RU2132878C1 - Method of gluconic acid salts and citric acid producing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention proposes a method of fermentation using possibilities of usual industrial strain-producers of citric acid to obtain two products by a single technological cycle. Gluconic acid salts and citric acid (as pure form or as salts), either one or another product are obtained depending on industry requirement. Method is based on fermentation of fungus Aspergillus niger in the presence of a single growth substance, carbohydrate source and mineral salt. After removal of product the process is carried out using another source of carbohydrate and mineral salt and conditions of process are changed also (pH value of medium, aeration regime). EFFECT: broadened assortment of production using the culture-producer Aspergillus niger. 7 cl, 1 tbl, 3 dwg

Description

 The invention relates to the microbiological industry, and in particular to a method for producing salts of gluconic acid (gluconates) and citric acid.

 The production of gluconic acid and citric acid salts is based on the fermentation of carbohydrate-containing media by various strains of molds under aerobic conditions, at a certain temperature, the regulated composition of the medium and the suppression of the development of extraneous microflora.

 A known method of producing sodium gluconate, providing for the fermentation of glucose by mold Asp. niger in the presence of corn extract as a growth substance and a source of manganese, pH about 6 and aeration of the medium about 0.5 volume of air per medium volume per minute [1]. Fermentation lasts 22 hours, the fungal biomass is not reused, and Asp reserves. niger remain unused. In addition, the biosynthesis conditions preclude the production of practically significant amounts of citric acid.

 There is also known a method for producing citric acid, which consists in the fermentation of Asp. niger sugar or molasses media in the presence of mineral salts containing zinc, pH below 3, aeration in the ratio of about 0.5 air volume per 1 medium volume per minute [2]. The specified method does not allow to obtain gluconic acid salts.

 The closest in technical essence is the method, which consists in growing seed from spores Asp. niger, in the presence of mineral salts and a corn extract containing manganese, and subsequent fermentation of glucose to gluconic acid salts at a pH of 6 to 7, aeration of less than 0.5 air volume per volume of medium per minute [3].

 When replacing manganese with zinc, introducing molasses or sugar medium, enhancing aeration, pH below 3, the same culture produces citric acid [3].

 The disadvantage of this method is that these biosynthesis products are obtained during different technological cycles, in different plants, using different strains of Asp. niger. Moreover, each of these industries is focused on the production of a single product.

 The aim of the invention is to provide a flexible method of fermentation, using the capabilities of ordinary industrial strains of producers of citric acid, to obtain during one technological cycle, using one strain and the same technological equipment as salts of gluconic acid and citric acid in pure form or in the form of salts, or of a product, depending on the needs of production.

 This goal is achieved by the fact that at the beginning of the process create conditions for the biosynthesis of gluconic acid salts: corn extract and mineral salts containing manganese are introduced into the inoculum, fermentation is carried out on glucose or glucose-containing syrup in the mode of periodic, continuous or detachable-refill fermentation while maintaining the pH from 5 to 7 and aeration not exceeding 0.5 volume of air per 1 volume of nutrient medium in min.

 After a sufficient amount of gluconate has been accumulated and the last portion of sugar has been disposed of, about 9 parts of the fermented solution together with the biomass are displaced from the reactor to the stage of gluconate separation and purification, and about 9 parts of molasses or sugar syrup containing zinc salts are added to the remaining culture, and aeration is increased to values exceeding 0.5 volume of air per 1 volume of medium per min. After the utilization of sugar, the process is stopped and citric acid is isolated in pure form or in the form of salts.

To isolate and purify gluconate, weaning and the main fermented solution after the first phase of biosynthesis are heated to 60-80 ^o C, incubated for 30 minutes, adjusted pH to 6.5-7.2. The biomass is separated, the filtrate is treated with activated carbon, filtered. The purified solution is evaporated in vacuo, slowly cooled. The precipitated salt crystals are separated and dried. Get gluconates reactive, food or pharmacopoeial purity with a yield of sugar on fermentation from 85 to 95%.

To isolate and purify citric acid and its salts after completion of the second phase of biosynthesis, the fermented solution is heated to 60-80 ^o C and incubated for 30 minutes, the precipitate is separated, the filtrate is clarified with activated carbon. The purified citric acid solution is evaporated to a content of 700-800 g / l, gradually cooled to 5-10 ^o C. The separated crystals are separated and dried. Citric acid of reactive, food or pharmacopoeial purity is obtained with a sugar yield of 80-90%.

 When preparing water-soluble salts of citric acid, before separating the biomass sediment, the fermented solution is neutralized with a suitable alkaline agent to a pH of 6.5 -7.2, treated with activated carbon, filtered, the filtrate is evaporated in vacuo and the citric acid salt crystallizes with gradual cooling. The crystals are separated and dried.

 Sparingly soluble salts are isolated after separation of the biomass directly when the pH of the fermented solution is adjusted to the optimum values and a precipitant is added, the salt precipitate is separated and washed on the filter until it reacts negatively to sulfates and chlorides, and dried. Reactive, pharmacopoeial or food grade citric acid salts are obtained.

 The proposed method is implemented on a semi-industrial installation as follows.

In a 100-liter reactor equipped with a stirrer and a sterile air supply system, 300 g of glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g of monosubstituted potassium phosphate, 30 g of diammonium phosphate and 10 l of artesian water are introduced. The pH was adjusted with phosphoric acid to 5 to 6. Sterilized at 125 ^° C for 30 minutes, cooled to 36-38 ^° C and inoculated with a suspension of 0.5 g of Asp spores. niger strain L 4.

Cultivation of seed is carried out for 24 hours at 37-38 ^o C, aeration of 0.1 - 0.5 volume of air per 1 volume of medium in min. Sterilized 80 l of glucose syrup with a content of 100-350 g / l. Gluconate biosynthesis is carried out at 34 - 36 ^o C, aeration of about 0.1 air volume per 1 volume of medium per min, maintaining the pH from 6 to 6.5 with chalk, lime, a solution of alkali, soda, potash or other neutralizing agent, depending on the desired reaction product.

After glucose utilization is completed, fermentation is carried out by a continuous or detachable-refill method, guided by the results of the analysis of residual sugar and gluconate and maintaining the pH with appropriate neutralizing agents. After utilization of the last portion of sugar, 75-80 l of the fermented solution is displaced to the stage of separation and purification of gluconate, 20 l of a sterile sugar solution containing 60 g / l of sucrose or 120 g / l of molasses, 2.5 g of zinc sulfate, 15 g are introduced magnesium sulfate, 15 g of monosubstituted potassium phosphate, 15 g of diammonium phosphate. With aeration of 7 l / min and a temperature of 36 - 37 ^o C, they stand for 3 hours, after which 60 l of a sterile solution of sucrose or molasses is introduced with a sugar concentration of about 250 g / l. Gradually, over the course of 24 hours, aeration is increased to 45-50 l / min. With such aeration and stirring, sucrose is utilized in 72 - 120 hours.

Example 1
Preparation of gluconate and calcium citrate (hereinafter citrate-salts of citric acid) 300 g of glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g of monosubstituted potassium phosphate, 30 g of diammonium phosphate are loaded into a 100 liter reactor 10 liters of artesian water. Adjust to pH 5.3 with phosphoric acid. Sterilized for 30 minutes at 125 ^° C., cooled to 38 ^° C. and inoculated with a suspension of 0.5 g of Asp.niger strain L 4 spores.

Grow seed for 24 hours in the following aeration mode:
- up to 3 hours - 1.7 l / min during the operation of the mixer for 1 min for each hour;
- from 3 to 6 hours - 3.5 l / min, the mixer is switched on for continuous operation;
- from 6 to 12 hours - 5 l / min;
- from 12 to 24 hours - 7 l / min.

80 l of syrup containing 140 g / l glucose and 200 g of quicklime are sterilized. For the entire biosynthesis period, aeration of 7 l / min is established, which corresponds to a ratio of 0.08 air volumes per 1 medium volume per minute, and the temperature is maintained at 35 ^o C. throughout the entire biosynthesis. A sample is taken for analysis every 3 hours and at a pH below 5 5 add 200 g of lime. With a residual glucose of 11 g / L, samples are taken for analysis every hour and at a glucose concentration of 2.3 g / L, the first phase of the synthesis is stopped. For this, 80 l of the fermented solution is displaced with sterile air to the stage of separation and purification of calcium gluconate. Instead, 20 l of sterile sugar syrup containing 600 g of sucrose, 1200 g of molasses, 2.5 g of zinc sulfate, 15 g of magnesium sulfate, 15 g of monosubstituted potassium phosphate, 30 g of diammonium phosphate, 19 l of artesian water are injected. With aeration of 7 l / min and stirring, they stand for 3 hours, after which another 60 l of syrup containing 250 g / l of sucrose is introduced. Increase aeration over 6 hours to 20 l / min after 12 hours to 45 l / min and continue biosynthesis, maintaining a temperature of 35 ^o C, up to 168 hours from the beginning of the sowing stage and achieve a sucrose concentration of 4 g / l. The contents of the reactor are heated to 80 ^o C, incubated for 30 minutes and transfer the solution to the stage of selection.

Isolation of Calcium Gluconate
The fermented solution from the first phase of biosynthesis is heated to 80 ^o C and incubated for 30 minutes Lime was adjusted to pH 6.5, heated for 30 minutes and filtered on a suction filter to remove biomass and sediment. The precipitate is washed with 20 l of hot demineralized water, adding wash water to the filtrate. The filtrate is clarified with 800 g of activated carbon, the charcoal is separated on a suction filter and washed with 10 l of hot demineralized water. The filtrate and washings were evaporated in vacuo to a calcium gluconate concentration of 210 g / l. Crystallized for 24 hours with gradual cooling to 20 ^° C. The precipitated crystals were separated in a centrifuge and dried at 50 ^° C. 6842.5 g of injectable calcium gluconate substance were obtained. The sugar yield on fermentation is 85%, and 59.55% is isolated.

Calcium Citrate Release
The contents of the reactor from the second phase of the process are filtered from biomass on a suction filter. The filtrate is clarified with 800 g of activated carbon. The coal is filtered off and washed with 10 l of demineralized water. Wash water and the filtrate are neutralized with lime to a pH of 6.8, incubated for 2 hours at 80 ^o C, filtered on a suction filter. Washed with hot demineralized water the precipitate of calcium citrate on the filter until the washings react negatively to sulfates and chlorides. The precipitate is dried at 60 ^o C. Receive 16820 g of calcium citrate tetrahydrate reactive purity with a yield of sugar on the allocation of 103.8%. The sugar yield of citric acid in fermentation is 85%.

Example 2
Obtaining calcium gluconate and citric acid
300 g of glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g of disubstituted potassium phosphate, 30 g of diammonium phosphate and 10 l of artesian water are loaded into a 100 l reactor. The pH was adjusted to 5.0 with phosphoric acid. They are sterilized for 30 minutes at 125 ^° C, cooled to 38 ^° C and inoculated with a suspension of 0.5 g of Asp.niger strain L4 spores.

Grow seed for 24 hours in the following aeration mode:
- up to 3 hours -1.7 l / min, during operation of the mixer 1 minute for each hour;
- from 3 to 6 hours - 3.5 l / min, the mixer is switched on for continuous operation;
- from 6 to 12 hours - 5 l / min;
- from 12 to 24 hours - 7 l / min.

80 l of syrup with a glucose content of 140 g / l and 200 g of quicklime are sterilized. For the entire biosynthesis period, aeration of 7 l / min is established and the temperature is maintained at 35 ^° C. Every 3 hours samples are taken for analysis and 200 g quicklime is added at a pH below 5.5. With residual glucose of 3 g / l, the process is transferred to the mode of weaning and topping up. For this, 10 l of the fermented solution is displaced to the isolation step and 19 l of sterile glucose syrup with a glucose concentration of 100 g / l is introduced instead. After 3 hours, when the concentration of residual glucose becomes 2.5 g / l, 10 l of the fermented solution are again weaned and topped up with 10 l of glucose syrup, 200 g of quicklime are simultaneously introduced. Thus, the process is conducted up to 75 hours from the beginning of the sowing stage, introducing a total of 80 l of syrup and weaning 80 l of the fermented solution. After that, 80 l of the fermented solution is displaced with sterile air to the isolation stage and 20 l of sugar solution is introduced instead, containing: 600 g of sucrose, 1200 g of molasses, 2.5 g of zinc sulfate, 15 g of magnesium sulfate, 15 g of monosubstituted potassium phosphate, 30 g diammonium phosphate, 19 l of artesian water. Withstand 3 hours with aeration of 7 l / min, after which another 60 l of sugar syrup containing 250 g / l of sucrose is introduced. Increase aeration to 20 l / min for 6 hours, then after 12 hours to 45 l / min and continue in this mode the process, maintaining the temperature of 35 ^o C, up to 219 hours from the start of cultivation. At a concentration of residual sugar of 3.2 g / l, the contents of the reactor are heated to 60 ^o C, incubated for 30 minutes and transferred to selection.

Isolation of Calcium Gluconate
To isolate calcium gluconate, the fermented solution from the first phase of biosynthesis is heated to 65 ^o C and incubated for 30 minutes Lime was adjusted to pH 6.7, filtered from the precipitate on a suction filter, the precipitate was washed with 50 l of hot demineralized water. Wash water and the filtrate clarify 2000 g of activated carbon, coal is washed with 10 l of demineralized water. Wash water and the filtrate are evaporated in vacuo to a concentration of calcium gluconate 210 g / L. Crystallized for 24 h, cooling to 20 ^o C. the Crystals are separated in a centrifuge and dried at 50 ^o C. Receive 11,700 g of substance injectable calcium gluconate. Sugar yield on fermentation 89%, on the allocation of 60%.

Isolation of Citric Acid
The fermented solution from the second phase of biosynthesis is filtered on a suction filter, washed with water, 800 g of activated carbon are added and filtered. The precipitate is washed with 5 l of demineralized water. The filtrate and washings were evaporated in vacuo to a citric acid content of 800 g / l. Crystallized upon cooling to 5 ^° C. for 8 hours. The crystals were separated in a centrifuge and dried at 50 ^° C. 10,530 g of reactive citric acid were obtained with a yield of 85.2% in sugar on fermentation, 65% being isolated.

Example 3
Preparation of Gluconate and Sodium Citrate 300 g of glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g of monosubstituted potassium phosphate, 30 g of diammonium phosphate and 10 l of artesian water are charged into a 100 liter reactor. Adjust to pH 5.2 with phosphoric acid. They are sterilized for 30 minutes at 125 ^° C, cooled to 38 ^° C and inoculated with a suspension of 0.5 g of Asp.niger strain L4 spores. Grow seed for 24 hours in the following aeration mode:
- up to 3 hours - 1.7 l / min, the stirrer is turned on for 1 min for every hour;
- from 3 to 6 hours -3.5 l / min, the mixer is switched on for continuous operation;
- from 6 to 12 hours - 5 l / min;
- from 12 to 24 hours - 7 l / min.

80 l of syrup containing 300 g / l glucose are sterilely administered. For the entire period of biosynthesis, aeration of 7 l / min is established. The pH is maintained at 6.5 with a 25% NaOH solution. Every 3 hours, samples are taken for analysis, residual glucose, pH, and the concentration of sodium gluconate are determined. If the pH drops below 6.5, add 200 g of a 25% NaOH solution. Upon reaching a glucose content of 2.3 g / l, 80 l of the fermented solution is displaced to the isolation step. Instead, 20 l of sterile sugar syrup containing 600 g of sucrose, 1200 g of molasses, 2.5 g of zinc sulfate, 15 g of magnesium sulfate, 15 g of monosubstituted potassium phosphate, 30 g of diammonium phosphate, 19 l of artesian water are injected. With aeration of 7 l / min and stirring, they stand for 3 hours, after which another 60 l of sugar syrup with a concentration of 250 g / l are introduced. Increase aeration over 6 hours to 20 l / min, after 12 hours - up to 45 l / min and continue biosynthesis, maintaining a temperature of 35 ^o C, up to 184 hours from the start of the sowing stage and residual sugar of 3.2 g / l. After that, the contents of the reactor are heated to 75 ^o C, incubated for 30 minutes and transferred to the stage of selection.

Isolation of Sodium Gluconate
The fermented solution from the first phase of biosynthesis is heated to 60 ^° C, held for 30 minutes, filtered from biomass on a suction filter, adjusted to pH 7.2 with NaOH, clarified with 800 g of activated carbon, filtered again on a suction filter, washed with 5 l of demineralized water. The filtrate and washings were evaporated in vacuo to a sodium gluconate content of 470 g / l. Crystallized for 8 hours upon cooling to 5 ^° C. 14587 g of reactive purity sodium gluconate were obtained with a yield of 86.5% in sugar on fermentation, 60% in isolation.

Isolation of sodium citrate
The contents of the reactor from the second phase of biosynthesis are filtered on a suction filter from biomass, the pH is adjusted to 6.9 with a NaOH solution, and 800 g of activated carbon are clarified. Coal is separated on a suction filter, washed with 5 l of demineralized water. The washings and the filtrate are evaporated in vacuo to a sodium citrate content of 420 g / l, crystallized for 6 hours while cooling to 5 ^° C. The crystals are separated in a centrifuge, dried at 60 ^° C.

 Receive 10,600 g of sodium citrate of reactive purity with a yield of sugar on the fermentation of 85.2%, on the allocation of 65.4%.

 Quantitative data on the above examples, as well as comparative characteristics with a known method are shown in the table.

Technical and economic effectiveness of the proposed method
Specialists know [4] that Asp.niger can be operated with high efficiency for 10-12 days. The maximum productivity of the culture reaches 3-8 days.

 FIG. 1 illustrates this fact by the dependencies: changes in total acidity (1), mass concentration of citric acid (2), sugar concentration (3) versus fermentation time.

 In this regard, the claimed method combines increasing the efficiency of biomass up to 2-4 days during the biosynthesis of gluconates and highly active synthesis of citric acid from 3 to 8 days.

 This is in addition to saving spore material (according to the table. Spore consumption per kg of commercial product is 2-3.5 times lower according to the claimed method).

 In addition, it is necessary to take into account the reduction by the present method of 2-3.5 times the proportion of the unproductive sowing stage of the process, which entails the saving of energy resources, raw materials and materials.

 Despite the rich potential of culture, large microbiological enterprises producing gluconates or citric acid are oriented toward a single product and suffer from uneven marketing of products. Citric acid plants have maximum sales in the summer. Gluconate production is less in terms of output, which is why production costs are high.

It should also be taken into account that microbiological production practically cannot combine different cultures in the same production areas, as this leads to mutual infection. In this case, the proposal considers expanding the range of products only by using the capabilities of the same culture. In the autumn-winter-spring period, citric acid plants can produce gluconates and citrates, the shelf life of which is from 3 to 10 years, in contrast to the short-term preservation of citric acid. acids (shelf life of food citric acid monohydrate 6 months). The production of gluconates, gluconic acid and gluconolactones can expand the range due to the release of citric acid and its salts in the summer
According to the claimed method, after the end of the first phase of biosynthesis, about 10 g / l of gluconate remains in the medium after preparation for the next phase. This gluconate is not a by-product and waste in the synthesis of citric acid, since it serves as a metabolite in this synthesis and is almost completely utilized by the end of the process (Fig. 2, dependence 1; dependences 2,3,4 reflect, respectively, changes in the concentrations of oxalic, malic succinic acid during biosynthesis). The content of citric acid in the amount of biosynthesis acids is 96-99% [4].

 In the initial stage of biosynthesis, manganese salt and corn extract are introduced, since it is known [3, 5] that in a concentration of 0.0001 to 0.0004 wt.% Manganese sharply reduces the rate of formation of citric acid. When moving to the second phase of the process, the biosynthesis of citric acid, the claimed method provides for dilution by weaning and adding 10-30 times and bringing the concentration of manganese to the optimum for the biosynthesis of citric acid.

It should also be noted the well-known peculiarity of calcium gluconate, which allows the process to be conducted at concentrations exceeding saturation. This compound is difficult to dissolve at 20 - 30 ^o C, but easily forms supersaturated solutions. The solubility of calcium gluconate at 30 ^o C is about 40 g / l [4], that is, above this concentration, the salt must be in the sediment, which can create difficulties in mass transfer. In practice, this does not happen. FIG. 3 illustrates that at a concentration below 100 g / l, calcium gluconate is in solution under biosynthesis conditions. This is evidenced by the uniform, regular course of the dependences 1-4 of the drug content in the culture fluid from the time of fermentation. Only after 106-110 g / l the analysis results become unreliable, which is explained by the presence in the analyzed sample of a greater or lesser amount of gluconate precipitate.

Claims (7)

 1. A method of producing salts of gluconic acid and citric acid, which consists in growing seed from Aspergillus niger spores in the presence of growth substances, sources of carbohydrates and mineral salts containing manganese or zinc, followed by fermentation while maintaining a certain pH value, aeration, isolation and purification obtained products, characterized in that during one technological cycle, using one strain Asp. niger, one portion of spores and the same technological equipment receive both gluconic acid salts, and citric acid in pure form or in the form of salts, or one or another product, depending on production needs, by fermentation in the presence of one growth substance, source carbohydrates and mineral salt with the transition after weaning to another source of carbohydrates and mineral salt, changing the process conditions, namely the pH of the medium and the aeration mode.  2. The method according to claim 1, characterized in that at the beginning of the process create conditions for the preparation of gluconic acid salts: corn extract and mineral salt containing manganese are introduced into the seed medium, the process is carried out on glucose or glucose-containing syrup in a batch, continuous or detachable mode -dolving fermentation while maintaining a pH of 5 - 7 and aeration not exceeding 0.5 volume of air per 1 volume of nutrient medium in min.  3. The method according to claims 1 and 2, characterized in that after the accumulation of a sufficient amount of gluconate and the end of sugar utilization, about 9 parts of the fermented solution with biomass are displaced from the reactor to the stage of separation and purification of gluconate, and about 9 parts of molasses are added to the remaining part sugar solution containing zinc salts, aeration is increased to values exceeding 0.5 volume of air per 1 volume of medium per min, after the utilization of sugar biosynthesis is stopped. 4. The method according to claims 1 to 3, characterized in that to obtain gluconates, weeds and the main fermented solution after the first phase of biosynthesis are heated to 60 - 80 ^o C, incubated for 30 minutes, adjusted to pH 6.5 - 7.2, separated the precipitate was clarified with activated carbon, filtered, the filtrate was evaporated in vacuo and crystallized upon cooling, the crystals were separated and dried. 5. The method according to claims 1 to 4, characterized in that to obtain citric acid, the fermented solution after the second phase of biosynthesis is heated to 60 - 80 ^o C, incubated for 30 minutes, the precipitate is separated, clarified with activated carbon, filtered, the filtrate is evaporated in vacuum, crystallize upon cooling, the crystals are separated and dried.  6. The method according to claims 1 to 5, characterized in that upon receipt of water-soluble citrates, the fermented solution is neutralized with an appropriate agent to a pH of 6.5 - 7.2, filtered, the filtrate is evaporated in vacuum and crystallized by cooling, the crystals are separated and dried.  7. The method according to claims 1 to 5, characterized in that in order to obtain sparingly soluble citrates, the biomass is separated, the filtrate is adjusted with an appropriate agent to optimal pH values, the precipitate formed is separated, washed to the absence of sulfates and chlorides in the washings and dried.

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