Marine yeasts and their applications in mariculture - yimg.com

Journal of Ocean University of China (Oceanic

and Coastal Sea Research)

Review

ISSN 1672-5182,July 30, 2006, Vol.5, No.3, pp.251-256 http: // u,ww. ouc. edu. cn/xbywb/ E-maih xbywb@ouc edu. cn

Marine Yeasts and Their Applications in Mariculture CHI Zhenming*, LIU Zhiqiang, GAO Lingmei, GONG Fang, MA Chunling, WANG Xianghong, and LI Haifeng UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, P. R. China

(Received May 12, 2005; accepted April 10,2006) Abstract The terrestrial yeasts have been receiving great attention in science and industry for over one hundred years because they can produce many kinds of bioactive substances. However, little is known about the bioactive substances of marine yeasts. In recent years, it has been found that marine yeasts have wide applications in mariculture and other fields. Therefore, marine yeasts, the bioactive substances from them and the applications of marine yeasts themselves and the bioactive substances they produced are reviewed in this paper. Key words

marine yeasts; bioactive substances; mariculture; single cell protein

and environmental protection.

1 Introduction Because yeasts can produce many bioactive substances, such as protein, amino acids, vitamin, polysaccharide, fatty acid, phospholipid, polyamine, astaxanthin, 13-carotenoid, trehalose, glutathione, superoxide dismutase, chitinase, amylase, phytase, protease, killer toxin and so on, they have been receiving much attention for many decades. In the last century, beer and wine industries which use the yeast Saccharomyces cerevisiae in the production had a yearly turnover of 100 billion dollars and created ten million jobs all over the world. At present, bioactive substances mainly come from terrestrial yeasts (Kurtzmam and Fell, 1998; Dijken, 2002). However, little is known about the bioactive substances from marine yeasts up to now. The oceans cover 71% of the surface of the earth, and there are abundant biotic resources, including yeast. Marine yeasts are those that can survive longer in sea water than in fresh water or can grow better in the medium prepared with sea water than in that prepared with distilled water. Therefore, the investment for the development of marine yeasts as a resource of bioactive substances and a gene resource is needed. If marine yeasts could be used for producing bioactive substances, seawater, the most abundant water resource in the world, could be used in fermentation, so that a large amount of freshwater could be saved. In our recent studies, it has been found that marine yeasts could produce bioactive substances with the potential application in mariculture, food, pharmaceutical, cosmetic, chemical industries * Corresponding author. Te1:0086-532-82032266 E-mail: zhenming@sdu, edu. cn

2 Single Cell Protein (SCP) and Amino Acids In the last 20 years the maricultural industry of China has been developing very rapidly with its cultured sea-food yield becoming the highest in the world. Therefore, the demand of feed of marine animals is increasing steadily. However, there is still a lack of feed which is composed of single cell protein from marine yeasts with high content of protein and other nutrients. A variety of microatgae such as Spirulina and Chlorella and brown algae are extensively used as feed for marine animals. However, they have some limitations for animal consumption. They have rigid cell walls. Their production is generally done outdoors and dependent on the climatic conditions. The algal culture is very easily contaminated by protozoa and bacteria. Some yeasts such as Saccharomyces cerevisiae, Candida utilis, Candida tropicalis and the species of genera Hansenula, Pichia and Torulopsis can be used for their single cell protein. They have many advantages over algae. For example, their protein contents account for up to 50% of the dry cell weight. Moreover, they can also supply the feed with the B-complex group vitamins, minerals and other components, which could stimulate the disease resistance of marine animals. They show a low level of nucleic acid content ( 9 . 7 % ) . Especially, yeast cells have higher concentration of essential amino acids such as lysine, methionine and leucine than algal cells. The digestion rate of single cell protein of yeast cells is high (generally above 80 % ). Such protein could replace fish powder. In general, yeast cells can grow very quickly in

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medium and the cell density in the fermenting medium is very high (over 108 cells m L - 1 ) , and the fermentation period is very short. Compared to algal cultivation, it is very easy to manage the large-scale yeast cell production in the fermentor. Because most yeasts are characterized by flocculation and the cell size of yeast cells is much bigger than that of bacterial cells, it is very easy to collect and concentrate the yeast cells from the liquid culture. Many yeasts can grow well on cheap products, such as molasses and corn starch which are widely available from sugar industry and agriculture. However, many relevant parahaeters indicate that the nucleic acid safety in algae is better than that in fungi and bacteria (Anupama and Ravindra, 2000). Fungal sources can be exploited as nutritive SCP if the nucleic acid content is considerably reduced to levels comparable to the nucleic acid content of algae. Moreover, it has been shown that two polyunsaturated fatty acids (PUFAs) 20:5n-3 and 22.6n-3 are absent in the yeast cells, and thus if the yeast strains are to be used in fish aquaculture, they would need to be supplemented with live algae rich in these PUFAs (Brown et al., 1996). Unfortunately, little is known about marine yeasts that have high protein content and could be used as aquafeed. Rhishipal and Philip ( 1 9 9 8 ) isolated 33 strains of marine yeasts from the coastal and offshore waters off Cochin in India and found that the isolates had the ability of using starch, gelatin, lipid, cellulose, urea, pectin, lignin, chitin and prawn-shell wastes. The isolates were inoculated into the prawn-shell wastes and SCP generation was recognized in the increase of protein content in the final products. After the transformation of the prawn-shell waste by strain M15 of Candida sp, the protein content of the final products was increased from 38.5% to 70.4%. This finding promises wide application in aquaculture, particularly as a feed supplement. Moreover, the prawnshell, a waste in shrimp processing becomes a valuable raw material in a novel industry aiming at the microbial transformation of prawn-shell waste into aquaculture feed. Such transformation will facilitate the abatement of pollutant and recycling of waste. Brown et al. (1996) evaluated the possibility of using marine yeasts Debaromyces hansenii ACM 4784, Dipodascus capitatus ACM 4779, Dipodascus sp. ACM 4780 as feed for bivalve auqaculture. They found that Debaromyces hansenii ACM 4784, Dipodascus capitatus ACM 4779 and Dipodascus sp. ACM 4780 contained 23 %, 32%, and 36% of crude protein, respectively, while Candida utilis ACM 4774 contained 42% of crude protein. They concluded that high protein content, high levels of carbohydrate and good amino acid composition characterized all marine yeasts, and high levels of saturated fats characterized some marine yeasts. However, all the marine yeast strains lacked the 2 0 . 5 n - 3 and 22:6n-3 fatty acids, making them

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unsuitable as a complete diet for larval raising. Recently, we have determined the contents of crude protein of 327 marine yeast isolates using Kjeldahl's method and found that strain YA03a, which was isolated from the surface of Sargassum from Penglai coast in Shandong province, had 56.38% of crude protein content on the basis of the dry cell weight.

3 Probiotics In recent years, there are many reports about the application of marine yeasts as probiotics in mariculture. Tovar et al. (2002) found that some polyamineproducing yeasts have a strong potential of adhesion to intestinal mucus, an important condition for probiotic efficiency. They also found that the production of polyamines by Debaryomyces hansenii HF1 ( D H ) , a yeast strain isolated from fish gut, was three times higher than that by Saccharomyces cerevisiae ( S C ) . Both strains adhered to the gut of sea bass larvae. When the yeasts were introduced into a compound diet, the colonization was effective in the larvae. The DH diet led to an increase in the amylase secretion in 27-day-old larvae in comparison with the control, and the secretion of amylase and trypsin was lower in the SC diet. The activity of brush border membrane enzymes was stimulated by DH diet and delayed by SC diet in comparison with the control. The survival of larvae was also increased in DH diet, but the growth rate was lower than that of the control. TovarRamirez et al. (2004) found that the incorporation of 1.1% of marine yeast Debaryomyces hansenii CBS 8339 in diets improved survival rate by 10 %, and reduced proportion of malformed sea bass larvae. Final mean weight of groups fed with 1.1% yeast was twice the others. Activity and concentrations of trypsin and lipase were higher in the two groups fed with yeast than in the control. This suggested that the pancreas matured faster in the two groups fed with the marine yeast than in the control. Activities of intestinal enzymes, such as alkaline phosphatase, animopeptidase N and maltase in the group fed 1.1% yeast were higher than those in the other two, revealing early development of intestinal digestions. Ampe and Thiery (1998) found that a monolayer structure had been formed by the marine yeasts which attached to the inner surface of the peritrophic matrix of fairy shrimp Branchinella spinosa. The organization of yeasts on the peritrophic matrix suggested a symbiotic association between these microorganisms and B. spinosa. The peritrophic matrix provides a mechanical protection of the midgut epithelium against microbial invasion. Gamaet al.(2001) found that the marine yeasts were capable of adhering to the intestinal muscosa, thus providing potential applications as a probiotic supplement for human and animal health. Marine yeasts have been regarded as safe and have been show-

CHI Z.M. et al.: Marine Yeaste and Their Applications in Mariculture ing a beneficial impact on biotechnological process. Bacterins and an extracellular gluean-producing yeast Acremonium dyosporii were applied by Vici el al. (2000) to protect the larvae of Macrobrachium rosenbergii from Vibrio. They found that the larvae fed with bacterins and yeast cell powder exhibited significantly higher survival rates than the untreated one challenged with Vibrio sp. ANM 708 and Photobacterium sp. AAC 727,

4 Vitamin C Vitamin C is essential for the normal growth and many physiological functions of fish. High level of dietary vitamin C as reported could increase the resistance to Edwardsiella tarda and E. ictaluri infection in channel catfish, to V . anguillarum in rainbow trout and to A . salmonicida and V . salmonicida in Atlantic salmon (Sakai, 1999). Many studies have shown that treatment with high dose of vitamin C increased the complement activity in catfish and Atlantic salmon. Vitamin C was also reported to activate macrophages in Atlantic salmon and turbot. Hardie et al. (1991) found that treatment of fish with high dose of vitamin C stimulated macrophage activation factor causing lymphocyte proliferation. These results showed that fish fed with high dose ( m o r e than 1000 mg kg- 1 ) of vitamin C have protective immune response (Hardie et al., 1993). At present, about 50% of vitamin C produced is used as vitamin additives. Other fields of use include food production (25 % ), beverage additive ( 15 % ), and feed production (10 % ). However, the biosynthesis of vitamin C in marine yeast has not been investigated so far. If the whole biosynthesis pathway of vitamin C from glucose exists in marine yeast, the aquafeed made from marine yeast will provide materials for the growth of fish and stimulate protective immune response of fish at the same time. Furthermore, these marine yeasts not only are of scientific significance in biochemistry and biotechnology, but also can be used for mass production of vitamin C. In one of our recent studies, the contents of vitamin C in 327 marine yeast isolates have been determined. We found that only one strain did not contain vitamin C. The contents of others were higher than S . cerevisiae. Strain YF12b isolated from the gut of Hexagrammos otakii contained nearly 10 mg vitamin C per 100 g dry cells. The profile of HPLC analysis from the extract of YF12b grown in YPD medium showed that only one peak appeared, which was identical to that of the standard vitamin C solution. This evidence strongly shows that strain YF12b could completely synthesize vitamin C from glucose.

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5 Glutathione Glutathione (7-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant non-protein thiol compound widely distributed in living orgahisms, especially in eukaryotic cells. GSH can serve as antioxidant, immunity booster, and detoxifier of higher eukaryotic organisms (Li et al., 2004). GSH is thus considered as one of the most powerful, versatile, and important self-generated defense molecules. It is necessary to develop an economical process for GSH production on a large scale. At present, fermentation for GSH production is the major commercial method used. S. cerevisiae and Candida utilis are the main producers of GSH. For example, the permeabilized cells of Saccharomyces cerevisiae IFO 0021 treated by sodium dodecylsulfate and 1]-1, 3-glucanase could produce 4320 mg L - 1 of glutathione (Li et al., 2 0 0 4 ) . GSH accounting for 9.5% of dry cell weight was obtained from S. cerevisiae by fermentation (Li et al., 2004). Although the physiological roles of GSH in human and animal tissues, plant cells and microbial cells have been extensively investigated, the physiological roles in marine animals and applications of GSH in mariculture have not been carried out yet to date. Recently, we have determined the glutathione contents of 327 marine yeast isolates in our laboratory and found that all the isolates examined contained glutathione, but the contents varied among different strains. Strain YA09b isolated from the surface of Gracilaria teJctorii accumulated GSH up to 15.43 mg g 1 dry cell weight, almost the same as S. cerevisiae did.

6 p-Carotenoid 1]-carotenoid is a natural pigment widely distributed in organisms, such as red yeasts, some bacteria and plants, but can not be synthesized in animals. In industry, 1]-carotenoid and astaxanthin can be used as the additives of food and feed. 1]-carotenoid also can serve as the precursor of vitamin A in mammals. In recent years, many types of carotenoid have aroused extensive interest because of their beneficial effects on human health. For instance, lycopene and astaxanthin have the strong abilities to eliminate singlet oxygen, preventing human from cancer and enhancing human immune responses. Lycopene exhibits superior antioxidant ability, inhibits lipid peroxidation, and participates in cooxidation reactions, such as the cooxidation of a carotenoid and a polyunsaturated fat. 1]-carotenoid may inhibit the production of the lipid peroxyl radical products of lipooxygenase. So 1]-carotenoid have extensive use in medicine, cosmetic, chemistry and food industries. Meanwhile, Rhodotorula spp. which can produce a large amount of 1]-carotenoid is widely ap-

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plied in mariculture in China, because the species in this genus can increase the yields of mariculture animals and raise their ability to resist diseases (Misawa and Shimada, 1998). In recent years, many studies on enhanced production of ~-carotenoid by mutants of Rhodotorula spp. and optimization of the fermentation processes for cultivation of Rhodotorula spp. were undertaken. For example, Bhosale and Gadre (2001) found that mutant derived from R. glutinis NCIM 3253 produced 76-fold more ~-carotene than the original. In the growth medium prepared with seawater, the total carotenoid content and dry cell mass were 86 mg L - 1 and 16 g L - 1, respectively, as compared to 70 mg L-1 and 12 g L-1 obtained with a medium prepared with distilled water. Recently, our laboratory has isolated 10 strains of marine Rhodotorula sp. from the hypersaline environments, and found that these yeasts grew well in seawater medium and had high yields of Q-carotenoid.

tant enzyme in the guts of marine animals. Among the amylase-producing yeasts, Saccharomycopsis fibuligera can secret the biggest amount of amylase and has the highest growth rate. We found that this yeast could convert starch into trehalose effectively. The trehalose yield could reach over 23 g (100 g dry cell weight) - 1 ( C h i e t al., 2003). However, little is known about amylase-producing marine yeasts. We analyzed the activities of amylase produced by 427 marine yeast isolates and found that strain N13d from deep sea sediment of the Pacific Ocean could produce an extracellular amylase. This strain was identified to be Aureobasidium pullulans according to its 18S rRNA sequence and biochemical approaches. Under the optimal condition, 244.03 units of amylase per mg protein were produced within 56 h of fermentation (Li et al., 2006) O.

7 Glucan

Alkaline protease in the intestine of marine animals can help digest protein in the feed and the activity of alkaline protease in the intestine regulates the use of components in the compound diet and shows the stage of development in marine animals. Therefore, alkaline protease in the guts of marine animals has received much attention in recent years (Chong et al., 2002). Recently we have determined the protease activities of 427 marine yeast isolates, and found that many of them could produce protease. Strain HN3.11 from sediment of Dongfeng saltern near Qingdao produced the highest yield of protease. The protease had the highest activity at pH 9.0 and 45 E . Under the optimal condition, 623.1 U mg- 1 protein (7.2 U m L - 1) of alkaline protease was produced in the culture within 30h (Chi et al., 2006) |

The immune stimulatory effects of glucan mainly from cell wall of yeasts in marine animals have been studied. The types of glucan used include t3-1, 3-glucan, Q-I, 6-glucan from S. cerevisiae and peptide-glucan, 13-1, 3-glucan from bacteria. One result has shown that intraperitoneal injection of 13-1, 3-glucan and ~-1, 6-gluca from cell walls of S. cerevisiae into Atlantic salmon led to enhanced resistance to V. anguillarum, V. salmonicida and Y. ruckeri (Robertsen et al., 1990). Chen and Ainsworth (1992) reported that catfish injected with yeast glucan showed increased resistance to E. ictaluri. Oral administration of yeast glucan in Atlantic salmon can improve its protection against infection of V . anguillarum and V . salmonicida (Raa et al., 1992). Tiger shrimp immersed in yeast glucan solution (0.5 and l m g m L - 1) showed increased protection against V. vulnificus infection (Sung et al., 1994). Yeast glucan also has adjuvant effects on marine animals and the abilities to enhance the lysozyme activity, complement activity and bacteria-killing activity of macrophages of marine animals and the production of superoxide by macrophages or hemocytes in some marine animals (Sakai, 1999). Q-l, 3-glucan from Schizophyllum commune had similar function in marine animals (Sakai, 1999). However, there have not been any reports about the immune stimulatory effects of glucan from marine yeasts on marine animals.

8 Amylase Starch is a common substrate for production of yeast cells on a large scale due to its low price and easy availability ( C h i e t al., 2003). Amylase is an impor-

9 Protease

10 Phytase Phytase catalyses the release of phosphate from phytate (myco-inositol hexakiphosphate), which is the main form of phosphorus predominantly existing in cereal grains, legumes and oilseeds. Phytase is found to have many applications in animal nutrition, human nutrition and synthesis of lower inositol phosphates (Haefner et al., 2005). Phytate accounts for 60 %-80 % of phosphorus found in plant-derived feedstuffs, such as corn meal, wheat flour and soybean meal. The phytate molecule cannot be absorbed in the O Li, H. F., Z. Chi, X. H. Wang, andC. L. Ma, 2006. Amylase production by the marine yeast Aureobasidium pullulans. J. Ocean Univ. Chin., (accepted). 0 Chi, Z., C. Ma, P. Wang, and H. F. Li, 2006. Optimizationof mediumand cultivationconditionsfor alkalineproteaseproductionby the marine yeast Aureobasidium pullulans. Bioresour. Technol., (in press).

CHI Z.M. et al.. Marine Yeaste and Their Applications in Mariculture digestive tract without enzymatic degradation by phytase. Generally, this degradation can occur in the digestive tract and/or in the feed before consumption. The microbial ecosystem in monogastric animals including marine animals is mainly located in the large intestines, so that most of the phosphate released from phytate is not absorbed, but excreted after release by microorganisms. Due to the low availability of phosphorus in plant-derived feedstuffs, diets for marine animals have been traditionally supplemented with inorganic phosphates, leading to excessive dietary phosphorus that is released into marine environments. Due to increasing maricultured animal density in many regions including China, the released phosphorus will result in accumulation of phosphate in marine environments, leading to eutrophication of sea water. Microbial phytase has been supplemented to marine animal diets. It was found that supplementation of microbial phytase also showed effects on other nutrients. For example, the improvement of Ca, Mg, Zn, Cu and Mn availability could be achieved in many trials. Besides improving the availability of minerals and trace elements, microbial phytase is also able to enhance protein digestibility by the degradation of phytate protein, phytate mineral protein complexes and phytateamino acids-complex and by enhancement of protease activity. As phytate can also bind starch and inhibit amylase, activity of phytase supplemented is also able to increase energy utilization in marine animals as well. If some marine yeasts can produce a large amount of phytase, they can be incorporated into the diets. As mentioned above, marine yeasts have many functions in marine animals. Therefore, the phytaseproducing marine yeasts will have many applications in mariculture. Recently, we have analyzed phytase activities in 423 marine yeast isolates and found that many of them could produce a large amount of phytase, some of which are cell-bound while others are extraeellular.

11 Killer Toxin Killer toxin produced by some yeast strains is a low molecular mass protein or glycoprotein toxin which kills sensitive cells of the same or related yeast genera without direct cell-cell contact (Magliani e t a l . , 1997). The killer strains themselves are immune to their own toxin, but remain susceptible to the toxins secreted by other killer yeasts. The killer phenotype is very common in occurrence and can be found both in natural yeast isolates and in laboratory yeast strain collections. Up to now, toxin-producing killer yeasts have been identified in genus Candida, Cryptococcus, Debaryomyces , Hanseniaspora , Hansenula , Kluyveromyces , Metschnikowia , Pichia , Saccharomyces , Ustilago, Torulopsis, Williopsis and Zygosaccha-

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romyces, indicating that the killer phenomenon is in-

deed widespread among yeasts. Killer determinants are either cytoplasmically inherited encapsulated dsRNA viruses, linear dsDNA plasmids or nuclear genes. In addition, analysis of killer toxins and their receptor-mediated mode of action have proved to be an effective means for investigating the molecular structure and the assembly of yeast and fungal cell walls. The analysis can also provide important information for combating yeast infections caused by certain human pathogenic strains of the yeasts Candida albicans and/or Sporothrix schenkii. During the last two decades, secreted killer toxins and toxin-producing killer yeasts have found several applications. For instance, in fermentation industries, killer yeasts have been used to combat contaminating wild-type yeasts which can occur during the production of wine, beer and bread. Killer yeasts have also been used as bio-control agents in the preservation of food, vegetable and fruit, in the bio-typing of medically important pathogenic yeasts and yeast-like fungi, in the development of novel antimycotics for the treatment of human and animal fungal infections, and finally in the field of recombinant DNA technology (Magliani et al., 1997). In recent years, it has been well documented that some marine yeasts are pathogenic to some marine animals. Like bacterial and virus disease, the yeast disease has caused big economic losses in maricultural industry in some regions of China (Xu, 2005; Xu and Xu, 2003). For example, an explosive epidemic disease which is called milky disease has happened in cultured Portunus trituberculatus since 2001 in Zhoushan, Zhejiang Province, China, leading to high mortality of this crab and great economic loss in this area. The pathogenic agent for the milky disease was found to be the yeast Candida oleophila. The purified yeast strain from the diseased parts of the marine animal can develop the same symptom in the muscle, heart and hepatopancreas of the infected marine animals in the challenging test. It was found that nystatin, benzalkonium bromide, formaldehyde and extract of goldthread root and garlic are active against the pathogenic yeast. However, the compounds with minimum inhibitory concentration (MIC) are toxic to the crab and it is impossible to apply the expensive antibiotics to the open sea. Sun and Sun (1998) found that the yeast Torulopsis mogii is the pathogen to some shrimp in China. Moore (2003) observed that the yeast Metschnikowia bicuspidate var. bicuspidate, a pat-hogenic yeast of aquatic invertebrates was capable of infecting aquaculture-reared, disease-free A r t e m i a . As discussed above, killer yeasts can be applied to control the growth of pathogenic yeasts in human, animal and plant. Marine killer yeasts which have high activity against the pathogenic yeast in marine animals

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were isolated by us. W e found that m a n y marine strains are very highly active against the pathogenic yeast Candida oleophila, which causes disease in crab. Strain Y F 0 7 b has the highest killing activity against the pathogenic yeast. How to control the pathogenic yeast in the diseased crab by the killer yeast is currently under our investigation.

Acknowledgements T h e authors t h a n k the National Natural Science F o u n d a t i o n of China for its providing financial support to this research ( No. 3 0 3 7 0 0 1 5 ) .

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