Preparation method of Thermus thermophilus fermentation lysate

Abstract

The invention discloses a preparation method of a thermophilic thermophilic bacteria fermentation lysate, which comprises the following steps: inoculating a seed culture medium with thermophilic thermophilic bacteria for culturing to prepare a seed bacteria solution; inoculating the seed bacterial liquid into an initial culture medium for culturing, adding a supplementary culture medium when residual sugar reaches 4-6g / L, maintaining the residual sugar at 4-6g / L, and fermenting for 30-40 hours to obtain fermentation liquor; and filtering the obtained fermentation liquor by using a ceramic membrane, cleaning by using 7 times of water volume until the conductivity of the supernate is 100 [mu] s / cm, diluting the bacterial suspension to OD = 20, and homogenizing at high pressure for 1-3 times to obtain the Thermus thermophilus lysate. According to the method, the activity of superoxide dismutase in the thermophilic thermophilic bacteria lysate is improved, and the stability of nucleic acid in the thermophilic thermophilic bacteria lysate at normal temperature is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for preparing fermentation lysates of thermophilic bacteria. Background Technology

[0002] Thermophilic bacteria Thermus Thermophilus It is a thermotolerant bacterium widely distributed in hot springs, geothermal soils, near submarine volcanoes, and manure. The unique "growth factors" in thermophilic bacteria are mainly composed of nucleic acids, vitamin A, vitamin B2, nicotinamide, beta-carotene, glutathione, and polyamino acids. Vitamins, glutathione, and polyamino acids act as cell growth factors, restoring and promoting cellular physiological functions. They can promote cell growth during the repair of chronic, non-therapeutic wounds, reduce skin inflammation, and resist sun damage.

[0003] Thermophilic bacteria contain specific hyperthermophilic proteins. For example, superoxide dismutase (SOD) in thermophilic bacteria has heat resistance and strong stability. It can withstand high temperatures above 90 °C and still has a strong DNA protection and repair ability.

[0004] In recent years, studies have found that it also contains active substances such as nicotinamide mononucleotide. Nucleic acids are essential substances for cell metabolism, which help nourish epidermal cell genes and repair damage, deeply moisturize the skin, and make the skin soft.

[0005] Therefore, achieving high-density fermentation of *Thermophilic thermophilus* is of great significance, as it can increase volumetric yield, shorten the culture cycle, thereby reducing culture costs and enhancing market competitiveness. Currently, there are no reports on improving the superoxide dismutase activity in *Thermophilic thermophilus* lysates or the stability of nucleic acids in the lysates at room temperature during the fermentation stage. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the object of this invention is to overcome the shortcomings of the prior art and provide a method for preparing fermentation lysate of thermophilic bacteria, comprising, Thermophilic bacteria were inoculated into seed culture medium and cultured to obtain seed culture solution; The seed culture was inoculated into the initial culture medium and cultured. When the residual sugar reached 4-6 g / L, feed culture medium was added to maintain the residual sugar at 4-6 g / L. Fermentation was carried out for 30-40 h to obtain the fermentation broth. The obtained fermentation broth was filtered through a ceramic membrane, washed with 7 times the volume of water, and the conductivity of the supernatant was 100 μs / cm. The bacterial suspension was diluted to OD=20 and homogenized under high pressure 1-3 times to obtain thermophilic thermophilic bacteria lysate. The initial culture medium formulation is as follows: peptone 5 g / L, yeast extract 5 g / L, CaSO4·2H2O 0.12 g / L, MgSO4·2H2O 0.2 g / L, NaCl 0.016 g / L, KNO3 0.21 g / L, Na2HPO4 0.22 g / L, sodium sulfite 50~150 mg / L; The thermophilic bacterium, with the suggested classification name Thermus thermophilus RTNB324001, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20252066, on September 19, 2025.

[0009] As a preferred embodiment of the preparation method described in this invention, the seed culture medium is formulated as follows: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0010] As a preferred embodiment of the preparation method described in this invention, the bacterial content (OD) in the seed culture is... 600 =6.0±0.2.

[0011] As a preferred embodiment of the preparation method described in this invention, the initial culture medium is cultured under the following conditions: temperature controlled at 55~75℃, ventilation at 5 L / h, dissolved oxygen cascade rotation speed to ensure DO ≥ 30%, pH adjusted to 7.0 with ammonia, and seed culture inoculation amount of 2%~15%.

[0012] As a preferred embodiment of the preparation method described in this invention, the feed culture medium is formulated as follows: 80 g / L peptone, 80 g / L yeast extract, 500 g / L glucose and 0.54 g / L MgSO4.

[0013] As a preferred embodiment of the preparation method described in this invention, the ceramic membrane is filtered with 7 times the volume of water, and the high-pressure homogenization pressure is 1200 MPa.

[0014] Another objective of the present invention is to overcome the shortcomings of the prior art and provide a method for improving the superoxide dismutase activity of thermophilic thermocide fermentation lysate, comprising culturing thermophilic thermocide in an initial culture medium to improve the superoxide dismutase activity of thermophilic thermocide fermentation lysate. The initial culture medium formula is as follows: 5 g / L peptone, 5 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, 0.22 g / L Na2HPO4, and 50~150 mg / L sodium sulfite.

[0015] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for improving the stability of nucleic acids in the fermentation lysate of Thermophilic Bacteria at room temperature, comprising culturing Thermophilic Bacteria in an initial culture medium to improve the stability of nucleic acids in the fermentation lysate of Thermophilic Bacteria at room temperature. The initial culture medium formula is as follows: 5 g / L peptone, 5 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, 0.22 g / L Na2HPO4, and 50~150 mg / L sodium sulfite.

[0016] Beneficial effects of this invention: (1) The present invention cultivates thermophilic bacteria in a 10 L fermenter and optimizes the fermentation temperature, inoculum size, dissolved oxygen level, feeding method, feeding medium and initial medium. It achieves high-density cultivation of thermophilic bacteria with OD600=86.1 in a 10 L fermenter, which helps to realize low-cost and high-efficiency industrial production.

[0017] (2) This invention improves the activity of superoxide dismutase in thermophilic thermophilic bacteria lysate.

[0018] (3) This invention improves the stability of nucleic acids in thermophilic thermophilic bacteria lysate at room temperature.

[0019] (4) The present invention uses a ceramic membrane to clean the fermentation broth of thermophilic bacteria obtained by fermentation to obtain bacterial turbidity, and high pressure homogenization to obtain thermophilic bacteria lysate, which helps to achieve low energy consumption and water consumption in industrial production, while reducing wastewater treatment costs. Detailed Implementation

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0021] Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl.

[0022] Fermentation medium: peptone 5 g / L, yeast extract 5 g / L, CaSO4·2H2O 0.12 g / L, MgSO4·2H2O 0.2 g / L, NaCl 0.016 g / L, KNO3 0.21 g / L, Na2HPO4 0.22 g / L.

[0023] Feeding medium: 80 g / L peptone, 80 g / L yeast extract, 500 g / L glucose and 0.54 g / L MgSO4.

[0024] Fermentation conditions: cultured in a 10L fermenter at a temperature of 65℃, pH 7.0, aeration 1 vvm, and DO ≥ 30%.

[0025] Method for superoxide dismutase (SOD) activity assay: A regression equation was prepared to determine the inhibition rate of pyrogallol (PR) autooxidation by a final concentration of standard SOD. The inhibition rate of PR by *Thermophilus thermophilus* lysate was then measured. The SOD activity of the *Thermophilus thermophilus* lysate was calculated based on the standard regression equation and the dilution factor of the sample solution. Experimental conditions were: PR concentration 60 mmol / L, system pH = 8.2 ± 0.01, and absorbance measured at 320 nm at an incubation temperature of 25 ℃.

[0026] Nucleic acid assay method: The absorbance was measured using a UV spectrophotometer with an absorption wavelength of 260 nm. The absorbance of 1 μg / ml DNA solution was 0.020.

[0027] The thermophilic bacterium used in the embodiments of this invention is recommended to be classified and named Thermus thermophilusRTNB324001. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20252066 and deposit date of September 19, 2025.

[0028] Example 1 Take the glycerol bacteria out of the -80℃ freezer and inoculate them into two 250 mL / 2L seed culture media. Incubate at 65℃ and 220 rpm for 24 h.

[0029] Inoculate 10% of the solution into a 10L fermenter, maintain a temperature of 65℃, aeration of 5 L / h, and a dissolved oxygen cascade rotation speed to ensure DO ≥ 30%. Adjust the pH to 7.0 with ammonia. When the residual sugar reaches 5.0 g / L, begin feeding with 80 g / L peptone, 80 g / L yeast extract, 500 g / L glucose, and 0.54 g / L MgSO4 to maintain the residual sugar at 4-6 g / L. After 40 hours of fermentation, the OD... 600 It reached a maximum of 86.1.

[0030] Comparative Example 1 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol-containing bacteria were removed from a -80℃ freezer and inoculated into two 250 mL / 2L seed culture media. The cultures were incubated at 60℃ and 220 rpm for 24 h. The bacterial count (OD) in the seed culture was measured. 600 =6.0±0.2.

[0031] The above seed culture was inoculated into a 10L fermenter at a 5% inoculum rate. Temperatures were controlled at 55℃, 60℃, 65℃, 70℃, and 75℃, with a ventilation rate of 5 L / h. Dissolved oxygen was maintained at a cascade rotation speed to ensure DO ≥ 10%. The pH was adjusted to 7.5 with 5 mol / L NaOH. OD was measured when residual sugars were depleted. 600 As shown in the table below:

[0032] Comparative Example 2 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of the -80℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65 ℃ and 220 rpm for 24 h.

[0033] Inoculation was carried out at 2%, 5%, 10%, and 15% of the culture medium into a 10L fermenter, respectively. The temperature was controlled at 65℃, the aeration rate was 5 L / h, the dissolved oxygen cascade speed was maintained to ensure DO ≥ 10%, and the pH was adjusted to 7.5 with 5 mol / L NaOH. The OD was measured when the residual sugar was depleted. 600 As shown in the table below:

[0034] Comparative Example 3 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of a -80 ℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65 ℃ and 220 rpm for 24 h.

[0035] A 10% inoculum was introduced into a 10L fermenter, and the temperature was controlled at 65℃ with a ventilation rate of 5 L / h. The dissolved oxygen cascade rotation speed was adjusted to DO of 10%, 20%, 30%, and 50%, respectively. The pH was adjusted to 7.5 with 5 mol / L NaOH, and the OD was measured when the residual sugar was depleted. 600 As shown in the table below:

[0036] Comparative Example 4 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of the -80℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65℃ and 220 rpm for 24 h.

[0037] Inoculate 10% of the feed into a 10L fermenter, maintain the temperature at 65℃, aeration at 5 L / h, and dissolved oxygen cascade speed to achieve DO of 30%. Adjust the pH to 7.5 with 5 mol / L NaOH. When residual sugar is depleted, add glucose at a final concentration of 10 g / L in a single feeding, and continue fermentation until residual sugar is depleted. At this point, measure the OD. 600 It is 36.6.

[0038] Comparative Example 5 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of the -80℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65℃ and 220 rpm for 24 h.

[0039] Inoculate 10% of the solution into a 10L fermenter, maintain the temperature at 65℃, aeration at 5 L / h, and dissolved oxygen cascade speed to achieve DO of 30%. Adjust the pH to 7.5 with 5 mol / L NaOH. When the residual sugar is 5 g / L, add 70% glucose mother liquor to maintain the residual sugar at 4-6 g / L. Stop fermentation when OD stops increasing. 600 It is 46.5.

[0040] Comparative Example 6 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of a -80 ℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65 ℃ and 220 rpm for 24 h.

[0041] Inoculate 10% of the solution into a 10L fermenter, maintain the temperature at 65℃, aeration at 5 L / h, and dissolved oxygen cascade speed to achieve DO of 30%. Adjust the pH to 7.5 with ammonia. When the residual sugar is 5 g / L, add a 70% glucose mother liquor to maintain the residual sugar at 4-6 g / L. Stop fermentation when OD stops increasing. At this point, OD... 600 It is 55.8.

[0042] Comparative Example 7 The fermentation medium consisted of 10 g / L peptone, 10 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, and 0.22 g / L Na2HPO4. Glycerol bacteria were taken out of the -80℃ freezer and inoculated into 250 mL / 2L seed medium. The culture was carried out at 65℃ and 220 rpm for 24 h.

[0043] Inoculate 10% of the culture into a 10L fermenter, maintain the temperature at 65℃, aeration at 5 L / h, and dissolved oxygen cascade speed to achieve DO of 30%. Adjust the pH to 7.5 with ammonia. When the residual sugar is 5 g / L, feed culture medium consisting of 50 g / L peptone, 50 g / L yeast extract, 500 g / L glucose, and 0.54 g / L MgSO4 is added to maintain the residual sugar at 4-6 g / L. Fermentation is stopped when the OD stops increasing. At this point, the OD... 600 It is 74.3.

[0044] Example 2 The obtained fermentation broth was filtered through a ceramic membrane and washed with 7 times the volume of water. The conductivity of the supernatant was 100 μs / cm. The bacterial suspension was diluted to OD=20 and homogenized three times under high pressure at 1200 MPa to obtain thermophilic thermophilic bacteria lysate.

[0045] Comparative Example 8 The obtained fermentation broth was centrifuged using a vertical centrifuge at a centrifugal force of 10,000 g. The fermentation broth needs to be diluted to an OD value of [missing value]. 600 =5 is required for centrifugation to obtain bacterial cells. The cells are then washed with 17 times the volume of fermentation broth water, and the obtained cells are diluted to OD0.05. 600High-pressure homogenization at 20 and 1200 MPa was performed once, twice, and three times, respectively, to obtain thermophilic thermocline lysates. The cell disruption rates are shown in the table below.

[0046] Example 10 Take the glycerol bacteria out of the -80℃ freezer and inoculate them into two 250 mL / 2L seed culture media. Incubate at 65℃ and 220 rpm for 24 h.

[0047] 10% inoculum was added to 10L fermenters containing initial sodium sulfite concentrations of 0, 50, 100, 150, and 200 mg / L, respectively. The temperature was controlled at 65 °C, aeration at 5 L / h, and dissolved oxygen cascade rotation speed to maintain DO ≥ 30%. The pH was adjusted to 7.0 with ammonia. Feeding was started when the residual sugar reached 5.0 g / L, consisting of 80 g / L peptone, 80 g / L yeast extract, 500 g / L glucose, and 0.54 g / L MgSO4, maintaining the residual sugar at 4-6 g / L. After 40 h of fermentation, the cell OD was measured. 600 SOD activity in thermophilic bacteria.

[0048] Example 11 The bacterial cells obtained from fermentation with sodium sulfite at a concentration of 100 mg / L (SOD activity of 434.6 U / mL) in Example 10, and the bacterial cells obtained from fermentation without sodium sulfite (SOD activity of 362.4 U / mL) in Example 10, were used to obtain lysates according to Example 2. The absorbance was measured at 260 nm, and the nucleic acid content was calculated to be approximately 2400 ppm. 20% butanediol and 1% hexanediol were added to prepare lysates with a nucleic acid content of 2000 ppm, respectively. The lysates were placed at room temperature for one month to compare the nucleic acid concentration.

[0049] One month later, the nucleic acid content in the lysate with high SOD activity was 1952 ppm, while the nucleic acid content in the lysate with low SOD activity was 1459 ppm.

[0050] 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 it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing fermentation lysate of thermophilic bacteria, characterized in that: include, Thermophilic bacteria were inoculated into seed culture medium and cultured to obtain seed culture solution; The seed culture was inoculated into the initial culture medium and cultured. When the residual sugar reached 4-6 g / L, feed culture medium was added to maintain the residual sugar at 4-6 g / L. Fermentation was carried out for 30-40 h to obtain the fermentation broth. The obtained fermentation broth was filtered through a ceramic membrane, washed with 7 times the volume of water, and the conductivity of the supernatant was 100 μs / cm. The bacterial suspension was diluted to OD=20 and homogenized under high pressure 1-3 times to obtain thermophilic thermocline lysate. The initial culture medium formulation is as follows: peptone 5 g / L, yeast extract 5 g / L, CaSO4·2H2O 0.12 g / L, MgSO4·2H2O 0.2 g / L, NaCl 0.016 g / L, KNO3 0.21 g / L, Na2HPO4 0.22 g / L, and sodium sulfite 50~150 mg / L; Thermophilic thermophile, suggested classification name: Thermus thermophilus RTNB324001 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252066, depositary institution: China Center for Type Culture Collection, deposit date: September 19, 2025.

2. The preparation method according to claim 1, characterized in that: The seed culture medium is formulated as follows: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl.

3. The preparation method according to claim 1 or 2, characterized in that: The bacterial content (OD) in the seed culture 600 =6.0±0.

2.

4. The preparation method according to claim 1, characterized in that: The initial culture conditions were as follows: temperature controlled at 55-75℃, ventilation at 5 L / h, dissolved oxygen cascade rotation speed to ensure DO ≥ 30%, pH adjusted to 7.0 with ammonia, and seed culture inoculation amount of 2%-15%.

5. The preparation method according to claim 1, characterized in that: The feed culture medium formula is: 80 g / L peptone, 80 g / L yeast extract, 500 g / L glucose and 0.54 g / L MgSO4.

6. The preparation method according to claim 1, characterized in that: The ceramic membrane is filtered with 7 times the volume of water, and the high-pressure homogenization pressure is 1200MPa.

7. A method for improving the superoxide dismutase activity of fermentation lysates of thermophilic bacteria, characterized in that: This includes culturing Thermophilus in an initial culture medium to increase the superoxide dismutase activity of Thermophilus fermentation lysate; The initial culture medium formulation is as follows: 5 g / L peptone, 5 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, 0.22 g / L Na2HPO4, and 50~150 mg / L sodium sulfite.

8. A method for improving the stability of nucleic acids in the fermentation lysate of thermophilic bacteria at room temperature, characterized in that: This includes culturing Thermophilus in an initial culture medium to improve the stability of nucleic acids in Thermophilus fermentation lysates at room temperature; The initial culture medium formulation is as follows: 5 g / L peptone, 5 g / L yeast extract, 0.12 g / L CaSO4·2H2O, 0.2 g / L MgSO4·2H2O, 0.016 g / L NaCl, 0.21 g / L KNO3, 0.22 g / L Na2HPO4, and 50~150 mg / L sodium sulfite.

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