Preparation Process By Pectinase Treatment And Application In Fermentation To Produce Vitamin B₁₂ Of Citrus Molasses

Citrus processing waste is a by-product of citrus processing,mainly with peel,pulp and seeds.There is about 5%⁸% sugar in fresh peel and 8.8%¹1% sugar in pulp,which can be made a high-sugar dark brown viscous liquid,called citrus molasses,after pressed and evaporated.Citrus molasses,with a large mount of monosaccharides,disaccharides,little polysaccharides and flavonoids,is a good source of fermented carbon.Based on this,this paper studied the enzymatic preparation of citrus molasses,the separation and extraction of sugar from citrus molasses and the application in fermentation of Vitamin B₁₂(VB₁₂).The contents and conclusions are as follows:?1?Preparation technology of pectinase treatment was used to clarify peel press liquor and pulp wash liquor with a large amount of insoluble substances,such as cell membrane fragments,from citrus processing waste.Taking light transmittance as index,using single factor experiments and L9?34?orthogonal experiments to opmitize the pectinase treatment conditions.The results showed that optimum conditions of pectinase treatment in peel press liquor were amount of pectinase 0.125 U/m L,pH value 4.0,reaction time 2.5 h and temperature 40?,with transmittance increased from 14.6% to 75.1%.The optimum conditions of pectinase treatment in pulp wash liquor were amount of pectinase 0.100 U/m L,pH value 4.0,reaction time 1.5 h and temperature 35?,with transmittance increased from 46.0% to 83.8%.?2?Rotary evaporation was used to liquor concentration and concentration curve was darwm with concentration time as abscissa and sugar concentration?soluble solid content?as ordinate,using the independent sample t-test to analyze the significance of concentration speed between liquor from enzyme treated and non-enzyme treated.The results showed that when concentration time was 100 min,the soluble solid content of peel press liquor were significant difference under the conditions of temperature 70±5?,rotation speed 80 r/min and vacuum degree?0.095±0.005 MPa.When concentration time was 50 min,the soluble solid content of pulp wash liquor were significant difference and increased with concentration time going on,under the conditions of temperature 50±2?,rotation speed 182 r/min and vacuum degree?0.098±0.002 MPa.?3?Crude polysaccharide extraction was investigated with respect to conditions of reflux degreasing and ethanol precipitation by response surface optimization.The results showed the highest crude polysaccharide extraction was 5.47%,under the optimum reflux degreasing conditions of degreaser dosage 20 times?V/V?,petroleum ether content 90%,reflux time 3.5 h and ethanol precipitation conditions of ethanol content 99%,ethanol dosage 8 times?V/V?,precipitating time 8 h.?4?Sugars of four samples,including enzyme treated pulp/peel molasses,non-enzyme treated pulp/peel molasses,were determined by DNS method and anthrone reagent method with extraction and isolution by crude polysaccharide extraction.The results showed that the sugar content of citrus molasses was mainly reduced sugar,sucrose,and little polysaccharides.The purity of citrus molasses made by pectin enzymatic method was increased.The content of reducing sugar was 32.21% in enzyme treated peel molasses,which accounted for more than 85% of its total sugar.The content of reducing sugar in enzyme treated pulp molasses was 22.19%,which was about 57% of its total sugar.?5?Enzyme treated peel molasses was use to produce Vitamin B₁₂(VB₁₂)with Propionibacterium shermanii as fermenting microbe.Taking VB₁₂ yield as index,under 30?,single factor experiments,Placket-burman experiments and response surface optimization were used to optimize fermentation medium composition and process technology.The results showed that the optimum composition of the fermentation medium was citrus molasses 22 °Brix,yeast extract 92 g/L,CoCl₂·6H₂O 40 mg/L,KH₂PO₄ 1.0 g/L,?NH₄?₂PO₄ 2.0 g/L,initial pH 6.9,DMB addition amount 50 mg/L.The influence order of each component on yield of VB₁₂ was initial pH>citrus molasses > yeast extract >?NH₄?₂PO₄ > CoCl₂·6H₂O > KH₂PO₄ > DMB addition amount.Under the optimum fermentation medium,fermentation conditions were optimized as inoculation amount 20%,volume of liquid 90 mL in 250 mL flask,DMB addition time 74 h and fermentation time 96 h,with VB₁₂ yield 28.0339 mg/L.