Physiological and Biochemical Characteristics and Antibiotic Susceptibility Tests
The 12 strains of lactic acid bacteria with antibacterial activity from the primary screening were tested for temperature, pH and NaCl tolerance. All strains were grown at temperatures 10°C, 30°C, at pH 4.0, 4.5, 5.0, 8.0 and 9, and at 3.0% NaCl. QZ11, QZ14,QZ21,QZ50 and QZ71 could grow at 5°C, QZ21, QZ50, QZ67, QZ81 could survive at 50°C, while all other strains cannot survive at 5°C, or 50°C.QZ14, QZ21, QZ50, QZ67, QZ71 can tolerate growth in a high salinity environment of 6.5% Nacl.QZ11, QZ14, QZ21, QZ50, QZ57, QZ71 and QZ81 can grow at pH 3.5 and and only two strains QZ50, QZ71 can survive in pH 3.0 environment. Taken together, the seven strains, strains QZ11, QZ14, QZ21, QZ50, QZ67, QZ71 and QZ81 had better environmental tolerance.
Identification of selected Strains
Physiological and biochemical tests were performed to identify QZ50 (Table 2). Strain QZ50 grew at 10, 15, and 45°C and weakly at 50°C. The strain grew in 3 and 6.5% NaCl and at pH 3–10. Strain QZ50 produced gas from glucose, and utilized ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, raffinose, and gentiobiose, but not glycerol, erythritol, L-arabinose, D-xylose, adonitol, β-methyl-xyloside, sorboseE, rhamnose, dulcitol, inositol, α-methyl-D glucoside, inulin, starch, glycogen, xylitol, D-turanose, D-tagatose, and L-arabitol. Moreover, it could not completely utilize D-arabitol and gluconate.
Table 2
Physiological and biochemical identification of strain QZ50
Character | QZ50 | Substrate | QZ50 | Substrate | QZ50 |
Fermentation type | Homo | Glycerol | - | Sorbitol | + |
Growth at temperature (°C): | | Erythritol | - | α-Methyl-D Glucoside | - |
10 | + | D- arabinose | - | Amygdalin | + |
15 | + | L- arabinose | - | Arbutin | + |
45 | + | Ribose | + | Esculin | + |
50 | w | D-xylose | | Salicin | + |
Growth in NaCl: | | Adonitol | - | Cellobiose | + |
3.00% | + | β-Methyl –Xyloside | - | Lactose | + |
6.50% | + | Galactose | - | Maltose | + |
Growth at pH: | | Glucose | + | Lactose | + |
3 | + | Fructose | + | Melibiose | w |
3.5 | + | MannosE | + | Sucrose | + |
4 | + | SorbosE | - | Trehalose | + |
4.5 | + | Rhamnose | - | Raffinose | + |
5 | + | Dulcitol | - | Gluconate | w |
9.5 | + | Inositol | - | Inulin | - |
10 | + | Mannitol | + | Glycogen | - |
Note: +, positive; -, negative; w, weakly positive; Homo, homofermentative. |
For the 16S rRNA gene identification of QZ50, a phylogenetic tree was constructed using a neighbor-joining method (Fig. 1). In the Lactobacillus cluster, strain QZ50 was grouped with L. pentosus, L. plantarum, and L. paraplantarum, but it could not be identified to a species level based on 16S rRNA gene sequence analysis. The amplification products gained from the recA gene was displayed in Fig. 2. Strain QZ50 and type strain L. plantarum subsp. plantarum JCM 1149T all produced 318 bp products. Thus, it was identified as L. plantarum subsp. plantarum. All the above strains with good antibacterial activity were identified, QZ11, QZ21, QZ71 and QZ81 were identified as Lactobacillus plantarum, QZ58 was identified as Lactococcus lactis, and QZ14, QZ71 were identified as Lactobacillus johnsonii.
Determination and Comparison of the performance of selected lactic acid bacteria.
The antibiotic susceptibility of the 12 selected strains with antibacterial activity was tested and the results were shown in Table 3. All the selected LAB strains were resistant to CN, CIP, CT, VA and P, while most strains exhibited sensitivity to AMP, TE and C. For erythromycin (E), the QZ11, QZ14, QZ21, QZ42,QZ57, QZ58 and QZ81 had sensitivity, while QZ29, QZ41, QZ50 and QZ67 were moderately resistant, and QZ71 was resistant to it. As to rifampicin (RD), QZ29, QZ41 QZ57, QZ58, QZ81 were resistant to it. Strain QZ57 was found to be resistant to most antibiotics except erythromycin and chloramphenicol.
Table 3
Profile of antibiotic susceptibility of selected LAB strains
Strain | AMP | CN | TE | CIP | E | CT | C | VA | P | RD |
QZ11 | S | R | S | R | S | R | S | R | R | S |
QZ14 | S | R | R | R | S | R | S | R | R | S |
QZ21 | S | R | S | R | S | R | R | R | R | S |
QZ29 | S | R | S | R | M | R | S | R | R | R |
QZ41 | S | R | S | R | M | R | S | R | R | S |
QZ42 | R | R | S | R | S | R | S | R | R | S |
QZ50 | S | R | S | R | M | R | S | R | R | S |
QZ57 | R | R | R | R | S | R | S | R | R | R |
QZ58 | R | R | S | R | S | R | S | R | R | R |
QZ67 | S | R | S | R | M | R | R | R | R | S |
QZ71 | S | R | S | R | R | R | S | R | R | S |
QZ81 | S | R | S | R | S | R | S | R | R | R |
Note: |
aThe concentrations of antibiotics are expressed in micrograms per disk (µg/disk), R, resistant; M, moderate resistance; S, susceptible |
bAmpicillin (AMP, 10 µg/disk), gentamicin (CN, 10 µg/disk), tetracycline (TE, 30 µg/disk), ciprofloxacin (CIP, 5 µg/disk), erythromycin (E, 15 µg/disk), colistin sulphate (CT, 10 µg/disk), chloramphenicol (C, 30 µg/disk), vancomycin (VA, 30 µg/disk), penicillin (P, 10 µg/disk), rifampicin (RD, 5 µg/disk). |
The surface hydrophobicity and agglutination of the Lactobacillus strains were measured. As shown in Fig. 3, the surface hydrophobicity of the representative strains of LAB ranged from 19–48%, with strains QZ21, QZ58, QZ34 and QZ50 showing high surface hydrophobicity, while the remaining the remaining 2 strains showed lower surface hydrophobicity. As shown in Fig. 4, the self-agglutination of LAB strains varied from 26.3–58.3%, with strains QZ71 and QZ50 showing significantly higher self-agglutination than the remaining 4 strains. The self-agglutination of 2 strains, QZ71 and QZ81, was significantly lower than that of the remaining strains.
The acid production capacity of the six selected strains of LAB is shown in the Fig. 5, there was no significant difference in acid production capacity, and strain QZ50 has the strongest acid production capacity, at 36 hours, the pH was reduced to 3.98. Combined with the above experimental results and analysis of acid production capacity and surface hydrophobicity and self-aggregation and bacterial inhibition, three strains of bacteria, QZ14 QZ50 and QZ71 were selected for simulated gastrointestinal survival experiments. As shown in the Fig. 6, all three strains of LAB survived after 7h incubation in the simulated gastrointestinal tract. After 3h of artificial gastric juice, the viable counts of the three strains of LAB decreased to different degrees, among which the viable counts of QZ14 and QZ71 were significantly reduced, indicating that these two strains were less tolerant to the artificial gastric juice. After transferring the lactic acid bacteria from the simulated gastric juice to the artificial intestinal fluid, the viable counts of QZ14, QZ71 and QZ50 were significantly reduced, and the viable counts of all three strains of LAB were significantly reduced compared with the initial ones after 4h incubation in the artificial intestinal fluid. Collectively, the strains of LAB were less affected by the artificial gastric fluid, especially ZX50 had no significant decrease in viable bacteria count after incubation in gastric fluid. In contrast, all strains showed a significant decrease of at least 0.89 log10 CFU/mL after incubation in intestinal fluid.
Optimization of media and culture conditions
Some more in-depth studies were performed on Strain QZ50, a broad-spectrum bacteriocin-producing strain with the highest antibacterial activity. The effects of different media, temperature, initial pH values, and inoculum amount on bacteriocin production are shown in Fig. 7. The highest inhibition zone diameter was obtained in MRS broth, at a temperature of 30°C, a pH value of 6.5, and an inoculum amount of 3%. The lowest inhibition zone diameter was obtained with M17 broth, at 25°C, a pH of 4.5, and a 1% inoculum amount. Compared with the conditions of MRS broth, 30°C, pH 6.5, the antimicrobial activity of strain QZ50 under other conditions was significantly different (P < 0.001). Compared with the inoculum amount of 3%, other inoculum amount except 2% was significantly different (P < 0.001).
Optimization of medium components
The effects of different C and N sources on bacteriocin production are respectively shown in Fig. 8 and Fig. 9. The antimicrobial activity of strain QZ50 due to different C sources was significantly different (P ≤ 0.001). As shown in Fig. 8, Glucose contributed to the highest inhibition zone diameter, while soluble starch contributed to no bacteriocin production. Lower inhibition zone diameters were obtained in a descending order as: fructose, sucrose, maltose, lactose, and cellobiose.
As shown in Fig. 9, yeast extract contributed to the highest diameter of inhibition, while inorganic N contributed to no bacteriocin production. Except ammonium citrate and ammonium chloride, the antimicrobial activity of strain QZ50 due to different N sources was significantly different (P ༜ 0.05). Lower inhibition zone diameters were obtained in a descending order as: tryptone, peptone, casein peptone and beef extract.
The effect of different stimulating factors on bacteriocin production is presented in Fig. 10(A). The diameter of inhibition of QZ50 was the highest with Tween 80 as surfactant. The antimicrobial activity of strain QZ50 due to different surfactants was significantly different (P ≤ 0.01). As shown in Fig. 10(B), the levels of stimulating factor in the culture medium had significant effect on bacteriocin production(P < 0.01). The diameter of inhibition of QZ50 was the highest with 2% v/v Tween 80 and the lowest with no Tween 80.