Culture medium and application thereof in culture of genetically modified lactococcus lactis

Abstract

The invention provides a culture medium and application thereof in culture of genetically modified lactococcus lactis. The culture medium comprises a carbon source, amino acid, nucleotide, inorganic salt and vitamin, the carbon source comprises glucose; the amino acids comprise alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; the nucleotide comprises adenine, guanine, uracil or xanthine; the inorganic salt comprises sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, monopotassium phosphate, dipotassium phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate and copper sulfate. The method can solve the problem of unstable fermentation culture of lactococcus lactis in the prior art, and is suitable for the field of lactococcus lactis culture.

Description

Technical Field

[0001] The present invention relates to the field of Lactococcus lactis culture, in particular to a culture medium and application thereof in the culture of genetically modified Lactococcus lactis. Background Art

[0002] Lactococcus lactis is widely favored in the food, biotechnology, and pharmaceutical industries for its excellent fermentation capacity and safety. In recent years, with the rapid development of genetic engineering technology, Lactococcus lactis has been genetically modified to have more functions, such as producing specific metabolites, expressing heterologous proteins, and serving as a vaccine carrier, greatly expanding its application range. However, genetic modification of Lactococcus lactis is often accompanied by changes in its nutritional requirements, which to some extent poses new challenges to traditional culture media.

[0003] Commonly used culture media for Lactococcus lactis, such as GM17 medium, are semi-synthetic media that combine ingredients from natural organic matter and chemical reagents to meet the basic nutritional needs of Lactococcus lactis. However, the composition of semi-synthetic culture media is complex and not fully defined, which not only leads to poor consistency and reproducibility between different batches of culture media, but also the volatility of natural ingredients may affect the growth and metabolic stability of microorganisms. In addition, due to the presence of complex organic matter, such as peptone and yeast extract, this culture medium is difficult to precisely control the type and concentration of nutrients, which may not provide an optimal growth environment for genetically modified Lactococcus lactis.

[0004] Genetically modified Lactococcus lactis may have higher requirements for certain nutrients or be more sensitive to the concentrations of certain ingredients, requiring culture media that can precisely meet these specific conditions. The imprecision of semi-synthetic culture media has led to a growing demand for fully synthetic culture media (Chemically Defined Media). Fully synthetic culture media are composed of known pure chemical components and can provide a highly controllable growth environment, avoiding the uncertainty and instability brought by natural ingredients. They are particularly suitable for precise nutritional regulation and large-scale production of genetically modified microorganisms.

[0005] The development of a fully synthetic culture medium suitable for genetically modified Lactococcus lactis can not only solve the shortcomings of semi-synthetic culture media in terms of consistency, precise regulation and production costs, but also promote the application of Lactococcus lactis in biopharmaceuticals, food additives, biocatalysts and other fields. It has important scientific research value and industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0007] Figure 1 Statistical graphs of glucose utilization in culture media 1 to 4 during the fermentation process according to Example 2 of the present application are shown. Summary of the Invention

[0008] The main purpose of the present invention is to provide a culture medium and its application in the cultivation of genetically modified Lactococcus lactis, so as to solve the problem of unstable fermentation culture of Lactococcus lactis in the prior art.

[0009] To achieve the above-mentioned object, according to a first aspect of the present invention, a culture medium is provided, which comprises a carbon source, amino acids, nucleotides, inorganic salts and vitamins; the carbon source comprises glucose; the amino acids comprise one or more of alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; the nucleotides comprise one or more of adenine, guanine, uracil or xanthine; the inorganic salts comprise one or more of sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate or copper sulfate; wherein, by weight, the glucose is present in an amount of 10 to 30 parts.

[0010] Furthermore, the vitamins include one or more of biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride or calcium pantothenate;

[0011] Preferably, the culture medium comprises: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate;

[0012] Preferably, the culture medium comprises: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, cysteine hydrochloride, glycine, asparagine, glutamic acid, proline, tyrosine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate;

[0013] Preferably, the culture medium comprises glucose, alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride, and calcium pantothenate.

[0014] Furthermore, the culture medium comprises, by weight, 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of arginine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, 0.001-1.0 parts of magnesium sulfate heptahydrate, 0.01-2.0 parts of potassium dihydrogen phosphate, and 0.01-2.0 parts of dihydrogen phosphate. 0.01-2.0 parts of potassium, 0.001-1.0 parts of cobalt chloride, 0.001-1.0 parts of manganese sulfate, 0.001-1.0 parts of calcium chloride, 0.001-1.0 parts of ferrous sulfate, 0.001-1.0 parts of copper sulfate, 0.001-1.0 parts of adenine, 0.001-1.0 parts of guanine, 0.001-1.0 parts of uracil, 0.001-1.0 parts of xanthine, 0.001-1.0 parts of biotin, 0.001-1.0 parts of niacin, 0.001-1.0 parts of pyridoxal hydrochloride, 0.001-1.0 parts of riboflavin, 0.001-1.0 parts of folic acid, 0.001-1.0 parts of thiamine hydrochloride, and 0.001-1.0 parts of calcium pantothenate;

[0015] Preferably, the culture medium comprises, by weight: 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, 0.01-1.0 parts of histidine. 0.01-1.0 parts, arginine 0.01-1.0 parts, cysteine hydrochloride 0.01-1.0 parts, glycine 0.01-1.0 parts, asparagine 0.01-1.0 parts, glutamic acid 0.01-1.0 parts, proline 0.01-1.0 parts, tyrosine 0.01-1.0 parts, sodium chloride 0.01-2.0 parts, potassium acetate 0.1-5.0 parts, zinc sulfate heptahydrate 0.001 -1.0 part, magnesium sulfate heptahydrate 0.001-1.0 part, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 part, manganese sulfate 0.001-1.0 part, calcium chloride 0.001-1.0 part, ferrous sulfate 0.001-1.0 part, copper sulfate 0.001-1.0 part, adenine 0.001-1.0 part, guanine 0.001-1.0 part of urea, 0.001-1.0 part of uracil, 0.001-1.0 part of xanthine, 0.001-1.0 part of biotin, 0.001-1.0 part of niacin, 0.001-1.0 part of pyridoxal hydrochloride, 0.001-1.0 part of riboflavin, 0.001-1.0 part of folic acid, 0.001-1.0 part of thiamine hydrochloride and 0.001-1.0 part of calcium pantothenate;

[0016] Preferably, the culture medium comprises, by weight, 10-30 parts of glucose, 0.01-1.0 parts of alanine, 0.01-1.0 parts of arginine, 0.01-1.0 parts of aspartic acid, 0.01-1.0 parts of cysteine hydrochloride, 0.01-1.0 parts of glutamic acid, 0.01-1.0 parts of glycine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of serine, 0.01-1.0 parts of threonine, 0.01-1.0 parts of tyrosine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, and 0.001-1.0 parts of magnesium sulfate heptahydrate. 1-2 parts, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 parts parts, uracil 0.001-1.0 parts, xanthine 0.001-1.0 parts, biotin 0.001-1.0 parts, niacin 0.001-1.0 parts, pyridoxal hydrochloride 0.001-1.0 parts, riboflavin 0.001-1.0 parts, folic acid 0.001-1.0 parts, thiamine hydrochloride 0.001-1.0 parts and calcium pantothenate 0.001-1.0 parts.

[0017] Furthermore, the culture medium further comprises a surfactant; preferably, the surfactant comprises one or more of Tween 80, Tween 60 or PEG6000, more preferably Tween 80; preferably, the surfactant is 0.1-20 parts by weight.

[0018] To achieve the above-mentioned object, according to a second aspect of the present invention, there is provided a use of the above-mentioned culture medium in culturing Lactococcus lactis or genetically modified Lactococcus lactis, the use comprising: activating target Lactococcus lactis and then inoculating the activated target Lactococcus lactis into the above-mentioned culture medium for fermentation to obtain a fermentation broth containing the target Lactococcus lactis; the target Lactococcus lactis includes genetically modified Lactococcus lactis; the genetically modified Lactococcus lactis is a strain in which bases in the genes of Lactococcus lactis have been deleted, added, or replaced using gene editing technology.

[0019] Furthermore, the pH of the fermentation culture is 6.2 to 6.6; preferably, the pH of the fermentation culture is 6.5.

[0020] Furthermore, the fermentation temperature is 28-35° C.; preferably, the fermentation time is 8 to 13 hours; preferably, the fermentation is anaerobic fermentation.

[0021] Furthermore, the number of viable Lactococcus lactis in the fermentation broth is ≥ 0.8 × 10 10 cfu / mL.

[0022] In order to achieve the above object, according to a second aspect of the present invention, there is provided a use of the above culture medium in preparing a Lactococcus lactis product.

[0023] Furthermore, the viable count of Lactococcus lactis in the Lactococcus lactis product is ≥ 6.0×10 11 cfu / g; preferably, the Lactococcus lactis product includes Lactococcus lactis dry powder, probiotic fermentation agent or probiotic preparation.

[0024] By applying the technical solution of the present invention, Lactococcus lactis is fermented and cultured using a fully synthetic culture medium for Lactococcus lactis containing a carbon source, amino acids, nucleotides, inorganic salts, and vitamins. This culture medium contains all necessary nutrients and can remain stable in multiple batches of fermentation tests, ensuring high consistency and repeatability under test conditions. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0026] Explanation of terms:

[0027] Chemically Defined Media: refers to a culture medium that is precisely designed and artificially prepared from pure chemical substances of known composition based on the types and quantities of nutrients required by the target culture.

[0028] As mentioned in the background art, the GM17 medium used in the prior art for fermentation and cultivation of Lactococcus lactis has limitations due to its semi-synthetic culture medium, which affects the stability and repeatability of Lactococcus lactis fermentation and cultivation, hindering the production and development of Lactococcus lactis products. Based on this, the inventors of this application attempt to provide a culture medium for Lactococcus lactis that overcomes the shortcomings of the aforementioned fermentation medium, making Lactococcus lactis more stable during fermentation and cultivation, and thus propose a series of protection schemes of this application.

[0029] In a first typical embodiment of the present application, a culture medium is provided, which comprises a carbon source, amino acids, nucleotides, inorganic salts and vitamins; the carbon source comprises glucose; the amino acids comprise one or more of alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; the nucleotides comprise one or more of adenine, guanine, uracil or xanthine; and the inorganic salt comprises one or more of glutamine, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Organic salts include one or more of sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, and copper sulfate; vitamins include biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride, and calcium pantothenate; wherein, by weight, glucose is 10-30 parts (including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 parts).

[0030] GM17 medium, a semi-synthetic medium commonly used for Lactococcus lactis, combines the advantages of natural organic compounds and chemical reagents. While these media incorporate the characteristics of natural organic compounds (such as peptone and yeast extract) and chemical reagents, they suffer from complex and difficult-to-determine compositions, leading to significant variability between batches and impacting the stability and reproducibility of experimental results.

[0031] The presence of natural ingredients in GM17 medium makes precise control of its nutritional profile difficult. The content and types of components such as proteins and polysaccharides vary depending on their source, which can affect the growth and metabolism of microorganisms or cells. Furthermore, microbial growth rates and yields in semi-synthetic media may be lower than those in fully synthetic media. The complexity of natural ingredients can lead to deficiencies or excesses of certain nutrients, limiting the growth potential of microorganisms.

[0032] Moreover, the preparation process of GM17 culture medium is relatively cumbersome, requiring pretreatment of natural ingredients and strict quality control to ensure safety and reliability, which undoubtedly increases the complexity and cost of the operation. The pretreatment step is not only time-consuming, but also requires additional resources to ensure that contaminants in the raw materials are effectively removed, while quality control is to maintain the consistency of the culture medium and avoid performance differences caused by different batches of raw materials. These directly or indirectly increase the cost of Lactococcus lactis fermentation production. In addition, since GM17 culture medium relies on animal-derived ingredients (such as beef extract, yeast extract, etc.), it has the risk of virus transmission. Although this risk is controllable under the strict monitoring of modern biotechnology, its potential impact on food safety and biopharmaceutical compliance cannot be ignored, which increases safety concerns in industrial production.

[0033] In contrast, the culture medium of this application is a fully synthetic culture medium (Chemically Defined Media) with completely clear ingredients. It replaces the peptone, soy peptone, casein peptone, yeast extract powder and beef extract powder in GM17 culture medium, covers the essential nutrients for Lactococcus lactis, helps technicians to control the culture environment more precisely, avoids the instability of traditional culture media, and ensures high consistency and repeatability of experimental conditions.

[0034] The present application selects glucose as the type of the sole carbon source of the present application because all Lactococcus lactis strains can efficiently utilize glucose (directly metabolized through the EMP pathway), a single carbon source reduces variables, facilitates the optimization of fermentation conditions (such as pH), and has the advantages of fast metabolic rate and simple regulation. When the carbon source is controlled within the above-mentioned range of portions, it can promote the growth of Lactococcus lactis, and the utilization rate of glucose is also maintained at a high level, avoiding the waste of resources and increase in culture costs caused by the increase of excessive carbon source.

[0035] The present application selects alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine as amino acids in the culture medium of the present application. These amino acids can replace the free amino acids and small peptides provided by complex nitrogen sources (such as peptone, yeast extract, tryptone, etc.) in semi-synthetic culture media. The amino acid composition of peptone / yeast extract is unclear and the batch differences are large. The fully synthetic culture medium eliminates the uncertainty in the fermentation process of Lactococcus lactis through precise proportioning.

[0036] This application selects adenine, guanine, uracil, or xanthine as nucleotides in the culture medium of this application. The reason is: mainly to replace the free nucleotides, nucleic acid derivatives, and their precursors provided by the complex components of semi-synthetic culture medium (such as yeast extract and peptone). Yeast extract contains nucleic acids (RNA / DNA), nucleotides (ATP, NAD+), and their degradation products (such as hypoxanthine), but the concentration and composition are unstable. Fully synthetic culture medium eliminates batch variability by explicitly adding nucleotides.

[0037] The present application selects sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, and copper sulfate as the inorganic salts of the present application. The reason is to meet the basic inorganic element requirements for the growth and metabolism of Lactococcus lactis while maintaining osmotic pressure, pH balance and enzyme activity.

[0038] The culture medium of the present application does not rely on animal-derived components, reduces the risk of viral contamination, ensures the safety of the culture process, and the clear composition reduces impurity interference, makes experimental data more reliable, and makes batch experiments more stable. In addition, the Lactococcus lactis culture medium of the present application can provide accurate nutritional support for the growth and metabolism of Lactococcus lactis, and when applied to the culture of Lactococcus lactis, significantly improves the fermentation efficiency of strains, and has low raw material cost, making it more suitable for large-scale industrial production.

[0039] In a preferred embodiment, the culture medium includes vitamins including one or more of biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride or calcium pantothenate; preferably, the culture medium includes: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate; preferably, the culture medium includes: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, cysteine hydrochloride, glycine, asparagine, glutamic acid, proline, tyrosine, chloride Preferably, the culture medium comprises glucose, alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, glutamine ... amino acid, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, cupric sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride, and calcium pantothenate.

[0040] Through a large amount of research and screening by the present inventors, it is found that the growth of Lactococcus lactis thalline shows significant dependence on valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, and arginine, and these amino acids are the key essential amino acids that limit thalline growth. The shortage of cysteine hydrochloride, glycine, asparagine, glutamic acid, proline, and tyrosine can significantly reduce thalline quantity, but it is not an absolutely essential amino acid for thalline growth, indicating that the thalline of Lactococcus lactis may possess certain compensatory metabolic pathways for these amino acids. Alanine, aspartic acid, glutamine, and serine are relatively weak to the promoting effect of thalline growth, and their impact may depend on the metabolic characteristics of specific culture conditions or bacterial strains. Therefore, in the process of configuring the culture medium of Lactococcus lactis, except the above-mentioned essential amino acids, the remaining amino acids can be flexibly adjusted and selected according to actual production and thalline characteristics.

[0041] In a preferred embodiment, the culture medium comprises, by weight: 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of arginine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, 0.001-1.0 parts of magnesium sulfate heptahydrate, 0.01-2.0 parts of potassium dihydrogen phosphate. 1-1.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 parts, uracil 0.001-1.0 parts, xanthine 0.001-1.0 parts, biotin 0.001-1.0 parts, niacin 0.001-1.0 parts, pyridoxal hydrochloride 0.001-1.0 parts, riboflavin 0.001-1.0 parts, folic acid 0.001-1.0 parts, thiamine hydrochloride 0.001-1.0 parts and calcium pantothenate 0.001-1.0 parts;

[0042] Preferably, the culture medium comprises, by weight: 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, 0.01-1.0 parts of histidine. 0.01-1.0 parts, arginine 0.01-1.0 parts, cysteine hydrochloride 0.01-1.0 parts, glycine 0.01-1.0 parts, asparagine 0.01-1.0 parts, glutamic acid 0.01-1.0 parts, proline 0.01-1.0 parts, tyrosine 0.01-1.0 parts, sodium chloride 0.01-2.0 parts, potassium acetate 0.1-5.0 parts, zinc sulfate heptahydrate 0.001 -1.0 part, magnesium sulfate heptahydrate 0.001-1.0 part, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 part, manganese sulfate 0.001-1.0 part, calcium chloride 0.001-1.0 part, ferrous sulfate 0.001-1.0 part, copper sulfate 0.001-1.0 part, adenine 0.001-1.0 part, guanine 0.001-1.0 part of urea, 0.001-1.0 part of uracil, 0.001-1.0 part of xanthine, 0.001-1.0 part of biotin, 0.001-1.0 part of niacin, 0.001-1.0 part of pyridoxal hydrochloride, 0.001-1.0 part of riboflavin, 0.001-1.0 part of folic acid, 0.001-1.0 part of thiamine hydrochloride and 0.001-1.0 part of calcium pantothenate;

[0043] Preferably, the culture medium comprises, by weight, 10-30 parts of glucose, 0.01-1.0 parts of alanine, 0.01-1.0 parts of arginine, 0.01-1.0 parts of aspartic acid, 0.01-1.0 parts of cysteine hydrochloride, 0.01-1.0 parts of glutamic acid, 0.01-1.0 parts of glycine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of serine, 0.01-1.0 parts of threonine, 0.01-1.0 parts of tyrosine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, and 0.001-1.0 parts of magnesium sulfate heptahydrate. 1-2 parts, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 parts parts, uracil 0.001-1.0 parts, xanthine 0.001-1.0 parts, biotin 0.001-1.0 parts, niacin 0.001-1.0 parts, pyridoxal hydrochloride 0.001-1.0 parts, riboflavin 0.001-1.0 parts, folic acid 0.001-1.0 parts, thiamine hydrochloride 0.001-1.0 parts and calcium pantothenate 0.001-1.0 parts.

[0044] The present application achieves precise control of the types of various nutrients in the culture medium and the precise ratio of the various nutrients within the above-mentioned range, thereby obtaining a culture medium capable of providing precise nutritional support for the growth and metabolism of Lactococcus lactis. This is beneficial for the full growth and reproduction of Lactococcus lactis during the fermentation and cultivation process, significantly improving the fermentation efficiency, and maintaining the viable cell count of the fermentation liquid finally obtained at a high level, which is beneficial to the development of the Lactococcus lactis industry.

[0045] In a preferred embodiment, the culture medium further comprises a surfactant; preferably, the surfactant comprises one or more of Tween 80, Tween 60 or PEG6000, more preferably Tween 80; preferably, the surfactant is 0.1-20 parts by weight.

[0046] The surfactant is used to reduce the surface tension of the culture medium, making the nutrients in the culture medium more evenly distributed, promoting the growth of microorganisms, and increasing the growth density of the bacteria. The present application controls the surfactant within the above-mentioned weight ratio to promote high-density fermentation of Lactococcus lactis, which is beneficial to the growth and reproduction of Lactococcus lactis in the present application.

[0047] In a second typical embodiment of the present application, there is provided a use of the above-mentioned culture medium in the cultivation of Lactococcus lactis or genetically modified Lactococcus lactis, the use comprising: activating the target Lactococcus lactis and then inoculating the target Lactococcus lactis into the above-mentioned culture medium for fermentation to obtain a fermentation broth containing the target Lactococcus lactis.

[0048] The genetically modified Lactococcus lactis strains are strains in which nucleotides in the genes of Lactococcus lactis have been deleted, added, or replaced using gene editing techniques. Such gene editing techniques include, but are not limited to, CRISPR / Cas9 and homologous recombination, i.e., modifications to the genome of Lactococcus lactis. Such modifications include, but are not limited to, modifications to genes encoding specific metabolites, genes encoding therapeutic proteins, and genes involved in metabolic pathways or signal transduction pathways.

[0049] The culture medium of the present application can be a universal, fully synthetic culture medium capable of culturing various metabolic types of Lactococcus lactis. The target Lactococcus lactis herein includes, but is not limited to, commercially available genetically modified Lactococcus lactis, such as CICC 20400, CICC 24337, or ATCC 19257, or genetically modified Lactococcus lactis obtained by gene editing by those skilled in the art, and is not specifically limited thereto.

[0050] In a preferred embodiment, the pH of the fermentation culture is 6.2-6.6; preferably, the pH of the fermentation culture is 6.5.

[0051] In a preferred embodiment, the fermentation temperature is 28-35°C (including but not limited to 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C). Preferably, the fermentation is anaerobic fermentation. Preferably, the rotation speed of the fermentation equipment is 100-200 rpm (including but not limited to 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 rpm).

[0052] In a preferred embodiment, the number of viable Lactococcus lactis in the fermentation broth is ≥ 0.8×10 10 cfu / mL.

[0053] By precisely controlling the fermentation culture conditions within the above range, the present application can enable the lactococcus lactis culture medium of the present application to function and cooperate in the process of culturing genetically modified lactococcus lactis or conventional lactococcus lactis to obtain excellent fermentation effects, promote high-density fermentation of lactococcus lactis, improve fermentation efficiency, and increase the number of viable bacteria in the final fermentation liquid. Compared with the system for lactococcus lactis fermentation culture based on GM17 culture medium, the raw material composition of the culture medium of the present application is clear and stable, and it can stably ferment and culture lactococcus lactis of various metabolic types and improve fermentation efficiency by precisely controlling the fermentation culture conditions. The number of viable bacteria in the final fermentation liquid is ≥0.8×10 10 cfu / mL, which is significantly higher than the viable bacterial count of the fermentation broth of the GM17 culture system. The fermentation culture method of the present application reduces the overall fermentation culture cost of Lactococcus lactis and is more suitable for promotion to industrial production.

[0054] The fermentation culture method of the present application comprises: activating the target Lactococcus lactis, inoculating the activated bacteria into the above-mentioned Lactococcus lactis culture medium (pH 6.5, sterilized at 121°C for 15 minutes), and performing anaerobic culture to obtain a Lactococcus lactis seed liquid; inoculating the Lactococcus lactis seed liquid into the above-mentioned Lactococcus lactis culture medium, controlling the above-mentioned fermentation conditions, and performing fermentation culture for 8 to 13 hours. When the alkali consumption decreases, the fermentation is terminated to obtain a high-density fermentation liquid containing Lactococcus lactis.

[0055] During the fermentation process of the present application, the pH can be controlled by adding a neutralizer, including but not limited to aqueous solutions of inorganic bases such as NaOH, KOH, Na2CO3 or ammonia solution. The anaerobic conditions of the present application can be maintained by bubbling nitrogen (0.02-0.05 vvm).

[0056] In a third typical embodiment of the present application, a use of the above-mentioned Lactococcus lactis culture medium in preparing a Lactococcus lactis product is provided.

[0057] In a preferred embodiment, the viable cell count of Lactococcus lactis in the Lactococcus lactis product is ≥ 6.0×10 11 cfu / g; preferably, the Lactococcus lactis product includes Lactococcus lactis dry powder, probiotic fermentation agent or probiotic preparation.

[0058] The above application includes preparing Lactococcus lactis dry powder using the high-density fermentation broth obtained by the above-mentioned Lactococcus lactis culture method, and the preparation method includes:

[0059] a. Bacteria concentration: The high-density fermentation broth is centrifuged at 8000-12000g to concentrate the bacteria;

[0060] b. Add protective agent: Add 1 to 4 times the protective agent solution to the bacterial concentrate and freeze-dry;

[0061] The protective agent solution comprises, but is not limited to, sucrose, lysine, arginine, polydextrose, maltodextrin and 100 mL of distilled water.

[0062] The total number of viable bacteria in the freeze-dried Lactococcus lactis powder can reach 6.0×10 11 cfu / g or above, and can maintain a relatively high level, and can be used in the preparation of probiotic fermentation agents or probiotic preparations.

[0063] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0064] Unless otherwise specified, the reagents in the examples of this application are all conventional commercially available products.

[0065] Example 1

[0066] Preparation of fully synthetic culture medium: glucose (5 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.1 part zinc sulfate heptahydrate), 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), The mixture comprises 0.5 parts of magnesium sulfate, 1.0 parts of potassium dihydrogen phosphate, 2.0 parts of potassium hydrogen phosphate, 0.001 parts of cobalt chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0067] Preparation of GM17 medium: 2.5 g peptone, 2.5 g casein peptone, 5 g soytone, 5 g beef extract powder, 2.5 g yeast extract powder, 0.5 g ascorbic acid, 0.25 g magnesium sulfate, 19 g sodium β-glycerophosphate pentahydrate, 5 g glucose, and 1000 mL distilled water.

[0068] Culture conditions:

[0069] a. Activation of the strain: Glycerol bacteria (ATCC 19257, manufacturer: Microbiologics; agent: Shanghai Baolu Biological; catalog number: 0152P) stored at -80°C were inoculated into GM17 liquid culture medium sterilized at 121°C for 15 minutes and incubated anaerobically at 30°C for 16 hours. This process was repeated twice to obtain an activated strain.

[0070] b. Culture medium preparation: Mix the prepared synthetic medium and GM17 medium, adjust the pH to 6.8, and sterilize at 121°C for 15 minutes.

[0071] c. Preparation of seed solution: Take the activated bacteria in step a and inoculate them into the culture medium prepared in b. Anaerobic culture was stopped at 30°C for 9 hours;

[0072] d. Inoculation and culture: The seed solution of step c was inoculated into the culture medium prepared in b and fermented under controlled fermentation conditions for 9h;

[0073] d. Fermentation conditions are controlled as follows: constant temperature cultivation at 30°C, rotation speed at 200 rpm, natural fermentation, and anaerobic fermentation. The anaerobic conditions can be nitrogen-filled at 0.02-0.05 vvm.

[0074] e. Terminate the fermentation when OD600 remains unchanged, obtain the fermentation broth of the strain, and detect the number of viable bacteria in the fermentation broth.

[0075] At the end of fermentation, the OD600 of the fermentation broth of the synthetic medium was 1.9, and the viable bacterial density reached 2.2×10 9 cfu / mL; GM17 culture broth OD600 1.7, viable bacterial density reached 2.0×10 9 cfu / mL.

[0076] Testing the OD600 of the fermentation broth can indirectly reflect the concentration of bacteria in the fermentation broth. The higher the OD600 value, the more bacterial cells there are in the fermentation broth.

[0077] Example 2

[0078] The target Lactococcus lactis strain of this example is the same as that of Example 1.

[0079] Medium 1: glucose (5 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0080] Medium 2: glucose (10 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0081] Medium 3: glucose (20 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0082] Medium 4: glucose (30 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0083] Medium 5: glucose (40 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0084] Culture conditions: a. Activation of the strain: Inoculate glycerol stock stored at -80°C into GM17 liquid culture medium sterilized at 121°C for 15 minutes, incubate anaerobically at 30°C for 16 hours, and subculture twice to obtain the activated strain.

[0085] b. Preparation of culture medium: Mix the prepared fully synthetic culture medium, adjust the pH to 6.8, and sterilize at 121°C for 15 minutes;

[0086] c. Preparation of seed solution: The activated bacteria in step a were inoculated into the medium prepared in step b and incubated anaerobically at 30°C for 9 hours; d. Inoculation and culture: The seed solution from step c was inoculated into the medium prepared in step b and fermented under controlled fermentation conditions for 9 hours;

[0087] d. Fermentation conditions were controlled as follows: constant temperature cultivation at 30°C, rotation speed at 200 rpm, and no pH control of the fermentation system during the fermentation process, maintaining anaerobic fermentation. The anaerobic conditions may include nitrogen flow at 0.02-0.05 vvm.

[0088] e. Terminate the fermentation when OD600 remains unchanged, obtain the fermentation broth of the strain, and detect the number of viable bacteria in the fermentation broth.

[0089] The culture medium of this example was used for cultivation. At the end of fermentation, the OD600 and viable bacterial count (unit: CFU / mL) of each fermentation medium are shown in Table 1.

[0090] Table 1

[0091] Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 OD600 2.0 2.3 2.5 4.0 3.1 Fermentation viable bacteria count <![CDATA[2.1×10 9 ]]> <![CDATA[2.5×10 9 ]]> <![CDATA[3.0×10 9 ]]> <![CDATA[4.2×10 9 ]]> <![CDATA[2.7×10 9 ]]>

[0092] The glucose utilization in culture medium 1 to 4 during the fermentation process of this example was detected. The results were as follows: Figure 1 shown.

[0093] from Figure 1 The results show that the glucose in medium 1 to medium 3 can be fully utilized, a small amount of glucose remains in medium 4, and a large amount of glucose remains in medium 5. The amount of glucose in medium 4 is appropriate.

[0094] Example 3

[0095] The target Lactococcus lactis strain of this example is the same as that of Example 1.

[0096] Based on the culture medium 4 in Example 2, an amino acid deficiency experiment was conducted. The added amino acid composition was divided into 22 groups, among which Group 1 added all 20 amino acids, Groups 2 to 21 each had one less amino acid component than Group 1, and Group 22 did not add any amino acids.

[0097] The culture medium of this example was used for culture. At the end of fermentation, the OD600 results of each fermentation medium are shown in Table 2.

[0098] Table 2

[0099] Group Amino acid addition OD600 1 20 amino acids in medium 4 3.9 2 Alanine deficiency 3.1 3 Arginine deficiency 0.6 4 Aspartate deficiency 3.2 5 Asparagine deficiency 2.8 6 L-cysteine hydrochloride 2.9 7 Glutamate deficiency 2.1 8 Glutamine deficiency 3.1 9 Glycine deficiency 2.9 10 Histidine deficiency 0.1 11 Isoleucine deficiency 0.2 12 Leucine deficiency 0.2 13 Lysine hydrochloride deficiency 0.4 14 Methionine deficiency 0.1 15 Phenylalanine deficiency 0.3 16 Proline deficiency 1.4 17 Serine deficiency 3.0 18 Threonine deficiency 0.3 19 Tryptophan deficiency 0.4 20 Tyrosine deficiency 2.5 21 Valine deficiency 0.1 22 Deficiency of all amino acids 0.1

[0100] The above results indicate that bacterial growth is significantly dependent on valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, and arginine, which can be considered as key essential amino acids for bacterial growth.

[0101] In contrast, the deficiency of cysteine hydrochloride, glycine, asparagine, glutamic acid, proline, and tyrosine will reduce the number of bacteria, but they are not absolutely essential amino acids for bacterial growth, indicating that the bacteria may have certain compensatory metabolic pathways.

[0102] In addition, the promoting effects of alanine, aspartic acid, glutamine and serine on bacterial growth were relatively weak, and their effects may depend on specific culture conditions or metabolic characteristics of the strain.

[0103] Example 4

[0104] The target Lactococcus lactis strain of this example is the same as that of Example 1.

[0105] On the basis of Example 3, an amino acid dosage investigation experiment was conducted. The amino acid portions, OD600 and viable bacterial counts in the specific experimental groups are shown in Table 3.

[0106] Table 3

[0107] Group Group 1 Group 2 Group 3 Group 4 Valine 1 serving 0.1 part 3 servings 1 serving Leucine 1 serving 0.1 part 3 servings 1 serving Isoleucine 1 serving 0.1 part 3 servings 1 serving Lysine 1 serving 0.1 part 3 servings 1 serving Methionine 1 serving 0.1 part 3 servings 1 serving Phenylalanine 1 serving 0.1 part 3 servings 1 serving Tryptophan 1 serving 0.1 part 3 servings 1 serving Threonine 1 serving 0.1 part 3 servings 1 serving Histidine 1 serving 0.1 part 3 servings 1 serving Arginine 1 serving 0.1 part 3 servings 1 serving Cysteine hydrochloride 1 serving 0.1 part 1 serving 0.1 part Glycine 1 serving 0.1 part 1 serving 0.1 part Asparagine 1 serving 0.1 part 1 serving 0.1 part glutamate 1 serving 0.1 part 1 serving 0.1 part Proline 1 serving 0.1 part 1 serving 0.1 part Tyrosine 1 serving 0.1 part 1 serving 0.1 part Alanine 1 serving 0.1 part 1 serving 0.1 part Aspartic acid 1 serving 0.1 part 1 serving 0.1 part Glutamine 1 serving 0.1 part 1 serving 0.1 part Serine 1 serving 0.1 part 1 serving 0.1 part OD600 4.0 2.1 1.8 3.7 Viable bacteria count CFU / mL <![CDATA[4.3×10 9 ]]> <![CDATA[1.8×10 9 ]]> <![CDATA[1.6×10 9 ]]> <![CDATA[3.7×10 9 ]]>

[0108] The results showed that, compared with the culture medium of group 1 and group 4, reducing the dosage of other non-essential amino acids had no significant inhibitory effect on the growth of Lactococcus lactis.

[0109] Comparison of the culture media in Group 2 and Group 4 showed that reducing the dosage of essential amino acids significantly inhibited the growth of Lactococcus lactis, indicating that these amino acids were the key limiting factors for its growth.

[0110] Comparison of the culture media in Group 1 and Group 3 showed that increasing the dosage of essential amino acids significantly inhibited the growth of Lactococcus lactis, indicating that the dosage of essential amino acids needed to be within an appropriate range.

[0111] In summary, the growth of Lactococcus lactis is highly dependent on the appropriate supply of specific amino acids. Excess or deficiency will affect its growth performance. Therefore, the concentration of amino acid addition needs to be optimized during the culture process.

[0112] Example 5

[0113] The target Lactococcus lactis strain of this example is the same as that of Example 1.

[0114] Culture medium: glucose (30 parts), amino acids (1 part alanine, 1 part arginine, 1 part aspartic acid, 1 part asparagine, 1 part cysteine hydrochloride, 1 part glutamic acid, 1 part glutamine, 1 part glycine, 1 part histidine, 1 part isoleucine, 1 part leucine, 1 part lysine hydrochloride, 1 part methionine, 1 part phenylalanine, 1 part proline, 1 part serine, 1 part threonine, 1 part tryptophan, 1 part tyrosine, 1 part valine), inorganic salts (1 part sodium chloride, 0.05 part potassium acetate, 0.005 part zinc sulfate heptahydrate, 0.01 part magnesium sulfate heptahydrate) 0.5 parts of iodine, 0.05 parts of sodium phosphate, 0.01 parts of potassium phosphate, 0.01 parts of potassium chloride, 0.005 parts of manganese sulfate, 0.01 parts of calcium chloride, 0.01 parts of ferrous sulfate, 0.005 parts of copper sulfate), nucleotides (0.05 parts of adenine, 0.05 parts of guanine, 0.05 parts of uracil, 0.05 parts of xanthine), vitamins (0.01 parts of biotin, 0.01 parts of niacin, 0.01 parts of pyridoxal hydrochloride, 0.01 parts of riboflavin, 0.01 parts of folic acid, 0.01 parts of thiamine hydrochloride, 0.01 parts of calcium pantothenate, 0.5 parts of Tween 80, and 1000 mL of distilled water.

[0115] Culture conditions:

[0116] a. Activation of the strain: Glycerol bacteria stored at -80°C were inoculated into GM17 liquid culture medium sterilized at 121°C for 15 minutes, and incubated anaerobically at 30°C for 16 hours. This was repeated twice to obtain the activated strain.

[0117] b. Preparation of culture medium: Mix the prepared fully synthetic culture medium, adjust the pH to 6.8, and sterilize at 121°C for 15 minutes;

[0118] c. Preparation of seed solution: Take the activated bacteria in step a and inoculate them into the culture medium prepared in b. Anaerobic culture was stopped at 30°C for 9 hours;

[0119] d. Inoculation and culture: The seed solution of step c was inoculated into the culture medium prepared in b and fermented under controlled fermentation conditions for 10h;

[0120] d. Fermentation conditions were controlled as follows: constant temperature at 30°C, a rotation speed of 100-200 rpm, and pH control (no control, 6.0, 6.5, or 7.0) to maintain anaerobic fermentation. The pH of the fermentation system was controlled by feeding a neutralizer, which in this example was an aqueous ammonia solution. Nitrogen was passed through the fermentation system at 0.4-1.0 lpm.

[0121] e. Termination of fermentation: When the alkali consumption is reduced, the fermentation is terminated to obtain a high-density fermentation broth of the strain. At the end of fermentation, the OD600 and viable counts of each fermentation medium are shown in Table 4.

[0122] Table 4

[0123] No regulation pH 6.0 pH 6.5 pH 7.0 OD600 4.0 7.3 10.2 8.4 Fermentation viable bacteria count CFU / mL <![CDATA[4.0×10 9 ]]> <![CDATA[8.0×10 9 ]]> <![CDATA[1.2×10 10 ]]> <![CDATA[8.9×10 9 ]]>

[0124] Example 6

[0125] All fermentation conditions were consistent with those in Example 5, the pH of the fermentation culture was 6.5, and batch fermentation culture of Lactobacillus lactis was carried out using the culture medium in Example 6 and GM17 culture medium. This was repeated three times. The viable cell count (CFU / mL) and OD600 of the fermentation broth of each batch of experiments were statistically analyzed, and the results are shown in Table 5.

[0126] Table 5

[0127]

[0128]

[0129] Example 7

[0130] Preparation of freeze-dried Lactococcus lactis powder:

[0131] The high-density fermentation broth of the strain obtained in the first batch of Example 6 was centrifuged at 8000 g to obtain concentrated bacterial cells. A protective agent solution (5 g sucrose, 1 g lysine, 1 g arginine, 2 g polydextrose, 3 g maltodextrin, and 100 mL distilled water) was added at a ratio of 1:4 between the concentrated bacterial cells and the protective agent solution. After mixing evenly, the mixture was placed in a freeze dryer and freeze-dried for 26 hours to obtain freeze-dried Lactococcus lactis powder. The viable bacterial count of the powder can reach 6.0 × 10 11 cfu / g or above, and can be directly used in probiotic fermentation agents or probiotic preparations.

[0132] Example 8

[0133] The fermentation culture conditions of this example are exactly the same as those of Example 5. The pH of the fermentation culture is 6.5. The commercially available recombinant Lactococcus lactis NZ9000 TyrL expression strain (supplier: Baosai Bio; product number: K2039) is fermented and cultured using the fully synthetic medium of this application. The experiment was repeated three times. The statistical results of the viable bacterial count (unit: CFU / mL) and OD600 of the fermentation broth for each batch of experiments are shown in Table 6.

[0134] Table 6

[0135]

[0136] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by utilizing the bacterial culture medium with clear composition and high stability in the present application and applying it to the fermentation culture of genetically modified Lactococcus lactis, a high-density Lactococcus lactis fermentation broth can be prepared, and the number of viable bacteria in the fermentation broth can be maintained at a high level, which is higher than the number of viable bacteria in the GM17 medium fermentation. The fermentation broth is subsequently freeze-dried to obtain bacterial powder with a high viable bacteria count, which is beneficial to the production and application of these genetically engineered strains in the pharmaceutical field.

[0137] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A culture medium, characterized in that The culture medium includes a carbon source, amino acids, nucleotides, inorganic salts and vitamins; The carbon source includes glucose; The amino acids include one or more of alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; The nucleotides include one or more of adenine, guanine, uracil or xanthine; The inorganic salt includes one or more of sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate or copper sulfate; Wherein, the glucose is 10-30 parts by weight.

2. The culture medium according to claim 1, characterized in that The vitamins include one or more of biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride or calcium pantothenate; Preferably, the culture medium comprises: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate; Preferably, the culture medium comprises: glucose, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, threonine, histidine, arginine, cysteine hydrochloride, glycine, asparagine, glutamic acid, proline, tyrosine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate; Preferably, the culture medium comprises glucose, alanine, arginine, aspartic acid, asparagine, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, sodium chloride, potassium acetate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, cobalt chloride, manganese sulfate, calcium chloride, ferrous sulfate, copper sulfate, adenine, guanine, uracil, xanthine, biotin, niacin, pyridoxal hydrochloride, riboflavin, folic acid, thiamine hydrochloride and calcium pantothenate.

3. The culture medium according to claim 2, characterized in that The culture medium comprises, by weight, 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of arginine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, 0.001-1.0 parts of magnesium sulfate heptahydrate, 0.01-2.0 parts of potassium dihydrogen phosphate, and 0.01-2.0 parts of dipotassium hydrogen phosphate. 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 parts, uracil 0.001-1.0 parts, xanthine 0.001-1.0 parts, biotin 0.001-1.0 parts, niacin 0.001-1.0 parts, pyridoxal hydrochloride 0.001-1.0 parts, riboflavin 0.001-1.0 parts, folic acid 0.001-1.0 parts, thiamine hydrochloride 0.001-1.0 parts and calcium pantothenate 0.001-1.0 parts; Preferably, the culture medium comprises, by weight: 10-30 parts of glucose, 0.01-1.0 parts of valine, 0.01-1.0 parts of leucine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of methionine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of tryptophan, 0.01-1.0 parts of threonine, and 0.01-1.0 parts of histidine. 0.01-1.0 parts, arginine 0.01-1.0 parts, cysteine hydrochloride 0.01-1.0 parts, glycine 0.01-1.0 parts, asparagine 0.01-1.0 parts, glutamic acid 0.01-1.0 parts, proline 0.01-1.0 parts, tyrosine 0.01-1.0 parts, sodium chloride 0.01-2.0 parts, potassium acetate 0.1-5.0 parts, zinc sulfate heptahydrate 0.00 1-1.0 parts, magnesium sulfate heptahydrate 0.001-1.0 parts, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 part of urea, 0.001-1.0 part of uracil, 0.001-1.0 part of xanthine, 0.001-1.0 part of biotin, 0.001-1.0 part of niacin, 0.001-1.0 part of pyridoxal hydrochloride, 0.001-1.0 part of riboflavin, 0.001-1.0 part of folic acid, 0.001-1.0 part of thiamine hydrochloride and 0.001-1.0 part of calcium pantothenate; Preferably, the culture medium comprises, by weight: 10-30 parts of glucose, 0.01-1.0 parts of alanine, 0.01-1.0 parts of arginine, 0.01-1.0 parts of aspartic acid, 0.01-1.0 parts of cysteine hydrochloride, 0.01-1.0 parts of glutamic acid, 0.01-1.0 parts of glycine, 0.01-1.0 parts of histidine, 0.01-1.0 parts of isoleucine, 0.01-1.0 parts of lysine, 0.01-1.0 parts of phenylalanine, 0.01-1.0 parts of serine, 0.01-1.0 parts of threonine, 0.01-1.0 parts of tyrosine, 0.01-2.0 parts of sodium chloride, 0.1-5.0 parts of potassium acetate, 0.001-1.0 parts of zinc sulfate heptahydrate, and 0.001-1.0 parts of magnesium sulfate heptahydrate. 0 parts, potassium dihydrogen phosphate 0.01-2.0 parts, dipotassium hydrogen phosphate 0.01-2.0 parts, cobalt chloride 0.001-1.0 parts, manganese sulfate 0.001-1.0 parts, calcium chloride 0.001-1.0 parts, ferrous sulfate 0.001-1.0 parts, copper sulfate 0.001-1.0 parts, adenine 0.001-1.0 parts, guanine 0.001-1.0 parts parts, uracil 0.001-1.0 parts, xanthine 0.001-1.0 parts, biotin 0.001-1.0 parts, niacin 0.001-1.0 parts, pyridoxal hydrochloride 0.001-1.0 parts, riboflavin 0.001-1.0 parts, folic acid 0.001-1.0 parts, thiamine hydrochloride 0.001-1.0 parts and calcium pantothenate 0.001-1.0 parts.

4. The culture medium according to claim 1, characterized in that The culture medium further comprises a surfactant; Preferably, the surfactant comprises one or more of Tween 80, Tween 60 or PEG6000, more preferably Tween 80; Preferably, the surfactant is present in an amount of 0.1-20 parts by weight.

5. Use of the culture medium according to any one of claims 1 to 4 in the cultivation of Lactococcus lactis or genetically modified Lactococcus lactis, characterized in that: The application comprises: activating the target Lactococcus lactis and inoculating the target Lactococcus lactis into a culture medium of the Lactococcus lactis according to any one of claims 1 to 4 for fermentation to obtain a fermentation liquid containing the target Lactococcus lactis; The target Lactococcus lactis includes genetically modified Lactococcus lactis; The genetically modified Lactococcus lactis is a strain in which the bases of the genes of the wild-type Lactococcus lactis are deleted, added or replaced by gene editing technology.

6. The use according to claim 5, characterized in that The pH of the fermentation culture is 6.2 to 6.6; Preferably, the pH of the fermentation culture is 6.

5.

7. The use according to claim 5, characterized in that The fermentation culture temperature is 28-35°C; Preferably, the fermentation culture time is 8 to 13 hours; Preferably, the fermentation is anaerobic fermentation.

8. The use according to claim 5, characterized in that The number of viable Lactococcus lactis in the fermentation broth is ≥ 0.8×10 10 cfu / mL.

9. Use of the culture medium according to any one of claims 1 to 4 in preparing a Lactococcus lactis product.

10. The use according to claim 9, characterized in that The Lactococcus lactis product includes Lactococcus lactis dry powder, probiotic fermentation agent or probiotic preparation.

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