Screening and biological characteristics of excellent lactic acid bacteria from Kefir

: Four excellent lactic acid bacteria were screened from Kefir. After morphological observation, physical and chemical detection, and 16SrDNA sequence comparison, three strains were preliminarily identified as Lactobacillus Kefir, and one strain was Lactobacillus acidophilus. Two strains were selected to study their biological characteristics. The results showed that the lactic acid bacteria isolated from Kefir had good probiotic properties, with a survival rate of more than 42% after artificial gastric juice digestion for 3 hours and more than 46% after artificial intestinal juice digestion for 6 hours; It has inhibitory effects on intestinal pathogenic bacteria such as Staphylococcus aureus and Escherichia coli; Does not produce hemolysis; There are different levels of resistance to antibiotics.


Introduction
Kefir is a fermented beverage with renowned probiotics that coexist in symbiotic association with other microorganisms in kefir grains.These gelatinous grains are a symbiotic culture of bacteria and yeast embedded in a polysaccharide matrix. Lactic acid bacteria, yeast and acetic acid bacteria are the primary microbial members of the sugary kefir grain.Amongst other contributions, species of lactic acid bacteria produce the exopolysaccharide matrix from which the kefir grain is formed, while yeast assists the bacteria by a nitrogen source that can be assimilated.This beverage consumption is associated with a wide array of nutraceutical benefits, including anti-inflammatory, anti-oxidative, anticancer,antimicrobial,antidiabetic,antihypertensive,andant i-hypercholesterolemic effects.
The microbial composition of kefir grains comprised 65 to 80 % of Lactic acid bacterias and the remaining portion was completed by yeasts.Lactic acid bacteria are gram positive bacteria that can ferment carbohydrates to produce organic acids such as lactic acid, and are widely found in fermented dairy products, pickles, silage, and other environments. Lactic acid bacteria are not only conducive to food fermentation, but also can improve the nutritional value of food and play a better regulatory role in the physiological functions of the human body .
In order to fully understand the functional characteristics of lactic acid bacteria in Kefir, lactic acid bacteria with excellent characteristics such as acid production and bacteriostasis were isolated from Kefir. Through morphological observation, biochemical a Guoab12@163.com b cgbatbatan@cmu.edu.ph property determination, and 16S rDNA strain identification, the bacteriostasis and gastrointestinal resistance of Kefir lactic acid bacteria were studied, and the safety of lactic acid bacteria in Kefir was evaluated from both hemolysis and drug resistance, To provide safe and high-quality lactic acid strains for the better development of Kefir milk drinks.

Materials and reagents
MC culture medium, blood agar plate, HKI013 EasylD lactic acid bacteria biochemical identification kit, antimicrobial susceptibility paper, Guangdong Huankai Company; MRS broth culture medium, Hangzhou Bess Company; Bacterial Genomic DNA Extraction Kit, Beijing Tiangen Company; 16S upstream and downstream primers, Shanghai Shenggong; Artificial gastric juice, artificial intestinal juice;, Hangzhou Microbiology Company. The rest are domestic analytical or chemical reagents.

Isolation and purification of lactic acid bacteria
Dip in Kefir fermentation broth and scribe on an MRS plate. Incubate at 37 ℃ for 48 hours. Use a sterilized toothpick to pick up single colonies and transfer them to MRS liquid culture medium for cultivation. The strains with negative contact enzyme reaction and positive Gram stain microscopic examination were preliminarily identified as lactic acid bacteria.

Preliminary screening
Inoculate the purified single bacterial colony in MRS liquid culture medium, incubate at 37 ℃ for 48 hours, measure the OD value at a wavelength of 600 nm, and measure the pH value of the fermentation broth of each strain. Select strains with higher OD values and lower pH values for subsequent experiments.

Double screening
Fully activated bacterial solution was added to MRS liquid culture medium at 3% inoculation amount, and incubated at 37 ℃ for 48 hours under constant temperature oscillation. The acidity was measured according to the method in GB/T 5413.34-2010 [5].

Observation of morphological characteristics
Observe the culture characteristics of lactic acid bacteria on MRS plate, such as the color, size, texture, and edge of a single colony.The Gram stain microscope is magnified 1000 times to examine the color, morphology, and arrangement of bacteria.

Biochemical identification
The lactic acid bacteria biochemical identification kit was used to conduct biochemical tests such as aescin hydrolysis test, xylose fermentation test, cellobiose fermentation test, arabinose fermentation test, maltose fermentation test, galactose fermentation test, mannose fermentation test, melibiose, raffinose fermentation test, cane sugar fermentation test, trehalose fermentation test, and determine whether the test is positive or negative based on color changes.

Amplification and sequence analysis of 16S rDNA
The genomic DNA of lactic acid bacteria was extracted using a bacterial genomic DNA extraction kit. The conserved sequence of the 16S rDNA of the bacteria was amplified using the 16S universal primers 27f and 1492r. The purified PCR product was sent to Shanghai Shenggong for sequencing. The sequencing results were compared with similar sequences in the NCBI database.

Study on the Tolerance of Lactic Acid Bacteria to Artificial Gastrointestinal Fluid
The lactic acid bacteria were resuspended in artificial gastric juice and artificial intestinal juice for 3 and 6 hours, respectively, and the bacteria were collected. The viable bacteria were counted according to the National Standard for Food Safety -Microbiological Examination of Food -Determination of Total Colony Count (GB47892-2022). The tolerance was calculated based on survival rate [4] The formula is as follows: Survival rate/%=lgN1/lgN0 × 100% In the formula, N1 -viable bacterial count after tolerance treatment (CFU/mL); N0 -viable bacterial count before tolerance treatment (CFU/mL).

Bacteriostatic study
The Oxford Cup method was used to determine the antibacterial activity of lactic acid bacteria against common pathogenic bacteria. The supernatant was centrifuged from the bacterial solutions of Escherichia coli and Staphylococcus aureus. The supernatant was treated in three ways: adjusting the pH to 6.0 with 1 mol/L NaOH, treating with catalase and trypsin, and then filtering for sterilization. Add the prepared pathogenic bacteria plate to an Oxford cup, place the plate under 37 ℃ for 48 hours, and measure the diameter of the bacteriostatic ring (mm) .

1) Hemolysis
Select the activated lactic acid bacteria and draw a line on a blood agar plate. Incubate at 37 ℃ for 48 hours to observe whether there are hemolytic circles. At the same time, use Staphylococcus aureus as a positive control for comparison .
2) Drug resistance testing Incubate the lactic acid bacteria liquid to 105~106 CFU/mL, evenly coat it on the MRS agar plate, paste vancomycin paper, erythromycin paper, chloramphenicol paper, tetracycline paper, gentamicin, amoxicillin paper, penicillin G paper, and streptomycin paper on the MRS agar plate, incubate at 37 oC for 48 hours, observe and measure the diameter of the bacteriostatic ring [8].
All trials were conducted three parallel trials, and the experimental data were statistically analyzed using Origin 8.5 and Excel 2016. Single factor analysis of variance was performed on the data of each group. P<0.05 indicates a significant difference between the data.

Screening of excellent lactic acid bacteria
Using Kefir as raw material, a total of 34 strains of lactic acid bacteria, named KFR-1, KFR-2, KFR-3, were obtained through isolation and purification, microscopic examination, and contact enzyme experiments. The strains with fermentation broth OD600 values above 1.

Observation of morphological characteristics
The colonies of selected KFR-11, KFR-23, and KFR-31 on MRS medium were small, white, round, and smooth and moist in surface; The colony of KFR-27 on MRS medium is small and its surface is smooth and moist. Four strains of lactic acid bacteria were Gram positive, and their bodies were all rod shaped, arranged in pairs or chains.

Biochemical identification
Using a biochemical identification kit for lactic acid bacteria, the ability of KFR-11, KFR-23, KFR-27, and KFR-31 to utilize carbon sources was tested. The results showed that the aescin hydrolysis test, cellobiose fermentation test, L-arabinose, maltose fermentation test, galactose fermentation test, mannose fermentation test, and fucose fermentation test were all positive; The xylose fermentation test, arabinose fermentation test, and raffinose fermentation test and sucrose fermentation test were all negative.

Amplification and sequence analysis of 16S rDNA
Using the DNA of lactic acid bacteria as a template, a fragment of about 1.5 kb was obtained by PCR amplification. The 16S rDNA sequencing results were submitted to NCBI for BLAST comparative analysis. The strains KFR-11, KFR-23, and KFR-31 isolated in this study have 99% homology with Lactobacillus kefir, and KFR-27 have 99% homology with Lactobacillus acidophilus. According to the colony characteristics, morphological characteristics, sugar fermentation tests, and 16S rDNA sequence analysis of lactic acid bacteria, KFR-11, KFR-23, and KFR-31 were identified as Lactobacillus kefir, and KFR-27 as Lactobacillus acidophilus. According to the acid production performance, KFR-11 and KFR-27 were selected as the research objects for biological characteristics research.

Study on Tolerance of Artificial Gastrointestinal Fluid
Kefir is generally consumed without heating. Most acid intolerant microorganisms from food are killed by the acid  Table 1. After 3 hours of digestion in artificial gastric juice, the survival rates of the two strains were 42% ± 0.49 and 58% ± 0.28, respectively. There was a significant difference between the two strains (P<0.05), with the survival rate of KFR-27 being greater than 50%. The survival rate of both strains of bacteria after 6 hours of digestion in artificial intestinal fluid was close to 50%, and there was no significant difference between the two strains (P>0.05).

Bacteriostatic study
The fermentation supernatant of both strains of lactic acid bacteria can inhibit Escherichia coli and Staphylococcus aureus, but not Pseudomonas aeruginosa. From Table 2, it can be seen that neutralization treatment and catalase treatment eliminate the impact of organic acids and hydrogen peroxide on the bacteriostatic effect of the indicator bacteria. Using trypsin to treat the fermentation broth of the two strains, the bacteriostatic effect was significantly reduced (P<0.05), and it was determined that the bacteriostatic substance contained bacteriocins with protein properties. The antibacterial effect of lactic acid bacteria fermentation supernatant on pathogens has potential application value. Lactic acid bacteria can maintain gastrointestinal balance by inhibiting the growth of pathogens, thereby improving human health. If the diameter is<9 mm and there is no antibacterial activity, it is marked as "-"; 9 mm ≤ diameter<12 mm, with antibacterial activity, marked as "+"; Diameter ≥ 12mm, strong antibacterial activity, marked as "++".

Safety evaluation results
As an edible probiotic strain, the strain isolated from Kefir was tested for potential adverse effects on human health. Analysis of antibiotic resistance and hemolytic activity of lactic acid bacteria is an important feature of their safe use.

Hemolysis
The agar around Staphylococcus aureus showed a green ring, while the agar around KFR-11 and KFR-27 did not show a green transparent ring. The strains with transparent hemolytic circles are β-Hemolytic or completely hemolytic, the green circle is α-Hemolytic or partially hemolytic. Only non hemolytic lactic acid bacteria can be used as potential probiotics in fermented foods. In this experiment, no hemolytic phenomenon was found in the screening of lactic acid bacteria.

Resistance
With the overuse of antibiotics, the problem of microbial resistance is becoming increasingly serious. Fermented products containing lactic acid bacteria may enter the human gut through the food chain, and drug resistance genes are transmitted to pathogenic bacteria in the body through molecular binding and other actions, posing a serious threat to human health. The sensitivity of the two strains of lactic acid bacteria tested to eight common antibiotics is divided into three levels according to the standards specified by the American Society for Clinical and Experimental Standards (CLSI): sensitive (S), moderately sensitive (I), and resistant (R). From Table 3

CONCLUSIONS
Some lactic acid bacteria in Kefir are naturally occurring or artificially added. Both Lactobacillus Swiss and Lactobacillus Kefir belong to the genus Lactobacillus, which can ferment to produce a variety of antibacterial substances, inhibiting and killing spoilage bacteria and pathogenic bacteria in fermented foods. Many lactobacilli can produce volatile substances that give fermented foods a special flavor. If lactic acid bacteria want to play a role in improving the structure of intestinal flora and inhibiting intestinal pathogenic bacteria, it is required to overcome the impact of physical and chemical factors in the stomach and intestines, and reach the stomach and intestines in a viable state. KFR-11 and KFR-27 have a certain survival rate after being treated with artificial gastric and intestinal fluids. According to the safety evaluation of the 2 strains of lactic acid bacteria, hemolysis will not occur. In this study, it was found that 2 strains of bacteria had no antibiotic resistance except for gentamicin. The two excellent lactic acid bacteria selected from Kefir in this experiment can withstand the stomach and intestinal environment, inhibit the growth of common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and provide a reference basis for the development and utilization of probiotics. Currently, the lactic acid bacteria used in fermented foods are basically wild type, which are generally consumed directly without secondary disinfection, and may carry out resistance gene transmission with normal intestinal flora or pathogenic bacteria. Before lactic acid bacteria are used as edible production strains, it is necessary to detect the drug resistance of the strains and prevent the spread of drug resistant genes.
11. Nv X,Yaru L,Chaoyu W.(2020) Evaluation of antibiotic resistance and analysis of resistance genes of lactic acid bacteria in traditional fermented foods.