Preparation method of trehalose
阅读说明：本技术 一种海藻糖的制备方法 (Preparation method of trehalose ) 是由 刘功良 杨松光 张敏倩 白卫东 梁景龙 吴玉莹 黄力 于 2019-12-03 设计创作，主要内容包括：本发明涉及海藻糖制备技术领域,具体公开了一种海藻糖的制备方法。所述的海藻糖通过蜂蜜接合酵母(Zygosaecharomyces mellis)LGL-2发酵制备得到；具体通过如下方法制备得到：将蜂蜜接合酵母(Zygosaecharomyces mellis)LGL-2接种在培养液中,在20～30℃下发酵84～108h,得发酵液；然后从发酵液中进行海藻糖的提取即得海藻糖。实验表明,采用蜂蜜接合酵母(Zygosaecharomyces mellis)LGL-2来发酵制备海藻糖可以显著提高菌种胞内海藻糖积累量,提高酵母菌发酵海藻糖的产量。(The invention relates to the technical field of trehalose preparation, and particularly discloses a preparation method of trehalose. The trehalose is prepared by fermenting LGL-2 of Zygosaccharomyces mellis; the preparation method specifically comprises the following steps: inoculating honey zygosaccharomyces mellis LGL-2 into a culture solution, and fermenting at 20-30 ℃ for 84-108 h to obtain a fermentation solution; and extracting trehalose from the fermentation liquor to obtain the trehalose. Experiments show that the trehalose prepared by fermenting LGL-2 with honey zygosaccharomyces mellis can obviously improve the intracellular trehalose accumulation of strains and improve the yield of trehalose fermented by yeasts.)
1. A preparation method of trehalose is characterized in that the trehalose is prepared by fermenting LGL-2 of honey-conjugated yeast (Zygosaecharomyces mellis).
2. The preparation method according to claim 1, which is specifically prepared by the following method:
inoculating honey zygosaccharomyces mellis LGL-2 into a culture solution, and fermenting at 20-30 ℃ for 84-108 h to obtain a fermentation solution; and extracting trehalose from the fermentation liquor to obtain the trehalose.
3. The method according to claim 2, wherein the sugar concentration of the culture solution is 300 to 500 g/L; most preferably, the sugar concentration of the culture broth is 400 g/L.
4. The preparation method according to claim 2, characterized in that the fermentation is carried out at 24-28 ℃ for 84-96 h; most preferably, fermentation is carried out at 24 ℃ for 96 h.
5. The method according to claim 2, wherein a honey-conjugated yeast (Zygosaecoralomyces mellis) LGL-2 is inoculated in a culture solution in the form of a seed culture solution; the inoculation amount of the inoculation liquid is 8-12% of the weight of the culture liquid; most preferably, the inoculum size of the inoculum is 10% by weight of the broth.
6. The method according to claim 5, wherein the inoculum is prepared by: adding 1-3 rings of activated honey zygosaccharomyces mellis LGL-2 into 8-15 mL of culture solution with sugar concentration of 300-500 g/L, culturing at constant temperature of 25-30 ℃ for 36-60 h, adding into 80-150 mL of culture solution with sugar concentration of 300-500 g/L, and continuously culturing at constant temperature of 25-30 ℃ for 36-60 h to obtain inoculation liquid.
7. The method according to claim 6, wherein the inoculum is prepared by: adding 2 rings of activated honey-conjugated yeast (Zygosaechomyces mellis) LGL-2 into 10mL of culture solution with sugar concentration of 400g/L, culturing at constant temperature of 28 ℃ for 48h, adding into 90mL of culture solution with sugar concentration of 400g/L, and continuously culturing at constant temperature of 28 ℃ for 48h to obtain the inoculation solution.
8. The method according to claim 2, wherein the extraction of trehalose from the fermentation broth comprises:
and centrifuging the fermentation liquor to obtain yeast paste, then placing the yeast paste into an oven for treatment, adding deionized water after the treatment, carrying out water bath for 15-40 min at 40-45 ℃, centrifuging, taking the liquid, concentrating and drying to obtain the trehalose.
9. The method according to claim 8, wherein the extraction of trehalose from the fermentation broth comprises:
centrifuging the fermentation liquor to obtain yeast paste, then placing the yeast paste into an oven at 80-100 ℃ for treatment for 40-80 min, and treating the yeast paste according to a solid-liquid mass-volume ratio of 1 g: adding 8-15 mL of deionized water, performing water bath at 40-45 ℃ for 15-40 min, centrifuging, taking liquid, concentrating and drying to obtain trehalose.
Technical Field
The invention relates to the technical field of trehalose preparation, and particularly relates to a preparation method of trehalose.
Background
Trehalose is a non-reducing disaccharide, has lower sweetness which is 45 percent of sucrose, and therefore has less damage to teeth; the trehalose is nontoxic, and can be degraded into glucose by trehalase in a human body to provide energy for the human body; in addition, the food additive has stable chemical properties and no reducibility, does not generate Maillard reaction even if being mixed and heated with amino acid, protein and the like, and is a good food additive.
Trehalose has the characteristic of stabilizing cell membranes and protein structures in biological cells, can protect the biological cells and bioactive substances from being damaged under the adverse environmental conditions of dehydration, high temperature, freezing, high osmotic pressure, toxic reagents and the like because hydrogen bonds are formed between the trehalose and the proteins or lipids so as to stabilize the conformations of the proteins or lipids under dehydration or certain adverse conditions, and meanwhile, the trehalose can influence the growth and toxicity of the cells by influencing the synthesis of cell walls, and the characteristics are that other saccharides such as sucrose, glucose and the like do not have in nature.
Because the trehalose has the functions, the trehalose can be used as a food additive to be mixed with other sweeteners in the field of foods to reduce the side effect of decayed teeth of the sweeteners and can also be used for preventing protein in the foods in a dry or frozen environment from being denatured; in the field of cosmetics, seaweed is considered as a new cosmetic raw material and is widely applied to skin cosmetics, the problem of skin dryness can be well relieved due to the moisture retention of the seaweed, and anhydrous trehalose can be used as a dehydrating agent of phospholipid and enzyme and is used in skin cream; in the medical field, the product can be used as a protective agent for biological products such as enzymes, antibodies, antibiotics and the like, and can also be prepared into derivatives for anticancer agents and antitumor agents; in the agricultural field, trehalose can enhance the stress resistance of crops, so that the salt and alkali resistant crops, frost-resistant fruits and vegetables and the like can be cultivated by a transgenic technology. Therefore, the deep research on the trehalose has important significance for improving the yield of the trehalose and reducing the production cost.
The production of trehalose by microbial fermentation is one of the preparation methods of trehalose; for example, Pen\37094, etc. (Pen\37094, Zen 37094, New Yong, the influence of high sugar stress on physiological metabolism of saccharomyces cerevisiae in an long term [ J ]. modern food technology 2011,27(4):397 and 399.) active dry yeast is used as an experimental material, the growth condition and the intracellular trehalose accumulation and metabolism rules of the dry yeast under the high sugar environment stress are researched, and the intracellular trehalose accumulation amount reaches 48.73mg/g finally. However, the trehalose produced by the existing microbial fermentation method has low accumulation of trehalose in the strain cells due to the problem of fermenting the strain, thereby influencing the yield of trehalose.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of trehalose. The preparation method uses honey conjugated yeast (Zygosaechycosis mellis) LGL-2 to prepare trehalose by fermentation; trehalose prepared by fermenting LGL-2 of honey-conjugated yeast (Zygosaccharomyces mellis) can significantly improve the accumulation of trehalose in the strain cells.
The technical problem to be solved by the invention is realized by the following technical scheme:
the invention provides a preparation method of trehalose, wherein the trehalose is prepared by fermenting LGL-2 of honey-conjugated yeast (Zygosaecharomyces mellis).
The honey zygosaccharomyces mellis LGL-2 is screened by a large amount of experiments by the inventor, and the strain is preserved in China center for type culture collection (the address is Wuhan university in Lodokana Lodoku, Wuhan, Hubei, China) within 12 months and 26 days in 2016; the preservation number is CCTCC No. M2016787.
The main morphological characteristics of the honey-conjugated yeast (Zygosaecharomyces mellis) LGL-2 are as follows: the cell morphology is characterized by oval shape, and the reproduction mode comprises bud reproduction and spore reproduction; the bacterial colony is thick, and the arch is great, and the bacterial colony color is light yellow, and the surface is smooth and more moist, and the line characteristic at bacterial colony edge is obvious. The honey zygosaccharomyces mellis LGL-2 is obtained by screening according to the following method:
(1) pouring 100 times diluted all-flower honey into a culture dish filled with a wort agar culture medium, culturing for 2d at a constant temperature of 28 ℃, observing the growth condition of the strain, selecting a single colony, observing under a microscope, and screening out the yeast according to the morphological characteristics of the yeast;
(2) marking the yeast screened in the step S1 in a culture dish filled with a wort agar culture medium with the sugar concentration of 70% (w/v), culturing for 2d under the constant temperature condition of 28 ℃, and observing the growth condition of the strain; selecting single colony of the grown strain, streaking and separating for 2 times until the single colony is separated; then placing the single colony under a microscope for observation, and screening pure honey-conjugated yeast (Zygosaccharomyces mellis) LGL-2 according to the morphological characteristics of the honey-conjugated yeast (Zygosaccharomyces mellis) LGL-2.
The wort agar culture medium in the step (2) is prepared by the following method: dissolving glucose, malt extract powder, deionized water and agar powder in boiling water bath, and stirring.
The wort agar medium with the sugar concentration of 70% (w/v) in the step (2) is prepared by the following method:
138g of glucose, 13g of malt extract powder, 100mL of deionized water and 2g of agar powder are weighed, dissolved in a boiling water bath and stirred continuously to obtain the agar powder.
The inventor researches show that the fermentation strain plays a decisive influence on the intracellular trehalose accumulation of the strain, and if the fermentation strain is not properly selected, the intracellular trehalose accumulation of the strain cannot be greatly improved even if the fermentation conditions are optimized and adjusted. The inventor indicates through a large number of experimental studies that trehalose prepared by fermenting honey-conjugated yeast (Zygosaccharomyces) LGL-2 by adopting trehalose can obviously improve the intracellular trehalose accumulation of strains; thereby improving the yield of the trehalose.
The inventor carries out molecular biological identification on 6 strains of honey zygosaccharomyces (1-6) obtained by separation in the related research process, and specific results are as follows:
the extracted total DNA of the yeast is taken as a template, universal primers NL-1 and NL-4 of a fungal 26SrDNAD1/D2 region are taken as upstream and downstream primers, a strain 26S rDNA fragment is amplified, and the electrophoresis detection result of a PCR product is shown in figure 1. The sequence length of the yeast ribosome large subunit 26SrDNAD1/D2 region is about 600bp, and as can be seen from figure 1, the DNA bands amplified by PCR are all 500-750, which indicates that the gene fragment of the 26SrDNA D1/D2 region is obtained.
The 26SrDNA sequence determination results of the strains are shown in Table 1, a 26SrDNAD1/D2 region sequence phylogenetic tree including a target strain and a control strain is constructed based on the 26SrDNA sequence determination results, the phylogenetic tree takes a 26SrDNAD1/D2 region sequence of milk-derived yeast (Pichia pastoris) as an outer group, the phylogenetic tree in figure 2 can show that strains 2-6 are clustered in a honey-junction yeast branch represented by Z.mellis ML343, the
TABLE 1 sequencing of 26SrDNA of strains
Preferably, the preparation method is specifically prepared by the following steps:
inoculating honey zygosaccharomyces mellis LGL-2 into a culture solution, and fermenting at 20-30 ℃ for 84-108 h to obtain a fermentation solution; and extracting trehalose from the fermentation liquor to obtain the trehalose.
More preferably, the sugar concentration of the culture solution is 300 to 500 g/L.
Most preferably, the sugar concentration of the culture broth is 400 g/L.
Further preferably, the preparation method comprises fermenting for 84-96 hours at 24-28 ℃.
Most preferably, fermentation is carried out at 24 ℃ for 96 h.
Further preferably, the honey-conjugated yeast (Zygosaechycodes mellis) LGL-2 is inoculated in the culture broth in the form of a seed-inoculating liquid; the inoculation amount of the inoculation liquid is 8-12% of the weight of the culture liquid.
Most preferably, the inoculum size of the inoculum is 10% by weight of the broth.
Further preferably, the inoculation liquid is prepared by the following method: adding 1-3 rings of activated honey zygosaccharomyces mellis LGL-2 into 8-15 mL of culture solution with sugar concentration of 300-500 g/L, culturing at constant temperature of 25-30 ℃ for 36-60 h, adding into 80-150 mL of culture solution with sugar concentration of 300-500 g/L, and continuously culturing at constant temperature of 25-30 ℃ for 36-60 h to obtain inoculation liquid.
Most preferably, the inoculum is prepared by the following method: adding 2 rings of activated honey-conjugated yeast (Zygosaechomyces mellis) LGL-2 into 10mL of culture solution with sugar concentration of 400g/L, culturing at constant temperature of 28 ℃ for 48h, adding into 90mL of culture solution with sugar concentration of 400g/L, and continuously culturing at constant temperature of 28 ℃ for 48h to obtain the inoculation solution.
Further, the inventors have found that trehalose is produced from honey-conjugated yeast (Zygosaechycodes mellis) LGL-2, and that factors such as the amount of inoculated strain, the sugar concentration of the culture broth, the fermentation temperature and the fermentation time have an important influence on the intracellular trehalose accumulation of the strain LGL-2 of honey-conjugated yeast (Zygosaechycodes mellis), and that the highest trehalose accumulation can be obtained when the factors such as the amount of inoculated strain, the sugar concentration of the culture broth, the fermentation temperature and the fermentation time are within the above-mentioned ranges.
Further preferably, the specific method for extracting trehalose from the fermentation broth comprises the following steps: and centrifuging the fermentation liquor to obtain yeast paste, then placing the yeast paste into an oven for treatment, adding deionized water after the treatment, carrying out water bath for 15-40 min at 40-45 ℃, centrifuging, taking the liquid, concentrating and drying to obtain the trehalose.
More preferably, the specific method for extracting trehalose from the fermentation broth comprises the following steps: centrifuging the fermentation liquor to obtain yeast paste, then placing the yeast paste into an oven at 80-100 ℃ for treatment for 40-80 min, and treating the yeast paste according to a solid-liquid mass-volume ratio of 1 g: adding 8-15 mL of deionized water, performing water bath at 40-45 ℃ for 15-40 min, centrifuging, taking liquid, concentrating and drying to obtain trehalose.
Has the advantages that: according to the invention, the trehalose is prepared by fermenting LGL-2 with honey-conjugated yeast (Zygosaechamomyces mellis) for the first time, and the accumulation amount of trehalose in strain cells can be obviously increased by preparing the trehalose by fermenting LGL-2 with the honey-conjugated yeast (Zygosaechamomyces mellis).
Drawings
FIG. 1 is a diagram showing the electrophoresis result of PCR amplification products of strain 26S rDNA.
FIG. 2 is a phylogenetic tree of honey junction yeast based on the 26S rDNNAD 1/D2 region sequence.
FIG. 3 is a standard curve of trehalose content measured by anthrone-sulfuric acid colorimetry.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
The culture solution is prepared according to the following method: 13g of wort is weighed, 54g, 78g, 98g, 118g and 138g of glucose are respectively added, and then the mixture is respectively dissolved in 100mL of deionized water to prepare culture solutions with sugar concentrations of 300g/L, 400g/L, 500g/L, 600g/L and 700 g/L.
The slant culture medium of the invention is prepared according to the following method: 13g of wort, 138g of glucose and 2g of agar powder are weighed by an electronic scale and dissolved in 100mL of deionized water to prepare the slant culture medium.
The 0.2% anthrone sulfuric acid reagent of the present invention was prepared according to the following method: 0.2g of anthrone is weighed, dissolved in a 100mL volumetric flask with 98% sulphuric acid, and the volume is fixed to the scale, shaken up and placed in a brown flask.
The activation method of the strain comprises the following steps: taking honey zygosaccharomyces mellis LGL-2 out of ultralow temperature refrigerator, and standing in 28 deg.C incubator for 30 min; a ring of yeast is picked up and streaked in a slant culture medium for 96h until colonies appear, and activated honey-conjugated yeast (Zygosaechamomyces mellis) LGL-2 is obtained.
The trehalose content is measured by adopting an anthrone-sulfuric acid colorimetric method: adding 0.2% anthrone-sulfuric acid reagent 4mL into 2mL of solution to be measured, mixing, heating in boiling water bath for 5min, cooling with tap water, and measuring OD with water as control625Trehalose content was determined according to the standard curve shown in FIG. 3.
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