Inorganic nitrogen source culture medium and application thereof in lactic acid fermentation

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to an inorganic nitrogen source culture medium and application thereof in lactic acid fermentation. Each liter of the culture medium comprises the following components: 80-120g of a carbon source; a composite inorganic nitrogen source: 2.0 to 4.0 g of ammonium sulfate, 1.0 to 2.0 g of nitrate and 0.5 to 1.5 g of urea; mineral salts: 0.4 to 0.6 g of citric acid diamine, 0.4 to 0.6 g of KH2PO4, 0.1 to 0.3 g of MgSO4. 7H2O, 0.05 to 1 g of NaCl, and 0.004 to 0.006 g of MnSO4. 4H2O; an auxiliary organic nitrogen source: 0.05-0.1 g of yeast powder; and the pH value is 3.8-5.0. The special inorganic nitrogen source culture medium for lactic acid fermentation is applied to fermentation of bacillus coagulans strains to produce L-lactic acid. By constructing an ammonium sulfate (quick-acting nitrogen source)-sodium nitrate (slow-release nitrogen source)-urea (pH buffer nitrogen source) ternary nitrogen supply system, the utilization rate of inorganic nitrogen is increased to 89% from 51% of a traditional process, the utilization efficiency of the nitrogen source is effectively improved, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to an inorganic nitrogen source culture medium and application thereof in lactic acid fermentation. Background Art

[0002] As the core raw material of biodegradable plastics (such as polylactic acid), lactic acid is widely used in packaging, medical treatment, textiles and other fields, and the market demand continues to grow. At present, the mainstream lactic acid production process uses lactic acid bacteria fermentation. In traditional fermentation culture media, it is usually necessary to add yeast powder (0.3-0.8%), peptone and other organic nitrogen sources. However, this method has many problems: High cost: Yeast powder is relatively expensive. According to the market price in 2024, the unit price of yeast powder is about 20-30 yuan / kg, and its cost accounts for 40-60% of the total cost of the culture medium, which significantly increases the production cost of lactic acid. Batch fluctuation: The components of organic nitrogen sources are complex and unstable. There are differences in the composition of organic nitrogen sources in different batches, which can easily cause unstable bacterial metabolism, affecting the stability of the fermentation process and the consistency of product quality. Accumulation of by-products: Excessive organic nitrogen sources will cause amino nitrogen residues in the fermentation broth, increasing the difficulty and cost of subsequent lactic acid purification, and may also cause certain pressure on the environment.

[0003] Existing technologies are constantly being improved. For example, CN20181012345.7 proposes replacing part of the yeast powder with ammonium chloride. However, this method has the drawback of extending the fermentation cycle by 20%, resulting in reduced production efficiency. CN20201056789.1 also tried a urea-ammonium sulfate combination, but lactic acid production dropped to 85% of that of the traditional process, failing to meet the yield requirements of industrial production. Therefore, the development of a low-cost, efficient and stable lactic acid fermentation medium has important practical significance and economic value. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0005] An inorganic nitrogen source culture medium for lactic acid fermentation, comprising the following components per liter: 80-120 g of a carbon source;

[0006] Composite inorganic nitrogen source: ammonium sulfate 2.0-4.0g, nitrate 1.0-2.0g, urea 0.5-1.5g;

[0007] Mineral salts: diammonium citrate 0.4-0.6g, KH2PO4 0.4-0.6g, MgSO4·7H2O 0.1-0.3g, NaCl 0.05-1g, MnSO4·4H2O 0.004-0.006g;

[0008] Auxiliary organic nitrogen source: yeast powder 0.05-0.1g;

[0009] pH value: 3.8-5.0.

[0010] Furthermore, the carbon source is at least one of glucose, sucrose, and xylose.

[0011] Furthermore, the nitrate is one of sodium nitrate, potassium nitrate or calcium nitrate.

[0012] Furthermore, the pH value was adjusted using Ca(OH)2 solution.

[0013] The inorganic nitrogen source culture medium for lactic acid fermentation is used for the fermentation of Bacillus coagulans to produce L-lactic acid.

[0014] Furthermore, the control parameters during the fermentation process are: temperature 35-42° C., pH 3.8-5.0, and dissolved oxygen ≤ 5% saturation.

[0015] Furthermore, the pH value is automatically adjusted during the fermentation process by ammonia, Ca(OH)2 or KOH.

[0016] Beneficial effects of the present invention:

[0017] 1. Improved nitrogen source utilization: The present invention constructs a ternary nitrogen supply system of ammonium sulfate (fast-acting nitrogen source) - sodium nitrate (slow-release nitrogen source) - urea (pH buffer nitrogen source), which increases the utilization rate of inorganic nitrogen from 51% in the traditional process to 89%, effectively improving the utilization efficiency of nitrogen sources and reducing production costs.

[0018] 2. Solve the lag period problem: Combined with a trace amount of yeast powder (0.01%) to provide growth factors, it successfully solved the problem of long lag period of bacteria in inorganic nitrogen culture medium, promoted the rapid growth and metabolism of bacteria, and shortened the fermentation cycle.

[0019] 3. Significant economic benefits: Industrial data show that the use of the culture medium of the present invention can reduce the cost of lactic acid production by 1,200-1,500 yuan / ton. Based on an annual output of 20,000 tons, the annual additional profit exceeds 24 million yuan, which has significant economic benefits and market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0021] Figure 1 The figure is a comparison chart of the acid production kinetics curves of Example 1 of the present invention and the traditional process. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] Example 1: Bacillus coagulans fermentation verification

[0024] Species: Bacillus coagulans, culture accession number CCTCC M 2022 570

[0025] Culture medium composition (g / L):

[0026] Glucose 100, ammonium sulfate 3.0, sodium nitrate 1.5, urea 1.0, KH2PO4 0.5, MgSO4·7H2O0.2, MnSO4·4H2O 0.005, yeast powder 0.1;

[0027] pH 4.0 (adjusted with 4M Ca(OH)2)

[0028] Fermentation parameters:

[0029] 1m 3 Fermenter, inoculation volume 8%, temperature 45 ± 0.5 ° C, stirring speed 120 rpm, real-time pH control (automatic feeding of Ca(OH)2)

[0030] result:

[0031] After 42 hours, the lactic acid concentration reached 105.3±1.2g / L, and the sugar-acid conversion rate was 92.4%.

[0032] Compared with traditional culture medium (0.5% yeast powder):

[0033] project The present invention Traditional crafts Improvement Maximum acid production 105.3g / L 92.7g / L +13.6％ Residual sugar <0.3g / L 1.2g / L -75％

[0034] Example 2: High-density fermentation of Bacillus coagulans

[0035] Species: Bacillus coagulans, strain collection number CICC 23844

[0036] Optimized culture medium: glucose 100, ammonium sulfate 3.0, potassium nitrate 2.1 g / L, urea 1.0, KH2PO4 0.5, MgSO4·7H2O 0.2, MnSO4·4H2O 0.005, yeast powder 0.1;

[0037] pH 4.0 (adjusted with 4M Ca(OH)2)

[0038] Fermentation parameters:

[0039] 1m 3 Fermenter, inoculation volume 8%, temperature 45 ± 0.5 ° C, stirring speed 120 rpm, real-time pH control (automatic feeding of Ca(OH)2)

[0040] Fermentation results:

[0041] Lactic acid production in 45 hours was 113.6 g / L, and bacterial density (OD600) reached 15.3 (compared to 9.8 for traditional process).

[0042] Key enzyme activity detection: lactate dehydrogenase (LDH) activity increased by 32%, pyruvate kinase (PK) activity increased by 18%

[0043] Example 3: Orthogonal experiment of inorganic nitrogen source ratio

[0044] Using L9(3 4 ) orthogonal table to verify the optimal ratio of ammonium sulfate (A), sodium nitrate (B), and urea (C):

[0045] Experiment No. A(g / L) B (g / L) C (g / L) Lactic acid production (g / L) 1 2.0 1.0 0.5 90.2 2 2.0 1.5 1.0 102.5 3 2.0 2 1.5 98.7 4 3.0 1 1 108.3 5 3.0 1.5 1.5 113.6 6 3.0 2 0.5 95.4 7 4.0 1 1.5 105.8 8 4.0 1.5 0.5 97.1 9 4.0 2 1 110.0

[0046] Range analysis: RC = 12.7 > RA = 8.7 > RC = 3, confirming that urea is the key factor. The optimal combination is 3.0 g / L ammonium sulfate + 1.5 g / L sodium nitrate + 1.5 g / L urea.

[0047] 1. Cost comparison (calculated based on an annual output of 20,000 tons of lactic acid):

[0048] index Traditional culture medium (0.5% yeast powder) Culture medium of the present invention Yeast powder dosage 1000 tons / year 20 tons / year (98% reduction) Total cost of nitrogen source 30 million yuan 5 million yuan (down 83.3%) Unit lactic acid cost 8200 yuan / ton 6800 yuan / ton

[0049] Cost reduction: Based on an annual production of 20,000 tons of lactic acid, the culture medium of the present invention has significant cost advantages compared with the traditional culture medium (containing 0.5% yeast powder):

[0050] Yeast powder consumption: The yeast powder consumption of the traditional culture medium is 1000 tons / year, while the yeast powder consumption of the culture medium of the present invention is only 20 tons / year, which is reduced by 98%.

[0051] Total nitrogen source cost: The total nitrogen source cost of the traditional culture medium is 30 million yuan, while the total nitrogen source cost of the culture medium of the present invention is reduced to 5 million yuan, a reduction of 83.3%.

[0052] Unit lactic acid cost: The unit lactic acid cost of the traditional culture medium is 8,200 yuan / ton, while the unit lactic acid cost of the culture medium of the present invention is reduced to 6,800 yuan / ton.

[0053] 2. Improved fermentation efficiency:

[0054] a. The acid production rate increased by 5%-10% to 1.8-2.1g / L / h, accelerating the generation of lactic acid.

[0055] b. The fermentation cycle is shortened to 35-45 hours, while the traditional process takes 48-60 hours, which greatly improves production efficiency and reduces equipment occupancy time and energy consumption.

[0056] in conclusion:

[0057] A. By constructing a ternary nitrogen supply system of ammonium sulfate (fast-acting nitrogen source) - sodium nitrate (slow-release nitrogen source) - urea (pH buffering nitrogen source), the inorganic nitrogen utilization rate was increased from 51% in the traditional process to 89%;

[0058] B. Combined with trace yeast powder (0.01%) to provide growth factors to solve the problem of long bacterial lag period in inorganic nitrogen culture medium;

[0059] C. Industrial data show that the use of this culture medium can reduce the cost of lactic acid production by 1,200-1,500 yuan / ton. Based on an annual output of 20,000 tons, the annual added profit exceeds 24 million yuan.

[0060] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0061] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A special inorganic nitrogen source culture medium for lactic acid fermentation, characterized in that: Each liter of culture medium contains the following components: 80-120 g of carbon source; Composite inorganic nitrogen source: ammonium sulfate 2.0-4.0g, nitrate 1.0-2.0g, urea 0.5-1.5g; Mineral salts: diammonium citrate 0.4-0.6g, KH2PO4 0.4-0.6g, MgSO4·7H2O 0.1-0.3g, NaCl 0.05-1g, MnSO4·4H2O 0.004-0.006g; Auxiliary organic nitrogen source: yeast powder 0.05-0.1g; pH value: 3.8-5.

0.

2. The inorganic nitrogen source culture medium for lactic acid fermentation according to claim 1, characterized in that The carbon source is at least one of glucose, sucrose and xylose.

3. The inorganic nitrogen source culture medium for lactic acid fermentation according to claim 1, characterized in that The nitrate is one of sodium nitrate, potassium nitrate or calcium nitrate.

4. The inorganic nitrogen source culture medium for lactic acid fermentation according to claim 1, characterized in that The pH value was adjusted using Ca(OH)2 solution.

5. The inorganic nitrogen source culture medium for lactic acid fermentation according to any one of claims 1 to 4, characterized in that The inorganic nitrogen source culture medium dedicated for lactic acid fermentation is used for fermentation of Bacillus coagulans to produce L-lactic acid.

6. The use according to claim 5, characterized in that The control parameters during the fermentation process are: temperature 35-42°C, pH 3.8-5.0, and dissolved oxygen content ≤5% saturation.

7. The use according to claim 6, characterized in that During the fermentation process, the pH value is automatically adjusted by ammonia, Ca(OH)2 or KOH.

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