| The feed of sea cucumber (Apostichopus japonicus) was respectively fermentedby four strains (Saccharomgces cerevisae, Candida utilis, Bacillus subtilis andGeotrichum candidum), which based on the safety to A. japonicus in certainconcentration range. S. cerevisae was the optimal fermentation strain through thestudy on the effects of fermentation. Forthermore, the fermentation condition with S.cerevisiae as starter was optimized, and the effects of sterilization of feed onfermentation and the degradation of macromolecular protein during the fermentationprocess were studied. Likewise, the effects of fermentation feed and feedingfrequency on the growth, energy distribution and immunity of sea cucumber wascompared. In addition, the technology of fermentation was used into the large-scareculturing of juvenile sea cucumber and the effects of fermentation feed o n thegrowth of juvenile sea cucumber were studied. The main content and results weresummarized as follows:1. An experiment was conducted to study the safety of four fermentation strains onsea cucumber A. japonicus, which lasted21days, and the initial weight ofexperimental sea cucumber was (4.66±0.50)g. The four strains were S. cerevisae, C.utilis, B. subtilis and G. candidum, and the concentrations of each strain were108cfu/ml,106cfu/ml,104cfu/ml. Sea cucumber was respectively soaked in thesebacterial suspensions. Through the comparison of adverse reactions, which includingvisceral ejection rate, mouth timidity rate and mortality rate, the results showed thatthe visceral ejection rates in B. subtilis with104cfu/ml and C. utilis with108cfu/mlwere significantly higher than that in the control (P<0.05); and the mouth timidityrates in B. subtilis with106cfu/ml and other three strains with108cfu/ml were significantly higher than that in the control (P<0.05); additionaly, the mortality ratesin each strain with108cfu/ml and B. subtilis and G. candidum with106cfu/ml weresignificantly higher than that in the control (P<0.05). Besides, the remaining groupsof each index were significantly different from the control (P>0.05). Through thecalculation, the highest concentration of fermentation stratin in aquaculture waterafter feeding was104cfu/ml. Thus, four fermentation strains were safe for seacucumber when the concentration was104cfu/ml, and all could be used to fermentfeed with this concentration.2. Effects of different microbes on fermenting feed for sea cucumber A. japonicuswere compared in order to select the optimal fermentation strain in this study.Saccharomgces cerevisae, Candida utilis, Bacillus subtilis and Geotrichum candidumwere respectively added into the compound feed against a control with adding equalsaline, which constituted five groups. The fermentation treatments were inoculatedwith10%(V/W) seed solution under the condition of25℃and70%water content,which lasted for5days in order to elucidate the optimal strain for fermenting effect.Physicochemical indexes and sensorial characteristics were measured per day duringthe fermentation process, including dry matter recovery (DMR), crude protein (CP),the percentage of amino acid nitrogen to total nitrogen (AA-N/tN), the percentage ofammonia nitrogen to total nitrogen (NH3-N/tN), and the ratio of fermentation strainsand vibrios to the total microbes, color, smell, viscosity. The results showed that DMR,CP and AA-N/tN of the S. cerevisae group reached the highest level on day3, but theratio of fermentation strain was second to C. utilis group. In addition, its NH3-N/tNand the ratio of vibrios were maintained in reasonable ranges, and the sensoryevaluation score including smell, color and viscosity was the highest in S. cerevisaegroup on day3. Therefore, S. cerevisae could be the optimal strain for the feedfermentation of sea cucumber in this study.3. The fermentation condition with S. cerevisiae as sea cucumber feed fermentationstarter was optimized in this study. The effects of water content of fermentationfeedstuff (on a dry matter basis,50%,60%70%,80%), fermentation temperature (20°C,25°C,30°C,35°C) and fermentation time (1d,2d,3d,4d,5d) on fermentationfeed of sea cucumber were considered. The fermentation treatments were inoculatedwith10%(V/W) S. cerevisiae seed solution. The evaluation indexes of fermentationincluded physicochemical indexes and sensorial characteristics of feed in thefermentation process. The results showed that the physicochemical indexes includingdry matter recovery (DMR), crude protein (CP) and the percentage of amino acidnitrogen to total nitrogen (AA-N/tN) reached the highest level, and the percentage ofammonia nitrogen to total nitrogen (NH3-N/tN) maintained in a reasonable range onday3in25°C,70%water content group; at the same time, the sensorialcharacteristics including smell, color and viscosity achieved the best level in thisgroup. Thses all indicated that S. cerevisiae can be used as one of candidate strains forsea cucumber feed fermentation, and the optimum technological parameters (watercontent, fermentation temperature and fermentation time) were70%,25°C and3days.The optimized parameters will provide some technical references for the reparation ofsea cucumber new type feed.4. The feed for sea cucumber was fermented, which followed the technologicalparameters optimized in the previous study, and the samples were taken every12hours in fermentation process. The study on degradation of macromolecular proteinwas conducted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and the concentration of separation gel and stacking gel were15%and5%. The samples of electrophoresis were prepared through extracting6samples inTris-HCl buffer and centrifuging. The changes of protein bands in the gel wereobserved after staining in coomassie brilliant blue R-250and decolored in glacialacetic acid. The results showed that macromolecular bands gradually disappeared withfermentation, but the quantity of micromolecular bands increased, which suggestedthat part of macromolecular protein was degraded into polypeptide, peptide and someother micromoleculars.5. The effects of sterilization of feed on fermentation were studied in this paper. Thefeed was sterilized under the condition of65°C and30minutes, and fermented with the optimum technological parameters. The experimental groups were respectivelyinoculated with10%(V/W) S. cerevisiae seed solution, which included the sterilizedand unsterilized. The controls did not inoculated with fermentation strain, and alsoincluded the sterilized and unsterilized. The physicochemical indexes and sensorialcharacteristics of feed were evaluated after fermentation within four groups. Thephysicochemical indexes included the ratio of fermentation strain and vibrios to thetotal microbes, dry matter recovery (DMR) and crude protein (CP); and the sensorialcharacteristics included color, smell and viscosity of fermentation feed. The resultsshowed that there were no significant differences between the two experimentalgroups in the ratios of fermentation strain and vibrios to the total microbes, DMR andCP (P>0.05), but respectively significantly higher than those in the two controls(P<0.05). Meanwhile, there were no significant differences between the twoexperimental groups in color, smell and viscosity (P>0.05). Thus, the effects ofpretreatment of feed on fermentation were not obvious under the semi-closedfermentation condition in this experiment.6. In order to investigated the effects of fermented feed and unfermented feed on thegrowth, energy distribution and immunity, three sizes of sea cucumber (4g,8g and20g) marked as A, B and C were respectively fed with these two different feed. After50-day feeding trial, the results showed that the special growth rate (SGR), feedconversion efficiency (FCE) and the final rate of viscera to body wall (FRVBW) ofsea cucumber fed with fermented feed were all higher than those fed withunfermented feed in size A, but the ingestion rate (IR) and feces production rate (FPR)were lower than those fed with unfermented feed. The changes of growth indexes insize B were same to those in size A; besides IR and FPR, the growth indexes of seacucumber fed with fermented feed were significantly higher than those fed withunfermented feed (P<0.05). FPR of sea cucumber fed with fermented feed was higherthan that fed with unfermented feed in size C; however, the cha nges of other growthindexes were same to those in size B. Likewise, fermentation feed could more easilypromote the growth of smaller sea cucumber. In energy distribution, the energy deposited in growth of three sizes sea cucumber fed with fermented feed were higherthan that fed with unfermented feed; and the energy deposited in growth wasgradually reduced with the increase of size when sea cucumber was fed withfermentation feed. Therefore, considering from the growth and save feed, the effectsof fermented feed in the farming of three sizes sea cucumber were better thanunfermented feed.7. A50-day feeding experiment was conducted to determine the effects of feedingfrequency on the growth, energy distribution and immunity of sea cucumber in thisstudy. The initial weight of sea cucumber was (11.18±0.46) g, and the feedingfrequency was once every two days, once a day and twice a day. The results showedthat the specific growth rate (SGR), feed conversion efficiency (FCE) and the finalrate of viscera to body wall (FRVBW) of sea cucumber fed once a day were allsignificantly higher than other frequencies (P<0.05), and the apparent digestive ratio(ADR) was decline with the increased feeding frequency, but the trend of fecesproduction rate (FPR) was rising. However, there were no significant differences iningestion rate (IR) of sea cucumber fed with different frequency (P>0.05). In addition,the energy deposited in growth when fed once a day was significantly higher thanother frequencies (P<0.05). Thus, the best feeding frequency was once a day underthe conditions of this experiment. Culturing with the optimum feeding frequency, thegrowth of sea cucumber could be effectively promoted and FCE could be effectivelyimproved.8. A35-day feeding trial was conducted to determine the effects of fermentation feedon the growth of juvenile sea cucumber in this study. The commercial feed wasfermented by S. cerevisiae followed the optimum technological parameters and thecontent of crude protein was significantly higher than the unfermented feed (P<0.05).The initial weight of juvenile sea cucumber was (3.15±0.05) kg/pond and the volumeof pond was7.5m3. The control was fed with unfermented feed, and26parallels wereset in each treatment. The results showed that the final weight, weight gain rate andspecial growth rate of juvenile sea cucumber fed with fermented feed were all significantly higher than those fed with unfermented feed (P<0.05), and the intestinalfilling degree of sea cucumber fed with fermented feed was better than that fed withunfermented feed. Hence, the feed was properly fermented could promote the healthyand rapid growth in the seeding of juvenile sea cucumber. |
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