Research On Microbial Ecology And Its Effect Mechanism On Silage Quality During Citrus Waste Ensiling

Citrus wastes are byproduct of citrus processing,about 1.05?10⁶ tons are produced in the Three Gorges Reservoir Region every year.Citrus waste is easy to decay and difficult for long-term preservation due to its high water and sugar content.A large number of citrus wastes are discarded at will and seriously pollute the environment.Citrus waste is a good raw material for silage because of its rich nutrition content.The existing ensiling methods are mainly underground or semi-above-ground,leading to the disadvantages of poor sealing.In addition,the climate of summer and autumn in the Three Gorges Reservoir Region is dominated by high temperature,high humidity and high rainfall.The silage is easily polluted by the surface runoff mixed with spoilage microorganisms.The spatial variation of citrus waste-silage quality in the bunker silo,is still not clear,that seriously restrict the improvement of silage quality and the popularization and application of ensiling technology.The purpose of this paper is to reuse citrus waste as new resource,and improve the existing rough ensiling method,reduce the degree of proteolysis and improve the quality of silage.In this paper,citrus waste,bran and vegetable were used as raw materials for silage.The temporal and spatial succession of microbial community structure and silage composition during ensiling were analyzed to explore the variation mechanism of silage quality;based on the spatial variation of protein composition and degree of proteolysis in the bunker silo,the promotion and inhibition on proteolysis were analyzed to explore the mechanism of protein hydrolysis in the silage;the edible safety of silage was established and evaluated by feeding model species zebrafish.The specific contents and results are as follows:The temporal and spatial variation of microbial richness,diversity,community composition and quantity in silage were studied by Illumina MiSeq sequencing technology,the effects of microbial inoculants on silage were analyzed and the ensiling process was evaluated by the succession of microbial community structure.The results showed that,in the natural ensiling process,the relative abundance of lactic acid bacteria（LAB）was less than 1.00%in raw materials,and rapidly increased to 72.72%on the 3rd day of ensiling,its final relative abundance was 92.70%.Among LAB,Pediococcus showed a trend of increase first and then decrease,the final abundance was lower than 1.00%.Lactobacillus was changed from OTU80 to OTU105 and OTU108during ensiling.A total of 6 genera were involved in spoilage bacteria,and the total relative abundance increased firstly and then decreased.The total relative abundance of spoilage bacteria in the final silage was 3.13%.The sum of relative abundance of LAB with microbial inoculants was at least 6.24%higher than that of natural ensiling,spoilage bacteria were well inhibited from the 3rd day of ensiling,and no spoilage bacteria could be detected at the end of ensiling.With the addition of bacterial inoculant A,Lactobacillusreplacedunclassified_fEnterobacteriaceaeand norank_cCyanobacteria and became the dominant bacterial genus in top layer and bottomlayer,witharelativeabundancegreaterthan82.66%.Unclassified_oSaccharomycetales replaced unclassified_fTrichocomaceae,and became the dominant fungal genus in top layer and bottom layer,with a relative abundance greater than 81.12%.The changes of bacterial and fungal communities in middle layer were significantly different from those in top layer and bottom layer.With the addition of bacterial inoculant B,Lactobacillus replaced norank_cCyanobacteria and norank_fMitochondria,became the dominant bacterial genus in the whole bunker silo,with a relative abundance greater than 97.49%,and gradually increased from the toptothebottom.Unclassified_fTrichocomaceaereplaced unclassified_oSaccharomycetales,became the dominant fungal genus in the whole bunker silo,with a relative abundance greater than 34.76%,and gradually increased from the top to the bottom.The total relative abundance of Saccharomycetaceae from top to bottom was lower than 25.91%,and gradually decreased from the top to the bottom.The changes of fungal community in middle layer were different from that in top layer and bottom layer.The temporal and spatial variation characteristics of organic carbon composition,organic nitrogen composition and the quantity of culturable microorganisms in silage were studied,and the relationship between silage composition,microbial community structure and environmental factors was quantitatively analyzed in order to explore the changing mechanism of silage composition.The results showed that water soluble carbohydrates（WSC）,soluble protein（SP）and residual oxygen were metabolised by aerobic microbes in raw material from the 3rd day to the 6th day of ensiling,a large number of enzymes were produced,crude fiber（CF）,pectin and protein were rapidly degraded under the action of enzymes,leading to WSC,SP,total water soluble amino acid（TWSAA）contents decreased firstly and then increased.WSC was transformed into organic acid by LAB,and the pH decreased rapidly in the early stage of ensiling.The decrease of pH and the consumption of residual oxygen also inhibited the growth of aerobic microorganisms.As a result,the secretion of enzyme decreased,and the degradation of CF,pectin and protein were slown down.The loss rates of total carbon（TC）,CP and dry matter（DM）all tended to be stable,and the substance content was basically stable at the 34th day of ensiling.The production of organic acids in the natural ensiling process were the results of the co-action of various microorganisms,and Lactobacillus dominated the reduction of pH in group A and group B.Protein degradation rates were the highest in natural ensiling,with 0.32%of protein degraded to TWSAA and 0.25%of protein completely degraded to NH₃-N.Weissella and Clostridium_sensu_stricto₁ were the main causes of high protein degradation rate.The quality of silage was improved by adding microbial inoculants.The pH value of silage gradually decreased from the top to the bottom,and from the outside to the center.The WSC content showed a trend of gradual decrease from the top to the bottom.SP content was the lowest in middle layer,while TWSAA content was the highest in middle layer.The content of NH₃-N gradually decreased from the top to the bottom,indicating that more SP was transformed into TWSAA in middle layer.The degradation rates of CF,pectin and CP all showed a trend of gradual decrease from the top to the bottom,leading to a decrease of DM loss rate from the top to the bottom.The lower the silage position,thebetterthesilagequality.Unclassified_oSaccharomycetalesand norank_oEurotiales played the main roles in the degradation of CF and pectin.Sphingobium,Brevundimonas,and fungi played major roles in CP degradation.The highest TWSAA content in middle layer was due to more SP was hydrolyzed by Sphingobium and Brevundimonas.The production of organic acids and the reduction of pH were induced by Lactobacillus and Pediococcus during ensiling.The total proportion of heterofermentative LAB in top layer（>87.92%）was higher than that in middle layer and bottom layer,resulting in a significantly higher content of acetic acid（AA）in top layer than the other two layers.Although the total relative abundance of LAB in middle layer was the lowest,the homofermentative LAB accounted for 92.87%of the total amount of LAB,so the ability of lactic acid（LA）production was not decreased.The spatial variation of temperature,moisture content and pH value inhibited the spoilage bacteria in the raw material and promoted the growth of beneficial bacteria in ensiling process.The quality of silage was determined by the composition of microbial community and environmental factors.In order to improve the quality of silage,the additive amount of LAB on the upper layer of bunker silo should be increased.The proteolysis degree of silage was systematic evaluated,and the relative roles of promotion and inhibition on proteolysis during ensiling were quantified among various factors.The causes of inhibition and promotion of proteolysis were analyzed through changes of microbial community.Finally,effective methods to reduce the degree of proteolysis in silage was proposed.The results showed that,the pH,and DM,TC and CP loss rates were less than 3.74,8.33%,8.72%,17.39%,respectively,demonstrating the success of ensiling,however,the CP loss rate was higher.The protein of silage were composed of SP,insoluble protein（ISP）,TWSAA and NH₃-N.The content of SP and ISP decreased,and the content of NH₃-N increased during ensiling.The content of SP in top layer and bottom layer decreased by 35.01%and 35.84%,the content of ISP in top layer and bottom layer decreased by 11.82%and 10.45%,while the content of NH₃-N in top layer and bottom layer increased by 93.33%and 22.54%,respectively.The largest loss rate of SP and ISP（56.26%and 14.34%,respectively）and the largest increase rate of NH₃-N（129.21%）were all appeared in middle layer.The loss rates of TWSAA in top layer and middle layer were 1.42%and 12.07%,respectively,while the increased rate of TWSAA in bottom layer was 11.24%.The largest loss rate of TWSAA appeared in middle layer.Acid protease（AP）was mainly microbial protease,which could tolerate the pH range of 3.44-3.77,leading both the hydrolysis of CP and SP and the hydrolysis of TWSAA.The decrease of pH and the increase of WSC,LA and AA significantly promoted AP activity,while the increase of moisture content and the loss of TC and DM inhibited AP activity.The first stage of proteolysis was mainly accelerated by AP activity.The increased moistrue,the loss of TC and DM promoted the second stage of proteolysis,while the increased WSC,LA and AA and the decreased pH inhibited the second stage of proteolysis.B-CC,OTU75 and OTU41 promoted the production and activity of AP,B-CC,OTU75 and F-CC promoted the first stage of proteolysis,OTU43and F-CC promoted the second stage of proteolysis,but B-CC and OTU75 inhibited the second stage of proteolysis.Proteolysis was strongest in middle layer,due to the relative abundance of B-CC（OTU315 and OTU317）and F-CC（OTU9,OTU72 and OTU100）were different from the other two layers,resulting in the highest degree of proteolysis.The main measure to reduce the proteolysis in silage was to increase the concentration of homofermentative LAB in raw materials,especially in middle layer.A method was developed to evaluate the edible safety of silage using zebrafish as a model system.Zebrafish were fed with Silage A and Silage B at different levels.After the breeding experiment,the changes of growth and development indicators,the health status of main organs and tissues,and intestinal immunity of zebrafish were detected to analyze the edible safety of citrus waste silage.The results showed that,the death rate of zebrafish was reduced by feeding the silage of 0.2%of body weight,and the condition factor and integrity of intestinal development of zebrafish were not affected by feeding silage.However,the death rates were increased,the silage conversion rates were reduced,the development of male liver,the growth rates of body length and weight were inhibited,the development of gonad,blood glucose concentration and protein content of muscle tissue were all prevented by feeding the silage of 2%of body weight.Silage played an active role in regulating the microbial community structure in the intestinal tract of zebrafish,significantly inhibiting the relative abundance of pathogenic bacteria Vibrio and improving the species diversity.However,the intestinal weight was decreased,the relative abundance of pathogenic bacteria Aeromonas was increased,the intestinal immunity was reduced,and the risk of infection was increased by feeding the silage of 0.2%of body weight.The suitable addition of citrus silage to livestock and poultry diet was 0.2%body weight.