CN115725441A - Bacillus tsukuni HJ2101 and application thereof in production of galactooligosaccharides - Google Patents

Abstract

The invention discloses a Zjut HJ2101 of Zjut of flower body and its application in producing galacto-oligosaccharide, wet thalli obtained by fermenting and culturing Zjut HJ2101 of flower body is used as catalyst, lactose is used as substrate, buffer solution with pH6.0-8.0 is used as reaction medium, conversion reaction is carried out under the conditions of 180-200rpm and 30-35 ℃, conversion solution containing galacto-oligosaccharide is obtained, and separation and purification are carried out to obtain galacto-oligosaccharide. The high-salt environment for the growth of the strain can effectively prevent the pollution of other microorganisms, save energy consumed by sterilization and the like, and the beta-galactosidase produced by the strain has salt tolerance and can still keep 50% of the initial enzyme activity within the range of 3-15% of salinity.Can produce beta-galactosidase under the condition of high salt concentration (the yield reaches 4.5U/g) _Thallus ) And can also be used for the synthesis of galactooligosaccharides.

Description

Bacillus tsukuni HJ2101 and application thereof in production of galactooligosaccharides

(I) technical field

The invention relates to the technical field of microbial separation and application, in particular to halophilic microorganisms, namely Bacillus dysaricus Takesii and application thereof in preparation of galactooligosaccharides by catalyzing lactose.

(II) background of the invention

Galacto-oligosaccharides (GOS) are important prebiotics that promote the growth of bifidobacteria and lactobacilli, and at the same time improve mineral absorption and regulate the immune system. As a functional oligosaccharide, GOS can be widely used in various fields, particularly in infant formula foods and bakery foods. Currently, galactooligosaccharides are mainly produced by enzymatic synthesis, i.e., a mixture of galactooligosaccharides is formed by decomposing lactose, a substrate, into galactosyl and glucosyl groups by using the hydrolytic activity of beta-Galactosidase (also called Lactase), and transferring the galactosyl group to different glycosyl receptors by using the transglycosylation activity of the enzyme.

The source of beta-galactosidase is very wide, both animals, plants and microorganisms exist, and the industrial application is mainly microbial sources, such as bacillus circulans, klebsiella oxytoca, escherichia coli, aspergillus oryzae and the like. Most microorganisms cannot survive under extreme conditions (such as high salt concentration, high temperature, strong acid and the like), cannot produce beta-galactosidase, and most of beta-galactosidase derived from microorganisms does not have transglycosylation activity, so that the application to GOS synthesis is difficult. The invention not only provides a strain for producing beta-galactosidase under the condition of high salt concentration, but also inspects the capability of utilizing the strain to carry out whole-cell catalysis on lactose to produce galactooligosaccharides, and provides a new reference for the enzymatic production of the galactooligosaccharides.

Disclosure of the invention

The invention aims to provide a halophilic new strain-Zjut HJ2101 capable of growing under the condition of high salt concentration and producing beta-galactosidase, and application thereof in production of galacto-oligosaccharide, and provides a new reference for enzymatic production of the galacto-oligosaccharide.

The technical scheme adopted by the invention is as follows:

the invention provides a halophilic new strain capable of growing under the condition of high salt concentration and producing beta-galactosidase, namely Zjut HJ2101 of Bacillus hwajinponensis, which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2022196, the preservation date of 2022 years, 3 months and 4 days, the address: china, wuhan university, post code 430072.

The invention also provides an application of the Zjut HJ2101 in the production of galactooligosaccharides, and the application comprises the following steps: taking wet thalli obtained by fermenting and culturing the Bacillus dysenteriae Zjut HJ2101 as a catalyst, taking lactose as a substrate, taking a buffer solution with the pH value of 6.0-8.0 as a reaction medium, carrying out conversion reaction (preferably 24-48 h) under the conditions of 180-200rpm and 30-35 ℃ to obtain a conversion solution containing galacto-oligosaccharide, and separating and purifying to obtain the galacto-oligosaccharide.

Preferably, the addition amount of the lactose is 100-300g/L, preferably 300g/L, calculated by the volume of the buffer solution; the dosage of the wet bacteria is 100-300g/L, preferably 200g/L calculated by the volume of the buffer solution.

Preferably, the buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.0.

Preferably, the catalyst is prepared as follows:

(1) Plate culture: inoculating Bacillus tsukuni HJ2101 on a plate culture medium, and culturing at 30 ℃ for 48-72h to obtain a plate culture strain; the plate culture medium consists of: 15g/L of lactose, 20g/L of anhydrous magnesium sulfate, 3g/L of trisodium citrate dihydrate, 10g/L of tryptone, 2g/L of KCl, 50g/L of NaCl, 20g/L of agar, pH 7.0 and deionized water as a solvent;

(2) And (3) proliferation culture: selecting a ring of strains after plate culture, inoculating the strains to a multiplication culture medium, and culturing for 24 hours in an incubator with the rotating speed of 200rpm and the temperature of 30 ℃ to obtain a multiplication bacterial liquid; the proliferation medium consists of: 15g/L of lactose, 20g/L of anhydrous magnesium sulfate, 3g/L of trisodium citrate dihydrate, 10g/L of tryptone, 2g/L of KCl, 50g/L of NaCl, 7.0 of pH and deionized water as a solvent;

(3) Fermentation culture: inoculating the enrichment bacterial liquid obtained in the step (2) into an enzyme-producing fermentation culture medium with the inoculation amount of 2% (v/v), culturing at the temperature of 30 ℃ for 36h at 200rpm, centrifuging the culture liquid at 8000rpm at the temperature of 4 ℃ for 10min, and collecting wet thalli; the enzyme-producing fermentation medium comprises the following components: 15g/L lactose, 5g/L tryptone, 10g/L yeast extract powder, 0.3g/L anhydrous magnesium sulfate and KH ₂ PO ₄ 0.05g/L, naCl 50g/L, pH 7.0, and deionized water as solvent.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a new strain for producing beta-galactosidase-Zjut HJ2101; the high-salt environment for the growth of the strain can effectively prevent the pollution of other microorganisms, save energy consumed by sterilization and the like, and in addition, the beta-galactosidase produced by the strain has salt tolerance and can still keep 50% of the initial enzyme activity within the range of 3-15% of salinity.

(2) The Bacillus tsukuni HJ2101 can produce beta-galactosidase (the yield reaches 4.5U/g) under the condition of high salt concentration (3% -15%, preferably 5%) _Thallus ) And can also be used for the synthesis of galacto-oligosaccharides.

Description of the drawings

FIG. 1 TLC chart of transformed product of Zjut HJ2101 B.tsugae. (in the figure, sample 1 is a galactose standard sample; sample 2 is a glucose standard sample; sample 3 is a lactose standard sample; sample 4 is a mixed standard sample of glucose, galactose and lactose; sample 5 is a commercially available galacto-oligosaccharide product (G909325, macklin); sample 6 is a wet-catalyzed lactose conversion sample of Bacillus tsunekii Zjut HJ2101; the concentrations of the standard samples are all 10 mg/mL).

FIG. 2 colony morphology of Bacillus tsunefarensis Zjut HJ 2101.

FIG. 3 gram stain of Zjut HJ2101 Bacillus tsugaensis.

FIG. 4 phylogenetic tree of Bacillus tsunekii Zjut HJ 2101.

FIG. 5 HPLC analysis of transformed product of Zjut HJ2101 strain of Bacillus tsunekii and mixed standard sample; 1 is a liquid phase diagram of a mixed standard sample of glucose, galactose and lactose; 2 is a liquid phase diagram of the transformed product of the Zjut HJ2101 strain of Bacillus tsunekii.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto:

the culture medium used in the examples of the present invention was as follows:

the enrichment medium consists of (g/L): anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, pH 7.0, and deionized water as solvent.

The composition of the screening plate culture medium is (g/L): anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, X-gal 0.024, agar 20 in solid culture medium, pH 7.0, and deionized water as solvent.

The composition of the screening fermentation medium is (g/L): lactose 10, anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, pH 7.0, and deionized water as a solvent.

The composition of the slant culture medium is (g/L): lactose 15, anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, agar 20 added to a solid medium, the pH value is 7.0, and the solvent is deionized water.

The plate medium composition was (g/L): lactose 15, anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, agar 20 added to a solid medium, the pH value is 7.0, and the solvent is deionized water.

The proliferation medium consists of (g/L): lactose 15, anhydrous magnesium sulfate 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, naCl 50, pH 7.0, and deionized water as a solvent.

The enzyme-producing fermentation medium consists of (g/L): lactose 15, tryptone 5, yeast extract powder 10, anhydrous magnesium sulfate 0.3 ₂ PO ₄ 0.05, naCl 50, pH 7.0, and deionized water as solvent.

Example 1 isolation of a β -galactosidase producing halophilic Strain Zjut HJ2101

1. Preliminary screening

Respectively putting 1g of soil around Tibet Paru sylvate lake in 10mL of sterilized NaCl aqueous solution with different concentrations (5%, 10%,15%, 25%) at 30 ℃, shaking for 30min and mixing uniformly in a shaking table with 200rpm, standing for 15min, respectively absorbing 1mL of soil suspension in 50mL of enrichment medium, and carrying out enrichment culture for 3-5 days at 30 ℃ and 200 rpm. Respectively sucking 100 μ L of the above enrichment culture solution, and diluting with sterile saline gradient to 10% ^-4 ，10 ^-5 ，10 ^-6 Respectively sucking 200 mu L of the bacterial suspension, uniformly coating the bacterial suspension on a screening plate culture medium, inverting the bacterial suspension in a constant temperature incubator at 30 ℃ for culture, picking blue single bacterial colonies after the bacterial growth, streaking and purifying the bacterial suspension on the plate culture medium, inverting the bacterial suspension in the incubator at 30 ℃ for culture for 2-3 days to obtain a pure culture, performing gram staining and microscopic examination on the separated bacterial strains, and then transferring the pure culture with different forms after microscopic examination to a slant culture medium for preservation.

2. Wet cell detection

And (2) selecting a ring of the strains preserved on the inclined plane in the step 1, inoculating the strains into 50mL of enrichment medium, activating the strains for 24h at 30 ℃ and 200rpm, inoculating the strains into 100mL of screening fermentation medium in an inoculation amount of 2% (v/v), culturing the strains for 24h at 30 ℃ and 200rpm, and centrifuging the fermentation liquor for 10min at 8000rpm and 4 ℃ to obtain wet thalli. 100mg of wet cells were collected, 400. Mu.L of 1mg/mL oNPG solution (colorless, 2-nitrophenyl-. Beta. -D-galactopyranoside) prepared from 0.1M, pH 7.0.0 disodium hydrogenphosphate-sodium dihydrogenphosphate buffer was added, and after 10min at 37 ℃, 0.5mL of 0.15M Na was added ₂ CO ₃ The reaction is stopped by aqueous solution, na is added in the control tube ₂ CO ₃ The aqueous solution was added with the same amount of wet cells and centrifuged at 8000rpm for 10min at room temperature. If the supernatant of the sample tube is yellow, the colorless oNPG (2-nitrophenyl-beta-D-galactopyranoside) is hydrolyzed into yellow oNP (o-nitrophenol), which proves the existence of beta-galactosidase in the wet thallus.

Example 2 screening of transglycosylation-active beta-galactosidase-producing strains

A loop of the beta-galactosidase-containing slant storage strain which is proved to have the beta-galactosidase exists by the step 2 of the example 1 is selected and inoculated into 50mL of multiplication culture medium, the slant storage strain is activated for 24h under the conditions of 30 ℃ and 200rpm and then inoculated into 100mL of screening fermentation medium by the inoculation amount of 2% (v/v), the slant storage strain is cultured for 36h under the same conditions, the fermentation liquid is centrifuged for 10min at 8000rpm and 4 ℃, cells are collected, and the wet thalli are obtained after washing and centrifugation for 3 times by using disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the same volume as the fermentation liquid and the pH value of 7.0. Adding lactose into 10mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 7.0 to make the concentration of the buffer solution be 0.3g/mL, adding 2g of wet thallus, converting at 200rpm and 30 ℃ for 36h, centrifuging at 8000rpm and normal temperature for 10min, and obtaining the supernatant as the transglycosylation reaction product.

The reaction products of the different strains were analyzed by Thin-Layer Chromatography (TLC) using activated Silica gel aluminium plates (Silica gel 60F254 Merck) spotted with 10. Mu.L capillaries, chromatographed to 1cm from the top using n-butanol: ethanol: water = 5. If a new oligosaccharide spot appears at the lower part of the lactose spot on a TLC plate of a reaction product of the strain, the beta-galactosidase produced by the strain has transglycosylation activity; otherwise, transglycosylation activity is absent. The β -galactosidase producing strain with high transglycosylation activity was selected according to the size of the spot corresponding to the oligosaccharide product on the TLC plate and labeled as strain Zjut HJ2101 (see sample 6 in FIG. 1).

Example 3 identification of the Strain Zjut HJ2101

1) Morphological observation

The strain Zjut HJ2101 is inoculated in a plate culture medium, after culturing for 48-72h at 30 ℃, the colony and the thallus morphology are observed, the colony is round, the surface is smooth, the edge is neat, and the color is milky white, and the result is shown in figure 2. The gram stain was purple, and the shape of the cells was short rod-like, and the results are shown in FIG. 3, which are positive bacilli.

2) Physiological and biochemical test

VITEK physiological and biochemical identification is carried out on the strain Zjut HJ2101 (see table 1), and the physiological and biochemical characteristics of the strain are identical with those of the Bacillus dysenteriae.

TABLE 1 physiological and biochemical identification table

( Note: in the table, (-) -, (+) indicates weak yin and weak positive )

3) 16S rRNA sequence analysis

The 16S rRNA gene sequence of the strain Zjut HJ2101 is completed by biological engineering (Shanghai) GmbH, the length of the 16S rRNA sequence of the strain Zjut HJ2101 is 1444bp, and the specific nucleotide sequence is shown as SQE ID NO. 1. The obtained 16S rRNA sequence of the strain is compared by a Genebank sequence, a phylogenetic tree is established by MEGA-X, the result is shown in figure 4, the strain Zjut HJ2101 is identified as Zjut HJ2101 of Bacillus aquaticus (Bacillus hwajinponensis) and is preserved in China center for type culture collection, the preservation number is CCTCC NO: M2022196, the preservation date is 3 months and 4 days 2022, and the address: china, wuhan university, zip code 430072.

SQE ID NO.1

CTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGAGATTTGGGAGCTTGCTCCCAAATCTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCCTGCAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGGTAATACATCGCACCGCATGGTGTGATGTTGAAAGTTGGCCTCTGGCTAACACTGCAGGATGGGCCCGCGGCGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGACGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGGTTCCACCCTCAGTGCTGAAGTTAACACATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTGGAGACAGGACGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGGGCAGCAACACCGCGAGGTGAAGCGAATCCCATAAAGCCGTTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTATGGAG.

Example 4 preparation of Wet cells of Bacillus Oxfordii Zjut HJ2101

(1) Plate culture

The Bacillus pumilus Zjut2101 is inoculated on a plate culture medium and cultured for 48-72h at 30 ℃ to obtain a plate culture strain.

(2) Proliferation culture

And (3) selecting a ring of the strains after the plate culture, inoculating the strains to a multiplication culture medium, and culturing for 24 hours in an incubator with the rotating speed of 200rpm and the temperature of 30 ℃ at constant temperature to obtain a multiplication bacterial liquid.

(3) Fermentation culture

The proliferated bacterial solution was inoculated into 100mL of an enzyme-producing fermentation medium at an inoculum size of 2% (v/v), cultured at 30 ℃ for 36 hours at 200rpm, and the culture solution was centrifuged at 8000rpm for 10min at 4 ℃ to collect wet cells.

Example 5 production of galactooligosaccharides by Whole cell catalysis of Bacillus Oxfordii Zjut HJ2101

(1) Lactose was added to 10mL of disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution having a pH of 7.0 to give a concentration of 0.3g/mL, 2g of the wet cells after fermentation according to example 4 was added, and the mixture was transformed at 200rpm at 30 ℃ for 36 hours to obtain a transformed solution containing galactooligosaccharides.

(2) Centrifuging 1mL of the transformation solution at 8000rpm at 4 deg.C for 10min, diluting the supernatant with mobile phase to 50 times, filtering with 0.22 μm microporous filter, and detecting the concentration of galactooligosaccharide by HPLC to determine that the yield of galactooligosaccharide in the transglycosylation reaction can reach 15% (as shown in FIG. 5). Centrifuging the residual conversion solution containing galacto-oligosaccharide at 8000rpm and 4 deg.C for 10min, collecting supernatant, adding anhydrous ethanol until ethanol concentration is 70%, mixing, centrifuging at 8000rpm for 10min, and discarding supernatant to obtain galacto-oligosaccharide.

The yield of galactooligosaccharides was calculated from the following formula:

[GOS]＝[Lactose] _i -[Lactose] _r -[Glicose]-[Galactose]

wherein: [ GOS ]]Is the galacto-oligosaccharide concentration in the reaction product; [ Lactose] _i Is the initial lactose concentration in the reaction product; [ Lactose] _r Is the residual lactose concentration of the reaction solution product; [ Glucose ]]Is the glucose concentration in the reaction product; [ Galactose]Is the concentration of galactose in the reaction product; y is _GOS The yield of galacto-oligosaccharides.

Chromatographic determination conditions: agilent1290 high performance liquid chromatography-Evaporative Light Scattering Detector (ELSD), column: NH (NH) ₂ P-50E 4.6mmi.D. multiplied by 250mm amino column; mobile phase acetonitrile: water =70 (v/v), flow rate: 1.0mL/min; temperature: 30 ℃; sample introduction amount: 10 μ L.

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.

Claims (6)

1. Zjut HJ2101 of Bacillus pumilus (Bacillus hwajinpoensis) is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2022196, the preservation date of 2022 years, 3 months and 4 days, the address: china, wuhan university, zip code 430072.

2. The use of the Bacillus tsunekii Zjut HJ2101 of claim 1 in the production of galactooligosaccharides.

3. The use according to claim 2, wherein said use is: taking wet thalli obtained by fermenting and culturing the Bacillus dysenteriae Zjut HJ2101 as a catalyst, taking lactose as a substrate and taking a buffer solution with pH of 6.0-8.0 as a reaction medium, carrying out conversion reaction under the conditions of 180-200rpm and 30-35 ℃ to obtain a conversion solution containing galacto-oligosaccharide, and separating and purifying to obtain the galacto-oligosaccharide.

4. The use according to claim 3, wherein the lactose is added in an amount of 100-300g/L based on the volume of the buffer; the dosage of the wet thalli is 100-300g/L by volume of the buffer solution.

5. The use of claim 3, wherein the buffer is a disodium phosphate-monobasic sodium phosphate buffer at pH 7.0.

6. The use of claim 3, wherein the catalyst is prepared by:

(1) Plate culture: inoculating Bacillus tsukuni HJ2101 on a plate culture medium, and culturing at 30 ℃ for 48-72h to obtain a plate culture strain; the plate culture medium consists of: 15g/L of lactose, 20g/L of anhydrous magnesium sulfate, 3g/L of trisodium citrate dihydrate, 10g/L of tryptone, 2g/L of KCl, 50g/L of NaCl, 20g/L of agar, pH 7.0 and deionized water as a solvent;

(2) And (3) proliferation culture: selecting a ring of strains after plate culture, inoculating the strains to a multiplication culture medium, and culturing for 24 hours in an incubator with the rotating speed of 200rpm and the temperature of 30 ℃ to obtain a multiplication bacterial liquid; the proliferation medium consists of: 15g/L of lactose, 20g/L of anhydrous magnesium sulfate, 3g/L of trisodium citrate dihydrate, 10g/L of tryptone, 2g/L of KCl, 50g/L of NaCl, 7.0 of pH and deionized water as a solvent;

(3) Fermentation culture: inoculating the proliferated bacterial liquid in the step (2) into an enzyme production fermentation culture medium in an inoculum size of 2% of volume concentration, culturing at the temperature of 30 ℃ for 36h at 200rpm, centrifuging the culture liquid for 10min at the temperature of 4 ℃ at 8000rpm, and collecting wet thalli; the enzyme-producing fermentation medium comprises the following components: 15g/L lactose, 5g/L tryptone, 10g/L yeast extract powder, 0.3g/L anhydrous magnesium sulfate and KH ₂ PO ₄ 0.05g/L, naCl 50g/L, pH 7.0, and deionized water as solvent.

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