The method of obtaining salts of gluconic acid and citric acid

 

(57) Abstract:

Proposed method of fermentation, using ordinary industrial strains-producers of citric acid to obtain in one technological cycle of the two products. Get a salt of gluconic acid and citric acid (in pure form or in the form of salts) or a particular product depending on the needs of production. The method is based on the fermentation of the fungus Asp. niger in the presence of one of the growth substance, source of carbohydrates and mineral salts with transition after weaning to another source of carbohydrates and mineral salt. In this changing process conditions (pH of the medium and mode of aeration). The method allows to extend the range of products manufactured using culture-producer Asp.niger 6 C.p. f-crystals, 1 tab., 3 Il.

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

The production of gluconic acid salts and citric acid based on the fermentation of carbohydrate-containing media of different strains of fungi in aerobic conditions, at a certain temperature, regulated the composition of the medium and suppressing the development of glucose mold fungus Asp. niger in the presence of corn extract as a growth substance and source of manganese, a pH of about 6 and aeration of the medium is about 0.5 volume of air to one volume of medium per minute [1]. Fermentation continues for 22 h, the biomass of the fungus is not re-used reserves Asp. niger remains unused. In addition, the conditions of biosynthesis preclude obtaining in almost significant quantities of citric acid.

Also known is a method of obtaining citric acid, which consists in fermenting Asp. niger sugar or molasses environments in the presence of mineral salts containing zinc pH below 3, the aeration in the ratio of about 0.5 volume of air to 1 volume of medium per minute [2]. This method does not allow to obtain a salt of gluconic acid.

The closest in technical essence is the method lies in the cultivation of inoculum of spores Asp. niger, in the presence of mineral salts and corn extract containing manganese, and subsequent fermentation of glucose to gluconic acid salts at a pH of from 6 to 7, aeration of less than 0.5 volume of air to 1 volume of medium per minute [3].

When replacing the manganese to zinc, the introduction of molasses or sugar environment, increase aeration, pH below 3 is the same culture that produces citric acid [3].

The aim of the invention is to provide a flexible method of fermentation, using ordinary industrial strains-producers of citric acid, to obtain within one technological cycle, using the same 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 objective is achieved in that in the beginning of the process and create conditions for the biosynthesis of gluconic acid salts: in the sowing medium injected corn extract and mineral salts containing manganese, fermentation leading to glucose or gluconeogenesis syrup in a periodic, continuous or detachable-topping-up fermentation while maintaining the pH from 5 to 7 and aeration of not more than 0.5 volume of air to 1 volume of the nutrient medium in minutes

Operating time after a sufficient number of gluconate and after disposal of the last portion of sugar to about 9 parts fermented restorable about 9 parts of molasses or sugar syrup, containing zinc salts, aeration increases to values greater than 0.5 volume of air to 1 volume of medium in the minutes After the utilization of sugar, the process stops and produce citric acid in pure form or in the form of salts.

For isolation and purification of the gluconate themy and primary fermented solution after the first phase of the biosynthesis heated to 60-80oC, incubated 30 min, pH adjusted to 6.5 to 7.2. The biomass is separated, the filtrate is treated with activated charcoal, filtered. The purified solution is evaporated in vacuum, cooled slowly. The crystals formed salt is separated and dried. Get gluconate reactive, food or Pharmacopoeia purity out of the sugar fermentation from 85 to 95%.

For isolation and purification of citric acid and its salts after completion of the second phase of the biosynthesis of the fermented solution is heated to 60-80oC and incubated for 30 min, the precipitate is separated, the filtrate lighten activated carbon. Purified citric acid solution evaporated to content 700 - 800 g/l, gradually cooled to 5-10oC. the crystals formed are separated and dried. Get citric acid reactive, food or Pharmacopoeia purity with the release of sugar 80-90%.

Upon receipt isout corresponding alkaline agent to a pH of 6.5 -7,2, treated with activated charcoal, filtered, the filtrate evaporated in vacuum and crystallized salt of citric acid with a gradual cooling. The crystals are separated and dried.

Sparingly soluble salts emit after separation of the biomass directly by bringing the pH of the fermented solution to the optimal values and the introduction of the precipitator, the precipitated salt is separated and washed on the filter until a negative reaction to sulfates and chlorides, and dried. Get a salt of citric acid reactive pharmacopoeial or food purity.

The proposed method is implemented on pilot plant as follows.

In a reactor with a volume of 100 l equipped with a stirrer system and a supply of sterile air, enter: 300 g glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g one-deputizing potassium phosphate, 30 g of diammonium phosphate and 10 l of artesian water. Bring the pH of the phosphoric acid to values from 5 to 6. Sterilized at 125oC 30 min, cooled down to 36-38oC and seeded with a suspension of 0.5 g of spores Asp. niger strain L 4.

Podkrashivanie seed are within 24 h at 37-38oC, aeration of 0.1 to 0.5 volume of air to 1 volume of medium in Sterile minutes enter 80 l of syrup glitch is m environment in mines, maintaining a pH of 6 to 6.5 chalk, lime, alkali, soda, potash or other neutralizing agent depending on the desired reaction product.

After the utilization of glucose fermentation continuously or detachable-topping-up method, based on the results of the analysis of residual sugar and gluconate and supporting relevant pH neutralizing agents. After disposing of the last portion of sugar displace the stage separation and purification gluconate 75-80 l fermented solution, instead impose 20 l sterile sugar solution containing 60 g/l sucrose, or 120 g/l molasses, 2.5 g of zinc sulfate, 15 g of magnesium sulfate, 15 g one-deputizing potassium phosphate, 15 g of diammonium phosphate. When aeration 7 l/min and a temperature of 36 - 37oC stand 3 hours, then enter 60 l of a sterile solution of sucrose or molasses concentration on sugar 250 g/L. Gradually within 24 h increase aeration to 45-50 l/min. At such aeration and mixing sucrose is utilized for 72 - 120 hours

Example 1

Getting gluconate and calcium citrate (hereinafter citrate salt of citric acid) In the reactor with a volume of 100 l load 300 g glucose, 90 g of corn extract, 14 g of sulfate. avodat to pH 5,3 phosphoric acid. Sterilized for 30 min at 125oC, cooled to 38oC and seeded with a suspension of 0.5 g of spores Asp.niger strain L 4.

Pokasivaut seed for 24 h at following aeration mode:

- up to 3 h of 1.7 l/min when the agitator 1 min for each hour;

- from 3 to 6 h 3.5 l/min, the stirrer includes a permanent job;

- from 6 to 12 h - 5 l/min;

- from 12 to 24 h - 7 l/min.

Sterile enter 80 l of syrup containing 140 g/l glucose and 200 g of unslaked lime. For the entire period of biosynthesis install aeration 7 l/min, which corresponds to a ratio of 0.08 volume of air to 1 volume of medium per minute, and support throughout the biosynthesis of the temperature of the 35oC. every 3 hours take samples for analysis and at pH below 5.5 add 200 g of lime. When the residual glucose 11 g/l samples for analysis are taken every hour and when the glucose concentration of 2.3 g/l the first phase synthesis ceased. For this 80 l fermented solution displace sterile air through the isolation and purification of calcium gluconate. Instead impose 20 l 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 one-deputizing potassium phosphate, 30 g of diammonium phosphate, 19 l Amigo 250 g/l sucrose. Increase aeration for 6 h to 20 l/min after 12 h for up to 45 l/min and are so on the biosynthesis, maintaining the temperature of the 35oC up to 168 h from the beginning seed stage and achieve concentrations of sucrose, 4 g/L. the Contents of the reactor are heated to 80oC, incubated for 30 min and transfer the solution to the extraction.

The selection of calcium gluconate

Fermented solution with the first phase of the biosynthesis heated to 80oC and incubated for 30 minutes Lime to bring the pH to 6.5, heated 30 min and filtered on a suction filter from biomass and sediment. The precipitate was washed with 20 liters of hot demineralized water, adding wash water to the filtrate. Lighten the filtrate 800 g of activated carbon, coal is separated on a suction filter and washed with 10 l of hot demineralized water. The filtrate and the washing water evaporated in vacuum to a concentration of calcium gluconate 210 g/L. Crystallized for 24 h with gradual cooling to 20oC. the Precipitated crystals are separated in a centrifuge and dried at 50oC. Get 6842,5 g substance injection of calcium gluconate. The output of sugar for fermentation 85% allocation 59,55%.

The selection of calcium citrate

The contents of the reactor with the second phase of the process is filtered from biomass to nobody. The washing water and the filtrate is neutralized with lime to pH of 6.8, incubated 2 h at 80oC, filtered on a suction filter. Washed with hot demineralized water, the precipitate of calcium citrate on the filter to the negative reaction of wash water to the sulfates and chlorides. The precipitate is dried at 60oC. Get 16820 g of calcium citrate tetrahydrate rocket clean out of the sugar allocation to 103.8%. The yield of citric acid on sugar fermentation 85%.

Example 2

Receiving calcium gluconate and citric acid

In a reactor with a volume of 100 l load 300 g glucose, 90 g of corn extract, 14 g of magnesium sulfate, 4 g of manganese sulfate, 15 g disubstituted potassium phosphate, 30 g of diammonium phosphate and 10 l of artesian water. Bring the pH to 5.0 with phosphoric acid. Sterilized for 30 min at 125oC, cooled to 38oC and seeded with a suspension of 0.5 g of spores Asp.niger strain P4.

Pokasivaut seed for 24 h at following aeration mode:

- up to 3 h of-1.7 l/min, when the agitator 1 min for each hour;

- from 3 to 6 h 3.5 l/min, the stirrer includes a permanent job;

- from 6 to 12 h - 5 l/min;

- from 12 to 24 h - 7 l/min.

Sterile enter 80 l of syrup with glucose 140 g/l and 200 g of unslaked lime. On Mesut samples for analysis and at pH below 5.5 add 200 g of unslaked lime. When the residual glucose 3 g/l, the process is switched to the mode of Achimov and Dolivo. For this 10 liters of fermented solution to displace the extraction and introduce instead of 19 l of sterile glucose syrup with glucose concentration of 100 g/l After 3 h, when the concentration of residual glucose becomes 2.5 g/l, again make weaning 10 liters of fermented solution and adding 10 l of glucose syrup, simultaneously impose on 200 g of unslaked lime. So are up to 75 h from the beginning seed stage, entering a total of 80 l of syrup and making weaning 80 l fermented solution. After that, 80 l of fermented solution displace sterile air through the selection and enter instead of 20 l of a sugar solution containing: 600 g of sucrose, 1200 g of molasses, 2.5 g of zinc sulfate, 15 g of magnesium sulfate, 15 g one-deputizing potassium phosphate, 30 g of diammonium phosphate, 19 l artesian water. Incubated 3 h with aeration 7 l/min, and then enter another 60 litres of sugar syrup, containing 250 g/l sucrose. Increase the aeration of 20 l/min for 6 h, then after 12 h up to 45 l/min and continue in this mode, the process, maintaining the temperature of the 35oC to 219 h after the beginning of cultivation. When the concentration of residual sugar 3.2 g/l the contents of the reactor load, the Oia

For the selection of calcium gluconate fermented solution with the first phase of the biosynthesis heated to 65oC and incubated for 30 minutes Adjusted with lime to a pH of 6.7, filtered from the precipitate by suction, the filter residue is washed with 50 liters of hot demineralized water. The washing water and the filtrate lighten 2000 g of activated carbon, coal washed with 10 l of demineralized water. The washing water and the filtrate evaporated in vacuum to a concentration of calcium gluconate 210 g/L. Crystallized for 24 h, cooling to 20oC. the Crystals are separated in a centrifuge and dried at 50oC. Get 11700 g substance injection of calcium gluconate. The output of sugar for fermentation 89% allocation 60%.

The selection of citric acid

Fermented solution with the second phase of the biosynthesis filtered on a suction filter, washed with water, add 800 grams of activated charcoal and filtered. Wash the precipitate with 5 l of demineralized water. The filtrate and the washing water is evaporated in a vacuum to the content of citric acid 800 g/L. Crystallized by cooling to 5oC for 8 hours, the Crystals are separated in a centrifuge and dried at 50oC. Get 10530 g citric acid reactive with the release of sugar in the fermentation of 85.2%, on the allocation of 65%.

Example 3
oC, cooled to 38oC and seeded with a suspension of 0.5 g of spores Asp.niger strain L4. Pokasivaut seed for 24 h at following aeration mode:

- up to 3 h of 1.7 l/min, the stirrer is turned on 1 min for each hour;

- from 3 to 6 h is 3.5 l/min, the stirrer includes a permanent job;

- from 6 to 12 h - 5 l/min;

- from 12 to 24 h - 7 l/min.

Sterile enter 80 l of syrup containing 300 g/l glucose. For the entire period of biosynthesis install aeration 7 l/min. pH support 6.5 25% NaOH solution. Every 3 hours take samples for analysis, determine the residual glucose, pH, concentration of sodium gluconate. If the pH becomes below 6.5, add 200 g of 25% NaOH solution. Upon reaching glucose 2.3 g/l 80 l fermented solution to displace the extraction. Instead impose 20 l 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 one-deputizing potassium phosphate, 30 g of diammonium phosphate, 19 l artesian water. When aeration 7 l/min and stirring incubated 3 h, the village is e 12 h up to 45 l/min and are so on the biosynthesis, maintaining the temperature of the 35oC to 184 h from the beginning seed stage and residual sugar 3.2 g/L. After that, the contents of the reactor are heated to 75oC, incubated for 30 min and passed through the selection.

The selection of sodium gluconate

Fermented solution with the first phase of the biosynthesis heated to 60oC, incubated 30 min, filtered from biomass on a suction filter, pH adjusted with NaOH to 7.2, lighten 800 g of activated carbon, again filtered on a suction filter, washed with 5 l of demineralized water. The filtrate and the washing water is evaporated in a vacuum to the content of sodium gluconate 470 g/L. Crystallized for 8 h under cooling to 5oC. Get 14587 g of sodium gluconate rocket clean out of the sugar fermentation 86,5% allocation 60%.

The selection of citrate

The contents of the reactor with the second phase of the biosynthesis filtered on a suction filter from biomass, pH adjusted to 6.9 with NaOH solution and fined 800 g of activated charcoal. The coal is separated on a suction filter, washed with 5 l of demineralized water. The washing water and the filtrate evaporated in vacuo to content citrate 420 g/l, crystallized for 6 h under cooling to 5oC. To the environment with the release of sugar in the fermentation of 85.2%, on selection of 65.4%.

The quantitative data shown in the examples and comparative characteristics with the known method shown in the table.

Technical and economic efficiency of the proposed method

Specialists known [4] that Asp.niger can be operated with high efficiency within 10-12 days. The maximum productivity of the culture reaches 3-8 day.

Fig. 1 illustrates this dependency: changes in the total acidity (1), the mass concentration of citric acid (2), the concentration of sugar (3) on fermentation time.

In this regard, the inventive method combines the effectiveness of biomass to 2-4 days in the period biosynthesis gluconate and high-level synthesis of citric acid with 3 to 8 days.

This is in addition to saving spore material (according to the table. consumption of spores per kg of end product is 2-3 .5 times lower than the claimed method).

In addition, it is necessary to take into account reduced by the present method is 2-3 .5 times the share of unproductive seed stage process that entails savings in energy, raw materials and materials.

Despite the rich potential of culture, large ICRI is radauth from uneven distribution of the products. Plants citric acid have a maximum sales in the summer. Production of gluconate smaller volumes of products, which production costs are high.

It is also necessary to consider that the microbiological production almost can combine different cultures in some manufacturing areas, as it leads to mutual infection. In this case, the proposal considers the expansion of the product range only due to the use of resources of one and the same culture In autumn-winter and spring plants citric acid can produce gluconate and citrate, the retention of which from 3 to 10 years, in contrast to short-term security of citric acid (the shelf life of food citric acid monohydrate 6 months). Production of gluconate, gluconic acid and gluconolactone can extend the range by producing citric acid and its salts in the summer

By the present method, after the first phase of the biosynthesis in the environment remains after preparation of the next phase of about 10 g/l gluconate. This gluconate is not a by-product and waste in the synthesis of citric acid, as it provides a metabolite in ageut, accordingly, the changing concentrations of oxalic, malic, succinic acid during biosynthesis). The content of citric acid in the amount of acid biosynthesis is 96-99% [4].

In the initial stage of the biosynthesis of injected salt of manganese and corn extract, as it is known [3, 5] that at a concentration of from 0.0001 to 0.0004 wt.% manganese reduces the rate of formation of citric acid. During the transition to the second phase of the process, the biosynthesis of citric acid by the present method provides for the dilution due to themof and Dolivo 10-30 times and the reduction of the manganese concentration to the optimum for the biosynthesis of citric acid.

It should also be noted a certain originality of calcium gluconate, allowing the process when its concentrations in excess of saturation. This connection is difficult dissolves at 20 - 30oC, but readily forms supersaturated solutions. The solubility of calcium gluconate at 30oC about 40 g/l [4], i.e. the higher the concentration of salt should be present in the sediment, which can create difficulties in mass transfer. In practice this does not happen. Fig. 3 illustrates that at concentrations below 100 g/l calcium gluconate in terms of biosynthesis is the solution. About this evidence is erimentale. Only after 106-110 g/l the results are unreliable because of the presence in the analyzed sample more or less sediment gluconate.

1. The method of obtaining salts of gluconic acid and citric acid, consisting in growing inoculum of spores of Aspergillus niger in the presence of growth substances, sources of carbohydrates and mineral salts containing manganese or zinc, subsequent fermentation while maintaining a certain pH, aeration, separation and purification of the products obtained, characterized in that during one process cycle, using the same strain of Asp. niger, one portion dispute and the same technological equipment get as salts of gluconic acid, and citric acid in pure form or in the form of salts, or a particular product depending on the needs of production by conducting the fermentation in the presence of one of the growth substance, source of carbohydrates and mineral salts with transition after weaning to another source of carbohydrates and mineral salt, changing the conditions of the process, namely the pH of the medium and aeration mode.

2. The method according to p. 1, characterized in that in the beginning of the process create conditions gladie manganese, the process is conducted on glucose or gluconeogenesis syrup in a periodic, continuous or detachable-topping-up fermentation while maintaining a pH of 5 to 7 and aeration of not more than 0.5 volume of air to 1 volume of the nutrient medium in minutes

3. The method according to PP.1 and 2, characterized in that the operating time after a sufficient number of gluconate and after the utilization of sugar, about 9 parts of the fermented solution to displace biomass from the reactor for the extraction and purification gluconate, and to the other add about 9 parts of molasses or sugar solution containing salts of zinc, aeration increase to values greater than 0.5 volume of air to 1 volume of medium per minute, after the utilization of sugar biosynthesis cease.

4. The method according to PP.1 to 3, characterized in that for obtaining gluconate themy and primary fermented solution after the first phase of the biosynthesis heated to 60 - 80oC, incubated 30 min, adjusted to pH 6.5 to 7.2, separating the precipitate, lighten activated charcoal, filtered, the filtrate evaporated in vacuum and crystallized upon cooling, the crystals are separated and dried.

5. The method according to PP.1 to 4, characterized in that for obtaining citric acid fermented solution of the data coal, filtered, the filtrate evaporated in vacuo, crystallized upon cooling, the crystals are separated and dried.

6. The method according to PP.1 to 5, characterized in that for obtaining water-soluble citrates fermented solution is neutralized by the corresponding agent to pH 6.5 to 7.2, filtered, the filtrate evaporated in vacuum and crystallized upon cooling, the crystals are separated and dried.

7. The method according to PP.1 to 5, characterized in that to get a sparingly soluble citrates biomass is separated, the filtrate is adjusted to the appropriate agent to the optimum pH, the precipitation is separated, washed out prior to the absence in the lavage of sulfates and chlorides, and dried.

 

Same patents:

The invention relates to the microbiological industry and relates to a technology of production of edible organic acids, namely citric acid, by the method of deep fermentation

The invention relates to the cleaning and bleaching fermented environments in directed biosynthesis of citric acid
The invention relates to a biotechnological production of food acids

The invention relates to the field of biotechnological synthesis of edible organic acids, in particular citric acid, and can be used at the enterprises of the biotechnology industry for the production of citric acid, intended for household use

The invention relates to biotechnology, namely, a process for the production of citric acid

The invention relates to the microbiological industry, and specifically to methods of production of citric acid

The invention relates to a new microbiological method for the terminal oxidation of the alkyl groups in the carboxyl group

The invention relates to the microbiological industry and relates to a technology of production of edible organic acids, namely citric acid, by the method of deep fermentation

The invention relates to the microbiological industry, and for the new strain - producer of the protein, which may find application as a protein fortifier food or feed additives in agriculture
Up!