TW202325854A - Method of producing natural type n-acetylglucosamine - Google Patents

Abstract

The present invention provides a method of producing N-acetylglucosamine, the method comprises the following steps: (a) providing Chitinibacter tainanensis; (b) inoculating Chitinibacter tainanensis into liquid medium which contains chitin, diammonium hydrogen phosphate and yeast extract, the concentration of the diammonium hydrogen phosphate is 1.9 mg/mL, the concentration of the yeast extract is 1.5 mg/mL; (c) make Chitinibacter tainanensis ferment under the 30 degree Celsius environment to degrade the chitin as N-acetylglucosamine. By the above method of producing N-acetylglucosamine, the yield of N-acetylglucosamine is thus enhanced.

Description

Production method of natural N-acetylglucosamine

The invention relates to a production method of N-acetyl glucosamine, in particular to a production method of N-acetyl glucosamine which uses microorganisms to produce natural N-acetyl glucosamine.

N-acetylglucosamine (N-acetylglucosamine, NAG) is an aminosugar (Aminosugar) that has important physiological functions in the human body. Or the synthesis of proteoglycan (Proteoglycan), the above-mentioned sugars are important materials that constitute the components of human cells.

Regarding the production process of NAG, the raw materials used in the current process are mainly glucose or chitin, among which chitin is more commonly used to manufacture NAG. In the process of producing NAG from chitin, it is mainly divided into the following three methods.

The first method is a chemical method, which is to hydrolyze chitin with strong acid at high temperature to produce glucosamine, then make the acid anhydride react with the aforementioned glucosamine, and then attach the reactant of the aforementioned action to acetyl to produce N - Acetyl Glucosamine. However, NAG produced by chemical methods may have the problem of residual chemical agents.

The second method is the enzymatic method, which uses enzymes such as endochitinase, exochitinase, and N-acetylglucosaminidase to hydrolyze chitin to produce NAG. The NAG produced by the enzyme method is natural NAG, which has the advantages of high safety, high purity and no chemical residues, but the production cost of NAG produced by the enzyme method is relatively high.

The third method is the biotransformation method, which uses microorganisms to decompose chitin into NAG. The NAG produced in this way is also natural NAG, and its production cost is relatively low.

However, when using the biotransformation method to produce NAG, the yield of this process will be affected by the growth conditions of the microorganisms and the fermentation conditions. For example, giving microorganisms an appropriate nitrogen source in a fermentation environment can provide microbial growth and decompose chitin into NAG. At present, the nitrogen sources generally used for microorganisms are tryptin and yeast extract, and how to provide a better nitrogen source formula to further increase the yield of the microbial production of NAG is still an unresolved problem.

The object of the present invention is to address the above-mentioned problem, and a kind of N-acetyl glucosamine production method is provided, and it comprises the following steps: (a) provide Chitinibacter tainanensis bacterial strain; (b) Chitinibacter tainanensis bacterial strain is inoculated in liquid culture medium, in this liquid culture medium Contains chitin, diammonium hydrogen phosphate and yeast extract, the concentration of the diammonium hydrogen phosphate in the liquid medium is 1.9 mg/mL, and the concentration of the yeast extract in the liquid medium is 1.5 mg/mL and (c) fermenting the Chitinibacter tainanensis strain in an environment at a temperature of 30° C. to decompose the chitin into N-acetylglucosamine.

As mentioned above, in the step (b), the weight percent concentration of the chitin in the liquid medium is 6%.

As mentioned above, the chitin in the liquid medium is α-chitin.

As mentioned above, the source of the α-chitin is shrimp shell.

As mentioned above, the source of the α-chitin is crab shell.

According to the production method of N-acetyl glucosamine mentioned above, the yield of natural N-acetyl glucosamine can be further improved by using a better nitrogen source formula.

In order to fully understand the purpose, features and effects of the present invention, the present invention will be described in detail through the following specific embodiments and accompanying drawings, as follows:

The N-acetylglucosamine production method of embodiment 1

The bacterial strain used in Example 1 is Chitinibacter tainanensis. The aforementioned strain of Chitinibacter tainanensis was obtained from Taiwan Patent No. I328041 by the Biological Resources Preservation Center of the Institute of Food Industry Development of the Republic of China, and its strain number is BCRC910256.

First, Chitinibacter tainanensis colonies were inoculated in 400 ml of LB medium, and after fermentation and culture at 30°C for 16 hours, five 10 ml inoculum solutions (concentration of about 1.7 x 10 ⁹ cfu /ml). Prepare five 500 ml conical flasks at the same time, and pour 100 ml of liquid BH medium (Difco Bushnell-Haas broth) into each conical flask, which contains α-chitin at a concentration of 6% by weight (This is just an example of the type of chitin provided in Example 1. In other embodiments, users can use other types of chitin according to their needs). Inoculate the above-mentioned five parts of 10 ml of bacteria liquid for inoculation into the above-mentioned five parts of liquid BH culture medium respectively, and the above-mentioned five parts of BH culture medium inoculated with Chitinibacter tainanensis bacterial strain are respectively used as the control group and the experimental group one to four, and in the culture medium of the control group. No additional nitrogen source was added; the medium of experimental group 1 was added with yeast extract (Yeast Extract) at a final concentration of 1.5 mg/ml; the medium of experimental group 2 was added with a final concentration of yeast extract of 3 mg/ml ; Add the yeast extract with a final concentration of 4.5 mg/ml to the medium of the experimental group three; add the yeast extract with a final concentration of 6 mg/ml to the medium of the experimental group four.

The above-mentioned samples of the control group and the experimental group were fermented and cultured at 30°C for 96 hours, so that the Chitinibacter tainanensis strain therein decomposed the chitin into N-acetylglucosamine. After the samples of the control group and the experimental group were cultured for 96 hours, the samples of the control group and the experimental group were taken for centrifugation (centrifugation speed was 10,000 rpm, and the centrifugation time was 10 minutes). After the centrifugation was completed, one of the samples of the control group and the experimental group was taken out. Supernatant, measure the refraction (Brix%) of the sample supernatant (Brix%) of the sample supernatant of the control group and the experimental group with a refractometer (ATAGO) (that is, measure the total concentration percentage of N-acetylglucosamine dissolved in the supernatant), Converted to the concentration of N-acetylglucosamine.

The experimental results are shown in Table 1 below. The results show that the addition of yeast extract to the medium can increase the yield of NAG produced by Chitinibacter tainanensis strains, and the increase in the yield of Chitinibacter tainanensis strains increases with the increase in the concentration of yeast extract promote. The above experimental results confirmed that giving microorganisms an appropriate amount of nitrogen source can improve the efficiency of microorganisms in decomposing chitin into NAG.

Table 1-Comparison of NAG production rate improvement percentage in each group group NAG concentration (mg/ml) NAG yield increase percentage (%)* control group 26 0 Experimental group one 31 19.2 Experimental group two 34 30.8 Experimental group three 36 38.5 Experimental group four 38 46.2 *NAG yield increase percentage (%) = (NAG concentration of the experimental group - NAG concentration of the control group) / NAG concentration of the control group × 100%

The N-acetylglucosamine production method of embodiment 2

The process flow of the N-acetylglucosamine production method of this embodiment 2 is roughly the same as that of embodiment 1, and the composition of the culture medium of the control group of this embodiment 2 is also the same as that of embodiment 1, the only difference is that the experimental group 1 to the experimental group of this embodiment 2 The type of nitrogen source added in group four is different from that in embodiment 1. The types and concentrations of nitrogen sources in the experimental group 1 to the experimental group 4 in this embodiment 2 are shown in Table 2 below.

Table 2-Nitrogen source types and concentrations in experimental group 1 to experimental group 4 The type of nitrogen source and the concentration of nitrogen source in the medium Experimental group one 1.0 mg/ml tryptone and 0.5 mg/ml yeast extract Experimental group two 2.0 mg/ml trypsin and 1.0 mg/ml yeast extract Experimental group three 3.0 mg/ml trypsin and 1.5 mg/ml yeast extract Experimental group four 4.0 mg/ml trypsin and 2.0 mg/ml yeast extract

The experimental results are shown in Table 3 below. The results show that adding tryptin and yeast extract to the medium can increase the yield of NAG produced by Chitinibacter tainanensis strains, and the increase in the yield of Chitinibacter tainanensis strains increases with tryptin and yeast The increase of the concentration of the bacterial extract increases. And when comparing the experimental results of this Example 2 with the experimental results of Example 1, it can be found that part of the yeast extract in the medium is replaced by trypsin, and the yield of the Chitinibacter tainanensis strain is improved to a better degree. The above experimental results confirmed that different types of nitrogen sources have different degrees of improvement in the efficiency of microbial production of NAG. In particular, the effect of adding tryptin as a nitrogen source is better than that of simply using yeast extract as a nitrogen source.

Table 3-Comparison of NAG production rate improvement percentage in each group group NAG concentration (mg/ml) NAG yield increase percentage (%)* control group 28 0 Experimental group one 35 25.0 Experimental group two 40 42.9 Experimental group three 44 57.1 Experimental group four 47 67.9 *NAG yield increase percentage (%) = (NAG concentration of the experimental group - NAG concentration of the control group) / NAG concentration of the control group × 100%

The N-acetylglucosamine production method of embodiment 3

The process flow of the N-acetylglucosamine production method of this embodiment 3 is roughly the same as that of embodiment 2, and the composition of the culture medium of the control group of this embodiment 3 is also the same as that of embodiment 2, the only difference is that the experimental group 1 to the experimental group of this embodiment 3 The type of nitrogen source added in group four is different from that in embodiment 2. The types and concentrations of nitrogen sources in Experimental Group 1 to Experimental Group 4 in Example 3 are shown in Table 4 below.

Table 4-Nitrogen source type and concentration in experimental group 1 to experimental group 4 The type of nitrogen source* and the concentration of nitrogen source in the medium Experimental group one 1.9 mg/ml of (NH ₄ ) ₂ HPO ₄ and 1.5 mg/ml of yeast extract Experimental group two 1.1 mg/ml of NH ₄ NO ₃ and 1.5 mg/ml of yeast extract Experimental group three 1.8 mg/ml of (NH ₄ ) ₂ SO ₄ and 1.5 mg/ml of yeast extract Experimental group four 0.8 mg/ml urea and 1.5 mg/ml yeast extract *The addition amount of inorganic nitrogen source is calculated based on the same nitrogen content

The experimental results are shown in Table 5 below. The results show that adding inorganic nitrogen sources and yeast extracts to the medium can improve the yield of NAG produced by Chitinibacter tainanensis strains, and the experimental results of Example 3 (experimental group one, containing 1.9 mg/ml (NH ₄ ) ₂ HPO ₄ and 1.5 mg/ml yeast extract) and the experimental results of Example 2 (experimental group three, containing 3.0 mg/ml trypsin and 1.5 mg/ml yeast When comparing the tryptic protein in the culture medium with diammonium hydrogen phosphate ((NH ₄ ) ₂ HPO ₄ ), it can be found that the yield of Chitinibacter tainanensis strain is improved to a greater extent. In addition, it can also be found from the experimental results of Example 3 that even among the four inorganic carbon sources, diammonium hydrogen phosphate is a better source of inorganic nitrogen source.

Table 5-Comparison of NAG production rate improvement percentage in each group group NAG concentration (mg/ml) NAG yield increase percentage (%)* control group 28 0 Experimental group one 45 60.7 Experimental group two 34 21.4 Experimental group three 33 17.9 Experimental group four 30 7.1 *NAG yield increase percentage (%) = (NAG concentration of the experimental group - NAG concentration of the control group) / NAG concentration of the control group × 100%

The N-acetylglucosamine production method of embodiment 4

First, prepare 160 grams of α-chitin powder, pour the above-mentioned chitin powder into 2 liters of BH medium, and mix evenly. The BH medium is also added with diammonium hydrogen phosphate at a final concentration of 1.9 mg/ml With 1.5 mg/ml of yeast extract. Pour the medium mixture of the above-mentioned chitin powder and BH medium into a 5-liter fermenter, and sterilize it with a general-purpose autoclave.

Then, prepare the bacterial liquid for inoculation according to the preparation method of the bacterial liquid for inoculation in Example 1. Take 100 ml of Chitinibacter tainanensis inoculation bacteria solution and inoculate it into the above-mentioned medium mixture, and ferment at a speed of 200 rpm, a temperature of 30°C, and an air flow of 2 L/min. The fermentation time is 165 hours. Take out the fermentation broth sample, and according to the method of embodiment 1, obtain the supernatant from the fermentation broth sample, and measure the inflection of the supernatant, the highest value in the NAG concentration of the supernatant measured at each time point can be It can reach 65 mg/ml, and if converted into NAG yield, it can reach 81.3% (NAG yield = NAG production amount/chitin addition amount x 100%).

The N-acetylglucosamine production method of embodiment 5

First, according to the method of Example 1, five parts of 10 ml bacteria solution for inoculation were prepared. Prepare five 500 ml Erlenmeyer flasks and five parts of experimental group liquid culture medium at the same time. The above five parts of experimental group liquid culture medium are based on BH medium as the base medium, and different sources of α-chitin (100% by weight in the medium) are added. concentration of 6%), diammonium phosphate (final concentration in medium 1.9 mg/ml) and yeast extract (final concentration in medium 1.5 mg/ml). The liquid culture medium of the above five experimental groups were respectively referred to as experimental group 1 to experimental group 5.

Then, according to the method of Example 1, five parts of 10 ml bacterial solution for inoculation were prepared. Prepare five 500 ml Erlenmeyer flasks and five parts of control group liquid culture medium at the same time. The above five parts of control group liquid culture medium are based on BH medium as the basal medium, and α-chitin from different sources is added (100% by weight in the culture medium) Min concentration is 6%), but no nitrogen source is added. The liquid culture medium of the above five experimental groups was used as control group 1 to control group 5 respectively.

Among them, experimental group 1 and control group 1 used the same α-chitin source; experimental group 2 and control group 2 used the same α-chitin source; experimental group 3 and control group 3 used the same α-chitin source; Group 4 and control group 4 used the same α-chitin source; experimental group 5 and control group 5 used the same α-chitin source. In Table 6 below, experimental group one and control group one are collectively referred to as group one, experimental group two and control group two are collectively referred to as group two, and experimental group three and control group three are collectively referred to as group three; experimental group Group 4 and control group 4 are collectively referred to as group 4, and experimental group 5 and control group 5 are collectively referred to as group 5.

The sources of α-chitin in groups 1 to 5 are shown in Table 6 below.

Table 6 - Sources and forms of α-chitin in groups 1 to 5 Source of α-chitin type group one shrimp shell pure white fine powder group two shrimp shell light red fine powder group three shrimp shell light yellow fine powder Group four crab shell Pure white powder, coarse particles Group five shrimp shell off-white fine powder

Next, inoculate the aforementioned five parts of 10 ml bacterial solution for inoculation into the liquid BH medium of experimental group 1 to experimental group 5, and the samples of experimental group 1 to experimental group 5 were shaken at 200 rpm in an environment of 30°C for 72 hours, so that the Chitinibacter tainanensis strain breaks down the chitin into N-acetylglucosamine. After the samples of the above experimental groups were fermented and cultured for 72 hours, according to the method of Example 1, supernatants were obtained from the samples of experimental groups 1 to 5, and the NAG refraction of the supernatants were measured.

At the same time, the above-mentioned five parts of 10 ml bacterial solution for inoculation were also inoculated into the liquid LB medium of the control group 1 to the control group 5, and the fermentation operation was carried out as the above-mentioned fermentation operation process of the experimental group 1 to the experimental group 5, to measure the control group. The NAG refraction of the supernatants from the first to the fifth control group.

The experimental results are shown in Figure 1. The black bars in Figure 1 represent the experimental group in the group, and the white bars represent the control group in the group. In groups 1 to 5, the experimental groups are all added with final concentration Diammonium hydrogen phosphate of 1.9 mg/ml and yeast extract with a final concentration of 1.5 mg/ml were used as nitrogen sources. Compared with the corresponding control group (using the same α-chitin source but not adding the above Nitrogen source), the NAG yields of each experimental group were increased. Based on the above experimental results, it can be known that no matter what α-chitin source is used as raw material, as long as the above-mentioned nitrogen source formula is added to the medium, the NAG yield of the production method of utilizing microorganisms to produce N-acetylglucosamine can be significantly improved. promoted. Moreover, it can be seen from the above experimental results that even if waste biomass materials such as shrimp shells or crab shells from different sources are used as chitin, as long as the medium contains the above-mentioned nitrogen source formula, Chitinibacter tainanensis strains can effectively produce NAG, while chitin The diversity of quality source selection will help to further reduce the production cost of NAG.

As mentioned above, with the above N-acetylglucosamine production method, this production method provides a better nitrogen source formula, that is, 1.9 mg/ml diammonium hydrogen phosphate and 1.5 mg/ml yeast extract, compared with In the conventional N-acetyl glucosamine production method using only BH medium, the above-mentioned N-acetyl glucosamine production method with better nitrogen source formula can further increase the yield of natural N-acetyl glucosamine.

The present invention has been disclosed above with preferred embodiments, but those skilled in the art should understand that the embodiments are only used to describe the present invention, and should not be construed as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to the embodiment should be included in the scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the scope of the patent application.

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Figure 1 shows the comparison results of the yields of NAG produced from different chitin sources in the N-acetylglucosamine production method of Example 5 of the present invention.

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Claims (8)

A method for producing N-acetylglucosamine, comprising the following steps: (a) provide Chitinibacter tainanensis strains; (b) Chitinibacter tainanensis bacterial strain is inoculated in liquid medium, and this liquid medium contains chitin, diammonium hydrogen phosphate and yeast extract, and the concentration of this diammonium hydrogen phosphate in this liquid medium is 1.9 mg/mL, The concentration of the yeast extract in the liquid medium is 1.5 mg/mL; and (c) Fermenting the Chitinibacter tainanensis strain in an environment at a temperature of 30° C. to decompose the chitin into N-acetylglucosamine. The method as claimed in item 1, wherein, in step (b), the weight percentage concentration of the chitin in the liquid medium is 6%. The method according to claim 2, wherein the chitin in the liquid medium is α-chitin. The method according to claim 3, wherein the source of the α-chitin is shrimp shells. The method according to claim 3, wherein the source of the α-chitin is crab shell. The method according to claim 1, wherein, in step (b), the chitin in the liquid medium is α-chitin. The method according to claim 6, wherein the source of the α-chitin is shrimp shells. The method according to claim 6, wherein the source of the α-chitin is crab shell.

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