CN1974755B - Yeast strain for producing erythritol - Google Patents

Abstract

The present invention relates to a yeast strain capable of converting glucose into erythritol, said strain having the following identifying characteristics: lack of motile spores; septa mycelium exists; asexual propagation; lack of kidney cells; conidia are optionally formed on the short dentition rather than the long stalk; lack of pycnidia; non-monopolar sprouting on broad base; apical shoot conidial chains; dark brown, thick-walled, chlamydospores; the ability to assimilate sucrose, glycerol and maltose; the inability to assimilate lactose; galactose is not fermented; and can grow at 25 ℃ -36 ℃.

Description

Yeast strains producing erythritol

This application is a divisional application with the filing date being July 21, 2000, the application number being 00121352.0, and the invention name being "yeast strain for producing erythritol".

Erythritol is a sugar alcohol, its sweetness is 60% to 80% of sucrose, but its caloric value is only about 1/10 of sucrose, and it will not cause dental caries. Unlike many sugar alcohols, it does not cause diarrhea. In addition, erythritol has excellent processing characteristics: it is heat stable; it does not react with amino groups and therefore does not cause browning of organic matter.

Erythritol can be found in lichens, leaves of long-fiber plants, fungi, fermented foods (such as wine and soy sauce) and microorganisms. Microorganisms that can produce erythritol include yeast strains of the genera Pichia, Candida, Torulopsis, Trichomonas, Moniliella, Aureobasidium, and Trichosporium.

The present invention relates to novel yeast strains capable of converting glucose to erythritol in a simple medium.

The yeast strains of the invention are characterized by the absence of motile or zoospores (i.e., spores with flagella); having septal mycelium (i.e., mycelium with septal walls); asexual reproduction (i.e., no kasogamy and meiotic reproduction); lack of reniform cells; conidia optionally form on short denticles (i.e., small tooth-like protrusions) rather than long stalks; lack of throwing conidia (i.e., forcibly released conidia); non-unipolar budding on a broad base (i.e., unipolar protrusions do not give rise to a proliferative process for new cells); terminal budding conidia chains (i.e., characterized by septa delimited by protocells previously, identifiable conidial primordial cells were markedly enlarged); dark brown, thick-walled sclerenchyma (i.e., asexual 1-compartmental spores each derived endogenously and solely from pre-existing part of the cell); fermentative (i.e., the ability to ferment at least one carbon source semi-anaerobically); can assimilate sucrose, glycerol, and maltose; cannot assimilate lactose; cannot ferment galactose; , 35°C and 36°C growth. Optionally, the yeast strains of the invention are also characterized by the ability to ferment sucrose, glucose (ie, D-glucose) or maltose; or the ability or inability to assimilate galactose.

The ability of a strain to ferment or assimilate a specific carbon source, to grow in a vitamin-free medium, and to grow at a specific temperature can be determined according to the methods described in the practical examples below.

Yeast strains characterized by the morphological traits described above and the physiological traits described in Tables 1, 2, 3, 4, 5 or 6 below are also intended to be included within the scope of the present invention. Examples thereof include, but are not limited to, 6 strains deposited on January 27, 2000 at the American Type Culture Collection (10801 University Blvd., Manassas, VA 20110-2209, USA). The deposit numbers of the deposited strains are PTA-1227, PTA-1228, PTA-1229, PTA-1230, PTA-1231 and PTA-1232. Mutants derived from the deposited strains are also within the scope of the present invention.

The yeast strain of the present invention is closest to Moniliella acetobuten. In fact, their above-mentioned morphological characters are consistent with M. acetobuten. On the other hand, their physiological characteristics differ little from M. acetobuten. For example, unlike M. acetobuten, the strains of the present invention cannot assimilate lactose. In addition, they were able to convert glucose to erythritol at a much higher rate than M. acetobuten in a simple medium (eg, only glucose and yeast extract).

Other features or advantages of the invention are apparent from the following detailed description (including the examples) and appended claims.

The yeast strains of the present invention may be isolated from natural sources, such as samples with high sugar content, such as honey, preserves and pollen. The strains were identified based on their ability to convert glucose to erythritol and their distinct morphological and physiological traits. The yeast strain of the present invention is capable of converting 1 g of glucose into at least 0.3 g of erythritol (ie conversion rate > 30%). The transformation rate was determined by culturing the strain in a 50 ml flask at 30°C for 6 days in 10 ml broth containing 30% glucose and 1% yeast extract with rotary shaking (initial cell density was 1×10 ⁵ cells/ml). Morphological traits were determined after 10 days of growth on 4% malt extract/0.5% yeast extract agar at 20°C. See Yeast, Taxonomic Research, eds. Kurtzman et al., 4th ed., p785, Elsevier, Amsterdam (1998). On the other hand, physiological traits are determined by the methods described in the Examples below.

Other yeast strains of the invention may be variants derived from strains isolated from natural sources. For example, these strains may be mutants obtained by UV irradiation, N-methyl-N'-nitrosoguanidine treatment, ethyl methanesulfonate treatment, nitrous acid treatment, acridine treatment and the like. They can also be recombinant strains produced by genetic engineering using cell fusion or recombinant DNA technology.

Those skilled in the art can obtain and utilize to the fullest extent the yeast strains of the present invention according to the following specific examples, which are given by way of illustration only and not to limit the disclosure thereof. All publications mentioned herein are incorporated herein by reference.

Example

The 370 samples were collected from honey, coarse pollen, processed pollen, candied fruit, fresh fruit, sugar mill wastewater and molasses. The collected samples were then cultured for 3 to 4 days in a medium containing 40% glucose and 1% yeast extract. Spread the culture on an agar plate containing 20% glucose and 1% yeast extract, and incubate in a 30°C incubator for 3 to 4 days. Select different bacterial strains according to the appearance of the colonies, inoculate the selected bacterial strains in broth containing 30% glucose and 1% yeast extract, and incubate them in a 30°C incubator for 4 to 5 days. The amount of erythritol in each supernatant was determined by HPLC and TLC to determine the ability of the isolated strains to produce erythritol.

Using a Hewlett Packard H4033A analyzer, set the temperature on the Ion-300 chromatographic column to 75° C., use 0.1 N sulfuric acid as the mobile phase, and perform HPLC analysis at a flow rate of 0.4 ml/min. TLC analysis was performed according to the method of Neissner et al. (Neissner et al., 1980, Manufacture, analysis and DC-separation of erythritol fatty acid esters, FETTE'SEIFEN'ANSTRICHMITTEL. 82:10-16). After rinsing Kieselgel 60F254 (Merck) with 4% boric acid, the gel should be heated in an incubator at 105° C. for 20 minutes before use. The diffusion solvent is ethyl ketone: acetone: water (volume ratio 100:10:10), and the chromogenic agent is KMnO ₄ dissolved in concentrated sulfuric acid.

Erythritol purified from the supernatant by HPLC or TLC was further purified by extraction, followed by drying under reduced pressure. According to the method of Shindou et al. (Shindou et al., 1989, identify the erythritol in various pulp by HPLC and GC-MS and carry out quantitative determination, Journal of Agricultural Food Chemistry, 37: 1474-1476) through further purified product and Erythritol standards were acetylated. The resulting samples were examined by GC-MS to determine if the repurified product was identical to the standard sample.

A total of 630 strains were isolated from 370 samples, 22 of which showed the ability to produce erythritol. 161 isolates were obtained from processed honey, processed pollen and molasses, from which 6 strains of erythritol producing bacteria were selected, and the conversion rate of erythritol was 0.5 to 1.5%. The low conversion rate may be due to the disappearance of the erythritol-producing microorganisms as they were heated and dried before the samples were collected. Three out of 26 strains, namely 440, 441 and 442, isolated from 49 honey samples (mostly unprocessed) were found to be able to produce erythritol from glucose at high conversion (>30%). None of the 66 strains isolated from sugar mill wastewater and mud samples showed the ability to produce erythritol. Three excellent erythritol-producing strains (conversion rate>30%) were isolated from coarse pollen and candied fruit samples, namely 166-2, 262-1 and 278-3.

In order to study the effect of glucose concentration on the production of erythritol, strain 166-2 was cultured at 30°C in a medium containing 1% yeast extract and 20%, 30% and 40% glucose at a speed of 150rpm. , 262-1 and 278-3 for 1 to 6 days (50 ml flask, 10 ml medium; initial cell density 1×10 ⁵ cells/ml). The amount of erythritol produced was then determined. The results showed that the three strains cultured in 30% glucose medium for 6 days (the standard method for determining the conversion rate of the yeast strains of the present invention) all showed a conversion rate of more than 30%. In addition, after 6 days of culture in a medium containing 40% glucose, the conversion rates of all three strains were about 30%. As for glucose consumption, strain 166-2 consumed all glucose on day 5, and strains 262-1 and 278-3 consumed all glucose on day 6 when cultured in a medium containing 30% glucose .

Studies with 0.5, 1.0 and 1.5 M KCl or NaCl added to 30% glucose medium showed that the increase in ion osmolarity decreased the transformation rate of all three strains. The study of the pH profile showed that: at pH4.0-7.0, the conversion rates of the three strains grown in 30% glucose medium were roughly the same. Conversion decreased at pH8. Strains 166-2, 262-1 and 278-3 were grown in 30% medium at 25°C, 30°C and 35°C, respectively. All 3 strains showed the highest conversion rate at 30°C and the lowest conversion rate at 25°C.

The effect of different media on erythritol production was also studied. When 30% glucose was replaced by 30% maltose, all three strains did not produce erythritol. Substitution of 30% glucose with 30% maltodextrin or 1% yeast extract with 6% corn extract or 6% soy flour significantly reduced conversion. In addition, the effect of different concentrations (0.5%, 0.75% and 1.0%) of yeast extract in the culture medium on the production of erythritol was also studied. The results showed that the conversion rate increased proportionally with the level of yeast extract. In addition, various minerals were added at different concentrations, namely MgSO ₄ ·7H ₂ O (0.02% to 0.1%), K ₂ HPO ₄ (0.001% to 0.02%), CaCl ₂ ·2H ₂ O (0.1% to 0.4% ) and CaCO ₃ (0.1% to 1%) did not significantly affect the conversion.

In addition to culturing in flasks as described above, strains 166-2, 262-1 and 278-3 were also cultured at 30°C in 5 liter fermentors containing 2 liters of 30% glucose/1% yeast extract medium. Culture for 6 to 7 days (rotation speed: 150 rpm; aeration: lvvm or volume/min/medium volume; initial cell density is 1×10 ⁵ cells/ml). Strains 166-2 and 262-1 had approximately completed conversion of glucose to erythritol by the end of day 5. Strain 278-3 converted glucose to erythritol at a slower rate and did not complete the conversion until day 7.

On day 7, the supernatant of each culture was collected by centrifugation, decolorized with charcoal, and passed through ion exchange resin (IRA-410:IRA-120B=2:1) to remove impurities. After concentration by evaporation and recrystallization, erythritol was obtained as clear white crystals. The structure of the obtained erythritol crystals was further confirmed by 1H and 13CNMR.

It has been found that at 30°C, in a 50ml flask, in 10ml of medium containing 30% glucose/1% yeast extract, each shake flask was cultured for 6 days at a rotating speed of 150rpm (initial cell density was 1×10 ⁵ cells/ ml), it was found that 166-2, 262-1, 278-3, 440, 441 and 442 strains could produce 98.7, 104.1, 117, 99, 97.8 and 102.6 g of erythritol per liter, respectively.

Determination of 166-2, 262-1, 278-3, 440, 441 according to the method provided by "Yeast, Taxonomic Research" (Kreger-van Rij et al., ed., 3rd edition, p76-101, Elsevier, Amsterdam (1994)) and the physiological traits of 442 strains.

Specifically, the fermentation of all sugars was tested in a 2% (w/v) solution in Durham tubes, the inoculum was from a 48-hour culture of malt extract agar, after inoculation, the cells were incubated in fermentation minimal medium at 25°C , see p78-79.

The aerobic utilization (assimilation) test of carbon-containing compounds was performed according to the method described in the section "1. Liquid medium assimilation test" on p.81-83. More specifically, a cell suspension was prepared with sterile tap water, and after inoculation, the cells were incubated at 25°C. A 10-fold concentrated medium was prepared by dissolving 6.7g of Bacto yeast nitrogen base and an appropriate amount of carbon-containing compounds equivalent to glucose (ie, the same amount of carbon as 5g of glucose) in 100ml of demineralized water. For simplicity, all positive reactions (ie 1+, 2+ and 3+) are denoted as "+".

The aerobic utilization (assimilation) test of nitrogen-containing compounds was performed according to the method described in the section "1. Assimilation in liquid medium" on pages 85-86. For simplicity, all positive reactions (ie 1+, 2+ and 3+) are denoted as "+".

Growth experiments in vitamin-free media were performed according to the method described in the section "3. Growth in vitamin-free media, vitamin requirements" on p. 86-87. For simplicity, all positive reactions (ie 1+, 2+ and 3+) are denoted as "+".

Growth experiments at various temperatures were performed according to the method described in the section "5. Growth at 37°C and other temperatures; maximum temperature for growth" on pages 88-89. A solid medium is used.

All test results are shown in Tables 1 to 6 below:

Table 1 Physiological characteristics of strain 166-2

Table 2 Physiological characteristics of strain 262-1

Table 3 Physiological characteristics of bacterial strain 278-3

Physiological characteristics of table 4 bacterial strain 440

Physiological characteristics of table 5 strain 441

Physiological characteristics of table 6 bacterial strain 442

According to the above description, those skilled in the art can easily determine the essential technical characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions . For example, although all yeast strains tested were able to grow in media containing only 30% glucose and 1% yeast extract, those strains that grew only in media supplemented with one or more nutrients were also included in this study. within the scope of the invention. Accordingly, other implementations are also within the claims.

Claims (6)

1. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1227. 2. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1228. 3. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1229. 4. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1230. 5. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1231. 6. A yeast strain deposited with the American Type Culture Collection under the accession number PTA-1232.