The Pilot Fermentation Strategies For Agaro-oligosaccharides Production By The Deep Sea Bacterium Flammeovirga Pacifica WPAGA1 And The Characterizations Of Related Enzymes

Alga oligosaccharides are the degradation products of algal polysaccharides?agar,algin,carrageenan,etc.?,mainly including agaro-oligosaccharides?AOS?,alginate oligosaccharides and carrageenan oligosaccharides.AOS are the hydrolysates of agar,which generally refer to oligosaccharides with the degree of 2²0.Compared with agar,AOS have many biological activities,such as antioxidation,anti-inflammatory,antitumor,intestinal probiotic proliferation,whitening and moisturizing.AOS have attractive prospects in the fields of medicine,health food,cosmetics,agriculture and so on.At present,AOS are mainly prepared from agar?polysaccharides?by chemical/biological degradation.The chemical method needs to consume a large amount of acid and alkali,thus not only causing environmental pollution and high cost,but also uneasy to control the production conditions.The molecular weight of AOS is uneven and chemical substances remain.The biological method has the advantages of stable molecular weight,low pollution and low cost,but the efficiency is low and the industrial production has not been realized.Therefore,it is significant to develop an efficient and environment-friendly production technology of AOS from Gracilaria lemaneiformis.A new species of bacterium Flammeovirga pacifica WPAGA1 was isolated from deep sea sediments in the western Pacific Ocean.The strain WPAGA1 has the ability to produce AOS using Gracilaria lemaneiformis powder as the sole carbon source.In the previous work,the optimal conditions of AOS production by the strain WPAGA1have been established in shaking flask.The preparation method of“one-step method”for AOS production was established for the first time.A variety of biological activities for AOS have been verified.However,there are some problems in the large-scale preparation,such as the expanding culture of strains and the preparation conditions with high viscosity.Therefore,the production methods must be optimized in the large-scale preparation.In this paper,based on the previous work,the expanded culture conditions of the strain WPAGA1 were further optimized.The optimization and control strategies of pilot-scale fermentation were built to solve the effect of viscosity on AOS production.The related enzyme genes and transcription levels in the process of AOS production from Gracilaria lemaneiformis by the strain WPAGA1 were analyzed at the genomic and transcriptome levels.The conditions for recombinant agarase Aga0950 were optimized,and the high activity agarase was obtained.The preservation effect of AOS was studied.The main results of this work are summarized as follows:?1?In order to solve the problem of difficult transfer culture,the expanded culture conditions and growth kinetics model of the strain WPAGA1 were established.The optimal medium of seeds in 10 L and 200 L fermenters was agar 1 g/L,yeast powder 1 g/L,peptone 5 g/L,NaCl 20 g/L.The culture conditions of seeds 10 L were temperature 32?,inoculum volume 3%?v/v?,agitation speed 150 r/min and aeration ratio 0.5 vvm;and the culture conditions in 200 L fermenter were temperature 32?,inoculum volume 3%?v/v?,agitation speed 100 r/min and aeration ratio 0.25 vvm.Logistic equation fitted the growth kinetics model of the strain WPAGA1 well.Under the optimized conditions,the yield and productivity of AOS reached the maximum at the later logarithmic stage?14¹6 h?,which were 1.52 g/L and 0.0362 g/L/h,respectively.?2?The fermentative system belonged to high viscosity fermentation using Gracilaria lemaneiformis as substrates,and viscosity was an important factor affecting AOS production.The factors affecting AOS yield and viscosity of fermentative system were studied.The optimal control strategies of for AOS production by the strain WPAGA1 in 200 L fermenter were put forward as follows:Gracilaria lemaneiformis powder concentration 20 g/L,C:N 5:1?peptone as nitrogen source?,NaCl 20 g/L and inoculum volume 3%?v/v?.The fermentation conditions were controlled at 37?,150 r/min and 0.375 vvm of aeration ratio.Under the above control conditions,the yield of AOS reached 1.69 g/L after 42 h fermentation,which was 45.7%higher than that before optimization.Using the principle of equal power consumption per unit volume of fermentation broth,the fermentation process of AOS production by strain WPAGA1 was established in 1 m³ fermenter.The yield of AOS reached 96.4%of that in 200 L fermenter and well reduced the effect of viscosity on fermentation performance.?3?Exploring the related enzymes in the process of AOS production by the strain WPAGA1 is beneficial to fermentation regulation.The whole genome sequencing showed that the strain WPAGA1 contained abundant genes related to polysaccharide degradation,especially 13 agarase genes.Transcriptomics analysis showed that the transcription levels of polysaccharide metabolism and galactose metabolism related genes were up-regulated using Gracilaria lemaneiformis as substrates.Among them,8 agarase genes were significantly up-regulated,and agarase gene aga0950 was up-regulated by 9.47 folds than that of control group.The high activity agarase Aga0950 was obtained by heterologous expression of aga0950 gene.The AOS yield is 1.5 g/L by Aga0950 hydrolyzed of 20 g/L Gracilaria lemaneiformis dry powder for3 h,and the end-products were neoagarotetraose and neoagarohexaose.The prediction of secondary structure and tertiary structure showed that the enzyme had a high proportion of?helix,which could improve the rigid structure had better thermal stability.The enzymatic properties showed that the optimum temperature of the enzyme was 50?and the enzyme had good thermal stability,which was consistent with the predicted results of enzyme structure.The optimum pH range of Aga0950was 4.0 to 10.0.Activities of Aga0950 were enhanced by Co²⁺,Mn²⁺and Fe³⁺,while were strongly inhibited by Cu²⁺.?4?In order to increase the expression of recombinant agarase Aga0950,the high-efficiency expression strategies were developed in a 7.0 L fermenter.In the pre-induction phase,exponential feeding strategy of glycerol with different specific growth rates???was applied.The results showed that the specific growth rate 0.2,was beneficial to cell growth and agarase expression.In the post-induction phase,continuous lactose feeding strategy?lactose of 1.0 g/?L·h??was used instead of IPTG induction.The activity of Aga0950 reached 115.3 U/mL after 17.5 h's induction,which was 4.97 times higher than that before optimization.?5?The activities of AOS produced by strain WPAGA1 and agarase Aga0950,were compared in regard to the fresh-keeping performance of litchi fruits.The results showed that the sound fruit rates of the two kinds of AOS were 57.65%and 62.85%respectively at 7 d storage,which was 26%higher than that of the control group.Compared with soluble solids,titratable acid,vitamin C,polyphenol oxidase and peroxidase activity,the results showed that the AOS groups were better than the control group.According to the sound fruit rate of 100%,shelf life was prolonged 24h than that of control group.