CS259439B1 - A process for the preparation of a mixture of fructose and gluconic acid and the addition of salts in a submerged culture of Aspergillus foetidus var. pallidus by converting a sucrose substrate - Google Patents

Abstract

Method for producing a mixture of fructose and gluconic acid and its salts in a freeze-dried culture of the fungus Aspergillus foetidus var. pallidus by converting a sucrose substrate. The spore inoculum of the CCF 1889 mutant of this strain is either directly inoculated into a production fermenter or previously into an inoculation tank in which a vegetative inoculum is prepared, with which the production fermenter is inoculated, after which the mutant is cultivated at a temperature of 25 to 30 °C at a pH of 5.0 to 7.0 in a solution containing 5 to 50% by weight of sucrose or products, intermediate products and waste from sucrose production, in particular affinade, raffinade, light and heavy juice, lees and molasses. The advantage of this method is that with the help of the high-production strain Aspergillus foetidus var. pallidus CCF 1889, higher yields are achieved, not only sucrose, but also highly concentrated sucrose substrates, intermediate products and waste materials from sucrose production up to 50% by weight can be processed. Both cultivation products form a mixture whose components can be easily separated, while the resulting fructose syrup contains almost no ballast admixtures of glucose and sucrose.

Description

The present invention relates to a process for the preparation of a mixture of fructose and gluconic acid and its salts in a submerged culture of Aspergilus foetidus var. pallidus by converting a sucrose substrate.

Both products, fructose and gluconic acid ev. its salts are desirable products in a number of industries. Fructose has recently been used mainly for its important organoteptic and dietetic properties * in the food industry, gluconic acid ev. its salts are used in the building industry, the paint industry, in the pharmaceutical industry and in numerous applications utilizing its anticorrosive complexing and dissolving effects.

Methods for the preparation of fructose syrups (i.e., a mixture of fructose with glucose) or pure fructose by conversion of glucose by the enzyme glucose isomerase or microorganisms having an increased content of this enzyme (yeast, lactic acid bacteria, streptomyces), usually in immobilized form are known (Barrett SP et al. U.S. Pat. No. 4,288,548, 1981; Heady RE U.S. Pat. No. 4,276,379, 1982; U.S. Pat. No. 4,317,880, 1982; Demner, K. et al., Chem. Sheets 78, 199-210 (1984). -glucose syrups ("Isomerose", "Isosyrup" "Cornsweet", "Isoswet"), obtained in this way usually from hydrolysates of starch raw materials, containing 42-55% fructose. or microorganisms with an increased content of this enzyme (yeast), again in immobilized form [Ajimifo Co., Jap. Pat. 82 163484; Jugnickel F., Ger. Pat. GDR 15 8040, 1982; et al, AO 189 302, 1982, Hradil J. et al, AO 204 643, 1982, Bareš M. et al, AO 187 815, 1981; Imail K. et al. Biotechnol. Bioeng. 25, 613-7 (1983); Hradil J., Svec F. Enzyme Microb. Technol. 3, 336-40 (1981), ibid. 3, 331-5/1981]. In these cases, the fructose content of the final product is about 50%.

In view of the use of these fructose-glucose syrups, the glucose content is essentially a ballast component and an attempt is made to minimize it. Due to the very similar chemical composition of both saccharides and similar physicochemical properties, however, their division is difficult. In contrast, a mixture of fructose and gluconic acid ev. their salts obtained according to the invention are much easier to separate by crystallization. By the process according to the invention both components can be obtained practically pure. Compared to a number of processes using immobilized enzymes, the process according to the invention is economically more advantageous, since the isolation, purification and immobilization of the respective enzyme is eliminated. Carbohydrate consumption per cell mass increase in the batch is very low compared to the production rate. severe carbohydrate conversion (90-95%). The efficiency of the process according to the invention is further increased by repeated use of the separated mycelium of the microorganism. The effects of the physicochemical parameters of the culture and the chemical composition of the culture medium are discussed in AO No. 250 277 concerning the use of the same microorganism to obtain gluconic acid and its salts by glucose conversion.

The essence of the process for producing fructose in addition to gluconic acid and its salts in a submerged culture of Aspergillus foetidus var. pallidus by converting a sucrose substrate according to the invention is that the spore inoculum of a mutant of this strain AN 166 is either directly inoculated into a production fermenter or previously in an inoculation tank in which a vegetative inoculum is prepared to inoculate the production fermenter and then cultured at 25 to 30 ° C and a pH of 5.0 to 7.0 in a solution containing 5 to 50 wt. sucrose or sucrose products, intermediate products and wastes, in particular affinades, raffinates, light and heavy juices, clergy and molasses. The culture of this strain is deposited in the Collection of Fungus Cultures of the Department of Botany, Charles University, Prague 2, Benátská 2 under the number CCF 1889.

After fermentation, the cell mass of the microorganism can be separated from the liquid portion of the culture medium and reused for the conversion of sucrose or sucrose products, intermediates and wastes, in particular affinades, raffinates, light and heavy juices, clergy and molasses.

A staple or vegetative inoculum is used to inoculate fermentation vessels. Its amount does not depend on the weight amount of the sucrose source, but only on the volume of the fermentation medium.

In the case of a spore inoculum, the amount of spores used by the microorganism washed with 0.1% Tween 80 solution is used such that their concentration in the medium at the beginning of the fermentation measured at 420 nm reaches E420 = 0.105 to 0.02.

The vegetative inoculum prepared by germination of spores at a concentration corresponding to E420 - = 0.05 to 0.2 is dosed into the fermentation medium in an amount of 3 to 10% of the total volume, corresponding to the starting dry matter at fermentation 0.05 to 0.1 mg / ml.

The preparation of fructose and gluconic acid salts according to the process of the invention is given in the following examples:

Example 1

Inoculum preparation: Aspergillus foetidus var. pallidus AN 166 growing 7 days on wort agar on 15 cm diameter Petri dishes, rinsing with 50 ml of 0,1% w / w. of a surfactant solution (Tween 80). The buckle suspension obtained from the dishes was inoculated with 150 L of copper in a fermentation tank. The volume of the spore suspension is chosen so that the initial concentra- tion of the spores in the medium corresponds to Εί20 == 0.01. After 12 hours of low aeration (0.1 volume of air per volume of medium per minute) without agitation, the spores germinate at 30 ^° C. Aeration is increased and mixing is started.

Preparation of fermentation medium. Prepare a sterile fermentation medium of a composition (% by weight):

sucrose,

0.025 KC1,

0.025 KH2PO4,

025 MgSO1. 7 H2O,

0.1Ca (NO3) 2.4H2O,

0.056 (NH 2 O 2 SO 3).

The pH correction during fermentation is carried out automatically by the addition of 20% by weight. NaOH, KOH, NHiOH or NaaCOo solution depending on which salt we want to obtain.

Fermentation: The fermentation is carried out in a stainless steel tank with a centrally placed turbine stirrer. Volume of fermentation medium 150 l, stirring 400 rpm. aeration during fermentation 0.5 volume of air per 1 volume of fermentation medium per minute, temperature 30 ° C, pH is maintained at 6.0. The fermentation time, including the germination phase, is 40 to 45 hours. The concentration of the respective gluconic acid salt is on average 19 to 20% by weight, fructose is 18 to 20% by weight. During this fermentation period, neither gluconic acid (its salt) nor fructose is further degraded.

Example 2

Inoculum preparation: worts from wort agar were seeded with propagation tanks with a medium volume of 2.5 <1 (the amount of spores was selected in the same manner as in Example 1) of the same composition as in Example 1, with only a reduced sucrose concentration of 5 % wt. The inoculated microorganism was cultured at 30 ° C, aerated with 0.1 volume of air per 1 volume of medium and stirrer speed 100 rpm. for 8 to 10 hours.

Fermentation: the obtained vegetative mycelium is inoculated with its own fermentation tank in an amount of 3 to 10% by volume. The fermentation parameters and the composition of the medium are the same as in Example 1. The yield of fructose and gluconic acid (gluconate) are the same. The fermentation time is 28 to 30 hours.

Example 3

Preparation of the inoculum can be carried out in both ways, i.e. as in Example 1 and 2.

Fermentation: Fermentation parameters and medium composition are the same as in Examples 1 and 2.

At the end of the fermentation, the mycelium is separated, which, without rinsing, is again returned to the medium containing (% by weight): 40 to 50 sucrose, 0.025 KH2PO4, 0.1 CaCl2. The conversion takes place under the same conditions as the fermentation itself. The conversion time is 18-23 hours, fructose yield 45-49%, gluconic acid (such as sodium gluconate) 49-50 percent based on sucrose.

Separated mycel can be used several times (10X average). The reversible use of mycelium is discontinued if the conversion rate falls below the desired limit or if severe contamination occurs.

Example 4

The same procedure as in Example 1 was followed using molasses as a source of sucrose.

Example 5

In the same manner as in Example 2, the intermediates from the production of sucrose were used as a carbon source, in particular affinades, raffinates, light and heavy juices and / or clergy.

Claims (2)

A method for producing a mixture of fructose and gluconic acid and its salts in a submerged culture of the song Aspergillus foetidus var. pallidus by converting a sucrose substrate, characterized in that the inoculum of CCF 1889 mutiaint strain of this strain is either inoculated directly into a production fermenter. or previously into an inoculation tank in which a vegetative inoculum is prepared to inoculate a production fermenter, after which the mutant is cultured at a temperature of 25 to 30 ° C and a pH of 5.0 to 7.0 in a solution containing 5 to 50% by weight of sucrose, or products, intermediates and wastes from the production of sucrose, in particular affinades, raffinates, light and heavy juices, clergy and molasses. 2. Method according to claim 1, characterized in that after fermentation the cell mass of the microorganism is separated from the liquid fraction of the culture medium and reused for the conversion of sucrose or sucrose products, intermediates and wastes, in particular affinades, raffinates, light and heavy juices. of clergy and molasses.