Selasa, 10 September 2013

Aspergillus oryzae

Jual Aneka Mikrobio

1. Historical backgrounds

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries, including soy sauce, sake, bean curd seasoning and vinegar production. Filamentous fungi generally have the ability to produce various and vast amounts of enzymes in a secretory manner. Among filamentous fungi, A. oryzae is known to have prominent potential for the secretory production of various enzymes. In addition, developments in genetic engineering technology have led to the application of A. oryzae in the production of industrial enzymes in modern biotechnology. Aspergillus oryzae was used for the first example of commercial production of heterogonous enzyme, the lipase for laundry detergent in 1988.1

1.1. Solid-state cultivation

One of the distinctive features of the use of A. oryzae in traditional Japanese fermentation is the use of solid-state cultivation (SSC) (rice grain, soybean and wheat bran). This style of fermentation is thought to have originated 3000–2000 years ago in China.2,3 The technology was imported into Japan during the Yayoi period (B.C. 10th–A.D. 3rd).2 Inocula from filamentous fungi for fermentation have been commercially available as koji since the 13–15th century (Heian and Muromachi period).4 This indicates that koji was cultivated without the knowledge that it is composed of a microorganism. Thus, the word, koji, indicates both the material fermented by A. oryzae in the form of SSC and the A. oryzae microorganism itself (koji mold). A key technology enabling the industrial production and distribution of A. oryzae was the production of conidiospores whilst keeping them alive and uncontaminated. Traditionally, this technology involved the use of hardwood leaves burned to white ashes in poor aeration. The conidiospores packed in paper bags were layered with the ashes between them in a box and stored. The technology was indispensable to the avoidance of contamination by other microorganisms in a period when desiccant or air conditioning was unavailable.4 This technology led to the discovery that leaf ashes added to steamed rice also allowed reliable production of conidiospores. Conidiospores prepared industrially for sake brewing are called Moyashi. Signboards with three Japanese characters, ‘moyashi’ (or fermentation starter), indicate the supplier of A. oryzae conidiospores for sake brewers (Fig. 1). It is currently known that alkaline pH, produced by the addition of the ash, prevents contamination by other microorganisms and that minerals contained with the ash enhance the formation of conidiospores.2,4,5 This technology has been applied to the production of Trichoderma spores as a biological pesticide by Akita Konno Co., Ltd, a biotech company whose origins stem from the traditional fermentation industry.

A historical signboard of a producer of A. oryzae conidiospores. Aspergillus oryzae conidiospores are industrially produced and are distributed to fermentation companies. Two suppliers were established ∼600 years ago (Muromachi period). No other suppliers were established before A.D. 17–18th. The figure shows a photograph of an original signboard, Kuro-ban (black stamp), prepared under the license of Koji-za, the association of A. oryzae conidiospores suppliers during the Muromachi period. Currently there are five major distributors in Japan supplying A. oryzae conidiospores to 4500 sake (Japanese alcoholic beverage, ca. 1900 brewers), miso (soybean paste, ca. 1200 brewers) and shoyu (soy sauce, ca. 1500 brewers) brewers in Japan, excluding several of the biggest soy-sauce companies. The three characters called Hiragana, which were originally developed in Japan; on the signboard have pronunciations, “mo”, “ya” and “shi” from top to bottom. Moyashi means the A. oryzae conidiospores used mainly by sake brewers.
The ability of secretory production of proteins is further enhanced in solid-state culture compared with submerged culture. For example, A. oryzae can produce ∼50 g of α-amylase from 1 kg of wheat bran, which is roughly equivalent to 1 L of liquid culture medium. The first example of the industrial application of SSC to enzyme production by A. oryzae was Taka-diastase, developed as a stomach medicine by Jokichi Takamine in 1894. Recently, solid-state fermentation systems using the industrial fungi A. oryzae and A. sojae have been introduced for the production of various industrial enzymes (cf. amylases, proteases, lipases) and speciality chemicals.6 In spite of the extensive use of SSC, much less is known about SSC than for submerged cultivation, which is widely used in modern biotechnology owing to its easier automatic handling. Gene expression analysis revealed important factors affecting secretion of enzymes in SSC, physical barrier and low water activity.7 Recently, practical approaches to liquid cultivation containing solid materials succeeded in the effective production of enzymes in a secretory manner allowing the degradation of raw materials with a similar efficiency to SSC.8 Analysis of the mechanism for the secretory production of enzymes in SSC may be important to the understanding of highly efficient secretion.

1.2. Isolation and identification of A. oryzae

Sequencing the genome of A. oryzae RIB40 (ATCC-42149) was completed in 2005.9 The sequenced strain is a wild-type strain, most similar to those used for sake brewing but still has the ability of strong production of proteases, which is one of the most important characteristics for soy-sauce fermentation. Aspergillus oryzae was first isolated from koji by H. Ahlburg in 1876 when he was invited to the Japanese Medical College. Its original name, Eurotium oryzae, was later renamed A. oryzae by F. Cohn because he found that it lacked the ability of sexual reproduction. It is said that A. oryzae is a domesticated species and, therefore, that A. oryzae may be found only in a domesticated form but not in nature. On the other hand, it has been stated, in a historical literature, that koji should be isolated from the mold growing on an ear of rice. Some reports about the isolation of A. oryzae from soil, plants and food10 support the natural isolation of A. oryzae for fermentation. However, these species might now be distributed in nature following domestication many years ago.
There are two scenarios to be considered for the origination of koji fermentation. In the first scenario, A. oryzae may have been isolated from nature independently in Japan. A literature describing naturally fermented alcoholic beverage was found in a historical document, Harima no Kuni Fudoki, edited in AD 715.2 Another historical document described that koji must be isolated from the mold growing on an ear of rice. This means that A. oryzae may have existed in nature before domestication and might be isolated from other dangerous species such as A. flavus through the method indicated in the historical literatures above. In the second scenario, A. oryzae may have been imported from China during the Yayoi period. The material used for the fermentation in East Asia was a block of hardened raw wheat or rice powder. The filamentous fungi that grew on the block is thought to have been Mucor or Rhizopus.2 On the other hand, the material used in the production of Japanese alcoholic beverage was steamed rice grain, thus, proteinous components in rice are considerably denatured. Mucor and Rhizopus, which have a low productivity of proteases allowing the assimilation of denatured proteins as nitrogen sources, grow poorly on steamed rice. On the contrary, A. oryzae, producing a considerable amount of proteases, could predominantly grow in an environment where a mixture of spores of these heterologous species exists. The names of the traditional fermented materials, which indicate yellow color, but not dark gray as Mucor and Rhizopus, also suggests that A. oryzae was first used in Japanese fermentation industries. Therefore, the second scenario may not be well supported.

1.3. Safety

The long history of extensive use of A. oryzae in food fermentation industries prompted industrial applications of A. oryzae to be listed as Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA) in the USA.11,12 The safety of this organism is also supported by World Health Organization (WHO).13 Although A. oryzae is genetically very close to A. flavus, which is known to produce the most potent natural carcinogen, aflatoxin, A. oryzae has no record of producing aflatoxin or any other carcinogenic metabolites.14 Fermented foods produced by A. oryzae including A. sojae, the close relative to A. oryzae in section Flavi, have been shown to be aflatoxin free.15,16 The two species, A. oryzae and A. flavus were traditionally distinguished based on morphological, physiological and culture-based characteristics.17 Recent DNA-based techniques have enhanced the potential for distinction.1820 Zhang et al. reported that homologs of aflatoxin biosynthesis gene cluster of A. oryzae were not expressed even under the conditions that are favorable to aflatoxin expression in A. flavus and A. parasiticus.21
We have received huge benefits from A. oryzae for thousands of years and may continue to receive them in the future. At the same time, we are facing various mysteries regarding A. oryzae: unique cultivation method, species origin, isolation and so forth, as described above. In spite of economical importance and scientific interest, the fact that A. oryzae forms multinucleate conidia and lacks a sexual life cycle have prevented extensive basic studies by conventional approaches. It is very interesting to see whether and to what extent genomics elucidates the subjects concerning ancestor, molecular mechanism for high secretion and safety, and the potential of A. oryzae in new fields where A. oryzae has never yet been applied.

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