Method for regulating high-yield heat shock protein of Thermus thermophilus, product and application

Abstract

The invention discloses a method for regulating high-yield heat shock protein of Thermus thermophilus as well as a product and application thereof. The method at least comprises the following steps: (1) low-temperature and high-oxygen starting: starting inoculated thermophilic thermophilic bacteria in a fermentation tank in a low-temperature and high-dissolved-oxygen environment to promote thalli of the thermophilic thermophilic bacteria to quickly enter a logarithmic phase; (2) temperature rise and oxygen reduction induction: synchronously and linearly reducing dissolved oxygen and raising the temperature; (3) heat shock circulation: carrying out periodic heat shock circulation, and repeatedly strengthening the cross stress signal; each heat shock cycle sequentially comprises a heating section, a maintaining section, a cooling section and a low-temperature section; and (4) carrying out low-temperature centrifugal collection, rapidly cooling after the step (3) is finished, and carrying out centrifugal collection, so as to obtain the heat shock protein-rich Thermus thermophilus sludge. The method disclosed by the invention can synchronously amplify the biomass of the thallus of the thermophilic thermophilic bacteria and the yield of the heat shock protein (HSP70).

Description

TECHNICAL FIELD

[0001] The present application relates to a method for regulating high-yield heat shock proteins of Thermus thermophilus, belonging to the technical field of biological medicine. BACKGROUND

[0002] Thermus thermophilus is an extreme thermophilic bacterium that can grow efficiently in the temperature range of 55-80℃, widely distributed in hot springs, hydrothermal vents and other high-temperature environments. Its genome, membrane lipid and enzyme system are highly "heat adapted": the GC content in DNA is as high as 68-72%, tRNA and rRNA contain a large number of GC base pairs; membrane lipids are mainly long-chain, high-saturation and branched fatty acids, which can maintain a liquid crystal state at high temperatures; the protein surface is rich in charged and aromatic amino acids, and the core is more tightly packed, thereby maintaining conformational stability at high temperatures. Thermus thermophilus has a high fermentation rate and high biomass, and can accumulate active substances such as heat-stable SOD, glutathione, B vitamins, polypeptides and nucleic acid derivatives in cells.

[0003] Thermus thermophilus-derived active substances have been widely used in the cosmetics industry, mainly including fermentation products (Thermus thermophilus ferment), heat-stable enzymes and photolyase (INCI: sr-Thermus Thermophilus Polypeptide-1).

[0004] The fermentation product is composed of small molecule amino acids, natural moisturizing factor (NMF), polypeptides, SOD, catalase, glutathione, nicotinamide mononucleotide (NMN) and trace elements Se / Zn, etc. The currently disclosed effects of the fermentation product include: Antioxidant: scavenging superoxide anion, hydroxyl radical, inhibiting lipid peroxidation; Whitening: inhibiting tyrosinase activity, reducing melanin production (in vitro IC50≈0.8 mg / mL); Repair: promoting fibroblast proliferation and collagen type I / IV expression, increasing cell survival rate by more than 30% after UVB damage; Anti-inflammatory: reducing IL-6 and TNF-α levels, relieving erythema and stinging.

[0005] The heat-stable enzyme is mainly superoxide dismutase (SOD), and the half-life of Thermus thermophilus SOD is >2h at 90℃, and it remains active in the pH range of 4-10. The main effects are antioxidant, anti-pollution and post-sun emergency, often combined with vitamin C and E to restore skin oxidation-reduction homeostasis.

[0006] The mechanism of action of photolyase is to repair cyclobutane pyrimidine dimers (CPD) in DNA by photoactivation, reversing UV-induced mutations. It is mainly used as a "DNA repair enzyme" active ingredient in cosmetics.

[0007] Heat-shock proteins (HSPs) are a class of molecular chaperones that are synthesized rapidly when cells are subjected to high temperatures or other stresses, helping proteins to fold correctly, preventing aggregation, repairing or degrading denatured proteins. According to molecular weight, they can be divided into HSP100, HSP90, HSP70, HSP60 and small HSP (sHSP) families. They bind to exposed hydrophobic fragments in an ATP-dependent or ATP-independent manner, helping new peptide chains to fold, transporting old proteins, and breaking down irreparable aggregates, thereby maintaining intracellular protein homeostasis and significantly improving cell tolerance to heat, oxidation, toxins and other stresses.

[0008] Heat-shock proteins are considered "protein-level molecular chaperones" in the cosmetics industry, forming a heat-oxidation double protective network on the skin surface: on the one hand, their oligomeric structure can encapsulate and stabilize collagen, enzymes and signal peptides that are susceptible to high temperature, blue light or free radical attack, reducing denaturation and aggregation caused by urban "thermal aging" and light pollution; on the other hand, small HSPs can penetrate the stratum corneum and help repair denatured DNA and mitochondrial proteins in cells, increasing the "self-healing" rate of post-sun and overworked skin, and thus are developed as core active ingredients in high-end skincare formulations for anti-thermal stress, anti-blue light, post-sun first aid, and medical aesthetic wound repair.

[0009] Although Thermus thermophilus is favored by the cosmetics industry due to its "extreme heat tolerance" in fermentation-based active ingredients, small heat-shock proteins are almost treated as "heat tolerance background values" in academic literature, patents and record information, and are not individually enriched, characterized or claimed for efficacy, resulting in a long-term gap in the "research without products, activity without applications" of this high-potential raw material that can quantify, record and target damaged proteins. SUMMARY

[0010] The purpose of the present application is to provide a method for regulating the high production of heat-shock proteins by Thermus thermophilus. It can simultaneously amplify the biomass of Thermus thermophilus and the yield of heat-shock proteins (HSP70). On the other hand, according to the method, a slurry product can be obtained at low cost and high activity, which can be used to prepare cosmetics with the effect of repairing thermal aging and oxidative damage.

[0011] The technical solution of the present application is a method for regulating the high production of heat-shock proteins by Thermus thermophilus, which at least comprises the following steps: ① Low-temperature high-oxygen start-up, low-temperature and high-dissolved oxygen environment is used to start the inoculated Thermus thermophilus in the fermenter, so as to make the Thermus thermophilus enter the logarithmic growth phase quickly; this step can make the Thermus thermophilus grow quickly to obtain high-density seed; ② Temperature rise and oxygen reduction induction, simultaneously linearly reduce the dissolved oxygen and increase the temperature; use the "temperature difference-oxygen difference" double signal to complete the HSP70 transcription switch; ③ Heat shock cycle, periodic heat shock cycle is carried out to repeatedly strengthen the cross-stress signal; each heat shock cycle includes a temperature rise section, a holding section, a temperature drop section and a low-temperature section; this step forms repeated protein denaturation-renaturation requirements, and continuously amplifies the accumulation of HSP70; ④ Low-temperature centrifugal collection, after step ③ is completed, quickly reduce the temperature and centrifugal collection to obtain Thermus thermophilus slurry rich in heat shock proteins.

[0012] As preferred, in the step ①, the inoculation amount of Thermus thermophilus is 10% volume percentage, the low-temperature temperature is controlled at 55℃, the dissolved oxygen is maintained at ≥ 80%, and the duration is 5-6H.

[0013] As preferred, in the step ②, the linear temperature rise speed is 0.5-1.0℃ / min, and the dissolved oxygen is reduced to 30%.

[0014] As preferred, in the step ③, the holding section controls the temperature to maintain at 78-85℃; the low-temperature section controls the temperature to maintain at 60℃.

[0015] As preferred, the cycle number of the step ③ is 4-8 times.

[0016] As preferred, in the step ④, the quick cooling speed is reduced to 30℃ within 5min.

[0017] As preferred, the tank pH of the step ① is maintained at 6.8-7.0.

[0018] As preferred, the inoculated strain in the step ① is Thermus thermophilus ZHH-88, which has been preserved in China Center for Type Culture Collection (CCTCC) on December 12, 2025, and the preservation number is CCTCC M 20252862.

[0019] The product obtained according to the foregoing method is the Thermus thermophilus slurry rich in heat shock proteins.

[0020] The application of the product in the preparation of cosmetics with the effect of repairing thermal aging and oxidative damage, by quantitatively adding the product, the cosmetics have the effect of repairing thermal aging and oxidative damage.

[0021] Compared with the prior art, the present application is based on a coupling fermentation process of "low-temperature high-oxygen rapid propagation-linear temperature rise and oxygen stress-periodic heat shock cycle", which pushes the Thermus thermophilus to a double peak of high cell concentration and high intracellular content of heat shock protein HSP70 by precisely controlling the timing changes of temperature and dissolved oxygen, obtains protein-level skin care raw materials with the functions of repairing thermal aging and oxidative damage, which can be quantitatively added, with low cost and high activity, significantly simplifies the subsequent purification steps, and meets the needs of the cosmetic industry for efficient, compliant and sustainable active substances. DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with examples, but not as the basis for limiting the present application.

[0023] Example 1: Strain and seed preparation 1.1 Strain: Thermus thermophilus ZHH-88 with high yield of HSP70, which was deposited in China Center for Type Culture Collection (CCTCC) on December 12, 2025, with the accession number CCTCC M20252862.

[0024] 1.2 Solid activation: TB plate (containing 0.8% yeast powder, 0.4% peptone, 0.2% NaCl, 2% agar, pH 7.2 by mass percentage) was incubated at 70°C for 24 h.

[0025] 1.3 Primary seed: a single colony was inoculated into 100 mL of TB medium, and incubated at 70°C and 200 rpm until OD600≈1.0.

[0026] 1.4 Secondary seed: 1 L of TB was inoculated with 5% (v / v) of the primary seed, and incubated under the same conditions until OD600≈2.5 (about 6 h), as the seed for the fermenter.

[0027] 2 Preparation of 5 L fermenter 2.1 Liquid loading amount: 3 L of optimized medium: yeast powder 12 g / L, peptone 8 g / L, NaCl 4 g / L, glycerol 5 g / L, KH2PO41 g / L, MgSO4•7H2O 0.5 g / L, FeCl3•6H2O 0.01 g / L, pH 7.2 (121°C sterilization for 20 min).

[0028] 2.2 Dissolved oxygen electrode, temperature electrode, pH electrode, feeding needle, sampling valve online sterilization.

[0029] 3 Inoculation and Phase-I low temperature high oxygen start-up 3.1 Inoculum: 10% (v / v).

[0030] 3.2 Control parameters: temperature 55 °C, agitation 400 rpm, aeration 3 L / min (about 1 vvm), dissolved oxygen linkage to keep ≥ 80%, pH 7.2 (automatic 2M NaOH / 2M HC1 supplement).

[0031] 3.3 Duration: 6 h.

[0032] 4 Phase-II stepwise temperature increase-oxygen decrease induction 4.1 Temperature: linearly increased to 75 °C in 30 min.

[0033] 4.2 Dissolved oxygen: linkage decreased to 30% (achieved by decreasing aeration to 1 L / min and agitation to 250 rpm).

[0034] 4.3 Feed start-up: fed-batch 50% (w / v) glucose-yeast powder (3:1) mixture at a rate of 8 mL / h.

[0035] 4.4 Duration: 2 h.

[0036] 5 Phase-III periodic heat-shock cycle (core strengthening phase) 5.1 Cycle procedure: - Temperature increase segment: 10 min from 75 °C → 80 °C; - Holding segment: 80 °C for 15 min; - Temperature decrease segment: 10 min from 80 °C → 60 °C; - Low temperature segment: 60 °C for 15 min; This is one cycle, 50 min in total, and 6 cycles are continuously executed (total 5 h).

[0037] 5.2 Dissolved oxygen: automatically controlled to fixed 30% during the cycle, and agitation linearly changed from 150 → 350 rpm automatically with temperature.

[0038] 5.3 pH: maintained at 7.2 ± 0.05.

[0039] 6 End and cell harvesting 6.1 Fermentation end point: the end of the 6th cycle (total fermentation time about 13 h).

[0040] 6.2 Rapid cooling: drop to 30℃ within 5 min by cooling water, stop stirring, stop aeration.

[0041] 6.3 Centrifugation: 4℃, 8000 x g, 10 min, collect the cell paste, store at low temperature.

[0042] Temperature control at each stage is shown in Table 1.

[0043] 7 Analysis 7.1 DCW: dry the cell paste at 105℃ to constant weight.

[0044] 7.2 HSP70 concentration: commercial human / bacterial HSP70 ELISA kit (cross- reaction <3%), unit: mg / g DCW.

[0045] 8 Test data 8.1 Data record: record the DCW, HSP70, etc. after the fermentation is completed.

[0046] Table 1. Temperature control table at each stage Table 2. Dry weight of the cell and the content of heat shock protein HSP70 after the fermentation is completed Example 2: Verify the marginal effect of faster temperature rise and less cycles on enzyme production compared with Example 1; the balance between acetic acid inhibition and HSP70 folding in slightly acidic environment.

[0047] 1 Equipment and basic parameters (same as Example 1) 1.1 5 L borosilicate glass jacketed tank, working volume: 3 L.

[0048] 1.2 Probe, electrode, blue light LED configuration same as Example 1.

[0049] 2 Medium (g / L - ): yeast powder 10 (same as Comparative Scheme 1, below.-16%), peptone 7 (-12%), glucose 6 (+20%, slow-release carbon source), NaCl 4, KH2PO4 1, MgSO4•7H2O 0.5, FeCl3•6H2O 0.01. Initial pH: 6.5 (-0.7 units, slightly acidic start) 3 Online control strategy 3.1 Inoculation and Phase-I low temperature and high oxygen start; 3.2 Inoculation amount: 10% (v / v); 3.3 Temperature: 55℃; 3.4 Dissolved oxygen set: 85% (+5%); 3.5 Agitation: 400 rpm → 450 rpm (+50 rpm); 3.6 Aeration: 3 L / min → 3.5 L / min (+0.5 L / min, ~1.15 vvm); 3.7 Duration: 5 h (-1 h, shorten the log phase); 4 Phase-II Ladder Temperature-Up - Oxygen-Down Induction 4.1 Temperature-up rate: 1 ℃ / min (+0.5 ℃ / min, faster temperature-up); 4.2 End-point temperature: 75 ℃; 4.3 Dissolved oxygen down to: 30%; 4.4 Feeding: 50% glucose-yeast powder (3:1), initial flow rate 10 mL / h (+25%).

[0050] 5 Phase-III Periodic Heat-Shock Cycle (Core Adjustment) 5.1 High-temperature segment: 82 ℃ (+2 ℃, stronger induction); 5.2 Holding time: 12 min (-3 min, shorten the damage); 5.3 Low-temperature segment: 60 ℃, 12 min (-3 min, symmetrically shorten); 5.4 Temperature-up / down time: 10 min / 10 min (unchanged); 5.5 Cycle number: 5 times (-1 time, total length 4 h 10 min); 5.6 Dissolved oxygen: fixed at 30%, agitation automatically varied with temperature 150-350 rpm.

[0051] 6 End and Harvest 6.1 Total fermentation time: 11.5 h (-1.5 h); 6.2 Rapid temperature-down: 30 ℃ within 5 min, rest same as Example 1.

[0052] 7 Analysis and Detection 7.1 Same as Example 1; additionally measure acetic acid, reducing sugar, FAD shedding rate.

[0053] 8, Experimental Results 8.1 DCW: 5.3 g / L -1 (reduced by 0.7 g / L relative to Example 1 -1 ); 8.2 HSP70: 24 mg / g -1 DCW (increased by 2 mg / g relative to Example 1-1 DCW, indicating that high temperature and short time are conducive to folding); 8.3 Acetic acid: 0.4 g / L - ¹ (0.2 g / L less than Example 1 - ¹, indicating that slightly acidic + short cycle can inhibit by-product); 8.4 Enzyme specific activity: HSP70 specific activity is about 8% higher than Example 1, and the total fermentation cycle is shortened by 11%.

[0054] Comparative Example 1: 5 L fermenter constant culture: 70 °C + 60% dissolved oxygen (the rest of the operation is exactly the same as the experimental group) 1 Strain and two-stage seed 1.1 Strain: high-yield HSP70 Thermus thermophilus ZHH-88, which was deposited in China Center for Type Culture Collection (CCTCC) on December 12, 2025, with the accession number CCTCC M20252862.

[0055] 1.2 Solid activation: TB plate (0.8% yeast powder, 0.4% peptone, 0.2% NaCl, 2% agar, pH 7.2) 70 °C static culture for 24 h.

[0056] 1.3 Primary seed: pick a single colony and inoculate into 100 mL TB medium, 70 °C, 200 rpm, to OD600≈1.0.

[0057] 1.4 Secondary seed: transfer 1 L TB at 5% (v / v) inoculation amount, and culture under the same conditions to OD600≈2.5 (about 6 h), as a fermenter seed.

[0058] 2 Tank preparation 2.1 Liquid loading amount: 3 L optimized medium: yeast powder 12 g / L, peptone 8 g / L, NaCl 4 g / L, glycerol 5 g / L, KH2PO4 1 g / L, MgSO4•7H2O 0.5 g / L, FeCl3•6H2O 0.01 g / L, pH 7.2 (121 °C sterilization for 20 min).

[0059] 2.2 Dissolved oxygen electrode, temperature electrode, pH electrode, feeding needle, sampling valve online sterilization.

[0060] 3 Inoculation 3.1 Inoculation amount 10% (v / v).

[0061] 3.2 Initial parameters: temperature set directly to 70 °C, agitation 350 rpm, aeration 2 L / min; dissolved oxygen electrode linkage, target value 60% (±5%), pH 7.2 ± 0.05.

[0062] 4 Steady-state culture throughout (no thermal shock) 4.1 Temperature: constant 70 °C, no ramp, no cycle.

[0063] 4.2 Dissolved oxygen: linked control at constant 60%; if dissolved oxygen varies, automatically increase according to the same strategy as in Example 1.

[0064] 4.3 Feed: glucose-yeast powder feed rate (50% mixed feed), initial flow rate 8 mL / h.

[0065] 4.4 Total culture time: about 13 h.

[0066] 5 Harvest and cooling 5.1 13 h cooling with cooling water, to 30 °C in 5 min.

[0067] 5.2 Centrifugation at 4 °C, 8,000 x g, 10 min, to collect the bacteria, stored at -80 °C.

[0068] 6 Test items and determination 6.1 DCW: oven dry the slurry at 105 °C to constant weight.

[0069] 6.2 HSP70 concentration: commercial human / bacterial HSP70 ELISA kit (adapted cross-reaction <3%), unit mg / g DCW.

[0070] 7 Test data 7.1 Data recording: record DCW, HSP70, etc. after fermentation is complete.

[0071] Table III. Temperature control table by phase Table IV. Dry weight of bacteria and heat shock protein HSP70 content after fermentation is complete Summary: both Example 1 and 2 have significant improvements in DCW and HSP70 concentration.

[0072] Efficacy experiment Conclusion: Using the product of Example 1, at an addition level of 0.1 % (w / w) in cosmetics, the survival rate of keratinocyte model at 43 °C can be increased by > 38 %, and protein carbonylation can be reduced by > 42 %, which is suitable for after-sun repair, anti-blue light, and medical aesthetic wound care. The effective concentration window is 0.05 - 0.5 % (w / w).

Claims

1. A method for regulating the high production of heat shock proteins in thermophilic bacteria, characterized in that, At least the following steps are included: ① Low temperature and high oxygen start-up: The thermophilic bacteria inoculated in the fermenter are started up in a low temperature and high dissolved oxygen environment to promote the rapid entry of thermophilic bacteria into the logarithmic growth phase. ② Induced by raising temperature and lowering oxygen, simultaneously and linearly reducing dissolved oxygen and raising temperature; ③ Heat shock cycle: Perform periodic heat shock cycles to repeatedly reinforce cross-stress signals; each heat shock cycle includes a heating phase, a holding phase, a cooling phase, and a low-temperature phase in sequence. ④ Low-temperature centrifugation collection: After step ③, the sample is rapidly cooled and centrifuged to obtain thermophilic thermophilic bacteria sludge rich in heat shock proteins.

2. The method for regulating the high production of heat shock proteins in thermophilic bacteria according to claim 1, characterized in that: In step ①, the inoculum amount of thermophilic thermophilic bacteria is 10% by volume, the low temperature is controlled at 55℃, the dissolved oxygen is maintained at ≥ 80%, and the duration is 5-6 hours.

3. The method for regulating the high heat shock protein production of thermophilic bacteria according to claim 1, characterized in that: In step ②, the linear heating rate is 0.5-1.0℃ / min, and the dissolved oxygen is reduced to 30%.

4. The method for regulating the high production of heat shock proteins in thermophilic bacteria according to claim 1, characterized in that: In step ③, the temperature of the maintenance section is maintained at 78-85℃; the temperature of the low-temperature section is maintained at 60℃.

5. The method for regulating the high production of heat shock proteins in thermophilic bacteria according to claim 4, characterized in that: The number of cycles for step ③ is 4-8.

6. The method for regulating the high heat shock protein production of thermophilic bacteria according to claim 1, characterized in that: In step ④, the rapid cooling rate is to reduce the temperature to 30°C within 5 minutes.

7. The method for regulating the high production of heat shock proteins in thermophilic bacteria according to claim 1, characterized in that: In step ①, the pH inside the tank is maintained at 6.8-7.

0.

8. The method for regulating the high production of heat shock proteins in thermophilic bacteria according to claim 1, characterized in that: The bacterial strain inoculated in step ① was Thermostylophora inerminosa ZHH-88, with the preservation number CCTCC M 20252862.

9. The product obtained by the method according to any one of claims 1-8, characterized in that: The product is a thermophilic bacterium sludge rich in heat shock proteins.

10. The use of the product of claim 9 in the preparation of cosmetics having the function of repairing thermal aging and oxidative damage.

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