《生物技术与人类》课程教学资源（经典阅读）Applied modern biotechnology for cultivation of Ganoderma

Appl Microbiol Biotechnol (2012)93:941-963 D0110.1007/s00253-011-3780-7 MINI-REVIEW Applied modern biotechnology for cultivation of Ganoderma and development of their products Xuan-Wei Zhou.Kai-Qi Su.Yong-Ming Zhang Received:21 October 2011/Revised:18 November 2011 /Accepted:21 November 2011/Published online:15 December 2011 C Springer-Verlag 2011 Abstract A white-rot basidiomycete Ganoderma spp.has (or Ganodermaceae)of Aphyllophorales.is one of the most long been used as a medicinal mushroom in Asia,and it has popular medicinal mushrooms in China,Japan,Korea,and an array of pharmacological properties for immunomodula- other Asian countries.It has been under modern biochemi- tory activity.There have been many reports about the bio- cal and pharmacological research during the last 30 years active components and their pharmacological properties.In (Gao et al.2006).However,in the earliest Chinese litera- order to analyze the current status of Ganoderma products, tures,this medicinal mushroom is not called "Lingzhi", the detailed process of cultivation of Ganoderma spp.and instead of "Rui Cao”(means auspicious herbs)or“Zhi” development of their products are restated in this review After the Han dynasty,the name of Lingzhi started to appear article.These include the breeding,cultivating,extracting in the ancient Chinese literatures.It should be noted that bioactive component,and processing Ganoderma products, Lingzhi mentioned in ancient Chinese literatures is different etc.This article will expand people's common knowledge from the Ganoderma described in fungal classification to- on Ganoderma,and provide a beneficial reference for re- day.Besides the Ganoderma and its relatives,Lingzhi de- search and industrial production. scribed in ancient Chinese literatures also included some fungi belonging to Polyporales and Agaricales,etc.,espe- Keywords Ganoderma spp.biotechnology.Breeding cially in Taoism history books(Zhou and Lin 1999). and cultivation.Development and utilizations. In the opinion of modern traditional Chinese medicine Ganoderma-based products.Quality control (TCM),Lingzhi presents three characters for prevention or treatment of diseases.Firstly,the usage of Lingzhi is without any toxicity and apparent absence of side effects;secondly, it has no pertinence on a special organ;and the last one is its Introduction improvement effects on normalization of the organ function. With the development of biotechnology,many researchers Ganoderma lucidum (Fr.)Karst (named as Lingzhi in China). have intensively studied the bioactive components of a species of basidiomycetes which belongs to Polyporaceae Lingzhi and many Lingzhi-based products.Modern phar- X.-W.Zhou·Y.-M.Zhang(☒ macological and clinical trials have demonstrated that College of Life and Environment Sciences, Lingzhi showed a significant effect on the prevention and Shanghai Normal University, treatment of various diseases.For example,the anti-cancer Shanghai 200234,People's Republic of China effects of Lingzhi were associated with triterpenes,polysac- e-mail:zhym@shnu.edu.cn charides,and fungal immunomodulatory proteins (FIPs)by X.-W.Zhou(☒)·K.-Q.Su the mechanisms of DNA polymerase inhibition,post- Plant Biotechnology Research Center,Shanghai Key Laboratory translation modification inhibition of the Ras oncoprotein, of Agrobiotechnology,School of Agriculture and Biology, or cytokine production stimulation(Sliva 2006;Ding et al. Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, 2009a,b;Ogbe et al.2011).Nowadays,there is an increas- Shanghai 200240,People's Republic of China ing public interest on the secondary metabolites of Lingzhi e-mail:xuanweizhou@sjtu.edu.cn for exploring new drugs or leading compounds.Therefore,a ②Springer

MINI-REVIEW Applied modern biotechnology for cultivation of Ganoderma and development of their products Xuan-Wei Zhou & Kai-Qi Su & Yong-Ming Zhang Received: 21 October 2011 /Revised: 18 November 2011 /Accepted: 21 November 2011 / Published online: 15 December 2011 # Springer-Verlag 2011 Abstract A white-rot basidiomycete Ganoderma spp. has long been used as a medicinal mushroom in Asia, and it has an array of pharmacological properties for immunomodula￾tory activity. There have been many reports about the bio￾active components and their pharmacological properties. In order to analyze the current status of Ganoderma products, the detailed process of cultivation of Ganoderma spp. and development of their products are restated in this review article. These include the breeding, cultivating, extracting bioactive component, and processing Ganoderma products, etc. This article will expand people’s common knowledge on Ganoderma, and provide a beneficial reference for re￾search and industrial production. Keywords Ganoderma spp. biotechnology . Breeding and cultivation . Development and utilizations. Ganoderma-based products. Quality control Introduction Ganoderma lucidum (Fr.) Karst (named as Lingzhi in China), a species of basidiomycetes which belongs to Polyporaceae (or Ganodermaceae) of Aphyllophorales, is one of the most popular medicinal mushrooms in China, Japan, Korea, and other Asian countries. It has been under modern biochemi￾cal and pharmacological research during the last 30 years (Gao et al. 2006). However, in the earliest Chinese litera￾tures, this medicinal mushroom is not called “Lingzhi”, instead of “Rui Cao” (means auspicious herbs) or “Zhi”. After the Han dynasty, the name of Lingzhi started to appear in the ancient Chinese literatures. It should be noted that Lingzhi mentioned in ancient Chinese literatures is different from the Ganoderma described in fungal classification to￾day. Besides the Ganoderma and its relatives, Lingzhi de￾scribed in ancient Chinese literatures also included some fungi belonging to Polyporales and Agaricales, etc., espe￾cially in Taoism history books (Zhou and Lin 1999). In the opinion of modern traditional Chinese medicine (TCM), Lingzhi presents three characters for prevention or treatment of diseases. Firstly, the usage of Lingzhi is without any toxicity and apparent absence of side effects; secondly, it has no pertinence on a special organ; and the last one is its improvement effects on normalization of the organ function. With the development of biotechnology, many researchers have intensively studied the bioactive components of Lingzhi and many Lingzhi-based products. Modern phar￾macological and clinical trials have demonstrated that Lingzhi showed a significant effect on the prevention and treatment of various diseases. For example, the anti-cancer effects of Lingzhi were associated with triterpenes, polysac￾charides, and fungal immunomodulatory proteins (FIPs) by the mechanisms of DNA polymerase inhibition, post￾translation modification inhibition of the Ras oncoprotein, or cytokine production stimulation (Sliva 2006; Ding et al. 2009a, b; Ogbe et al. 2011). Nowadays, there is an increas￾ing public interest on the secondary metabolites of Lingzhi for exploring new drugs or leading compounds. Therefore, a X.-W. Zhou : Y.-M. Zhang (*) College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, People’s Republic of China e-mail: zhym@shnu.edu.cn X.-W. Zhou (*) : K.-Q. Su Plant Biotechnology Research Center, Shanghai Key Laboratory of Agrobiotechnology, School of Agriculture and Biology, Fudan–SJTU–Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China e-mail: xuanweizhou@sjtu.edu.cn Appl Microbiol Biotechnol (2012) 93:941–963 DOI 10.1007/s00253-011-3780-7

942 Appl Microbiol Biotechnol (2012)93:941-963 number of bioactive constituents have been isolated from quality has an effect not only on the yield of Lingzhi-based Lingzhi,including small molecule compounds,polysacchar- product but also its quality.Therefore,selection of a good ides,proteins,enzyme,polysaccharide-protein complexes, Lingzhi strain is a very important task.At any time,high etc.(Zhong and Xiao 2009;Xu et al.2010a,b;Ferreira et al. production and good quality are always the principal goals 2010;Xu et al.2011).Because of the unique pharmacolog- for agriculturally important crops,without the exception of ical function and apparent absence of side effects,it has medicinal mushrooms.There are a lot of breeding methods. attained a reputation in the East and some African countries such as mass selection,programmed mutation,cross-breeding as the ultimate herbal substance.Now Lingzhi has been and transgenic breeding,etc.Up to now,with the development added not only to the Chinese Pharmacopoeia(Zhou et al. of modern biotechnology,the protoplast fusion techniques 2007a)but also to the American Herbal Pharmacopoeia and widely applied on mushroom breeding have made greater Therapeutic Compendium (Sanodiya et al.2009). progress.Transgenic engineering techniques applied on the In an overview of previous literatures,there was a larger medicinal mushroom breeding are a technological innovation collection of papers on Lingzhi's bioactive components and on the molecular level.However,the selection and transgenic their pharmacological properties,and a number of reviews breeding are more objective and promising,and have made a had appeared on these aspects (Luo and Lin 2002;Shiao lot of progress from theory to practice in the last 20 years.The 2003:Yuen and Gohel 2005:Zhou et al.2007a,b:Sanodiya breeding strategy of Lingzhi is summarized in Fig.1. et al.2009;Olaku and White 2011;Xu et al.2011).The reason why Lingzhi draws so much attention is because it is Artificial selection Artificial selection,also known as selec- a potential pharmacological macrofungi (Sanodiya et al. tive breeding,is a primitive breeding method,which uses 2009)and it plays an important role in disease prevention artificial means to choose superior strains from nature,for and treatment in folk medicine;meanwhile,modern phar- biologically obtaining the new species and reproducing it macological tests have also demonstrated some actions and selectively.The basic methods of artificial selection were properties of Lingzhi,including immunomodulating,induc- the tissue separation and spore separation methods for obtain- ing cytokine production,anti-allergic,anti-radiation,anti- ing the pure strain,followed by optimization of this strain. tumor,anti-inflammatory,anti-parasitic,anti-oxidant, and then the required strain is obtained (Chen and Su 2008). benefiting on the cardiovascular system,respiratory system, In fact,during the procedure of mushroom production,tissue endocrine and metabolic systems,etc.(Wasser 2002;Gao et separation method is often employed in getting the strain al.2004;Hong et al.2004;Zhou et al.2007a;Mahajna et al. because Lingzhi spore is hard to germinate (Lin and Zhou 2009).However,Lingzhi products processed by bioactive 1999a).Artificial selection is more commonly used in the components depend on the upstream cultivation,such as the breeding of other edible mushrooms than in Lingzhi fruiting bodies,mycelia,and culture broths;meanwhile, Lingzhi-based products also rely on the downstream pro- Mutation breeding Mutation breeding is a new and more cess.In summary,the qualitative and quantitative differ- effective method compared to artificial selection method, ences in the chemical composition of Lingzhi products are which changes genes of the strains,and achieves genic dependent on the strain,origin,extracting process,and recombination.A general procedure of mutation breeding cultivation conditions (Mizuno 1995:Zhou and Lin 1999: involves the following steps:selection of original strain- Zhou et al.2008a:McKenna et al.2002).In this review preparation of spore (or protoplast)suspension liquid article,the cultivation methods and conditions of Lingzhi viable count and mutagenizing-spreading plate for culti- have been overviewed.Subsequently,the current status of vation-picking up strain and inoculation-initial screen- products process would be addressed.Finally,problems and ing→slope culture→re-screening→selection of superior prospects were analyzed and viewed.This review is benefi- strain.The protoplast is usually chosen for mutation breeding cial to researchers and producers (Li et al.2001),and the increased bioactive components,such as polysaccharides (Gao et al.2008),triterpenoids (Li et al. 2001),and organic germanium (Dong et al.2009),are Cultivation of Ganoderma species regarded as the breeding objectives.Mutation breeding could not only increase strain mutation rate through simple opera- Wild Lingzhi is difficult to collect and to control its quality.In tion but also provide genetic markers for further cross- 1970,a Chinese technician used"spore separation cultivation breeding and cell fusion breeding.However,it also has some method"to successfully cultivate Lingzhi.From then on. disadvantages.For example,the mutation generation is ran- artificial cultivation of Lingzhi has been available in China. dom and the work on selecting the mutant is complicated,etc Breeding of Lingzhi The good quality of Lingzhi strain is Cross-breeding Cross-breeding technology is the most the precondition or key for Lingzhi production.The strain widely used and effective breeding method in breeding the ②Springer

number of bioactive constituents have been isolated from Lingzhi, including small molecule compounds, polysacchar￾ides, proteins, enzyme, polysaccharide–protein complexes, etc. (Zhong and Xiao 2009; Xu et al. 2010a, b; Ferreira et al. 2010; Xu et al. 2011). Because of the unique pharmacolog￾ical function and apparent absence of side effects, it has attained a reputation in the East and some African countries as the ultimate herbal substance. Now Lingzhi has been added not only to the Chinese Pharmacopoeia (Zhou et al. 2007a) but also to the American Herbal Pharmacopoeia and Therapeutic Compendium (Sanodiya et al. 2009). In an overview of previous literatures, there was a larger collection of papers on Lingzhi’s bioactive components and their pharmacological properties, and a number of reviews had appeared on these aspects (Luo and Lin 2002; Shiao 2003; Yuen and Gohel 2005; Zhou et al. 2007a, b; Sanodiya et al. 2009; Olaku and White 2011; Xu et al. 2011). The reason why Lingzhi draws so much attention is because it is a potential pharmacological macrofungi (Sanodiya et al. 2009) and it plays an important role in disease prevention and treatment in folk medicine; meanwhile, modern phar￾macological tests have also demonstrated some actions and properties of Lingzhi, including immunomodulating, induc￾ing cytokine production, anti-allergic, anti-radiation, anti￾tumor, anti-inflammatory, anti-parasitic, anti-oxidant, benefiting on the cardiovascular system, respiratory system, endocrine and metabolic systems, etc. (Wasser 2002; Gao et al. 2004; Hong et al. 2004; Zhou et al. 2007a; Mahajna et al. 2009). However, Lingzhi products processed by bioactive components depend on the upstream cultivation, such as the fruiting bodies, mycelia, and culture broths; meanwhile, Lingzhi-based products also rely on the downstream pro￾cess. In summary, the qualitative and quantitative differ￾ences in the chemical composition of Lingzhi products are dependent on the strain, origin, extracting process, and cultivation conditions (Mizuno 1995; Zhou and Lin 1999; Zhou et al. 2008a; McKenna et al. 2002). In this review article, the cultivation methods and conditions of Lingzhi have been overviewed. Subsequently, the current status of products process would be addressed. Finally, problems and prospects were analyzed and viewed. This review is benefi￾cial to researchers and producers. Cultivation of Ganoderma species Wild Lingzhi is difficult to collect and to control its quality. In 1970, a Chinese technician used “spore separation cultivation method” to successfully cultivate Lingzhi. From then on, artificial cultivation of Lingzhi has been available in China. Breeding of Lingzhi The good quality of Lingzhi strain is the precondition or key for Lingzhi production. The strain quality has an effect not only on the yield of Lingzhi-based product but also its quality. Therefore, selection of a good Lingzhi strain is a very important task. At any time, high production and good quality are always the principal goals for agriculturally important crops, without the exception of medicinal mushrooms. There are a lot of breeding methods, such as mass selection, programmed mutation, cross-breeding and transgenic breeding, etc. Up to now, with the development of modern biotechnology, the protoplast fusion techniques widely applied on mushroom breeding have made greater progress. Transgenic engineering techniques applied on the medicinal mushroom breeding are a technological innovation on the molecular level. However, the selection and transgenic breeding are more objective and promising, and have made a lot of progress from theory to practice in the last 20 years. The breeding strategy of Lingzhi is summarized in Fig. 1. Artificial selection Artificial selection, also known as selec￾tive breeding, is a primitive breeding method, which uses artificial means to choose superior strains from nature, for biologically obtaining the new species and reproducing it selectively. The basic methods of artificial selection were the tissue separation and spore separation methods for obtain￾ing the pure strain, followed by optimization of this strain, and then the required strain is obtained (Chen and Su 2008). In fact, during the procedure of mushroom production, tissue separation method is often employed in getting the strain because Lingzhi spore is hard to germinate (Lin and Zhou 1999a). Artificial selection is more commonly used in the breeding of other edible mushrooms than in Lingzhi. Mutation breeding Mutation breeding is a new and more effective method compared to artificial selection method, which changes genes of the strains, and achieves genic recombination. A general procedure of mutation breeding involves the following steps: selection of original strain→ preparation of spore (or protoplast) suspension liquid→ viable count and mutagenizing→spreading plate for culti￾vation→picking up strain and inoculation→initial screen￾ing→slope culture→re-screening→selection of superior strain. The protoplast is usually chosen for mutation breeding (Li et al. 2001), and the increased bioactive components, such as polysaccharides (Gao et al. 2008), triterpenoids (Li et al. 2001), and organic germanium (Dong et al. 2009), are regarded as the breeding objectives. Mutation breeding could not only increase strain mutation rate through simple opera￾tion but also provide genetic markers for further cross￾breeding and cell fusion breeding. However, it also has some disadvantages. For example, the mutation generation is ran￾dom and the work on selecting the mutant is complicated, etc. Cross-breeding Cross-breeding technology is the most widely used and effective breeding method in breeding the 942 Appl Microbiol Biotechnol (2012) 93:941–963

Appl Microbiol Biotechnol (2012)93:941-963 943 Fig.1 The breeding strategy of Ganoderma species Set up the breeding Define the regions and types that objectives of Lingzhi Lingzhi being origin from Identification,purification and construction of gene pools of Lingzhi Conventionality Radiating and Cell engineering Gene engineering breeding mutant breeding breeding breeding Evaluation of the agronomic traits,physiological and biochemical characteristic,genetic rule of generation ★ Generation area Comparison of Identification Selection of species area area breeding area Multi-site test,demonstration base,popularization and transformation new species of edible or medicinal mushroom.The principle protoplast-regenerating and culturing the fusion-detecting of cross-breeding is to achieve genetic recombination and selecting fusion.In the 1970s,this method has been through haploid mating,and then strains from generation widely applied in basidiomycetes breeding (Ferenczy et al. with parent's good traits are selected.After the 1980s,cross- 1974).At the beginning of the 1980s,it has been used in breeding technology was widely used in the breeding edible mushroom breeding in some Asian countries.Initially, researches of edible fungi in China and other Asian most of the researches focused on isolation and preparation of countries (Zhao and Chang 1993;Chiu et al.2005).The Lingzhi protoplast(Choi et al.1987;Li and Li 1999;Chen et Lingzhi spores are difficult to germinate under artificial al.2007a,b),and then it was gradually applied in breeding the conditions,so the monokaryotic strains cannot be obtained, new strains by fusing intragenus protoplast(Park et al.1988) which is necessary for the breeding of the Lingzhi.As a and intergeneric protoplast (Yoo et al.2002).Some research- result,the hybridization process of Lingzhi is restricted. ers also successfully obtained the new variety of Lingzhi by Therefore,the protoplast monokaryogenesis method to ob- protoplast monokaryogenesis method (Wu et al.2009a). tain the new strain are used in practice (Wu et al.2009a). There have been some reports about artificial cross-breeding Genetic engineering breeding Genetic engineering is a tech- where most of them were selected using protoplast as mate- nological innovation in the area of molecular biology.Using rials(Chiu et al.2005). this technology,a DNA sequence from one species can be isolated and then transferred to another.Meanwhile,the Cell fusion breeding Cell fusion breeding is an important good characters of donor strain can be expressed in host part of modem biotechnology and is also a significant leap strain,which will become a high-production and good- on genetic breeding.In the fusion of cell protoplasts,the quality strain.This novel breeding method provides a new different genotypes of protoplasts from various organisms breeding solution for edible or medicinal mushroom,espe- are fused with each other,which is induced by the fusion cially for those limited by conventional breeding means. agent after breaking the cell wall.In that case,the fusions The general procedure of genetic engineering breeding is could make the cell genomes from different genus mix effec- presented as follows:selecting donor strains-separating tively,which produces a whole set of genetic exchange and gene-genes reconstruction in vitro-transfer the gene into restructures to generate a new individual(Tan et al.2005).A the recipient cell-reproduction and expression of recom- common method of cell fusion engineering breeding involves binant DNA-selection of new individual.Transformation the following procedure:selecting parent strain-ensuring is the key to Lingzhi genetic engineering breeding.Up to genetic markers of parent strain-isolating protoplast from now,six kinds of transformation methods have been applied parent strain-regenerating and culturing protoplast-fusing for filamentous fungi,which includes protoplast-mediated ②Springer

new species of edible or medicinal mushroom. The principle of cross-breeding is to achieve genetic recombination through haploid mating, and then strains from generation with parent’s good traits are selected. After the 1980s, cross￾breeding technology was widely used in the breeding researches of edible fungi in China and other Asian countries (Zhao and Chang 1993; Chiu et al. 2005). The Lingzhi spores are difficult to germinate under artificial conditions, so the monokaryotic strains cannot be obtained, which is necessary for the breeding of the Lingzhi. As a result, the hybridization process of Lingzhi is restricted. Therefore, the protoplast monokaryogenesis method to ob￾tain the new strain are used in practice (Wu et al. 2009a). There have been some reports about artificial cross-breeding where most of them were selected using protoplast as mate￾rials (Chiu et al. 2005). Cell fusion breeding Cell fusion breeding is an important part of modern biotechnology and is also a significant leap on genetic breeding. In the fusion of cell protoplasts, the different genotypes of protoplasts from various organisms are fused with each other, which is induced by the fusion agent after breaking the cell wall. In that case, the fusions could make the cell genomes from different genus mix effec￾tively, which produces a whole set of genetic exchange and restructures to generate a new individual (Tan et al. 2005). A common method of cell fusion engineering breeding involves the following procedure: selecting parent strain→ensuring genetic markers of parent strain→isolating protoplast from parent strain→regenerating and culturing protoplast→fusing protoplast→regenerating and culturing the fusion→detecting and selecting fusion. In the 1970s, this method has been widely applied in basidiomycetes breeding (Ferenczy et al. 1974). At the beginning of the 1980s, it has been used in edible mushroom breeding in some Asian countries. Initially, most of the researches focused on isolation and preparation of Lingzhi protoplast (Choi et al. 1987; Li and Li 1999; Chen et al. 2007a, b), and then it was gradually applied in breeding the new strains by fusing intragenus protoplast (Park et al. 1988) and intergeneric protoplast (Yoo et al. 2002). Some research￾ers also successfully obtained the new variety of Lingzhi by protoplast monokaryogenesis method (Wu et al. 2009a). Genetic engineering breeding Genetic engineering is a tech￾nological innovation in the area of molecular biology. Using this technology, a DNA sequence from one species can be isolated and then transferred to another. Meanwhile, the good characters of donor strain can be expressed in host strain, which will become a high-production and good￾quality strain. This novel breeding method provides a new breeding solution for edible or medicinal mushroom, espe￾cially for those limited by conventional breeding means. The general procedure of genetic engineering breeding is presented as follows: selecting donor strains→separating gene→genes reconstruction in vitro→transfer the gene into the recipient cell→reproduction and expression of recom￾binant DNA→selection of new individual. Transformation is the key to Lingzhi genetic engineering breeding. Up to now, six kinds of transformation methods have been applied for filamentous fungi, which includes protoplast-mediated Set up the breeding objectives of Lingzhi Define the regions and types that Lingzhi being origin from Identification, purification and construction of gene pools of Lingzhi Conventionality breeding Radiating and mutant breeding Cell engineering breeding Gene engineering breeding Evaluation of the agronomic traits, physiological and biochemical characteristic, genetic rule of generation Generation area Comparison of species area Identification area Selection of breeding area Multi-site test, demonstration base, popularization and transformation Fig. 1 The breeding strategy of Ganoderma species Appl Microbiol Biotechnol (2012) 93:941–963 943

944 Appl Microbiol Biotechnol (2012)93:941-963 transformation (PMT),agrobacterium-mediated transforma- cultivation.In the developed countries and developing tion,electroporation,biolistic transformation,restriction countries,the effects of mushroom breeding rely on the enzyme-mediated integration (REMD),and lithium acetate, amount of cultivation test for verification.In any case,from etc.(Zhou et al.2010).Most of these methods have suc- the viewpoint of the study level of mushroom genetics,the cessfully worked in breeding of Lingzhi (Park et al.1991: method of DNA special analysis cannot be applied directly Sun et al.2001a:Kim et al.2004a). to explain and decide on the detection of strain quality and For example,Li et al.(2004)constructed fungal expres- other problems relative to the strain quality (Zhang et al. sion plasmid pAN7-1(6.7 kb),which carried the promoters 2005a,b).Today,the quality standards of Lingzhi strains of hph(hygromycin phosphate dehydrogenase)gene from have been addressed in some provinces in China where the Escherichia coli and the gpd(glyceraldehyde-p-dehydroge- Lingzhi could be produced on a large scale (Fujian Bureau nase)gene from Aspergillus nidulans.The plasmid could of Quality and Technical Supervision 2002;Anhui Bureau express hygromycin B resistance in fungal culture and of Quality and Technical Supervision 2004).The local achieve the transformation of Lingzhi protoplasts with standards were used for the evaluation of Lingzhi strains. 60%polyethylene glycol (PEG)4000.So it provided a In different opinions,TCM fingerprint generates from foundation for transforming Lingzhi protoplast by the different names,such as chemical fingerprints and biologi- PMT method (Li et al.2004).Kim et al.(2004a,b)reported cal fingerprint,spectral fingerprint and chromatographic the studies on genetic transformation and mutant isolation fingerprint,etc.From the view of modern biotechnology based on REMI technology in G.lucidum.They constructed analysis,it includes the DNA fingerprint,protein finger- a plasmid pJS205-1(6.5 kb)carrying the resistance gene of print,and chemical fingerprints.At present,there are many geneticin (geneticin,an aminoglycoside antibiotic)and studies about chemical fingerprints of Lingzhi.However, phosphinothricin.After using restriction enzyme EcoRV, chromatography methods were popular and were studied Notl,and Xhol,plasmid pJS205-1 was transformed into in depth during the past decade (Xing et al.2004;Huang the Lingzhi protoplasts.Then a series of mutants were et al.2004a,b;Zhang et al.2009;Chen et al.2010a,b; obtained and its preliminary identification on biochemical Dejaegher and Heyden 2010).With the development of characteristics was observed (Kim et al.2004a).For exam- molecular biology technology,DNA fingerprint technology ple,Li and Chen(2002)used a Ti plasmid vector containing was firstly introduced to fungi to research genetic diversity an exogenous gene to transform the exogenous gene into the related to the genus,species,strain,etc.(Anderson et al fungus protoplasts and made it stably replicated and expressed 1987;Hwang and Kim 1995).Subsequently,it was intro- (Li and Chen 2002).Zhang et al.(2011)over-expressed rice duced to variety identification and authentication in Lingzhi OsUgp2 gene in Ganoderma sinensis and increased the con- (Hseu et al.1996;Shi et al.2008;Wu et al.2009b;Zheng et tent of intracellular and extracellular polysaccharide(EPS)in al.2009).Some scholars tried to use the function gene such G.sinensis (Zhang et al.2011).In all,gene engineering as FIPs to identify Lingzhi (Zhou et al.2008b).However, techniques could contribute to the new vigor for using sec- there are many different opinions about how to build an ondary engineering to breed Lingzhi. effective fingerprint(Gottlieb et al.2000;Zhou et al.2005). The total strategies are shown in Fig.2(Zhou et al.2006). Setting up the fingerprint of good quality species It is cru- DNA fingerprint has two main purposes:identification of cial for both growers and researchers to understand the the TCM authenticity and analysis of the TCM quality.The features and qualities of the best strains.The choice of a DNA molecular marker technology,which DNA fingerprint proper strain can determine success or failure.Therefore,the depends on,can be divided into three groups:(1)restriction analysis and evaluation of the strain qualities have always fragment length polymorphism(RFLP)and other technolo- been the focus of the producer and researcher of medicinal gies,in which the core is based on the electrophoretic mushroom.Up to now.in China.the evaluated content of techniques and molecular hybridization techniques(Tanksley medicinal mushroom(including Lingzhi)includes six major et al.1989;Deragon and Landry 1992);(2)DNA fingerprints indices of determinations and assays,which are the appear- and DNA sequencing technology such as random amplified ance of mycelia or strains,microorganism examination, polymorphic DNA (RAPD)(Bardakci 2001),simple se- microbe testing,determination of the growing speed,the quence repeats (SSR),arbitrarily primed polymerase chain esterase isozyme analysis,and the cultivated character.The reaction(AP-PCR),etc.,which depend on the electrophoretic abovementioned cultivated character includes the shape of techniques and PCR techniques;and(3)amplified fragment fruit bodies,mycelia growing speed,vegetative stage (and length polymorphism (AFLP)(Vos et al.1995),sequence reproductive stages),yield,etc. characterized amplified regions(SCAR).direct amplification The existing knowledge and technique for identification of length polymorphism(DALP),RFLP-PCR,RAPD-PCR, of strain qualities are based on the cultivation test.determi- and the latest gene chip diagnostic technique,which combine nation,and assay of agronomic traits in the course of the two abovementioned techniques.Of course,all kinds of ②Springer

transformation (PMT), agrobacterium-mediated transforma￾tion, electroporation, biolistic transformation, restriction enzyme-mediated integration (REMI), and lithium acetate, etc. (Zhou et al. 2010). Most of these methods have suc￾cessfully worked in breeding of Lingzhi (Park et al. 1991; Sun et al. 2001a; Kim et al. 2004a). For example, Li et al. (2004) constructed fungal expres￾sion plasmid pAN7-1 (6.7 kb), which carried the promoters of hph (hygromycin phosphate dehydrogenase) gene from Escherichia coli and the gpd (glyceraldehyde-p-dehydroge￾nase) gene from Aspergillus nidulans. The plasmid could express hygromycin B resistance in fungal culture and achieve the transformation of Lingzhi protoplasts with 60% polyethylene glycol (PEG) 4000. So it provided a foundation for transforming Lingzhi protoplast by the PMT method (Li et al. 2004). Kim et al. (2004a, b) reported the studies on genetic transformation and mutant isolation based on REMI technology in G. lucidum. They constructed a plasmid pJS205-1(6.5 kb) carrying the resistance gene of geneticin (geneticin, an aminoglycoside antibiotic) and phosphinothricin. After using restriction enzyme EcoRV, NotI, and XhoI, plasmid pJS205-1 was transformed into the Lingzhi protoplasts. Then a series of mutants were obtained and its preliminary identification on biochemical characteristics was observed (Kim et al. 2004a). For exam￾ple, Li and Chen (2002) used a Ti plasmid vector containing an exogenous gene to transform the exogenous gene into the fungus protoplasts and made it stably replicated and expressed (Li and Chen 2002). Zhang et al. (2011) over-expressed rice OsUgp2 gene in Ganoderma sinensis and increased the con￾tent of intracellular and extracellular polysaccharide (EPS) in G. sinensis (Zhang et al. 2011). In all, gene engineering techniques could contribute to the new vigor for using sec￾ondary engineering to breed Lingzhi. Setting up the fingerprint of good quality species It is cru￾cial for both growers and researchers to understand the features and qualities of the best strains. The choice of a proper strain can determine success or failure. Therefore, the analysis and evaluation of the strain qualities have always been the focus of the producer and researcher of medicinal mushroom. Up to now, in China, the evaluated content of medicinal mushroom (including Lingzhi) includes six major indices of determinations and assays, which are the appear￾ance of mycelia or strains, microorganism examination, microbe testing, determination of the growing speed, the esterase isozyme analysis, and the cultivated character. The abovementioned cultivated character includes the shape of fruit bodies, mycelia growing speed, vegetative stage (and reproductive stages), yield, etc. The existing knowledge and technique for identification of strain qualities are based on the cultivation test, determi￾nation, and assay of agronomic traits in the course of cultivation. In the developed countries and developing countries, the effects of mushroom breeding rely on the amount of cultivation test for verification. In any case, from the viewpoint of the study level of mushroom genetics, the method of DNA special analysis cannot be applied directly to explain and decide on the detection of strain quality and other problems relative to the strain quality (Zhang et al. 2005a, b). Today, the quality standards of Lingzhi strains have been addressed in some provinces in China where the Lingzhi could be produced on a large scale (Fujian Bureau of Quality and Technical Supervision 2002; Anhui Bureau of Quality and Technical Supervision 2004). The local standards were used for the evaluation of Lingzhi strains. In different opinions, TCM fingerprint generates from different names, such as chemical fingerprints and biologi￾cal fingerprint, spectral fingerprint and chromatographic fingerprint, etc. From the view of modern biotechnology analysis, it includes the DNA fingerprint, protein finger￾print, and chemical fingerprints. At present, there are many studies about chemical fingerprints of Lingzhi. However, chromatography methods were popular and were studied in depth during the past decade (Xing et al. 2004; Huang et al. 2004a, b; Zhang et al. 2009; Chen et al. 2010a, b; Dejaegher and Heyden 2010). With the development of molecular biology technology, DNA fingerprint technology was firstly introduced to fungi to research genetic diversity related to the genus, species, strain, etc. (Anderson et al. 1987; Hwang and Kim 1995). Subsequently, it was intro￾duced to variety identification and authentication in Lingzhi (Hseu et al. 1996; Shi et al. 2008; Wu et al. 2009b; Zheng et al. 2009). Some scholars tried to use the function gene such as FIPs to identify Lingzhi (Zhou et al. 2008b). However, there are many different opinions about how to build an effective fingerprint (Gottlieb et al. 2000; Zhou et al. 2005). The total strategies are shown in Fig. 2 (Zhou et al. 2006). DNA fingerprint has two main purposes: identification of the TCM authenticity and analysis of the TCM quality. The DNA molecular marker technology, which DNA fingerprint depends on, can be divided into three groups: (1) restriction fragment length polymorphism (RFLP) and other technolo￾gies, in which the core is based on the electrophoretic techniques and molecular hybridization techniques (Tanksley et al. 1989; Deragon and Landry 1992); (2) DNA fingerprints and DNA sequencing technology such as random amplified polymorphic DNA (RAPD) (Bardakci 2001), simple se￾quence repeats (SSR), arbitrarily primed polymerase chain reaction (AP-PCR), etc., which depend on the electrophoretic techniques and PCR techniques; and (3) amplified fragment length polymorphism (AFLP) (Vos et al. 1995), sequence characterized amplified regions (SCAR), direct amplification of length polymorphism (DALP), RFLP-PCR, RAPD-PCR, and the latest gene chip diagnostic technique, which combine the two abovementioned techniques. Of course, all kinds of 944 Appl Microbiol Biotechnol (2012) 93:941–963

Appl Microbiol Biotechnol(2012)93:941-963 945 Fig.2 Formation strategy of DNA fingerprint of Ganoderma Component identification Ganoderma Activity (revised from Zhou et al.2006) Pesticide residue detection lucidum analysis DNA extraction The choice of Sample selection target gene Expasion of DNA amplification target gene Fingerprint DNA sequencing Restriction endonuclease enzyme digestion Specific-primer PCR Fingerprint molecular marker methods have its advantages and disadvan- Artificial cultivation of Lingzhi fruit bodies It is necessary to tages (Jones et al.1997).Any kind of marker technologies meet the requirement of Lingzhi fruit body growth,which cannot overall analyze all the medicinal materials.The selec- includes the nutrients components and the environmental con- tion of methods must lie on the actual need,different features, ditions.Meanwhile,we must also master the culture methods. and advantages of the molecular marking techniques.How- The nutrition conditions of Lingzhi growth mainly include ever,the fingerprint techniques are better or advanced,but not carbon resources and nitrogen resources,together with inor- exact.When selecting the specific technologies,the popularity ganic salts and growing factors.The Lingzhi growth generally of the technology and the cost of samples analysis also should uses organic carbon resources such as sugar,starch,cellulose. be taken into consideration(Wu et al.2009b;Zhou et al.2006; hemicelluloses,and lignin.The glucose and sucrose are usu- Blundeoll 2006;Urbanelli et al.2007). ally used in culture of mycelia,the sawdust of broad-leaved tree (Jiang 2001;Xia et al.2003;Liao and Xiao 2006),and the agricultural by-products,such as cotton seed husk,straw,and corn cob,in cultivation of fruit bodies (Wei et al.2005;Yan Cultivation procedures 2000).Some small molecular weight compounds,such as amino acid,urea,nitrogen,etc.,can be utilized by Lingzhi The methods of Lingzhi cultivation can be divided into two mycelia.During the process of Lingzhi cultivation,yeast major types,including liquid-state cultivation (LSC)and powder and peptone are usually added in the media of cultur- solid-state cultivation (SSC).The SSC also can be divided ing mycelia,while wheat bran,corn powder,coarse powders into two methods based on the used raw materials.which are of rice bran,ammonium sulfate,urea,etc.are used in the called as the log (or basswood)cultivation and substituted media of culturing fruit bodies.When the rate of carbon to cultivation.At the beginning of the artificial cultivation of nitrogen is 15-45:1 in the substrate,the Lingzhi mycelia may this valuable medicinal mushroom,only four species was grow well.The appropriate rate of carbon to nitrogen on the used for trials,which included G.lucidum (Leyss.Ex.Fr.) substrate for cultivation of fruit bodies is 30-40:1 (Lin and Karst,G.lucidum (Leyss.Ex.Fr)Karst vat.,G.japonicum Zhou 1999b;Han et al.2003;Wu et al.2008). (Fr.)Lloyd,and G.capense (Lloyd)Teng (Zhou and Lin Besides carbon and nitrogen resources,Lingzhi growth 1999).Through more than 40 years of development,the also needs inorganic salts and other inorganic elements,in- cultivation techniques have achieved significant progress. cluding kalium,natrium,calcium,magnesium,phosphorus, Currently,the various methods are widely used for commer- sulfur,zinc,etc.Among them,phosphorus,kalium,and mag- cial production (Hou and Liao 2009;Zhou et al.2010). nesium are the three main nutrition elements.Their ②Springer

molecular marker methods have its advantages and disadvan￾tages (Jones et al. 1997). Any kind of marker technologies cannot overall analyze all the medicinal materials. The selec￾tion of methods must lie on the actual need, different features, and advantages of the molecular marking techniques. How￾ever, the fingerprint techniques are better or advanced, but not exact. When selecting the specific technologies, the popularity of the technology and the cost of samples analysis also should be taken into consideration (Wu et al. 2009b; Zhou et al. 2006; Blundeoll 2006; Urbanelli et al. 2007). Cultivation procedures The methods of Lingzhi cultivation can be divided into two major types, including liquid-state cultivation (LSC) and solid-state cultivation (SSC). The SSC also can be divided into two methods based on the used raw materials, which are called as the log (or basswood) cultivation and substituted cultivation. At the beginning of the artificial cultivation of this valuable medicinal mushroom, only four species was used for trials, which included G. lucidum (Leyss. Ex. Fr.) Karst, G. lucidum (Leyss. Ex. Fr) Karst vat., G. japonicum (Fr.) Lloyd, and G. capense (Lloyd) Teng (Zhou and Lin 1999). Through more than 40 years of development, the cultivation techniques have achieved significant progress. Currently, the various methods are widely used for commer￾cial production (Hou and Liao 2009; Zhou et al. 2010). Artificial cultivation of Lingzhi fruit bodies It is necessary to meet the requirement of Lingzhi fruit body growth, which includes the nutrients components and the environmental con￾ditions. Meanwhile, we must also master the culture methods. The nutrition conditions of Lingzhi growth mainly include carbon resources and nitrogen resources, together with inor￾ganic salts and growing factors. The Lingzhi growth generally uses organic carbon resources such as sugar, starch, cellulose, hemicelluloses, and lignin. The glucose and sucrose are usu￾ally used in culture of mycelia, the sawdust of broad-leaved tree (Jiang 2001; Xia et al. 2003; Liao and Xiao 2006), and the agricultural by-products, such as cotton seed husk, straw, and corn cob, in cultivation of fruit bodies (Wei et al. 2005; Yan 2000). Some small molecular weight compounds, such as amino acid, urea, nitrogen, etc., can be utilized by Lingzhi mycelia. During the process of Lingzhi cultivation, yeast powder and peptone are usually added in the media of cultur￾ing mycelia, while wheat bran, corn powder, coarse powders of rice bran, ammonium sulfate, urea, etc. are used in the media of culturing fruit bodies. When the rate of carbon to nitrogen is 15–45:1 in the substrate, the Lingzhi mycelia may grow well. The appropriate rate of carbon to nitrogen on the substrate for cultivation of fruit bodies is 30–40:1 (Lin and Zhou 1999b; Han et al. 2003; Wu et al. 2008). Besides carbon and nitrogen resources, Lingzhi growth also needs inorganic salts and other inorganic elements, in￾cluding kalium, natrium, calcium, magnesium, phosphorus, sulfur, zinc, etc. Among them, phosphorus, kalium, and mag￾nesium are the three main nutrition elements. Their Component identification Pesticide residue detection Ganoderma lucidum Activity analysis Sample selection DNA extraction The choice of target gene DNA amplification Expasion of target gene Fingerprint DNA sequencing Fingerprint Specific-primer PCR Restriction endonuclease enzyme digestion Fig. 2 Formation strategy of DNA fingerprint of Ganoderma (revised from Zhou et al. 2006) Appl Microbiol Biotechnol (2012) 93:941–963 945

946 Appl Microbiol Biotechnol (2012)93:941-963 appropriate concentration in the media is 100-150 mg/L. development.it is at 3.000 to 50.000 lx.At the condition Although some of the elements have existed in the raw mate- of light level about 3,000-10,000 Ix,the stip and cap (pileus) rials,the substrates are still added the Caso,KH2PO4,and shape are normal.The optimum light level is at 15,000 to MgSO4,especially the amount of usage CaSO4 is at most up 50,000 Ix.The optimum pH value for mycelial running is 5.0 to 1%of total substrate weight.It is a significant reason that to 5.5 (Zhou and Lin 1999;Hou and Liao 2009). calcium sulfate may adjust the pH value of substrate,change Artificial cultivation of Lingzhi has a history of more the substrate porosity,increase the air flow,fix the nitrogen, than 40 years.Many methods of cultivating Lingzhi derive and enhance the mounts of calcium and sulfur elements.In the from the name of cultivation raw materials (log cultivation, development process of Lingzhi,the growth factors are the sawdust cultivation,substituted cultivation,etc.),the pack- necessary components,including vitamin B,B6,and biotin, aging forms of raw materials(bag or bottle),or cultivation which are related to Lingzhi metabolism.Because of these location (outdoor cultivation,indoor cultivation.bionics growth factors existing in the natural substrates,they do not wild cultivation,etc.).For example,based on the cultivation necessarily need to be added into the substrates.In addition, raw materials,basswood cultivation technology is actually clean well water should be used in large-scale cultivation of designed to culture mushroom using basswood as raw mate- Lingzhi.It is necessary that the water is not contaminated with rials (Zhang et al.2004).The substituted cultivation tech- undesirable pollutants.The appropriate content of water nology was defined as using the sawdust of hardwood, should be 65-70%(Zhou and Lin 1999:Han et al.2003). cotton seed husk,or foot materials of farm crops to cultivate Lingzhi is found more frequently in subtropical regions mushroom(Zhang and Wang 2010).The word"cultivation than in the temperate zones.It is an annual mushroom, technology"may mean different things,in different people growing on a wide variety of dead and dying trees.The and countries.However,regarding the choice of what kind growth parameters (Lin 1996;Chang and Miles 2004)are of method or raw materials to use,the production of the summarized mainly to include four aspects:temperature, basic process is roughly the same.Similarly,as to other humidity,air,and light.Firstly,temperature for mycelia cultivated edible mushrooms,the process for producing growth ranges from 15 to 35 C and the optimum temper- Lingzhi fruiting bodies can be divided into two major ature is 25 to 30 C:for primordial initiation,it ranges from stages.The first stage is the preparation of various raw 18 to 25 C;and for fruiting body development,the range is materials,which involves the selection of mother spawn, from 24 to 28 C.Under the condition of 20 C or lower,the cultivation method,and cultivation relative to materials.The fruit body would become yellow and stops growing.In the second stage is the performance of cultivation,including the procedure of fruit body development,it is actually not planting spawn,stock culture,fruiting culture,and utiliza- necessary to be stimulated with temperature difference. tion of the growth substrates for mushroom production. The higher temperature differences easily cause macroce- Currently,the methods adopted for commercial production phalic Lingzhi.Secondly,humidity is a term for the amount shows a variety of forms,which mainly include the wood of water vapor in the air or in the substrates.During the log,short basswood segment,tree stump,sawdust bag,and process of Lingzhi cultivation,the water content in sub- bottle procedures (Mayzumi et al.1997:Lin and Zhou strates should be maintained at 60%to 65%level.Relative 1999a:Erkel 2009:Chen 1999:Han et al.2008). humidity for the mycelial running is within 60%to 70% Because some methods are not beneficial for the protec- level.the primordial initiation is within 85%to 90%level. tion of the ecological system,Lingzhi cultivation using and the fruit body development is with 85%to 95%humid- wood log is not adopted in production practice.Here,we ity level in the environmental air.Finally,in the different only restate the basic procedure related to substituted culti- development periods,Lingzhi have the different require- vation.In the research of substituted cultivation of Lingzhi, ments on the rate of oxygen and carbon dioxide.Lingzhi the research content often focuses on the following aspects: mycelial running do not need oxygen.But during the fruc- selection of substrate and its formulation.cultivation models tification period,good ventilation is necessary.When the (bag or bottle,inside or outside),sterilization and inocula- concentration of COz is below 0.1%,the fruit body would tion,cultivation and administration of mycelia,and admin- be abnormally developed.When the concentration of CO2 is istration of the stage of fruit body development.Briefly, above 0.1%,the fruit body would be normally developed. spawn is transferred onto a sterile solid-state substrate, Similar to other mushrooms,Lingzhi do not require the which frequently consists of a mixture of hardwood saw- various types and amount of light to grow.Because of the dust,wheat bran,and other supplemental substances,and is light for Lingzhi mycelial running having an inhibiting incubated under dark conditions until colonization of sub- effect,colonizing substrate should be kept under dark con- strate is achieved.After this,incubation under the weak light ditions to make sure the substrate does not pin prematurely. condition and at increased oxygen level is carried out to During primordia initiation of Lingzhi growth,the light develop the primordial initiation.Once the desired primor- level is required at 500 to 1,000 Ix;for fruit body dial initiation is achieved,the conditions are again altered to ②Springer

appropriate concentration in the media is 100–150 mg/L. Although some of the elements have existed in the raw mate￾rials, the substrates are still added the CaSO4, KH2PO4, and MgSO4, especially the amount of usage CaSO4 is at most up to 1% of total substrate weight. It is a significant reason that calcium sulfate may adjust the pH value of substrate, change the substrate porosity, increase the air flow, fix the nitrogen, and enhance the mounts of calcium and sulfur elements. In the development process of Lingzhi, the growth factors are the necessary components, including vitamin B1, B6, and biotin, which are related to Lingzhi metabolism. Because of these growth factors existing in the natural substrates, they do not necessarily need to be added into the substrates. In addition, clean well water should be used in large-scale cultivation of Lingzhi. It is necessary that the water is not contaminated with undesirable pollutants. The appropriate content of water should be 65–70% (Zhou and Lin 1999; Han et al. 2003). Lingzhi is found more frequently in subtropical regions than in the temperate zones. It is an annual mushroom, growing on a wide variety of dead and dying trees. The growth parameters (Lin 1996; Chang and Miles 2004) are summarized mainly to include four aspects: temperature, humidity, air, and light. Firstly, temperature for mycelia growth ranges from 15 to 35 °C and the optimum temper￾ature is 25 to 30 °C; for primordial initiation, it ranges from 18 to 25 °C; and for fruiting body development, the range is from 24 to 28 °C. Under the condition of 20 °C or lower, the fruit body would become yellow and stops growing. In the procedure of fruit body development, it is actually not necessary to be stimulated with temperature difference. The higher temperature differences easily cause macroce￾phalic Lingzhi. Secondly, humidity is a term for the amount of water vapor in the air or in the substrates. During the process of Lingzhi cultivation, the water content in sub￾strates should be maintained at 60% to 65% level. Relative humidity for the mycelial running is within 60% to 70% level, the primordial initiation is within 85% to 90% level, and the fruit body development is with 85% to 95% humid￾ity level in the environmental air. Finally, in the different development periods, Lingzhi have the different require￾ments on the rate of oxygen and carbon dioxide. Lingzhi mycelial running do not need oxygen. But during the fruc￾tification period, good ventilation is necessary. When the concentration of CO2 is below 0.1%, the fruit body would be abnormally developed. When the concentration of CO2 is above 0.1%, the fruit body would be normally developed. Similar to other mushrooms, Lingzhi do not require the various types and amount of light to grow. Because of the light for Lingzhi mycelial running having an inhibiting effect, colonizing substrate should be kept under dark con￾ditions to make sure the substrate does not pin prematurely. During primordia initiation of Lingzhi growth, the light level is required at 500 to 1,000 lx; for fruit body development, it is at 3,000 to 50,000 lx. At the condition of light level about 3,000–10,000 lx, the stip and cap (pileus) shape are normal. The optimum light level is at 15,000 to 50,000 lx. The optimum pH value for mycelial running is 5.0 to 5.5 (Zhou and Lin 1999; Hou and Liao 2009). Artificial cultivation of Lingzhi has a history of more than 40 years. Many methods of cultivating Lingzhi derive from the name of cultivation raw materials (log cultivation, sawdust cultivation, substituted cultivation, etc.), the pack￾aging forms of raw materials (bag or bottle), or cultivation location (outdoor cultivation, indoor cultivation, bionics wild cultivation, etc.). For example, based on the cultivation raw materials, basswood cultivation technology is actually designed to culture mushroom using basswood as raw mate￾rials (Zhang et al. 2004). The substituted cultivation tech￾nology was defined as using the sawdust of hardwood, cotton seed husk, or foot materials of farm crops to cultivate mushroom (Zhang and Wang 2010). The word “cultivation technology” may mean different things, in different people and countries. However, regarding the choice of what kind of method or raw materials to use, the production of the basic process is roughly the same. Similarly, as to other cultivated edible mushrooms, the process for producing Lingzhi fruiting bodies can be divided into two major stages. The first stage is the preparation of various raw materials, which involves the selection of mother spawn, cultivation method, and cultivation relative to materials. The second stage is the performance of cultivation, including the planting spawn, stock culture, fruiting culture, and utiliza￾tion of the growth substrates for mushroom production. Currently, the methods adopted for commercial production shows a variety of forms, which mainly include the wood log, short basswood segment, tree stump, sawdust bag, and bottle procedures (Mayzumi et al. 1997; Lin and Zhou 1999a; Erkel 2009; Chen 1999; Han et al. 2008). Because some methods are not beneficial for the protec￾tion of the ecological system, Lingzhi cultivation using wood log is not adopted in production practice. Here, we only restate the basic procedure related to substituted culti￾vation. In the research of substituted cultivation of Lingzhi, the research content often focuses on the following aspects: selection of substrate and its formulation, cultivation models (bag or bottle, inside or outside), sterilization and inocula￾tion, cultivation and administration of mycelia, and admin￾istration of the stage of fruit body development. Briefly, spawn is transferred onto a sterile solid-state substrate, which frequently consists of a mixture of hardwood saw￾dust, wheat bran, and other supplemental substances, and is incubated under dark conditions until colonization of sub￾strate is achieved. After this, incubation under the weak light condition and at increased oxygen level is carried out to develop the primordial initiation. Once the desired primor￾dial initiation is achieved, the conditions are again altered to 946 Appl Microbiol Biotechnol (2012) 93:941–963

Appl Microbiol Biotechnol(2012)93:941-963 947 aid in the fruiting body development(shown in Fig.3).The Liquid-state (submerged)fermentation of Lingzhi mycelia entire growth of Lingzhi from spawn running to cropping in Liquid-state fermentation (LSF)of mushroom.also known artificial cultivation is different from the method of cultiva- as liquid culture or submerged fermentation,is a process of tion used.In general,it should take on average approximately culturing microorganism in liquid media,but not on the 90-150days. surface of liquid media.Preparation of mushroom mycelium It directly affects the Lingzhi product's quality to admin- in submerged culture was initially developed during the ister the development process of fruit body.In the overview 1970s.At that time,only lower fungi (fungi that are not of previous publications,the cultivation procedures were from basidiocarps)were successfully cultured in fermentors roughly the same;the difference was only diverse from for economical production of various natural products(Yang different cultivation methods or raw materials (Chen 1999: and Liau 1998).The main principle is that the liquid medi- Chi 2005:Erkel 2009:Hossain et al.2009:Xiao et al.2010). um is added to the fermentation tank or flask.When sterile Up to now,in the Lingzhi production practice in China,the air is led to reactor,it will increase the dissolved oxygen preparation of substrate,including substrate formulations, content in the medium to improve respiratory metabolism of making the synthetic logs in bag cultivation and substrate the mushroom mycelium.At the same time,the mushroom sterilization etc.,have appeared in the textbook of some mycelium is agitated or oscillated,and appropriate external vocational schools or popular science books (Zhang 1998; conditions are controlled so that mushroom cells grow in the Liu and Jiang 2007).Meanwhile,the cultivation processes liquid depths of breeding,producing a lot of the mycelium of Lingzhi have also been described in these books.Such or metabolites.Another review indicated that there were few questions regarding the crucial stages in Lingzhi cultivation, investigations into the development of high fungi(basidio- how to speed up the spawn running,how to control the mycetes)bioprocesses (Fang and Zhong 2002a,b). environmental factors,and how to produce Lingzhi with Compared with the artificial cultivation of Lingzhi fruit caps have been introduced in various media,including mag- bodies,LSF holds obvious advantages.Firstly,the advan- azines,popular science books,DVD video,TVetc.Growers tage of LSF over the traditional,multinational cultivation of will benefit from concentrating on the step-by-step instruc- fruit body is the reduction in the time spent to obtain the tion on how to cultivate it. product of interest (Mshandete and Mgonja 2009).The production of fruit bodies takes at least 3-5 months,while Artificial cultivation of Lingzhi mycelia Both the liquid- reasonable amounts of ganoderic acids(GAs)and polysac- state fermentation and solid-state fermentation are popular charides can be obtained by submerged fermentation after for the production of Lingzhi mycelia. 3-5 weeks only (Wagner et al.2003).Secondly,the myce- lium grown in LSF holds high stability and standardization in which the environmental conditions (temperature,dis- solved oxygen,pH,etc.)are easier to control.This is im- portant not only for producing the desired product but also might be beneficial for producing mushroom-based medi- cines and nutraceuticals.The products obtained by this method are easy to achieve,with higher quality standards and safety (Wasser and Weis 1999).Thirdly,advantage of mycelia culturing in LSF is to provide a new method for scalable production and increased yields of biologically active compounds.By LSF method,the producer can in- Development stage crease the yield of protein rich in essential amino acids and vitamins serving as functional foods compared with the yield of these components in carpophores of the standard Bud-developing stage fruiting basidiomycetes(Friel and McLoughlin 2000).Thus, Growth stage production of fungus metabolites through LSF method has received a lot of attention because of short time cultivation, high productivity,fewer chances of contamination,and easy recovery of producing metabolites (Huang and Liu 2008; Kim et al.2007). In general,the methods of artificial cultivation typically Bud-breaking stage in liquid media involve five stages:(1)selection of Lingzhi Maturity stage strain;(2)preparation of culture maintenance medium for Fig.3 The developmental process of Lingzhi fruit body different culture phases;(3)inoculation;(4)cultivation of ②Springer

aid in the fruiting body development (shown in Fig. 3). The entire growth of Lingzhi from spawn running to cropping in artificial cultivation is different from the method of cultiva￾tion used. In general, it should take on average approximately 90–150 days. It directly affects the Lingzhi product’s quality to admin￾ister the development process of fruit body. In the overview of previous publications, the cultivation procedures were roughly the same; the difference was only diverse from different cultivation methods or raw materials (Chen 1999; Chi 2005; Erkel 2009; Hossain et al. 2009; Xiao et al. 2010). Up to now, in the Lingzhi production practice in China, the preparation of substrate, including substrate formulations, making the synthetic logs in bag cultivation and substrate sterilization etc., have appeared in the textbook of some vocational schools or popular science books (Zhang 1998; Liu and Jiang 2007). Meanwhile, the cultivation processes of Lingzhi have also been described in these books. Such questions regarding the crucial stages in Lingzhi cultivation, how to speed up the spawn running, how to control the environmental factors, and how to produce Lingzhi with caps have been introduced in various media, including mag￾azines, popular science books, DVD video, TV etc. Growers will benefit from concentrating on the step-by-step instruc￾tion on how to cultivate it. Artificial cultivation of Lingzhi mycelia Both the liquid￾state fermentation and solid-state fermentation are popular for the production of Lingzhi mycelia. Liquid-state (submerged) fermentation of Lingzhi mycelia Liquid-state fermentation (LSF) of mushroom, also known as liquid culture or submerged fermentation, is a process of culturing microorganism in liquid media, but not on the surface of liquid media. Preparation of mushroom mycelium in submerged culture was initially developed during the 1970s. At that time, only lower fungi (fungi that are not from basidiocarps) were successfully cultured in fermentors for economical production of various natural products (Yang and Liau 1998). The main principle is that the liquid medi￾um is added to the fermentation tank or flask. When sterile air is led to reactor, it will increase the dissolved oxygen content in the medium to improve respiratory metabolism of the mushroom mycelium. At the same time, the mushroom mycelium is agitated or oscillated, and appropriate external conditions are controlled so that mushroom cells grow in the liquid depths of breeding, producing a lot of the mycelium or metabolites. Another review indicated that there were few investigations into the development of high fungi (basidio￾mycetes) bioprocesses (Fang and Zhong 2002a, b). Compared with the artificial cultivation of Lingzhi fruit bodies, LSF holds obvious advantages. Firstly, the advan￾tage of LSF over the traditional, multinational cultivation of fruit body is the reduction in the time spent to obtain the product of interest (Mshandete and Mgonja 2009). The production of fruit bodies takes at least 3–5 months, while reasonable amounts of ganoderic acids (GAs) and polysac￾charides can be obtained by submerged fermentation after 3–5 weeks only (Wagner et al. 2003). Secondly, the myce￾lium grown in LSF holds high stability and standardization in which the environmental conditions (temperature, dis￾solved oxygen, pH, etc.) are easier to control. This is im￾portant not only for producing the desired product but also might be beneficial for producing mushroom-based medi￾cines and nutraceuticals. The products obtained by this method are easy to achieve, with higher quality standards and safety (Wasser and Weis 1999). Thirdly, advantage of mycelia culturing in LSF is to provide a new method for scalable production and increased yields of biologically active compounds. By LSF method, the producer can in￾crease the yield of protein rich in essential amino acids and vitamins serving as functional foods compared with the yield of these components in carpophores of the standard fruiting basidiomycetes (Friel and McLoughlin 2000). Thus, production of fungus metabolites through LSF method has received a lot of attention because of short time cultivation, high productivity, fewer chances of contamination, and easy recovery of producing metabolites (Huang and Liu 2008; Kim et al. 2007). In general, the methods of artificial cultivation typically in liquid media involve five stages: (1) selection of Lingzhi strain; (2) preparation of culture maintenance medium for Fig. 3 The developmental process of Lingzhi fruit body different culture phases; (3) inoculation; (4) cultivation of Appl Microbiol Biotechnol (2012) 93:941–963 947

948 Appl Microbiol Biotechnol (2012)93:941-963 strain in Erlenmeyer flasks,seeding tank,and fermentor. (IPS),and 582 mg/L for GA.In recent years,Chinese respectively;and(5)harvest of Lingzhi production.In the scientists have reported that they produced G.lucidum process of Lingzhi mycelia LSF,it is very important to 730 mycelia in a 500-L automatic stainless steel fer- select and control the appropriate fermentation conditions, mentor.The results showed that after a fermentation such as strain,amount of inoculation,temperature,pH,air time of 70 h,there was no obvious germination and flow,stirring rate,etc.The determination of the indices of lock-like concentration on the mycelia wall.There was the filamentous morphology,concentration,nutrients con- a little mycelium dissolved with no other bacteria.The sumption,and the outward appearance and viscosity of consistence of G.lucidum clump is about 30%.EPS broth could be used as the final quality standard of control and IPS is 3.5 g/L and 4.8 g/L,respectively (Wei et al. fermentation(Zhao 2002;Sanchez 2004;Wang et al.2007a, 2007a,b).Other reported studies of fermentation with b,c). G.lucidum were carried out in volumes of 10 m'with However,a multi-objective analysis and research was the fermentation liquid 7.5 m,the average production presented in the previous study of the Lingzhi LSF. cycle 150 h,and pH reduced from 6.5 to 3.5.The Some aimed simply to produce biomass,with no con- results showed that after the mycelia fermented,the cern for its composition.Others aimed to maximize the average weight of dry powders in every fermentor is production of either GAs or polysaccharides,and to 66.1 kg by spray drying,the average recovery 8.76 kg/ understand how different variables affect their produc- m',the content of pure polysaccharide 68.5 g/kg in the dry tion.Among these studies,many researchers studied powders,and the weight of the pure polysaccharide 4.225 kg Lingzhi LSF to obtain substances and special metabolic in every fermentor(Hu 2006). products (food,medicine or industrial enzyme,etc.) In addition,the cost-benefit ratio of liquid culture (Fang and Zhong 2002a;Yang 2005;Chen et al. and solid culture technologies for spawn production 2008;Songulashvili et al.2007).Also,other researchers were investigated by some researchers.The results dem- focused on the parameters in Lingzhi LSF including onstrated that liquid culture technology for spawn pro- culture media (Sun et al.2000),initial pH (Fang and duction is obviously more advantageous,which is Zhong 2002b),culture times and temperature,etc.(Wei reflected in better spawn quality,reduction of contami- et al.2007a,b;Xia et al.2007;Zhang et al.2008a,b; nation,increased efficiency,reduction of cost,and in- Liang 2011).In summary,in the process of culturing creased production stability.For this technology to be Lingzhi by LSF method,the suitable carbon and nitro- broadly used,it is key to improve the incubator shakers, gen resources are corn flour and soybean meal with fermentation tanks,and inoculation equipment.Efforts concentrations of 3%and 2.5%,respectively.The opti- to facilitate researches according to the demand of a mum cultivation conditions were as follows:initial pH 4.5- dynamic market would promote the application of this 5.0,rotation speeds 120-150 rpm,100-120 mL medium/ technology in the fast-growing mushroom spawn pro- 500 mL Erlenmeyer flask at 25 C.When the diameter of duction industry (Guo and Liu 2011).Based on the mycelium pellet was 0.85-0.9 mm in the process of culturing character of Lingzhi mycelium having bio-enrichment with Erlenmeyer flask,the biomass of Lingzhi mycelia is the of mineral nutrition,some researchers also focused their highest(Dong et al.2004;Zhu et al.2009). interest on the accumulation of some minerals by sub In spite of having achieved significant progress in merged fermentation,which include the selenium- Lingzhi LSF,the applications of LSF techniques have enriched Lingzhi (Xie et al.1996;Shen and Yu 2008; an interval to industrialization production.In other Ling et al.2008),the calcium-enriched Lingzhi (Gao et words,the successful example in large-scale culture al.2007).the zinc-enriched Lingzhi (Liu et al.2005: Lingzhi by LSF is extraordinary.In the previous review Wei et al.2010),the iron-enriched Lingzhi (Miao and articles,the researchers greatly summarized the pub- Lv 2007),and so on.The abovementioned studies pro- lished data on submerged fermentation with Lingzhi vide a foundation of development of functional food (Wagner et al.2003;Sanodiya et al.2009).In this using mineral nutrition enrichment Lingzhi as raw mate- review article,only one report of a large-scale fermen- rials (Wang et al.2001;Mao and Ma 2009). tation was described in which Ganoderma tsugae was cultivated in tanks with a volume of 20 m'.All other Solid-state fermentation of Lingzhi mycelia In contrast to reported studies of LSF with Lingzhi were done only in LSF,solid-state fermentation(SSF),also known as SSC,is volumes of 10 L or less.By estimating results general- the cultivation of microorganisms under controlled condi- ly,half the studies were undertaken in Erlenmeyer tions in the absence of free water.The production examples flasks and others were in fermentors.However,the best of SSF include the industrial enzymes (Wu et al.2000), yields reported to date are 22.1 g/L for biomass,1.71 g/ biofuels,biopesticide and nutrient-enriched animal feeds. L for EPS,2.49 g/L for intercellular polysaccharides etc.(Habijanic and Berovic 2000;Sun et al.2007;Chen et ②Springer

strain in Erlenmeyer flasks, seeding tank, and fermentor, respectively; and (5) harvest of Lingzhi production. In the process of Lingzhi mycelia LSF, it is very important to select and control the appropriate fermentation conditions, such as strain, amount of inoculation, temperature, pH, air flow, stirring rate, etc. The determination of the indices of the filamentous morphology, concentration, nutrients con￾sumption, and the outward appearance and viscosity of broth could be used as the final quality standard of control fermentation (Zhao 2002; Sánchez 2004; Wang et al. 2007a, b, c). However, a multi-objective analysis and research was presented in the previous study of the Lingzhi LSF. Some aimed simply to produce biomass, with no con￾cern for its composition. Others aimed to maximize the production of either GAs or polysaccharides, and to understand how different variables affect their produc￾tion. Among these studies, many researchers studied Lingzhi LSF to obtain substances and special metabolic products (food, medicine or industrial enzyme, etc.) (Fang and Zhong 2002a; Yang 2005; Chen et al. 2008; Songulashvili et al. 2007). Also, other researchers focused on the parameters in Lingzhi LSF including culture media (Sun et al. 2000), initial pH (Fang and Zhong 2002b), culture times and temperature, etc. (Wei et al. 2007a, b; Xia et al. 2007; Zhang et al. 2008a, b; Liang 2011). In summary, in the process of culturing Lingzhi by LSF method, the suitable carbon and nitro￾gen resources are corn flour and soybean meal with concentrations of 3% and 2.5%, respectively. The opti￾mum cultivation conditions were as follows: initial pH 4.5– 5.0, rotation speeds 120–150 rpm, 100–120 mL medium/ 500 mL Erlenmeyer flask at 25 °C. When the diameter of mycelium pellet was 0.85–0.9 mm in the process of culturing with Erlenmeyer flask, the biomass of Lingzhi mycelia is the highest (Dong et al. 2004; Zhu et al. 2009). In spite of having achieved significant progress in Lingzhi LSF, the applications of LSF techniques have an interval to industrialization production. In other words, the successful example in large-scale culture Lingzhi by LSF is extraordinary. In the previous review articles, the researchers greatly summarized the pub￾lished data on submerged fermentation with Lingzhi (Wagner et al. 2003; Sanodiya et al. 2009). In this review article, only one report of a large-scale fermen￾tation was described in which Ganoderma tsugae was cultivated in tanks with a volume of 20 m3 . All other reported studies of LSF with Lingzhi were done only in volumes of 10 L or less. By estimating results general￾ly, half the studies were undertaken in Erlenmeyer flasks and others were in fermentors. However, the best yields reported to date are 22.1 g/L for biomass, 1.71 g/ L for EPS, 2.49 g/L for intercellular polysaccharides (IPS), and 582 mg/L for GA. In recent years, Chinese scientists have reported that they produced G. lucidum 730 mycelia in a 500-L automatic stainless steel fer￾mentor. The results showed that after a fermentation time of 70 h, there was no obvious germination and lock-like concentration on the mycelia wall. There was a little mycelium dissolved with no other bacteria. The consistence of G. lucidum clump is about 30%. EPS and IPS is 3.5 g/L and 4.8 g/L, respectively (Wei et al. 2007a, b). Other reported studies of fermentation with G. lucidum were carried out in volumes of 10 m3 with the fermentation liquid 7.5 m3 , the average production cycle 150 h, and pH reduced from 6.5 to 3.5. The results showed that after the mycelia fermented, the average weight of dry powders in every fermentor is 66.1 kg by spray drying, the average recovery 8.76 kg/ m3 , the content of pure polysaccharide 68.5 g/kg in the dry powders, and the weight of the pure polysaccharide 4.225 kg in every fermentor (Hu 2006). In addition, the cost–benefit ratio of liquid culture and solid culture technologies for spawn production were investigated by some researchers. The results dem￾onstrated that liquid culture technology for spawn pro￾duction is obviously more advantageous, which is reflected in better spawn quality, reduction of contami￾nation, increased efficiency, reduction of cost, and in￾creased production stability. For this technology to be broadly used, it is key to improve the incubator shakers, fermentation tanks, and inoculation equipment. Efforts to facilitate researches according to the demand of a dynamic market would promote the application of this technology in the fast-growing mushroom spawn pro￾duction industry (Guo and Liu 2011). Based on the character of Lingzhi mycelium having bio-enrichment of mineral nutrition, some researchers also focused their interest on the accumulation of some minerals by sub￾merged fermentation, which include the selenium￾enriched Lingzhi (Xie et al. 1996; Shen and Yu 2008; Ling et al. 2008), the calcium-enriched Lingzhi (Gao et al. 2007), the zinc-enriched Lingzhi (Liu et al. 2005; Wei et al. 2010), the iron-enriched Lingzhi (Miao and Lv 2007), and so on. The abovementioned studies pro￾vide a foundation of development of functional food using mineral nutrition enrichment Lingzhi as raw mate￾rials (Wang et al. 2001; Mao and Ma 2009). Solid-state fermentation of Lingzhi mycelia In contrast to LSF, solid-state fermentation (SSF), also known as SSC, is the cultivation of microorganisms under controlled condi￾tions in the absence of free water. The production examples of SSF include the industrial enzymes (Wu et al. 2000), biofuels, biopesticide and nutrient-enriched animal feeds, etc. (Habijanic and Berovic 2000; Sun et al. 2007; Chen et 948 Appl Microbiol Biotechnol (2012) 93:941–963

Appl Microbiol Biotechnol(2012)93:941-963 949 al.2010b).In recent years,the SSC of mycelia has led considered as the new turning point of modern biolog- to a wide range of applications at the laboratory scale ical technology and the new channel of the new drug R because information from SSC can be applied to more D of TCM (Zhuang et al.2007).Based on inventor's commonly used liquid-state cultivation (Maldonado and opinion,the bidirectional SSF has two characteristics. Strasser de Saad 1998;Mahapatra and Banerjee 2009). The used nutrient substance contains the medicinal SSC has also been frequently utilized in preliminary herbs with multiple active compositions,instead of the tests for cultivating microorganisms under experimental one that consists of only farming byproducts.The prod- conditions because it requires less time and labor than ucts formed by this technique are fungous substance LSF. containing herbs which was produced by a medicinal Though there is very little information about the produc- fungous substance (Zhuang 1991,1995,2002;Zhuang tion of Lingzhi in SSF,it is not difficult to find some basic et al.2007).What is the medicinal fungi bidirectional researches under experimental conditions,such as substrate SSF technology?The key of this technology is that the components,pH,and temperature,etc.Among these stud- medicinal mushroom strains are cultured in the special ies,the most praiseworthy work is an experiment conducted substrate,which consisted of CMM or medicine slag as by Habijanic and Berovic.They carried out the Lingzhi medicinal substrate instead of the traditional nutritious fermentation in a horizontal stirred tank reactor with a total substrate cultured.The fermentation products are known volume of 30 L with suitable conditions (Habijanic and as the medicinal fungal substance.In the fermentation Berovic 2000).Their eminent work showed a positive as- process,while the medicinal substrate provided nutrients pect for satisfactory rates of growth and exopolysaccharide for fungal growth,it was also affected by the enzyme production by favorable conditions.In their study,the sub- produced from fungi.Hence,the tissues and compo- strate consisted of beech sawdust,olive oil,(NH4)2S04, nents of this medicinal substrate would be changed KH2PO4,CaCl22H2O,MgSO47H2O,FeSO47H2O,and and produce new functional components,so the bio- distilled water,which provide the optimal substrates for chemical process of medicinal fungi and herbs in fer- the production of immunostimulatory animal feed supple- mentation substrate hold "bidirectional",which present ments because the whole fermented substrate is used as the the perfect combination with medicinal fungi and CMM product.In other previous reports,Hsieh and Yang (2004) (Zhang et al.2005a,b;Zhuang and Hong 2006;Zhuang used soy residue for the Lingzhi SSF.In their experiment, etal.2004). the SSF was conducted in three types of containers:test Since the invention of the technique,Lingzhi is the most tube,500-mL flask,and sterilizable polypropylene plas- preferred species to study by Chinese scientists.For exam- tic bag.The highest rate of mycelial growth of 6 mm/day was ple,based on the purpose of drug research and development, observed in the medium of carbon/nitrogen(C/N)ratio of Chen and Chen reported their research results.In their 80 using test tubes.However,a growth rate of 7.5 mm/ experiment,SSF was applied for medicinal fungi by fer- day was found at the C/N ratio of 70-80 in the 500-mL menting G.lucidum with Radix astragali containing medi- flasks (Hsieh and Yang 2004).Tea waste (TW)is used um.G.lucidum was fermented in ordinary medium,CMM- as a new supplement for substrate mixtures in Lingzhi containing medium (containing Radix astragali),and cultivation.Peksen and Yakupoglu determined the selenium-rich CMM-containing medium,respectively.The effects of sawdust (S)-based substrates supplemented polysaccharide contents of fermentation products from the with TW at the various levels (75S:25TW,80S:20TW, three kinds of culture media were investigated at different 85S:15TW,and 90S:10TW)and G.lucidum strains on times,and the changes were compared.The results showed the yield,biological efficiency (BE),and the chemical that the polysaccharide contents of fermentation products composition of fruiting bodies in SSF.The result from the three kinds of culture media were 4.65%.3.76% showed that the significant differences were found and 4.50%,respectively,and their relative standard deviation among substrates regarding yield and BE,while yield were 1.61%,1.99%,and 1.86%,respectively.By observing and BE of the strains were not different (Peksen and the changes of the contents of polysaccharide,protein,and Yakupoglu 2009). total saponin in fermentation products from the CMM- The combination of modern biotechnology and tradi- containing medium at different times,it was found that the tional fermentation technology of Chinese materia 28th fermentation day was the time when secondary metabo- medica (CMM)provides a broad space for the rapid lism was the most active,and it should be the fermented development of fermentation technology of CMM.It is terminal point(Chen and Chen 2004).For the same purpose, interesting that a new type of fermentation technique, many combinations of Lingzhi and other medicinal herbs, known as the bidirectional SSF technology,has emerged such as Lingzhi with Astragalus membranaceu (Huang qi) in China after the 1990s.Along with the theoretical and (Zhu et al.2010),Radix glycyrrhizaze (Gan cao)(Zhu et al. technical development of the engineering science,it was 2009),and Radix astragali (also called as Huang qi in ②Springer

al. 2010b). In recent years, the SSC of mycelia has led to a wide range of applications at the laboratory scale because information from SSC can be applied to more commonly used liquid-state cultivation (Maldonado and Strasser de Saad 1998; Mahapatra and Banerjee 2009). SSC has also been frequently utilized in preliminary tests for cultivating microorganisms under experimental conditions because it requires less time and labor than LSF. Though there is very little information about the produc￾tion of Lingzhi in SSF, it is not difficult to find some basic researches under experimental conditions, such as substrate components, pH, and temperature, etc. Among these stud￾ies, the most praiseworthy work is an experiment conducted by Habijanic and Berovic. They carried out the Lingzhi fermentation in a horizontal stirred tank reactor with a total volume of 30 L with suitable conditions (Habijanic and Berovic 2000). Their eminent work showed a positive as￾pect for satisfactory rates of growth and exopolysaccharide production by favorable conditions. In their study, the sub￾strate consisted of beech sawdust, olive oil, (NH4)2SO4, KH2PO4, CaCl2·2H2O, MgSO4·7H2O, FeSO4·7H2O, and distilled water, which provide the optimal substrates for the production of immunostimulatory animal feed supple￾ments because the whole fermented substrate is used as the product. In other previous reports, Hsieh and Yang (2004) used soy residue for the Lingzhi SSF. In their experiment, the SSF was conducted in three types of containers: test tube, 500-mL flask, and sterilizable polypropylene plas￾tic bag. The highest rate of mycelial growth of 6 mm/day was observed in the medium of carbon/nitrogen (C/N) ratio of 80 using test tubes. However, a growth rate of 7.5 mm/ day was found at the C/N ratio of 70–80 in the 500-mL flasks (Hsieh and Yang 2004). Tea waste (TW) is used as a new supplement for substrate mixtures in Lingzhi cultivation. Peksen and Yakupoglu determined the effects of sawdust (S)-based substrates supplemented with TW at the various levels (75S:25TW, 80S:20TW, 85S:15TW, and 90S:10TW) and G. lucidum strains on the yield, biological efficiency (BE), and the chemical composition of fruiting bodies in SSF. The result showed that the significant differences were found among substrates regarding yield and BE, while yield and BE of the strains were not different (Peksen and Yakupoglu 2009). The combination of modern biotechnology and tradi￾tional fermentation technology of Chinese materia medica (CMM) provides a broad space for the rapid development of fermentation technology of CMM. It is interesting that a new type of fermentation technique, known as the bidirectional SSF technology, has emerged in China after the 1990s. Along with the theoretical and technical development of the engineering science, it was considered as the new turning point of modern biolog￾ical technology and the new channel of the new drug R & D of TCM (Zhuang et al. 2007). Based on inventor’s opinion, the bidirectional SSF has two characteristics. The used nutrient substance contains the medicinal herbs with multiple active compositions, instead of the one that consists of only farming byproducts. The prod￾ucts formed by this technique are fungous substance containing herbs which was produced by a medicinal fungous substance (Zhuang 1991, 1995, 2002; Zhuang et al. 2007).What is the medicinal fungi bidirectional SSF technology? The key of this technology is that the medicinal mushroom strains are cultured in the special substrate, which consisted of CMM or medicine slag as medicinal substrate instead of the traditional nutritious substrate cultured. The fermentation products are known as the medicinal fungal substance. In the fermentation process, while the medicinal substrate provided nutrients for fungal growth, it was also affected by the enzyme produced from fungi. Hence, the tissues and compo￾nents of this medicinal substrate would be changed and produce new functional components, so the bio￾chemical process of medicinal fungi and herbs in fer￾mentation substrate hold “bidirectional”, which present the perfect combination with medicinal fungi and CMM (Zhang et al. 2005a, b; Zhuang and Hong 2006; Zhuang et al. 2004). Since the invention of the technique, Lingzhi is the most preferred species to study by Chinese scientists. For exam￾ple, based on the purpose of drug research and development, Chen and Chen reported their research results. In their experiment, SSF was applied for medicinal fungi by fer￾menting G. lucidum with Radix astragali containing medi￾um. G. lucidum was fermented in ordinary medium, CMM￾containing medium (containing Radix astragali), and selenium-rich CMM-containing medium, respectively. The polysaccharide contents of fermentation products from the three kinds of culture media were investigated at different times, and the changes were compared. The results showed that the polysaccharide contents of fermentation products from the three kinds of culture media were 4.65%, 3.76%, and 4.50%, respectively, and their relative standard deviation were 1.61%, 1.99%, and 1.86%, respectively. By observing the changes of the contents of polysaccharide, protein, and total saponin in fermentation products from the CMM￾containing medium at different times, it was found that the 28th fermentation day was the time when secondary metabo￾lism was the most active, and it should be the fermented terminal point (Chen and Chen 2004). For the same purpose, many combinations of Lingzhi and other medicinal herbs, such as Lingzhi with Astragalus membranaceu (Huang qi) (Zhu et al. 2010), Radix glycyrrhizaze (Gan cao) (Zhu et al. 2009), and Radix astragali (also called as Huang qi in Appl Microbiol Biotechnol (2012) 93:941–963 949

950 Appl Microbiol Biotechnol (2012)93:941-963 Chinese)(He 2010),etc.,containing medium have been in- pharmaceuticals have a long history and have received vestigated in the SSF (Gu et al.2005). considerable attention in recent years.Importantly,pre- In other reports,based on the purpose of functional food vious research reports discovered that the water-soluble research and development,Lingzhi production by SSF using polysaccharides from Lingzhi characterized more than comn substrate was investigated,and the fermentation con- 20 types and strongly inhibited tumor growth(Zhou et ditions were optimized through single factor and orthogonal al.2007a).The major biologically active polysacchar- tests.Meanwhile,the content of polysaccharide reducing ides from Lingzhi are glucans,whose basic structure is B-1-3 sugar,protein,and amino acid was determined.The results D-glucopyronan with 1 to 15 U of B-1-6 monoglucosyl side showed that the size of crushed granule,strain,time, chains and with(1→3)β，(1→4)β，and/or(1→6)βlink- temperature,and inoculum size had a significant effect ages.The bioactive polysaccharides differ greatly in their on the content of polysaccharide,reducing sugar,pro- composition and consequently in chemical structure,and tein,and amino acid.The optimum fermentation con- one common feature is their relatively high molecular weight ditions were 10 mesh granularity,12%inoculum size, which has an average molecular weight of 10+6 Da(Huie and 28 C fermentation temperature,and 20-day fermenta- Di 2004;Chang and Lu 2004;Zhong et al.2009). tion time.The content of polysaccharide was 21.97 mg/ The basic extraction methods for Lingzhi polysacchar- g under the optimized cultivation conditions (Gao ides are the water extract-alcohol precipitation method.So 2007).In addition,more than ten beans as substrate many researches focused their attention on some parameters supplements were applied for SSF of Lingzhi.These related to extraction such as extraction of temperature,time, researches provided a foundation for further develop- and rate of raw materials to water(Song et al.2008;Chen et ment of health food (Zhang et al.2006;Wei et al. al.2009).However,the water extraction-alcohol precipita- 2009;You 2009).In addition,Lingzhi (mushroom)cul- tion methods have some disadvantages,such as long extrac- tivation can also help to convert agricultural and forest tion time,low extraction rate,high extraction temperature, wastes into useful matter and reduce pollution in the etc.,and so previous researchers have done a lot of work on environment. comparing the different extracting methods.Based on dif- ferent cell-wall broken methods,the extraction methods can be divided into three ways:ultrasonic extraction method (Zhang et al.2007,2010),microwave extraction methods, Development of Ganodermna-based products and enzymatic method (Zhu et al.2004;Lu 2009;Huang and Ning 2010).A previous review article presented a There has been a recent upsurge of interest in Lingzhi, standard methodology involved in the extraction of Lingzhi which is not only a health food rich in polysaccharides polysaccharide (Huie and Di 2004).Actually,this is one of and triterpenoids but also a source of biologically active the extraction methods of water-insoluble polysaccharides compounds of medicinal value.Many of Lingzhi or its from "Lingzhi".This method is based on the attack of the extracts may be processed as the complementary medicine/ enzyme on the polysaccharide substrates,composed of cel- dietary supplements(DSs)for anti-cancer,anti-viral,immu- lulose and lignin.For information,it has been reported that nopotentiating,hypocholesterolemic,and hepatoprotective enzyme hydrolysis reaction could be enhanced by ultrasonic agents(Paterson 2006;Zheng 2011).The bioactive com- waves. pounds are extractable from either the Lingzhi mycelium or fruiting body and represent important components of the Triterpenoids/triterpenes Other main bioactive components expanding Lingzhi biotechnology industry. from Lingzhi are triterpenoids/triterpenes of which pharma- cological effects have been demonstrated that are well Extraction and purification of bioactive components Lingzhi known as antioxidative,immunomodulating and anti- contains numerous bioactive components,such as polysac- tumor,etc.Major triterpenoids isolated from Lingzhi are charides,ergosterols,various proteins,unsaturated fatty different types of GA.There are dozens of GAs that have acids,vitamins,and minerals (Zhou et al.2007a,b).In been isolated and characterized.Among them,GA-A,GA- previous publications,numerous studies all focused on the B,and GA-C are the best representatives.The bitterness of following three kinds of biactiove components,i.e.,poly- Lingzhi mainly originated from GAs.Now,there are more saccharides,triterpenoids,and functional proteins. than 130 oxygenated triterpenes(mostly lanostane-type tri- terpenes)that have been isolated from the fruiting bodies, Polysaccharides Lingzhi polysaccharide is one type of bio- spores,mycelia,and broths.The basic chemical structure is active components isolated from Lingzhi and has a wide range based on the ground structure of lanosterol,which is an of physicochemical properties.The bioactive polysaccharides important intermediate in the biosynthetic for steroid and (including protein/peptide bound polysaccharides)as triterpene in microorganisms and animals(Chang and Buswell ②Springer

Chinese) (He 2010), etc., containing medium have been in￾vestigated in the SSF (Gu et al. 2005). In other reports, based on the purpose of functional food research and development, Lingzhi production by SSF using corn substrate was investigated, and the fermentation con￾ditions were optimized through single factor and orthogonal tests. Meanwhile, the content of polysaccharide reducing sugar, protein, and amino acid was determined. The results showed that the size of crushed granule, strain, time, temperature, and inoculum size had a significant effect on the content of polysaccharide, reducing sugar, pro￾tein, and amino acid. The optimum fermentation con￾ditions were 10 mesh granularity, 12% inoculum size, 28 °C fermentation temperature, and 20-day fermenta￾tion time. The content of polysaccharide was 21.97 mg/ g under the optimized cultivation conditions (Gao 2007). In addition, more than ten beans as substrate supplements were applied for SSF of Lingzhi. These researches provided a foundation for further develop￾ment of health food (Zhang et al. 2006; Wei et al. 2009; You 2009). In addition, Lingzhi (mushroom) cul￾tivation can also help to convert agricultural and forest wastes into useful matter and reduce pollution in the environment. Development of Ganoderma-based products There has been a recent upsurge of interest in Lingzhi, which is not only a health food rich in polysaccharides and triterpenoids but also a source of biologically active compounds of medicinal value. Many of Lingzhi or its extracts may be processed as the complementary medicine/ dietary supplements (DSs) for anti-cancer, anti-viral, immu￾nopotentiating, hypocholesterolemic, and hepatoprotective agents (Paterson 2006; Zheng 2011). The bioactive com￾pounds are extractable from either the Lingzhi mycelium or fruiting body and represent important components of the expanding Lingzhi biotechnology industry. Extraction and purification of bioactive components Lingzhi contains numerous bioactive components, such as polysac￾charides, ergosterols, various proteins, unsaturated fatty acids, vitamins, and minerals (Zhou et al. 2007a, b). In previous publications, numerous studies all focused on the following three kinds of biactiove components, i.e., poly￾saccharides, triterpenoids, and functional proteins. Polysaccharides Lingzhi polysaccharide is one type of bio￾active components isolated from Lingzhi and has a wide range of physicochemical properties. The bioactive polysaccharides (including protein/peptide bound polysaccharides) as pharmaceuticals have a long history and have received considerable attention in recent years. Importantly, pre￾vious research reports discovered that the water-soluble polysaccharides from Lingzhi characterized more than 20 types and strongly inhibited tumor growth (Zhou et al. 2007a). The major biologically active polysacchar￾ides from Lingzhi are glucans, whose basic structure is β-1-3 D-glucopyronan with 1 to 15 U of β-1-6 monoglucosyl side chains and with (1→3)-β, (1→4)-β, and/or (1→6)-β link￾ages. The bioactive polysaccharides differ greatly in their composition and consequently in chemical structure, and one common feature is their relatively high molecular weight which has an average molecular weight of 104–6 Da (Huie and Di 2004; Chang and Lu 2004; Zhong et al. 2009). The basic extraction methods for Lingzhi polysacchar￾ides are the water extract–alcohol precipitation method. So many researches focused their attention on some parameters related to extraction such as extraction of temperature, time, and rate of raw materials to water (Song et al. 2008; Chen et al. 2009). However, the water extraction–alcohol precipita￾tion methods have some disadvantages, such as long extrac￾tion time, low extraction rate, high extraction temperature, etc., and so previous researchers have done a lot of work on comparing the different extracting methods. Based on dif￾ferent cell-wall broken methods, the extraction methods can be divided into three ways: ultrasonic extraction method (Zhang et al. 2007, 2010), microwave extraction methods, and enzymatic method (Zhu et al. 2004; Lu 2009; Huang and Ning 2010). A previous review article presented a standard methodology involved in the extraction of Lingzhi polysaccharide (Huie and Di 2004). Actually, this is one of the extraction methods of water-insoluble polysaccharides from “Lingzhi”. This method is based on the attack of the enzyme on the polysaccharide substrates, composed of cel￾lulose and lignin. For information, it has been reported that enzyme hydrolysis reaction could be enhanced by ultrasonic waves. Triterpenoids/triterpenes Other main bioactive components from Lingzhi are triterpenoids/triterpenes of which pharma￾cological effects have been demonstrated that are well known as antioxidative, immunomodulating and anti￾tumor, etc. Major triterpenoids isolated from Lingzhi are different types of GA. There are dozens of GAs that have been isolated and characterized. Among them, GA-A, GA￾B, and GA-C are the best representatives. The bitterness of Lingzhi mainly originated from GAs. Now, there are more than 130 oxygenated triterpenes (mostly lanostane-type tri￾terpenes) that have been isolated from the fruiting bodies, spores, mycelia, and broths. The basic chemical structure is based on the ground structure of lanosterol, which is an important intermediate in the biosynthetic for steroid and triterpene in microorganisms and animals (Chang and Buswell 950 Appl Microbiol Biotechnol (2012) 93:941–963