Method for producing lactic acid by fermentation using sucrose as main carbon source

Abstract

Provided is a method for producing lactic acid by fermentation using sucrose as a main carbon source. The method comprises a seed culture step and a fermentation culture step. The seed culture step comprises: inoculating a seed culture medium with rhizopus oryzae spores for culture to form a seed liquid, wherein the seed culture medium comprises 40-60.0 g / L of sucrose, 1.8-2.2 g / L of urea, 0.15-0.3 g / L of KH2PO4, 0.15-0.3 g / L of MgSO4·7H2O, 0.005-0.01 g / L of ZnSO4·7H2O, and 8-12 g / L of CaCO3 <sb / >. The fermentation culture step comprises: inoculating a fermentation medium with the seed liquid for fermentation culture, wherein the fermentation medium comprises 120-150 g / L of sucrose, 8-12% of molasses, 0.83-1.05 g / L of a nitrogen source, 0.5-0.7 g / L of KH2PO4, 0.2-0.4 g / L of MgSO4·7H2O, 0.005-0.01 g / L of ZnSO4·7H2O, 0.3-0.5 g / L of FeSO4·7H2O, 3%-5% of biochar, and 50-70 g / L of CaCO3 <sb / >.

Description

Method for fermenting lactic acid with sucrose as main carbon source TECHNICAL FIELD The present application relates to a method for fermenting lactic acid with sucrose as main carbon source. BACKGROUND Lactic acid is prepared by fermentation method with glucose, starch or milk as raw material, and through microbial fermentation (lactic acid bacteria, filamentous fungi, etc.). There are many microorganisms in nature that can produce L-lactic acid, but only some species of Rhizopus in molds, Lactobacillus, Streptococcus and Bacillus can be applied to industry. However, bacteria have complex nutritional requirements, such as amino acids, vitamins and inorganic salts, etc., while the nutritional requirements of Rhizopus are relatively simple, only inorganic nitrogen and a small amount of other inorganic salts are needed. At present, the raw materials used for lactic acid fermentation by Rhizopus are mainly glucose or starchy raw materials. For example, in the Chinese patent application No. 200710170658.7 entitled "Continuous production method of L-ammonium lactate based on Rhizopus", a continuous production method of L-ammonium lactate based on Rhizopus is disclosed, which comprises the following steps: (1) Seed culture: spores of any one of Rhizopus are inoculated into seed culture medium for aerobic liquid culture, and the components of the seed culture medium include carbon source, nitrogen source, inorganic salt and water. In the seed culture medium, the concentration of phosphate is 0.8-1.2 g / L; (2) The product obtained in step (1) is inoculated into fermentation medium for aerobic liquid fermentation culture, and ammonia water or ammonia gas is added during the culture to control the pH of the culture system to 5-8, then the feeding medium is added, and the fermentation product is obtained, and then the target product ammonium lactate is collected from the fermentation product. The fermentation medium uses liquefied starch, ammonium sulfate, KH2PO4, MgSO4·7H2O and ZnSO4·7H2O. With the continuous rise in the price of glucose or starchy raw materials, the production cost of L-lactic acid is high, and the cost of related products is also increasing. Although there are some studies on L-lactic acid fermentation using crude raw materials in China, the yield of L-lactic acid is very low, and the price of the produced L-lactic acid does not have market competitiveness. Based on the above problems, the applicant has studied the above problems and produced the present case. TECHNICAL PROBLEM The purpose of the present application is to provide a method for fermenting lactic acid with sucrose as main carbon source, which can reduce cost, is convenient to obtain materials and has high lactic acid yield. TECHNICAL SOLUTION In order to achieve the above object, the present application adopts the following technical solutions: A method for fermenting lactic acid with sucrose as a main carbon source, comprising a seed culture step and a fermentation culture step,

[0011] The seed culture step is to inoculate Rhizopus oryzae spores into a seed culture medium to form a seed liquid, and the seed culture medium comprises 40-60.0 g / L sucrose, 1.8-2.2 g / L urea, 0.15-0.3 g / L KH2PO4, 0.15-0.3 g / L MgSO4·7H2O, 0.005-0.01 g / L ZnSO4·7H2O, 8-12 g / L CaCO3, and the balance of water. The fermentation culture step is to inoculate the above seed liquid into a fermentation culture medium for fermentation culture, and the fermentation culture medium comprises 120-150 g / L sucrose, 8-12% molasses by mass percentage, 0.83-1.05 g / L nitrogen source, 0.5-0.7 g / L KH2PO4, 0.2-0.4 g / L MgSO4·7H2O, 0.005-0.01 g / L ZnSO4·7H2O, 0.3-0.5 g / L FeSO4·7H2O, 3-5% biochar by mass percentage, 50-70 g / L CaCO3, and the balance of water. As a preferred mode of the present application, the biochar is obtained by pyrolysis of bagasse or peanut shell in a reaction furnace, the pyrolysis temperature is 300-500℃, the pyrolysis time is 30-60 min, and the particle size of the biochar is 2mm-4mm. As a preferred mode of the present application, the Rhizopus oryzae is Rhizopus oryzae CICC 3038. As a preferred mode of the present application, the seed liquid is inoculated into the fermentation culture medium at a mass percentage of 8-12%. As a preferred mode of the present application, the fermentation culture has a culture time of 68-72h, the temperature is controlled at 28-30℃ in 0-28h, and then the temperature is controlled at 30-32℃, the culture is stirred at a speed of 200rpm-400rpm, the volume fraction of dissolved oxygen in the fermentation culture medium is controlled at 30%-60%, and the PH is controlled at 5.0-6.0 by using ammonia water. As a preferred mode of the present application, the nitrogen source is one of urea and ammonium sulfate or a mixture of the two. As a preferred mode of the present application, the initial concentration of sucrose in the fermentation culture medium is 50g / L, and the remaining sucrose is divided into two parts, which are added at 29-32h and 48-52h after the start of fermentation, respectively. Advantages After the technical scheme of the present application is adopted, the Rhizopus has strong fermentation sucrose acid production capacity with sucrose as the main carbon source. The sucrose yield is large and the source is sufficient; it is safe and non-toxic, and is a renewable resource. The use of sucrose fermentation to produce lactic acid can reduce the production cost of lactic acid, expand the application field of lactic acid, on the one hand, and on the other hand, it can make full use of natural resources, effectively improve the economic benefit of sugar factory, and develop sugar industry faster and better. The addition of FeSO4·7H2O in the fermentation medium increases the reducing power of the fermentation system, helps the electron system transmission, and promotes the biological catalytic reaction of sugar-pyruvic acid-lactic acid. The addition of biochar in the fermentation medium has a certain promoting effect on the material conversion in the microbial fermentation process, can become the growth carrier of Rhizopus oryzae, stabilize the growth density of Rhizopus oryzae in the fermentation system, and promote the smooth progress of biological catalytic reaction. [Corrected according to Rule 91 04.03.2025][deleted] [Corrected according to Rule 91 04.03.2025][deleted] Embodiments of the present application In order to better understand the technical scheme of the present application, the following embodiments are described in more detail. In the present application, the strain is selected as the Rhizopus oryzae CICC 3038 of the China Industrial Microorganism Preservation Center as the starting strain, and the starting strain is stored in the PDA culture medium. The PDA culture medium is prepared in the following manner: 200g of peeled potatoes are cut into small pieces, 1L of water is added, boiled for 30min, filtered with double-layer gauze to take the clear liquid, water is added to supplement the water loss due to evaporation, 20g of glucose, 15g of agar and natural pH are added. The starting strain is taken out, and after 10 times of streaking and subculturing on the sucrose culture plate under the condition of 28-30℃, it is used as the fermentation strain. The sucrose culture medium on the sucrose culture plate includes: sucrose 40-60.0g / L, urea 1.8-2.2g / L, KH2PO4 0.15-0.3g / L, MgSO4·7H2O 0.15-0.3g / L, ZnSO4·7H2O 0.005-0.01g / L, CaCO3 8-12g / L, agar powder 16-18g / L, and the balance of water. Seed liquid culture: spores were picked from sucrose culture plate, inoculated a certain amount of spores into seed culture medium, the initial spore concentration was 1.0 x 10 spores / mL, the rotation speed was 180 rpm, and the spores were cultured at 30°C for 14 h to reach the logarithmic growth phase. The seed culture medium contains the following components: sucrose 50 g / L, urea 2 g / L, KH2PO4 0.15-0.3 g / L, MgSO4·7H2O 0.25 g / L, ZnSO4·7H2O 0.008 g / L, CaCO3 10 g / L, and the rest is water. Example 1 Fermentation medium: sucrose 120 g / L, urea 1.8 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.3 g / L, ZnSO4·7H2O 0.008 g / L, CaCO3 50 g / L, and the rest is water. The cultured Rhizopus oryzae seed liquid was inoculated into the fermentation medium at a 10% inoculation amount, and cultured at 28°C for 28 h, and then the fermentation temperature was adjusted to 32°C. The initial concentration of sucrose was 50 g / L, and the remaining amount was added in two equal parts at 32 h and 52 h after the start of fermentation. The fermentation was stirred at 300 rpm for 72 h, the dissolved oxygen volume fraction was controlled at 30%, and the pH was controlled at 5.0 using diluted ammonia water. The lactic acid yield reached 63.7 g / L. Example 2 Fermentation medium: sucrose 120 g / L, molasses 10 g / L, urea 1.8 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.3 g / L, ZnSO4·7H2O 0.008 g / L, FeSO4·7H2O 0.3 g / L, biochar 3% (by mass), CaCO3 50 g / L, and the rest is water. (In the examples, the molasses used is sugarcane molasses, and the same applies below) The cultured Rhizopus oryzae seed liquid was inoculated into the fermentation medium at a 10% inoculation amount, and the culture conditions were the same as in Example 1. The difference between Example 2 and Example 1 is the composition of the fermentation medium, and the lactic acid yield is increased by 18% (by mass) compared to Example 1. Example 3 Fermentation medium: sucrose 130 g / L, urea 2.0 g / L, KH2PO4 0.6 g / L, MgSO4·7H2O 0.35 g / L, ZnSO4·7H2O 0.01 g / L, CaCO3 60 g / L, and the rest is water. The well-cultured Rhizopus oryzae seed liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and was fermented at 30°C for 28h, and then the fermentation temperature was adjusted to 32°C. The initial concentration of sucrose was 50g / L, and the remaining amount was evenly divided into two times, and the remaining sucrose was added at 30h and 48h after the start of fermentation, respectively. The culture was stirred at a rotation speed of 300rpm for 68h, the dissolved oxygen volume fraction of the fermentation liquid was controlled at 30%, and the pH was controlled at 5.3 by using ammonia water diluted by 2 times. The lactic acid yield reached 68.9g / L. Example 4 The fermentation medium: sucrose 130g / L, molasses 12g / L, urea 2.0g / L, KH2PO40.6g / L, MgSO4·7H2O 0.35g / L, ZnSO4·7H2O 0.01g / L, FeSO4·7H2O 0.3g / L, biochar 4%, CaCO3 60g / L, and the balance of water. The well-cultured Rhizopus oryzae seed liquid was inoculated into the modified fermentation medium at an inoculation amount of 10%, and the culture conditions were the same as in Example 3. The difference between Example 4 and Example 3 was that the components of the fermentation medium were different, and the lactic acid yield was increased by 20%. Example 5 The fermentation medium: sucrose 150g / L, urea 2.0g / L, KH2PO4 0.6g / L, MgSO4·7H2O 0.35g / L, ZnSO4·7H2O 0.01g / L, CaCO3 60g / L, and the balance of water. The well-cultured Rhizopus oryzae seed liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and was fermented at 30°C for 28h, and then the fermentation temperature was adjusted to 32°C. The initial concentration of sucrose was 50g / L, and the remaining amount was evenly divided into two times, and the remaining sucrose was added at 30h and 48h after the start of fermentation, respectively. The culture was stirred at a rotation speed of 300rpm for 68h, the dissolved oxygen volume fraction of the fermentation liquid was controlled at 30%, and the pH was controlled at 5.3 by using ammonia water diluted by 2 times. The lactic acid yield reached 68.9g / L. Example 6 The fermentation medium: sucrose 150g / L, molasses 12g / L, urea 2.0g / L, KH2PO4 0.6g / L, MgSO4·7H2O 0.35g / L, ZnSO4·7H2O 0.01g / L, FeSO4·7H2O 0.3g / L, biochar 4%, CaCO3 60g / L, and the balance of water. The well-cultured Rhizopus oryzae seed liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and the culture conditions were the same as in Example 5. The difference between Example 6 and Example 5 was that the components of the fermentation medium were different, and the lactic acid yield was increased by 15%. In the above embodiment, the biochar is obtained by pyrolysis of bagasse in a reaction furnace, the pyrolysis temperature is 400 DEG C, the pyrolysis time is 45 min, and the particle size of the biochar is 3 mm. The protection scope of the present application is not limited to the above embodiment, and any similar transformation thereof is deemed to be within the protection scope of the present application. Industrial applicability The present application has the advantages of low cost, convenient material selection and high lactic acid yield. [Corrected according to Rule 91 04.03.2025][Deleted]

Claims

1. A method for producing lactic acid by fermentation using sucrose as a main carbon source, comprising a seed culture step and a fermentation culture step, characterized by: The seed culture step is to inoculate the spores of Rhizopus oryzae into a seed culture medium to form a seed liquid, the seed culture medium comprises 40-60.0 g / L sucrose, 1.8-2.2 g / L urea, 0.15-0.3 g / L KH2PO4, 0.15-0.3 g / L MgSO4·7H2O, 0.005-0.01 g / L ZnSO4·7H2O, 8-12 g / L CaCO3, and the balance is water; the fermentation culture step is to inoculate the seed liquid into a fermentation culture medium to carry out fermentation culture, the fermentation culture medium comprises 120-150 g / L sucrose, 8-12% molasses by mass percentage, 0.83-1.05 g / L nitrogen source, 0.5-0.7 g / L KH2PO4, 0.2-0.4 g / L MgSO4·7H2O, 0.005-0.01 g / L ZnSO4·7H2O, 0.3-0.5 g / L FeSO4·7H2O, 3-5% biochar by mass percentage, and 50-70 g / L CaCO3.

2. The method for producing lactic acid by fermentation using sucrose as the main carbon source as described in claim 1, characterized in that: The biochar is obtained by pyrolysis of bagasse or peanut shell in a reaction furnace, the pyrolysis temperature is 300-500 DEG C, the pyrolysis time is 30-60 min, and the particle size of the biochar is 2 mm-4 mm.

3. A process for the fermentation of sucrose as the main carbon source to lactic acid according to claim 2, characterized in that: The Rhizopus oryzae is Rhizopus oryzae CICC 3038.

4. The method for producing lactic acid by fermentation using sucrose as the main carbon source as described in claim 3, characterized in that: The seed liquid is inoculated into the fermentation culture medium at 8-12% by mass percentage.

5. The process for the fermentation of sucrose as the main carbon source to lactic acid according to claim 4, characterized in that: The fermentation culture is cultured for 68-72 h, the temperature is controlled at 28-30 DEG C in 0-28 h, and then the temperature is controlled at 30-32 DEG C, the culture is stirred at a speed of 200 rpm-400 rpm, the volume fraction of dissolved oxygen in the fermentation culture medium is controlled at 30%-60%, and the pH is controlled at 5.0-6.0 by using ammonia water.

6. The process for the fermentation of sucrose as the main carbon source to lactic acid according to claim 5, characterized in that: The nitrogen source is one of urea and ammonium sulfate or a mixture of the two.

7. A process for the fermentation of sucrose as the main carbon source to lactic acid according to claim 6, characterized in that: The initial concentration of sucrose in the fermentation culture medium is 50 g / L, and the remaining sucrose is evenly divided into two parts, which are added at 29-32 h and 48-52 h after the start of fermentation, respectively.