Study On Cultivation And Cryoprotectant Of Bifidobacterium Bifidum

Bifidobacterium is a kind of physiological bacteria which is benefit to thebody,it now widely used in functional foods. While the probiotic powder mainlyimported from abroad now, its high price and dependence on foreign countriesgreatly restricted the development of probiotic products in our country. Thestudy optimized the medium, feeding mode, anti-freezing factors andcryoprotectants on Bifidobacterium bifidum BB01, obtained medium of highdensity, cryoprotectants of high survival rate and the the powder of high activity.The effect of carbohydrate and prebiotic on growth of B. bifidum wasstudied by single-factor test and Plackett-Burman(PB)test. The results showedthat lactose, glucose, oligofructose, xylo-oligosaccharides, galacto-oligosaccharide and inulin could promote the growth of Bifidobacterium bifidum.Among them, lactose, oligofructose and inulin were main factors. Then throughthe climb test and Central Composite Design (CCD) response surface analysis,we got the compound carbon source and prebiotics: lactose1.6%, inulin0.26%,oligofructose0.22%. The optimized medium, in which the number of viable cellsreached (2.17±0.06)×10⁹cfu/mL.On the base of optimized carbon source and prebiotics, we used PB designto study peptone, beef extract, yeast extract and casein hydrolyzate, derived thatthe first three organic nitrogen were main factors. Then by climb test and CCDresponse surface analysis, we got the nitrogen source composition: peptone0.9%,yeast extract0.3%, beef extract0.7%. The number of viable cells in optimizedmedium was (2.49±0.06)×10⁹cfu/mL, which was65.46%higher than MRS(lactose) medium.The experiment also researched in the effect of feeding time and neutralizeron growth of B. bifidum, discovered that adding1mol/L NaOH after13hincubation could prolong the logarithmic phase and increase the number ofviable cells of B. bifidum in the culture medium significantly. The viable countof bacteria reached (3.52±0.046)×10⁹cfu/mL at20h in maximum, which was29.26%higher than the maximum at18h in the control group. The effect of anti-freeze factors including NaCl, sorbitol, mannitol,glutamic acid, sodium glutamate and betaine on growth of feeeze-drying ofB. bifidum was studied by single-factor test, PB design and Box-Behnken test.Thougu the experiments, we got the anti-freeze factors: NaCl0.74%, sorbitol0.08%, glutamic acid0.02%. The survival rate reached75.2±0.4%in thiscondition, which was32.65%higher than the control group.On the base of optimized medium, response surface method was used inB. bifidum freeze-drying cryoprotectant optimization to get the prebioticcryoprotectant as follows: XOS7%, inulin13%, stachyose11%, the survival ratewas88.7±1.4%, which was15.33%higher than the control group; and the bestcombination of amino acids and salts protective agent: glycine5.5%, arginine4.5%, sodium bicarbonate0.8%, ascorbic acid2.3%, the survival rate was87.8±2.3%, which was15.83%higher than the control group. Because of theadding of cryoprotectants, the solid content increased and then the powder outputcorresponding increased.On the base of above experiments, anti-freeze factors were taken in themedium and seven kinds of cryoprotectants and skim milk were inspected again.Finally obtained the cryoprotectant combination as: glycine5.5%, sodiumbicarbonate0.8%, XOS7%, arginine4.5%, skim milk25%. In this condition,the survival rate was90.37±1.9%, the number of viable cells per unit powderwas (2.78±0.13)×10¹¹cfu/g, and the powder output was6.67g/L.The optimized medium could increase the number of viable cells in thebroth and prolong the growth cycle effectively; with the use of anti-freeze factorsand cryoprotectants, not only reduced the cost of the freeze-dried production,improved output of powder, but also raised survival rate and viable count perunit, which laid the technical foundation for further industrial production.