Super-Concentrated Incubation And Biological Changes Of Lactobacillus Bulgaricus

Lactobacillus bulgaricus (L.b) is widely adopted in food fermentation, the production of lactic acid in industry, animal feeds and the field of medical care. The preparation of lactic acid bacteria is depended on the technique of super-concentrated incubation.There are some methods of super-concentrated incubation, inculding method of neutralization,method of buffer salt method,method of adsorption,method of dialysis,continuous incubation,bioreactor of membrane. In these methods,the bioreactor of membrane is a new, high performance method developed in these years, but the application is confined because of expensive equiments and being complicated to make. If we want to obtain the lactic acid bacteria with high density and activity, the bacteria must be cultured in suitable culture conditions, adopted suitable media and methods. If the changes of biochemical metabolism of all kinds of materials and the ultramicrostructure can be mastered, the real super-concentrated incubation of L.b can become ture.The condition of L.b for super-concentrated incubation was achieved by single factor experiment (temperature, the initial pH and the methods of culture) ,and the results were :41℃, the initial pH 6.6, few oxygen.The main nutritive materials (glucose, peptone, power beef, power of yeast) , salts (diammonium citrate, dibasic potassium phosphate, sodium acetate , MgSO₄·7H₂O, MgSO₄·7H₂O) and growth factors (glutamic acid, mash, the juice of tomato) in MRS culture of L.b for super-concentrated incubation were respectively optimized by orthogonal experiments. And the results were (in 1 000mL medium): peptone 15g, powder of beef 10g, powder of yeast 7.5g, glucose 30g, diammonium citrate 2.25g, dibasic potassium phosphate I.5g, MgSO₄·7H₂O 0.48g , MnSO₄·4H₂O 0.35g, sodium acetate 6g ,glumatic acid 0.5g, mash 80mL, the juice of tomato 50mL, tween -80 1mL , pH 6.6 , water 1 000mL, sterilizing at 121℃ for 15min. The method of micro-filtration membrane and the second culture were suitful for super-concentrated incubation of L.b at 22h, and the glucose and peptone in medium for the second culture was optimized.The maximal density of bacteria was 1.001 × 10¹⁰ cfu/mL at 28h. The changes of amino acid, gluose, protein, triglyceride, cholesterol, lactic acid, LDH in culture and LDH, lypase, amino acid in cells were detected by kits respectively. And these indexes had the relations with the growth of L.b, the micro-filtration membrane and the second culture. The changes of figures and contents of L.b were systematically observed by negative staining and ultramicrotomy TEM at the different time (14h, 22h, 28h, 32h in culture) during super-concentrated incubation.The apoptosis of L.b were detected by In situ cell death detection kit, fluorescent at the different time (14h, 22h, 28h ,32h in culture) during super-concentrated incubation.They were closely related to the growth of L.b, energy changes and the regularities of lactic acid making. The cell wall, contents and apoptosis of the cellshad little changes when the lactic acid content was lower, plentiful nutritive materials and low cell density during early logarithmic growth phase; The cell walls were severely crimple, dissolvation, the shape and quantity of the contents of L.b had secerely changes and the apoptosis of bacteria were more and serious when the lactic acid content was higher during decline phase.The super-concentrated incubation of L.b has being achieved in this experiment.The regularities of biochemistry metabolism of L.b in super-concentrated incubation are the same as the fundamental biochemistry metabolism.They all decompose glucose, protein and triglyceride and form the bacteria, generate metabolic waste.The changes of ultramicrostructure of L.b has closely relation to the growth of L.b ,the lactic acid making and the energy metabolism. The apoptosis of L.b probably exist during super-concentrated incubation.