CN112501049B - Lactobacillus kefir producing transglycosylation active beta-galactosidase and method for producing galactooligosaccharides by using prepared beta-galactosidase - Google Patents

Lactobacillus kefir producing transglycosylation active beta-galactosidase and method for producing galactooligosaccharides by using prepared beta-galactosidase Download PDF

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CN112501049B
CN112501049B CN202011173142.XA CN202011173142A CN112501049B CN 112501049 B CN112501049 B CN 112501049B CN 202011173142 A CN202011173142 A CN 202011173142A CN 112501049 B CN112501049 B CN 112501049B
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关波
胡有贞
韩明明
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Xinjiang Chuangyi Biotechnology Co ltd
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Abstract

The invention discloses a bacterial strain for producing transglycosylation active beta-galactosidase, which is characterized in that: the classification name is Lactobacillus kefir L6, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO of M20191030, and the preservation date is 12 months and 9 days in 2019.

Description

Lactobacillus kefir producing transglycosylation active beta-galactosidase and method for producing galactooligosaccharides by using prepared beta-galactosidase
Technical Field
The invention relates to the food biotechnology field, in particular to a bacterial strain for producing transglycosylation active beta-galactosidase and a method for producing galactooligosaccharides by using the same in a wider temperature range
Background
Galacto-oligosaccharides (GOS) are a mixture of oligosaccharides containing one or more galactosyl moieties linked to the glucose termini, or a mixture of oligosaccharides formed from galactose units alone (galactose disaccharide, galactose trisaccharide, etc.), a prebiotic that modulates the intestinal microflora to confer a health benefit to the host. The existing research shows that the GOS can promote beneficial bacteria (such as lactobacillus and bifidobacterium) in the intestinal tract of the human body to exert the functions to the maximum extent; promoting intestinal peristalsis; reducing the incidence of intestinal disease; the formation of harmful substances such as phenol in the digestive tract is delayed, so that the harmful substances are difficult to rapidly expand through blood, and the life of a patient with renal failure is prolonged; reducing the genetic disease incidence of infants and the like.
Due to its unique processing property, low calorie, and relatively stable to acid and heat, galactooligosaccharide can be used as a naturally occurring Human Milk Oligosaccharide (HMOS), and so on, and at present, infant formula milk powder added with galactooligosaccharide to simulate physiological effects in human milk as much as possible is on the market in large quantities. It is estimated that global consumption of GOS is around 20000 t/year and increases at a rate of 10% -20% per year. The GOS has a wide development prospect both for functional food development and for assistance of body functions.
Currently, the obtaining way of GOS is very limited due to the special complex structure, and the probiotic characteristics of the galactooligosaccharides with different types of glycosidic bonds and polymerization degrees are different. The naturally occurring GOS content in food products is generally very low and is now mainly synthesized by enzymatic methods: prepared by reacting beta-galactosidase (EC 3.2.1.23) with transglycosylation activity and lactose with certain concentration. Commercial β -galactosidase preparations of synthetic GOS are now mainly derived from Aspergillus oryzae (Aspergillus oryzae), Kluyveromyces lactis (Kluyveromyces lactis) and Bacillus circulans (Bacillus circulans).
Although galacto-oligosaccharides synthesized by beta-galactosidase from different sources are on the market, the galacto-oligosaccharides synthesized by the enzyme method still have the problems of low GOS yield, more lactose residues, high preparation cost and the like. For example, commercial galactooligosaccharide Oligomat55 derived from Aspergillus oryzae of Yokogaku GmbH, Kyowa Volvori, Netherlands, commercial GOS Vivinal derived from Bacillus circulans, and a bowling galactooligosaccharide powder product of bowling Bio Inc., China all contained about 10-20% lactose residue. In addition, the obtained GOS has different linkage types and polymerization degrees depending on enzyme sources, and in order to produce high-purity galactooligosaccharides, the enzymatically synthesized galactooligosaccharides should be further subjected to decolorization, desalting, filtration, etc., and finally concentrated into GOS liquid or dried to obtain GOS powder. Therefore, the main factors restricting the large-scale development of GOS at present are: (1) how to obtain beta-galactosidase with excellent performance, and (2) high separation and purification cost of GOS.
In conclusion, screening high transglycosylation activity, researching and utilizing or modifying the catalytic reaction for regulating beta-galactosidase so as to solve the common technical problems of the function development and application of the beta-galactosidase at present in the aspects of GOS synthetic yield and optimized product molecular structure. Researchers at home and abroad screen and obtain beta-galactosidase with transglycosylation activity from Lactobacillus strains such as Bacillus megaterium (Bacillus megaterium), Enterobacter agglomerans (Enterobacter agglomerans), Kluyveromyces fragilis (Kluyveromyces fragilis), and Bifidobacterium infantis (Bifidobacterium infantis) of different sources, Bifidobacterium bifidum (Bifidobacterium bifidum), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus pentosus (Lactobacillus pentosus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus plantarum (Lactobacillus plantarum), and Streptococcus thermophilus (Streptococcus thermophilus) in succession. However, the related characteristics of the synthesis of GOS by beta-galactosidase derived from Lactobacillus kefir (Lactobacillus kefir) in camel milk have not been reported.
Disclosure of Invention
The invention aims to enrich the sources of the transglycosylation active beta-galactosidase enzyme-producing strain and lay the foundation for further developing and applying the beta-galactosidase produced by the strain to efficiently synthesize GOS, and particularly provides the strain for producing the transglycosylation active beta-galactosidase and a method for producing galactooligosaccharides by using the beta-galactosidase in a wider temperature range. Beta-galactosidase prepared by lactobacillus kefiri L6 can catalyze and synthesize galacto-oligosaccharide products mainly comprising transfer disaccharide and transfer trisaccharide by using lactose as a substrate.
In order to achieve the purpose, the invention is implemented according to the following technical scheme:
the first purpose of the invention is to provide a bacterial strain for producing the beta-galactosidase with transglycosylation activity, the temperature range of the catalytic reaction of the beta-galactosidase produced by the bacterial strain is wider, and the optimal reaction temperature is 55 ℃; the relative enzyme activity can be kept above 50% at 45-70 ℃. The classification name is Lactobacillus kefiri (Lactobacillus kefir) L6, and the strain is preserved in the China center for type culture Collection. Address: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, postcode: 430072, with the preservation number: CCTCC NO of M20191030, and the preservation date is 12 months and 9 days in 2019.
The second purpose of the invention is to provide a method for preparing beta-galactosidase by fermentation, and the bacterial strain for producing the transglycosylation activity beta-galactosidase comprises the following specific steps:
(1) seed culture
Seed culture medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
seed culture conditions: culturing the preserved L.kefiri L6 at 30-38 deg.C and 100-250rpm with shaking for 12-24h to activate the strain;
(2) fermentation culture
Fermentation medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
fermentation conditions are as follows: taking the inoculation amount of the activated lactobacillus kefiri L6 as 1-10% of a fermentation culture medium, and carrying out fermentation culture for 12-24h at 30-38 ℃ and with the shaking rotation speed of 100-300 rpm to produce fermentation liquor;
(3) post-fermentation treatment
And (3) freezing and centrifuging the fermentation liquor obtained in the step (2), collecting wet thalli, preparing bacterial suspension by using 0.05M phosphate buffer solution with the pH value of 6.5, and then performing ultrasonic crushing at the temperature of 0 ℃ under the ice bath condition, wherein the ultrasonic crushing is stopped for 10s every 5s, the ultrasonic power is 150W, and the total time of ultrasonic crushing is 15 min. The obtained suspension is a crude enzyme solution of beta-galactosidase, and the enzyme activity is up to 65.7U/mL through detection.
Further, a method for producing beta-galactosidase by fermentation, which uses the bacterial strain for producing the transglycosylation active beta-galactosidase, and comprises the following specific steps:
(1) seed culture
Seed culture medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
seed culture conditions: culturing Lactobacillus kefiri L6 at 30-38 deg.C and 100-250rpm with shaking for 12-24h to activate the strain;
(2) fermentation culture
Fermentation medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
fermentation conditions are as follows: the inoculation amount of the lactobacillus kefiri L6 is 1% -10% of the fermentation culture medium, and the beta-galactosidase is produced by fermentation culture for 12-24h at 30-38 ℃ and the shaking rotation speed of 100-300 rpm;
(3) post-fermentation treatment
After the fermentation liquor is centrifuged and wet thalli is collected, the wet thalli is subjected to isovolumetric resuspension by using 0.05M phosphate buffer solution with the pH value of 6.5, 1mg/mL lysozyme is added for reaction for 3-4h, the thalli is moved into a beating tube containing 200 mu M acid-washed glass beads, a cell crusher is beaten (0-4 ℃, 48 seconds of beating, 58 seconds of stopping, 320 seconds of beating and 4 times of beating), the cell crusher is centrifuged at 8000rpm for 10min, the obtained supernatant is crude enzyme liquid, and the enzyme activity is detected to reach 43.8U/mL.
As a preferable scheme of the invention, in the step (3), the bacterial suspension is subjected to ultrasonic crushing under the condition of ice bath at 0 ℃, the ultrasonic crushing is stopped for 10s every 5s, the ultrasonic power is 150W, and the ultrasonic crushing time is 5 min.
The third purpose of the invention is to provide a method for producing galacto-oligosaccharides by catalyzing transglycosylation reaction in a wider temperature range by using the beta-galactosidase crude enzyme liquid prepared by the method for preparing beta-galactosidase through fermentation, which comprises the following specific steps:
adding the beta-galactosidase crude enzyme solution into a 45% (w/v) lactose solution to catalyze a transglycosylation reaction, wherein the reaction conditions are as follows: adding 2-4U of enzyme, reacting at 45-75 deg.C for 2-24h to obtain enzyme reaction solution containing 6.93-31.50% (w/w) galacto-oligosaccharide.
Compared with the prior art, the L6 strain of the lactobacillus kefir (L.kefiri) disclosed by the invention is derived from camel milk, and has the advantages of good safety, genetic stability, high growth speed and easiness in culture. The beta-galactosidase prepared by fermentation by using a fermentation medium which is composed of lactose as a carbon source, nitrogen sources such as yeast extract powder, peptone and beef extract powder, inorganic salts and the like has a wide catalytic reaction temperature range and an optimal reaction temperature of 55 ℃; the relative enzyme activity can be kept above 50% at 45-70 ℃; in addition, when the pH value of the enzyme is 7.0, the relative enzyme activity is highest, and within the range of pH 6.0-9.0, the relative enzyme activity can be kept above 60%; and Mg2+Has obvious activation effect on the enzyme, and the enzyme activity is not influenced by other metal ions such as Na+、K+、Ba2+、Fe2+、Fe3+Etc. Adding the prepared beta-galactosidase enzyme solution into 45% (w/v) lactose solution, carrying out catalytic reaction for 4h at 65 ℃, wherein the yield of the synthesized transglycosylation product GOS can reach 31.51% (w/w), and the transfer disaccharide and the transfer trisaccharide are respectively 13.51% (w/w) and 13.85% (w/w). At present, the study on the characteristics of GOS synthesized by Lactobacillus kefir-derived beta-galactosidase has not been reported. Therefore, the kefir separated and sieved from the camel milk by the inventionLactobacillus (l.kefiri) L6 has significant advantages in that it catalyzes transglycosylation reactions to produce galactooligosaccharides over a wide range of conditions.
Drawings
FIG. 1 is a phylogenetic analysis of L6 of Lactobacillus kefir (L.kefiri) producing transglycosylating active beta-galactosidase.
FIG. 2 is a graph showing the effect of temperature on beta-galactosidase enzyme activity.
FIG. 3 is a graph showing the effect of pH on beta-galactosidase enzyme activity.
FIG. 4 shows the effect of metal ions on beta-galactosidase enzyme activity.
FIG. 5 shows TLC analysis of the Galactooligosaccharide (GOS) products formed at different reaction temperatures.
FIG. 6 shows the effect of fermentation time on beta-galactosidase enzyme activity.
FIG. 7 is an HPLC analysis of galactooligosaccharides synthesized by transglycosylation-active beta-galactosidase produced by Lactobacillus kefiri L6.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The strain for producing the transglycosylation active beta-galactosidase, provided by the embodiment, is classified and named as Lactobacillus kefir (Lactobacillus kefir) L6, and is preserved in China center for type culture collection with the preservation number: CCTCC NO of M20191030, and the preservation date is 12 months and 9 days in 2019. The transglycosylation active beta-galactosidase producing lactobacillus kefir (l.kefiri) L6 has the following biological characteristics:
morphological characteristics: gram-positive bacilli do not form spores, form 1-2mm circular colonies on MRS medium, have regular edges, and are milky on the surface.
Physiological and biochemical characteristics: can ferment lactose and glucose, and can not ferment arabinose, galactose, rhamnose, maltose, mannose, xylose and sucrose. The starch hydrolysis test, the gelatin liquefaction test and the hydrogen sulfide production test were all negative.
The length of the nucleotide sequence of 16S rRNA of Lactobacillus kefir (L.kefiri) L6 for producing the transglycosylation active beta-galactosidase is 1070 bases, as shown in the sequence <210>, and the specific nucleotide and amino acid sequence table is as follows:
<210>1
<211>1070
<212> nucleotide sequence
<213> Lactobacillus kefiri (Lactobacillus kefiri) L6
<220>
<221>misc_feature
<223>16S rRNA gene nucleotide sequence
<400>1
GTGCATGCGGCTGCCTATACATGCAGTCGAACGCGTTTCCGTTTATTGATTTTAGA GTGCTTGCATTTAGAATGATTTAACACGAAACGAGTGGCGAACTGGTGAGTAACACG TGGGTAACCTGCCCTTGAAGTAGGGGATAACACTTGGAAACAGGTGCTAATACCGTAT AACAACCAAAACCACATGGTTTTGGTTTAAAAGATGGCTTCGGCTATCACTTTAGGAT GGACCCGCGGCGTATTAGCTTGTTGGTAAGGTAATGGCCTACCAAGGCAATGATACGT AGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTA CGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGC CGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACAGGT GTCAGAGTAACTGTTGACATCTTGACGGTATCCAACCAGAAAGCCACGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTA AAGCGAGCGCAGGCGGTTTCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGA AGTGCATCGGAAACCAGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTG TAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGG TCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTG GTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGC TGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCA AGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTATTCGATGCTACGC GAAGAACCTTACAGGTCTTGACATCTCTGCACCTAGAGATAGCGTCCCTTCGGGACA GATGACAGGTGGTGCATGGTGTCGTCAGCCTCGTGTCGTGAGATG。
The beta-galactosidase producing lactobacillus kefir (l.kefiri) L6 of this example was isolated and screened from camel milk collected from west gobi town of shawan county, Uygur autonomous region, Xinjiang, and the specific procedures for isolating and screening the beta-galactosidase producing lactobacillus kefir (l.kefiri) L6 of this example are as follows.
Example 1: isolation and screening of beta-galactosidase producing transglycosylation activity
Taking an appropriate amount of sample in an equal part in a superclean workbench, shaking up in a conical flask filled with sterilized normal saline, namely 10-1The sample dilution of (1). Gradient dilution to 10 with sterile saline in test tubes-5Separately, 100. mu.L of the diluted solution (10) was taken-3、10-4、 10-5) Uniformly coating the mixture on an MRS primary screening plate which takes lactose as a unique carbon source and contains X-gal, culturing for 1-2 days at 37 ℃, carrying out primary screening by using blue and white colonies, and selecting blue colonies to purify for three times under the same culture conditions. The primary screening strains are respectively preserved by adopting a slant method and a glycerol method. And carrying out microscopic examination, gram staining and other morphological characteristic observation on the separated strain.
MRS medium plate (g/L) comprises 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 18g of agar powder and 1000mL of distilled water, the pH value is 6.2-6.4, and the sterilization is carried out for 15min at 121 ℃.
Selecting a blue single colony on an activated plate, inoculating the blue single colony in 20mL of MRS liquid culture medium, culturing at 37 ℃ and 160rpm for 24h, centrifuging the cultured fermentation liquor at 4 ℃ and 8000rpm for 10min, collecting cells, washing with 0.05M phosphate buffer solution with pH 6.5 for 2-3 times, performing isovolumetric resuspension, adding 1mg/mL lysozyme for reaction for 3h, transferring the solution into a beating tube containing 200 mu M acid-washed glass beads, beating with a cell breaker (0-4 ℃, beating for 48 seconds, stopping for 58 seconds, totally 320 seconds, beating for 4 times), centrifuging at 8000rpm for 10min, adding 50 mu L of obtained supernatant into 30% lactose solution, performing water bath reaction at 40 ℃ for 12h, and centrifuging at 12000rpm for 5min, wherein the supernatant is a transglycosylation reaction product.
Performing Thin Layer Chromatography (TLC) on reaction products of different strains, performing TLC analysis by using activated Silica gel aluminum plate Silica gel 60No.553(Merck company), spotting with a microsyrin, developing with n-butanol, anhydrous ethanol and water at ratio of 5:3:2 as developing agents, performing chromatography to a position 1cm away from the top end of the Silica gel aluminum plate, taking out, air drying, performing spray color development with a color developing agent (aniline-dianiline-monophosphate-acetone), baking in an oven at 80 ℃ for 15min, and screening to obtain the strains L1, L4, L6, L10 and L11 for producing the transglycosylation active beta-galactosidase according to the size of spots corresponding oligosaccharide products on the TLC plate.
Example 2: identification of beta-galactosidase strain producing transglycosylation activity
Further, the strain L.kefiri-L6 screened in example 1 was identified.
(1) Morphological observation and physiological and biochemical test
Inoculating the strain L.kefiri-L6 into an MRS culture medium, culturing for 48h at 37 ℃, and enabling the strain L.kefiri-L6 to form a circular colony with the diameter of 1mm on the MRS culture medium, wherein the edge of the circular colony is neat, and the surface of the circular colony is milky white; the rod shape is observed by a microscope, and gram staining is positive. Physiological and biochemical characteristic analysis of the strain L.kefiri-L6 shows that the strain can ferment lactose and glucose, and cannot ferment arabinose, galactose, rhamnose, maltose, mannose, xylose and sucrose; the starch hydrolysis test, the gelatin liquefaction test and the hydrogen sulfide production test were all negative.
(2) Cloning of 16S rRNA and sequence analysis thereof
Extracting genome DNA of a strain producing beta-gal, carrying out PCR amplification by using universal primers 27f and 1492r of a bacterial 16S rRNA gene, identifying a PCR result by agarose gel electrophoresis, and carrying out bidirectional sequencing on the universal primers 27f and 1492r of a PCR product sent to the company of Biotechnology (Shanghai) GmbH to obtain the sequence <210> as described above. The sequencing result is subjected to homology comparison analysis from NCBI-blast, and the comparison result shows that the similarity of the 16S rRNA gene sequence of the strain L6 and the 16S rRNA gene sequences of a plurality of strains of Lactobacillus kefiri reaches 99%. And establishing a phylogenetic tree according to the related species sequences in the database. The evolutionary distance is calculated by adopting a Neighbor-join method, a two-parameter method of p-distances and Kimura-2 parameters is adopted in MEGA 6.0 software to construct, a Bootstrap method is selected to evaluate the stability of branching and clustering of the evolutionary tree, the population classification condition of the enzyme-producing strains is obtained by repeating 1000 times, and the results are shown in figure 1, and the screened strains L1, L4, L6, L10 and L11 are classified into one with Lactobacillus kefir (Lactobacillus kefir). Strain L6 was therefore identified as l.kefiri L6.
Example 3:
the method for producing beta-galactosidase by fermentation of a transglycosylation active beta-galactosidase producing strain L.kefiri L6 comprises the following specific steps:
(1) seed culture
Seed culture medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
seed culture conditions: culturing Lactobacillus kefiri L6 at 30-38 deg.C and 100-250rpm with shaking for 12-24h to activate the strain;
(2) fermentation culture
Fermentation medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
fermentation conditions are as follows: the inoculation amount of the lactobacillus kefiri L6 is 1% -10% of the fermentation culture medium, and the beta-galactosidase is produced by fermentation culture for 12-24h at 30-38 ℃ and the shaking rotation speed of 100-300 rpm;
(3) post-fermentation treatment
And (2) freezing and centrifuging the fermentation liquor, collecting wet thalli, preparing a bacterial suspension by using 0.05M phosphate buffer solution with the pH value of 6.5, and then carrying out ultrasonic crushing at the temperature of 0 ℃ under an ice bath condition, wherein the ultrasonic crushing is stopped for 10s every 5s, the ultrasonic power is 150W, the total time of the ultrasonic crushing is 15min, and the obtained suspension is the crude beta-galactosidase enzyme solution. Adding 50 mu L of oNPG (prepared from 0.05M phosphate buffer solution with pH 6.5) and 50 mu L of crude enzyme solution into a hole of an enzyme-labeled plate, reacting at 37 ℃ for 10min, adding 200 mu L of 0.5M sodium carbonate solution to stop the reaction, standing for 5min to obtain obvious yellow, measuring the light absorption value at the wavelength of 420nm by using an enzyme-labeled meter, calculating the enzyme activity by using an oNPG standard curve method, and detecting that the enzyme activity reaches 65.7U/mL.
Example 4
The method for producing beta-galactosidase by fermentation of a transglycosylation active beta-galactosidase producing strain L.kefiri L6 comprises the following specific steps:
(1) seed culture
Seed culture medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
seed culture conditions: culturing Lactobacillus kefiri L6 at 30-38 deg.C and 100-250rpm with shaking for 12-24h to activate the strain;
(2) fermentation culture
Fermentation medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
fermentation conditions are as follows: the inoculation amount of the lactobacillus kefiri L6 is 1% -10% of the fermentation culture medium, and the beta-galactosidase is produced by fermentation culture for 12-24h at 30-38 ℃ and the shaking rotation speed of 100-300 rpm;
(3) post-fermentation treatment
After the fermentation liquor is centrifuged and wet thalli are collected, the wet thalli are subjected to isovolumetric resuspension by using 0.05M phosphate buffer solution with the pH value of 6.5, 1mg/mL lysozyme is added for reaction for 3-4h, the mixture is moved into a beating tube containing 200 mu M acid-washed glass beads, a cell disruptor is beaten (0-4 ℃, 48 seconds, 58 seconds of stopping, 320 seconds and 4 times of beating), the mixture is centrifuged at 8000rpm for 10min, and the obtained supernatant is the crude enzyme solution. Adding 50 mu L of oNPG (prepared from 0.05M phosphate buffer solution with pH 6.5) and 50 mu L of crude enzyme solution into a hole of an enzyme-labeled plate, reacting at 37 ℃ for 10min, adding 200 mu L of 0.5M sodium carbonate solution to stop the reaction, standing for 5min to obtain obvious yellow, measuring the light absorption value at the wavelength of 420nm by using an enzyme-labeled meter, and calculating the enzyme activity by using an oNPG standard curve method. The enzyme activity reaches 43.8U/mL through detection.
Example 5
Ammonium sulfate fractional precipitation purification of the beta-galactosidase obtained in example 3 or 4, the specific steps are as follows:
taking 10mL of the crude beta-galactosidase obtained in example 4-5, slowly adding ammonium sulfate solid corresponding to 40% saturation in ice bath state at 4 ℃ and slowly stirring to avoid air bubbles generated by protein denaturation, standing overnight, centrifuging at 4 ℃ for 30min at 3000g, collecting precipitate, adding 0.05M phosphate buffer (pH 7.0) to dissolve, dialyzing in a 14kDa dialysis bag overnight to obtain primarily purified beta-galactosidase.
Example 6
Effect of temperature on the Primary purified beta-galactosidase obtained in example 5
50 mu L of oNPG (prepared by 0.05M phosphate buffer solution with pH 6.5) and 50 mu L of the primarily purified beta-galactosidase obtained in the example 5 are added into a hole of an ELISA plate, the mixture reacts for 10min at the conditions of 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃, 200 mu L of 0.5M sodium carbonate solution is added to stop the reaction, obvious yellow is seen after the mixture is kept still for 5min, the absorbance value at the wavelength of 420nm is respectively measured by an ELISA reader, and the enzyme activity is calculated by an oNPG standard curve method. The result is shown in figure 2 in detail, the optimum catalytic temperature of the beta-galactosidase produced by the strain L.kefiri L6 is 55 ℃, the temperature range of the catalytic reaction of the beta-galactosidase is wide, and the beta-galactosidase can keep more than 50% of relative enzyme activity at 45-70 ℃. At 75 ℃, the relative enzyme activity is reduced to below 20 percent.
Example 7
Effect of pH on the Primary purified beta-galactosidase obtained in example 5
Respectively taking 50 mu L of phosphate buffer solutions with different pH values to respectively prepare oNPG solutions with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, adding 50 mu L of the primarily purified beta-galactosidase obtained in the embodiment 5 into a hole of an enzyme-labeled plate, reacting for 10min at55 ℃, adding 200 mu L of 0.5M sodium carbonate solution to terminate the reaction, standing for 5min to show obvious yellow, respectively measuring the light absorption value at the wavelength of 420nm by using an enzyme-labeled instrument, and calculating the enzyme activity by using an oNPG standard curve method. The result is shown in figure 3 in detail, the activity of beta-galactosidase produced by the strain L.kefiri L6 is higher within the range of pH6.0-9.0, the relative enzyme activity can be kept above 60%, and when the pH is 7.0, the relative enzyme activity of the beta-galactosidase is highest. The enzyme activity is basically lost when the pH is 3.0-4.0.
Example 8
Effect of Metal ions on the Primary purified beta-galactosidase obtained in example 5
1mmol of NiCl is respectively taken2、MgCl2、BaCl2、KCl、NaCl、AlCl3、CaCl2、FeCl3、FeCl2And (2) preparing an oNPG solution by using a metal ion salt solution, adding 50 mu L of the primarily purified beta-galactosidase obtained in the embodiment 5 into a hole of an enzyme-labeled plate, reacting for 10min at55 ℃, adding 200 mu L of 0.5M sodium carbonate solution to terminate the reaction, standing for 5min to obtain obvious yellow, respectively measuring the light absorption values at the wavelengths of 420nm by using an enzyme-labeled instrument, and calculating the enzyme activity by using an oNPG standard curve method. The results are shown in FIG. 4, Mg2+Has obvious activation effect on the enzyme, and the relative enzyme activity is about 193.30 percent of that of a control. Ni2+、Ca2+And Al3+Has obvious inhibition effect on enzyme activity, and other metal ions such as Na+、K+、Ba2+、Fe2+、 Fe3+Has no obvious influence on the enzyme activity.
Example 9
The embodiment provides a method for producing galactooligosaccharides by using a beta-galactosidase crude enzyme solution prepared by the method for producing beta-galactosidase through fermentation, which comprises the following specific steps:
the crude enzyme solution of beta-galactosidase obtained in example 3-4 was added to a 45% (w/v) lactose solution to perform a catalytic reaction under the following conditions: adding enzyme 2-4U, reacting at 45-75 deg.C for 2-24h to obtain enzyme reaction solution containing galacto-oligosaccharide.
The transglycosylation reaction product solution is diluted moderately, is developed by using an activated Silica gel plate Silica gel 60No.553(Merck company) and a capillary spotting technology to sample 10 mu L, and n-butanol, absolute ethyl alcohol and water are 5:3:2 as developing agents, is chromatographed to a position 1cm away from the top end of the Silica gel plate, is taken out and dried, is sprayed and developed by using a color developing agent (aniline-bis-aniline-monophosphate-acetone), is baked in an oven at 80 ℃ for 15min, and various sugars show different colors and are compared with standard sugars, and the result is shown in figure 5 and figure 5: 1 is standard sugar (lactose, galactose, glucose mixture); 2-8 are sequentially corresponding to transglycosylation reaction products with the transglycosylation reaction temperature of 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃. The results of the yield of galactooligosaccharide and the content of residual lactose in the reaction solution after different reaction times of enzyme catalysis are shown in FIG. 6.
The reaction solution was diluted with triple distilled water to a sugar concentration of 1%, filtered through a 0.22 μm filter and subjected to HPLC sample analysis, the sample volume was 20 μ L, the Column chromatography was Hi-Plex Na Column 300mm × 7.7mm (Agilent), the mobile phase was triple distilled water, the flow rate was 0.2mL/min, the Column temperature was 80 ℃, and the sample volume was 20 μ L. By combining TLC analysis and HPLC analysis, the yield of galactooligosaccharide in the transglycosylation reaction liquid can reach 31.51% (w/w), wherein the transfer disaccharide and the transfer trisaccharide are respectively 13.51% (w/w) and 13.85% (w/w), and the oligosaccharide above the transfer trisaccharide accounts for 4.15% (w/w), and the results are shown in FIG. 7. in FIG. 7, the peak with the retention time of 52.040min is galactose, the retention time of 48.393min is glucose, 40.612min is lactose and transfer disaccharide, 34.530min is galactooligosaccharide trisaccharide, and the peak between 16.223min and 30.052min is the galactooligosaccharide component above the trisaccharide.
The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.
<110> river university
<120> Lactobacillus kefiri and method for producing galacto-oligosaccharide by using beta-galactosidase prepared by Lactobacillus kefiri
<141>
<160> 3
<210> 1
<211> 1070
<212> nucleotide sequence
<213> Lactobacillus kefiri (Lactobacillus kefiri) L6
<220>
<221> misc_feature
<223>16S rRNA gene nucleotide sequence
<400> 1
GTGCATGCGGCTGCCTATACATGCAGTCGAACGCGTTTCCGTTTATTGATTTTAGAGTGCTTGCATTTAGAATGATTTAACACGAAACGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTGCCCTTGAAGTAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACCAAAACCACATGGTTTTGGTTTAAAAGATGGCTTCGGCTATCACTTTAGGATGGACCCGCGGCGTATTAGCTTGTTGGTAAGGTAATGGCCTACCAAGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACAGGTGTCAGAGTAACTGTTGACATCTTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCAGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTATTCGATGCTACGCGAAGAACCTTACAGGTCTTGACATCTCTGCACCTAGAGATAGCGTCCCTTCGGGACAGATGACAGGTGGTGCATGGTGTCGTCAGCCTCGTGTCGTGAGATG
<210> 2
<211> 604
<212> amino acid sequence
<213> lactase controlling 10_947
<220>
<221> misc_feature
<223> lactase contig10_947 amino acid sequence
<400> 1
MLYPLTTPSRTLLDLSGLWKFMIDEEINPIDPKKPLPIEDVISVPASFNDQTASKKIREH
NGFVWCETNFDIAKPLRSQRLVLRFGSATHEAWVYLNGKEITHHKGGFTPFEVEINDYLK
DSNNRLTVKLSNILDYTTLPVGNYSETRDKDGHIQRHVDENFDFFNYAGLQRPVKIYSTP
KDYIGDITVVPDVDLEKSNADIHIKVATEGHFDEVKVTLLNQDGVQVASGTGVDQTLQIV
DAHLWQPLHAYLYTAKVDAIENGEVVDTYSEEFGVRKIEVKNAQFLINGKPFYFKGFGKH
EDSYVHGRGFNEPVNVLDIHLMKDMGANSFRTSHYPYSEEMMRLCDREGIVVIDETPAVG
LMTSFEFDVSMLESDDYQDDTWTKLKTAEAHRQVIKEIIDRDKNHACVAMWSIANEAATF
SKGAYEYFKPLFDEAHELDPQKRPCTYTSIMMANPKTDNCISLVDVIALNRYYGWYVGNG
DLETAERATRDELKEYSEKFPDKPIMYTEYGADTIPGLHSNYDEPFSEEFQEDYYRMASK
VFDEFPHFVGEQLWNFADFQTKFGIQRVQGNKKGIFTRSREPKMVVRYLKNRWTNIPNFD
YKKK
<210> 3
<211> 629
<212> amino acid sequence
<213> lactase controlling 49_2123
<220>
<221> misc_feature
<223> lactase contig49_2123 amino acid sequence
<400> 1
MEPDIKWLDDPQNFRVGMLPAHSDHQFYQSYDEMKNDDSSYVQSLNGQWQFAFSKDPMHR
IKDFYTADCDSRHFDTIKVPQHIEFAGYDKFHYINTMYPWEGKVYRRPAYALDQNDKEAG
QFSEAADNPVGQYIKKFNLNSTFKDKKVRVRFDGVEKAMYVWLNGHFLGYAEDSFTPSEF
DLTPYLQDGENTLAVEVFKLSTAAWLEDQDMFRFFGIFRDVSLIAQPEAHIEDLSIKPTL
SSDLKNGTLNVETKMTVNKSAISAKVTVNDADGNSVYAESQPIHDSVSLKDVAFKDVHLY
DNHDPYLYQLNIELDDKDGNILEIVPYKFGFRKIEINQDKVITLNGKRLVINGVNRHEWD
CHSGRVVSVDDMKADIQTFKKNNINAVRTCHYPDHTLWYHLCDINGIYVMAENNLESHGT
WQKLGAIEPSYNVPGSVPQWKAAVVDRARNNYEMLKNHASILFWSLGNESYAGDDMQAME
EFYKSVDPTRLVHYEGVVHNRKYEDVISDVESRMYASPDEIKKYLDNHPKKPFMLCEYMH
DMGNSLGGMNSYTDLIDKYPMYAGGFIWDFIDQAIEVNDEVTGKKVLRYGGDFDDRMSDY
EFSGDGLMFADRTEKPAMQEVKYYYGQHK

Claims (5)

1. Transglycosylation activityβ-a strain of galactosidase characterized in that: it is classified and named as Lactobacillus kefiri (A)Lactobacillus kefiri) L6, deposited in China center for type culture Collection with the deposit number: CCTCC NO of M20191030, and the preservation date is 12 months and 9 days in 2019.
2. Fermentation productionβ-a galactosidase process, characterized in that: comprises the following steps:
(1) seed culture
Seed culture medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
seed culture conditions: lactobacillus kefir deposited according to claim 1L. kefiri L6 is subjected to shaking culture at the rotation speed of 100-250rpm at the temperature of 30-38 ℃ for 12-24h to activate the strain;
(2) fermentation culture
Fermentation medium: 20g of lactose, 10g of peptone, 10g of beef extract powder, 5g of yeast extract powder and K2HPO45g, 2g diammonium hydrogen citrate, 5g sodium acetate and MgSO4·7H2O0.58 g, Tween-801 mL, MnSO40.25g, 1000mL of distilled water, pH 6.2-6.4, sterilizing at 121 ℃ for 15 min;
fermentation conditions are as follows: the lactobacillus kefir activated in the step (1) is treatedL. kefiri L6 is inoculated on a fermentation medium, the inoculation amount is 1-10 percent of the fermentation medium, and fermentation culture is carried out for 12-24h at 30-38 ℃ and the oscillation rotation speed of 100-300 rpm to obtain fermentation liquor;
(3) post-fermentation treatment
Freezing and centrifuging the fermentation liquor obtained in the step (2), collecting wet thalli, preparing bacterial suspension by using 0.05M phosphate buffer solution with pH of 6.5, crushing at 0-4 ℃, and crushingThe obtained suspension isβ-crude galactosidase enzyme solution.
3. The fermentative production of claim 2β-a galactosidase process, characterized in that: the crushing adopts ultrasonic crushing, the ultrasonic crushing is stopped for 10s every 5s, the ultrasonic power is 150W, and the total time of the ultrasonic crushing is 15 min.
4. The fermentative production of claim 2β-a galactosidase process, characterized in that: the crude enzyme liquid in the step (3) is subjected to fractional precipitation and purification by 40 percent of ammonium sulfate to obtain primarily purified enzyme liquidβ-a galactosidase enzyme.
5. A method for producing galactooligosaccharides, characterized by: prepared by the process of claim 2βPreparing a crude galactosidase enzyme solution, which comprises the following specific steps:
obtained by the process of claim 2βAdding the crude galactosidase enzyme solution into a 45% w/v lactose solution to catalyze a transglycosylation reaction, wherein the reaction conditions are as follows: adding enzyme 2-4U, reacting at 45-75 deg.C for 2-24h to obtain enzyme reaction solution containing galacto-oligosaccharide.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101687897A (en) * 2007-05-11 2010-03-31 曼纳泰克公司 Processing of natural polysaccharides by selected non-pathogenic microorganisms and methods of making and using the same
CN109679864A (en) * 2018-12-13 2019-04-26 石河子大学 A method of it producing the bacterial strain for turning glycosyl active p-galactosidase and produces galactooligosaccharide with the enzyme

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060099623A1 (en) * 2000-11-28 2006-05-11 Matthew Glenn Polynucleotides and polypeptides isolated from lactobacillus and methods for their use
WO2004076615A2 (en) * 2003-02-27 2004-09-10 Bioneer A/S Immunomodulating probiotic compounds
EA028325B1 (en) * 2010-11-23 2017-11-30 Кр. Хансен А/С Use of glycosidase in preparation of a milk product
KR101589466B1 (en) * 2015-05-21 2016-02-01 주식회사 쎌바이오텍 Use of bifidobacterium longum cbt bg7 strain for promoting growth and nutraceutical composition for promoting growth comprising the same
EP3414317A2 (en) * 2016-02-12 2018-12-19 Probiotical S.p.A. Use of bacterial strains belonging to the species lactobacillus kefiri in paediatrics to generate and/or maintain a state of homeostasis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101687897A (en) * 2007-05-11 2010-03-31 曼纳泰克公司 Processing of natural polysaccharides by selected non-pathogenic microorganisms and methods of making and using the same
CN109679864A (en) * 2018-12-13 2019-04-26 石河子大学 A method of it producing the bacterial strain for turning glycosyl active p-galactosidase and produces galactooligosaccharide with the enzyme

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
beta-galactosidase [Lactobacillus kefiri];NCBI;《Genbank database》;20151108;WP_056981475.1 *
beta-glucuronidase [Lactobacillus kefiri];NCBI;《Genbank database》;20161031;WP_056982029.1 *
Evaluation and Determination of Lactase Activity on Various Lactic Acid Bacteria isolated from Kefir by using HPLC;Jeong, Dana等;《Journal of Dairy Science and Biotechnology》;20161231;第34卷(第4期);231-237 *
开菲尔产品微生态、成分和功能活性的研究进展;高鑫等;《食品与发酵工业》;20160303;第42卷(第03期);243-250 *
开菲尔菌粒及其发酵乳的研究进展;姜志辉等;《肠外与肠内营养》;20180619;第25卷(第04期);252-256 *
开菲尔菌群的益生功效及其在发酵食品中的应用进展;何雪畅等;《食品工业》;20161020;第37卷(第10期);220-223 *
西藏乳及发酵乳制品中乳酸菌与酵母菌分布;罗章等;《食品工业科技》;20130601;第34卷(第11期);392-395 *
驼乳中产转糖基活性β-半乳糖苷酶乳酸菌的筛选鉴定及其酶学特性分析;韩明明等;《食品科学》;20201229;第42卷(第24期);74-83 *

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