Method for co-producing microbial polysaccharide and beta-carotene through mixed bacteria fermentation

Abstract

The invention discloses a method for co-production of microbial polysaccharide and beta-carotene by fermentation of mixed bacteria, and belongs to the technical field of bioengineering.The microbial polysaccharide and beta-carotene are co-produced by fermentation of bacillus velezensis LT-2 and Exiguobacterium acetyl-38, the fermentation process preferably uses sucrose as a carbon source and yeast extract powder as a nitrogen source, and the fermentation process preferably uses sucrose as a carbon source and Exiguobacterium acetyl-38 as a nitrogen source. Microbial polysaccharide and beta-carotene are obtained through fermentation, and the yield of the microbial polysaccharide and the yield of the beta-carotene in the co-production process reach 12.54 g / L and 1.28 g / L respectively.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and more specifically, relates to a method for co-producing microbial polysaccharides and beta-carotene by mixed bacteria fermentation. Background Art

[0002] Microbial polysaccharides are highly active macromolecular substances produced by microorganisms during the fermentation process. They have multiple biological functions, such as antioxidant, antiviral, anti-tumor and immune enhancement, and have broad application prospects in the fields of health care products and medicine. In addition, microbial extracellular polysaccharides have thickening and emulsifying properties and are widely used in the food industry.

[0003] β-carotene is an orange-yellow fat-soluble compound and the most stable natural pigment that exists in nature. Studies have shown that β-carotene has nutritional and health-care effects and antioxidant effects. It can also be converted into vitamin A in the body under certain conditions, and has the effects of protecting vision, preventing night blindness and dry eyes. Therefore, it has a wide range of applications in food, cosmetics, medicine and other fields.

[0004] Microbial polysaccharides have multiple biological activities and have great application prospects in the fields of food and medicine. However, both the fermentation process and the extraction and separation process of microbial polysaccharides require a long time. For example, fermentation requires several days or even weeks to reach the maximum yield of polysaccharides, and the multiple precipitation, centrifugation, purification and other steps in the extraction process are also very time-consuming, resulting in high production costs and low production efficiency. The main methods for producing β-carotene are chemical synthesis, natural product extraction and microbial fermentation, but the products produced by chemical synthesis have low activity and the products produced by natural product extraction have high costs. β-carotene produced by microbial fermentation has high activity, high yield and strong stability.

[0005] After searching, there is no report on the co-production of microbial polysaccharides and β-carotene by mixed fermentation of Bacillus velez and Exiguobacterium acetylicum. In addition, the production of microbial polysaccharides and pigments reported so far has the problems of high production cost and complicated extraction steps, making industrial production difficult. The present invention reports for the first time a method for co-production of microbial polysaccharides and β-carotene by mixed fermentation of Bacillus velez and Exiguobacterium acetylicum and a new extraction method, which reduces the production cost of microbial polysaccharides and β-carotene, combines the extraction steps to simplify the time cost, and also increases the final yield of microbial polysaccharides and β-carotene. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a method for co-producing microbial polysaccharides and β-carotene by mixed bacteria fermentation, which can directly ferment to obtain the two products, avoiding the tediousness of separate fermentation and effectively increasing the yield of the two products.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for co-producing microbial polysaccharides and beta-carotene by mixed bacteria fermentation is characterized in that Bacillus velezensis LT-2 and Exiguobacterium acetylicum LT-38 are inoculated into a fermentation medium for aerobic culture to prepare microbial polysaccharides and beta-carotene.

[0009] Specifically, Bacillus velezensis LT-2 and Exiguobacterium acetylicum LT-38 were activated on LB solid medium, transferred to seed liquid, and then the seed liquid was inoculated into fermentation medium for aerobic culture.

[0010] It should be noted that the Bacillus velezensis LT-2 described in the present invention has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M2019904 and a deposit date of November 7, 2019.

[0011] It should be noted that the E. acetylicum LT-38 described in the present invention has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20231647 and the deposit date being September 7, 2023.

[0012] Preferably, the fermentation medium comprises the following components: 10-50 g / L of carbon source, 5-25 g / L of nitrogen source, 0.001-8 g / L of metal salt, 15 g / L of Tween 80, pH value 6.0-8.0, and the solvent is water.

[0013] Preferably, the carbon source is any one or a combination of Jerusalem artichoke powder, sucrose, sugarcane juice, lactose, maltose, fructose and glucose.

[0014] More preferably, the carbon source is sucrose, and the added amount of the sucrose is 30 g / L.

[0015] Preferably, the nitrogen source is any one or a combination of peanut meal, soy protein isolate, yeast extract, peptone, tryptone, beef extract, ammonium sulfate, urea, ammonium chloride, and ammonium nitrate.

[0016] More preferably, the nitrogen source is yeast extract powder, and the addition amount of the yeast extract powder is 15 g / L.

[0017] Preferably, the metal salt is one or a combination of magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, manganese sulfate, and calcium chloride.

[0018] More preferably, the potassium hydrogen phosphate, calcium chloride, magnesium sulfate and manganese sulfate are 0.001-10 g / L; the calcium chloride is preferably 0.3-0.6 g / L, the magnesium sulfate is preferably 0.2-0.6 g / L, and the manganese sulfate is preferably 0.002-0.004 g / L.

[0019] Preferably, the fermentation medium comprises the following components: 20-40 g / L sucrose, 10-20 g / L yeast extract, 0.3-0.6 g / L calcium chloride, 0.2-0.6 g / L magnesium sulfate, 0.002-0.004 g / L manganese sulfate, 15 g / L Tween 80, and the balance is water, and the initial pH of the fermentation medium is adjusted to 6.5-7.5 using sodium hydroxide.

[0020] More preferably, the fermentation medium comprises the following components: 30 g / L sucrose, 15 g / L yeast extract, 0.5 g / L calcium chloride, 0.4 g / L magnesium sulfate, 0.003 g / L manganese sulfate, 15 g / L Tween 80, and the balance is water, and the initial pH of the fermentation medium is adjusted to 7.0 using sodium hydroxide.

[0021] Preferably, the inoculation volumes of Bacillus velezensis LT-2 and Exiguobacterium acetylicum LT-38 are 0.8% (v / v) to 1.2% (v / v) and 0.8% (v / v) to 1.2% (v / v).

[0022] Specifically, the inoculation volume ratio of the Bacillus velezensis LT-2 and the E. acetylicum LT-38 is (0.8-1.2):1.

[0023] More preferably, the inoculation volumes of Bacillus velezensis LT-2 and Exiguobacterium acetylicum LT-38 are 1% (v / v) and 1% (v / v).

[0024] In some preferred embodiments, the activation on LB solid medium is to inoculate three zones of the strain from the glycerol tube stored at -80°C on LB solid medium and place it in a 30°C incubator for 24 hours for activation.

[0025] Preferably, the LB solid culture medium comprises: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract powder, 20 g / L agar powder, and pH 6.5.

[0026] In some preferred embodiments, the transfer to the seed liquid is to aseptically scrape 1 to 2 loops of bacterial mud from the activated strain into the liquid seed culture medium using an inoculation loop, and culture the culture medium on a shaker at 30°C and 200 rpm for 12 hours.

[0027] Preferably, the liquid seed culture medium comprises: 15-25 g / L sucrose, 5-15 g / L tryptone, 5-15 g / L sodium chloride, 3-8 g / L yeast extract powder, and a pH of 6.0-7.0.

[0028] More preferably, the liquid seed culture medium comprises: 20 g / L sucrose, 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract powder, and a pH of 6.5.

[0029] In some preferred embodiments, the aerobic culture is specifically to place the fermentation liquid on a shaker at a temperature of 32° C. and a rotation speed of 200 rpm, culture for 12 to 24 hours, and then stop the fermentation.

[0030] The present invention also provides a method for the combined extraction of microbial polysaccharides and β-carotene, comprising the following steps:

[0031] S11. The fermentation broth was centrifuged to obtain bacterial precipitate and fermentation supernatant respectively;

[0032] S12. adding methanol solution (pH=3) to the bacterial precipitate, ultrasonically assisted extraction for 15 to 25 minutes, extracting overnight, and centrifuging to obtain a crude pigment extract;

[0033] S13. The fermentation supernatant in step S11 is concentrated to 1 / 6 to 1 / 4 of the original volume, and 1 / 2 to 1 / 4 volume of Sevag reagent is added to the supernatant, and the protein is removed by shaking, and the mixture is repeated once to obtain a deproteinized supernatant;

[0034] S14. The pigment in the deproteinized supernatant is adsorbed by a pretreated macroporous resin at 30° C. for 24 h, and the macroporous resin adsorbing the pigment and the deproteinized and depigmented supernatant are separated by solid-liquid separation;

[0035] S15. Elute the macroporous resin adsorbing the pigment in step S14, mix the eluted pigment-containing eluate and the pigment crude extract in step S12 to obtain the total pigment crude extract, and then concentrate it, extract it, perform column chromatography, and freeze-dry it to obtain β-carotene; add 3 to 5 times of anhydrous ethanol to the deproteinized and depigmented supernatant in step S14, centrifuge to obtain the precipitate, and freeze-dry it to obtain microbial polysaccharides.

[0036] Specifically, the Sevag reagent in step S13 is chloroform and n-butanol in a volume ratio of 4:1.

[0037] Specifically, the pretreated macroporous resin in step S14 is a macroporous resin immersed in 5% (v / v) HCl for 6 hours to fully swell, and then washed with water until neutral; then immersed in 5% (v / v) NaOH for 6 hours to fully swell, and then washed with water until neutral; then immersed in 95% ethanol for 24 hours, the macroporous resin is washed with 95% ethanol until the solution is transparent, and then washed with water until there is no odor to obtain the pretreated macroporous resin.

[0038] Specifically, the macroporous resin includes one or a combination of AB-8, D101, DM-2, NAK-9 or D4020.

[0039] Specifically, the mass volume ratio of the pretreated macroporous resin and the deproteinized supernatant in step S14 is 3:100.

[0040] Specifically, the absorbance of pigments in deproteinized supernatants and deproteinized depigmented supernatants was measured, and the sugar content was measured at 490 nm using the phenol-sulfuric acid method.

[0041] Specifically, according to polysaccharide retention rate (%) = M 后 / M 前 ×100% to calculate the polysaccharide retention rate; where M 前 is the sugar content in the deproteinized supernatant, M 后 It is the sugar content in the deproteinized and depigmented supernatant.

[0042] Specifically, according to the pigment absorption rate (%) = (A 前 -A 后 ) / A 前 ×100% to calculate the pigment absorption rate; where A 前 is the absorbance of the deproteinized supernatant, A 后 is the absorbance value of the deproteinized and depigmented supernatant.

[0043] Preferably, the macroporous resin is D4020.

[0044] Specifically, the concentration in step S15 is to concentrate the total pigment crude extract into a paste; the extraction is to add 2 times the amount of ethyl acetate for extraction; the column chromatography is to use an eluent with a volume ratio of petroleum ether and acetone of 3:2 for elution, a silica gel mesh of 200-300 mesh, and filter through a 0.22 μm filter membrane.

[0045] Specifically, the β-carotene is detected by high performance liquid chromatography, chromatographic column: Sepax C18; mobile phase: acetonitrile: ammonium acetate (85:15); flow rate: 1.00 mL / min; injection volume: 10 μL; column temperature: 30°C.

[0046] Specifically, the microbial polysaccharide is prepared by adding 3 mL of 72% sulfuric acid to 0.1 g of microbial polysaccharide, mixing, and water bathing at 32°C for 60 minutes. After taking out, double distilled water is added to dilute to 5%, and mixed. After being treated at 121°C for 1 hour, the mixture is taken out and cooled, and the pH is adjusted to 6.5 to obtain a fermentation product hydrolyzate. The results show that the mixed bacteria fermentation product has a total of one monosaccharide component, glucose.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention provides a method for co-producing microbial polysaccharides and beta-carotene by mixed bacteria fermentation. One fermentation medium can produce two products at the same time, greatly reducing the production cost, combining and simplifying the polysaccharide and pigment extraction steps, making the operation simple, shortening the growth cycle, and increasing the final extraction amount. In the small batch shaking table culture stage, after 24 hours of fermentation, the final extraction amount of microbial polysaccharides is 12.54 g / L, and the final extraction amount of beta-carotene is 1.28 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The process route diagram of the mixed bacteria fermentation co-production of microbial polysaccharides and β-carotene of the present invention;

[0050] Figure 2 The effect of carbon source type on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation;

[0051] Figure 3 The effect of carbon source concentration on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation;

[0052] Figure 4 The effect of organic nitrogen source types on the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation;

[0053] Figure 5 The effect of organic nitrogen source concentration on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation;

[0054] Figure 6 The effect of inorganic nitrogen source types on the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation;

[0055] Figure 7 The effect of pH on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation;

[0056] Figure 8 The effect of temperature on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation;

[0057] Fig. 9The effects of different types of mixed bacteria fermentation on the co-production of β-carotene (mixed bacteria combination 1: Bacillus alantiba LP-1 and Exiguobacterium acetylicum LT-38; mixed bacteria combination 2: Coxsackia coli LT-1 and Exiguobacterium acetylicum LT-38; mixed bacteria combination 3: mixed fermentation of Bacillus velez LT-2 and Exiguobacterium acetylicum LT-38). DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0059] Figure 1 This is a flow chart of the mixed bacteria fermentation co-production of microbial polysaccharides and β-carotene of the present invention;

[0060] LB solid medium: tryptone 10g / L, sodium chloride 10g / L, yeast extract powder 5g / L, agar powder 20g / L, pH 6.5;

[0061] Liquid seed culture medium: sucrose 20g / L, tryptone 10g / L, sodium chloride 10g / L, yeast extract powder 5g / L, pH 6.5;

[0062] The seed liquid Bacillus velezensis LT-2 and the seed liquid E. acetylicum LT-38 described in the examples and comparative examples were prepared by the following method:

[0063] Bacillus velezensis LT-2 and E. acetylicum LT-38 from glycerol tubes preserved at -80°C were inoculated into LB solid culture medium by three-zone streaking, and the culture was placed in a 30°C incubator for 24 hours for activation. For the activated strains, 1 to 2 loops of bacterial mud were scraped out using an inoculation loop under sterile conditions and placed in a liquid seed culture medium, which was placed on a shaker at 30°C and 200 rpm for culturing for 12 hours to obtain seed liquid Bacillus velezensis LT-2 and seed liquid E. acetylicum LT-38.

[0064] Specifically, the Bacillus velezensis LT-2 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO:M2019904 and a deposit date of November 7, 2019.

[0065] The acetylicobacterium E.acetylicum LT-38 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO:M20231647 and a deposit date of September 7, 2023.

[0066] Seed liquid Bacillus alantiba LP-1 and seed liquid Coxsackie LT-1 (deposited in China Center for Type Culture Collection, with a deposit number of CCTCC No: M2017850 and a deposit date of December 29, 2017) were prepared according to the same method as above.

[0067] The microbial polysaccharides and β-carotene described in each embodiment and comparative example are obtained by the following steps:

[0068] S11. The fermentation broth was centrifuged to obtain bacterial precipitate and fermentation supernatant respectively;

[0069] S12. adding methanol solution (pH=3) to the bacterial precipitate, ultrasonically assisted extraction for 20 min, then extracting overnight, centrifuging, and obtaining a crude pigment extract;

[0070] S13. The fermentation supernatant in step S11 was concentrated to 1 / 5 of the original volume, and 1 / 3 volume of Sevag reagent was added to the supernatant, and the protein was fully removed by shaking, and the mixture was repeated once to obtain a deproteinized fermentation product solution supernatant;

[0071] S14., using pretreated macroporous resin D4020 (the macroporous resin is soaked in 5% (v / v) HCl for 6 hours to fully swell, and then washed with water to neutral; then soaked in 5% (v / v) NaOH for 6 hours to fully swell, and then washed with water to neutral; then soaked in 95% ethanol for 24 hours, the macroporous resin is washed with 95% ethanol until the solution is transparent, and then washed with water until there is no odor to obtain the pretreated macroporous resin) to adsorb the pigment in the deproteinized supernatant, adsorb at 30°C for 24 hours, and separate the solid and liquid to obtain the macroporous resin adsorbing the pigment and the deproteinized and depigmented supernatant; the mass volume of the pretreated macroporous resin and the deproteinized supernatant is The volume ratio is 3:100; S15. The macroporous resin adsorbing the pigment described in step S14 is eluted, and the eluted pigment-containing eluate and the pigment crude extract described in step S12 are mixed with the total pigment crude extract, and then concentrated to a paste, extracted with 2 times the volume of ethyl acetate, column chromatography (eluted with an eluent of petroleum ether and acetone in a volume ratio of 3:2, silica gel mesh number of 200-300 mesh, filtered through a 0.22μm filter membrane), and freeze-dried to obtain β-carotene; add 3-5 times anhydrous ethanol to the deproteinized and depigmented supernatant described in step S14, centrifuge to obtain the precipitate, and freeze-dry to obtain microbial polysaccharides.

[0072] Example 1 Optimization of carbon source types required for co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0073] This example illustrates the effect of different carbon sources on the yield of microbial polysaccharides and β-carotene produced by mixed fermentation. 1% of seed liquid Bacillus velezensis LT-2 and 1% of seed liquid E. acetylicum LT-38 were inoculated into a fermentation medium containing 20 g / L Jerusalem artichoke powder, sucrose, lactose, maltose, fructose and glucose as the sole carbon source (other ingredients: 10 g / L tryptone, 0.5 g / L calcium chloride, 15 g / L Tween, pH 6.5), and cultured in a shaking incubator at 30°C and 200 rpm for 24 h. The results are shown in FIG. Figure 2 As shown, it can be seen that when sucrose is used as the carbon source, the microbial polysaccharide yield and β-carotene yield are both high, the microbial polysaccharide extraction amount is as high as 5.57 g / L, and the β-carotene extraction amount is as high as 0.53 g / L.

[0074] Example 2 Optimization of the carbon source concentration required for the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0075] This example illustrates the effect of different carbon source concentrations on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation. Seed liquid Bacillus velezensis LT-2 was inoculated at 1% and seed liquid E. acetylicum LT-38 was inoculated at 1% in fermentation medium with sucrose concentrations of 10, 20, 30, 40 and 50 g / L (other ingredients: tryptone 10 g / L, calcium chloride 0.5 g / L, Tween 15 g / L, pH 6.5), and cultured at 30°C, 200 rpm for 24 h. The results are shown in FIG. Figure 3 As shown, when the selected sucrose concentration is 30g / L and 40g / L, the microbial polysaccharide production and β-carotene production of the strain are both high. Considering the fermentation cost, a sucrose concentration of 30g / L was selected as the carbon source concentration of the fermentation medium. At this time, the microbial polysaccharide extraction amount reached 7.10g / L, and the β-carotene extraction amount reached 0.61g / L.

[0076] Example 3 Optimization of organic nitrogen source types required for co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0077] This example illustrates the effect of different organic nitrogen sources on the yield of microbial polysaccharides and β-carotene produced by mixed bacteria fermentation. Seed liquid Bacillus velezensis LT-2 with an inoculum amount of 1% and seed liquid E. acetylicum LT-38 with an inoculum amount of 1% were inoculated into a fermentation medium containing 10 g / L peanut meal, soy protein isolate, yeast extract, peptone, tryptone, and beef extract as the only organic nitrogen source (other ingredients: 30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, pH 6.5), and cultured at 30°C, 200 rpm for 24 h. The results are as follows: Figure 4 As shown, it can be seen that when yeast extract powder is used as the organic nitrogen source, the microbial polysaccharide yield and β-carotene yield are both high, with the microbial polysaccharide extraction amount being 8.56 g / L and the β-carotene extraction amount being 0.81 g / L.

[0078] Example 4 Optimization of organic nitrogen source concentration required for co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0079] This example illustrates the effect of different yeast extract concentrations on the yield of microbial polysaccharides and β-carotene produced by mixed fermentation. Seed liquid Bacillus velezensis LT-2 was inoculated at 1% and seed liquid E. acetylicum LT-38 was inoculated at 1% in fermentation medium with yeast extract concentrations of 5, 10, 15, 20, and 25 g / L (other ingredients: 30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, pH 6.5), and cultured at 30°C, 200 rpm for 24 h. The results are shown in FIG. Figure 5 As shown, the selected yeast extract concentration is above 15 g / L, and the strain's microbial polysaccharide production and β-carotene production are both high. Considering the cost, a yeast extract concentration of 15 g / L is selected as the nitrogen source concentration of the fermentation medium. At this time, the microbial polysaccharide extraction amount is 9.06 g / L, and the β-carotene extraction amount is 1.12 g / L.

[0080] Example 5 Optimization of the types of inorganic nitrogen sources required for the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0081] This example illustrates the effect of different inorganic nitrogen sources on the yield of microbial polysaccharides and β-carotene produced by mixed fermentation. 1% of seed liquid Bacillus velezensis LT-2 and 1% of seed liquid E. acetylicum LT-38 were inoculated into a fermentation medium containing no inorganic nitrogen source but 5 g / L ammonium chloride, ammonium sulfate, urea and ammonium nitrate (other ingredients: 30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, 15 g / L yeast extract, pH 6.5), and cultured at 30°C with shaking at 200 rpm for 24 h. The results are shown in FIG. Figure 6 As shown, when no inorganic nitrogen source was added, the microbial polysaccharide production and β-carotene production were both higher.

[0082] Example 6 Optimization of pH required for co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0083] This example illustrates the effect of different pH values ​​on the co-production of microbial polysaccharides and β-carotene by mixed bacteria fermentation. Seed liquid Bacillus velezensis LT-2 was inoculated at 1% and seed liquid E. acetylicum LT-38 was inoculated at 1% in fermentation medium (30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, 15 g / L yeast extract) with pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0 (adjusted by hydrogen chloride and sodium hydroxide), respectively. The culture was shaken at 30°C and 200 rpm for 24 h. The results are shown in FIG. Figure 7 As shown, it can be seen that when the pH is 7.0, the biopolysaccharide yield and β-carotene yield are both high, the microbial polysaccharide extraction is as high as 9.76 g / L, and the β-carotene extraction is as high as 1.21 g / L.

[0084] Example 7 Optimization of the temperature required for the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0085] This example illustrates the effect of different temperatures on the yield of microbial polysaccharides and β-carotene produced by mixed bacteria fermentation. 1% of seed liquid Bacillus velezensis LT-2 and 1% of seed liquid E. acetylicum LT-38 were inoculated into fermentation medium (30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, 15 g / L yeast extract, pH 7.0) at 26°C, 28°C, 30°C, 32°C and 34°C, respectively. The culture was shaken at 200 rpm for 24 h. The results are shown in FIG. Figure 8As shown, it can be seen that when the temperature is 32°C, the microbial polysaccharide yield and β-carotene yield are both high, the microbial polysaccharide extraction amount is as high as 11.54 g / L, and the β-carotene extraction amount is as high as 1.27 g / L.

[0086] Example 8 Optimization of the types and concentrations of metal salts required for the co-production of microbial polysaccharides and β-carotene by mixed bacterial fermentation

[0087] This example illustrates the effect of different metal salts and concentrations on the yield of microbial polysaccharides and β-carotene produced by mixed bacteria fermentation. Seed liquid Bacillus velezensis LT-2 with an inoculum of 1% and seed liquid E. acetylicum LT-38 with an inoculum of 1% were inoculated into fermentation medium containing the following metal salt concentrations (30 g / L sucrose, 0.5 g / L calcium chloride, 15 g / L Tween, 15 g / L yeast extract, pH 7.0), and cultured in a shaking table at 32°C and 200 rpm for 24 h. The results of a single factor variable experiment are as follows:

[0088] Ferrous sulfate: 0.10, 0.20, 0.30, 0.40, 0.50 g / L and control group, the optimal concentration of ferrous sulfate was 0 g / L (no addition);

[0089] Magnesium sulfate: 0.2, 0.4, 0.6, 0.8 and 1.0 g / L and control group, the optimal concentration of magnesium sulfate was 0.4 g / L;

[0090] Manganese sulfate 0.001, 0.002, 0.003, 0.004 and 0.006 g / L and control group, the optimal concentration of manganese sulfate was 0.003 g / L;

[0091] Calcium chloride: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60 g / L and control group, the optimal concentration of calcium chloride was 0.50 g / L.

[0092] Potassium dihydrogen phosphate: 1, 2, 3, 4, 5, and 6 g / L and the control group, the optimal concentration of potassium dihydrogen phosphate was 0 g / L (no addition);

[0093] Potassium hydrogen phosphate: 3, 6, 9, 12, and 15 g / L and the control group, the optimal concentration of potassium hydrogen phosphate was 0 g / L (no addition);

[0094] Under the optimal metal salt and concentration, the extraction amount of microbial polysaccharides reached 12.54 g / L; the extraction amount of β-carotene was 1.28 g / L.

[0095] Example 9 Optimization of macroporous resin types for extraction of microbial polysaccharides and β-carotene

[0096] This example illustrates the effect of different macroporous resin types on the extraction of microbial polysaccharides and β-carotene. The fermentation broth after fermentation in Example 8 was extracted using different macroporous resins. The polysaccharide retention rate (%) was calculated by 后 / M 前 ×100% to calculate the polysaccharide retention rate; where M 前 is the sugar content in the deproteinized supernatant, M 后 is the sugar content in the deproteinized and depigmented supernatant; specifically, according to the pigment absorption rate (%) = (A 前 -A 后 ) / A 前 ×100% to calculate the pigment absorption rate; where A 前 is the absorbance of the deproteinized supernatant, A 后 is the absorbance value of the deproteinized and depigmented supernatant. The specific results are shown in Table 1:

[0097] Table 1 Effect of different macroporous resins on the results

[0098] Macroporous resin Pigment absorption rate (%) Polysaccharide retention rate (%) AB-8 54.64±1.01 78.11±1.00 D101 62.32±1.25 64.63±1.02 DM-2 65.43±1.09 71.72±1.71 NAK-9 60.66±1.52 69.44±0.79 D4020 72.82±0.79 75.09±1.11

[0099] It can be seen from Table 1 that when the macroporous resin is D4020, the effect is best.

[0100] Comparative Example 1

[0101] In this comparative example, the seed solution of Bacillus velezensis LT-2 and the seed solution of E. acetylicum LT 38 were inoculated at an inoculum amount of 1% into a fermentation medium (sucrose 30 g / L, yeast extract 15 g / L, calcium chloride 0.5 g / L, magnesium sulfate 0.4 g / L, manganese sulfate 0.003 g / L, Tween 80 15 g / L), and the initial pH of the fermentation broth was adjusted to 7.0 using sodium hydroxide. The fermentation was carried out separately and placed on a shaking table at a temperature of 32° C. and a rotation speed of 200 rpm. After fermentation and cultivation for 24 hours, the fermentation broth was respectively extracted with microbial polysaccharides and β-carotene according to the above-mentioned extraction method. The final total extraction amounts of microbial polysaccharides and β-carotene in the two groups of products were 10.46 g / L and 0.98 g / L, which were significantly lower than 12.54 g / L and 1.28 g / L in the embodiment.

[0102] Comparative Example 2

[0103] The fermentation and extraction methods in this comparative example are the same as those in Example 8, except that the seed liquid of Bacillus alantiba LP-1 and the seed liquid of Exiguobacterium acetylicum LT-38 are used for fermentation.

[0104] Comparative Example 3

[0105] The fermentation and extraction methods in this comparative example are the same as those in Example 8, except that the seed liquid of Bacillus velezensis LT-1 and the seed liquid of Exiguobacterium acetylicum LT-38 are used for fermentation.

[0106] Effects of different mixed bacteria fermentation on β-carotene production Fig. 9 As shown in the figure, it can be seen that other strains and E. acetylicum LT-38 could not further increase the production of carotene.

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for co-producing microbial polysaccharides and β-carotene by mixed bacterial fermentation, characterized in that: Bacillus velezensis LT-2 and Exiguobacterium acetylicum LT-38 were inoculated into a fermentation medium for aerobic culture to prepare microbial polysaccharides and beta-carotene.

2. The method according to claim 1, characterized in that: The fermentation medium comprises the following components: 10-50 g / L of carbon source, 5-25 g / L of nitrogen source, 0.001-8 g / L of metal salt, 15 g / L of Tween 80, pH value of 6.0-8.0, and the solvent is water.

3. The method according to claim 1, characterized in that: The Bacillus velezensis LT-2 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2019904 and a deposit date of November 7, 2019; the E. acetylicum LT-38 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20231647 and a deposit date of September 7, 2023.

4. The method according to claim 1, characterized in that: The carbon source is any one of Jerusalem artichoke powder, sucrose, sugarcane juice, lactose, maltose, fructose, and glucose, or a combination of several of them; the nitrogen source is any one of peanut cake powder, soy protein isolate, yeast extract powder, peptone, tryptone, beef extract, ammonium sulfate, urea, ammonium chloride, and ammonium nitrate, or a combination of several of them; the metal salt is any one of magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, manganese sulfate, and calcium chloride, or a combination of several of them.

5. The method according to claim 1, characterized in that: The fermentation medium comprises the following components: 20-40 g / L sucrose, 10-20 g / L yeast extract, 0.3-0.6 g / L calcium chloride, 0.2-0.6 g / L magnesium sulfate, 0.002-0.004 g / L manganese sulfate, 15 g / L Tween 80, and the balance is water. The initial pH of the fermentation medium is adjusted to 6.5-7.5 by using sodium hydroxide.

6. The method according to claim 5, characterized in that: The fermentation medium comprises the following components: 30 g / L sucrose, 15 g / L yeast extract, 0.5 g / L calcium chloride, 0.4 g / L magnesium sulfate, 0.003 g / L manganese sulfate, 15 g / L Tween, and the balance is water. The initial pH of the fermentation medium is adjusted to 7.0 using sodium hydroxide.

7. The method according to claim 1, characterized in that: The inoculation volumes of the Bacillus velezensis LT-2 and the Exiguobacterium acetylicum LT-38 were 1% (v / v) and 1% (v / v).

8. The method according to claim 1, characterized in that: The temperature of the aerobic culture is 31.5-32.5°C.

9. A method for the combined extraction of microbial polysaccharides and β-carotene, characterized in that: The steps include: S11. Centrifuging the fermentation broth obtained by the method according to any one of claims 1 to 8 to obtain bacterial precipitate and fermentation supernatant respectively; S12. adding methanol solution to the bacterial precipitate, ultrasonically assisted extraction for 15 to 25 minutes, extracting overnight, and centrifuging to obtain a crude pigment extract; S13. The fermentation supernatant in step S11 is concentrated to 1 / 6 to 1 / 4 of the original volume, and 1 / 2 to 1 / 4 volume of Sevag reagent is added to the supernatant, and the protein is removed by shaking, and the mixture is repeated once to obtain a deproteinized supernatant; S14. The pigment in the deproteinized supernatant is adsorbed by a pretreated macroporous resin at 30° C. for 24 h, and the macroporous resin adsorbing the pigment and the deproteinized and depigmented supernatant are separated by solid-liquid separation; S15. Elute the macroporous resin adsorbing the pigment in step S14, mix the eluted pigment-containing eluate and the pigment crude extract in step S12 to obtain the total pigment crude extract, and then concentrate it, extract it, perform column chromatography, and freeze-dry it to obtain β-carotene; add 3 to 5 times of anhydrous ethanol to the deproteinized and depigmented supernatant in step S14, centrifuge to obtain the precipitate, and freeze-dry it to obtain microbial polysaccharides.

10. The method according to claim 9, characterized in that: The macroporous resin in step S14 includes one or a combination of AB-8, D101, DM-2, NAK-9 or D4020.

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