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Enterococcus raffinosus TCCC11660 as the original strain was mutated by atmospheric and room temperature plasma breeding system. According to the lethality rate and positive mutation rate, the best mutagenesis conditions were confirmed: power 120W, air flow 10 L/min, distance 3 mm, and mutagenic time 90 s. The experiment screened a mutant A7 with high yield of γ-aminobutyric acid (GABA). Compared with the original strain, GABA yield was increased by 7% by liquid shake flask fermentation. The result of continuous passage culture shows that mutant A7 has a stable performance on producing GABA, and thus mutant A7 is a genetic mutation strain. Then fermentation conditions were optimized, and the final yield of 27.08% is higher than the original production.
Key words: ARTP; γ-aminobutyric acid; Enterococcus raffinosus; fermentation conditions optimizing
γ-aminobutyric acid (GABA) is a type of nonprotein amino acid which is widely distributed in animals and plants. It originates from glutamic acid through catalytic conversion via glutamate dehydrogenase, and is a type of inhibitory neurotransmitter on mammals' brain and spinal cord. Reportedly, GABA has many important physiological functions such as improving brain function, lowering blood pressure, anti-convulsion, analgesia, improving reproductive capability, mental stability, promoting long-term memory, promoting the secretion of growth hormone, and activation of renal and liver functions [2-4].
GABA production methods mainly include chemical synthesis and biosynthesis of plant enrichment. Chemical synthesized by-products are difficult to be purified, with high production cost and low safety. Although plant enrichment can be operated simply, the yield is low, and it is difficult to be applied in industrial production. Biosynthesis generally uses escherichia coli, but it has hidden troubles in existence safety and hygiene . In biosynthesis, the glutamic acid decarboxylase (GAD, Glutamate decarboxylase, EC 126.96.36.199) is the only enzyme to take one catalytic glutamic acid or sodium salt as the substrate off the carboxy generation GABA. Glutamic acid especially a sodium salt (MSG) has been mass-produced through the fermentation method at relatively low price, so the use of microbial body of GAD catalytic glutamic acid off is an effective strategy in GABA production.
Reportedly, plasma has a significant effect on bacteria's cytoderm, cytomembrane and proteins, and long-time processing can partly or completely break the bacteria's cytoderm, and release intracellular proteins. This is because active particles produced by plasma can break the structure of cells and also can cross cytoderm inside to break the bonds of genes and proteins, thus leading most microorganism to death. But a little microorganism will repair to survive by automatic self-repair system via ARTP. ARTP mutagenesis system has many advantages such as low temperature jet flow, uniform plasma, non-vacuum unit, easy operation, low cost, and obvious effect of biomacromolecules and cells[6-7]. In this research, a high-GABA-yield strain which was screened by a former research group from sauerkraut (a type of traditional fermentation food) was used as the original strain and mutagenized via ARTP to get a strain with a higher GABA yield and to investigate its fermentation conditions.
1 Materials and Methods
1.1Strain and media
1.1.1 Strain: Enterococcus raffinosus TCCC11660
Improved MRS solid medium for lactobacillus' separating and screening; MRS solid medium for lactobacillus' slant culturing and culture preservation; MRS seed medium for lactobacillus' seed culturing; improved MRS liquid medium for lactobacillus' submerged fermentation(g/L): sucrose, 40; yeast extract powder,30; NaAc,4; K2HPO4 , 2; Al2(SO4)3 , 0.3; MgSO4·7H2O , 0.2 ; (NH4)2SO4 , 0.6 ; Tween -80, 2; pH 5.5, Sterilization on 121oC for 15 min.
1.2 experimental methods
1.2.1 ARTP mutagenesis methods
Utilized ARTP machine to mutagenize the strains. This study used helium as the working gas, operating conditions as shown in table 1.
Power input (W)
Treatment distance (mm)
Gas flow rate (L/min)
Treatment time (s)
10 μL of culture broth with OD600=1
Table 1 Operating conditions using ARTP
1.2.2 Screening mutant
188.8.131.52 Primary screening
Puting the mutagenized sheet to 1ml normal saline in 1.5ml EP tube, diluting 105 times . Then pipetting 100 uL bacterium to primary screening solid medium and making the coating uniformity. Culturing 1 day on 30 oC,selecting strains who have better transparent zones/colony diameter to the liquid seed medium.
184.108.40.206 Second screening
Puting the activated liquid seed medium(inoculation size :10%) to the fermentation medium, fermenting stationary for 3 days and then assaying the yield by HPLC, selecting a high-GABA-yield strain.
1.2.3 Optimizing of fermentation conditions
Inoculation size, temperature, pH, substrate concentration, oscillation and static are processed single factor experiments in the process of fermentation, determining the optimal fermentation conditions.
1.2.4 Experiment in 5-Lfermentor
Puting the activated liquid seed medium(inoculation size :10%) to 5-L fermentor containing 3L fermention medium. Sampling every 2 h, assaying the change of substrate, GABA, cell dry weight and the residual sugar in the fermented liquid.
1.2.5 GABA determination methods
Pretreating sample: suction 1mL fermentation broth to 1.5mL centrifuge tube, centrifuging 10 min on 8000 r/min, diluting ten times, then pipetting 10 μL supernatant to 1.5mL centrifuge tube, adding 100 μL derived buffer, 100 μL derivative agent (DNFB), 790 μL constant volume buffer, uniformly mixed, water bath on 60 °C for 1 hour. Filtering with the 0.45 μm cellulose acetate membrane filters, using the refrigerator to keep the filtrate on 4 °C. Gradient elution conditions: Agilent ZORBAX StableBond C18 column, column temperature 30 °C, ultraviolet detection wavelength 360 nm, flow velocity 0.8 mL/min, sample amount 10 μL, mobile phase A: water, mobile phase B: acetonitrile, mobile phase D: sodium acetate solution.
2 Results and Discussion
2.1 Assay of fatality rate of ARTP mutagenesis
The study founds that plasma has a significant effect on the bacterial cell wall, cell membrane and protein, the processing time sustained will lead to total or partial cell wall broken, and release the cell protein. This is because the plasma produced energetic particle can destroy the cellular structure can also through the cell wall to cells, interrupt gene, protein key, etc., which can lead to most microbial death. But after a few ARTP to illuminate from microbes by itself to be automatic repair system repair live, and in this process produce gene mutation. Therefore, choose the right ARTP operating conditions to realize the rapid microbial mutation.
Fig.1 Variaton of the lethal rate with the ARTP treatment time.
As showed in figure 1,plasma has a strong lethality of Enterococcus raffinosus, when ARTP mutagenesis for 90 second, fatality rate reached 90ï¼… and for 120 additional 100%.Mutation is randomness, the relationship of fatality rate and a positive mutation is not abundantly clear. It depends on the mutation method and characteristics of strains itself. In the modern breeding theory, when fatality rate at the range of 90%-95%, progressive mutation to the top, therefore, the best mutagenic time is 90 seconds with a fatality rate of 90.2%.
2.2 Screening mutant
Put the mutant strains to the initial dense medium for spreading on well-distributed, culturing 1 day on 30oC, observe its growing states. Selecting 30 strains which has better transparent colony diameter.
Take 30 strains to liquid fermentation medium for the second screening. Fermenting 3 days by standing shake flask with 30 oC, assaying the yield by HPLC.
Fig.2 The GABA yield of the 30 mutants and the original strain (incubation for 72 h)
As showed in figure 2, we can know that mutant A7 has the highest yield, its yield of GABA increases by 15% over the contrast.
2.3 Genetic stability
For 10 times continuous passage cultures of mutant A7, liquid state fermentation for 3 days, assay its yield of GABA via HPLC to research its genetic stability. It can be studied from Table 2 that after 10 times continuous passage cultured, the difference value of GABA yield in any two of the generations are within 10%, it is indicated that mutant A7 has a stable performance on producing GABA, it can be used as original strain in following experiments.
Table 2 The GABA concentration of the mutants (g/L)
2.4 Optimizing of fermentation conditions
2.4.1 Effect of inoculum size on yield of GABA
Bacteria in fermention plays an important role on the yield and conversion of GABA, high inoculation size will lead to faster growth of the bacteria, the more consumption of nutrients so that affects the efficiency of transformation . Low inoculum size will result the slow growth of the bacteria, lesser mass of the bacteria, extended cycle.The bacterias will be activated with different inoculum size (v/v) of fermentation medium, standing fermenting for 3 d on 30 oC.
Fig.3 The yield of GABA and OD600 with different inoculums size
As shown in figure 3, when inoculum size was 10% (v/v), although bacteria mass is not the highest, but the yield of GABA is highest; when inoculum size was 15% (v/v), bacteria mass increased obviously, but the yield of GABA is not high. It is probablely that the overgrowth of bacteria leads to the shortened time of transformation ,so the yield of GABA is not high. Therefore, the optimal fermentation inoculum size is 10% (v/v).
2.4.1 Effect of temperature on yield of GABA
The optimal temperature is extremely important to enzyme activity, because the optimal temperature range of the mostly enzymes is exceedingly small. The reaction rate of enzyme increases with temperature improving and metabolism accelerating, leads to the production time in advance, but high temperature will lead to loss of enzyme activity , affects the final production, so it is needed to accurately control the temperature. Needing to pay attention to is the optimal growth temperature and the optimal fermentation temperature are not necessarily the same. Enterococcus raffinosus can grow under 10 oC and 45 oC . And according to some reports in the literature, the optimum temperature of the GAD of Enterococcus raffinosus is 30 oC. The study on fermentation temperature was investigated.
Experimenting optimal fermentation temperature of producing GABA with Enterococcus raffinosus mutant A7, an 10% of inoculum, stationary fermenting for 3 days at 30 °C, result as showed in figure 3.
Fig.4 Effect of temperature on yield of GABA in fermentation broth
As shown in figure 4 , when fermentation temperature is 30 °C, Enterococcus raffinosus mutant A7 has the highest GABA yield (42.08 g/L), so the optimal fermentation temperature of mutant A7 is 30 °C.
2.4.2 Effect of initial pH on yield of GABA
Researching on the optimal initial pH of the medium, as an inoculation of 10%, Stationary fermentation for 3 days on 30 °C. When initial pH of the medium is 5.5 has the highest GABA yield of 46 g/L, so the optimal initial pH of mutant A7 is 5.5, result as shown in figure 4.
Fig.5 Effect of initial pH on yield of GABA in fermentation broth
2.4.3 Effect of adding substrate on yield of GABA
As MSG is low cost and solubility is better than glutamic acid,so this experiment utilized MSG as the fermentation substrate and researches the effect of different substrate concentration on yield of GABA. The research shows that when substrate concentration is 10% has the highest GABA yield, with continuing increasing substrate concentration the yield decreases by degrees (figure 5), because of high substrate concentration may inhibit strain's growing and feedback inhibit the GAD enzyme, and that MSG's concentration in the fermentation broth increased will adverse to following extracting, so the optimal substrate concentration of mutant A7 is 10%.
Fig.5 Effect of substrate concentration on yield of GABA in fermentation broth
2.4.4 Effect between shake and stationary fermentation on yield of GABA
Enterococcus raffinosus is a facultative anaerobe: the bacteria needs oxygen to multiply and for its metabolism to work properly. A lack of oxygen results in the accumulation of products of anaerobic metabolism. This experiment compared the effects of shaken and stationary fermentation on the yield of GABA and confirmed the best fermentation process. Fermentation that was initially shaken for 24 hours and then stationary for the following 48 hours yielded up to 48g/L of GABA. This was higher than any other fermentation process, so the research used a fermentation process of shaking for 24 hours followed by stationary for 48 hours.
Fig.6 Effect between shake and stationary fermentation on yield of GABA in fermentation brothï¼ˆ1: Stationary fermentation for 72 h; 2: First stationary fermentation for 24 h, and then shake flask fermentation for 48 h; 3: First shake flask fermentation for 24 h, and then stationary for 48 h; 4: Shake flask fermentation for 72 hï¼‰
2.5 5-L fermentor
It was reported in the literature that optimum pH for glutamic acid decarboxylase enzyme activity is 4.5, but neutral pH conditions are optimal for the growth of E. coli. A pH of 4.5 is conducive to the enzyme catalyzed reactions in the fermentation process but is not suitable for the growth of bacteria cells. Through experiments conducted on the demurrage and logarithmic phase of thalli growth, it is known that the pH value plays an important role in the growth rate. So, in a 5-L fermenter the pH for bacteria growth was controlled, where it was maintained at 6.5 initially and then after 14 h reduced to 4.5.
Fig.7 the fermentation process
As shown in Fig. 7, from 0 to the 14th hour, bacteria mass was increased rapidly, and after 14 hour entered a stationary phase. From 0 to the 20th hour, sucrose was consumed rapidly, and after the 20th hour, the consumption rate was reduced gradually. The initial volume of feed substrate addition was 50 g/L, and when the concentration drop to 10 g/L during fermentation, the substrate was supplied by a feed batch, in consideration of the tolerance of bacteria to the substrate. Depending on the three additions of substrate, the concentration of GABA reached 125.46 g/L at the end of fermentation, and the glutamic acid used as substrate was nearly converted into GABA in the end, with a conversion rate of 96.4%.
GABA is very low in nature, so it is very difficult to mass-extract from plant and animal tissues. Currently, the main methods of GABA production include chemical synthesis and biosynthesis. In contrast with the low safety and serious environmental pollution in chemical synthesis, the yield of GABA production by plant enrichment is very low and microbial fermentation will have more development prospects.
E. raffinosus as normal intestinal bacterium in human and animals has high tolerance, high colonization ability, and high resistance. It can be used in fermented food or as probiotics added to animal feed to prevent livestock bowel disease and improve animal growth. In this study, a strain was isolated by our laboratory from pickled vegetables which were sold in Dandong City of Liaoning Province. Our laboratory conserved independent intellectual property rights of the high GABA yield E. raffinosus TCCC11660 to get higher yield of GABA by ARTP. We screened a mutant strain A7 after mutagenesis, and increased GABA yield by 7% compared to the original strain. We confirmed that the GAD of Lactic acid bacteria decarboxylated glutamic acid to produce GABA, and the optimal action temperature of the GAD of Lactic acid bacteria and the optimum growth temperature are closed. The optimum growth temperature of strain A7 is 30 °C, which benefits the accumulation of GABA. In addition, the optimal pH of the GAD of bacteria is generally between 3.5 and 5.5 [8-12], with the optimal pH of strain A7 at 5.5. The optimal initial concentration of substrate (glutamate) is 10%, the GABA production will not be increased by increasing the substrate concentration, because it also will increase the residual substrate concentration, which is not conducive to the late extraction. Oscillation culture in the bacteria breeding stage and standing culture in the accumulation stage of GABA are more beneficial for GABA accumulation. Under the above optimal fermentation conditions, the shaker production of GABA reached 61 g/L, which was increased by 27.08% (initial yield, 48g/L), and the concentration of GABA reached 125.46 g/L in the 5-L fermentor. This study reports the use of ARTP to screen a mutant strain with high GABA yield, providing the new culture resources for future GABA biosynthesis and development. The mutant strain also can be used for the development of rich GABA food and probiotics strains of animal feed and thus has a better application prospect.