Purification And Applications Of Bacteriocin Produced By Lactobacillus Acidophilus In Milk

Lactobacillus acidophilus is Gram-positive facultative anaerobes. It's the main probiotics in human intestine. And it has great significance on human health as safe food-grade microorganisms(GRAS). Milk and dairy are popular food with abundant nutrients, but they are easy to spoil. So people have been paying attention to use natural preservatives. Bacteriocins of Lactobacillus acidophilus have become the focus in recent years. Based on the previous study, bacteriocin Lactobacillin of Lactobacillus acidophilus is as the research object. The fermentation conditions of bacteriocins Lactobacillin was firstly optimized, then the specific route of purification was determined, the bacteriocin Lactobacillin was identified and the bioinformatics of the protein sequence was analyzed. Finally, the bacteriocin Lactobacillin was used for biological preservative in dairy. The main contents and conclusions are shown below:1. The fermentation conditions of bacteriocin Lactobacillin produced by Lactobacillus acidophilus were optimized through single factor experiment and orthogonal experiment using bacteriocin antibiotic titer(AU/mL) as evaluation index. The effects of initial pH, incubation temperature, incubation time and initial inoculum were studied. The results showed that the optimal fermentation conditions were as follows: initial pH 6.5, incubation temperature 34 ?, inoculum 2.5% and incubation time 28 h. Under these optimal conditions, the bacteriocin antibiotic titer increased by 2 folds and was reached up to 247.49±1.05 AU/mL.2. The bacteriocin Lactobacillin produced by Lactobacillus acidophilus was purified and identified. The fermented supernatant of Lactobacillin was precipitated by ammonium sulphate, ultrafiltrated by 1k ultrafiltration membranes. Then the Lactobacillin extract was subjected to strong anion exchanged chromatography with HiTrapTM CaptoTM Q, and gel permeation chromatography with SuperdexTM 75 10/300 GL for further purification. Finally, molecular weight of Lactobacillin was determined by Tricine-SDS-PAGE, amino acid sequence of Lactobacillin was analyzed by Matrix Assisted Laser Desorption/Ionization Time of Flight(MALDI- TOF/MS) and High Performance Liquid Chromatography with Mass Spectrometry(HPLC- MS/MS), and the bioinformatics of the protein sequence were explored. The result indicated that the specific activity of Lactobacillin was 11068.42 AU/mg, increased by 12.24 fold and the yield was 9.49%; the molecular weight of Lactobacillin was about 6 ku; moreover, the amino acid sequences of Lactobacillin was mlggvlsrltswddfgdvwfsrlqseaearslgwrrspwqaeplaagvrpfyitqengnkga, unstability index of Lactobacillin was 78.06%, no obvious transmembrane structure was present in the Lactobacillin, the alpha helix and random coil had a high percentage in which their secondary structure and tertiary structure contained alpha helix, extended strand, beta turn and random coil. The results of Lactobacillin provided an important reference for further identification.3. With the study of the influence of bacteriocin Lactobacillin produced by Lactobacillus acidophilus on the shelf life of fresh milk, the hazard analysis critical control point(HACCP) system in the application of bacteriocin Lactobacillin in sterilized milk has been established. After the bacteriocin Lactobacillin was purified by anion exchanged chromatography and gel chromatography, the protein concentration of Lactobacillin was 0.019 mg/mL, so 50 ?g/mL bacteriocin Lactobacillin was added to fresh milk and sterilized milk to analyzed the antibacterial activity, acidity, sensory characteristics at 4 ?. The experimental group adding Lactobacillin was compared with the control group without adding Lactobacillin. The result indicated that when the fresh milk adding Lactobacillin was stored 4 days, the number of bacteria was 4.5×106 cfu/mL, the acidity was 20 0T, the fresh milk was solidified by boiling, the colour of the fresh milk was cyan, it began to spoilage, however the fresh milk without adding Lactobacillin was stored 2 days, the indexs was the same with the above; when sterilized milk adding Lactobacillin was stored 40 days, the number of bacteria was 2.3×105 cfu/mL, the acidity was 25 0T, the fresh milk precipitated by boiling, the colour of the fresh milk was too light, it appeared sour smell, but the sterilized milk without adding Lactobacillin was stored 34~36 d, the indexs was similar to the above. So the shelf life of fresh milk was extended 2 d, and the shelf life of sterilized milk was extended 2~4 d at 4 while maintaining the original flavor, color, texture and other sensory characteristics of products. To ?ensure the safety of the product, all hazard factors of sterilized milk were analysed which would possibly affect the product quality in processing, the critical control points were set up, a pattern of preventive measures and control methods were formulated.