Identification and Mutagenesis of Lactic Acid Bacteria from Chinese Sauerkraut

Yajing CHAI,Hao SHI,Ri NA

College of Physical Science and Technology,Inner Mongolia University,Hohhot 010021,China

Identification and Mutagenesis of Lactic Acid Bacteria from Chinese Sauerkraut

Yajing CHAI,Hao SHI,Ri NA*

College of Physical Science and Technology,Inner Mongolia University,Hohhot 010021,China

In order to analyze the fermentation properties of lactic acid bacteria in Chinese sauerkraut and to improve acid production,21 samples of Chinese sauerkraut from Inner Mongolia and Northeast China were collected and isolated with a Man-Rogosa-Sharpe(MRS)culture.Sixteen strains of lactic acid bacteria were identified by combining both phenotype and genotype methods.After activation, the 16 strains were inoculated into the MRS medium with a concentration of 4% and then incubated at 37℃.The pH and the absorbance of the culture were measured.The activated strains were then mutagenized in a field of 4 KV/cm mutation, with dosages administered within 20 minutes and 30 minutes,respectively.The variation curves of the pH and the absorbance of the culture were determined.The experimental results showed that the lactic acid bacteria isolated from the soup were identified asLactobacillusand the acid production of the bacteria was significantly improved by the mutagenesis of the corona electric field.

Chinese sauerkraut;Lactic acid bacteria;Identification;Corona electric field;Mutagenesis

S uan Caior Chinese sauerkraut, a traditional ingredient in Chinese cuisine,is well known for its high nutritional value and strong after taste.It is made from fermenting cabbage with lactic acid bacteria.The dish,which is known for increasing the appetite,is said to have originated from Northeast China,but it is also found in places such as;Hebei, Shanxi,Shaanxi,Gansu,and Ningxia. The key microorganism of the fermentation process of Chinese sauerkraut is lactic acid bacteria.Therefore,the fermentation quality of the bacteria affects the taste and quality of Chinese sauerkraut.During the curing process nitrite is produced,which is toxic and harmful[1-2].By fermenting the cabbage with pure lactic acid bacteria,nitrite can be dramatically reduced so that the dish is safer to eat[3].In addition to reducing nitrite,lactic acid bacteria has many other benefits;it regulates intestinal microflora,it contains beneficial antioxidant properties,aids the digestive system,lowers cholesterol and improves immunity[4].

A corona discharge is an electrical discharge brought on by the ionization of a fluid surrounding a conductor that is electrically charged.To achieve this, a needle thin electrode is placed into the subject fluid.Next,an electrical charge is introduced through the electrode at a low voltage and is slowly increased at a steady pace.Once a substantial amount of charge has gathered around the electrode,the surrounding area will become ionized. At this point,a discharge phenomenon occurs and halation around the needle can be seen;which is how the term‘corona discharge’was derived[5-6].In recent years,this method has been adopted for treating environmental problems in China.Utilizing corona discharge technology in microorganism mutagenesis is a relatively new method needing further analysis.

The objective of this research was to identify lactic acid bacteria in Chinese sauerkraut and obtain quality lactic acid bacteria through corona discharge mutagenesis.This research will provide a foundation for developing and industrializing good quality of starters from Chinese sauerkraut, silage and yogurt.

Materials and Methods

Materials and reagents

SamplesChinese sauerkraut was collected from 12 cities;the Inner Mongolia Autonomous Region,Qiqihaer in the Heilongjiang Province, Suihua,Benxi in the Liaoning Province,and Tonghua in the Jilin Province.

ReagentsPeptone,yeast,Tween commercial,K2HPO4,MgSO4·7H2O, agar,calcium carbonate,Erie nonfat yogurt,Central Kay productsglucose, citric acid diamine,anhydrous sodium acetate,2×Tap PCR Master Mix, agarose,nucleic acid dyes.

Instruments and equipments

For this research,the following instruments and equipment were used: a Mettler-Toledo laboratory p H meter, a high-speed refrigerated centrifuge, an incubator,a clean table,a UV spectrophotometer(U-2900),an electrophoresis and a PCR amplifier.

Methods

Isolation and purification of lactic acid bacteriaIsolation and purification of lactic acid bacteria[7-14].On a sterile bench,21 numbered sauerkraut samples with a volume of 20 ul were prepared.Next,they were diluted by water into nine different concentration gradients(each of which was ten-fold dilution).The fifth,sixth,seventh, eighth and ninth gradients were 10 ml of the gradients were used to spread culture on Man-Rogosa-Sharpe (MRS)[15].They were sealed with paraffin and incubated at 37℃for 72hrs.Out of the colonies tested,theones qualitatively showing an obvious calcium circle were selected.Every isolated colony was purified after a streak culture.The purified strains were inoculated into MRS to culture for two generations,and were then preserved at 4℃.

Determination of acidogenicity and growth curveStrains were inoculated into the MRS fluid medium with a quantity of 4%for incubation at 37℃after mobilization by skin milk broth. The pH variations and growth characteristics of the colonies(5 colonies per sauerkraut soup)were qualitatively assessed.Before the 20 hours,the samples were tested every 2 h.After 30 hours,the samples were tested every 4 h.Using the blank medium as the control,the absorbency was measured at 620 nm and the pH value was measured using a pH meter.An acidogenicity rate curve and a growth curve were drawn;the incubation time was the x-coordinate and the absorbency and pH values were the y-coordinates.

Test of corona discharge mutagenesis

StrainsThe lactic acid bacteria were inoculated into the MRS fluid medium with a quantity of 4%after activation.It was then incubated at 37℃.The mediums were centrifuged at 900 r/m for 15min and the precipitation from the colonies was collected.The colonies were mixed and suspended with sterilized saline(0.85%),re-centrifuged,and washed two or three times with saline.

Preparation of bacteria suspension

The bacteria were suspended with sterilized saline(0.85%)to recreate the concentration of the original bacteria suspension.The suspension was transferred into triangle bottles containing glass beads.A bacteria suspension was created by vibrations from a rotary sharker at 200 rpm for 15 min,which was then preserved at 4℃.

Corona discharge mutagenesisThe two 9.2 cm MRS-agar plates, each with a volume of 15 ml,were sealed with paraffin.The voltage applied in this research was 3.4 KV,and the durations of treatment were 20 min and 30 min,respectively.The distance between the top and bottom was d= 0.85 cm.The frequency was 1 000 Hz. The sterile bench remained open.The experiment time began when the high voltage voltmeter reached a designated number(potential gradient).When the duration reached the designated time,the voltage was slowly reduced to zero.The mutated strains were preserved at 4℃.

Analyses and identification of strains’16SrRNA PCR after mutationThe same methods of analyses and identification used in 16SrRNA PCR of the original stains were applied here.

Determination of acidogenicity and growth curve of mutated bacteria

For the isolation and growth of the strains,10滋l of the strain suspension were mixed with 990滋l water.The eighteenth,nineteenth,twentieth, twenty-first and twenty-second gradients were chosen and 10 milliliters were seeded into MRS-agar plates, and incubated at 37℃.After a 72 h incubation period,colonies with obvious calcium circle in a short time[16]were seeded into MRS fluid medium with a quantity of 4%.Based on dates, pH variation and growth curves were drawn.

Results and Analyses

Isolation and purification of lactic acid bacteria

Sixteen lactic acid bacteria samples were isolated and purified out of the 21 samples.They were numbered from 1 to 16.To distinguish the acidproduction bacteria from other bacteria,the character of strain numbers 8 and 10 were identified as hoar,flat uplift,surface gross,clear,obvious of calcium circle.The other strains’character were ivory,surface gross, opacity,obvious of calcium circle.

Results of 16SrRNA PCR analysis

Fig.1 and Fig.2 demonstrate the initial and processed strains of the sequence analysis of the DNA,approximately 800 bq forward,indicated Lactobacillus.

Result of Corona discharge mutagenesis and activation

The strains were numbered from 1Y to 16Y after corona discharge mutagenesis.The colonies with obvious calcium circles in a short amount of time were sifted to be used for further experimentation.The initial strains started to curdle in skim milk after approximately 24 hours,and completed this process after approximately 48 hours.The strains used after mutation started to curdle after approximately 18 hours in skim milk,and completed this process after approximately 36 hours.The curd time was reduced because of mutagenesis,which increased its acid-production rate.

The strains’pH variation and growth curve after mutation

32 strains’growth curveBacteria growth for the different strains was similar.All 16 of the initial strains entered the log phase at approximately 3 hours.Strain No.10 and No.16 entered the stationary phase after 16 hours Strain No.3,No.5 and No.7 entered the stationary phase after 22 hours. The remaining strains also entered the stationary phase after approximately 22 hours.Strain No.7,however,hadan OD value of 12 in the stationary phase,while other OD values were 10.

Strain No.15Y entered the log phase at approximately 4 hours. Strains No.1Y,No.2Y,No.3Y,No.4Y, No.6Y,No.7Y,No.8Y,No.9Y,No.10Y, No.11Y,and No.16Y entered the log phase at approximately 6 hours. Strains No.5Y,No.12Y,No.13Y,and No.14Y entered the log phase at 8 hours.All of the 16 strains entered the stationary phase at approximately 22 hours.In the stationery phase,the OD values of No.3Y,No.8Y,and No.10Y were higher than others.

Fig.3 demonstrates the growth curves of the 4 strains after corona discharge mutation and if the gene functioned properly,the growth rate increased when compared with the initial strains.Strain No.10Y had an OD value that was steady in the stationary phase,whereas strain No.16Y had an OD value that was higher than before, indicating that the mutation functioned properly.

Acidogenicity of 32 strains and lactic acid bacteria from Yi li yogurt

The pH of the 16 initial strains’broth with fermentation time decreased,especially the pH of strains No.1,No.2, No.12,No.14,and No.16 were similar at 22 hours,with the pH measuring the minimum value.The pH of No.3,No.4, No.5,No.6,No.7,No.8,No.9,No.11, No.13,and No.15 was similar and at the 26 hours,the pH reached a minimum value.The pH of No.10 reached a minimum value at 18 hours The pH of No.1,No.2,No.3,No.4,No.5,No.9, No.12,No.13,and No.14 were 3.5 to 4.0.The pH values of No.6,No.7,No. 8,No.10,No.11,No.15,and No.16 were 4.0 to 4.5.Combined with the growth curves and the period of minimum pH values,the OD of No.2,No.3, and No.9 were still 10 to 12,indicating that they had a better quality of acid resistance when compared to the other strains[17].

The pH of 16 processed strains’broth with a decreased fermentation time had similar amounts of acid production.The pH of 16 processed strains’broth decreased after 4 hours, arrived at a minimum pH value of 4 at 26 hours.The pH value most of the time was between 3.5 and 4.0 and after 26 hours the pH remained close to 4.The OD of the processed strains were similar after 26 hours the strains demonstrated a steady growth curve. Without the addition of acid products, the pH was less than 4,causing the lactic acid bacteria to stop growing. Basic on its biochemistry character,indicated the strains were lactic acid bacteria.

The strains were seeded into Yili yogurt broth.The pH decreased quickly after 4 hours,continually decreasing until arriving at a minimum of 3.9 after 24 hours.

Fig.4 shows the difference in the acid production in the initial strains and the processed strains for the 4 colonies.Two types of MRS fluid mediums’pH curve,and acidogenicity for 6Y,12Y,14Y and 16Y increased. Results showed that the processed strains’pH value was lower than the initial strains’pH value.Furthermore, the pH value continued to decrease after processing,indicating that the strains mutated properly.

Discussions

For this research,21 samples of Chinese sauerkraut were obtained from Inner Mongolia and Northeast China,16 lactic acid bacteria were Lactobacillus,the ability of acid-production from different areas was different,the processed lactic acid bacteria ability of acid-production was better than before.

The pH of the media and the absorbance value of the culture solution were compared between the initial curve and the processed curve. Comparisons made at times based on the biochemistry character indicated the initial and processed strains were lactic acid bacteria.To ensure the results of phenotypic characterization, the 16SrRNA was fully sequenced, analysed and identified by comparison with initial strains,and all of the strains were Lactobacillus.

The experiment indicated corona discharge mutagenesis is a new method of mutagenesis where the quality of acid production is improved; therefore,providing new methodologies for researching microbial mutation breeding.Both of the processed strains demonstrated a significant increase in acid production and growth rate.However,there was ability lack of acid production from Yili yogurt.Compared to the initial strain,the lactic acid bacteria’s acid production was very different between Chinese sauerkraut soup from Inner Mongolia,Northeast China and Yili yogurt.Furthermore,it was found that an increase in the quality of acid production is necessary in order to benefit possible local economic development.

Research regarding mutation breeding leaves much to be desired and will require a significant amount of time and work to be fully understood. Future work includes proving the effectiveness of corona discharge mutagenesis,as well as improving the quality of the mutant strains.

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Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

Supported by National Natural Science Foundation of China.

*Corresponding author.E-mail:nari6363@sina.com

Received:May 3,2015 Accepted:June 16,2015