at 45 ℃, and the enzyme activity is less than 30% of the original enzyme activity.
Example 5
Optimum reaction pH and pH stability of Lactobacillus plantarum3-2 acid inulinase
The procedure for preparing acid inulinase was the same as in example 2, and the optimum reaction pH was determined by preparing buffers of different pH values, pH 3.5, 4.0, 4.5 and 5.0 were acetate buffers, pH 5.5, 6.0, 6.5 and 7.5 were phosphate buffers, pH 8.0, 8.5 and 9 were Tris-HCl buffers, and pH 9.5, 10.0, 10.5 and 11 were carbonate buffers. Reacting 100 mu L of acid inulase with 200 mu L of inulin solution prepared by buffering at different pH values at 30 ℃ for 30min, immediately putting into boiling water to terminate the reaction, measuring the enzyme activity by using a DNS method, and calculating the relative enzyme activity of each measurement group by taking the highest enzyme activity of the measurement results as 100%. 3 replicates were set for each pH. And (3) measuring the pH stability, namely respectively mixing 50 mu L of acid inulase with 50 mu L of buffer solutions with different pH values, standing at 4 ℃ for 1h, adding 200 mu L of inulin solution, reacting at 30 ℃ for 30min, immediately putting into boiling water to stop the reaction, measuring the enzyme activity by using a DNS method, and calculating the relative enzyme activity of each measurement group by taking the highest enzyme activity of the measurement result as 100%. 3 replicates were set for each pH.
As shown in FIG. 4, the optimum reaction pH of the Lactobacillus plantarum3-2 inulinase was 4.5, and the enzyme activity was maintained at 90% or more relative to the enzyme activity in the acidic range of pH4.5 to 6.5, and at 65% or more relative to the enzyme activity in the range of pH 3.5 to 8.0. As shown in FIG. 5, the Lactobacillus plantarum3-2 inulase has a wide pH tolerance range, the enzyme activity is not affected by the pH value within the pH range of 3.5-6.0, can keep more than 95% of the activity, can keep more than 65% of the activity within the pH range of 6.5-8.5, but the enzyme activity is sharply reduced when the pH value is higher than 8.5.
Example 6
Degrading fructan substances by using Lactobacillus plantarum3-2 fermentation liquor
Standing and fermenting Lactobacillus plantarum3-2 at 30 ℃ for 1-2 days to prepare fermentation liquor, and degrading fructan substances by utilizing the fermentation liquor. Taking 2% inulin type fructan as a substrate, mixing 100 mu L fermentation liquor with 200 mu L substrate, reacting for 1h at 35 ℃, taking the fermentation liquor which is heated and inactivated in a boiling water bath as a blank control, and detecting the concentration of reducing sugar in the product to be 310 mu mol/L by using a DNS method, thereby proving that the Lactobacillus plantarum3-2 fermentation liquor has the capability of degrading fructan substances.
Example 7
Inulin degradation by Lactobacillus plantarum3-2 fermentation liquor
Standing and fermenting Lactobacillus plantarum3-2 at 30 ℃ for 1-2 days to prepare fermentation liquor, and degrading inulin by using the fermentation liquor. Mixing 100 mu L of fermentation liquor with 200 mu L of substrate by taking 2% inulin as a substrate, reacting for 1h at 35 ℃, taking the fermentation liquor subjected to heating inactivation in a boiling water bath as a blank control, and detecting that the concentration of reducing sugar in the product is 307 mu mol/L by using a DNS method, thereby proving that the Lactobacillus plantarum3-2 fermentation liquor has the capacity of degrading inulin.
Example 8
The acidic inulinase produced by Lactobacillus plantarum3-2 is utilized to degrade fructan substances.
The preparation method of the acid inulase is the same as that of the example I, and the acid inulase is used for degrading fructan substances. Taking 2% inulin type fructan as a substrate, mixing 100 mu L acid inulase and 200 mu L substrate, reacting for 1h at 35 ℃, taking enzyme inactivated by heating in boiling water bath as a blank control, and detecting the concentration of reducing sugar in the product to be 658 mu mol/L by using a DNS method, thereby proving that the acid inulase generated by Lactobacillus plantarum3-2 has the capability of degrading fructan substances.
Example 9
Degrading inulin by using acid inulase generated by Lactobacillus plantarum 3-2.
The preparation method of acid inulinase is the same as that of the example I, and the acid inulinase is used for degrading inulin. Taking 2% inulin as a substrate, mixing 100 mu L of acid inulase with 200 mu L of substrate, reacting for 1h at 35 ℃, taking enzyme inactivated by heating in boiling water bath as a blank control, and detecting the concentration of reducing sugar in the product to be 698 mu mol/L by using a DNS method, thereby proving that the acid inulase generated by lactobacillus plantarum3-2 has the capacity of degrading inulin.
Example 10
Preparation of high fructose syrup by using acid inulinase produced by Lactobacillus plantarum3-2
The procedure for the preparation of acid inulinase was the same as in example 1. Acid inulase is put in an ice bath at 4 ℃, salting out is carried out by using ammonium sulfate with the saturation of 40% and then centrifuging at 4 ℃ to remove impure proteins, a small amount of precipitate is generated, and only a small amount of inulase activity exists, which indicates that most of inulase still remains in fermentation liquor, when salting out is continued by using ammonium sulfate with the saturation of 80%, enzyme activity hardly exists in supernate, which indicates that most of target proteins are precipitated, and target proteins are dissolved by using buffer solution to obtain primary pure enzyme solution. Hydrolyzing inulin with the primary pure enzyme solution to prepare high fructose syrup. Mixing 100 mu L of primary pure enzyme solution and 200 mu L of substrate by taking 2% inulin as a substrate, reacting for 10h at 35 ℃, and detecting a hydrolysate by using thin-layer chromatography (TLC), wherein a developing agent is n-butyl alcohol, isopropanol, acetic acid and water which are mixed according to a volume ratio of 7:5:1:2, and a color developing agent is 2% diphenylamine acetone solution, 2% aniline acetone solution and 85% phosphoric acid which are mixed according to a ratio of 5:5: 1.
The thin-layer chromatography result shows that the standard products of inulin and fructose are dyed red, glucose is dyed blue, the migration distance of inulin is lower than that of fructose and glucose, the fructose is equivalent to the glucose, and almost 100 percent of inulin is hydrolyzed into fructose by the acid-producing inulase of Lactobacillus plantarum3-2, which is the same as the hydrolysis result of the positive control of Leuconostoc citreum 1-1 inulase, which shows that the acid-producing inulase of Lactobacillus plantarum3-2 can be used for preparing high-fructose syrup.
The present invention is not limited to the above-described technical solutions, and any modifications to the present invention, including various simple modifications to the technical solutions, fall within the scope of the present invention.
It should be noted that the various features described in the foregoing detailed description may be combined in any suitable manner without contradiction, and various possible combinations will not be further described in order to avoid unnecessary repetition.
In addition, various embodiments of the present invention can be arbitrarily combined with each other, and the same should be regarded as the disclosure of the present invention as long as the idea of the present invention is not violated.
Sequence listing
<110> Nanning Zhongnuo bioengineering, LLC
<120> lactobacillus plantarum producing acid inulase and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1476
<212>DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum 3-2)
<400>1
gggcgtgggg cgggcctata catgcaagtc gaacgagctc tggtattgat tggtgcttgc 60
atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca 120
gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg 180
gtccgagctt gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct 240
agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg 300
gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360
cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt 420
aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480
taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540
gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600
gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac 660
agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720
ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt 780
agataccctg gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc 840
ttcagtgctg cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa 900
actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960
acgcgaagaa ccttaccagg tcttgacata ctatgcaaat ctaagagatt agacgttccc 1020
ttcggggaca tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080
gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattaag ttgggcactc 1140
tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200
cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc gaactcgcga 1260
gagtaagcta atctcttaaa gccattctca gttcggattg taggctgcaa ctcgcctaca 1320
tgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380
ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagtcgg tggggtaacc 1440
tttagaacca gccgcctaag gtgaccaaga atcgct 1476
Claims (7)
1. Lactobacillus plantarum3-2 producing inulinase, and is characterized in that the sequence is as follows:
the strain is preserved in China center for type culture Collection in 2019, 11 and 25 months, and the preservation number is CCTCC NO: m201997.
2. Use of Lactobacillus plantarum3-2 according to claim 1 for the production of acid inulase, characterized in that it comprises the following steps:
(1) seed culture
Streaking Lactobacillus plantarum3-2 into an MRS solid culture medium, and performing static culture at the temperature of 30 ℃ until a single colony appears; the formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization at the pH of 6.6-6.8 and the temperature of 121 ℃ for 15 min;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
when in use, a glucose stock solution with the final concentration of 2% is added;
(2) fermentation culture
Streaking a single colony in an MRS solid culture medium into a solid fermentation culture medium, and performing static culture at the temperature of 30 ℃ until the single colony appears; selecting a single colony which normally grows on a solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at the temperature of 30 ℃ to obtain a fermentation liquid; the solid fermentation medium comprises the following components:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% glucose stock solution: preparing 200g/L glucose solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
(3) preparation of acid inulinase
Absorbing 10mL of fermentation liquor, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, keeping precipitate, adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate, suspending the precipitate, adding 200 mu L of 100mg/mL lysozyme, standing for 2h at 30 ℃, adding 0.12g of quartz sand, grinding for 240s at-30 ℃ in a high-throughput tissue grinder, centrifuging for 30min at 4 ℃ and 13000r/min after cell breaking, and taking supernatant, namely the acid inulase.
3. Use of Lactobacillus plantarum3-2 according to claim 2 for the production of acid inulase, characterized in that it comprises the following steps:
(1) seed culture
Streaking Lactobacillus plantarum3-2 into an MRS solid culture medium, and performing static culture at the temperature of 30 ℃ until a single colony appears; the formula of the MRS solid culture medium is as follows:
the formula of the MRS solid culture medium is as follows:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 20g of tween-801 mL of agar and 1000mL of distilled water, and carrying out high-pressure sterilization for 15min under the conditions of pH 6.6-6.8 and 121 ℃;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
when in use, the inulin stock solution with the final concentration of 2% is added;
(2) fermentation culture
Streaking a single colony in an MRS solid culture medium into a solid fermentation culture medium, and performing static culture at the temperature of 30 ℃ until the single colony appears; selecting a single colony which normally grows on a solid fermentation culture medium, inoculating the single colony into a liquid fermentation culture medium, and standing and fermenting for 1-2 days at the temperature of 30 ℃ to obtain a fermentation liquid; the solid fermentation medium comprises the following components:
the liquid fermentation medium comprises the following components in parts by weight:
10g of beef extract, 5g of yeast powder, 10g of peptone, 2g of dipotassium phosphate, 2.0g of diammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1mL of tween-801, 1000mL of distilled water, 6.6-6.8 of pH, and autoclaving at 121 ℃ for 15 min;
20% inulin stock solution: preparing 200g/L inulin solution with ultrapure water, and autoclaving at 121 deg.C for 20 min;
adding inulin solution with final concentration of 2% when in use;
(3) preparation of acid inulinase
Absorbing 10mL of fermentation liquor, centrifuging for 5min at 4 ℃ and 10000r/min, discarding supernatant, keeping precipitate, adding 400 mu L of acetic acid buffer solution of 0.2mol/L, pH 5.0.0 into the precipitate, suspending the precipitate, adding 200 mu L of 100mg/mL lysozyme, standing for 2h at 30 ℃, adding 0.12g of quartz sand, grinding for 240s at-30 ℃ in a high-throughput tissue grinder, centrifuging for 30min at 4 ℃ and 13000r/min after cell breaking, and taking supernatant, namely the acid inulase.
4. Use of Lactobacillus plantarum3-2 according to claim 2 for the production of acid inulase, characterized in that: the optimum reaction temperature is 35 ℃, and the optimum reaction pH is 4.5.
5. Use of Lactobacillus plantarum3-2 according to claim 2 for the production of acid inulase, characterized in that: application of fermentation liquor of Lactobacillus plantarum3-2 in degrading fructan substances.
6. Use of Lactobacillus plantarum3-2 according to claim 2 for the production of acid inulase, characterized in that: application of fermentation liquor of Lactobacillus plantarum3-2 in degrading inulin.
7. Use of a fermentation broth of Lactobacillus plantarum3-2 according to claim 2 for the production of acid inulase, characterized in that acid inulase is used for the production of high fructose syrup.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110804575A (en) * | 2019-12-11 | 2020-02-18 | 广西大学 | Leuconostoc citreum for producing acid inulase and application thereof |
| CN113481278A (en) * | 2021-06-15 | 2021-10-08 | 四川省食品检验研究院 | Method for simultaneously determining activity of sucrase and activity of fructanase |
| CN113832072A (en) * | 2021-10-20 | 2021-12-24 | 江南大学 | Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof |
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| CN110804575A (en) * | 2019-12-11 | 2020-02-18 | 广西大学 | Leuconostoc citreum for producing acid inulase and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804575A (en) * | 2019-12-11 | 2020-02-18 | 广西大学 | Leuconostoc citreum for producing acid inulase and application thereof |
| CN113481278A (en) * | 2021-06-15 | 2021-10-08 | 四川省食品检验研究院 | Method for simultaneously determining activity of sucrase and activity of fructanase |
| CN113481278B (en) * | 2021-06-15 | 2022-04-12 | 四川省食品检验研究院 | Method for simultaneously determining activity of sucrase and activity of fructanase |
| CN113832072A (en) * | 2021-10-20 | 2021-12-24 | 江南大学 | Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof |
| CN113832072B (en) * | 2021-10-20 | 2023-10-27 | 江南大学 | Lactococcus lactis subspecies lactate with inulin utilization capacity and application thereof |
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