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INTRODUCTION:

Lactic acid is produced by humans, animals, plants and microorganisms . Lactic acid molecule has two optical active isomers, D(-) and L(+) forms [3]. Lactic acid is an organic acid with a wide variety of industrial applications.  The most important application as a preservative and acidulant in foods [3], as a prosthetic device, controlled delivery of drugs in pharmaceutical agents, as a precursor for production of polymers like polylactic acid [4] and as a moisture agents in cosmetics.

Optically pure lactic acid is important for the production of polylactide, because the physical properties of the polylactide are dependent on the stereochemistry of individual lactic acid molecule [5]. The L(+) form of lactic acid is used for food and drug industry, because human body is only adapted to assimilate this form and only produced L-Lactate dehydrogenase [5].    The most effective way for L-lactic acid syntheses is through biosynthesis rather than chemical processes [6, 7]. In fact, the only source for producing optically pure lactic acid isomers is microbial fermentation [2].

Certain types of microorganisms such as Lactobacillus spp. and Rhizopus spp. are capable of producing lactic acid in high concentrations. Rhizopus spp., especially R. oryzea, produce L(+)-lactic acid from glucose , starch and molasses, in the presence of CaCO3.

The objectives of this study were to screen several Lactobacilli and R. oryzae strains to determine optical active isomer producers of lactic acid in the fermentation broth and to provide a complete profile of substrate utilization by these micro-organisms.

Summary:

On the heterodoxy medical treatment, Tibet the person drinks,paint Tibet snow lotus and can cure many skin diseases after fermenting the liquid, try after drinks the most to feel the spirit,the appetite function become good, can cure the liver cancer.Ferment the milk and can repress the cause germ on the cultural heritage, promote a germs of bowel stabilized;Repress the tumor, reduce the creation of guiding the cancer thing;Activate the immune system, produce various cell hormones;Move to precipitate in addition to the cholesterol, reduce the cholesterol content in blood;Improve the lactose and don't bear the disease.The lactobacillus has stable germ inside the bowel mutually, regulating the host immunity, and the effect of the anti- tumor.

For confirming its effect reliability, make use of the liver cancer cell,the skin cell and the immunity cell as research objects, inquire in to ferment the liquid to influence to it respectively and the physiology function, also carry on the germ to analyze mutually besides, to understand it to contain the germ category do not, and compare it to sell the difference of the yoghurt with the city.

Find after the experiment the protein extracts the thing to repress the result to the liver cancer cell, and therewith the density enlarges the cell livability more low, the oil extracts the thing and protein to extract the skin cell influence for thing not obvious, both all repress the inflammation reaction result in the immunity cell.The germ analyzes the aspect mutually, the germ grows and ferments in the liquid, the quantity of the lactobacillus and yeast fungus all has no obvious variety, and the lactobacillus is the advantage germ of your.

The research motive:

The detection really reaches the skin condition improvement,the spirit condition to become the good effect, besides, can cure the liver cancer, but have no science due to being widely known it to ferment the liquid according to, experiment its influence to the liver cancer cell,the skin cell and the immunity cell.

The lactobacillus is omnipresent in the natural environment, lying in the food,livestock husbandry,agriculture,medicine and especially turning with the environmental protection etc. realm, all have the prominent position, general food special features fermented by lactobacillus is:Will be able to ferment the sugar to turn into the lactic acid and the organic acid, decline 2 low pHs and improve the taste, increase the nutrient, prolong the food expiry day and raise human body immunity function etc..

Ferment the milk article to be thought to have to the human body the advantage for long time biggest, for in order to repress the cause germ, the physiology in the body effect promote a germs of bowel stabilized, being the lactobacillus to become the advantage germs inside the bowel, will become the living creature film on the bowel wall cell, can expel the cause germ and other miscellaneous germ to stick to attaching in up.And the lactobacillus will produce in the metabolism process various suppress the germ material, it ferments the output organic acid of carbohydrate,such as the acetic acid and lactic acid,, the pH value that can reduce the environment, reduce to guide the cancer products to get, the lactobacillus can reduce the bowel inside harm the metabolism of the germ activity,break to guide the cancer thing formation,

reduce mutation source activity and repress the tumor cell growth;Activate the immune system, produce various cell hormones;Move to precipitate in addition to the cholesterol, reduce the cholesterol content in blood, the liver is small in the pH through the decomposition cholesterol and output salt in 6.0:00 because the deliquescence degree descends, and with the bowel way its cholesterol precipitates totally and ejects the body outside;Improve the lactose and don't bear the disease, ferment the β half lactose inside the lactobacillus in the milk germ body.After human body eating into 12 bowelses, because of looking like the most suitable function condition(37 ° Cs,pHs 7.0)and present to activate the appearance, can resolve lactose as the glucose and the half lactose.

Besides, the eligibility chooses the germ of having the particular medical treatment effect to grow, being used for mollify to have loose bowels or constipation of phenomenon, raise the bowel way's utilization for calcium ,iron, and defer the body aging,the anti- radiates, preventing and curing the function of the germ and the fungi vagina etc..In the livestock husbandry, the lactobacillus may have the living creature utilization and the nourishment value that the exaltation ferments the thick forage, the machine that reduces the night soil bad smell and fertilizer turns, raising the potentials, such as the forage taste eating and the prevention of the disease...etc., the lactobacillus has already had the initial result in the research of the forage.

The research result of the lactobacillus:

After the lactobacillus exploitation carbohydrate carries on fermenting, in addition to born lactic acid, some germ grows to also produce much carbohydrate with many high mark sons, the part secretes to the germ body the outside, becoming the  film or the sticky quality thing that becomes to swim to leave, so have the afterbirth outside many carbohydrate(exopolysaccharide it calls.

These polymer in ferment in the milk, can increase the sticky degree of the product, reduce the phenomenon , then protect the curd structure, to resist the machine in the manufacturing process injury, in addition to to help of the product quality, these have another the carbohydrate to have already been confirmed to have as well the living creature activity, can by strengthens the immunity of the host to defend the function, with attain the anti-cancer purpose.

After the Metchnikoff of 1908 put forward the edible lactobacillus(Lactobacilli) germ body, can prolong the theories of the life, the relevant benefit gets the physiology effect and nourishment of the germ is inquired into extensively namely.Be close to several decades of research in, find continuously the benefit gets the germ to have stable germ inside the bowel mutually, regulating the host immunity, and the effect of the anti- tumor.

These effects find in the cell out of the body experiment with huge bite the cell stub(RAW 264.7) to carry on activating to experiment with the assistance a T cell and the Bifidobacterium, find all Bifidobacterium all can activate effectively huge bite the cell to secrete the TNF-α and IL-6, and is more high to activate the result with the germ stub density more obvious.There is another scholar pointing out L.acidophilus the LA1 can stimulate effectively huge bite the cell RAW 264.7, the creation cell hormone.

In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum during Pressure Treatment

ABSTRACT:

Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions.

Milk buffer:

The milk buffer was chosen to contain the same amounts of minerals and lactose as whey from rennet casein; the buffer contained the following compounds (in grams per liter): KCl, 1.10; MgSO4·7H2O, 0.71; Na2HPO4·2H2O, 1.87; CaSO4·2H2O, 1.00; CaCl2·2H2O, 0.99; citric acid, 2.00; and lactose, 52.00. The pH was adjusted to 6.5, 6.0, 5.0, and 4.0 with KOH (1 M).

Bacterial strain and culture conditions:

Lactococcus lactis subsp. cremoris MG1363 was grown at 30°C in M17 broth supplemented with 1% glucose (GM17 broth). Lactobacillus plantarum TMW1.460 was grown at 30°C in MRS medium.

Labeling of cells with cFSE for pH in determination:

The fluorescence method developed by Breeuwer et al. (8) was adapted to Lactococcus lactis subsp. cremoris MG1363 and Lactobacillus plantarum. Harvested cells were washed and resuspended in 50 mM HEPES buffer, pH 8.0. Subsequently, the cells were incubated for 15 min at 30°C in the presence of 10.0 µM cFDASE, washed, and resuspended in 50 mM potassium phosphate buffer, pH 7.0. To eliminate nonconjugated cFSE, glucose (final concentration, 10 mM) was added and the cells were incubated for an additional 30 min at 30°C. The cells were then washed twice, resuspended in corresponding milk buffer, and placed on ice until required.

Effect of the buffer pH (pHex) on the pH in:

The pHin of energized cells of Lactococcus lactis and Lactobacillus plantarum were measured in HEPES, MES, and citrate buffer with pHex values ranging from 4.0 to 8.0 at atmospheric pressure (Fig. 1). Both lactic acid bacteria decreased their pHin values as a function of the pHex. The observed values of pHin of Lactobacillus plantarum were slightly lower than the corresponding values of Lactococcus lactis. Both organisms maintained an pHin between values of 6.3 and 8.0 at a buffer pHs of 4.0 and 8.0, respectively. Consequently, under acidic conditions (pHex < 5) the pH across the cell membrane was maintained at around 2.0 whereas under alkaline conditions it was dissipated.

FIG. 1. pHin of Lactococcus lactis subsp. cremoris MG1363 cells (•) and Lactobacillus plantarum TMW1.460 cells ( ) in the presence of 10 mM glucose at different values of pHex. The assays were performed at 30°C and 0.1 MPa pressure. The buffers were prepared from citric acid (50 mM), MES (50 mM), and HEPES (50 mM). The pH was adjusted with either NaOH or HCl. Symbols represent means ± standard deviations of results from two independent experiments.

FIG. 2. Calibration of the assay for pHin determination using Lactococcus lactis at pHs of 8.0, 7.0, and 4.0 at a pressure of 300 MPa. To equilibrate pHex and pHin values, 1 µM concentrations (each) of valinomycin and nigericin were added prior to treatment. Shown is the ratio of fluorescence intensities measured at 485 and 410 nm. The shaded area indicates the pressure ramp up time (90 s from 0.1 to 300 MPa). The pH values indicated on the left axis indicate measured pH values at ambient pressure; the pH values indicated on the right axis indicate calculated pH values at 300 MPa.

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C18 SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C18 column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml-1 for cyclo(L-Phe-L-Pro) and 7.5 mg ml-1 for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.

FIG. 3. Antifungal compounds isolated from supernatant of L. plantarum MiLAB 393 grown in MRS broth.

Lactobacillus plantarum WCFS1

DNA walk

Cumulative TA skew analysis

Cumulative GC skew analysis

A = 917'537

C = 737'079

G = 734'000

T = 919'658

ACGT = 3'308'274