Effect of yeast culture supplementation on nutrient intake, digestibility and growth performance of

The Fascinating World of Fermented FoodsFermentation is one of the oldest and most widespread food preservation techniques known to humanity.This ancient process,which involves the transformation of food by microorganisms,has given rise to a diverse array of flavors,textures,and nutritional benefits.From tangy sauerkraut to creamy yogurt,fermented foods are enjoyed across cultures and have become a staple in many diets.This essay delves into the fascinating world of fermented foods,exploring their history,the science behind fermentation,and the various health benefits they offer.A Brief History of FermentationFermentation has been practiced for thousands of years,with evidence of fermented foods dating back to ancient civilizations.The earliest records of fermentation can be traced to the Neolithic period,around 7000-6600BCE,when people began to ferment grains and fruits to produce alcoholic beverages.Ancient Egyptians,Greeks,and Romans also embraced fermentation,using it to make bread,wine,and cheese.Throughout history,fermentation has played a crucial role in food preservation,allowing communities to store food for extended periods. This was particularly important before the advent of refrigeration. Fermented foods also became integral to cultural and religious practices, with many traditional recipes being passed down through generations. The Science of FermentationFermentation is a metabolic process in which microorganisms such as bacteria,yeast,and molds convert sugars and other carbohydrates into alcohol,acids,and gases.This process not only preserves food but also enhances its flavor,texture,and nutritional value.There are several types of fermentation,each involving different microorganisms and resulting in distinct products:Lactic Acid Fermentation:This type of fermentation is carried out by lactic acid bacteria,which convert sugars into lactic acid.It is responsible for the tangy taste and extended shelf life of foods like yogurt, sauerkraut,kimchi,and pickles.Alcoholic Fermentation:Yeasts,particularly Saccharomyces cerevisiae, convert sugars into alcohol and carbon dioxide.This process is used to produce alcoholic beverages such as beer,wine,and sake,as well as leavened bread.Acetic Acid Fermentation:Acetic acid bacteria convert alcohol into acetic acid,resulting in the production of vinegar.This type of fermentation is used to make various types of vinegar,including apple cider vinegar and balsamic vinegar.Mold Fermentation:Certain molds,such as Aspergillus oryzae and Penicillium roqueforti,are used in the fermentation of foods like soy sauce,miso,and blue cheese.These molds contribute to the unique flavors and textures of these products.Health Benefits of Fermented FoodsFermented foods are not only delicious but also offer a range of health benefits.The fermentation process enhances the nutritional profile of foods and introduces beneficial microorganisms,known as probiotics, which support gut health.Some of the key health benefits of fermented foods include:Improved Digestion:Probiotics in fermented foods help maintain a healthy balance of gut bacteria,which is essential for proper digestion. They can alleviate symptoms of digestive disorders such as irritable bowel syndrome(IBS)and reduce bloating and gas.Enhanced Nutrient Absorption:Fermentation breaks down complex compounds in food,making nutrients more bioavailable.For example, the fermentation of dairy products increases the availability of calcium and B vitamins.Boosted Immune System:A healthy gut microbiome is closely linked to a strong immune system.Probiotics in fermented foods can enhance the body's ability to fight off infections and reduce inflammation. Reduced Risk of Chronic Diseases:Regular consumption of fermented foods has been associated with a lower risk of chronic diseases such as heart disease,diabetes,and certain cancers.The antioxidants and anti-inflammatory compounds produced during fermentation contribute to these protective effects.Mental Health Benefits:Emerging research suggests that gut health is connected to mental health through the gut-brain axis.Probiotics in fermented foods may help alleviate symptoms of anxiety and depression by promoting a healthy gut microbiome.Popular Fermented Foods Around the WorldFermented foods are enjoyed in various forms across different cultures, each with its unique flavors and traditions.Here are some popular fermented foods from around the world:Yogurt:A staple in many diets,yogurt is made by fermenting milk with lactic acid bacteria.It is known for its creamy texture and tangy flavor.Sauerkraut:This German delicacy is made by fermenting shredded cabbage with salt.The result is a tangy,crunchy,and probiotic-rich food.Kimchi:A traditional Korean dish,kimchi is made by fermenting vegetables,usually cabbage and radishes,with chili peppers,garlic, ginger,and fish sauce.It is known for its spicy and pungent flavor.Kombucha:A fermented tea beverage,kombucha is made by fermenting sweetened tea with a symbiotic culture of bacteria and yeast(SCOBY).It is fizzy,tangy,and slightly sweet.Tempeh:Originating from Indonesia,tempeh is made by fermenting soybeans with a mold called Rhizopus.It has a firm texture and nutty flavor,making it a popular plant-based protein source.Miso:A staple in Japanese cuisine,miso is a fermented soybean paste used to flavor soups,sauces,and marinades.It has a rich,umami flavor.Cheese:Various types of cheese,such as blue cheese,cheddar,and brie, are made through fermentation.The process involves the action of bacteria and molds,contributing to the distinct flavors and textures of each cheese.ConclusionThe fascinating world of fermented foods offers a rich tapestry of flavors, textures,and health benefits.From ancient preservation techniques to modern culinary delights,fermentation has played a vital role in shaping our diets and cultures.By embracing fermented foods,we can enjoy their unique tastes and reap the numerous health benefits they provide. Whether it's a spoonful of tangy yogurt,a bite of spicy kimchi,or a sip of fizzy kombucha,fermented foods continue to captivate and nourish people around the world.。

红曲与酵母共培养对红曲菌产酯的影响刘桂君;尚宏忠;李艳敏;高畅;刘红霞;朱婷婷【摘要】The effects of yeast on ethyl acetate-producing capability of red starter（Monascus） were studied.The yield of ethyl acetate of 6 red starter strains by liquid culture ranked in decreasing sequence as follows：M4M5M3M1M2M6.However,the yield of ethyl acetate of 6 red starter strains by liquid co-culture with S.cerevisiae or ester-producing yeast ranked in decreasing sequence as follows： M5M1M2M3M4M6.The results suggested that co-culture of red starter strains with yeast strains had great effects on ethyl acetate yield.In liquor-making practice,it was found that ethyl acetate content in base liquor by co-fermentation of red starter strains and yeast strains increased greatly than that in base liquor by common fermentation.Besides,tasting results also suggested that base liquor by co-fermentation of red starter strains and yeast strains got improved in aroma and taste.%主要研究酵母对红曲产乙酸乙酯能力的影响,6株红曲单菌株液体培养乙酸乙酯产量大小关系为M4〉M5〉M3〉M1〉M2〉M6;红曲、酿酒酵母Y001和生香酵母Y005液体共培养,乙酸乙酯产量大小关系为M5〉M1〉M2〉M3〉M4〉M6;结果表明,与酿酒酵母、生香酵母共培养对红曲乙酸乙酯产量影响较大。

Heterologous protein expression in the methylotrophic yeastPichia pastorisJoan Lin Cereghino,James M.Cregg*Department of Biochemistry and Molecular Biology,Oregon Graduate Institute of Science and Technology,20000N.W.Walker Road,Beaverton,OR97006-8921,USAReceived25July1999;accepted4September1999AbstractDuring the past15years,the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins.The increasing popularity of this particular expression system can be attributed to several factors,most importantly:(1)the simplicity of techniques needed for the molecular genetic manipulation of P.pastoris and their similarity to those of Saccharomyces cerevisiae,one of the most well-characterized experimental systems in modern biology;(2)the ability of P.pastoris to produce foreign proteins at high levels,either intracellularly or extracellularly;(3)the capability of performing many eukaryotic post-translational modifications,such as glycosylation,disulfide bond formation and proteolytic processing;and(4)the availability of the expression system as a commercially available kit.In this paper,we review the P.pastoris expression system:how it was developed,how it works,and what proteins have been produced.We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.ß2000Federation of European Microbiological Societies.Published by Elsevier Science B.V.All rights reserved.Keywords:Foreign gene expression;Heterologous protein production;Methylotrophic yeast;Pichia pastoris;Alcohol oxidase1gene promoter;Protein secretionContents1.Introduction (46)1.1.Pichia pastoris as an experimental organism (46)1.2.Methanol metabolism (46)1.3.AOX1promoter (46)1.4.Molecular genetic manipulation (47)2.Construction of expression strains (47)2.1.Expression vectors (48)2.2.Alternative promoters (48)2.3.Selectable markers (48)2.4.Host strains (49)2.5.Integration of expression vectors into the P.pastoris genome (50)2.6.Generating multicopy strains (50)2.7.High cell density growth in fermenter cultures (50)3.Post-translational modi¢cation of secreted proteins (52)3.1.Secretion signal selection (52)3.2.O-Linked glycosylation (53)3.3.N-Linked glycosylation (53)4.Conclusions (53)0168-6445/00/$20.00ß2000Federation of European Microbiological Societies.Published by Elsevier Science B.V.All rights reserved.PII:S0168-6445(99)00029-7Acknowledgements .........................................................58References ...............................................................581.Introduction1.1.Pichia pastoris as an experimental organismThirty years ago,Koichi Ogata ¢rst described the ability of certain yeast species to utilize methanol as a sole source of carbon and energy [1].The methylotrophs attracted immediate attention as potential sources of single-cell pro-tein (SCP)to be marketed primarily as high-protein ani-mal feed.During the 1970s,Phillips Petroleum Company developed media and protocols for growing Pichia pastoris on methanol in continuous culture at high cell densities (s 130g l 31dry cell weight,Fig.1)[2].Unfortunately,the oil crisis of the 1970s caused a dramatic increase in the cost of methane.Concomitantly,the price of soybeans,the major alternative source of animal feed,fell.As a result,the economics of SCP production from methanol were never favorable.In the following decade,Phillips Petroleum contracted with the Salk Institute Biotechnology/Industrial Associ-ates,Inc.(SIBIA,La Jolla,CA)to develop P.pastoris as an organism for heterologous protein expression.Re-searchers at SIBIA isolated the gene and promoter for alcohol oxidase,and generated vectors,strains,and corre-sponding protocols for the molecular genetic manipulation of P.pastoris .The combination of the fermentation meth-ods developed for the SCP process and the alcohol oxidase promoter's strong,regulated expression e¡ected surpris-ingly high levels of foreign protein expression.In 1993,Phillips Petroleum sold its P.pastoris expression system patent position to Research Corporation Technologies (Tucson,AZ),the current patent holder.In addition,Phil-lips Petroleum licensed Invitrogen Corporation (Carlsbad,CA)to sell components of the system,an arrangement that continues under Research Corporation Technologies.1.2.Methanol metabolismThe conceptual basis for the P.pastoris expression sys-tem stems from the observation that some of the enzymes required for methanol metabolism are present at substan-tial levels only when cells are grown on methanol [3,4].Biochemical studies showed that methanol utilization re-quires a novel metabolic pathway involving several unique enzymes [3].The enzyme alcohol oxidase (AOX)catalyzes the ¢rst step in the methanol utilization pathway,the ox-idation of methanol to formaldehyde and hydrogen per-oxide (Fig.2).AOX is sequestered within the peroxisome along with catalase,which degrades hydrogen peroxide to oxygen and water.A portion of the formaldehyde gener-ated by AOX leaves the peroxisome and is further oxi-dized to formate and carbon dioxide by two cytoplasmic dehydrogenases,reactions that are a source of energy for cells growing on methanol.The remaining formaldehyde is assimilated to form cel-lular constituents by a cyclic pathway that starts with the condensation of formaldehyde with xylulose 5-monophos-phate,a reaction catalyzed by a third peroxisomal enzyme dihydroxyacetone synthase (DHAS).The products of this reaction,glyceraldehyde 3-phosphate and dihydroxyace-tone,leave the peroxisome and enter a cytoplasmic path-way that regenerates xylulose 5-monophosphate and,for every three cycles,one net molecule of glyceraldehyde 3-phosphate.Two of the methanol pathway enzymes,AOX and DHAS,are present at high levels in cells grown on methanol but are not detectable in cells grown on most other carbon sources (e.g.,glucose,glycerol,or ethanol).In cells fed methanol at growth-limiting rates in fermenter cultures,AOX levels are dramatically induced,constitut-ing s 30%of total soluble protein [5,6].1.3.AOX1promoterThere are two genes that encode alcohol oxidase in P.pastoris :AOX1and AOX2;AOX1is responsible for a vast majority of alcohol oxidase activity in the cell[7^9].Fig.1.High cell density culture of P.pastoris .The centrifuge bottle on the left shows a P.pastoris culture grown in a £ask to a density of 1OD 600unit.The bottle on the right contains a sample of the strain grown in a fermenter to a density of 130g l 31dry cell weight (V 500OD 600units).J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6646Expression of the AOX1gene is controlled at the level of transcription [7^9].In methanol-grown cells,V 5%of poly(A) RNA is from AOX1;however,in cells grown on most other carbon sources,AOX1message is undetect-able [10].The regulation of the AOX1gene appears to involve two mechanisms:a repression/derepression mech-anism plus an induction mechanism,similar to the regu-lation of the Saccharomyces cerevisiae GAL1gene.Unlike GAL1regulation,the absence of a repressing carbon source,such as glucose in the medium,does not result in substantial transcription of AOX1.The presence of meth-anol is essential to induce high levels of transcription [7].1.4.Molecular genetic manipulationTechniques required for the molecular genetic manipu-lation of P.pastoris ,such as DNA-mediated transforma-tion,gene targeting,gene replacement,and cloning by functional complementation,are similar to those described for S.cerevisiae .P.pastoris can be transformed by electro-poration,a spheroplast generation method,or whole cell methods such as those involving lithium chloride and polyethylene glycol 1000[11^14].As in S.cerevisiae ,P.pas-toris exhibits a propensity for homologous recombination between genomic and arti¢cially introduced DNAs.Cleav-age of a P.pastoris vector within a sequence shared by the host genome stimulates homologous recombination events that e¤ciently target integration of the vector to that ge-nomic locus [15].Gene replacements occur at lower fre-quencies than those observed in S.cerevisiae and appear to require longer terminal £anking sequences to e¤ciently direct integration [14].P.pastoris is a homothallic ascomycetous yeast that can also be manipulated by classical genetic methods [10,16].Unlike homothallic strains of S.cerevisiae ,which are dip-loid,P.pastoris remains haploid unless forced to mate.Strains with complementary markers can be mated by subjecting them to a nitrogen-limited medium.After 1day on this medium,cells are shifted to a standardminimal medium supplemented with nutrients designed to select for complementing diploid cells (not self-mated or non-mated parental cells).The resulting diploids are stable as long as they are not subjected to nutritional stress.To obtain spore products,diploids are returned to the nitrogen-limited medium,which stimulates them to proceed through meiosis and sporulation.Spore products are handled by random spore techniques rather than micromanipulation,since P.pastoris asci are small and di¤cult to dissect.Yet most standard classical genetic ma-nipulations,including mutant isolation,complementation analysis,backcrossing,strain construction,and spore analysis,can be accomplished.2.Construction of expression strainsExpression of any foreign gene in P.pastoris requires three basic steps:(1)the insertion of the gene into an expression vector;(2)introduction of the expression vec-tor into the P.pastoris genome;and (3)examination of potential expression strains for the foreign gene product.A variety of P.pastoris expression vectors and host strains are available.A generalized diagram of an expression vec-tor and a list of possible vector components are shown in Fig.3and Table 1,respectively.More detailed informa-tion on vectors and strains can be found elsewhere [17,18].In addition,the DNA sequence of many vectors can be found at the Invitrogen website ().Table 2shows a list of commonly used P.pastoris hoststrains.Fig.2.The methanol pathway in P.pastoris .1,alcohol oxidase;2,cata-lase;3,formaldehyde dehydrogenase;4,formate dehydrogenase,5,di-hydroxyacetone synthase;6,dihydroxyacetone kinase;7,fructose 1,6-bi-phosphate aldolase;8,fructose1,6-bisphosphatase.Fig.3.General diagram of a P.pastoris expression vector.YFG,`Your Favorite Gene;'*,sites for cassette ampli¢cation.J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^66472.1.Expression vectorsAll expression vectors have been designed as Escherichia coli/P.pastoris shuttle vectors,containing an origin of rep-lication for plasmid maintenance in E.coli and markers functional in one or both organisms.Most expression vec-tors have an expression cassette composed of a 0.9-kb fragment from AOX1composed of the 5P promoter se-quences and a second short AOX1-derived fragment with sequences required for transcription termination [19].Be-tween the promoter and terminator sequences is a site or multiple cloning site (MCS)for insertion of the foreign coding sequence.In the native AOX1gene,the alcohol oxidase open reading frame (ORF)is preceded by an un-usually long 5P untranslated region (116nt)[8].Generally,the best expression results are obtained when the ¢rst ATG of the heterologous coding sequence is inserted as close as possible to the position of the AOX1ATG.This position coincides with the ¢rst restriction site in most MCSs.In addition,for secretion of foreign proteins,vec-tors are available where in-frame fusions of foreign pro-teins and the secretion signals of P.pastoris acid phospha-tase (PHO1)or S.cerevisiae K -mating factor (K -MF)can be generated.2.2.Alternative promotersAlthough the AOX1promoter has been successfully used to express numerous foreign genes,there are circum-stances in which this promoter may not be suitable.For example,the use of methanol to induce gene expression may not be appropriate for the production of food prod-ucts since methane,a petroleum-related compound,is one source of methanol.Also,methanol is a potential ¢re haz-ard,especially in quantities needed for large-scale fermen-tations.Therefore,promoters that are not induced by methanol are attractive for expression of certain genes.Alternative promoters to the AOX1promoter are the P.pastoris GAP ,FLD1,PEX8,and YPT1promoters.2.2.1.P GAPBoth northern and reporter activation results indicate that the P.pastoris glyceraldehyde 3-phosphate dehydro-genase (GAP )gene promoter provides strong constitutive expression on glucose at a level comparable to that seen with the AOX1promoter [20].GAP promoter activity lev-els in glycerol-and methanol-grown cells are approxi-mately two-thirds and one-third of the level observed for glucose,respectively.The advantage of using the GAP promoter is that methanol is not required for induction,nor is it necessary to shift cultures from one carbon source to another,making strain growth more straightforward.However,since the GAP promoter is constitutively ex-pressed,it is not a good choice for the production of proteins that are toxic to the yeast.2.2.2.P FLD1The FLD1gene encodes a glutathione-dependent form-aldehyde dehydrogenase,a key enzyme required for the metabolism of certain methylated amines as nitrogen sour-ces and methanol as a carbon source [21].The FLD1pro-moter can be induced with either methanol as a sole car-bon source (and ammonium sulfate as a nitrogen source)or methylamine as a sole nitrogen source (and glucose as a carbon source).After induction with either methanol or methylamine,P FLD1is able to express levels of a L -lacta-mase reporter gene similar to those obtained with metha-nol induction from the AOX1promoter.The FLD1pro-moter o¡ers the £exibility to induce high levels of expression using either methanol or methylamine,an inex-pensive nontoxic nitrogen source.2.2.3.P PEX8,P YPT1For some applications,the AOX1,GAP ,and FLD1promoters may be too strong,expressing genes at too high a level.There is evidence that,for certain foreign genes,the high level of expression from P AOX1may over-whelm the post-translational machinery of the cell,causing a signi¢cant proportion of foreign protein to be misfolded,unprocessed,or mislocalized [22,23].For these and other applications,moderately expressing promoters are desir-able.Toward this end,the P.pastoris PEX8and YPT1promoters may be of use.The PEX8gene encodes a per-oxisomal matrix protein that is essential for peroxisome biogenesis [24].It is expressed at a low but signi¢cant level on glucose and is induced modestly when cells are shifted to methanol.The YPT1gene encodes a GTPase involved in secretion,and its promoter provides a low but constit-utive level of expression in media containing either glu-cose,methanol,or mannitol as carbon sources [25].2.3.Selectable markersAlthough classical and molecular genetic techniques are generally well-developed for P.pastoris ,few selectable marker genes have been described for the molecular genet-ic manipulation of the yeast.Existing markers are limited to the biosynthetic pathway genes HIS4from either P.pastoris or S.cerevisiae ,ARG4from S.cerevisiae ,and the Sh ble gene from Streptoalloteichus hindustanus which confers resistance to the bleomycin-related drug zeocin [11,26,27].The stable expression of human type III colla-gen illustrates the need for multiple selectable markers inTable 1Relevant components of vectors used for protein expression in P.past-orisSecretion signals none,PHO1,K -MF,SUC2,PHA-EMarker genes ADE1,ARG4,G418,HIS4,URA3,Zeo r PromotersAOX1,GAP,FLD1,PEX8,YPT1See text for explanation of di¡erent elements.J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6648P.pastoris[28].The production of collagen requires the coexpression of prolyl4-hydroxylase,a central enzyme in the synthesis and assembly of trimeric collagen.Since prol-yl4-hydroxylase is an K2L2tetramer,the L subunit of which is protein disul¢de isomerase(PDI),three markers ^Arg,His,and zeocin resistance^were necessary to co-express all three polypeptides in the same P.pastoris strain.Recently,a new set of biosynthetic markers has been isolated and characterized:the P.pastoris ADE1(PR-ami-doimidazolesuccinocarboxamide synthase),ARG4(argini-nosuccinate lyase),and URA3(orotidine5P-phosphate de-carboxylase)genes[29].Each of these selectable markers has been incorporated into expression vectors.In addition, a series of host strains containing all possible combina-tions of ade1,arg4,his4,and ura3auxotrophies has been generated(Table2).2.4.Host strainsAll P.pastoris expression strains are derived from NRRL-Y11430(Northern Regional Research Laborato-ries,Peoria,IL).Most have one or more auxotrophic mu-tations which allow for selection of expression vectors containing the appropriate selectable marker gene upon transformation.Prior to transformation,all of these strains grow on complex media but require supplementa-tion with the appropriate nutrient(s)for growth on mini-mal media.2.4.1.Methanol utilization phenotypeMost P.pastoris host strains grow on methanol at the wild-type rate(Mut ,methanol utilization plus pheno-type).However,two other types of host strains are avail-able which vary with regard to their ability to utilize meth-anol because of deletions in one or both AOX genes. Strains with AOX mutations are sometimes better pro-ducers of foreign proteins than wild-type strains[30^32]. Additionally,these strains do not require the large amounts of methanol routinely used for large-scale fer-mentations of Mut strains.KM71(his4arg4aox1v:: SARG4)is a strain where AOX1has been partially deleted and replaced with the S.cerevisiae ARG4gene[15].Since the strain must rely on the weaker AOX2for methanol metabolism,it grows slowly on this carbon source (Mut s,methanol utilization slow phenotype).Another strain,MC100-3(his4arg4aox1v::SARG4aox2v:: Phis4),is deleted for both AOX genes and is totally unable to grow on methanol(Mut3,methanol utilization minus phenotype)[9].All of these strains,even the Mut3strain, retain the ability to induce expression at high levels from the AOX1promoter[32].2.4.2.Protease-de¢cient host strainsSeveral protease-de¢cient strains^SMD1163(his4pep4 prb1),SMD1165(his4prb1),and SMD1168(his4pep4)^ have been shown to be e¡ective in reducing degradation of some foreign proteins[23,33].This is especially noticeable in fermenter cultures,because the combination of high cellTable2P.pastoris host strainsStrain Genotype Reference Auxotrophic strainsY-11430wild-type NRRL aGS115his4[11]GS190arg4[16]JC220ade1[16]JC254ura3[16]GS200arg4his4[11]JC227ade1arg4[29]JC304ade1his4[29]JC305ade1ura3[29]JC306arg4ura3[29]JC307his4ura3[29]JC300ade1arg4his4[29]JC301ade1his4ura3[29]JC302ade1arg4ura3[29]JC303arg4his4ura3[29]JC308ade1arg4his4ura3[29] Protease-de¢cient strainsKM71v aox1::SARG4his4arg4[7]MC100-3v aox1::SARG4v aox2::Phis4his4arg4[9]SMD1168v pep4::URA3his4ura3[38]SMD1165prb1his4[38]SMD1163pep4prb1his4[38]SMD1168kex1::SUC2v pep4::URA3v kex1::SUC2his4ura3[34]a Northern Regional Research Laboratories,Peoria,IL.J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^6649density and lysis of a small percentage of cells results in a relatively high concentration of these vacuolar proteases. An additional protease-de¢cient strain SMD1168v pe-p4::URA3v kex1::SUC2his4ura3was recently devel-oped to inhibit proteolysis of murine and human endo-statin.Kex1protease can cleave carboxy-terminal lysines and arginines.Therefore,the deletion strain was generated to inhibit carboxy-terminal proteolysis.After40h of fer-mentation,puri¢cation of intact endostatin was achieved [34].Unfortunately,these protease-de¢cient cells are not as vigorous as wild-type strains with respect to PEP4.In addition to lower viability,they possess a slower growth rate and are more di¤cult to transform.Therefore,the use of protease-de¢cient strains is only recommended in situa-tions where other measures to reduce proteolysis have yielded unsatisfactory results.2.5.Integration of expression vectors into the P.pastorisgenomeExpression vectors are integrated into the P.pastoris genome to maximize the stability of expression strains. This can be done in two ways.The simplest way is to restrict the vector at a unique site in either the marker gene(e.g.,HIS4)or the AOX1promoter fragment and then to transform it into the appropriate auxotrophic mu-tant.The free DNA termini stimulate homologous recom-bination events that result in single crossover-type integra-tion events into these loci at high frequencies(50^80%of His transformants).The remaining transformants have undergone gene conversion events in which only the marker gene from the vector has integrated into the mu-tant host locus without other vector sequences. Alternatively,certain P.pastoris expression vectors can be digested in such a way that the expression cassette and marker gene are released,£anked by5P and3P AOX1 sequences.Approximately10^20%of transformation events are the result of a gene replacement event in which the AOX1gene is deleted and replaced by the expression cassette and marker gene.This disruption of the AOX1 gene forces these strains to rely on the transcriptionally weaker AOX2gene for growth on methanol[31],and,as a result,these strains have a Mut s phenotype.These gene replacement strains are easily identi¢ed among trans-formed colonies by replica-plating them to methanol and selecting those with reduced ability to grow on methanol. As mentioned previously,the potential advantage of Mut s strains is that they utilize less methanol and sometimes express higher levels of foreign protein than wild-type (Mut )strains,especially in shake-£ask cultures[15].2.6.Generating multicopy strainsOptimization of protein expression often,but not al-ways,includes the isolation of multicopy expression strains.A strain that contains multiple integrated copies of an expression cassette can sometimes yield more heter-ologous protein than single-copy strains[22,35].Three approaches lead reliably to multicopy expression strains in P.pastoris.As shown in Fig.4,the¢rst ap-proach involves constructing a vector with multiple head-to-tail copies of an expression cassette[23].The key to generating this construction is a vector which has an expression cassette£anked by restriction sites which have complementary termini(e.g.,Bam HI-Bgl II,Sal I-Xho I combinations).The process of repeated cleavage and reinsertion results in the generation of a series of vectors that contain increasing numbers of expression cas-settes.A particular advantage to this approach,especially in the production of human pharmaceuticals,is that the precise number of expression cassettes is known and can be recovered for direct veri¢cation by DNA sequencing.A second method utilizes expression vectors that con-tain the P.pastoris HIS4and the bacterial Tn903kan r genes.The bacterial kanamycin resistance gene also con-fers resistance to the related eukaryotic antibiotic G418 [36].The level of G418resistance can be roughly corre-lated to vector copy number.P.pastoris must¢rst be transformed to His prototrophy;then multicopy trans-formants are screened by replica-plating to plates contain-ing G418.This method results in a subset of colonies enriched for those containing multiple expression vector copies.However,the vector copy number varies greatly; thus,a signi¢cant number(50^100)of transformants must be subjected to further analysis of copy number and ex-pression level.By this approach,strains carrying up to30 copies of an expression cassette have been isolated[35].A third approach to constructing multicopy strains in-volves the use of a vector with the bacterial Sh ble gene, which confers resistance to the antibiotic zeocin[27].Un-like G418selection,strains transformed with expression cassettes containing the zeocin marker can be selected di-rectly by resistance to the drug.Additionally,populations of transformants can be enriched for multicopy expression cassette strains simply by plating on increased concentra-tions of zeocin in the selection plates.Also,because the Sh ble gene can serve as a selectable marker in both bacteria and yeast,these expression vectors are compact and con-venient to use.However,as with the G418selection,most transformants resistant to high levels of zeocin do not contain multiple vector copies,and numerous transform-ants must be screened for ones that do.2.7.High cell density growth in fermenter culturesP.pastoris is a poor fermenter,a major advantage rel-ative to S.cerevisiae.In high cell density cultures,ethanol (the product of S.cerevisiae fermentation)rapidly builds to toxic levels which limit further growth and foreign protein production.With its preference for respiratory growth,P.pastoris can be cultured at extremely high den-J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^66 50sities (500OD 600U ml 31)in the controlled environment of the fermenter with little risk of `pickling'itself.Fermenta-tion growth is especially important for secreted proteins,as the concentration of product in the medium is roughly proportional to the concentration of cells in culture.An-other positive aspect of growing P.pastoris in fermenter cultures is that the level of transcription initiated from the AOX1promoter can be 3^5times greater in cells fed meth-anol at growth-limiting rates compared to cells grown in excess methanol.Thus,even for intracellularly expressed proteins,product yields are signi¢cantly higher from fer-menter cultured cells.Also,methanol metabolism utilizes oxygen at a high rate,and expression of foreign genes is negatively a¡ected by oxygen limitation.Only in the con-trolled environment of a fermenter is it feasible to monitor and adjust oxygen levels in the culture medium.A hallmark of the P.pastoris system is the ease with which expression strains scale-up from shake-£ask to high-density fermenter cultures.Although some foreign pro-teins have expressed well in shake-£ask cultures,expres-sion levels are typically low compared to fermenter cul-tures.Considerable e¡ort has gone into the optimization of heterologous protein expression techniques,and de-tailed fed-batch and continuous culture protocols are available [23,37^39].In general,strains are grown initially in a de¢ned medium containing glycerol as its carbon source.During this time,biomass accumulates but heter-ologous gene expression is fully repressed.Upon depletion of glycerol,a transition phase is initiated in which addi-tional glycerol is fed to the culture at a growth-limiting rate.Finally,methanol or a mixture of glycerol and meth-anol is fed to the culture to induce expression.Thecon-Fig.4.Scheme for construction of vectors with multiple copies of a foreign gene expression cassette (from [22]).J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6651centration of foreign protein is monitored in the culture to determine time of harvest.The growth conditions for P.pastoris are ideal for large-scale production of heterologous protein,because the me-dium components are inexpensive and de¢ned,consisting of pure carbon sources(glycerol and methanol),biotin, salts,trace elements,and water.This medium is free of unde¢ned ingredients that can be sources of pyrogens or toxins and is therefore compatible with the production of human pharmaceuticals.Also,since P.pastoris is cultured in media with a relatively low pH and methanol,it is less likely to become contaminated by most other microorgan-isms.3.Post-translational modi¢cation of secreted proteinsA major advantage of P.pastoris over bacterial expres-sion systems is that the yeast has the potential to perform many of the post-translational modi¢cations typically as-sociated with higher eukaryotes,such as processing of sig-nal sequences(both pre and prepro type),folding,disul¢de bridge formation,certain types of lipid addition,and O-and N-linked glycosylation.3.1.Secretion signal selectionForeign proteins expressed in P.pastoris can be pro-duced either intracellularly or extracellularly.Because this yeast secretes only low levels of endogenous proteins, the secreted heterologous protein constitutes the vast ma-jority of total protein in the medium(Fig.5).Therefore, directing a heterologous protein to the culture medium can serve as a substantial¢rst step in puri¢cation.However, due to protein stability and folding requirements,the op-tion of secretion is usually reserved for foreign proteins that are normally secreted by their native hosts.In many cases,researchers simply need to take advantage of the pre-made expression cassettes available from Invitrogen. Using selected P.pastoris vectors,researchers can clone a foreign gene in frame with sequences encoding either the native signal,the S.cerevisiae K-factor prepro peptide, or the P.pastoris acid phosphatase(PHO1)signal. Although several di¡erent secretion signal sequences, including the native secretion signal present on heterolo-gous proteins,have been used successfully,results have been variable.The S.cerevisiae K-factor prepro peptide has been used with the most success.This signal sequence consists of a19-amino acid signal(pre)sequence followed by a66-residue(pro)sequence containing three consensus N-linked glycosylation sites and a dibasic Kex2endopep-tidase processing site[40].The processing of this signal sequence involves three steps.The¢rst is the removal of the pre signal by signal peptidase in the endoplasmic retic-ulum.Second,Kex2endopeptidase cleaves between Arg-Lys of the pro leader sequence.This is rapidly followed by cleavage of Glu-Ala repeats by the Ste13protein[41].The e¤ciency of this process can be a¡ected by the surround-ing amino acid sequence.For instance,the cleavage e¤-ciencies of both Kex2and Ste13proteins can be in£uenced by the close proximity of proline residues.In addition,the tertiary structure formed by a foreign protein may protect cleavage sites from their respective proteases.The S.cerevisiae K-MF prepro signal sequence is the classical and most widely used secretion signal(see Table 3,expressed proteins).In some cases,it is a better secre-tion signal for expression in P.pastoris than the leader sequence of the native heterologous protein.In a study concerning the expression of the industrial lipase Lip1 from Candida rugosa,the e¡ect of heterologous leader sequences on expression and secretion was investigated [42].It was found that the native Lip1p leader sequence allowed for secretion but somehow hampered expression. Either the K-factor pre or prepro signal was adequate for both secretion and expression,but the highest level of lipase secretion was from a clone with the full prepro sequence.This clone produced two species of secreted pro-tein.A small percentage was correctly processed to the mature protein.However,a majority of the product con-tained four additional N-terminal amino acids.Variability in the amino terminus is commonly seen with heterologous proteins secreted by P.pastoris using the K-factor prepro leader.In some cases,the standard K-MF or PHO1secretion signals have not worked,so synthetic leaders have been created.Martinez-Ruiz et al.[43]made mutations in the native leader to reconstruct a more e¤cient Kex2p recog-nition motif(Lys-Arg).This aided in secretion of the ri-bosome-inactivation protein K-sarcin from the mold As-pergillus giganteus.Another more drastic solution was to create an entirely synthetic prepro leader.For the expres-sion of human insulin,a synthetic leader and spacer se-quence was found to improve secretion and protein yield[44].Fig.5.Secreted expression of human serum albumin.7.5%SDS-PAGE of25-W l sample of culture supernatant from a P.pastoris strain(GS-HSA#4141)expressing human serum albumin.Cells were induced in BMMY(bu¡ered methanol-complex medium)for0,12,24,48,and ne M contains molecular mass markers(kDa).J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^66 52。

酵母菌发酵实验设计English Answer:The experiment on yeast fermentation can be designed as follows:1. Purpose: To investigate the effect of different sugar concentrations on yeast fermentation.2. Materials:Yeast culture.Different sugar solutions (e.g., glucose, fructose, sucrose)。

Water.Test tubes.Thermometer.Stopwatch.3. Procedure:a. Prepare different sugar solutions with varying concentrations. For example, prepare solutions with 5%, 10%, and 15% sugar concentrations.b. Label the test tubes accordingly.c. Add equal amounts of yeast culture to each test tube.d. Add the respective sugar solution to each test tube, ensuring that the sugar concentration is consistent.e. Mix the contents of each test tube gently.f. Place the test tubes in a warm environment (around 37°C) and start the stopwatch.g. Observe the fermentation process by noting the formation of bubbles or gas production.h. Record the time it takes for each sugar concentration to show signs of fermentation.i. Repeat the experiment multiple times to ensure accuracy and reliability of the results.j. Analyze the data collected and draw conclusions regarding the effect of sugar concentration on yeast fermentation.中文回答：酵母发酵实验可以设计如下：1. 目的，研究不同糖浓度对酵母发酵的影响。

热应激对奶山羊的影响及营养调控措施刘烨;周建川;彭点懿;何健;侯振;崔喜忠【摘要】热应激是近年来影响畜牧业发展的重要因素之一,尤其是对处于半开放养殖的奶山羊产业,夏季高温引起的热应激导致奶山羊产业损失巨大.本文综述热应激对奶山羊的影响和营养调控措施,以期引起生产和科研人员对奶山羊热应激危害重视,为夏季预防、缓解奶山羊热应激提供理论依据.【期刊名称】《现代畜牧兽医》【年(卷),期】2017(000)011【总页数】4页(P20-23)【关键词】奶山羊;热应激;营养调控;综述【作者】刘烨;周建川;彭点懿;何健;侯振;崔喜忠【作者单位】四川铁骑力士集团冯光德实验室,四川绵阳621006;四川铁骑力士集团冯光德实验室,四川绵阳621006;四川铁骑力士集团冯光德实验室,四川绵阳621006;四川铁骑力士集团冯光德实验室,四川绵阳621006;四川铁骑力士集团冯光德实验室,四川绵阳621006;四川铁骑力士集团冯光德实验室,四川绵阳621006【正文语种】中文【中图分类】S827近年来,全球气候持续性升温,根据政府间气候变化专门委员会(IPCC)此前预测,到本世纪末全球地表温度将再提升1.5～4.5℃.连续高温及空气潮湿极易引起家畜,尤其是自身代谢较快的奶畜热应激.哺乳动物都存在一个温度"舒适域",机体存在一个温度调控机制使体温维持在"舒适域"附近,一旦高温环境下体温偏离了这个区域,就会产生热应激,导致畜禽生理机能受损,对生产性能造成巨大影响.热应激影响畜禽生理机能的机制及缓解热应激的影响成为当今研究的热点.关于热应激对奶山羊生理生产影响及危害的研究不多,本文综述了奶山羊热应激的评估标准,热应激对奶山羊生理生产影响及缓解奶山羊热应激营养调控新措施.热应激是由环境高温刺激引起的机体自身调节反应,可能涉及神经系统、内分析系统及免疫系统等一系列活动.引起机体热应激的因素很多,包括环境因素及动物因素等.环境因素引发奶山羊热应激的主要因素,不同研究中对奶山羊热应激评估标准不同,多数热应激环境设定参考奶牛的热应激评价.农业部2013年发布《奶牛热应激评价技术规范》,通过牛舍环境温湿度指数(Temperature humidity index,THI)、呼吸频率及直肠温度三方面对遭受不同程度热应激的成年健康荷斯坦奶牛进行热应激评价.有研究指出奶山羊具有较宽的温度"舒适域"和较高的耐热性,加之奶山羊相对体表比小,自身代谢需求低,因此奶山羊较奶牛对热应激的承受能力更强[1].THI是目前比较通用的评价动物热应激程度指标.THI包括环境温度和相对湿度,是判断动物热应激程度的常用环境指标.不同研究选用奶山羊热应激评估标准不同(见表1).有研究指出THI并没有考虑日光照射等因素,实际的热应激指数远高于THI,因此对半露天放牧家畜产业THI并不完全准确[2].除环境因素外,不同品种奶山羊对热应激的抵抗能力不同.Brown等[3]研究对比阿尔卑斯山羊和努比亚山羊对热应激抵抗能力差异,结果表明在THI=79环境下连续饲喂5周显著降低了阿尔卑斯奶山羊的产奶量,但对努比亚奶山羊产奶量影响不大.Lallo等[4]也得到了相似的结果.究其原因可能是不同品种奶山羊之间体温调节能力和抗热应激能力不同.高温地区当地奶山羊品种对本地环境有较强的适应性,而外来引入品种奶山羊可能易受热应激影响,而且不同品种间奶山羊形态差异也会对耐热性和体温调节起到重要作用[5].影响奶山羊耐热因素很多,THI并不能准确、直观反映出奶山羊是否处于热应激状态,在评估奶山羊热应激还应参考奶山羊其他生理指标变化,如直肠温度和呼吸速率等.马燕芬等[7]采用8只泌乳中后期奶山羊,研究热应激对奶山羊生理指标影响.结果表明热应激奶山羊呼吸频率从43次/min提高至131次/min,且直肠温度提升了近2.0℃.王立志[13]也得到类似的结果.Hamzaoui等[14]研究不同THI下山羊生理指标变化,结果表明当THI在77～85,其直肠温度较对照组上升0.58℃,呼吸速率提高48次/min,饮水量和水分蒸发量分别上升了77%和207%.究其原因可能是热应激增加了奶山羊耗水量,增加的饮水量主要通过出汗和呼吸蒸发并伴随热量散失,奶山羊喘气与出汗不同,喘气散发水分和热量不会导致机体失去矿物质盐,当外界温度超过30℃通过此途径散发热占奶山羊散发热量的80%以上[16].外界环境温度升高刺激奶山羊外周热感受器传递抑制性神经冲动至下丘脑内食欲中枢,抑制外侧食欲中枢,导致采食量下降,因此热应激显著降低奶山羊采食量,外界温度超过40℃,采食量下降21%～35%[14-15,17].降低采食和反刍也是反刍动物减少自身产热的有效方式.也有研究表明,热应激通过降低奶山羊采食时间和采食速度调整采食量,对采食次数影响不显著[18].Hamzaoui等[15]研究发现热应激能够显著提高奶山羊DM和ADF消化率.这与Hirayama等[19]在母羊上的研究相近.其原因可能是热应激导致采食量下降,营养物质经过消化道的速率下降,消化率升高[20].热应激对奶山羊消化率的影响结果不一,马燕芬等[7]研究结果表明热应激导致奶山羊瘤胃形态结构发生变化,消化吸收能力下降,DM、CP、NDF和ADF等消化率显著下降.许啸等[12]研究发现热应激显著降低奶山羊瘤胃干物质降解率(P<0.05),热应激降低奶山羊采食量和饲料转化效率.Hamzaoui等[14]研究发现热应激并不影响泌乳前期奶山羊DM、OM、NDF 和ADF等消化率.不同奶山羊品种、健康状况及泌乳阶段可能是导致消化率结果不同的主要原因.热应激对奶山羊生产性能影响的报道不多.劳雪芬[21]研究发现热应激使奶山羊奶产量下降近10%.尽管热应激对奶牛和奶山羊采食量的影响相近(21%～35%),但是热应激对奶牛奶产量的影响(27%～33%)远高于其对奶山羊的影响(3%～13%)[22-23].马燕芬等[7]研究发现热应激会显著降低奶山羊奶产量、乳蛋白和乳脂含量.这与Hamzaoui等[14]结论一致.然而Hamzaoui等[15]研究结果发现热应激并未影响奶山羊奶产量.这可能与试验设定条件及奶山羊泌乳阶段有关.NRC[24]指出在热应激状态下山羊的维持需要升高了30%.热应激降低奶山羊采食量,能量摄入量下降,无法满足日常生产需要,尤其对于泌乳早期奶山羊.应激会降低泌乳早期奶山羊奶产量9%,乳脂率下降12%;泌乳晚期奶山羊奶产量下降3%,乳脂率下降1%[14].营养调控是缓解奶山羊热应激的常用方法之一,近年来研究人员尝试使用多种新型添加剂来缓解奶山羊热应激.许啸等[12]通过日粮添加有机铬研究热应激奶山羊瘤胃发酵指标变化,结果表明日粮添加有机铬显著提高热应激奶山羊瘤胃乙酸、丁酸浓度,降低丙酸浓度,平衡瘤胃发酵模式,缓解热应激带来不良影响.Wang等[25]研究补饲100 IU维生素E和15 g酵母培养物对奶山羊热应激的缓解作用,结果表明维生素E和酵母培养物不影响热应激奶山羊采食量,但能够显著降低奶山羊直肠温度及门静脉和颈动脉内毒素浓度,减少因内毒素吸收引起的机体氧化损伤.中草药能够显著减低机体白细胞介素及其他激素水平,缓解热应激对家畜的危害[26-27].劳雪芬[21]研究日粮添加女贞子粉对奶山羊热应激缓解作用,结果表明日粮添加女贞子粉能提高热应激奶山羊奶产量,增加热应激奶山羊红细胞数和血红蛋白含量,缓解奶山羊热应激状态.黎智峰[28]对山羊饲喂不同含量的苦丁茶提取物,发现添加600 ppm 苦丁茶提取物能够提升热应激山羊抗氧化能力和瘤胃pH值,降低热应激山羊直肠温度和呼吸频率.近年来,我国奶山羊产业发展迅猛,热应激严重降低奶山羊生产性能,损害机体健康,成为制约我国奶山羊产业发展主要问题之一.如何准确评估奶山羊热应激程度,正确了解热应激奶山羊健康和生产性能的危害,是解决奶山羊热应激难题的基础.尽管奶山羊和奶牛主要生理构造类似,但缓解奶牛热应激营养调控措施是否同样适用于奶山羊产业还有待进一步研究.【相关文献】[1]Silanikove N,Koluman N.Impact of climate change on the dairy industry in temperate zones:Predications on the overall negative impact and on the positive role of dairy goatsinadaptationtoearthwarming[J].Small Ruminant Research,2015,123(1):27-34.[2]Silanikove N.Adaptation of domesticated ruminant to harsh environments[J].2006[3]Brown D L,Morrison S R,Bradford G E.Effects of Ambient Temperature on Milk Production of Nubian and Alpine Goats[J].Journal of Dairy Science,1988,71(9):2486-2490.[4]Lallo C H,Paul I,Bourne G.The rmoregulation and performance of British Anglo-Nubian and Saanen goats reared in an intensive system in Trinidad[J].Tropical AnimalHealth&Production,2012,44(3):491.[5]Maia A S C,Silva R G D,Nascimento S T,et al.Thermoregulatory responses of goats in hot 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果寡糖在牛生产中的研究与应用吴峰洋;霍珊珊;李冲;高丽晓;贾丽楠;谷子林【摘要】This article reviewed the effect of fructooligosaccharide on growth performance and immune performance of cattle,to rovide reference for its further application in cattle production.%本文就果寡糖对牛生长性能和免疫性能的改善作用作一综述,为果寡糖在牛生产中的进一步应用提供参考.【期刊名称】《中国饲料》【年(卷),期】2015(000)021【总页数】3页(P9-11)【关键词】果寡糖;生长性能;免疫性能【作者】吴峰洋;霍珊珊;李冲;高丽晓;贾丽楠;谷子林【作者单位】河北农业大学动物科技学院,河北保定 071001;河北农业大学动物医学院,河北保定 071001;河北农业大学动物科技学院,河北保定 071001;河北农业大学动物科技学院,河北保定 071001;河北农业大学动物科技学院,河北保定 071001;河北农业大学动物科技学院,河北保定 071001;河北省山区农业工程技术研究中心,河北保定 071001【正文语种】中文【中图分类】S816.7果寡糖又称作黄金双歧因子糖、低聚果糖等，是一类由D-果糖分子通过β-1，2糖苷键与蔗糖连结而成的低聚糖的总称，在饲料中主要以寡果三糖、寡果四糖和寡果五糖的形式添加。

研究表明，果寡糖不仅能提高畜禽对营养物质的吸收利用效率，改善畜禽的生产性能，还能促进畜禽肠道微生物区系平衡，选择性地增殖肠道有益菌，抑制有害菌，提高畜禽的抗病能力。

目前，对果寡糖的相关研究主要集中在单胃动物上，在反刍动物上的应用研究报道较少。

本文就果寡糖对牛生长性能和免疫性能的改善作用作一综述，为果寡糖在牛生产中的进一步应用提供参考。

浙江大学学报（农业与生命科学版）48（4）：517~524，2022Journal of Zhejiang University (Agric.&Life Sci.)http ：///agr E -mail ：zdxbnsb @日粮添加硒代蛋氨酸锌对泌乳盛期奶牛泌乳性能和血浆生化指标的影响陈一1，冀飞2，刘建新1，王迪铭1*（1.浙江大学动物科学学院奶业科学研究所，杭州310058；2.美国金宝动物营养（国际）有限公司，美国明尼苏达州伊登普雷里市55344）摘要本试验旨在研究日粮中添加硒代蛋氨酸锌对泌乳盛期奶牛泌乳性能和代谢状态的影响。

选取60头健康的中国荷斯坦经产泌乳盛期奶牛，根据泌乳时间、体质量、胎次和产奶量相近的原则，每4头条件相近的奶牛为一个区组，区组内随机分成4组，每处理组15头奶牛。

4个处理组分别为对照组（基础日粮，不添加硒代蛋氨酸锌）和硒代蛋氨酸锌添加组（在基础日粮中分别添加含0.1、0.2、0.3mg/kg Se 的硒代蛋氨酸锌，按干物质量计）；预饲期2周，正式试验期12周。

结果显示：1）随着硒代蛋氨酸锌添加量的增加，奶牛的奶产量、能量校正乳、乳脂产量、乳蛋白产量、乳糖产量和饲料效率呈线性增加（P <0.01）。

2）随着硒代蛋氨酸锌添加量的增加，奶牛乳脂含量、乳糖含量和乳中硒含量呈线性或二次增加，乳蛋白含量呈线性降低（P <0.05）。

3）随着硒代蛋氨酸锌添加量的增加，奶牛血浆中谷胱甘肽过氧化物酶活性、过氧化氢酶活性呈线性或二次增加（P <0.05），而血浆中丙二醛含量呈线性或二次降低（P <0.01）。

综上所述，日粮中添加硒代蛋氨酸锌能够有效改善泌乳盛期奶牛的生产性能和抗氧化能力，提示来源于硒代蛋氨酸锌的硒能被泌乳奶牛有效利用并发挥其生物学效应，是奶牛日粮的有效硒源。

关键词硒代蛋氨酸锌；荷斯坦奶牛；生产性能；泌乳盛期；抗氧化能力中图分类号S 823.91文献标志码A引用格式陈一,冀飞,刘建新,等.日粮添加硒代蛋氨酸锌对泌乳盛期奶牛泌乳性能和血浆生化指标的影响[J].浙江大学学报(农业与生命科学版),2022,48(4):517-524.DOI:10.3785/j.issn.1008-9209.2021.06.292CHEN Yi,JI Fei,LIU Jianxin,et al.Effect of dietary supplementation of Zn -L -selenomethionine on lactation performance and plasma biochemical indexes of dairy cows in peak lactation period[J].Journal of Zhejiang University (Agriculture &Life Sciences),2022,48(4):517-524.Effect of dietary supplementation of Zn -L -selenomethionine on lactation performance and plasma biochemical indexes of dairy cows in peak lactation periodCHEN Yi 1,JI Fei 2,LIU Jianxin 1,WANG Diming 1*(1.Institute of Dairy Science,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China;2.Zinpro Animal Nutrition (International)Inc.,Eden Prairie 55344,Minnesota,USA )AbstractThe objective of this study was to investigate the effect of dietary supplementation of Zn -L -selenomethionine (Zn -L -SeMet)on lactation performance and metabolic status of dairy cows at peak lactation period.Sixty multiparous Chinese Holstein dairy cows at peak lactation were selected.The cows were divided intoDOI ：10.3785/j.issn.1008-9209.2021.06.292基金项目：中央高校基本科研业务费专项资金（2021FZZX001-32）。

肖敏敏，邢馨月，刘文光，等. 耐酒精高产L-乳酸菌株的筛选及发酵培养基优化[J]. 食品工业科技，2023，44（8）：135−143. doi:10.13386/j.issn1002-0306.2022040147XIAO Minmin, XING Xinyue, LIU Wenguang, et al. Screening of an Alcohol Tolerant and High-yield L-lactic Acid Strain and Optimization of Culture Medium[J]. Science and Technology of Food Industry, 2023, 44(8): 135−143. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022040147· 生物工程 ·耐酒精高产L-乳酸菌株的筛选及发酵培养基优化肖敏敏1，邢馨月1，刘文光2，孟祥慧1，魏姗姗1，张天笑1，王玉华1，李　侠1,*（1.吉林农业大学食品科学与工程学院，吉林长春 130118；2.华信检测技术有限公司，吉林长春 130000）摘　要：为提高L-乳酸产量，降低L-乳酸的生产成本，该研究经过筛选、驯化获得一株耐酒精且高产L-乳酸的菌株鼠李糖乳杆菌AK-0779。

使用玉米酒糟代替部分酵母粉作为菌株AK-0779发酵培养基的氮源。

在单因素实验基础上，对葡萄糖添加量、酵母粉添加量和玉米酒糟添加量进行三因素三水平响应面优化试验。

结果表明，最适发酵培养基为：葡萄糖添加量9.80%，玉米酒糟添加量0.98%，酵母粉添加量1.72%，L-乳酸产量为78.91 g/L ，糖酸转换率为80.52%。

与酵母粉完全充当氮源产L-乳酸82.36 g/L 相比，产量无显著差异，说明玉米酒糟能有效代替部分酵母粉作为发酵培养基的氮源，降低L-乳酸生产成本。

Journal of Environmental Sciences 20(2008)94–100E ffects of culture conditions on ligninolytic enzymes and protease productionby Phanerochaete chrysosporium in airXIONG Xiaoping,WEN Xianghua ∗,BAI Yanan,QIAN YiState Key Joint Laboratory of Environmental Simulation and Pollution Control,Department of Environmental Science and Engineering,Tsinghua University,Beijing 100084,China.E-mail:xxp02@Received 21March 2007;revised 22April 2007;accepted 28April 2007AbstractThe production of ligninolytic enzymes and protease by Phanerochaete chrysosporium was investigated under di fferent culture conditions.Di fferent amounts of medium were employed in free and immobilized culture,together with two kinds of medium with di fferent C /N ratios.Little lignin peroxidase (LiP)(<2U /L)was detected in free culture with nitrogen-limited medium (C /N ratio:56/2.2,in mmol /L),while manganese peroxidase (MnP)maximum activity was 231and 240U /L in 50and 100ml medium culture,respectively.Immobilized culture with 50ml nitrogen-limited medium gave the highest MnP and LiP production with the maximum values of 410and 721U /L separately on the day 5;however,flasks containing 100ml nitrogen-limited medium only produced less MnP with a peak value of 290U /paratively,carbon-limited medium (C /N ratio:28/44,in mmol /L)was adopted in culture but produced little MnP and LiP.Medium type had the greatest impact on protease rge amount of protease was produced due to glucose limitation.Culture type and medium volume influence protease activity corporately by a ffecting oxygen supply.The results implied shallow immobilized culture was a possible way to gain high production of ligninolytic enzymes.Key words :protease;culture conditions;ligninolytic enzymes;Phanerochaete chrysosporiumIntroductionThe white rot fungus Phanerochaete chrysosporium has been extensively studied because of its powerful ligni-nolytic enzymes.These enzymes,mainly including lignin peroxidase (LiP)and manganese peroxidase (MnP),are secreted during the secondary metabolism triggered by carbon,nitrogen or sulfur limitation (Je ffries et al .,1981;Tien and Kirk,1983,1988).They have been demonstrated to play a crucial role in lignin degradation and showed great potential in paper industry.At the same time,more and more researches have revealed that the ligninolytic enzymes are nonspecific enzymes and can assist in the degradation of a wide variety of recalcitrant organic pol-lutants,such as polycyclic aromatic hydrocarbons (PAHs),pesticide and dyes,as has been reviewed by Cameron et al .(2000).This raised the interest of study in ligninolytic enzymes production.To realize application of ligninolytic enzymes,a large production of the biocatalysts at low cost is needed (Ca-baleiro et al .,2002).However,most laboratory studies have been conducted in pure oxygen or in an oxygen-enriched environment (Dosoretz et al .,1990a;Zhen and Yu,1998),which increased the production cost.An ef-fective synthesis of ligninolytic enzymes in air would imply lower cost and greater feasibility essential for their*Corresponding author.E-mail:xhwen@.large scale production (Yu et al .,2006).So far,successful culture of P .chrysosporium with high ligninolytic enzymes production in air has rarely been reported.On the other hand,significant losses of enzyme activity occurred during all cultivations,which prevent enzymes accumulation in crude fermentation product.The simultaneous secretion of proteolytic enzymes (protease)could have caused the low stability of produced peroxidases although di fferent viewpoints still exist (Cabaleiro et al .,2001,2002;Chung et al .,2005;Dass et al .,1995;Dosoretz et al .,1990b,c;Jimenez et al .,2003;Pascal et al .,1993).Further study about protease production and its relationship with ligni-nolytic enzymes production in di fferent culture conditions is needed to assist in directing fermentation process design.In the present report,the fermentation was carried out in batches under air atmosphere.Di fferent volumes of both carbon-limited and nitrogen-limited medium were tested in both free and immobilized cultures.Protease and ligninolytic enzymes were measured during the whole fer-mentation ter,the e ffects of culture conditions,including culture type (free or immobilized),medium species (carbon-limited or nitrogen-limited)and volumes,on protease production,as well as the relationship between protease and ligninolytic enzymes,were discussed based on the results.No.1Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air951Materials and methods1.1StrainPhanerochaete chrysosporium strain BKM-F-1767 (ATCC24725)was maintained at37°C on PDA plates. 1.2CarriersPolyurethane foam cubes of5-mm per side(Dongfang Polyurethane Foam Co.,Beijing,China)were employed as the support in immobilized cultures.Prior to use,they were treated by boiling for10min and washing thoroughly three times with distilled water.After that,the carriers were dried at room temperature overnight and autoclaved at121°C for20min(Couto et al.,2002a).1.3Culture conditionsThe nitrogen-limited medium was prepared based on that described by Tien and Kirk(1988)with10g/L glucose as carbon source,except that the dimethylsuccinate was replaced by20mmol/L acetate buffer(pH4.5).Veratryl alcohol1.5mmol/L was introduced at the beginning of cul-tures and no surfactant was added(Couto and Ratto,1998). Seven-day-old spores were harvested in sterilized water,filtered through glass-wool and adjusted to absorbance of0.5at650nm.This spore suspension(about2.5×106 spores/ml)was used for inoculation(Urek and Pazarlioglu, 2004).In carbon-limited medium,C/N ratio was altered from 56/2.2(in mmol/L nitrogen-limited medium)to28/44(in mmol/L)(Yu et al.,2005)and the other components were the same.1.3.1Free culturesTwo series of medium volume(100ml,50ml)were adopted in250-ml Erlenmeyerflasks,100-ml and50-ml. When100ml medium was added,4ml spore suspension (maintained before)was used for inoculum and half of that was added in theflasks with50ml medium.Cultures were incubated in air at37°C in a rotary shaker with an agitation speed of160r/min.1.3.2Immobilized culturesIn immobilized culture,1.8g carriers were added in 250-ml Erlenmeyerflask containing100ml medium.After addition,the carriers were in critical immerged status. Comparably,0.9g carriers were added inflask containing 50ml medium.The inoculum size was the same as that adopted in free culture.1.4Analytical methodsLignin peroxidase(LiP)activity was measured as de-scribed by Tien and Kirk(1988),with one unit defined as1µmol veratryl alcohol oxidized to veratraldehyde per minute.Manganese peroxidase(MnP)activity was measured spectrophotometrically by the method of Paszczynski et al. (1988),using Mn2+as the substrate.One unit was defined as the amount of enzyme that oxidized1µmol Mn2+per minute.Protease activity was measured with azocoll(Sigma Chemical Co.,USA)as the substrate in50mmol/L acetate buffer,as described by Dosoretz et al.(1990b). Nitrogen ammonium content was determined by the phenol-hypochlorite method at625nm as described by Weatherburn(1967),using ammonium sulfate as a stan-dard.Reducing sugars were determined by dinitrosalicylic acid method at540nm as described by Ghose(1987), using D-glucose as a standard.2Results2.1Free cultures2.1.1Cultured with different volumes of N-limitedmediumIn the250-ml Erlenmeyerflasks with50or100ml nitrogen-limited medium,hyphal pellets were formed since the day2.After the pellets grew up to about5mm in diameter,spurs began to appear and MnP activity emerged in the medium ever since.Different volumes of medium inflasks led to different nutrition consumption rates(Fig.1).On one hand,am-monium nitrogen was totally consumed in2d when50 ml medium was added,but it was depleted on day3in cultures with100ml medium.On the other hand,faster average glucose consumption rate was found in50ml-medium cultures,which are0.407and0.845g/(L·d)during the primary metabolism phase(0–4d)and secondary metabolism phase(5–9d).In100ml-medium culture, glucose consumption rates are0.337and0.810g/(L·d) during these two phases.In these two cultures,protease activity curves were approximately the same with maximum value of around 2.4U/ml on the day6.However,the time when MnP ar-rived its maximum and its peak value were quite different. Fermented with50ml medium,MnP activity was detected since the day3and peaked on the day4with a value of 231U/L.It sharply decreased after that.While in culture containing100ml medium,MnP appeared since the day4 and reached its maximum value of240U/L on the day7. Very few LiP activities were detected in these two culture systems(less than2U/L).In both cultures,after the peak MnP activity the medium became more and more viscous because of the secretion of extracellular polysaccharide and the hyphal pellets began to disaggregate.2.1.2Cultured with different volumes of C-limitedmediumIn C-limited medium free cultures,hyphal pellets also formed since the day2,but they persisted for a longer time compared to those in N-limited medium free culture mentioned above.Ammonium nitrogen all reached its minimum on the day4and kept stable thereafter,but glucose was depleted more rapidly when50ml medium was employed(Fig.2).In spite of different volumes added in theflasks,protease96XIONG Xiaoping et al.V ol.20Fig.1Glucose,ammonium nitrogen concentration and MnP,LiP,protease activity curves during the free culture with N-limited medium (C /N ratio is 56/2.2,in mmol /L).In 250-ml Erlenmeyer flask,100ml (a)and 50ml (b)medium were added,respectively.Fig.2Glucose,ammonium nitrogen concentration and MnP,LiP,protease activity curves during the free culture with C-limited medium (C /N ratio is 28/44,in mmol /L).In 250-ml Erlenmeyer flask,100ml (a)and 50ml (b)medium were added,respectively.activity curves showed the same trend during the culture.When P .chrysosporium entered the secondary metabolism period,protease activity began to increase (Fig.2),which finally arrived maximum (10U /ml for 100ml medium culture and 7U /ml for 50ml medium culture)when glucose concentration in the culture broth was low (less than 1g /L).MnP and LiP activities were low no matter 50ml or 100ml medium was added in culture.MnP activity was less than 10U /L and LiP was less than 2U /L.2.2Immobilized cultures2.2.1Cultured with di fferent volumes of N-limitedmediumSince the immersion status of the support in the medium had e ffect on ligninolytic enzymes production (Yu et al .,No.1E ffects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air 972005),in 50and 100ml nitrogen-limited medium,0.9and 1.8g polyurethane foam carriers were added,respectively.This made both of these two culture systems in critical immersed conditions.Fewer mediums in the flask promoted ammonium nitro-gen and glucose consumption by fungus.In 50ml-medium system ammonium nitrogen was depleted on the day 1while it cost 2d for complete nitrogen consumption in 100ml-medium system.Glucose disappeared at a rate of 1.372g /(L ·d)during the whole fermentation process in 50ml-medium flask,which is faster than the rate of 0.911g /(L ·d)in 100ml-medium flask (Fig.3).Comparing results got in free and immobilized cultures,we can see that immobilization greatly improved nutrition take-in speed.Di fference was also found in MnP,LiP and protease activity in these two systems.Maximum MnP activity of 410U /L was reached on the day 5in flasks containing 50ml medium,while in 100ml-medium culture,321U /L MnP was produced on the day 10.MnP in the latter system was more stable compared to former MnP activity,although its peak value was less.The highest LiP activity was attained in culture system with 50ml medium.On the day 5,it reached 721U /L.However,the other system did not produce LiP at all.Protease activity curve in 50ml-medium system showed 2peaks (1.43U /ml on the day 2,and 0.8U /ml on the day 8),which were nominated primary protease peak and secondary protease peak in time sequence (Rothschild et al .,1999).Actually it was very low compared to that in 100ml-medium system.In the 100ml medium system,protease activity was higher than 3U /ml since the day 3.The viscosity of the culture broth gradually increased with the culture age for extracellular polysaccharide pro-duction as happened in free culture.2.2.2Cultured with di fferent volumes of C-limitedmediumIn the immobilized culture,carbon-limited medium was also employed to replace nitrogen-limited medium.As an e ffect of immobilization,nutrition disappeared quickly in the medium.Glucose was depleted on the day 2and ammonium nitrogen reached its minimum at the same time in both systems (Fig.4).Under these culture conditions,ligninolytic enzymes productions were comparatively low.In 50ml-medium flasks the maximum MnP activity of 33U /L was achieved and maximum LiP was 37U /L.Less MnP and LiP were produced in 100ml-medium culture,their maximum was 22U /L and 3.1U /L,respectively (Fig.4).With carbon-limited medium,the culture broth kept clear from the beginning till the end of the culture.3Discussion3.1Ligninolytic enzymes productionTo promote ligninolytic enzymes production in large scale,harvesting these products during fermentation in air is undoubtedly of great significance.MnP formation was generally less a ffected by oxygen level (Rothschild et al .,1999)and its production can be easily achieved in flasks or reactors with commonly used nitrogen-limited medium when exposed to air (Couto et al .,2001).This viewpoint is also demonstrated in the present research because of high MnP activity in all cultures with nitrogen-limited medium.However,LiP was seldom produced with the same medium without pure oxygen exposure (Rothschild et al .,1999;Couto et al .,2002b).In this report,the influ-ences of medium type,culture type and mediumvolumeFig.3Glucose,ammonium nitrogen concentration and MnP,LiP,protease activity curves during the immobilized culture with N-limited medium (C /N ratio is 56/2.2,in mmol /L).In 250-ml Erlenmeyer flask;(a)100ml medium and 1.8g polyurethane carriers;(b)50ml medium and 0.9g polyurethane carriers were added,respectively.98XIONG Xiaoping et al.V ol.20 Fig.4Glucose,ammonium nitrogen concentration and MnP,LiP,protease activity curves during the immobilized culture with C-limited medium(C/N ratio is28/44,in mmol/L).In250-ml Erlenmeyerflask,(a)100ml medium and1.8g polyurethane carriers,(b)50ml medium and0.9g polyurethane carriers were added,respectively.on ligninolytic production were studied.Meanwhile,by employing shallow immobilized culture we synchronously got high level of MnP and LiP production in air with nitrogen-limited medium.When different amounts of medium were employed in cultures,the transfer of oxygen from the air to biomass could be influenced(Couto et al.,2000).As the oxygen transfer speed may be the main limitation for fungus growth in liquid culture,the metabolite including ligni-nolytic enzymes and its deactivators could be released at different time.In our result,the free culture with50 ml nitrogen-limited medium gave maximum MnP activity earlier and MnP activity decreased more quickly(Fig.1). Zhang(1999)reported similar phenomenon for LiP when 50and90ml medium were employed separately in250-ml flasks.In immobilized culture,the polyurethane foam helped the fungus stretch out its hypha.This made it easier to assimilate nutrition and secret metabolites compared to the structure of fungal pellets.Actually,ammonium nitrogen and glucose consumption rates were higher when fungus hyphal was immobilized under all culture conditions in our experiment.Higher MnP activity was attained with the favor of carriers in both50and100ml nitrogen-limited medium.What is more,much LiP was produced in immobilized50ml-medium culture(Fig.3).On the other hand,immobilization can effectively reduce the shear stress which is reported to have inverse effect on LiP production and stability.The only high LiP production in immobilized50ml-medium culture reconfirmed the crucial importance of shear stress control and oxygen supply during LiP production process.The results also implied that culture under shallow immobilized conditions was a possible way to gain high activity of ligninolytic enzymes in air.Nitrogen-limited medium seems to be a better substrate because in these cultures higher ligninolytic enzymes production was achieved.However,because of the exces-sive glucose,production of polysaccharide during the late fermentation period made the broth viscous(Rothschild et al.,1999).It could hamper the diffusion of oxygen and other nutrient,production and secretion of ligninolytic en-zymes would be inhibited in succession.In order to make ligninolytic enzymes production more stable or realize its accumulation in nitrogen-limited culture,measures should be taken to avoid the influence of polysaccharide.3.2Protease productionSince protease is a factor which may influence ligninolytic enzymes stability during the culture of P. chrysosporium,studying the effect of culture conditions on protease production is helpful to direct fermentation process aiming at stable enzymes production.Of all the three factors involved in this research,medium type has the greatest effect on protease production.In all culture conditions when medium type was the only difference,protease production was always higher in C-limited system than that in N-limited system.The highest protease activity was achieved in immobilized culture with 100ml C-limited medium inflask.In addition,protease curves were about the same with the same medium in free culture.This result implies P.chrysosporium secreted more protease in response to glucose starvation although both glucose and ammonium limitation could stimulate protease secretion.Another common feature for C-limited system is that theNo.1Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air99protease maximum appeared when glucose was complete-ly consumed and all protease curves have no obvious peaks for primary and secondary protease.So far as we know, there is no report about protease production when cultured with C-limited medium.In free culture with N-limited medium and pure oxygenflushing,Dosoretz et al.(1990b) tried three kinds of initial glucose concentration.Their results showed that when glucose was depleted protease concentration increased,which was regarded as secondary protease.However,Dass et al.(1995)employed a medium containing excessive nitrogen source in their research and they suggested protease produced during the whole process was primary protease.So,we cannot determine which type the protease secreted in C-limited system belongs.The culture type and medium volume might act corpo-rately because they both can influence oxygen supply to biomass which was reported to be a factor for protease secretion(Dosoretz et al.,1990b;Zhen and Yu,1998). Immobilization favored biomass growth,which also in-creased nutrition,including glucose,ammonium nitrogen and oxygen consumption rate,as well as oxygen demand. Fewer medium inflask means more effective oxygen transfer with the same shaking speed.In immobilized culture,more oxygen is needed and100ml medium system cannot meet this requirement.Fungus was in status of oxygen starvation(measured DO concentration was about 0.2mg/L)and subsequently secreted more protease(Figs.3 and4).Less difference in free cultures with50ml medium and100ml medium was found because less oxygen was needed(Figs.1and2).The protease in immobilized culture with50ml N-limited medium had the lowest activity(1.4U/ml)in our experiment,which is also the lowest in all the re-ports we have found.Dosoretz et al.(1990b)measured the primary and secondary protease maximum activity in submerged liquid culture,which was about6U/ml. This could have been caused by pure oxygenflushing as increased oxygenation simultaneously increased protease activity(Dosoretz et al.,1990b).This also can explain why protease was above20U/ml in solid-state culture (Cabaleiro et al.,2002).Another comparatively low pro-tease activity(3.5U/ml)was observed in free culture of P.chrysosporium under air atmosphere.These results indicate that protease production can be minimized with proper culture conditions in air.3.3Relationship between protease and ligninolytic en-zymesAlthough the culture conditions affected both ligninolyt-ic enzymes and protease production,relationship between protease and ligninolytic enzymes was discussed in many research reports.Protease wasfirstly found to cause LiP degradation by Dosoretz et al.(1990b,c)and secondary protease was used in his experiment.Another research proved that the primary protease could totally denatured LiP(Pascal et al., 1993).In our result,high LiP production was only realized in the immobilized culture with50ml N-limited medium, where the lowest primary and secondary protease activity was detected.This indicated that protease produced during the culture is an important factor which reduces LiP production and it needs to be regulated to achieve higher LiP production in P.chrysosporium fermentation.It is reported that protease could be inhibited with addition of substances,such as PMSF(phenyl methane-sulfongl fluoride)and glucose(Dosoretz et al.,1990b)and conse-quently ligninolytic enzymes could be reproduced in the culture(Yu et al.,2005).Our experimental results proved that protease secretion could be controlled by adopting proper culture conditions,which subsequently led to high LiP production.The relationship between protease and MnP is more complicated.Cabaleiro et al.(2001)presented the view-point that MnP activity is inhibited by extracellular protease with the fact that MnP was more stable when protease activity was inhibited.However,recently another viewpoint came up that thefirst peak of extracellular protease helped MnP secretion by participating in hy-phal autolysis steps,while the protease produced during late idiophase would play a role in the decline of MnP (Jimenez et al.,2003).Although the MnP and protease were reversely correlated in some cultures(e.g.Fig.1b), MnP still increased when protease activity was high(e.g. Fig.1a).It implies that protease isn’t a main factor for MnP production and stability.AcknowledgementsThis work was supported by the National Natural Sci-ence Foundation of China(No.50478010). 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(Aerobic Yeast Propagation)Drying9-28-200110-31-2001 11-28-200112-5-2001达农威酵母培养物中含有上百种成分。

上图是其水溶性成分的高压液相色谱的图谱。

样本在280nm 下进行的分析。

There are hundreds of compounds in Diamond V yeast culture. This HPLC graph of the water extract shows peaks throughout the trace. The sample was analyzed at 280 nm.达农威酵母培养物的代谢产物种类列于下图。

这些代谢产物可改善肠道微生物区系和改变免疫状态。

是一种真正的含有许多有效成分的复杂产品。

The nature of Diamond V yeast culture is shown here. Many of thesecompounds may contribute to improved gut microflora and changes in immune status. It’s truly a complex product with many contributing components.and Kolver. 2001.DVRAMM, 2002. 1987. J. Dairy Sci. 70:2063-2068.M. elsdeniiand Martin. 1997n = 1,300Partial TotalHemi Yoon and Garrett. 1998Screen size, mm From Harrison et al., 2006Parker, 1994. MAFF fact sheet。