Breeding And Application Of High-yielding Non-starch Polysaccharide Enzyme Strains

Rationales:Non-starch polysaccharides(NSP)are a general term for all polysaccharide carbohydrates in plants except starch,which are difficult to digest and absorb by monogastric animals such as pigs.With the development of new alternative feeds such as distillers’ lees and bran,NSP content in feed increases significantly,which seriously affects the digestion and absorption of feed nutrients,and may even cause functional damage or lesions of digestive organs,leading to metabolic disorders.At present,domestic and foreign countries tend to use enzyme preparations to solve this problem.Enzyme preparations are added to feed to degrade part of macromolecular substances,so as to improve the digestibility of pigs and alleviate digestive problems.However,the use of NSP enzyme preparations has not obtained a unified standard,and different feed raw materials need to reasonably formulate different enzyme preparations to achieve better application effects.In the process of developing the new type of alternative feed,the research group found that germinated feed can effectively secrete protease,amylase and other substances to promote the digestion and absorption rate of pigs,but the degradation ability of NSP is minimal,and there are relatively few studies in this regard.It is necessary to prepare NSP complex enzyme scientifically to provide an effective NSP complex enzyme preparation for germinating feed.Objective:1.Screening strains that can efficiently produce non-starch polysaccharide enzymes;2.Scientific preparation of NSP enzyme complexes for germinating feeds.Methods:1.The germinated feed was processed by the biomimetic digestive system of monogastric animal.The residue was collected and used as the substrate to cultivate the microbial flora in a suitable environment.The microbial flora was isolated and purified,and then selectively cultured to screen out the strains with better degradation effect of nonstarch polysaccharide.The strains were identified by microscopic observation and molecular identification.2.The optimal addition amount,enzymolysis temperature and enzymolysis time of non-starch polysaccharides were determined by single-factor text and response surface design.The obtained enzyme preparations were compared with commercially available enzyme preparations added to feeds separately,and the effectiveness of the enzyme preparations was determined by measuring the change in dry matter digestibility and energy change of the feeds in vitro.3.Single factor experiment and response surface design were used to determine the optimal addition amount,enzymolysis temperature and enzymolysis time of each nonstarch polysaccharide enzyme in complex enzymes.The obtained enzyme preparation and commercially available enzyme preparation were added to the feed to determine the effectiveness of the enzyme preparation by measuring the changes of dry matter digestibility and energy in vitro.Results:1.The bacterial community is selectively cultured,and the best cellulase and pectinase producing strain is Penicillium strain ZT-7.The strain with high β-glucanase production capacity is Aspergillus ZT-5.ZT-10 of Penicillium is the best xylanase producing strain.The strain with high mannanase production capacity is Aspergillus ZT-12.2.The optimal culture conditions for cellulase producing strain ZT-7 is determined as follows: bran 3.0%,peptone 3.0%,p H 8.0,temperature 29.0℃,inoculum 3.0%,incubation time 5.3 d;The highest enzyme activity is 76.42 U/m L under these conditions.The optimal culture conditions for pectinase producing strain ZT-7 is: mixed feed 3.5%,yeast extract 3.0%,p H 8.0,temperature 29.6℃,inoculum 2.0%,incubation time 3.0 d.The highest enzyme activity is 174.53 U/m L under these conditions;the highest enzyme activity is 174.53 U/m L under these conditions,The optimal culture conditions for pectinase-producing strain ZT-7 is: 3.5% mixed feed,3.0% yeast extract,p H 8.0,29.6°C,2.0% inoculum,and 3.0 d.The highest enzyme activity is 174.53 U/m L under these conditions;the optimal culture conditions for β-glucan-producing strain ZT-5 is: 5.0%bran,3.0% peptone,p H 8.0,29.9°C,4.0% inoculum,and 4.0 d.The highest enzyme activity is 126.42 U/m L under these conditions.The optimal culture conditions for xylanase producing strain ZT-10 is: xylan 3.1%,ammonium sulfate 4.0%,p H 7.0,temperature 27.8°C,inoculum 2.3%,incubation time 3.0 d.The highest enzyme activity is584.12 U/m L under these conditions;the optimal culture conditions for mannanase producing strain ZT-12 is: konjac mannan 3.0%,peptone 3.0%,p H 8.0,temperature29.9°C,inoculum 4.0%,incubation time 4.0 d.The highest enzyme activity is 584.12U/m L under these conditions.The optimal culture conditions for ZT-12 is: konjac mannan3.0%,peptone 3.2%,p H 8.0,incubation temperature 30.0°C,inoculum 2.8%,and incubation time 2.2 d.The highest enzyme activity is 490.05 U/m L under these conditions.3.The optimum enzyme is determined by single-factor and response surface design as: cellulase 5424 U/kg,pectinase 4291 U/kg,β-glucanase 2830 U/kg,xylanase 1444 U/kg,and mannanase 1374 U/kg.The optimum enzymolysis temperature is 55℃ and the optimum enzymolysis time is 10 h.The results of in vitro simulation of digestion showed that the in vitro dry matter digestibility(IVDMD)was higher than that of commercial enzyme,and the residue calorific value was lower than that of commercial NSP enzyme,indicating that the enzyme had good application effect.Conclusion:In this experiment,four high-efficiency non-starch polysaccharide enzyme strains were screened,and an NSP compound enzyme preparation was prepared through optimization of culture conditions and screening of enzyme profiles.Through in vitro simulation digestion verification,it was determined that the enzyme preparation had high use value,which provided technology for the application of germinated feed.