Study On Activity Of Probiotics And Harmful Bacteria In Vitro And Molecular Modification Of Black Rice Anthocyanin

This paper mainly studied on extraction, purification, effects on the activity of intestinal probiotics and harmful bacteria in vitro and molecular modification of anthocyanins from black rice. First, crude anthocyanins were extracted from black rice by acidic hot water. Ultrafiltration membranes were used to remove the macromolecules in the crude anthocyanins, such as proteins, polysaccharides and starch. Then, the anthocyanins were further purified and concentrated by macroporous resin. The proliferative effect on probiotics and the inhibiting effect on harmful bacteria in vitro of the black rice anthocyanins were also studied. Anthocyanins are found to be unstable under the natural conditions due to the effect of pH, temperature, light and other factors. Therefore, molecular modification of the purified anthocyanins was adopted to improve the stability. The main contents and results are as follows:1. Study on the optimal extraction conditions of black rice anthocyanins. Firstly, the effects of several main factors (solid-liquid ratio, extraction temperature, pH and extraction time) on the extraction yield of black rice anthocyanins were determined through single-factor experiments. On the basis of that, orthogonal experiments (L9(34)) were applied to optimize the extraction condition. The optimal extraction conditions were solid-liquid 1:18, extraction temperature 50℃, pH2.2, extraction time 2.5h.Under the optimal condition, the highest extraction yield of anthocyanins was 4.32‰.2. Study on purification technology of black rice anthocyanins. Firstly, membrane separation method was used for anthocyanins purification preliminarily to eliminate most of proteins, polysaccharide, starch, etc. Then, the resulting permeate was subjected to macroporous adsorption resins (AB-8, X-5, HP-20, DM-301 and D101) to further purify the anthocyanins in terms of adsorptivity and desorptivity. The optimal operation conditions were resin kind of X-5, adsorption equilibrium time of 3h, pH value of 3, the temperature of 30℃, the adsorption capacity of 1.5 times the volume of resin, flow rate of 2.0mL/min, and the optimal eluent of 80% ethanol. The color value of purified anthocyanin was 66.1, and the product was dark purple powder. The UV-vis spectroscopy analysis showed that the maximum absorption peak was centered at 515nm. The HPLC analysis shows that the purified anthocyanin has four components, and the main component is cyanidin.3. Study on the proliferative effect of the black rice anthocyanins on probiotics. Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum and Lactobacillus acidophilus were selected as the model probiotics in the present work. The bacterial number and pH of the cultivation media were determined for evaluating the effect of proliferation. The results showed that anthocyanins can stimulate the proliferation of Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum and Lactobacillus acidophilus significantly. For Bifidobacterium adolescentis and Bifidobacterium infantis, the growth-promoting effects of anthocyanins were the best when treated with 0.5%anthocyanins. Whereas,1.5% and 1.0% were the optimal concentrations for proliferation of Bifidobacterium bifidumand lactobacilli, respectively. Furthermore, the pH values of the media decreased during the cultivation with certain amount of anthocyanins (≤1.5%). The results indicated that the probiotics can utilize the anthocyanins and produce phenolic acids. These results indicated that the anthocyanins can stimulate the proliferation of probiotics and have potential to be exploited as novel prebiotics.4. Study on the inhibitory effect of the black rice anthocyanins on harmful bacteria. Antibacterial activity of black rice anthocyanins experiment was conducted using filter paper method. The results showed that black rice anthocyanins had certain inhibitory effects on human harmful bacteria of S.aureus and E.coli, the minimum inhibitory concentrations were 1.Omg/mL and 0.5mg/mL, respectively.5. Study on the molecular modification of black rice anthocyanins. Anthocyanins reacted with oxalic acid in tri-sodium citrate and NaH2PO4 buffer solution (pH 3). The structure of modified product was determined by UV and IR spectroscopy. Single-factor experiments in terms of the effects of main factors on the esterification yield of black rice anthocyanins and were orthogonal experiments (L9(34)) were designed to optimize the esterification condition, investigated through single-factor experiments. On the basis of that, The optimal esterification conditions were mass ratio of oxalic acid and anthocyanins of 9:1, liquid-solid ratio of 20:1, esterification temperature of 65℃, reaction time of 3 h. Under these conditions, the resulting esterification yield was 40.4%. IR analysis showed that a new ester functional group was found in the structer of the modificated anthocyanins without destroying the original structure.6. Stabilities of black rice anthocyanins and esterified anthocyanins were studied. Results showed that the stability of esterified anthocyanins had been greatly improved. Compared with the un-esterified anthocyanins, the stability of esterified anthocyanins was improved significantly when subjected to high temperatures, light and high pH.