不同微生物发酵饲料影响海参(刺参)
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J.Ocean Univ.China(Oceanic and Coastal Sea Research) D0I 10.1007/sl1802—015.2591—3 ISSN 1672—5182,2015 14(5):873—880 tp.'//www.oue.edu.cn/xb),wb/ E-mail.'xbywb@OUC.edu.cn Effects of Diferent Microbes on Fermenting Feed for Sea Cucumber(Apostichopusjaponicus) JIANG Yan1)J2)WANG Yingeng )’MAI Kangsen¨ZHANG Zheng , ,,,LIAO Meijie .and RONG Xiaojun 1)Key Laboratory fMaroiculture fMionisty rEducation ofChina,Ocean University fChiona,Qingdao 266003, . ina 2)Yellow Sea Fisheries Research Institute,Chinese Academy fFioshery Sciences,Qingdao 26607 1, R.China (Received Janua ̄21,2014;revised April 24,2014;accepted May 27,2015) @Ocean University of China.Science Press and Spring-Verlag Berlin Heidelberg 201 5 Abstract The effects of djflferent microbes on fermenting feed for sea cucumber(Apostichopus]aponicus)were compared to select the optimal fermentation strain in this study.Saccharomgces cerevisae.Can 如utilis.Bacillus subtilis and Geotrichum canc ̄dum were independently added into the experimental compound feed,while only saline was mixed with the control feed.The fermentation treatments were inoculated with 10%seed solution under the condition of 25 and 70%water content which lasted or 5 days to felucidate the optimal microbe strain for fermenting effect.Physicochemical indexes and sensorial characteristics were measured per day during the fermentation.The indexes included dry nlatter recovery fDMR),crude protein(CP),the percentage of amino acid nirtogen to total nitrogen fAA—N/tN),the percentage of ammonia nitrogen to total nitrogen(NH3-N/tN).and the ratio of fermentation strains and vibrios to the total microbes,color,smel1 and viscosity.The results showed that DMR.CP and AA.N/tN of the cerevisae group reached the highest level on day 3.but the ratio of fermentation strain was second to e utilis cerevisae could be the optimal strain for the group.In addition。its NH3—N/f and the ratio of vibrios were maintained at low levels。and the sensory evaluation score including smel1.color and viscosity was the highest in cerevisae group on day 3.Therefore. feed fermentation for sea cucumber.This research developed a new production method of fermentation feed for sea cucumber. Key words Apostichopusjaponicus;fermentation feed;strain;physicochemical index;sensorial characteristic mals(Choe,1963;Yingst,1976;Moriarty,1982;Zhang 1 Introduction Pf ,..1 995).In some studies.sea cucumber showed sig— niifcant growth by feeding乙 v lactuca.Laminaria/a— As it is good for human health,the sea cucumber ponica,Sargassum thunbergii,Sargassum polycystum (Apostichopus japonicus)is a commercially important and sea mud(Battaglene et a1..1999;Zhu et a1.。2007;Liu echinoderm in Asia(Sun et a1.,2004;Okorie el a1., ,.,20 1 01.Powdered macroalgae, thunbergii and 2008).The trend of exploitation was increasing and the polycystum,were traditionally considered to be the best natural population of sea cucumber had been decimated choice.As A.japonicas culure itndustry grows.the he macroalgal demand for feed is also in. in some areas fHamel et a1.,200 1:Conand,2004;Uthicke, quantity of tng.The resources of these two macroalgal species 2004).To meet the consumption requirements,the stock creasienhancement and aquaculture programs for A.japonicas decreased dramatically and the price became vcry high since the expansion of sea cucumber farming and the have been encouraged(Conand,2004;Yuan el a1.,2006). ductive value and remarkable economic benefit in mari. culture in China.Its culure area is more than 1 t50000 hectares.while the annual production was 1 40000 tons and the output value was more than 30 billion RMB A.japonicus is a single breeding with the highest pro— mass use of these two macroalgae(Yuan,2005).Fur. thermore.the仃aditional powdered feed can easily make he water turbid and the feed would gradualtly deteriorate leading to the outbreak of various diseases(Wang et a1.. 2005:Wang et a1.,2006).Some researchers began to ind substiftutes or partial substitutes for thunbergii and rMOAC.20121. The holothurians which have deposit feeding habits usually ingest the sedimentary organic matter,including bacteria,protozoa,diatoms,and detritus of plants and ani. Corresponding author.Tel:0086—532—85841732 polycystum(Yuan et a1.,2005;Liu et a1.,2010:Seo gf口,. 20 ll、.However,some research has not been sue. cessfu1 because of several limitations;therefore.a new— style feed need be developed for sea cucumber.Fermen. tation feed has a considerable market potentia1. The application of fermentation feed in animal hus. E-mail:wangyg@ ysfri.ac.ca 鱼Springer 874 JIANG 口,./ Ocean fv.C ina(Oceanic andCoastalSeaResearch)2015 14:873—880 lowed the air to enter into the container, bandry is prevalent(Jensen and Mikkelsen,1 998;Kim ton,which alef a1.,200l:Brooks,2003:Li gf 0,.,2003;Kobashi et a1., and the aerobic fermentation could be conducted.The 2008;Guo et a1..2009),but relatively rare in aquacul— ture(Chen,2005;Luo et a1.,2004;Demeckovfi et a1., 2002;Zhang et a1.,20 1 1 1.Previously,some researchers used fermented algal powder or fermented fo・rmula feed in sea cucumbers farming and the utilization rate of feed 口,..20121. Saccharomgces cerevisae,Candida utilis,Bacillus subtilis and Geotrichum candidum could produce a vari. ety of nutrients.such as protein,vitamin among others. cerevisae.C utilis and B.subtilis are probiotics for sea fermentation conditions including water content(50%、 and temperature(25℃1 were on the basis of the pre. 1iminary experiments.The process of treating utilis.B subtilis and G candidum was the same as that of S.cere— visae.In the control group,only 300 mL saline(2%)was after stirring every day at certain time. Table 1 Formulation of basal fc:ed in fermentation on a ner.The samples were collected has been effectively improved(Yuan et aL.2006;Jiang added into the contaidry matter basis Ingredient Concentration(g kg一 ) cucumber.These four strains have been widely used in the fermentation researches(Wang et a1..2006;Liu et a1.. 2010:Yun et a1.,2010:Luo et a1.,2011:Yang et a1., 20 ll 1 and the results were positive.In this research these four strains were studied to select the optimal one ofr developing fermentation feed for sea cucumber. 2 Materials and Methods 2.1 Bacterial Strain and Cultivation cerevisae(CICC l2511,e utilis(CICC 31188),B. subtilis(CICC 1 0073)and G.Calldidum(CICC l 3 1 5、 were bought from China Center of Industrial Culture Collection fBeijing,China).These four strains were sc. 1ected through induced salt tolerance in their own solid medium,respectively. S.cerevisae and C.utilis were cultured in YPD me. dium at 37℃.B.subtilis was cultu【red in PDA medium and G candidum was cultured in TSB medium at 28℃. Two percent salt was added into YPD.PDA and TSB media.These strains were independently cultured in their own solid medium for 48h,48h,36h and 36h in digital biochemicalincubators.Meanwhile.four kinds of microbial suspension with 1 0 cfu mL were prepared with 2%saline and colonies of these four strains.They were inoculated to their own liquid medium with the proportion of 1 0%.respectively,and cultured to the logarithmic phase in vibration cultivating box with 1 20 r min~.Then.these four fermentation liquors were con centrated into 1 0 cfu mL and used as the seed solution ofr feed fermentation. 2.2 Preparing Fermentation Feed with Diflferent Microbes This experiment consisted of five groups,each was treated in triplicate.The first group was fermented with S.cerevisae;the second with utilis;the third with B. subtil ̄;the fourth with G c硎cfidum.and the fifth as control without any exogenetic microbe. During the fermentation experiments.7 1iters auto. claved sea water were added to 3 kg feed fTab1e 1 1 in the plastic container of 1 3 1iters.jnoculated 300 mL cere— visae seed solution.fermented at 25℃for 5 days and stirred once per day.The fermentation containers were enclosed by two.1ayer medical gauze and absorbent cot. 鱼Springer Laminariajaponica powder 100 Ulva lactuca powderb 200 Sargassum polycystum powder。 450 Soybean mealb 80 Scallop side powderb l50 Shell powderb 20 Proximate composition Crude protein f%1 23.34±O.25 Crudelipid(%) 3.42士0.17 Moisture r%、 5.54±0_21 Notes: produced by Qingdao Haichang Biological Technology Co.,Ltd.,Qingdao,Shandong Province,China; produced by Qingdao Ruizi Rare Marine Animal Culture&Development Co. Ltd.,Qingdao,Shandong Province,China. 2.3 The Evaluation Index Dr、,Mattcr Recovery(DMR1:The DMR was calcu一 1ated according to the weight of dry matter content be. ofre and after fermentation(Zhuang et a1.,2006). Determination of Crude Protein,Total Nitrogen and Amino Acid:The fermentative samples were dried at 65 oC and smashed.The crude protein fCP1 and total ni. trogen(tN)were analyzed by Kjeldahl method(UDK142 automatic distillation unit;VELP,Usmate MB,Italy), and amino acid(AA1 by amino acid automatic analyzer fHITACHI LP.2000.Japan). Ammonia Nitrogen(NH3-N):Estimation of NH1~N was conducted following the hypobromite oxidation method rICS 07.060 A45). Microbes in Fermentation:The amount of microbes vibrios and fermentation strains of each fermentation group were tested on TSB,TCBS and the fermentation strain’s culture medium. Sensorial Evaluation:The sensorial characteristics of fermentation feed include color.smell and viscosity. Feed samples were assessed by a panel of ten researchers using a score range of 1.00(worst)to 5.00(best)(Oner 口f.,1993:Tamang and Nikkuni 1996;Omafuvbe et a1.。2002).The data obtained were subjected to statistical analysis.The characteristics of good fermentation feed include an acid aroma,grayish brown。soft in texture and easy to mash be een the ifngers(Ikenebomeh.1 989). 2.4 Statistical Analysis The data from each treatment were analyzed by SPSS 1 6.0 for windows and used analysis of variance 876 JIANG eta1 /j.Ocean Univ.China(OceanicandCoastalSeaResearch)2015 14:873—880 cerevisiae group,G.candidum group and the control cere— visiae group.In fermentation.The ASP,GLU,HIS are the main amino acids in these fermentation feed.The trend of GLU was a first increase and then decrease ex— peared on the third day in cerevisiae group.The trend of HIS was a first increase and later decrease and the G candidum group had a similar trend in AA.N/tN third day in cerevisiae group.There were significant ghest level was on day 3 in (Fig.1c).The highest level f53.84%1 appeared on the group.and the hidifferences among S.cerevisiae group and other four of AA—N/tN was also significantly different from other significant differences on AA.N| by the interactions of n all treatments were irregular.The groups on the third day fP<0.05).and the highest value changes Of ASP idays within s cerevisiae Rroup(P<0.05).There were cept Can da utilis,and the highest values(1.24%)ap- fermentation strains and fermentation time <0.05). The amino acid composition of the fermented feed was represented in Table 2 and Table 3.The total amount of amino acids increased at the beginning and men decreased during the fermentation process in highest values(1.90%)appeared on the third day in cerevisiae group.Meanwhile.Other 1ow.content amino acids were also reached the highest level,f.e.,GLY,SER, AI A and SO on. Table 2 The amino acid composition of the feed independently fermented by Saccharomgees cerevisae, Candida utilis and Bacillus subtilis in 5 days on a dry matter basis Table 3 The amino acid composition of the feed independently fermented by Geotrichum candidum and the control in 5 days on a dry matter basis 垒Springer JIANG 口,-/ Ocean Univ.China(Oceanic andCoastalSeaResearch)20l5 14:873.880 877 signiifcantly diferent <O.05) 3.1.4 NH3-N/tN Both of the fermentation strains and fermentation time 3.1.6 The ratio of vibrios to total microbes Fermentation strains and time affected the ratio of vi— affected NH3-N/tN values signiifcantly <0.05).The NH1.N/tN values in all five groups increased along with the time.the lower values appeared in the first day in brios to total microbes signiifcantly <0.05).The ratio of vibrios to total microbes had the same trend in all five each fermentation strain and the lowest f0.079%1 was in groups(Fig.1 n.The lowest value was in cerev iae the third day.It was significantly different from cerevisiae group(Fig.1 d).It was signiifcantly lower group on he rest of fermentation strains on the same day r尸<0.05) than other four groups on the first day <0.05)and tthere was no significant difference between the first and and there was no significant difference among the first day,third day and the fourth day within cerevisiae the second day(O.082%)within cerevisiae group P> 05).There were signiifcant differences on 0.05).The interactions of fermentation strains and fer. group <0.the ratio of vibrios to total microbes by the interactions mentation time were signiifcantly diferent <0.05). of fermentation strains and fermentation time <0.05). 3.1-5 The ratio of the fermentation strains to total microbes There were signiicantf differences on the ratio of the fermentation strains to total microbes by fermentation 3.2 Sensorial Evaluation The sensorial changes of fermentation feed were shown in 6le 4.In each fermentation group.the sen— sory evaluation scores increased at the beginning and then decreased during the fermentation.In each day,the sensory evaluation scores of the samples fermented by 3 were higher than those in other days.The highest score strains and fermentation time(P<0.05)、respectively. The same trend was observed in cerevisiae group and C utilis group about the ratio of fermentation strain to in C utilis group.the third day,signiicantlfy higher than rains were different.Generally,scores in day t0ta1 microbes(Fig.1e、.The highest value was observed different stthe remaining four groups at this time ved in S.cerevisiae group on day 3.There <0.05)and was obsericant differences between day 3 and day 4 each day within C utilis group <0.05).The interac. were no signiftions of fermentation strains and fermentation time were in cerevisiae group >0.05),and the highest score Table 4 Sensory evaluation of fermentation feed by different strains Notes:Values aremean: ̄SD :lO);Meansinthe same rowwithdifferent superscripts are significantlydiferent(尸<O.O5) was not significantly diferent with C utilis on day 3 study,the similar results have been observed that aU of he DMR values were beltow 1 0O%.In addition.the fer— mentation container was semi—closed.and part of water >0.05). 4 Discussion 4.1 DMR-CP and AA-N/tN Some raw materials would be consumed and some would be evaporated.which may be the reason that the DMR was lower on day 1 and higher at the later time. With the longer period of fermentation.the feed started to decaN which lead to the dry matter reduction.The loss Of DMR depends on fermentation process and tim— gases would be generated along with the growth of mi- crobes.Thus the dry matter of fermentation feed would ing.In this study,the highest DMR(98.1 9%)appeared on day 3 in cerevisiae group.This result was different be reduced generally(Zhang and Chu,1 996).In this 垒Springer 878 jl NG et d}.}J.Ocean Univ.China(oceanic andCoastalSea Research)2015 14:873—880 from previous researches(Zhang et a1.,2007;Lv et a1., the fermentation time extended the growth of fermenta. rains entered the decay period and went to decline 20ll:Liu et a1..20I1).The diference mav be associated tion stwith the structure of the container,as closed fermenta— tion containers were used in other studies. gradually,and the harmful microbes grew more strongly (Wang and Lin,1 998).In this study,an increasing trend of NH1一N/tN was found during the fermentation process CP content of yeast cells is high,accounting for about 50%Of the dry matter.Therefore some yeast cells such as s cerevisiae and c.utilis、were used directly as die— in cerevisiae group.The NH1一N/tN content was lower than other groups except on day 5.and the lowest con— was 0.079%on day I. tary protein or were used to produce dietary protein(Lu tentPf a1.。2007;Liu el a1.,2009;Wang P a,.,2011).In the process of growth and metabolism,some strains could degrade macromolecular organic matter into micro— molecules and generate some usefu1 metabolites.such as 4.3 Sensorial Characteristics The aromatic substances produced in fermentation by yeasts can improve bOth feed palatability and feed intake peptides,amino acids,and vitamins,and so on(Chen, 2005;Zhou et a1. 2007;Jia et a1.,2009).In this study, the content of some amino acids。such as GL SER GLU and HIS.changed at different fermentation time and the highest leve1 was observed on day 3 in cere— visiae group.Meanwhile,the total amount of amino ac— ids was also the highest.A study showed that CP content of soybean meal could improve l2.1%through fermen. ration by yeast and Aspergillus oryzae at 28℃for 72h (Mo and Huang,2007).In addition,yeast could produce l mg mL alanine through fermentation at 28℃for 72 h (Wang el a1..1 992).In this sutdy,the results showed that CP(25.24%1 and AA—N/tN(53.84%1 were the highest on day 3 in S.cerevisiae group.Meanwhile,S.cerevisiae was the dominate microbe and its abundance among microbes was higher than that observed on other days. This result illustrated that fermentation strain had stronger growth and metabolism on day 3 than on other days. 4.2 Microbes and NH3-N/tN Franklin f l 993)reported that the number of E.colf in calf feces could be significantly reduced when the diet containing yeast cel1 walls.Yeast could grow and repro. duce in both aerobic and anaerobic environments(And. 1id el a1..1995:Chi and Gao,1999).The production ef- ifciency of yeast in aerobic environment was ten times higher than that in anaerobic environment.Therefore, fermentation was commonly conducted in ventilation conditions(Wang and Lin.1 998).The pH value de. creased in the later fermentation period,which indicated that the cells began to degrade and the growth of yeast has entered the decline phase.In this situation、the fer. mentation process should be ended(Wang and Lin 1998). The growth of cerevisiae was fast in early fermen. tation days.The ratio of the strain among the tota1 mi crobes was 33.62%on day 3 and was higher than B. subtilis group.G candidum group and control group. Additionally,the ratio of vibiros was only 1.82%on day 3 and lower than othcr four groups.This showed that the metabolites and nutrient competition could efficiently inhabit the growth and reproduction of vibiros. With elongation of fermentation time,the ammonia nitrogen content in all experiment groups presented an increasing trend(Lv et a1..20 1 1:0mafuvbe.2002).As 垒Springer (Yun et a1..2010 .The acid aroma appeared in cere. visiae group and C.utilis group.but lasted longer in cerevisiae group.The highest sensory evaluation score appeared in cerevisiae group on day 3,when color was grayish brown(close to sea mud),the viscosity was in idea1 range.and the acid aroma was stronger than at other days.In addition.thc ratios of fermentation strains were higher in cerevisiae and C.utilis group.which may be the reason that the sensorial characteristics were betteron day 3. 5 ConCIuSionS In comparison,the physicochemica1 indexes DMR CP and AA.N/tN reached the highest level in S.cere v&ae group on day 3.where NH1.N/tN was maintained at a 1ow 1eve1.Meanwhile.the ratio of fermentation strain to total microbes in cerevisae group was higher than those in B.subtilis group and G.caHdidum group. but the ratio of vibrios was lower than other four groups. At the same time.sensorial characteristics of cere. visde group including smell and color achieved its best condition.Thus cerevisae could be applied for feed fermentation for sea cucumber.The optima1 condition is to ferment at 25℃for 3 days with 70%water in the culture medium. Acknowledgements This study was financially supported by the Nationa1 High Technology Research and Development Program, China(20 1 2AA 1 0A4 1 21。the National Natural Science Foundation of China(No.312020161.the National Key Technology R&D Program(20 1 2BAD 1 7B03).Agricul— ture Seed Improvement Project of Shandong Province, Specia1 Funds for Technology R&D Program in Re. search Institutes(20 l1 EG 1 342 1 9),and Strategic Emerg. ing Industry Cultivation Project of Qingdao f 1 3—4-1—65一 hy). References Andlid,T.,Jufirez,R.V,and Gustafsson,L.,1995.Yeast colo— nizing the intestine of rainbow trout(Salmo gairdneri)and turbot(Scophtalmus maximus).Microbial Ecology,30: 321—334. 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