Food Business News Network, also known as benzoic acid, mordenite pure white scales or needles, light or slight odor benzoin or benzaldehyde odor. Benzoic acid is a food preservative commonly used in countries all over the world. It is easily excreted in the body with urine, does not accumulate, has low toxicity and low price, and currently occupies most of the domestic preservative market. However, since benzoic acid is an organic acid type food preservative, it can effectively inhibit the growth of microorganisms only in its molecular state. Therefore, benzoic acid is only suitable for application under acidic conditions (pH<4.5), and the antibacterial effect is most at pH3. Well, the scope of use is greatly limited. In order to break the restriction of environmental conditions on the antibacterial activity of benzoic acid, scholars improved the bacteriostatic pH range and antimicrobial metabolism by esterification modification of benzoic acid molecules, thereby improving their antibacterial properties. In this paper, a series of esters such as ethyl benzoate, propyl ester and butyl ester were synthesized by direct esterification method. The inhibitory effects of these esters on the mixed bacteria of cow's milk were studied.
Materials and Methods
1.1 Instrument and medicine stainless steel portable pressure steam sterilizer; 722s visible spectrophotometer; ZD-85A air bath constant temperature oscillator; DHG-9077A electric heating constant temperature blast drying oven.
Beef extract, peptone, sodium chloride, glucose, hydrochloric acid, sodium hydroxide, benzoic acid, sodium benzoate, methanol, ethanol, propanol, butanol: analytically pure; fresh milk: commercially available.
1.2 Experimental bacteria
Bovine lactic acid mixed bacteria.
1.3 Experimental medium
The beef paste peptone culture solution has the following composition: peptone 1%, beef extract 0.5%, sodium chloride 0.5%, glucose 1%, pH: 7.2-7.5, and sterilization at 120 °C for 20 min.
1.4 Esterification reaction
Benzoic acid and the corresponding alcohol were placed in a three-necked flask equipped with a reflux apparatus in a certain molar amount (mol), and slowly heated. After the benzoic acid was completely dissolved, the corresponding alcohol and catalyst were added, and the mixture was slowly heated and refluxed for 4 hours. After completion of the reaction, the mixture was washed with a saturated brine and a 5% sodium carbonate solution, and dried over anhydrous magnesium sulfate, and the corresponding fractions were collected by distillation to obtain a succinic acid as a colorless oily liquid.
1.5 Antibacterial experiment
1.5.1 Determination of minimum inhibitory concentration
Add benzoic acid and benzoate to different concentrations and add to the test tube containing 10 mL of broth medium, then add 0.1 mL of bovine lactic acid mixed bacteria, mix well, and make 2 other control tubes (blank and bacteria). The cells were cultured in a biochemical incubator at 37 °C for 24 h, and the degree of turbidity of each tube was compared by visual inspection to find the minimum inhibitory concentration of each preservative.
1.5.2 Determination of antibacterial activity
The inhibition ability of the synthesized product on the growth of the mixed bacteria of bovine lactis was determined by spectrophotometry. Add a certain amount of bovine lactic acid mixed bacteria to the medium, put them into a 250 mL flask, fill each bottle with 150mL, add the antiseptic dose of the minimum inhibitory concentration, seal the bottle mouth with the sterilized cotton cloth, and put it in the mixture. The bacteria are most suitable for growth at a growth temperature (37 °C), A560 mm is measured every 6 hours, and the antibacterial activity IR of the preservative is calculated.
Results and discussion
Product properties
At room temperature, the product is a colorless or light yellow transparent liquid with a special aroma. The refractive index nd of each product is: ethyl benzoate 1.507, propyl benzoate 1.4972, butyl benzoate 1.4940.
Minimum inhibitory concentration of preservative
After incubating for 24 h in a 37 ° C gas bath thermostat, the tube was taken out, and the degree of turbidity was observed with the naked eye, and the minimum inhibitory concentration was recorded. The minimum inhibitory concentration of each food preservative is shown in Table 1.
Effect of pH on the antibacterial effect of preservatives
The temperature of the fixed culture solution was 37 ° C. Under the minimum inhibitory concentration of each food preservative, the antibacterial activity of the food preservative was studied at pH 4~8. The results of the absorbance change curve with time are as follows: Figure 1~4 Shown.
It can be seen from Fig. 1 to Fig. 6 that under the condition of pH 4~6, benzoic acid, sodium benzoate and phenylpotassate have good antibacterial effect at the minimum inhibitory concentration of preservative added; at pH 7~9, Benzoic acid and sodium benzoate have almost no bacteriostatic effect; while benzoate still shows good antibacterial effect, especially the growth of mixed bacteria of ethyl benzoate and propyl benzoate is inhibited in 72 hours after inoculation. Low level, and with the increase of R alkyl carbon chain in benzoate, the stronger the lipophilicity, so the water solubility of butyl benzoate is lower than that of ethyl benzoate and propyl ester, at the minimum inhibitory concentration The anti-corrosion dose of the actual antiseptic effect is also small, so that the antibacterial strength is lower than that of ethyl benzoate and propyl ester.
Benzoic acid and sodium benzoate exist in a molecular state in an acidic environment, and are more likely to penetrate into the cell body through the microbial cell membrane to inhibit bacteria. When the pH is high, most of them exist in a dissociated state, and it is difficult to act through the microbial cell membrane, so benzene Formic acid and sodium benzoate are suitable for use in acidic foods. In the range of pH 4~9, ethyl benzoate and propyl benzoate are present in the medium in the molecular state, so the antiseptic effect is not affected by pH, and can be used in a wide range of pH.
Comparison of antibacterial rate of preservatives
The inhibition rate of benzoic acid, sodium benzoate and phenylpotassate against mixed bacteria at 37 °C, pH 7.2 was calculated, and the inhibition rate of benzoic acid, phenylpotassate and benzoate on mixed bacteria was changed with culture time. The curve is drawn as Figure 7.
Before 6h, the added preservative did not fully function, the microbial growth was fast, and the inhibition rate was low. After 6h, with the increase of the dissolution amount of each preservative, the antiseptic effect gradually reflected, and the preservative was inhibited at 12h. The highest bacterial rate, the strongest bacteriostatic effect is ethyl benzoate and propyl benzoate, which can maintain nearly 90% inhibition rate after 12h; butyl benzoate due to the growth of alkyl carbon chain, pro The stronger the oiliness, the lower the water solubility of butyl benzoate is lower than that of ethyl benzoate and propyl ester. The antiseptic effect of the actual antiseptic effect is also small at the minimum inhibitory concentration, and it plays a role in the water-based medium. It is relatively small, so the inhibition rate of butyl benzoate begins to decrease after 12h; the inhibition rate of benzoic acid and sodium benzoate is the weakest.
in conclusion
Solubility is the premise that preservatives play an antibacterial role. The antibacterial effect of benzoate increases significantly with the degree of esterification and the length of ester chain, but the solubility of butyl benzoate is better than that of ethyl benzoate and benzoic acid. The low propyl ester affects its antibacterial activity, so the antiseptic effect of the actual antiseptic effect at the minimum inhibitory concentration is also small, and its antibacterial effect is worse.
The antibacterial strength of benzoic acid was significantly enhanced after esterification. The antibacterial range was not affected by pH, and the stability of bacteriostatic was greatly improved. It has certain development and application prospects.
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