| Nanx wull is a kind of microbial fermented meat products more than two thousand years. The raw material is fresh pork with rice noodles, salt and other spices in anaerobic fermentation by microorganism under the natural condition. At present it mainly popular in China’s Chongqing, Sichuan, Guizhou, Guangxi, Hunan and other places. It is a kind of living carnivorous product for Dong, Miao, Dai and Maonan minority, Fermented sour meat contains abundant food culture connotation and wide range of edible. In this paper, a home-made fermented sour meat is choosed as raw material to select the lactic acid bacteria that can lower cholesterol. The stains were tested through the physiological and biochemical and molecular biological methods. The stains’ physiological activity was tested in a vitro simulation digestion environment to find out the mechanism of lowering cholesterol. The work of this paper is to provide the basic research data function for industrial production of fermented meat, and to provide the original strain and basic research support for the research and application of cholesterol-reducing lactic acid bacteria.1. Screening and identification of lowering cholesterol lactic acid bacteria1.1Screening of lowering cholesterol lactic acid bacteriaThe lactic acid bacteria were isolated from the calcium-MRS culture medium by plate streaking of separation and purification methods with the homemade fermented sour meat as raw materials.18lowering cholesterol lactic acid bacteria strains were selected from the egg yolk liquid. Among them, the rate of degradation of cholesterol of the SRI and SR10are more than30%, accounting for11.11%of the proportion of the total. There is only one strain’s cholesterol lowering rate below10%, accounting for5.57%of the proportion of the total. The cholesterol lowering rates of the rest strains are in10%～30%. The two highest and lowest cholesterol lowering rates respectively of SR10and SRI8are32.78%and7.17%. The results show that the lactic acid bacteria grown in the fermented meat have the ability to reduce cholesterol, but the cholesterol-lowering ability of different strains are vairous.After the first screening,8strains were selected:SRI, SR3, SR4, SR7, SR9, SR10, SR12and SRI7which have high lowering cholesterol ability to screen again, and then measured the cholesterol lowering rate are more than20%. Among the8strains, there are four best strains:SR10, SR12, SRI and SR7, which have high lowering cholesterol rate. The cholesterol source of them are egg yolk. The other four high cholesterol-lowering ability stains:SR10, SR1,SR4and SR12used the cholesterol micelles as the source. There are protein and lecithin in the egg yolk that may affect the strains’cholesterol-lowering effect. Combine with the preliminary screening and secondary screening results, the SR10and SRI in cholesterol micelles with high cholesterol-lowering ability, were choosed as the experimental strains in the following research. The cholesterol-lowering rates are respectively35.73%and31.89%1.2The identification of lowering cholesterol lactic acid bacteriaThe SRI and SR10with high cholesterol-lowering ability are identified and identification results show that the screening strains grown well, the colony is round, opaque, milky white, and with neat edge,wetting smooth surface and central bulge. Observation under oil microscope, the cells presented rod-shaped, show purple(G+) after gram staining. The results of peroxidase test, indole test and sugar alcohol test were compared with the "Berger’s Bacterial Identification Manual", and combined with morphological observation results. We preliminary choose the SRI and SR10strains as the Lactobacillus.The gene fragments1476bp and1480bp respectively of the SR1and SR10are gained through the16SrRNA molecular biology method. The operation steps including extraction, amplification and sequencing. Sequencing results are compared with the GenBank gene pool. Compared with the gene sequence of the digestion lactobacillus16SrRNA in the gene pool, the homology is99%. Considerate the morphological and physiological and biochemical identification results, the SRI and SR10are both digested lactobacillus.2Study on physiological characteristics of the cholesterol-lowering lactic acid bacteria in the fermented sour meat2.1Study on biological properties of the screening cholesterol-lowering lactic acid bacteriaThe test results show that the optima pH for SRI and SR10growth is6.0-7.0, the worst growth PH for SRI is3.4, the highest growth PH is10.2. The lowest growth PH for SR10is2.8, the highest growth PH is10.2. the optimum growth temperature for SRI and SR10both were30℃. The most suitable for growth temperature and Ph are in accord with other most acid bacteria.From the growth curve of SRI and SR10, SRI stain will enter in the logarithmic stage from the growth stage about4hours, and then transit in stable phase about10hours. The SR10strains need6hours to in the logarithmic phase from the growth stage and12hours culture to the stable period. The rate of acid production and growth density of the two stains are positive correlation. The pH value is reduced during the culture process, especially in the transition stage of the growth stage to the logarithmic phase. When the bacteria enter the stable period, the pH value maintain relatively, just only slightly decreased. The lowest pH value of the growth curve of SRI and SR2are both about4.2.2Study on Simulation results of digestive tolerance of lactic acid bacteriaAccording to the acid resistance test, the survival rates of SRI are30.11%,38.55%and45,61%respectively culture6hours in condition of pH2.0,2.5and3.0. In same conditions, the survival rates of SR10are38.02%,48.53%and58.47%. It demonstrates that the SRI and SR10both have strong acid resistant ability and the SR10have stronger acid resistant ability than SR1. The screening strains have strong survival rate under acidic conditions.According to the bile salt resistance experiment, the SRI and SRI strains can grow normally in long time in the0.1%and0.2%bile salt conditions after8hours cultivation. The growth of SRI and SR10are inhibited in the0.3%bile salt condition, after8hours, the survival rate of SRI and SR10are43.01%and39.73%. It demonstrated that the screening strains have strong bile salt resistance. There are20kinds of antibiotic to do susceptibility test, put the antibiotics into the inoculation culture medium plate, and measure the maximum and minimum bacteriostatic ring diameter of SRI. They are respectively doxycycline with37.85mm diameter and kanamycin with7.85mm diameter. The maximum and minimum diameter of bacteriostatic ring of SR10are respectively benzyl penicillin with38mm diameter and gentamicin with7.44diameter. It demonstrated that antibiotics have different inhibition effect to screening strains, different antibiotics have various inhibition effect.3. Preliminary study on the cholesterol-lowering mechanism of screening lactic acid bacteria3.1Analysis of cholesterol ability of the screening strains change with timeAnalysis of assimilation and precipitation of the digestion Lactobacillus SR10, the assimilation of cholesterol lowering effect are respectively14.98%and20.89%after12hours and24hours, at the same moment, the precipitation of cholesterol lowering rates are respectively9.42%and12.80%. It demonstrates that the assimilation and precipitation are worked together during the bacterial growth, and the assimilation of cholesterol lowering effect is more obvious than the precipitation of cholesterol-lowering effect. The experiments in this paper confirm that assimilation and precipitation are both contribution to the bacteria cholesterol-lowering effect, and assimilation plays a main role in the whole process.3.2Study on the enzyme source and enzymatic activity of the cholesterol-lowering lactic acid bacteriaExtracellular and intracellular enzyme activity and protein content were measured by purifying the crude enzyme liquid with70%saturation of ammonium sulphate solution through TritonX-100. The enzymatic activity, protein content and specific activity of the extracellular and intracellular fluid are respectively176.2U/ml and305.3U/ml,2.45mg/ml and0.49mg/ml and70.5and611.8. The enzymatic activity of intracellular fluid is1.7times of the extracellular crude enzyme solution. The extracellular fluid protein concentration is5times of intracellular crude enzyme solution. The intracellular enzyme specific activity is8.7times of the extracellular enzyme activity. It demonstrated that the cholesterol-lowering effect enzymes are mainly from intracellular, and the intracellular enzymes play a major role in the cholesterol-lowering process. |
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