TWI669392B - Novel lactobacillus brevis and the application thereof - Google Patents

Abstract

The invention provides an isolated Lactobacillus brevis NO.03, which can produce γ-aminobutyric acid, and the yield of γ-aminobutyric acid reaches 62.5 g/L, and γ - The conversion rate of aminobutyric acid is 93.28%.

Description

Novel Lactobacillus brevis and its application

The present invention relates to a short Lactobacillus strain, a culture medium for culturing a short Lactobacillus strain, and a Lactobacillus strain, in particular to a method for increasing the yield of γ-aminobutyric acid and converting the γ-aminobutyric acid. Up to 93.28% of applications.

Γ-aminobutyric acid (GABA) is widely found in microorganisms, animals and plants. Studies have found that the physiological functions of γ-aminobutyric acid include lowering blood pressure, promoting sleep, anti-depression, regulating blood sugar, improving epilepsy, improving Alzheimer's disease, and regulating immune function. In recent years, γ-aminobutyric acid has been widely used in various fields of food, biotechnology, and medicine, and is particularly used in the field of promoting the sleep by being added to health foods. Therefore, mass production of γ-aminobutyric acid by industrial means is an important issue.

At present, the industrial production mode of γ-aminobutyric acid includes a chemical synthesis method and a microbial fermentation method. The chemical synthesis method has a high speed and a high yield, but generates a large amount of harmful substances, and the technology for removing harmful substances is complicated, high in cost, poor in safety, and difficult to ensure 100% removal of harmful substances. Therefore, γ produced by chemical synthesis method Aminobutyric acid will not be used in the food industry.

Microbial fermentation is currently the most commonly used to industrially produce γ-aminobutyric acid. The method, for example, industrially produces γ-aminobutyric acid by a method of lactic acid bacteria fermentation. In the method for producing γ-aminobutyric acid by fermentation of lactic acid bacteria, lactic acid bacteria convert glutamic acid (Glu) into γ-aminobutyric acid by using glutamate decarboxylase in vivo, which has a cost. Low, easy to produce, and so on. However, in the current method for producing γ-aminobutyric acid by the lactic acid bacteria fermentation method, the lactic acid bacteria and the culture medium used cannot significantly increase the production rate and the conversion rate, resulting in limited production per unit time and unit space. Therefore, it is necessary to find a lactic acid bacteria strain which is more suitable for the production of γ-aminobutyric acid, and with a medium more suitable for the fermentation production of γ-aminobutyric acid, thereby improving the production rate of γ-aminobutyric acid as a whole. And conversion rate.

It is an object of the present invention to provide a strain of Lactobacillus mil strain which is capable of fermentatively producing a high concentration of γ -aminobutyric acid.

It is an object of the present invention to provide a strain of Lactobacillus which can significantly increase the conversion rate of glutamic acid to γ -aminobutyric acid.

A second object of the present invention is to provide a medium for use in the field of lactic acid bacteria fermentation which can significantly increase the yield of γ -aminobutyric acid.

Another object of the present invention is to provide a production method capable of remarkably increasing the production rate of γ -aminobutyric acid.

Another object of the present invention is to provide a production method capable of remarkably improving the conversion ratio of γ -aminobutyric acid.

Another object of the present invention is to provide a method for producing a high concentration of γ-aminobutyric acid by a simple batch fermentation method.

In a preferred embodiment, the present invention provides a Lactobacillus brevis having 16S ribosomal DNA (16S rDNA), the 16S ribosomal DNA comprising SEQ ID NO: 1; At least one glutamate decarboxylase allows the S. cerevisiae strain to convert sodium glutamate in a culture solution to gamma-aminobutyric acid under batch fermentation conditions ( γ-Aminobutyric acid), in which the concentration of γ-aminobutyric acid is increased by at least 50 g/L during the batch fermentation, and the conversion rate of sodium glutamate to γ-aminobutyric acid is converted. More than 90%; among them, the optimal fermentation temperature of the short Lactobacillus strain is 25 to 35 degrees.

In a preferred embodiment, the Lactobacillus brevis can convert the sodium glutamate in the culture solution to γ-aminobutyric acid under the batch fermentation condition, so that the concentration of γ-aminobutyric acid is in the At least 60g/L is added during batch fermentation.

In a preferred embodiment, the S. cerevisiae strain has the ability to produce a metabolite that inhibits tyrosinase activity.

In a preferred embodiment, the short lactobacillus strain is deposited in the Food Industry Development Institute of the Corporation, and the deposit number is BCRC910771.

In a preferred embodiment, the optimal fermentation temperature of the S. cerevisiae strain is greater than or equal to 28 degrees Celsius and less than 32 degrees Celsius.

In a preferred embodiment, the optimum fermentation temperature of the Lactobacillus strain is 25 to 37 degrees Celsius.

In a preferred embodiment, the optimum fermentation temperature of the S. cerevisiae strain is 28 to 32 degrees Celsius.

In a preferred embodiment, the optimum fermentation temperature of the short Lactobacillus strain is Celsius 29~31 degrees.

In a preferred embodiment, the optimum fermentation temperature of the S. cerevisiae strain is 29.5 to 30.5 degrees Celsius.

In a preferred embodiment, the optimal fermentation temperature of the S. cerevisiae strain is 30 degrees Celsius.

In another preferred embodiment, the present invention provides a use of a medium for preparing γ-aminobutyric acid by fermentation of a lactic acid bacteria, the medium comprising: monosodium glutamate; a carbon source; a nitrogen source; Calcium carbonate; and Manganese sulfate.

In another preferred embodiment, the medium further comprises polysorbate 80 (Tween 80).

In another preferred embodiment, the lactic acid bacterium is Lactobacillus brevis.

In another preferred embodiment, the 16S ribosomal DNA of the Lactobacillus brevis comprises SEQ ID NO: 1; the Lactobacillus sinensis strain has at least one glutamate decarboxylase, and the Lactobacillus brevis strain The sodium glutamate in a culture solution can be converted into γ-aminobutyric acid under a batch fermentation condition, thereby making the concentration of γ-aminobutyric acid in the fraction At least 50g/L is added during batch fermentation, and the conversion rate of sodium glutamate to γ-aminobutyric acid is more than 90%; wherein the optimum fermentation temperature of the strain is 25~ 35 degrees.

In another preferred embodiment, the Lactobacillus brevis is deposited with the Food Industry Development Institute of the Corporation and has a registration number of BCRC910771.

In still another preferred embodiment, the present invention provides a method for producing γ -aminobutyric acid, comprising: inoculating a short Lactobacillus strain to a culture solution containing calcium carbonate, manganese sulfate, and polysorbate 80; The culture is incubated for a predetermined length of time; a fermentation broth comprising gamma -aminobutyric acid is obtained; in a further preferred embodiment, the broth has a pH of 3.5 to 7.0.

In still another preferred embodiment, the pH of the culture solution is between 4.0 and 4.5.

In still another preferred embodiment, the culture solution further comprises pyridoxal phosphate (PLP) of greater than 0 μM and less than or equal to 100 μM.

Figure 1: Thin layer chromatogram of the fermentation broth after culturing the lactic acid bacteria in the Lactobacillus medium.

Figure 2A: High performance liquid chromatogram of a gamma-aminobutyric acid standard.

Fig. 2B is a high performance liquid chromatogram of the fermentation broth after the lactic acid bacteria numbered NO. 03 is cultured in the Lactobacillus medium.

Fig. 2C is a high performance liquid chromatogram of the fermentation broth after culturing the lactic acid bacteria numbered NO. 03 in a Lactobacillus medium containing 1% sodium glutamate.

Figure 3: Bar graph of γ-aminobutyric acid concentration in fermentation broth at different temperatures.

Fig. 4A is a bar graph and a line graph of γ-aminobutyric acid concentration, total bacterial count, and pH value in a medium containing different carbon sources in a culture medium containing different carbon sources.

Fig. 4B is a bar graph and a line graph of the concentration of γ-aminobutyric acid, the total number of bacteria, and the pH value in the fermentation broth of the culture medium containing different concentrations of glucose.

Fig. 5A is a bar graph and a line graph of γ-aminobutyric acid concentration, total bacterial count, and pH value in a fermentation broth in a medium containing different nitrogen sources.

Fig. 5B is a long bar graph and a line graph of γ-aminobutyric acid concentration, total bacterial count, and pH value in a fermentation medium containing a different concentration of yeast extract cultured in a medium containing different concentrations of yeast extract. .

Fig. 6 is a bar graph showing the concentration of γ-aminobutyric acid in the fermentation broth in a medium containing different trace elements in the culture of the short Lactobacillus strain NO.03.

Fig. 7 is a long bar graph and line graph of γ-aminobutyric acid concentration, total bacterial count, and pH value in a medium containing different combinations of trace elements in a culture medium containing different combinations of trace elements. .

Figure 8: The strain of γ-aminobutyric acid, total bacterial count, and pH in the fermentation broth is cultured in a medium with different pH values (pH). line chart.

Figure 9: Culture of Lactobacillus brevis strain NO.03 in medium containing different concentrations of Pyridoxal phosphate (PLP), γ-aminobutyric acid in fermentation broth Bar graph and line chart of concentration, total number of bacteria, and pH.

Figure 10: The larvae strain NO.03 is cultured in a medium containing different concentrations of sodium glutamate. The γ-aminobutyric acid concentration and the residual amount of glutamate in the fermentation broth are long and condensed. Figure.

Figure 11: The strain Lactobacillus sp. NO.03 was cultured in 2 liters of medium containing 650 mM sodium glutamate. The yield of γ-aminobutyric acid and the residual amount of sodium glutamate in the fermentation broth varied with fermentation time. Bar chart and line chart.

Figure 12: Bar graph of the effect of the fermentation broth of the short Lactobacillus strain NO. 03 on tyrosinase activity.

Figure 13: Bar graph of the effect of the fermentation broth of the short Lactobacillus strain NO.03 on the chelating ability of ferrous ions.

Figure 14: Bar graph of the effect of the fermentation broth of the short Lactobacillus strain NO.03 on the chelation ability of copper ions.

Figure 15: Bar graph of the effect of the fermentation broth of the short Lactobacillus strain NO.03 on reducing power.

Figure 16: Bar graph of the total phenolic content in the fermentation broth of the short Lactobacillus strain NO.03.

Figure 17: Bar graph of the effect of the fermentation broth of the short Lactobacillus strain NO.03 on the DPPH free radical scavenging capacity.

The following is a detailed description of embodiments of the invention, as well as the techniques and features of the invention. The present invention is not intended to limit the invention, and any changes and modifications made by those skilled in the art without departing from the spirit and scope of the invention are included in the scope of the invention.

Experiment 1: Screening of lactic acid bacteria strains capable of producing γ-aminobutyric acid from the intestinal tract of aquatic animals

Experiment 1-1: Screening of Lactic Acid Bacteria from the Intestines of Aquatic Animals

Buy Priacanthus macracanthus , Chanos chanos, Perca fluviatilis, Thunnus thynnus, Psenopsis anomala , from Kaohsiung Nanzhao and Zuoying Twilight Markets. Oysters ( Ospreoida rafinesque ), Epheppus orbis , Ctenopharyngodon idellus , and Penaeus monodon . Tissue was harvested using a sterile bag gastrointestinal tract of aquatic animals, iron stomach machine (Smasher; label) After homogenization, the sample with 5g of the genus Lactobacillus 25mL medium ^{(Lactobacilli MRS broth; BD Difco TM} ) mixed at 37 ℃ for the Cultivate for 24 hours. Then, screening was carried out in the following four experiments, and a total of 56 strains of lactic acid bacteria were screened. Among them, the lactic acid bacteria strain refers to the following four kinds of experiments, including four characteristics of the strain: (1) cultured in the LAMVAB-A selective medium, can produce a transparent ring; (2) is a Gram-positive strain; (3) The results of the catalyst test were no bubble generation; and (4) the oxidase test results showed no color change.

I. Screening with LAMVAB-A selective medium: The bacterial solution was applied to LAMVAB-A selective medium and cultured in an anaerobic environment at 37 ° C for 48 hours to observe the colony week. Whether it has a transparent ring. A single colony having a transparent ring was selected, and a single colony was isolated and purified, and then stored in a 4 ° C environment with Lactobacilli MRS agar slant culture-medium.

The method of preparation of the above-described LAMVAB-A: the powder medium Lactobacillus 52.2g ^{(Lactobacilli MRS powder; BD Difco TM} ), 0.25g cysteine hydrochloride (Cysteine HCL), 2.5g of calcium carbonate, and dissolved 40g agar solution The mixture was sterilized in 1 L of distilled water (Distilled Water) at 121 ° C for 15 minutes in an autoclave, and the pH was adjusted to 5.0 with 1 M hydrochloric acid. After cooling to 50 ° C, 1.5 mL of vancomycin hydrochloride was added and mixed well (see Hartemink et al., 1997. Journal of Microbiological Methods; 29(2): 77-84).

II. Screening by Gram staining: Gram staining was performed, and the color and shape of the strain were examined by high-magnification oil mirror. The strain was observed to be a blue-purple positive strain or a pink negative strain.

III. Screening by Catalase test: A single colony was placed on a glass slide, and a few drops of 3% hydrogen peroxide solution were dropped to observe the presence or absence of bubbles. A bubble is produced as a positive reaction; otherwise, a negative reaction.

IV. Screening by Oxidase test: A 1% concentration of tetramethyl-p-phenylenediamine dihydrochloride reagent was dropped on a single colony of the strain to be tested, and the color change was observed. If the colony is dark blue, it is a positive reaction; if the colony has no color change, it is a negative reaction.

The γ-aminobutyric acid contained in the fermentation broth obtained by the fermentation of the above 56 strains of lactic acid bacteria was evaluated by thin layer chromatography (TLC). The results showed that the fermentation broth obtained by fermentation of 56 strains of lactic acid contained γ-aminobutyric acid. From this, it is understood that the 56 strains of lactic acid have the ability to produce γ-aminobutyric acid. See Table 1 for the number of the 56 strains of lactic acid.

The above 56 strains of lactic acid were separately cultured in Lactobacillus medium and cultured at 37 ° C for 96 hours. Then, the γ-aminobutyric acid content in the fermentation broth obtained by fermentation of 56 strains of lactic acid bacteria was measured by high performance liquid chromatography (HPLC), and the experimental procedure was as follows.

The fermentation broth obtained after fermentation of 56 strains of lactic acid was derivatized. The solution preparation method required for derivatization is as follows: 40 mg of o-phthalaldehyde (OPA) or OPA is dissolved in 1 mL of methanol to prepare an o-phthalaldehyde solution. 1 mL of o-phthalaldehyde solution, 25 mL of 0.1 M TBE buffer, and 100 μL of 2-mercaptoethanol were uniformly mixed to prepare an o-phthalaldehyde standard solution. Then, 0.5 mL of the o-phthalaldehyde standard solution and 0.1 mL of the sample to be tested (that is, the fermentation liquid obtained by fermenting the lactic acid strain) were uniformly mixed and allowed to stand for one minute. After filtering through a 0.22 μm filter (PES Syringe Filter), 20 μL of the filtrate was poured into a column of a high performance liquid chromatography. 0.1 M sodium acetate (Sodium acetate; pH 6.7) was used as mobile phase A and methanol (Methanol) as mobile phase B, and detected by a UV 340 nm detector.

The results showed that the lactic acid strains numbered NO.02 and NO.03 isolated from the gastrointestinal tract of the large spine and the vaginal strain isolated from the gastrointestinal tract of the grass carp were numbered NO.41 and γ of the lactic acid strain of NO.45. The yield of aminobutyric acid was the highest, 785mg/L, 1024mg/L, 730mg/L, and 813mg/L, respectively. Among them, the γ-aminobutyric acid produced by the lactic acid strain numbered NO.03 was the highest.

Experiment 2: Identification and analysis of the characteristics of lactic acid bacteria

Experiment 2-1: Analysis of the ability of lactic acid bacteria to produce γ-aminobutyric acid

In this experiment, the ability of lactic acid bacteria to produce γ-aminobutyric acid was analyzed by thin layer chromatography (TLC). The experimental group was divided into NO.02 group, NO.02' group, NO.03 group, NO.03' group, NO.41 group, NO.41' group, NO.45 group, and NO.45' group; There were also 2 groups of control groups, namely γ-aminobutyric acid group and sodium glutamate group, a total of 10 groups.

The preparation method of the developing solution required in the thin layer chromatography analysis experiment step: uniformly mixing acetic acid (Acetic acid), n-butanol (1-Butanol), and distilled water (Distilled Water) in a ratio of 3:5:2. An additional 1% ninhydrin (Ninhydrin) coloring agent is added as a developing solution. Then, the experiment was conducted in the following manner.

Group NO.02: the number of lactic acid bacteria inoculated NO.02 Lactobacillus medium ^{(Lactobacilli MRS broth; BD Difco TM} ) , and cultured at 37 [deg.] C anaerobically for 48 hours and centrifuged (5,000 × g, 10 minutes) 0.7 μL of the supernatant was dropped onto a thin layer of the color layer, and subjected to chromatography using a developing solution at 105 °C.

NO.02' group: The lactic acid bacteria numbered NO.02 was inoculated into Lactobacillus medium containing 1% sodium glutamate (MSG), cultured in an anaerobic environment at 37 ° C for 48 hours, and centrifuged (5,000 × g, 10 minutes), 0.7 μL of the supernatant was dropped onto a thin layer of color layer, and subjected to chromatography using a developing solution at 105 °C.

Group NO.03: The lactic acid bacteria numbered NO.03 was inoculated into Lactobacillus culture medium, cultured in an anaerobic environment at 37 ° C for 48 hours, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was dropped to a thin The layered layer was subjected to chromatography using a developing solution at 105 °C.

NO.03' group: Inoculate lactic acid bacteria numbered NO.03 in 1% glutamic acid Sodium (Monosodium Glutamate, MSG) Lactobacillus culture medium, cultured in an anaerobic environment at 37 ° C for 48 hours, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was dropped to a thin layer of color layer, at 105 Chromatography was carried out using a developing solution at °C.

Group NO.41: The lactic acid bacteria numbered NO.41 was inoculated into Lactobacillus culture medium, cultured in an anaerobic environment at 37 ° C for 48 hours, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was dropped to a thin The layered layer was subjected to chromatography using a developing solution at 105 °C.

NO.41' group: The lactic acid bacteria numbered NO.41 was inoculated into Lactobacillus medium containing 1% sodium glutamate (MSG), cultured in an anaerobic environment at 37 ° C for 48 hours, and centrifuged (5,000 × g, 10 minutes), 0.7 μL of the supernatant was dropped onto a thin layer of color layer, and subjected to chromatography using a developing solution at 105 °C.

Group NO.45: The lactic acid bacteria numbered NO.45 was inoculated into Lactobacillus culture medium, cultured in an anaerobic environment at 37 ° C for 48 hours, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was dropped to a thin The layered layer was subjected to chromatography using a developing solution at 105 °C.

NO.45' group: The lactic acid bacteria numbered NO.45 was inoculated into Lactobacillus medium containing 1% sodium glutamate (MSG), cultured in an anaerobic environment at 37 ° C for 48 hours, and centrifuged (5,000 × g, 10 minutes), 0.7 μL of the supernatant was dropped onto a thin layer of color layer, and subjected to chromatography using a developing solution at 105 °C.

γ-Aminobutyric acid group: γ-aminobutyric acid standard (ChromaDex, Inc.; ASB-00001674-025) was dissolved in distilled water, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was taken. The mixture was dropped onto a thin layer of chromatography, and subjected to chromatography using a developing solution at 105 °C.

Sodium glutamate group: Sodium glutamate standard (Taiwan Weiwang Co., Ltd.) was dissolved in distilled water, centrifuged (5,000 × g, 10 minutes), and 0.7 μL of the supernatant was dropped to a thin layer of color layer. At 105 Chromatography was carried out using a developing solution at °C. ,

The results in Fig. 1 show that the NO.03' group contains more γ-aminobutyric acid than the other 7 groups.

Based on the results of Table 1 and Figure 1, it is known that the lactic acid bacteria numbered NO.03 has the best ability to produce γ-aminobutyric acid without adding 1% glutamate; and the lactic acid bacteria numbered NO.03 Compared to the γ-aminobutyric acid production without the addition of sodium glutamate, the γ-aminobutyric acid production of the lactic acid bacteria numbered NO.03 appeared to increase more significantly when 1% glutamate was added. Subsequent confirmation by quantitative analysis will confirm whether the γ-aminobutyric acid production of the lactic acid bacteria numbered NO.03 is significantly increased when 1% glutamate is added.

Experiment 2-2: Analysis of γ-aminobutyric acid production of lactic acid bacteria numbered NO.03 with the addition of 1% sodium glutamate

Quantitative analysis of γ-aminobutyric acid content in fermentation broth of NO.03 group and NO.03' group in experiment 2-1 by high performance liquid chromatography, experimental method and experiment 1 high performance liquid chromatography the same.

2A is a high performance liquid chromatogram of the γ-aminobutyric acid standard; FIG. 2B is a high performance liquid chromatogram of the fermentation broth after the lactic acid bacteria numbered NO. 03 is cultured in the lactic acid bacteria medium; FIG. 2C A high-performance liquid chromatogram of the fermentation broth after the lactic acid bacteria numbered NO.03 was cultured in a lactic acid bacteria medium containing 1% sodium glutamate. From the above experimental results, it is known that the lactic acid bacteria numbered NO.03 is compared with the γ-aminobutyric acid yield when no glutamate is added, and the lactic acid bacteria numbered NO.03 is added with 1% glutamate. The yield of γ-aminobutyric acid does increase significantly.

Experiment 2-3: Identification of lactic acid bacteria numbered NO.03

Entrusted Source International Biotechnology Co., Ltd. to be numbered NO.03 Identification of strains of lactic acid bacteria, in which strain identification was carried out by means of 16S rDNA sequence. First, the DNA of the lactic acid bacteria numbered NO.03 was extracted using a kit material (Genomic DNA Purification Kit, manufactured by Boon Co., Ltd.), and then the following primers were used to carry out a polymerase chain reaction (PCR). Copy 16S rDNA of lactic acid bacteria numbered NO.03. Then, the sequencing operation of the 16S rDNA of the lactic acid bacteria numbered NO.03 was carried out. Among them, the meaning of M in the primer is adenine (abbreviated as A) or (cytosine (C).

27F primer: 5'-AGAGTTTGATCMTGGCTCAG-3’

1492R primer: 5’-CGGTTACCTTGTTACGACTT-3’

The 16S rDNA sequence of the lactic acid bacteria numbered NO.03 is shown in SEQ ID NO: 1 of the Sequence Listing. By comparison with the 16S rDNA sequence in the database, the lactic acid bacteria numbered NO. 03 is Lactobacillus brevis , so the inventor referred to this strain as the short Lactobacillus strain NO.03.

Experiment 3: Effect of different culture temperatures on the yield of γ-aminobutyric acid

During the fermentation of lactic acid bacteria, the temperature should be controlled at the optimum fermentation temperature of the strain, so that the fermentation can proceed smoothly. Among them, the optimum fermentation temperature refers to the temperature most suitable for the growth of the strain or the most suitable for the production of fermentation products (please refer to Chapter 7 of Section 2 of the "Amino Acid Fermentation Production Technology" published in 2008, edited by Deng Maocheng, published by China Light Industry Press) .

The experiment was divided into 12 groups, which were named as 25°C group, 25°C′ group, 30°C group, 30°C′ group, 35°C group, 35°C′ group, 37°C group, 37°C′ group according to the culture temperature. 40 ° C group, 40 ° C ' group, 45 ° C group, and 45 ° C ' group.

The short Lactobacillus strain NO.03 was inoculated into Lactobacillus culture medium, and cultured in an anaerobic environment at 25 ° C, 30 ° C, 35 ° C, 37 ° C, 40 ° C, or 45 ° C for 96 hours to obtain a 25 ° C group. , 30 ° C group, 35 ° C group, 37 ° C group, 40 ° C group, or 45 ° C group of fermentation broth.

Short Lactobacillus strain NO.03 was inoculated into Lactobacillus medium containing 550M sodium glutamate (MSG), which was disgusting at 25 ° C, 30 ° C, 35 ° C, 37 ° C, 40 ° C, or 45 ° C, respectively. The cells were cultured for 96 hours in an oxygen atmosphere to obtain a fermentation broth of 25 ° C 'group, 30 ° C' group, 35 ° C' group, 37 ° C ' group, 40 ° C ' group, or 45 ° C ' group.

The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results in Figure 3 show that the 25 ° C group, the 25 ° C 'group, the 30 ° C group, the 30 ° C ' group, the 35 ° C group, the 35 ° C ' group, the 37 ° C group, the 37 ° C ' group, the 40 ° C group, the 40 ° C ' group The γ-aminobutyric acid contents of the 45 °C group and the 45 °C' fermentation broth were 923±104 mg/L, 17669±144 mg/L, 1,203±35 mg/L, 21,936±134 mg/L, and 781±108 mg/ L, 16840 ± 31 mg / L, 703 ± 9 mg / L, 14722 ± 103 mg / L, 342 ± 7 mg / L, 10779 ± 111 mg / L, 113 ± 9 mg / L, and 311 ± 8 mg / L. From this, it is understood that the temperature optimum for the production of γ-aminobutyric acid by fermentation of the short Lactobacillus strain NO. 03 is 25 ° C or more and less than 37 ° C regardless of whether or not sodium glutamate is added. Moreover, as is apparent from the results of FIG. 3, the short Lactobacillus strain NO. 03 can produce the maximum amount of γ-aminobutyric acid at a fermentation temperature of about 30° C. (for example, greater than or equal to 28 degrees Celsius and less than 32 degrees Celsius). Therefore, unlike the lactic acid bacteria generally isolated from dairy products, which has the optimum fermentation temperature of 37-45 ° C, the short Lactobacillus strain NO.03 isolated from the fish gut has an optimum fermentation temperature of about 30 ° C (for example, greater than or A novel feature equal to 28 degrees Celsius and less than 32 degrees Celsius reduces the energy costs required in the fermentation process. The subsequent experiment in this case will use 30 °C as the fermentation condition of the short lactobacillus strain NO.03.

Experiment 4: Effect of medium composition on the yield of γ-aminobutyric acid

Experiment 4-1: Carbon Source Test

The experiment was divided into 12 groups, named as glucose group, sucrose group, lactose group, galactose group, maltose group, glycerol group, molasses group, granulated sugar group, brown sugar group, dextrin group 1, dextrin according to the added carbon source. 2 groups and starch group.

The media formulations of each group contained 1% (w/v) carbon source, 1% yeast extract (Yeast extract), and 1% sodium glutamate, and the total volume of the medium was 9 mL. Among them, the carbon source added in the glucose group is glucose (Glucose), the carbon source added in the sucrose group is sucrose, the carbon source added in the lactose group is lactose (Lactose), and the carbon source added in the galactose group is Galactose, the carbon source added to the maltose group is maltose, the carbon source added to the glycerol group is glycerol (Glycerol), and the carbon source added to the molasses group is molasses (Molasses, purchased from Taiwan Sugar Co., Ltd.) Ltd.), the carbon source added to the sugar group is Refined Golden Sugar (purchased from Fengnianfenghe Co., Ltd.), and the carbon source added to the brown sugar group is Brown Sugar, and the carbon added in the dextrin group 1 The source is dextrin 1 (product number DE 8-10, purchased from Lishun Enterprise Co., Ltd.), dextrin 2 (product number DE 10-12, purchased from Lishun Enterprise Co., Ltd.), The carbon source added to the starch group is starch (Starch).

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results of Fig. 4A show that in the fermentation broth of the glucose group, the sucrose group, the lactose group, the galactose group, the maltose group, the glycerol group, the molasses group, the granulated sugar group, the brown sugar group, the dextrin group 1, the dextrin group 2 and the starch group, The content of γ-aminobutyric acid was 5,488±122 mg/L, 3,530±394 mg/L, 2,776±156 mg/L, 4,888±224 mg/L, 5,233±393 mg/L, 3,454±289 mg/L, and 5,291±231 mg/L, respectively. 4,003±263mg/L, 4,254±45mg/L, 1,296±19mg/L, 2,262±600mg/L, and 2,776±237mg/L, among which the γ-aminobutyric acid content in the glucose group is the highest, so the follow-up experiment The optimum concentration of glucose will be further tested using glucose as a source of carbon.

Experiment 4-2: Carbon source optimum concentration test

The experiment was divided into 0% group, 0.5% group, 1.0% group, 1.5% group, 2.0% group, 2.5 group, and 3.0% group. The medium formulation of each group was approximately the same as the glucose group in Experiment 4-1, the only difference being that the concentration of glucose was 0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, or 3.0%. The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group.

The results of Figure 4B show that the γ-aminobutyric acid content in the 0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5, and 3.0% fermentation broths was 5,178 ± 62 mg/ L, 5,081±61 mg/L, 5,424±127 mg/L, 4,896±30 mg/L, 4,596±48 mg/L, 4,108±8 mg/L, and 3,935±149 mg/L. Among them, the content of γ-aminobutyric acid was the highest in the 1% group, and it was found that when the glucose concentration exceeds 1%, the glucose effect (Crabtree effect) occurs and the yield of γ-aminobutyric acid is inhibited, so the subsequent experiment will be 1 The glucose concentration of % is used as the medium condition.

Experiment 4-3: Nitrogen source test

The experiment is divided into 12 groups, named as ammonium thiocyanate group and nitric acid according to the added nitrogen source. Aluminum group, ammonium nitrate group, ammonium molybdate group, ammonium persulfate group, phosphodiamine group, urea group, trypsin group, soybean peptone group, beef extract group, peptone group, and yeast extract group.

The medium formulations of each group contained 1% glucose, 1% (w/v) nitrogen source, and 1% sodium glutamate, and the total volume of the medium was 9 mL. Among them, the nitrogen source added to the ammonium thiocyanate group is Ammonium thiocyanate, the nitrogen source added to the aluminum nitrate group is aluminum nitrate, and the nitrogen source added to the ammonium nitrate group is ammonium nitrate ( Ammonium nitrate), the ammonium source added to the ammonium molybdate group is Ammonium molybdate, the nitrogen source added to the ammonium persulfate group is Ammonium persulfate, and the nitrogen source added to the phosphodiamine group is Diammonium hydrogen phosphate, the nitrogen source added to the urea group is Urea, the nitrogen source added to the trypsin group is Tryptone, and the nitrogen source added to the soy peptone group is soybean peptone. (Soya peptone), the nitrogen source added to the beef extract group is beef extract, the nitrogen source added to the peptone group is peptone, and the nitrogen source added to the yeast extract group is yeast extract.

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results of Fig. 5A show that ammonium thiocyanate group, aluminum nitrate group, ammonium nitrate group, ammonium molybdate group, ammonium persulfate group, phosphodiamine group, urea group, trypsin group, soybean peptone group, beef extract The γ -aminobutyric acid content in the fermentation broth of the group, peptone group and yeast extract group were 300±14 mg/L, 181±35 mg/L, 214±12 mg/L, 172±31 mg/L, 204±22 mg, respectively. /L, 197±5 mg/L, 197±3 mg/L, 3,948±104 mg/L, 2,318±383 mg/L, 4,690±125 mg/L, 5,148±212 mg/L, and 4,778±31 mg/L. Among them, the γ-aminobutyric acid content in the fermentation broth of the trypsin group, the soy peptone group, the beef extract group, the peptone group, and the yeast extract group was higher, and the γ-amine in the fermentation broth of the peptone group was further The content of butylbutyric acid is the highest. However, the cost of peptone is high, and the price of yeast extract is relatively low. Based on the cost in industrial production and the applicability of the food industry, subsequent experiments will use yeast extract as a nitrogen source to further explore its optimum concentration.

Experiment 4-4: Optimum concentration test of nitrogen source

The experiment was divided into 0.5% group, 1.0% group, 1.5% group, 2.0% group, 2.5 group, 3.0% group, and 3.5% group. The medium formulation of each group was approximately the same as the yeast extract group in Experiment 4-3, the only difference being that the concentration of the yeast extract was 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, or 3.5. %. The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group.

The results of Fig. 5B show that the γ-aminobutyric acid content of the fermentation broth of 0.5% group, 1.0% group, 1.5% group, 2.0% group, 2.5% group, 3.0% group, and 3.5% group was 5,267±308 mg/ L, 5, 305 ± 168 mg / L, 5, 624 ± 198 mg / L, 5, 962 ± 60 mg / L, 6, 464 ± 24 mg / L, 6, 298 ± 282 mg / L, and 6,692 ± 22 mg / L. Among them, the content of γ-aminobutyric acid in the 2.5% fermentation broth was the highest, so the follow-up experiment will use 2.5% yeast extract as the optimum medium for producing γ-aminobutyric acid.

Experiment 4-5: Effect of different single trace elements on the yield of γ-aminobutyric acid

The experiment is divided into 19 groups, named as calcium carbonate group, calcium chloride group, chromium chloride group, chromium trichloride group, cobalt chloride group, dipotassium hydrogen phosphate group, sulfuric acid according to the added single trace elements. Ferric ammonium group, ferrous sulfate group, magnesium sulfate group, manganese chloride group, manganese sulfate group, potassium carbonate group, potassium chloride group, potassium iodide group, potassium sulfate group, sodium acetate group, polysorbate 20 group, poly 80 groups of sorbitol ester and zinc sulfate group.

The medium formulations of each group contained 1% glucose, 2.5% yeast extract, 1% sodium glutamate, and 2 ppm trace elements, and the total volume of the medium was 9 mL. Among them, the trace element added in the calcium carbonate group is calcium carbonate (Calcium carbonate), the trace element added in the calcium chloride group is calcium chloride (Calcium chloride), and the trace element added in the chromium chloride group is chromium chloride ( Chromium chloride), the trace element added in the chromium trichloride group is Chromium trichloride, and the trace element added in the cobalt chloride group is Cobalt chloride, added in the dipotassium hydrogen phosphate group. The trace element is Dipotassium hydrogen phosphate, the trace element added to the ammonium iron sulfate group is Ferric ammonium sulfate, and the trace element added to the ferrous sulfate group is Ferrous sulfate. The trace element added in the magnesium sulfate group is magnesium sulfate (Magnesium sulfate), the trace element added in the manganese chloride group is Manganese chloride, and the trace element added in the manganese sulfate group is Manganese sulfate. The trace element added in the potassium carbonate group is Potassium carbonate, and the trace element added in the potassium chloride group is Potassium chloride, and the trace amount added in the potassium iodide group. The element is Potassium iodide, the trace element added in the potassium sulfate group is Potassium sulfate, the trace element added in the sodium acetate group is sodium acetate, and the trace amount added in the polysorbate 20 group. The element is polysorbate 20 (Tween 20), the trace element added in the polysorbate 80 group is polysorbate 80 (Tween 80), and the trace element added in the zinc sulfate group is zinc sulfate (Zinc sulfate).

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results of Fig. 6 show that the calcium carbonate group, the calcium chloride group, the chromium chloride group, the chromium trichloride group, the cobalt chloride group, the dipotassium hydrogen phosphate group, the ammonium iron sulfate group, the ferrous sulfate group, the magnesium sulfate group , manganese chloride group, manganese sulfate group, potassium carbonate group, potassium chloride group, potassium iodide group, potassium sulfate group, sodium acetate group, polysorbate 20 group, polysorbate 80 group, or zinc sulfate group fermentation liquid The contents of γ-aminobutyric acid were 8,556±28 mg/L, 8,412±294 mg/L, 7,508±272 mg/L, 7,715±316 mg/L, 7,079±178 mg/L, 6,443±202 mg/L, and 6,763±6 mg, respectively. /L, 6,138±43mg/L, 7,688±95mg/L, 7,367±170mg/L, 8,943±68mg/L, 7,192±225mg/L, 5,738±727mg/L, 6,756±265mg/L, 6,985±572mg/L 7,144±25mg/L, 8,033±66mg/L, 8,571±16mg/L, or 7,533±28mg/L. It can be seen that the γ-aminobutyric acid content of the calcium carbonate group, the calcium chloride group, the manganese sulfate group, and the polysorbate 80 group fermentation liquid is the highest. The effect of calcium carbonate, manganese sulfate, polysorbate 80 and combinations thereof on the content of γ-aminobutyric acid will be discussed later.

Experiment 4-6: Effect of different trace element combinations on the yield of γ-aminobutyric acid

The experiment was divided into 7 groups, named calcium carbonate group, manganese sulfate group, polysorbate 80 group, calcium carbonate and manganese sulfate group, calcium carbonate and polysorbate 80 groups, manganese sulfate and Group 80 of polysorbate, and 80 groups of calcium carbonate, manganese sulfate and polysorbate.

The medium formulation of each group contained 1% glucose, 2.5% yeast extract, 1% sodium glutamate, and at least one trace element, and the total volume of the medium was 9 mL. Among them, calcium carbonate The trace element added in the group was 2 ppm of calcium carbonate, the trace element added in the manganese sulfate group was 2 ppm of manganese sulfate, and the trace element added in the polysorbate group was 2 ppm of polysorbate 80, calcium carbonate and manganese sulfate. The trace elements added in the group were 2 ppm calcium carbonate and 2 ppm manganese sulfate, and the trace elements added in the calcium carbonate and polysorbate 80 group were 2 ppm calcium carbonate and 2 ppm polysorbate 80, manganese sulfate and poly sorbitol. The trace elements added in the alcohol ester 80 group were 2 ppm manganese sulfate and 2 ppm polysorbate 80, and the trace elements added in the calcium carbonate, manganese sulfate, and polysorbate 80 groups were 2 ppm of calcium carbonate and 2 ppm of manganese sulfate. And 2 ppm of polysorbate 80.

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results in Figure 7 show that calcium carbonate group, manganese sulfate group, polysorbate 80 group, calcium carbonate and manganese sulfate group, calcium carbonate and polysorbate 80 groups, manganese sulfate and polysorbate 80 groups, and carbonic acid The increase of γ-aminobutyric acid in the fermentation broth of calcium, manganese sulfate and polysorbate 80 groups was 2,540±187mg/L, 1,918±166mg/L, 1,395±69mg/L, 6,014±374mg/L, 1,889 ±59 mg/L, 4,335±92 mg/L, and 7,089±19 mg/L. Among them, the increase of γ-aminobutyric acid in the fermentation broth of calcium carbonate, manganese sulfate and polysorbate 80 was the highest, and the increase of γ-aminobutyric acid in the fermentation broth of calcium carbonate and manganese sulfate was second. Moreover, the results of Fig. 7 show that calcium carbonate and manganese sulfate have a synergistic effect on the function of increasing the γ-aminobutyric acid content by fermentation of S. cerevisiae NO. 03, and have unpredictable effects.

Experiment 4-7: Effect of initial pH of medium on yield of γ-aminobutyric acid

Divided into 8 groups, the initial pH values of the medium were named 3.5 group, 4.0 group, 4.5 group, 5.0 group, 5.5 group, 6.0 group, 6.5 group, and 7.0 group.

The media formulations of each group contained 1% glucose, 2.5% yeast extract, 1% sodium glutamate, 2 ppm calcium carbonate, 2 ppm manganese sulfate, and 2 ppm polysorbate 80, and the total volume was 9 mL, the difference was The inventors used sulfuric acid or sodium hydroxide to adjust the initial pH of the medium in groups 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 to set the initial pH of each medium. The values are in the order of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0.

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results in Figure 8 show that the contents of γ-aminobutyric acid in the fermentation broth of group 3.5, group 4.0, group 4.5, group 5.0, group 5.5, group 6.0, group 6.5, and group 7.0 were 764±20 mg/L, 22,839, respectively. ±280mg/L, 23,407±519mg/L, 18,230±122mg/L, 14,554±619mg/L, 12,903±458mg/L, 12,608±106mg/L, and 12,401±606mg/L; pH values are 3.2, 6.2, respectively. 6.65, 6.94, 7.03, 7.08, 7.15, and 7.2. Among them, the content of γ-aminobutyric acid in the fermentation broth of group 4.0 and group 4.5 was significantly higher than that of other groups. It can be seen that the initial pH of the culture solution is 4.0~4.5, which can significantly increase the γ in the fermentation broth. - Aminobutyric acid content.

Experiment 4-8: Effect of phosphoric acid over furfural on the yield of γ-aminobutyric acid

The experiment was divided into 7 groups. The groups were named as 0 group, 10 group, 20 group, 30 group, 40 group, 50 group and 100 group according to the addition amount of pyridoxal-5-phosphate. The medium formulations of each group contained 1% glucose, 2.5% yeast extract, 1% sodium glutamate, 2 ppm calcium carbonate, 2 ppm manganese sulfate, 2 ppm polysorbate 80, and phosphoric acid than furfural. It is 4.5 and the total volume is 9 mL. Among them, the concentrations of phosphate in the 0, 10, 20, 30, 40, 50, and 100 media were 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 100 μM.

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. After serially diluting each group of fermentation broth, the total number of bacteria in the fermentation broth was measured by plate counting method, and the total number of bacteria was converted into the logarithm of the total number of bacteria based on the base ₁₀ (Log ₁₀ total number of bacteria). . The pH of each fermentation broth was measured using a pH pH meter (Suntex-2200, Taiwan). The content of γ-aminobutyric acid in each fermentation broth was quantitatively analyzed by high performance liquid chromatography.

The results in Figure 9 show that the γ-aminobutyric acid contents in the fermentation broth of group 0, group 10, group 20, group 30, group 40, group 50 and group 100 were 23,147±183 mg/L and 26,688±157 mg/L, respectively. 26,316±633 mg/L, 25,708±451 mg/L, 24,750±311 mg/L, 23,481±277 mg/L, and 22,437±570 mg/L. Among them, the γ-aminobutyric acid content in the fermentation broth of 10 groups, 20 groups, and 30 groups was higher, and the γ-aminobutyric acid content in the fermentation broth of the group of 10 μM phosphoric acid compared with furfural was the highest. . Subsequently, 10 μM phosphoric acid to furfural was used as a medium condition for industrial production of γ-aminobutyric acid.

Experiment 4-9: Effect of sodium glutamate on the yield of γ-aminobutyric acid

The experiment was divided into 13 groups, and each group was named 250 groups according to the amount of sodium glutamate added. 300 groups, 350 groups, 400 groups, 450 groups, 500 groups, 550 groups, 600 groups, 650 groups, 700 groups, 750 groups, 800 groups, and 850 groups. The medium formulation of each group contained 1% glucose, 2.5% yeast extract, sodium glutamate, 2 ppm calcium carbonate, 2 ppm manganese sulfate, 2 ppm polysorbate 80, and 10 μM pyridoxal phosphate. The initial pH of the medium. The volume was 4.5, and the volume of each medium was 2 LmL, and culture was carried out in a 3 L Erlenmeyer flask. Among them, the concentration of sodium glutamate in the medium of 250 groups, 300 groups, 350 groups, 400 groups, 450 groups, 500 groups, 550 groups, 600 groups, 650 groups, 700 groups, 750 groups, 800 groups, and 850 groups The order is 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, and 850 mM.

The short Lactobacillus strain NO.03 was inoculated into each group of medium, and cultured in an anaerobic environment at 30 ° C for 96 hours to obtain a fermentation broth of each group. High-performance liquid chromatography was used to quantitatively analyze the content of γ-aminobutyric acid and residual sodium glutamate in each fermentation broth.

The results in Figure 10 show that γ in the fermentation broth of 250 groups, 300 groups, 350 groups, 400 groups, 450 groups, 500 groups, 550 groups, 600 groups, 650 groups, 700 groups, 750 groups, 800 groups, and 850 groups. - Aminobutyric acid content is 22,927 ± 2,653 mg / L, 25,858 ± 1,374 mg / L, 30,006 ± 1,149 mg / L, 32,222 ± 1,356 mg / L, 36,311 ± 1,762 mg / L, 38,395 ± 1,234 mg / L, 43,457 ±1,398 mg/L, 45,094±1,385 mg/L, 48,802±2,873 mg/L, 47,266±746 mg/L, 46,037±1,005 mg/L, 45,983±2,033 mg/L, and 45,185±3,385 mg/L; glutamine The residual amount of sodium is 495±16mg/L, 501±8mg/L, 555±39mg/L, 966±82mg/L, 1,092±41mg/L, 1,704±102mg/L, 1,072±4mg/L, 1,914± 61 mg/L, 2,008±63 mg/L, 6,528±749 mg/L, 10,705±108 mg/L, 12,770±121 mg/L, and 17,798±1,302 mg/L. The experimental results show that the short Lactobacillus sp. NO.03 was cultured with the addition of 650 mM sodium glutamate. In the medium, the γ-aminobutyric acid content in the fermentation broth is the highest.

Experiment 4-10: Mass production of γ-aminobutyric acid by batch fermentation

The experimental medium formulation contains 1% glucose, 2.5% yeast extract, 650 mM sodium glutamate, 2 ppm calcium carbonate, 2 ppm manganese sulfate, 2 ppm polysorbate 80, and 10 μM pyridoxal phosphate, the initial pH of the medium. The volume was 4.5, and the volume of each medium was 2 L, and culture was carried out in a 3 L Erlenmeyer flask.

Short Lactobacillus strain NO.03 was inoculated into the medium, cultured at 30 ° C in an anaerobic environment, and sampled at different time points. The content of γ-aminobutyric acid and sodium glutamate residue in the fermentation broth samples at the time point were quantitatively analyzed by high performance liquid chromatography.

The results in Figure 11 show the content of γ-aminobutyric acid in the fermentation broth after fermentation for 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, and 112 hours. They were 141±101mg/L, 370±55mg/L, 7,897±212mg/L, 31,317±4,484mg/L, 40,468±262mg/L, 38,605±851mg/L, 53,854±1,007mg/L, 48,301±721mg/ L, 51, 793 ± 309 mg / L, 57, 771 ± 1,095 mg / L, 62, 523 ± 3, 088 mg / L, 57, 592 ± 1, 303 mg / L, 59, 230 ± 628 mg / L, and 58,800 ± 1,515 mg / L, and residual glutamate The amounts were 75,290±1,011 mg/L, 67,077±600 mg/L, 61,103±2,595 mg/L, 31,450±435 mg/L, 13,991±479 mg/L, 10,025±655 mg/L, 7,875±113 mg/L, 5,045±113 mg. /L, 2,853±73 mg/L, 1,081±19 mg/L, 1,006±73 mg/L, 982±84 mg/L, 945±24 mg/L, and 901±46 mg/L.

The above mg/L unit was converted to mM unit, and the conversion ratio of γ-aminobutyric acid was calculated according to the following formula: conversion rate = (γ-aminobutyric acid yield / amount of added glutamate) *100%

The above γ-aminobutyric acid conversion rate formula is cited in the open literature of 2012 (Zhang, Y.; Song, L.; Gao, Q.; Yu, SM; Li, L.; Gao, NF, The two-step Biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells. Applied microbiology and biotechnology 2012, 94, (6), 1619-1627.).

It can be seen from Table 2 that the γ-aminobutyric acid content in the fermentation broth is as high as 606.3 mM after the culture of the short lactobacillus strain NO.03 in a 2 L culture medium supplemented with 650 mM sodium glutamate, and sodium glutamate ( The residual amount of MSG) is only 5.4 mM, and the conversion rate is as high as 93.28%. The conversion rates of different groups are shown in Table 2.

Experiment 5: Analysis of tyrosinase inhibition ability and antioxidant capacity of short Lactobacillus strain NO.03

Experiment 5-1: The ability of short Lactobacillus strain NO.03 to inhibit tyrosinase

The experiment was divided into a control group and a fermentation broth group. The control group was tested with a phosphate buffer solution to measure the activity when tyrosinase was not inhibited, and the fermentation broth group was subjected to a fermentation broth of the experiment 4-10 for 72 hours. test.

The phosphate buffer solution is prepared by dissolving 14.34 g of disodium hydrogen phosphate in 200 mL of distilled water to obtain a disodium hydrogen phosphate solution; and dissolving 5.52 g of sodium dihydrogen phosphate in 200 mL of distilled water to obtain a sodium dihydrogen phosphate solution. 122.5 mL of disodium hydrogen phosphate solution and 127.5 mL of sodium dihydrogen phosphate solution were separately mixed, and the volume was adjusted to 500 mL, and the pH was adjusted to 6.8 by a pH pH meter, which was a phosphate buffer solution.

Mix the test solution (phosphate buffer solution, or fermentation solution of Experiment 4-10 for 72 hours), phosphate buffer solution of pH 6.8, and tyrosine solution, and then add tyrosinase for 20 minutes to produce Enzyme browning intermediate o-dopachrome. Since the maximum absorption light wavelength of the enzymatic browning intermediate o-dopachrome is 475 nm, the absorbance at 475 nm is detected by an ultraviolet/visible spectrometer to separately measure the enzyme browning intermediates of the control group and the fermentation liquid group. O-dopachrome content.

Tyrosinase relative activity calculation formula: tyrosinase relative activity (%) = (B / A) * 100%

A: The enzymatic browning intermediate o-dopachrome content of the control group

B: Enzymatic browning intermediate o-dopachrome content in the fermentation broth

The results in Figure 12 show that the fermentation broth obtained after fermentation of the short Lactobacillus strain NO.03 (stock solution) reduces the relative activity of tyrosinase to 36% and has a whitening effect.

Experiment 5-2: Antioxidant effect of fermentation strain of NO.03

This experiment will be based on the following five test methods, namely ferrous ion chelation ability test, copper ion chelation ability test, reducing power measurement, total phenol content determination, and 1,1-diphenyl-2-trinitrobenzene The free radical scavenging ability test of 1(1,1-Diphenyl-2-picrylhydrazyl, DPPH) was used to analyze the antioxidant effect of the fermentation broth of NO.03.

Ferrous ion chelation ability test: This experiment is divided into control group, fermentation liquid group, 1/10 times fermentation liquid group, and 1/100 times fermentation liquid group. The control group was tested with ethylenediaminetetraacetic acid (EDTA) solution. The fermentation broth group, 1/10 times fermentation broth group, and 1/100 times fermentation broth group were 1x, 1/10 times, and 1/100, respectively. The fermentation broth of Experiment 4-10 fermentation for 72 hours was tested.

Among them, the ethylenediaminetetraacetic acid solution is prepared by dissolving 5 mg of ethylenediaminetetraacetic acid in 10 mL of distilled water to obtain a solution of 500 ppm ethylenediaminetetraacetic acid.

The test solution (ethylenediaminetetraacetic acid solution, or the fermentation broth of Experiment 4-10 for 72 hours), 2 mM ferric chloride, and methanol were uniformly mixed, and then 5 mM philoxine (Ferrozine) was added, and after complete reaction, After centrifugation at 3000 rpm for 5 minutes, the supernatant was taken, and the absorbance at 562 nm was measured by an ultraviolet/visible spectrometer to measure the content of the red wrong compound produced by the chelation of ferrous ions and phenazine at each group. The lower the content of the wrong compound (i.e., the lower the absorbance value), the more excellent the ferrous ion chelation ability of the sample.

Using the percentage of absorbance reduction, the ability of each group to chelate ferrous ions is determined. The calculation method is as follows: ferrous ion chelation rate (%) = [1- (absorbance value of the experimental group or control group / blank control) Group of light absorption Value)]*100%

The results of Figure 13 show that the ferrous ion sequestration rates of the control group, the fermentation broth group, the 1/10 times fermentation broth group, and the 1/100 broth fermentation broth were 99.67%, 97%, 97.67%, and 76.67%, respectively. It shows that the fermentation broth of the short Lactobacillus strain NO.03 has good ferrous ion chelation ability.

Copper ion chelation ability test: This experiment is divided into control group, fermentation liquid group, 1/10 times fermentation liquid group, and 1/100 times fermentation liquid group. The control group was tested with ethylenediaminetetraacetic acid solution, and the fermentation broth group, 1/10 times fermentation broth group, and 1/100 times fermentation broth group were 1 times, 1/10 times, and 1/100 times respectively. -10 Fermentation of fermentation for 72 hours was tested.

Among them, the ethylenediaminetetraacetic acid solution is prepared by dissolving 5 mg of ethylenediaminetetraacetic acid in 10 mL of distilled water to obtain a solution of 500 ppm ethylenediaminetetraacetic acid.

The test solution (ethylenediaminetetraacetic acid solution, or the fermentation broth of Experiment 4-10 for 72 hours) and the copper sulfate solution are uniformly mixed, and then pyrocatechol violet is added, and after complete reaction, ultraviolet The light/visible spectrometer measures the absorbance at 632 nm.

Using the percentage of absorbance reduction, the copper ion chelation ability of each group of liquid to be tested is judged by the following method: copper ion chelation rate (%) = [1 - (absorbance of experimental group or control group / blank control group) Absorbance value)]*100%

The results of Figure 14 show that the copper ion chelation ability of the control group, the fermentation broth group, the 1/10 times fermentation broth group, and the 1/100 times fermentation broth group were 98%, 92.33%, 74.67%, and 33%, respectively. It shows that the fermentation broth of the short Lactobacillus strain NO.03 has good copper ion chelating ability.

Reducing power measurement: This experiment is divided into control group, fermentation liquid group, 1/10 times fermentation broth Group, and 1/100 times fermentation broth. The control group was tested with vitamin C solution, and the fermentation broth group, 1/10 times fermentation broth group, and 1/100 times fermentation broth group were fermented by 1×, 1/10 times, and 1/100 times respectively. The 72 hour fermentation broth was tested.

The preparation method of the vitamin C solution is as follows: 1 mg of vitamin C is dissolved in 1 mL of a methanol solution, that is, a vitamin C solution of 1 mg/mL.

Mix the test solution (vitamin C solution, or fermentation broth of Experiment 4-10 for 72 hours), 0.2 M phosphate buffer solution, and 1% red blood salt solution (ie, potassium ferricyanide solution), and react at 50 ° C. After 20 minutes, it was rapidly cooled to room temperature, then 10% trichloroacetic acid solution was added, mixed uniformly and allowed to act for 10 minutes. The mixed solution, distilled water, and 0.1% ferric chloride solution were uniformly mixed, and the absorbance at 700 nm was measured by an ultraviolet/visible spectrometer to measure the content of iron ferrocyanide (Prussian blue), wherein iron ferrocyanide was obtained. The higher the content (the higher the absorbance value), the stronger the reducing power of the sample.

The absorbance values were used to judge the reducing power of each group.

The results of Fig. 15 show that the absorbance values of the control group, the fermentation broth group, the 1/10-fold fermentation broth group, and the 1/100-fold fermentation broth sample prepared in the above manner were 2.2462±0.1425, 2.516±0.0671, and 0.521±, respectively. 0.0683, and 0.1017±0.001, it can be seen that the fermentation broth of the short Lactobacillus strain NO.03 has a good reducing power effect.

Determination of total phenolic content: This experiment is divided into control group, fermentation broth group, 1/10 times fermentation broth group, and 1/100 times fermentation broth group. The control group was tested with gallic acid solution, and the fermentation broth group, the 1/10-fold fermentation broth group, and the 1/100-fold fermentation broth group were 1 time, 1/10 times, and 1/100, respectively. The fermentation broth of Experiment 4-10 fermentation for 72 hours was tested.

Among them, the preparation method of the gallic acid solution is: 5 mg of gallic acid is added to 5 mL of distilled water, and the mixture is uniformly mixed.

Mix the test solution (gallic acid solution, or the fermentation broth of Experiment 4-10 for 72 hours), 1N o-molybdate phenol indicator (Folin-Ciocalteau's Phenol Reagent), and 7.5% sodium carbonate solution. After 2 hours, the absorbance at 760 nm was measured by an ultraviolet/visible spectrometer.

The percentage of the absorbance was used to determine the total phenolic content of each group.

The results of Figure 16 show that the absorbance values of the control group, the fermentation broth group, the 1/10-fold fermentation broth group, and the 1/100-fold fermentation broth group were 0.457±0.0035, 0.5463±0.0123, 0.140±0.0025, and 0.0857±0.0004, respectively. It shows that the fermentation broth of the short Lactobacillus strain NO.03 has a high concentration of total phenolic compounds.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability test: This experiment is divided into control group, fermentation liquid group, 1/10 times fermentation Liquid group, and 1/100 times fermentation broth. The control group was tested with vitamin C solution, and the fermentation broth group, 1/10 times fermentation broth group, and 1/100 times fermentation broth group were fermented by 1×, 1/10 times, and 1/100 times respectively. The 72 hour fermentation broth was tested.

The preparation method of the vitamin C solution is: taking 1 mg of vitamin C in 1 mL of methanol solution, that is, 1 mg/mL of vitamin C solution.

Mix the test solution (vitamin C solution, or the fermentation broth of Experiment 4-10 for 72 hours) and 0.25 mM DPPH ethanol solution, and let it stand at room temperature for 20 minutes in the dark, and measure 517 nm with UV/Vis spectrometer. The absorbance value. Among them, DPPH is a kind of free radical with good stability, and has a strong absorbance near 517 nm. Therefore, the lower the absorbance value, the stronger the scavenging effect of the reactant on DPPH free radical.

Antioxidant capacity is presented as a percentage of scavenging activity (%), calculated as follows: DPPH free radical scavenging rate = (1 - absorbance of experimental group or control group / absorbance of blank control group) * 100%

The results of Fig. 17 show that the DPPH free radical scavenging ability of the control group and the 1/100 times fermentation broth group are 100% and 99%, respectively, indicating that the fermentation broth of the short Lactobacillus strain NO.03 has good free radical scavenging ability.

Based on the above experimental results of Figures 13 to 17, it can be seen that the fermentation broth of the short Lactobacillus strain NO.03 can (1) effectively chelate ferrous ions and copper ions, slow down oxidation, and (2) oxidize the generated free radicals. Reduction, conversion to less toxic or non-toxic substances, (3) use of total phenolic compounds (antioxidant components) to help free radicals undergo redox, conversion to less toxic or non-toxic substances, and (4) scavenging free radicals, It is shown that the fermentation broth obtained after fermentation of the short Lactobacillus strain NO.03 has good antioxidant capacity.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the claims of the present invention. Therefore, various other modifications or retouchings, etc., which are not departing from the spirit of the present invention, should be included in the present invention. Within the scope of the patent application.

【Biomaterial Storage】

Domestic registration information [please note according to the registration authority, date, number order]

Institute of Food Industry Development, 106/02/14, registration number BCRC910771

Foreign deposit information [please note according to the country, organization, date, number order]

no.

The following sequence is represented from left to right by the term of the 5' end to the 3' end.

<110> National Kaohsiung University of Ocean Technology

<120> Novel Lactobacillus brevis and its application

<160> 1

<210> 1

<211> 1438

<212> Deoxyribonucleic acid

<213> Lactobacillus brevis

<400> 1

Claims (9)

A Lactobacillus brevis having a 16S ribosomal DNA (16S rDNA) comprising SEQ ID NO: 1; the Lactobacillus strain is isolated from the gastrointestinal tract of an aquatic animal, the aquatic The animal is selected from the group consisting of Priacanthus macracanthus , Perca fluviatilis, Thunnus thynnus, Psenopsis anomala , and grass carp ( Ctenopharyngodon idellus ); Converting sodium glutamate in a culture solution to γ-aminobutyric acid under batch fermentation, thereby increasing the concentration of γ-aminobutyric acid in the batch fermentation process Adding at least 50g/L, and the ability to convert sodium glutamate to gamma-aminobutyric acid with a conversion rate of more than 90%, and having the fermentation broth in the batch fermentation conditions The bacterium has a capacity of at least 10 ⁸ CFU/ml; wherein the optimal fermentation temperature of the strain is 25-35 degrees, and the optimal fermentation pH of the strain is shorter than 4.0-4.5. The short Lactobacillus strain according to claim 1, which has the sodium glutamate in the culture solution converted to γ-aminobutyric acid under the batch fermentation condition, so that γ-aminobutyric acid is obtained. The concentration is increased by at least 60 g/L during the batch fermentation. A strain of Lactobacillus ssp. as described in claim 1 which has the ability to produce a metabolite which inhibits tyrosinase activity. For example, the short lactobacillus strain mentioned in the first paragraph of the patent application is deposited in the Food Industry Development Research Institute, and the registration number is BCRC910771. A medium for preparing γ -aminobutyric acid by fermentation of a short lactobacillus strain as described in claim 1, wherein the medium comprises: monosodium glutamate; carbon source; nitrogen source; carbonic acid Calcium carbonate; and Manganese sulfate. The use of the medium described in claim 5, further comprising polysorbate 80 (Tween 80). The use of the Lactobacillus sp. is deposited in the Food Industry Development Institute of the Corporation, and the deposit number is BCRC910771. A method for producing γ-aminobutyric acid, comprising: inoculating a strain of the short larvae according to claim 1 of the patent application to a culture solution containing calcium carbonate, manganese sulfate, and polysorbate 80; a predetermined length of time; and obtaining a fermentation broth comprising γ -aminobutyric acid. A short lactobacillus strain is deposited in the Food Industry Development Research Institute and has a registration number of BCRC910771.