A starch culture medium for extracting heart and liver powder, its preparation and application

Abstract

This invention relates to the field of bacterial culture medium technology, and more particularly to a heart and liver extract starch culture medium and its preparation and application. This invention provides a method for culturing *Alternaria fintelii*. Alistipes finegoldii Heart-liver infusion and starch (HLS) medium is a simple formulation with readily available components and low overall cost. Its preparation and operation are convenient, making it suitable for widespread use. *Alternaria fintelii* can be cultured using this medium. Alistipes finegoldii Its rapid growth, high upper limit of culture density, and high viability rate are beneficial for further research and application of this bacterium.

Description

Technical Field

[0001] This invention relates to the field of bacterial culture medium technology, and in particular to a heart and liver extract starch culture medium and its preparation and application. Background Technology

[0002] Fenrir's other branch ( Alistipes finegoldii It is an obligate anaerobic Gram-negative bacillus that is a symbiotic bacterium in the gut; it is a straight or slightly curved rod-shaped bacterium with a diameter of 0.2-0.9 µm and a length of 0.5-4 µm, with rounded ends.

[0003] Currently, the cultivation of *Alistipes finegoldiiaugments* mainly relies on brain-heart extract (BHI) liquid medium containing 10% fetal bovine serum (FBS), with the addition of 0.05% L-cysteine ​​as a reducing agent to maintain an anaerobic environment (*Alistipes finegoldiiaugments the efficacy of immunotherapy against solid tumors. Wu ZY, Wu QW, et al.). However, there are shortcomings in using this medium for culturing *Alistipes finegoldiiaugments*. On the one hand, *Alistipes finegoldiiaugments* grows slowly and at low density in this medium, making it difficult to meet the needs of large-scale preparation. Studies have indicated that the growth of this bacterium in liquid medium is quite difficult and may require specific supplementary additives to obtain the ideal cell yield. On the other hand, FBS is the most expensive component of the medium, and its price fluctuates wildly. The price of high-quality FBS can reach tens of thousands of yuan per liter, accounting for 60% to 80% of the total cost of serum-containing medium. Moreover, the collection of FBS involves the slaughter of pregnant cows, raising ethical controversies and supply chain instability issues.

[0004] Therefore, there is an urgent need to develop a low-cost liquid culture medium suitable for the efficient amplification of Alternaria finniculum. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for culturing *Fernandezova fentanyl*. Alistipes finegoldii Heart liver infusion and starch (HLS) medium.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a heart and liver extract starch culture medium comprising the following components at mass concentrations: 29-49 g / L heart and brain extract powder, 3-5 g / L bovine liver extract powder, 3-5 g / L water-soluble starch, 90-150 mL / L α-MEM solution, 5 mL / L fetal bovine serum and 1 mL / L 50% (w / v) L-cysteine ​​solution.

[0007] This invention targets existing *Fernella fenestration* species. Alistipes finegoldii The culture medium formulation has been improved, reducing the content of high-cost components (fetal bovine serum), resulting in lower preparation costs and effectively reducing the overall culture cost of the strains. Cultivation in this medium can increase the culture density of *Alternaria fintelii*, accelerating its growth rate.

[0008] Preferably, the heart and liver extract starch culture medium comprises the following components in mass concentrations: 39 g / L brain and heart extract powder, 4 g / L bovine liver extract powder, 4 g / L water-soluble starch, 120 mL / L α-MEM solution, 5 mL / L fetal bovine serum and 1 mL / L 50% (w / v) L-cysteine ​​solution.

[0009] Preferably, the pH value of the culture medium is 7.0-7.2.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned heart and liver extract starch culture medium, comprising the following steps: Weigh out brain and heart extract powder, bovine liver extract powder, and water-soluble starch, add distilled water and mix well, then sterilize and cool. Add α-MEM solution, fetal bovine serum and L-cysteine ​​solution, mix well, and prepare the heart and liver extract powder starch culture medium.

[0011] Thirdly, the present invention provides the above-mentioned heart and liver extract starch culture medium for culturing *Alternaria fentanilii*. Alistipes finegoldii Applications in [the context of the text].

[0012] Fourthly, the present invention provides a method for culturing *Alternaria finigens* using the above-mentioned heart and liver extract starch culture medium. Alistipes finegoldii The method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Alistipes finegoldii The bacterial culture was inoculated into the heart and liver extract starch culture medium and cultured anaerobically.

[0013] Preferably, the anaerobic culture conditions are 37°C, 80% N2, 10% CO2, and 10% H2.

[0014] The beneficial effects of this invention are as follows: This invention provides a method for culturing *Finniella fenestration*. Alistipes finegoldii Heart-liver infusion and starch (HLS) medium is a simple formulation with readily available components and low overall cost. Its preparation and operation are convenient, making it suitable for widespread use. *Alternaria fintelii* can be cultured using this medium. Alistipes finegoldii Its rapid growth, high upper limit of culture density, and high viability rate are beneficial for further research and application of this bacterium. Attached Figure Description

[0015] Figure 1 Comparison of growth curves of Alternaria finigens cultured in different culture media.

[0016] Figure 2 Peak growth rates of *Alternaria fintelii* in different culture media ( =0.0005).

[0017] Figure 3 To ensure that *Alternaria finniculum* grows to the same OD in different culture media 600nm The time required for the value (ns indicates no significant difference) =0.0064; =0.0003).

[0018] Figure 4 The viability of Alternaria fintelii at the end of logarithmic growth in different culture media (ns indicates no significant difference).

[0019] Figure 5 Comparison of the quality of *Alternaria finnifolia* sludge harvested from 6L culture medium in a small-scale trial ( =0.0428). Detailed Implementation

[0020] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0021] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0022] The sources of the raw materials used in this invention are shown in Table 1.

[0023] Table 1: List of reagents required for preparing HLS medium and BHI+10%BHI medium Example 1 An embodiment of the heart and liver extract starch culture medium (hereinafter referred to as HLS culture medium) of the present invention, the formulation (taking 1L as an example) and preparation method are as follows: Weigh 29g of brain and heart extract powder, 3g of bovine liver extract powder, and 3g of water-soluble starch. Add 904mL of distilled water and mix well. Then autoclave at 121℃ for 15min. After cooling to 60℃, add 90mL of α-MEM solution, 5mL of fetal bovine serum and 1mL of 50% L-cysteine ​​solution, mix well, and prepare 1L of heart and liver extract powder starch culture medium.

[0024] Example 2 An embodiment of the heart and liver extract starch culture medium of the present invention, the formulation (taking 1L as an example) and preparation method are as follows: Weigh 39g of brain and heart extract powder, 4g of bovine liver extract powder, and 4g of water-soluble starch. Add 874mL of distilled water and mix well. Then autoclave at 121℃ for 15min. After cooling to 60℃, add 120mL of α-MEM solution, 5mL of fetal bovine serum and 1mL of 50% L-cysteine ​​solution, mix well, and prepare 1L of heart and liver extract powder starch culture medium.

[0025] Example 3 An embodiment of the heart and liver extract starch culture medium of the present invention, the formulation (taking 1L as an example) and preparation method are as follows: Weigh 49g of brain and heart extract powder, 5g of bovine liver extract powder, and 5g of water-soluble starch. Add 844mL of distilled water and mix well. Then autoclave at 121℃ for 15min. After cooling to 60℃, add 150mL of α-MEM solution, 5mL of fetal bovine serum, and 1mL of 50% L-cysteine ​​solution. Mix well to prepare 1L of heart and liver extract powder starch culture medium.

[0026] Comparative Example 1 A comparative example of the heart and liver extract starch culture medium of the present invention, the formulation (taking 1L as an example) and preparation method are as follows: Weigh 39g of brain heart extract powder, add 899mL of distilled water and mix well. Then autoclave at 121℃ for 15min. After cooling to 60℃, add 100mL of fetal bovine serum and 1mL of 50% L-cysteine ​​solution, mix well, and prepare 1L of culture medium (hereinafter referred to as BHI+10%FBS medium).

[0027] Comparative Example 2 The comparative example of the heart and liver extract starch culture medium of the present invention differs from that of Example 2 only in that α-MEM solution is not added, and water is used to make up the volume. All other aspects are the same as in Example 2.

[0028] Test Example 1 The growth of *Alternaria finniculum* was tested in different culture media (Examples 1-3, Comparative Example 1).

[0029] Dissolve *Feniceria fenestrated* in 1 mL of culture medium. Alistipes finegoldii (DSMZ 17242, hereinafter the same) lyophilized powder was inoculated into 14 mL of culture medium and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) to revive the bacterial strain. Subculture was then performed continuously at a ratio of 1:50. After the third generation, the OD of the bacterial culture was measured using Nanodrop. 600nm Value. When the bacterial concentration in the culture of *Fernandezius fenestration* reaches OD... 600nmWhen the concentration is 1.0, inoculate the bacterial culture at a ratio of 1:100 into 15 mL of fresh culture medium and mix well. OD of bacterial culture was measured every 2 hours for 20-26 hours after inoculation. 600nm The values ​​were used to obtain the peak growth range of *Alternaria finniculum* in various culture media.

[0030] The test results are shown in Table 2.

[0031] Table 2 The results showed that the HLS culture medium used in Examples 1-3 was superior to that used in Comparative Example 1. Among them, the culture medium formulation in Example 2 showed the best performance, and therefore, it was used for subsequent tests.

[0032] Test Example 2 The growth of *Alternaria finniculum* was tested in different culture media (Comparative Example 2).

[0033] Dissolve *Feniceria fenestrated* in 1 mL of culture medium. Alistipes finegoldii (DSMZ 17242, hereinafter the same) lyophilized powder was inoculated into 14 mL of culture medium and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) to revive the bacterial strain. Subculture was then performed continuously at a ratio of 1:50. After the third generation, the OD of the bacterial culture was measured using Nanodrop. 600nm Value. When the bacterial concentration in the culture of *Fernandezius fenestration* reaches OD... 600nm When the concentration is 1.0, inoculate the bacterial culture at a ratio of 1:100 into 15 mL of fresh culture medium and mix well. OD of the bacterial culture was measured once 20 hours after inoculation. 600nm value.

[0034] The results showed that Comparative Example 2 had an OD of 20 hours after inoculation. 600nm The value was 1.55, lower than that of Example 2 and Comparative Example 1.

[0035] In the absence of α-MEM, the culture effect of Comparative Example 2 was significantly affected and worse than that of Comparative Example 1. Therefore, α-MEM is a key component in HLS medium that can replace fetal bovine serum.

[0036] While fetal bovine serum (FBS) contains various small-molecule nutrients required for bacterial culture, it also contains components that inhibit bacterial growth, such as complement and antibodies. These substances may lead to inhibited bacterial growth or even death. Furthermore, FBS contains a large amount of transferrin, a natural iron chelator, which may affect the iron uptake by anaerobic bacteria. α-MEM, on the other hand, is rich in small-molecule nutrients that can meet the needs of bacterial growth and does not contain bacterial inhibitors such as complement, antibodies, and transferrin. Therefore, it can be used as a substitute for FBS. When combined with the addition of bovine liver extract powder and soluble starch, it can provide abundant heme, ferritin, nicotinamide, and carbon sources for bacterial growth, thereby reducing the high cost associated with using FBS.

[0037] Test Example 3 The growth of *Alternaria fenestrated* was tested in different culture media (Example 2, Comparative Example 1).

[0038] Dissolve *Feniceria fenestrated* in 1 mL of culture medium. Alistipes finegoldii (DSMZ 17242, hereinafter the same) lyophilized powder was inoculated into 14 mL of culture medium and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) to revive the bacterial strain. Subculture was then performed continuously at a ratio of 1:50. After the third generation, the OD of the bacterial culture was measured using Nanodrop. 600nm Value. When the bacterial concentration in the culture of *Fernandezius fenestration* reaches OD... 600nm When the OD value is 1.0, inoculate the bacterial culture at a ratio of 1:100 into 15 mL of fresh culture medium and mix well. Then, add 200 μL of the inoculation solution to the well for OD measurement. 600nm The culture was placed in a dedicated 96-well plate with 6 wells. After covering the plate, it was placed in the plate for anaerobic incubation (80% N2, 10% CO2, 10% H2). The OD of the bacterial culture in each well was measured every 10 minutes. 600nm The growth curve of *Alternaria fenestrated* in the culture medium was obtained by measuring the value up to 48 hours after inoculation.

[0039] The bacterial concentration after resuscitation reached OD. 600nmA 1:100 inoculum of *C. fingraecum* was inoculated into 15 mL of fresh culture medium and mixed thoroughly. The culture was then anaerobic at 37°C (80% N2, 10% CO2, 10% H2). After 18 hours of inoculation (according to the measured growth curve, *C. fingraecum* reached the late logarithmic growth phase), the cells were collected by centrifugation at 25°C and 6000 × g for 15 min using a NACHT B-20R centrifuge. The cells were then washed with PBS. After washing the bacterial cells twice with PBS, resuspend them in 2 mL of PBS. Then, add 6 μL of a pre-mixed 1:1 ratio of 3.34 mM SYTO9 solution and 20 mM propidium iodide solution. After staining in the dark for 15 min, use a Beckman Coulter Cytoflex LX 19-color flow cytometer to detect SYTO9 staining and propidium iodide staining at excitation / emission wavelengths of 488 nm / 525 nm and 488 nm / 690 nm, respectively. The sample flow rate was controlled at approximately 300 particles / second, and the viability was calculated using the following formula: Viability = (Number of 1-propidium iodide-positive bacteria / Number of SYTO9-positive bacteria) × 100%.

[0040] The results are as follows Figure 1 As shown, *C. fentanyl* can grow in both culture media. The adjustment period after inoculation is similar in length, with both entering the early logarithmic growth phase approximately 6 hours after inoculation and simultaneously entering the logarithmic growth phase at approximately 10 hours. At approximately 14 hours after inoculation, the growth curve of *C. fentanyl* in HLS medium is steeper than that in BHI+10% FBS medium, indicating that *C. fentanyl* grows more rapidly in HLS medium during this period. At approximately 18 hours after inoculation, *C. fentanyl* enters the late logarithmic growth phase and reaches the stationary phase at 20 hours. After reaching the stationary phase, the bacterial density of *C. fentanyl* in HLS medium reaches its peak (OD). 600nm =2.50), compared to the peak bacterial density (OD) of *Alternaria fenestrated* in BHI+10% FBS medium during the same period. 600 nm =1.75) is 42.86% higher.

[0041] Further analysis of the growth curves of *Alternaria fintelii* in the two culture media, such as... Figure 2 As shown, the growth rate (i.e., OD) of *Alternaria fintelii* in HLS medium. 600 nm The peak value of the growth rate (0.0054 / min) was significantly higher than that of the peak value of growth rate in BHI+10%FBS medium (0.0038 / min), by about 42.11%, indicating that HLS medium significantly improved the liquid culture growth performance of Alternaria fintelii.

[0042] This improvement in growth performance is also reflected in the fact that the bacterial density of *Cephalomyces fintels* reaches a specific OD value. 600nm The required culture time ( Figure 3 In HLS medium, the bacterial density of *Alternaria fintelii* reached OD0.05. 600 nm The culture times required for values ​​of 1.2 and 1.8 were 14.61 hours and 16.61 hours, respectively, which were approximately 6.76% and 23.91% faster than those in BHI+10%FBS medium (15.67 hours and 21.83 hours, respectively). This effectively shortened the culture time for *Alternaria fintelii* to reach specific bacterial densities, indicating that HLS medium has significant potential to shorten the process time in *Alternaria fintelii* culture.

[0043] According to the statistical results of the viable bacterial rate at the end of the logarithmic growth phase, as shown in Table 3, Figure 4 As shown, when using HLS medium for the culture of *Alternaria fintelii*, the viability rate reached 99.97% at the end of the logarithmic growth phase, which was not significantly different from that using BHI+10% FBS medium (p>0.9999). This indicates that using HLS medium can effectively increase the bacterial density of *Alternaria fintelii* while ensuring the viability rate, resulting in a superior culture effect for *Alternaria fintelii* and making it suitable for higher density *Alternaria fintelii* culture experiments.

[0044] Table 3: Comparison of viability of *Alternaria finneri* at the end of logarithmic growth in different culture media Test Example 4: Small-scale culture According to the formulations of Example 2 and Comparative Example 1, 6L of HLS medium and BHI+10% FBS medium were prepared for large-volume culture of Alternaria fintelii.

[0045] Dissolve the lyophilized powder of *Alternaria finniculum* in 1 mL of culture medium and inoculate it into 14 mL of culture medium. Then, revive the strain by anaerobic culture at 37°C (80% N2, 10% CO2, 10% H2). Subsequently, inoculate it into 200 mL of culture medium at a ratio of 1:50 for further expansion culture.

[0046] The *Alternaria fintelii* culture obtained from the resuscitation and propagation of the bacterial strain was inoculated into 6 L of culture medium at a ratio of 1:50 and cultured anaerobicly at 37 °C (80% N2, 10% CO2, 10% H2). After 24 h of culture, the bacterial cells were collected by centrifugation at 4000 × g for 1 h at 25 °C using an Eppendorf 5910R centrifuge, and the mass of the bacterial precipitate was weighed using a balance.

[0047] The weighing results of the three experiments are shown in Table 4 and Figure 5As shown, at a small-scale culture scale (gL), the average mass of mycelial sludge collected per batch using HLS medium was 36.60g, while using BHI+10%FBS medium only yielded 32.34g. The comparison shows that HLS medium culture increased the collected mycelial sludge quantity by 13.17%, and the difference was statistically significant (p=0.0428). This indicates that when conducting small-scale *Alternaria fintelii* culture, using HLS medium can effectively increase the bacterial culture density, thereby increasing the amount of mycelial sludge collected. Therefore, at the small-scale scale, the culture effect of using HLS medium for *Alternaria fintelii* is superior to that of BHI+10%FBS medium.

[0048] Table 4: Quality of *Alternaria finigera* slime harvested from different culture media in small-scale *Alternaria finigera* culture Test Example 5: Cost Statistics of Biologics The cost of culturing *Finnobrinus fenestration* in Example 2 and Comparative Example 1 was statistically analyzed.

[0049] Table 5: Cost Statistics of Culture in HLS Medium and BHI+10% FBS Medium The statistical results are shown in Table 5. Due to the optimization of the culture medium formula, the content of fetal bovine serum in HLS culture medium was reduced to 1 / 20 of that in BHI+10%FBS culture medium. The preparation cost of HLS culture medium was only 78.55 yuan / L, compared with the cost of BHI+10%FBS culture medium of 451.75 yuan / L, the cost of culture medium was reduced by about 82.6%.

[0050] Meanwhile, due to the superior culture effect of HLS medium for *Alternaria fintelii*, the unit culture cost of HLS medium was only 12.88 yuan / g during small-scale culture, compared to 76.18 yuan / g for BHI+10%FBS medium. Considering the culture effect, the cost advantage of HLS medium is more significant, with a reduction of 83.1% in unit culture cost. This indicates that HLS medium is not only significantly superior to the original BHI+10%FBS medium in terms of culture performance for *Alternaria fintelii*, but also has a significant advantage in culture cost, effectively reducing the production cost of live bacterial biological agents prepared using *Alternaria fintelii*.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A starch culture medium for extracting heart and liver powder, characterized in that, The product contains the following components at the following concentrations: 29-49 g / L brain and heart extract powder, 3-5 g / L bovine liver extract powder, 3-5 g / L water-soluble starch, 90-150 mL / L α-MEM solution, 5 mL / L fetal bovine serum, and 1 mL / L 50% (w / v) L-cysteine ​​solution.

2. The heart and liver extract starch culture medium as described in claim 1, characterized in that, The components include the following mass concentrations: 39 g / L brain and heart extract powder, 4 g / L bovine liver extract powder, 4 g / L water-soluble starch, 120 mL / L α-MEM solution, 5 mL / L fetal bovine serum and 1 mL / L 50% (w / v) L-cysteine ​​solution.

3. The heart and liver extract starch culture medium as described in claim 1, characterized in that, The pH value of the culture medium is 7.0-7.

2.

4. A method for preparing a heart and liver extract starch culture medium as described in any one of claims 1-3, characterized in that, Includes the following steps: Weigh out brain and heart extract powder, bovine liver extract powder, and water-soluble starch, add distilled water and mix well, then sterilize and cool. Add α-MEM solution, fetal bovine serum and L-cysteine ​​solution, mix well, and prepare the heart and liver extract powder starch culture medium.

5. The heart and liver extract starch culture medium as described in any one of claims 1-3 for culturing *Alternaria finigens*. Alistipes finegoldii Applications in [the field].

6. A method for culturing *Alternaria finigens* using the heart and liver extract starch culture medium according to any one of claims 1-3. Alistipesfinegoldii The method is characterized by, The steps include: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Alistipesfinegoldii The bacterial culture was inoculated into the heart and liver extract starch culture medium and cultured anaerobically.

7. The method as described in claim 6, characterized in that, The anaerobic culture conditions were 37℃, 80% N2, 10% CO2, and 10% H2.

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