Dong Xinrong Jian Cao

The research on microbial functional oil at home and abroad was summarized from the important factors affecting the synthesis of microbial oil, the preparation of microbial oil, the qualitative analysis of microbial oil, the physical and chemical indexes and quality indexes of products, and the production of functional oil by microorganisms.

Microorganisms; Functional grease; manufacture

Classification number ts 2 18+438+0

Functional fats and oils from microorganisms

Dong Xinrong Jian Cao

(Department of Bioengineering, Zhengzhou Grain College, Zhengzhou 450052)

In this paper, the production of oils and fats by microorganisms, the important influencing factors in the production process, the qualitative analysis, physical and chemical properties and quality characteristics of products, and the application of microorganisms in the preparation of functional oils and fats at home and abroad were reviewed. The production of functional fats and oils from microorganisms was also studied.

Keywords microorganism; Functional fats and oils; produce

Preface of 0

Microbial oil is also called single cell oil. Under certain conditions, many microorganisms, such as bacteria, mold, yeast and algae, can produce a lot of oil in bacteria. Some dry-based bacteria contain more than 70% oil, which has similar fatty acid composition to ordinary vegetable oil (see table 1) [1]. The study of microbial oil began during the First World War. In order to solve the shortage of oil resources at that time, Germany used endospores to produce oil, and then the United States began to produce microbial oil, but it did not realize industrialization. It was not until the eve of World War II that German scientists screened out strains suitable for deep culture and began to industrially produce microbial edible oil in Germany.

Table 1 Fatty acid composition of some microbial oils and fats

The strain12: 014: 016: 016:18: 018.

Candida 0-TR 32- 15448-

Cryptococcus cerevisiae

Cryptococcus terrestris-tr 36 1 14 36 8 tr

rhodotorula

(Rhdotorula glatiuis)- 18 1660 1224∶0 1%

Irregular rhythm16261552656a 20 ∶ 07%

Endopora lipolytica -2 25 70 17 47 5 1

Ergosterone 23 62198-18:1-hydroxyl 42%

Fusarium moniliforme-114-130421

Mucor miehei-1 20 4 6 48 16 5a

Rhizopus arrhizus tr118412916tra

Chlamydomonas amyloliquefaciens-30.72.60.84.135.37.5b16: 216.2%

16∶3 2.7%

Mortierella alpina

Mucor bb bb bb150

20∶3 3%

20∶4 30%

20∶5 15%

Description: A-γ- linolenic acid; B- unknown; tr-trace； Br- branched acid; -oh carboxylic acid

Compared with the production of animal and vegetable oils and fats, the production of microbial oils and fats has many advantages: 1, strong adaptability of microorganisms, fast propagation speed and short production cycle; 2. The raw materials needed for microbial growth are rich and varied, especially the wastes produced in agricultural and sideline products, food industry and paper industry, such as sulfite pulp, wood saccharification liquid, waste sugar liquid and waste liquid from starch manufacturing. This also protects the environment; 3. Microbial production of oil and fat is labor-saving, free from the influence of site, climate and season, and can be produced continuously all year round. 4. It is especially suitable for developing some functional oils and fats by using the characteristics of great changes in the composition of different strains and culture media products. Such as oils rich in oleic acid, gamma-linolenic acid, arachidonic acid, EPA, DHA, squalene, dicarboxylic acid and cocoa butter substitutes. In addition, due to the growth of population, the contradiction between the demand for oil and the serious shortage of natural resources is increasingly acute, and it is more practical to open up a new oil source-microbial oil [2, 3].

At present, the technical feasibility of microbial oil production is not too great, mainly economic feasibility. Microbial oil production is affected by many factors, and oil-producing strains are limited. Only those microorganisms with high oil content and high oil conversion rate on a dry basis are of use value. At present, the oil content of dry-based microorganisms screened is generally 30% ~ 60%, and a few are 70% ~ 80%. The oil conversion rate is generally 15%, and individual strains reach 20% ~ 25%. Therefore, the economic value of general microbial oil can not compete with vegetable oil, and the research on microbial oil mainly focuses on the production of special nutritional oil and special industrial oil with high economic value by microorganisms. The main nutrients of this kind of oil are few or even nonexistent in natural animal and vegetable oils, but they have great physiological functions and special uses, so we collectively call them microbial functional oils. At present, Japan has realized microbial oil extraction to produce cocoa butter and yeast fermentation to produce cocoa butter substitute. γ -linolenic acid oil produced by molds appeared in Japan and Britain, and fungi and algae strains rich in arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, carbonic acid and castor oil were also found [1, 6,21]. As a necessary supplement to animal and vegetable oils, microbial functional oils and fats play an increasingly important role in promoting human health, so it is of great significance to study microbial functional oils and fats.

1 is an important factor affecting microbial oil synthesis.

C/N ratio 1. 1 medium.

The yield of oil is determined by the product of cell oil content and cell harvest. Microbial oil production process can be divided into two stages: cell proliferation stage and oil production stage. The C/N ratio of the medium used in these two stages is different, and the nitrogen nutrition in the cell proliferation period is relatively high to obtain enough bacterial cells. In the oil production period, after obtaining enough bacterial cells, carbon nutrients are added to create conditions for bacteria to accumulate a large amount of oil [9].

1.2 pH value

The optimum pH value of oil production varies with the type of microorganism, ranging from 3.5 to 6.0 for yeast and neutral to alkaline for mold. When Aspergillus nidulans was cultured at pH 2.8 ~ 7.4, the oleic acid content increased with the increase of pH value. However, the closer the initial pH value of the nutrient medium of the cultivated oil-bearing yeast is to neutrality, the higher the lipid content of the cells in the stable phase [7].

1.3 inorganic salts and trace elements

Generally speaking, for fungi, increasing the amount of inorganic salts and trace elements can improve the oil production speed and oil production. Carrid et al.' s research on Aspergillus nidulans showed that the oil content could be increased from 25% ~ 26% (generation rate 6.7 ~ 7.9) to 5 1% (generation rate 17.2) by adjusting the proportion of Na+, Mg2+, so42- and PO43-in plasma. An experiment of oil production by oil yeast proved that increasing the concentration of iron ions in the culture medium can accelerate the synthesis of oil, while increasing the concentration of zinc ions (some strains need vitamin B) can increase the accumulation.

1.4 temperature

The optimum temperature for oil generation is mostly around 25℃. Temperature can affect the composition and content of oil, and the content of unsaturated fatty acids will increase when the culture temperature is low.

1.5 culture time

Culture time is also very important for the synthesis of oil. Such as Aspergillus Niger, Aspergillus oryzae, Rhizopus, Rhodotorula and Saccharomyces cerevisiae, the optimum culture time is 3 days, 7 days, 7 days, 5 days and 6 days respectively. Insufficient culture time, the total number of microbial cells can not reach the maximum, which affects the oil output; If the culture time is too long, individual microorganisms will deform and autolyse, and the oil formed will be difficult to collect in the culture medium, which will also affect the oil yield.

1.6 spore number

When there are too many spores in the bacterial growth period, the oil yield of single cell may be low. Excessive accumulation of oil in cells will make bacteria lose their ability to proliferate. Therefore, when cultivating oil-producing bacteria, the optimum spore number should be achieved to maintain the proliferation ability and oil-producing physiological state of bacteria.

1.7 oxygen supply

Microorganisms need oxygen to synthesize oil and unsaturated fatty acids from substrate sugars, so they must provide enough oxygen.

1.8 plus

Adding intermediates of fatty acid synthesis or dicarbonyl compounds that can form intermediates, such as ethanol, acetate and acetaldehyde, can increase the oil content.

Preparation of microbial oil

2. 1 strain selection

The strain used to produce microbial oil requires the following conditions:

After improvement, (1) has the ability to synthesize oil or fat, with a large accumulation of oil, stable oil content above 50% and oil conversion rate not lower than 15%.

(2) Agricultural and sideline products, industrial wastewater and wastes can be utilized.

(3) Strong reproduction, not easy to be polluted by miscellaneous bacteria, and easy to precipitate, filter and separate oil.

(4) It has good flavor, is harmless to eat and easy to digest and absorb.

(5) When it is used in industrial production, it can adapt to industrial deep culture and the device is simple [4, 5]. In addition, different strains, different culture conditions and different products. See table 1 and table 2 for the fatty acid composition, types and triglyceride composition of some strains' oils.

Table 2 Stereospecificity Analysis of Microbial Oils

Grease sn14: 016: 016:17: 017:18: 016544.

Mycobacterium1189tr2760-7111

snegmatis 2 7 57 13 2 1 6 9- 1 tr tr 1 tr

3 1 7 7 tr tr 16 18-6 7 7 18 7

(oil yeast)13148-46110-

Fatty bacteria 2- 12-889

lipoferus 3 6 29 13 - - 9 37 6 - - -

- -

2.2 culture medium

The culture medium to be prepared includes slant culture medium, seed liquid culture medium, basic shake flask culture medium, fermentation culture medium, etc. Inclined culture medium is a common culture medium for this strain. The composition of seed culture medium and basic culture medium has little change, mainly to stabilize the characteristics of strains; Fermentation medium should increase the proportion of carbon source, reduce the proportion of nitrogen source, and increase the aeration rate at the same time, so that bacteria can fully synthesize oil [28, 29].

The carbon sources used in the preparation are lactose, glucose, fructose, sucrose, paraffin, waste molasses, pulp industry wastewater, wood hydrolysate, starch factory wastewater and so on. Nitrogen sources include ammonium salt, urea, nitrate, amino acids, yeast water, corn steep liquor, etc. Inorganic salts include KH2 PO4, MgSO4, CaCl2, etc. Auxin includes yeast extract, peptone, etc. If we want to improve the strain by mutation, we need to prepare a mutation medium. The mutagens used are nitrosoguanidine, N- methyl -N- nitrosoguanidine, diethyl sulfate, ultraviolet ray, laser and ion beam.

2.3 Cultivation methods

2.3. 1 strain activation

The preserved strains were transferred to slant culture medium and cultured at 28℃ for 4 days.

2.3.2 Preparation of Seed Liquid

The activated strains were washed with a small amount of sterile water, placed in a triangular flask with seed liquid culture medium, and cultured for 2 ~ 5 d at 24 ~ 30℃ and rotation speed 150 ~ 300 r/min. The culture temperature, time and oscillation speed depend on the type and number of strains. Usually, the liquid content of seed liquid culture medium is 65438+ 0/5 of that of triangular flask.

2.3.3 Shake flask culture

Use a triangular flask with the same volume as (2), filled with 1/5 volume of seed liquid culture medium, and inoculate 2 ~ 3 ml of seed liquid. The temperature and rotation speed are the same as (2), and the culture time is longer than (2)1~ 2 d.

2.3.4 Large tank fermentation

The volume of the liquid is 2/3 of that of the filler, the inoculation amount is 5%, the tank pressure is 0.5 kg/cm2, the stirring speed is increased to 2 times, and the tank temperature is the same as above. Sometimes, in order to gradually induce lipid production, the method of three-stage fermentation can be adopted, so that the nutrient supply of the culture medium tends to gradually increase the carbon source, reduce the nitrogen source, increase the aeration rate, and the pH value gradually approaches the optimal value of microbial synthesis of lipid.

2.4 Cell collection

After microscopic examination, the cultured bacteria were filtered with filter cloth (gauze and cool cloth), washed with distilled water for three times, weighed wet, some wet bacteria were dried at 60℃, weighed dry, and the moisture content of wet bacteria was determined. When a large number of bacteria are collected, centrifugation is used.

2.5 strain pretreatment and bacterial oil extraction before oil extraction

Microbial oils exist in tough cell walls, and some of them exist in the form of lipoproteins and lipopolysaccharides, so bacteria must be pretreated before oil extraction. There are four main pretreatment methods: (1) dry cell grinding method (grinding cells with sand); (2) Cooking dry bacteria with dilute hydrochloric acid (* * * cooking decomposes cells to facilitate oil extraction); (3) autolysis of the strain (2-3 days at 50℃); (4) Bacterial protein denaturation method (denature the binding protein with ethanol or propanol) [10]. In addition, there are methods of using high pressure homogenization, ball milling, expansion, high osmotic pressure and other treatments to break bacteria.

Organic solvents used for oil extraction mainly include ether, isopropyl ether, chloroform, ether-ethanol, petroleum ether, chloroform-methanol, etc. After extraction, the solvent was recovered by vacuum distillation.

3 qualitative analysis of microbial oil

After Sudan black staining, the fat particles in the bacteria are blue-purple or blue-gray, while the bacteria are red. According to the size of fat particles, the fat content can be preliminarily judged, and it can also be used to determine the best oil production time [5].

Determination of various physical and chemical indexes and quality indexes of microbial oil

Using AOCS method, the analysis indexes mainly include the following aspects: (1) refractive index; (2) specific gravity; (3) transparency; (4) sense of smell and taste; (5) moisture; (6) acid value; (7) peroxide value; (8) the price of iodine; (9) color; (10)2 80℃ experiment; (1 1) fatty acid composition; (12) triglyceride; (13) unsaponifiable matter.

5. Using microorganisms to produce functional oil.

By means of cell fusion and cell mutation, microorganisms can produce high-nutrient oils or oils composed of certain fatty acids that are more in line with human needs than animal and vegetable oils [27]. Now described as follows:

5. 1 oleic acid and linoleic acid

Linoleic acid is an essential fatty acid for human body, which can be converted into γ -linolenic acid needed by human body by δ 6 dehydrogenase. Although this oil is very common in plants, only safflower oil and sunflower oil have linoleic acid content above 70%. It is reported that the content of linoleic acid produced by the filamentous fungus pholiota adiposa cultured with cellulose as carbon source is as high as 71.8% ~ 76.3% [11]. It has been reported abroad that the oil rich in oleic acid and linoleic acid is industrially produced by endosporum lipolyticum. Oleic acid and linoleic acid often coexist in microbial oils, accounting for 65% ~ 78% of the total fat, which is very similar to many vegetable oils. In addition, the analysis results of physical and chemical properties such as melting point, refractive index, specific gravity, acid value, peroxide value, saponification value and iodine value are also close to those of vegetable oil [9].

The whole cell fatty acid analysis of 38 Candida strains showed that the oleic acid content of these yeasts was 34% ~ 69%, linoleic acid content was 5% ~ 34%, and palmitoleic acid content of some strains was 65,438+05.9% [2,3]. Oil synthesized by oil-producing yeast Rhodotorula sp. And Trichosporon. It is mainly composed of oleic acid, and its fatty acid composition is similar to that of olive oil and rapeseed oil in common vegetable oil [2, 13].

5.2 γ -linolenic acid

GLA exists naturally in a small amount, only in milk fat and seeds of special wild plants. The existence and activity of human △ 6 dehydrogenase are often influenced by obesity, cancer, virus infection, aging and other healthy nutritional factors, which makes linoleic acid unable to be converted into GLA and PG (prostaglandin) unable to be synthesized smoothly, thus leading to arteriosclerosis, thrombosis, diabetes and so on. Therefore, the oil rich in GLA is a kind of health oil [6544

Traditionally, GLA is mainly extracted from the seed oil of evening primrose. In 1948, Bernhard and Albercht first identified the fungus CLA from the mycelium fat of Rhizopus brucei, and its content reached 16%. Nugtern proved that its structure was similar to that of evening primrose seed oil GLA. /kloc-in 0/9 and 64 years, it was found that strains of phycomycetes contained GLA, but did not contain α -linolenic acid. Recently, Morio Hiramo of the Bioengineering Laboratory of Ona Cement Company in Japan and Yunki M iura of the Bioengineering Department of Tokyo Agricultural Technology University cultivated spirulina and Chlorella. NKG4240 produces GLA, and its content can reach10% of total fatty acids [15].

In the production of GLA by fermentation, in 1985, Osama Suzuki and others used Mortierella, Mortierella Raman and Mortierella parviflora for fermentation culture with high concentration of glucose (60 ~ 400 g/L) as carbon source, and the oil content of the bacteria reached 35% ~ 70%, of which GLA accounted for 3% ~/kloc-. 1987, longyikundao produced GLA by fermentation of Hyphantria cunea, and the GLA content reached 18%. Fusarium javanicum is used in Britain, and glucose produced by wheat starch is used as fermentation medium. After purification, the product reached the edible standard, and the content of γ -triacetin was as high as 65438 06%. John &; The output of Starge Co., Ltd. reached100 t/a [4,26]. Since 1986, UK SSL by Factory of Sturge Biochemicals and Japan Photochemistry Company have listed microbial GLA products, which are mainly used in medicine, health food, functional drinks and advanced cosmetics [1 1].

Shanghai Institute of Industrial Microbiology used M 102 strain to produce GLA in a 500L fermentor, and the GLA content reached 8%. In 1993, the Department of Biology of Nankai University took Mortierella As3.34 10 as the original strain, and obtained a mutant strain by ultraviolet mutagenesis. The College of Bioengineering of Fujian Normal University took Mortierella as 3.3 14 1998 as the starting strain, and produced GLA in 1 0L fermentor. The yield of bacteria was 29.3%, the oil content was 44.7% and the GLA content was 9.44%. After combined mutation by ultraviolet rays, diethyl sulfate and nitrosoguanidine, the strain was subjected to tertiary fermentation in a 60m3 fermentor, and its oil content was as high as 79. 2% [ 16].

5.3 Arachidonic acid

AA is traditionally derived from fish oil, but its content is extremely low, generally below 0.2%(W/W). AA and eicosapentaenoic acid (EPA) are important intermediates of arachidonic acid metabolism, and their status in nutrition and medicine has attracted worldwide attention, mainly because the metabolites PG, TX and LT of eicosapentaenoic acid have physiological functions of regulating vascular obstruction, thrombosis, wound healing, inflammation and allergy [1]. 1990 Buranova et al. found that several Mortierella can aggregate eicosatrienoic acid (DGLA) and AA and produce EPA under certain conditions. Since 1980s, microbial oils with high GLA and AA contents have been put into industrial production in Japan, Britain, France, New Zealand and other countries, and AA fermented products have been put on the market in Japan and Britain [17]. A Mortierella sp. A high-yield strain was obtained by UV mutagenesis, and the AA yield reached 0.83 g/L. The study also pointed out that the mycelium cultured in different time (3-5 days) was aged at room temperature for 65438±05d, and the total lipid content of the mycelium increased from 65438 08% ~ 30% to 36% ~ 465438 0%. The content of AA in mycelium increased from 1. 1% ~ 2.6% to 2.6% ~ 3.7% [18].

5.4 Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

Natural EPA and DHA are usually abundant in marine animals and marine phytoplankton. EPA and DHA belong to ω-3 polyunsaturated fatty acids (PUFA), and their physiological functions are: (1) prevention and treatment of atherosclerosis, thrombosis and hypertension. (2) Treating asthma, arthritis, periodic migraine, psoriasis and nephritis. (3) Treating breast cancer, prostate cancer and colon cancer. At present, the commercial source of ω-3 PUFA is marine fish and its oil. The composition and content of ω-3 PUFA in fish oil vary with fish species, seasons and geographical locations. In order to improve its oxidative stability, most fish oils often have to undergo hydrogenation, blending and other steps, thus destroying EPA and DHA.

Shimizu et al. proposed in 1988 that Mortierella alpina is a potential source of EPA production. When it grows at the low temperature of 65438 02℃, it can accumulate more than 65438 05% EPA. Thraustochytrium is a marine fungus with DHA content of 34%. Many seaweeds can produce oils with high EPA and DHA content. Some algae in Chrysophyta, Chrysophyta, Chrysophyta, Phaeophyta, Chlorophyta, Cryptophyta and Euascidia all contain high EPA. The content of DHA in dinoflagellate is high, while the content of EPA and DHA in dinoflagellate is high [15].

The composition of culture medium, ventilation, light intensity, temperature and culture time play an important role in the synthesis and accumulation of PUFA such as EPA and DHA. The amount of nitrogen sources affects the ratio of saturated fatty acids to unsaturated fatty acids. Insufficient light will increase the synthesis of ω-6 fatty acids and inhibit the synthesis of ω-3 fatty acids, and the concentration of microbial PUFA will reach the maximum at the end of logarithmic growth period or the beginning of stable period. In addition, the selection of strains by genetic engineering may greatly increase the potential of algae and fungi to produce PUFA such as EPA and DHA, and EPA in algae oil has greater oxidative stability than fish oil, without the smell and taste of fish oil.

Table 3 Microorganisms Producing Cocoa Butter-like Compounds

Bacterial dry biomass

Pc/g.l- 1 lipid content

Amount/%fatty acid composition /% 1, 3- di-saturated and 3- unsaturated

And glyceride content/%

16∶0 18∶0 18∶ 1

Rhodotorula

(Rhodosporidium globosum)12.8 59.8 25.012.7 46.4 47.9

Rhodotorula brilliant 8.0 35.8 29.81.8 35.5 32.8

Endopora lipolytica 8.510.425.12.347.130.6

Mortierella cerevisiae 5.0 33.7 27.9 12.7 48.0

Mortierella 4.5 565 438+0.3 25.1.1.16.6 44.4

Mortierella

Eel) 3.213.428.116.640.8

5.5 Long chain dibasic acid

Long-chain dibasic acids are widely used in industry, and are raw materials and intermediates for producing polymers, powder coatings, plasticizers, lubricants, spices and pesticides. Short-chain dicarboxylic acids below C 10 exist widely in nature and are easy to synthesize, while long-chain dicarboxylic acids above C 1 1 hardly exist and are difficult to synthesize. Many microorganisms can obtain C 1 1 ~ C 18 saturated and unsaturated dibasic acids by fermentation, among which Japan is the most widely used and has achieved good results.

5.6 Squalene

Squalene resources are also very scarce, mainly in the liver oil of deep-sea whales and sharks, and the contents of olive oil and rice bran oil are also high. Squalene has antioxidant effect in oil, but it becomes an oxygen promoter after complete oxidation. Its hydride is an excellent cosmetic matrix and lubricant for precision machinery such as watches and clocks. Using decane as carbon source, the squalene content in the fermented oil of Mortierella fuciformis can reach 50 mg/L.

5.7 generation cocoa butter

Cocoa butter is one of the most valuable oils in the world. Natural cocoa butter is obtained from cocoa beans by washing, peeling and hydraulic extraction. Its triglyceride composition is POS52%, SOS 19% and POP6%. Natural cocoa butter has the characteristics of good flavor, not easy to be oxidized and decomposed by lipolytic enzymes, suitable processing viscosity and easy demoulding. , has become an indispensable oil component in making chocolate. Due to the shortage of natural cocoa butter and its high price, many kinds of cocoa butter and cocoa butter substitutes have appeared. The preparation of cocoa butter by microorganisms includes two aspects: (1) using microbial enzymes as catalysts to catalyze the transesterification of oils and fats to achieve the required triglyceride composition of cocoa butter. Cocoa butter can be prepared by this method. (2) Cultivating microbial strains to produce cocoa butter-like and cocoa butter substitutes with physical and chemical properties close to cocoa butter or triglyceride composition.

The research on the production of oil by Rhodotorula gracilis K-76 and Rhodotorula globosa L- 103 by Moscow Industrial Research Institute showed that the yield of triglyceride with oleic acid in the 2-position was very high, and the physical and chemical properties of the separated oil were similar to those of cocoa butter, olive oil and cottonseed oil. Cocoa butter and its substitutes are produced in the Netherlands by 14 yeast varieties belonging to Candida, Candida and Rhodotorula. A high-yield strain was obtained by N- methyl -N- nitrosoguanidine mutation. After culture, the oil content reached 30%, and 95% triglycerides were P 37.6%, S 14.3%, O 37. In Canada, yeast is cultured in deproteinized whey, and cocoa butter-like products with similar triglyceride composition and content to cocoa butter are obtained by adding the required crystal form, which can be marketed without separation, and the products are uniform and stable [22].

The research on microbial synthesis of cocoa butter is the most in Japan. In a patent, lactic acid bacteria and bifidobacteria are inoculated into a mixture made of oil, fermented milk powder and sugar, and whole milk powder, sucrose, lactose, phospholipids, spices, citric acid and natural pigments are added in proportion. After fermentation at a temperature below 45℃, the product tastes like yogurt through sensory inspection, and can be used in food instead of cocoa butter [23].

Another patent is to cultivate a kind of Candida sensitive to erucic acid and its derivatives in shake flask. After culture, the triglyceride composition of Candida is 18.6%, POS is 39.0% and SOS is 14.6%, which can be used as cocoa butter [24]. Another patent uses Mortierella to produce cocoa butter substitutes, and the effect is also very remarkable [25].

To sum up, the research of microbial functional oils and fats is the development direction of 2 1 century, which will make the scope of oil industry wider, and also make microorganisms apply to more extensive and important fields.

About the author: Dong Xinrong: female, 1973, born with a master's degree.

Authors: Department of Bioengineering, Zhengzhou Grain College, Zhengzhou 450052.

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Date of receipt: 1999-06-25