CN105586367A - Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses - Google Patents

Abstract

The invention discloses a method for fermenting and producing citric acid based on adding glucoamylase in the pH response stage, which includes mixing, liquefying, inoculating and fermenting starchy raw materials to obtain a final fermentation liquid, and also includes in the fermentation process, when the fermentation liquid When the pH drops to 2.0~3.0, the step of adding glucoamylase in equal amounts in stages, the added in equal amounts every 2~8h, the total amount of glucoamylase added is 40~1000U/ g residual non-glucose amount. The method of the present invention can solve the problem of the loss of glucoamylase activity in the middle and later stages of citric acid fermentation, effectively shorten the fermentation period, increase the fermentation conversion rate, reduce the residual sugar amount of the final fermentation liquid, reduce waste water discharge, and can offset the self-secreted glucosidase activity at the same time, Reducing the production of non-fermentable sugars has important economic and environmental benefits.

Description

A method for producing citric acid by adding saccharification enzyme to ferment and produce citric acid based on pH response stage

technical field

The invention belongs to the technical field of fermentation engineering, and in particular relates to a method for producing citric acid by adding glucoamylase to ferment and produce citric acid based on the pH response stage.

Background technique

Citric acid is an important organic acid with multiple functions. It is widely used in food, medicine, chemical industry and other fields. It is the edible organic acid with the largest output and consumption in the world. The global output exceeds 1.7 million tons. At the same time, citric acid has excellent biological characteristics, and has great application potential in emerging industries such as biopolymerization, drug delivery, and cell culture, and its demand is growing at a rate of 5% per year.

Citric acid is often obtained by liquid submerged fermentation, and the traditional batch fermentation method is still used in industrial production. Because Aspergillus niger has the advantages of rich enzyme system, wide range of substrates, and high yield, it is the most important strain for citric acid fermentation. Aspergillus niger itself can secrete glucoamylase, and generally adopts the method of simultaneous saccharification and fermentation. However, during the production of citric acid by Aspergillus niger fermentation, with the continuous accumulation of synthesized citric acid, the pH of the fermentation broth will drop significantly, and the activity of glucoamylase will be gradually inhibited, resulting in high residual sugar in the final fermentation mash and a long fermentation period.

Prior Art Some researches have been done on saccharification during citric acid fermentation. The invention patent of application number 201010513719.7 "A method of adding glucoamylase to ferment and prepare citric acid" discloses a process of adding glucoamylase after cooling down to 40~60°C after liquefaction and before inoculation. Part of the glucoamylase is inactivated, resulting in a low utilization rate of glucoamylase; the invention patent of application number 201110162278.5 "a method for producing citric acid by fermentation" discloses adding glucoamylase during normal temperature control at 36~38°C after inoculation and fermentation; The invention patent of application number 201210009294.5 "a method for preparing citric acid by fermentation" discloses that glucoamylase is added to the fermentation medium within 0-8 hours after the citric acid fermentation strain is inoculated.

The above methods are based on the common guiding idea that high concentration of small molecular sugars (such as glucose) can be obtained at the early stage of fermentation by adding glucoamylase, but they do not solve the problem of glucose supply caused by the significant drop in pH and loss of glucoamylase activity in the later stages of fermentation. The sugar consumption rate of the strain is not coordinated, the synthesis rate of citric acid is slowed down, the fermentation cycle is long, and the residual sugar content is high. At the same time, it brings new problems: there is glucose in the medium itself in the early stage of fermentation, and the glucoamylase secreted by itself can already meet the nutritional needs of the fermentation strain. Excessively high concentration of glucose will inhibit the growth of the strain and prolong the lag period of fermentation, which is not conducive to lemon. Rapid synthesis of acids.

Contents of the invention

The purpose of the present invention is to provide a method for fermenting citric acid by adding glucoamylase in the pH response stage, which can solve the problem of loss of glucoamylase activity in the middle and later stages of citric acid fermentation, effectively shorten the fermentation cycle, increase the fermentation conversion rate, and reduce the final fermentation rate. The amount of residual sugar in the fermentation broth.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for producing citric acid by adding glucoamylase to ferment and produce citric acid based on the pH response stage, comprising: slurrying, liquefying, inoculating and fermenting starchy raw materials to obtain a final fermentation liquid; From 2.0 to 3.0, the step of adding glucoamylase in equal amounts in stages.

Specifically, the step-by-step equal addition is equal addition at intervals of 2 to 8 hours.

Specifically, the total amount of glucoamylase added is determined based on the residual non-glucose content in the fermentation broth (the difference between total residual sugar and residual grapes), and the total amount added is 40-1000 U/g residual non-glucose content .

Specifically, the starchy raw material includes at least one of corn flour, wheat flour, tapioca flour, sweet potato flour, starch, molasses, and wheat.

Preferably, the present invention specifically comprises the following steps:

(1) Mix the starch raw material powder and water evenly according to the ratio of 1:1.5~1:4, adjust the pH to 5.8~6.0, and then add high-temperature α-amylase, the amount of amylase added is 15~50U/ g powder;

(2) The mixture liquid obtained in step (2) is sprayed twice, the temperature of the first injection is 95~105°C, and the temperature of the second injection is 120~130°C. After passing the iodine test, the liquefied mixed liquid is obtained, and then the liquefied mixed liquid is Obtain liquefied supernatant through plate and frame filtration;

(3) mixing the liquefied mixed liquid obtained in step (2) with water, adding a nitrogen source, adjusting the total sugar of the mixed liquid to 6-12%, and the total nitrogen to 0.2%-0.4%, to obtain a seed culture medium;

(4) inoculating the seed culture medium in step (3) with the Aspergillus niger spore suspension to obtain mature seed liquid;

(5) mixing the liquefied mixed liquid and liquefied clear liquid obtained in step (2) with water, adjusting the total sugar of the mixed liquid to 16-18%, and the total nitrogen to 0.05-0.15%, to obtain a fermentation medium;

(6) Inoculate the mature seed liquid obtained in step (4) into the fermentation medium obtained in step (5), and cultivate for 20-32 hours;

(7) When the pH of the fermentation broth of the above fermentation medium drops to 2.0~3.0, add an equal amount of glucoamylase to it at intervals of 2~8h, and the total amount of addition is 40~1000U/g residual non-glucose.

Unless otherwise specified, the percentages mentioned above are percentages by weight (wt%).

The present invention has the following beneficial technical effects:

Based on the physiological characteristics of fermentation strains and the synthesis characteristics of citric acid, the present invention adopts the method of adding glucoamylase in stages and equal amounts according to the residual non-glucose amount when the pH of the fermentation liquid drops to a specific range, so as to coordinate the supply of glucose and the consumption of glucose in the whole fermentation process. achieve balance optimization. On the one hand, it can not only realize the efficient use of its own glucoamylase, but also ensure the rapid growth of bacteria in the early stage of fermentation; on the other hand, adding glucoamylase in an appropriate amount can effectively compensate for the loss of its own vitality, and cooperate with the citric acid fermentation process to ensure that the lemons in the middle and late stages of fermentation The rapid accumulation of acid can counteract the activity of self-secreted glucosidase and reduce the production of non-fermentable sugar. Application results show that the invention can shorten the fermentation period, increase the fermentation conversion rate, reduce the residual sugar in the final fermentation liquid, reduce waste water discharge, and reduce waste water COD _cr , which has important economic and environmental benefits.

detailed description

The present invention will be further described below by specific examples.

In the following examples and comparative examples, the assay method of total sugar and reducing sugar adopts the film titration method, the mensuration of citric acid adopts the NaOH titration of concentration 0.1429mol/L, and the spore count adopts a hemocytometer; if no special instructions, all adopt this method. Commonly used equipment and process methods in the field.

The raw materials and reagents used are commercially available general products unless otherwise specified.

Example 1

Mix cornflour and water evenly at a ratio of 1:1.5, adjust the pH to 5.8, then add high-temperature α-amylase, the addition amount is 50U/g cornflour; two spray liquefaction, the temperature of the first spray is 105 ° C, the second The injection temperature is 130°C, and the iodine test is qualified to obtain a liquefied mixed liquid, and part of the liquefied mixed liquid is filtered through a plate frame to obtain a liquefied clear liquid; the liquefied mixed liquid is mixed with water evenly, and a certain amount of ammonium sulfate is added to prepare a seed medium (total sugar 6%, total Niger 0.25%), inoculate the seed medium with the mature Aspergillus niger spore suspension, increase the stirring speed to 800rpm when culturing for 8 hours, and co-cultivate for 27 hours to obtain mature seed liquid; mix the liquefied mixed liquid, liquefied clear liquid and water to prepare fermentation culture base (total sugar 16%, total nitrogen 0.05%); inoculate the mature seed liquid into the fermentation medium for fermentation culture; when the pH of the fermentation liquid drops to 3.00, add glucoamylase, the total amount added is 40U/g residual non-glucose content, every Add 3840 U of glucoamylase in equal amounts at intervals of 4 hours, and end the fermentation when the concentration of reducing sugar is lower than 0.5%. The fermentation period was measured to be 52 hours, the citric acid content was 16.1%, and the residual sugar was 1.2%.

Example 2

Mix cornflour and water evenly in a ratio of 1:4, adjust the pH to 6.0, then add high-temperature α-amylase, the addition amount is 15U/g cornflour; two sprays are liquefied, the temperature of the first spray is 95°C, and the second spray is liquefied. The injection temperature is 120°C, and the iodine test is qualified to obtain a liquefied mixed solution, and part of the liquefied mixed solution is filtered through a plate frame to obtain a liquefied clear solution; the liquefied mixed solution is mixed with water evenly, and a certain amount of ammonium sulfate is added to prepare a seed medium (total sugar 10%, total Niger 0.30%), the mature Aspergillus niger spore suspension was inoculated into the seed medium, cultivated for 26 hours, and the mature seed liquid was obtained; the liquefied mixed liquid, the liquefied clear liquid and water were mixed to prepare a fermentation medium (total sugar 16.8%, total nitrogen 0.08% ); Inoculate the mature seed liquid into the fermentation medium for fermentation; when the pH of the fermentation liquid drops to 2.52, add glucoamylase, the total amount of addition is 100U/g residual non-glucose, add 16000U of glucoamylase in equal amounts every 4h, when the reduction Fermentation ends when the sugar concentration is lower than 0.5%. The fermentation period was measured to be 53 hours, the citric acid content was 16.9%, and the residual sugar was 0.9%.

Example 3

Mix cassava flour and water evenly at a ratio of 1:2.5, adjust the pH to 5.9, then add high-temperature α-amylase, the addition amount is 25U/g cassava flour; two sprays are liquefied, the temperature of the first spray is 100°C, and the second spray is liquefied. The injection temperature is 125°C, and the iodine test is qualified to obtain a liquefied mixed liquid, and part of the liquefied mixed liquid is filtered through a plate frame to obtain a liquefied clear liquid; the liquefied mixed liquid is mixed with water evenly, and a certain amount of ammonium sulfate is added to prepare a seed medium (total sugar 12%, total Niger 0.40%), the mature Aspergillus niger spore suspension was inoculated into the seed medium, cultivated for 30 hours, and the mature seed liquid was obtained; the liquefied mixed liquid, the liquefied clear liquid and water were mixed to prepare a fermentation medium (total sugar 16.7%, total nitrogen 0.12% ); inoculate the mature seed liquid into the fermentation medium for fermentation; when the pH of the fermentation liquid drops to 2.00, add glucoamylase, the total amount of addition is 300U/g residual non-glucose, and add 48000U of glucoamylase in equal amounts every 8h. Fermentation ends when the sugar concentration is lower than 0.5%. The measured fermentation period is 55h, the citric acid content is 16.5%, and the residual sugar is 1.2%.

Example 4

Mix cassava flour and water evenly at a ratio of 1:2.5, adjust the pH to 5.8, then add high-temperature α-amylase, the addition amount is 25U/g cassava flour; two sprays are liquefied, and the temperature of the first spray is 100°C, and the second spray is liquefied. The injection temperature is 125°C, and the iodine test is qualified to obtain a liquefied mixed liquid, and part of the liquefied mixed liquid is filtered through a plate frame to obtain a liquefied clear liquid; the liquefied mixed liquid is mixed with water evenly, and a certain amount of ammonium sulfate is added to prepare a seed medium (total sugar 12%, total Niger 0.40%), the mature Aspergillus niger spore suspension was inoculated into the seed medium, cultivated for 32 hours to obtain mature seed liquid; the liquefied mixed liquid, liquefied clear liquid and water were mixed to prepare a fermentation medium (total sugar 17.5%, total nitrogen 0.12% ); Inoculate the mature seed liquid into the fermentation medium for fermentation; when the pH of the fermentation liquid drops to 2.60, add glucoamylase, the total amount of addition is 500U/g residual non-glucose, and add 50400U of glucoamylase in equal amounts every 6h. Fermentation ends when the sugar concentration is lower than 0.5%. The measured fermentation period is 63h, the citric acid content is 17.4%, and the residual sugar is 1.6%.

Example 5

Mix cassava flour and water evenly at a ratio of 1:3.5, adjust the pH to 5.9, then add high-temperature α-amylase, the addition amount is 30U/g cassava flour; two sprays are liquefied, the temperature of the first spray is 98°C, and the second spray is liquefied. The injection temperature is 127°C, and the iodine test is qualified to obtain the cassava liquefaction mixture, which is then filtered through a plate and frame to obtain the cassava liquefaction clear liquid. Mix cornflour and water evenly according to the ratio of 1:2.5, adjust the pH to 5.8, then add high-temperature α-amylase, the addition amount is 20U/g cornflour; two spray liquefaction, the temperature of the first spray is 105 ℃, the second The injection temperature is 125°C, and the iodine test is qualified to obtain a corn liquefaction mixture, and part of the corn liquefaction mixture is filtered through a plate frame to obtain a corn liquefaction clear liquid. Mix the corn liquefaction mixture with water evenly, add a certain amount of ammonium sulfate to prepare a seed medium (total sugar 8%, total nitrogen 0.28%), inoculate the mature Aspergillus niger spore suspension into the seed medium, and cultivate for 29 hours to obtain mature seeds mixed liquid of corn liquefaction, clear liquid of corn liquefaction, clear liquid of cassava liquefaction and water to prepare a fermentation medium (total sugar 18.0%, total nitrogen 0.15%); mature seed liquid was inoculated into the fermentation medium for fermentation; when the fermentation liquid When the pH drops to 2.15, add glucoamylase, the total amount added is 1000U/g residual non-glucose, 36800U glucoamylase is added every 3 hours in equal amounts, and the fermentation ends when the reducing sugar concentration is lower than 0.5%. The measured fermentation period is 67h, the citric acid content is 17.7%, and the residual sugar is 2.2%.

Example 6

Mix cornflour and water evenly according to the ratio of 1:3.0, adjust the pH to 5.8, then add high-temperature α-amylase, the addition amount is 34U/g cornflour; two spray liquefaction, the temperature of one spray is 96 ℃, the second spray The injection temperature is 123°C, and the iodine test is qualified to obtain a corn liquefaction mixture, which is then filtered through a plate frame to obtain a corn liquefaction clear liquid. Mix wheat flour and water evenly according to the ratio of 1:2.0, adjust the pH to 6.0, then add high-temperature α-amylase, the addition amount is 23U/g wheat flour; the second injection is liquefied, the temperature of the first injection is 95 ℃, and the temperature of the second injection At 128°C, pass the iodine test to obtain a wheat liquefaction mixture, and then filter through a plate frame to obtain a wheat liquefaction clear liquid. Mix the corn liquefaction mixture with water evenly, add a certain amount of ammonium sulfate to prepare a seed medium (total sugar 10%, total nitrogen 0.32%), inoculate the mature Aspergillus niger spore suspension into the seed medium, and cultivate for 28 hours to obtain mature seeds mixed liquid of corn liquefaction, clear liquid of corn liquefaction, clear liquid of wheat liquefaction and water to prepare a fermentation medium (total sugar 17.2%, total nitrogen 0.09%); mature seed liquid was inoculated into the fermentation medium for fermentation; when the fermentation liquid Add glucoamylase when the pH drops to 2.05, add glucoamylase when the pH of the fermentation broth drops to 2.05, add the total amount of 70U/g residual non-glucose, add 5600U of glucoamylase in equal amounts every 5h, when the concentration of reducing sugar is lower than 0.5 % to end the fermentation. The measured fermentation period is 63h, the citric acid content is 17.1%, and the residual sugar is 1.3%.

Comparative example (prior art)

Mix cornflour and water evenly at a ratio of 1:2.5, adjust the pH to 5.8, then add high-temperature α-amylase, the addition amount is 40U/g cornflour; two sprays are liquefied, the temperature of the first spray is 98°C, and the second spray is liquefied. The injection temperature is 130°C, and the iodine test is qualified to obtain a liquefied mixed liquid, and part of the liquefied mixed liquid is filtered through a plate frame to obtain a liquefied clear liquid; the liquefied mixed liquid is mixed with water evenly, and a certain amount of ammonium sulfate is added to prepare a seed medium (total sugar 12%, total nitrogen 0.25%), inoculate the mature Aspergillus niger spore suspension into the seed medium and cultivate for 28 hours to obtain mature seed liquid; mix the liquefied mixed liquid, liquefied clear liquid and water to prepare a fermentation medium (total sugar 16.6%, total nitrogen 0.85%) ; Inoculate the fermentation medium with the mature seed liquid for fermentation and culture, and end the fermentation when the reducing sugar concentration is lower than 0.5%. The measured fermentation period is 64h, the citric acid content is 16.2%, and the residual sugar is 2.8%.

The test indexes of Examples 1-6 are compared with the comparative examples, and the results are shown in the table below.

Table 1 embodiment 1～6 and comparative example each test index comparative result

It can be seen from Table 1 that in Examples 1-6, the specific pH range (pH2.0-3.0) of the fermentation broth responds to the addition of glucoamylase in equal amounts in stages, which can not only ensure the rapid growth of the bacteria in the early stage of fermentation, but also ensure the rapid growth of the bacteria in the fermentation stage. The rapid accumulation of citric acid in the middle and later stages shortens the fermentation cycle, improves the fermentation conversion rate and fermentation index, and effectively reduces the residual sugar content of the final fermentation liquid.

The above descriptions are only preferred implementations of the present invention, and the present invention is not limited to the above examples. It can be understood that other improvements and changes directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims (6)

1. A method for adding glucoamylase fermentation to produce citric acid based on the pH response stage, comprising starchy raw materials being subjected to pulping, liquefaction, inoculation and fermentation, to obtain a final fermentation liquid, characterized in that: also included in the fermentation process, When the pH of the fermentation broth drops to 2.0~3.0, the step of adding glucoamylase in equal amounts in stages. 2. The method for producing citric acid according to claim 1, characterized in that: the equal addition in stages is equal addition at intervals of 2 to 8 hours. 3. The method for producing citric acid according to claim 1, characterized in that: the added total amount of the glucoamylase is based on the residual non-glucose content (the difference between residual total sugar and residual grapes) in the fermented liquid Sure. 4. The method for producing citric acid according to claim 3, characterized in that: the total amount of glucoamylase added is 40-1000U/g residual non-glucose content. 5. The method for producing citric acid according to claim 1, characterized in that: said starchy raw material comprises at least one of corn flour, wheat flour, cassava flour, sweet potato flour, starch, molasses, and wheat. 6. the method for producing citric acid described in any one of claim 1～5, is characterized in that specifically comprising the following steps: (1) Mix the starch raw material powder and water evenly according to the ratio of 1:1.5~1:4, adjust the pH to 5.8~6.0, and then add high-temperature α-amylase, the amount of amylase added is 15~50U/ g powder; (2) The mixture liquid obtained in step (2) is sprayed twice, the temperature of the first injection is 95~105°C, and the temperature of the second injection is 120~130°C. After passing the iodine test, the liquefied mixed liquid is obtained, and then the liquefied mixed liquid is Obtain liquefied supernatant through plate and frame filtration; (3) mixing the liquefied mixed liquid obtained in step (2) with water, adding a nitrogen source, adjusting the total sugar of the mixed liquid to 6-12%, and the total nitrogen to 0.2%-0.4%, to obtain a seed culture medium; (4) inoculating the seed culture medium in step (3) with the Aspergillus niger spore suspension to obtain mature seed liquid; (5) mixing the liquefied mixed liquid and liquefied clear liquid obtained in step (2) with water, adjusting the total sugar of the mixed liquid to 16-18%, and the total nitrogen to 0.05-0.15%, to obtain a fermentation medium; (6) Inoculate the mature seed liquid obtained in step (4) into the fermentation medium obtained in step (5), and cultivate for 20-32 hours; (7) When the pH of the fermentation broth of the above-mentioned fermentation medium drops to 2.0~3.0, add an equal amount of glucoamylase to it at intervals of 2~8h, and the total amount of addition is 40~1000U/g residual non-glucose; (8) When the concentration of reducing sugar in the above-mentioned fermentation medium is lower than 0.5%, the fermentation is terminated; Unless otherwise specified, the percentages mentioned above are percentages by weight (wt%).