Microbial agent for producing caproic acid through co-culture as well as preparation method and application of microbial agent

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent for producing caproic acid through co-culture as well as a preparation method and application of the microbial agent. According to the specific technical scheme, the compound microbial agent comprises caproic acid bacteria and combined lactobacillus. According to the novel compound microbial agent provided by the invention, compared with single culture of caproic acid bacteria, after co-culture, the yield of caproic acid can be remarkably increased. In the co-culture process, the links of pH adjustment and lactic acid purification can be omitted, the production cost is reduced, and the production process is simplified. The compound microbial agent is mainly used for preparing or optimizing pit mud, increasing the content of caproic acid in the pit mud and promoting aging of a new pit. The compound microbial agent has the advantages of being simple in preparation process, short in culture period, low in cost and energy consumption and good in stability, and has wide market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a co-cultured caproic acid-producing microbial agent and a preparation method and application thereof. Background Art

[0002] Usually, cellars that are more than 10 years old can produce high-quality liquor. This is because, during the long-term production and domestication, the bacteria from the soil in the cellar mud are basically suppressed, the proportion of beneficial bacteria occupies the dominant ecological niche, and a cellar mud microbial community structure that is conducive to the production of aroma (acids, esters and alcohols) is formed in the cellar mud. However, the long-term maintenance of cellars is time-consuming and labor-intensive, and the costs are high. Therefore, how to quickly remove the bacteria in the loess in the newly built cellars, increase the number of beneficial bacteria, form a high-quality bacterial community as soon as possible, and promote the aging of new cellars is a problem that needs to be solved urgently.

[0003] Ethanol and lactic acid have good antibacterial effects and can inhibit the growth of bacteria in the pit. Yeast, lactic acid bacteria and some anaerobic Clostridium can produce metabolites such as ethanol, lactic acid and acetic acid. If ethanol- or lactic acid-producing microorganisms are co-cultured with caproic acid-producing microorganisms, the metabolites can be exchanged and complemented between the different microorganisms, achieving synergistic growth and enhanced metabolism, thereby improving the overall metabolic efficiency.

[0004] Therefore, the research on the mixed culture system of caproic acid bacteria has attracted much attention. At present, studies have shown that co-culturing caproic acid bacteria with yeast, lactic acid bacteria, butyric acid bacteria, actinomycetes, etc. may promote the synthesis of caproic acid. Co-culturing caproic acid bacteria and yeast can increase the production of caproic acid by more than 1 times, but yeast is an aerobic bacteria and it is difficult for it to adapt to the anaerobic environment required for cellar mud production / optimization. Lactic acid bacteria are facultative anaerobes that can adapt to the anaerobic environment of cellar mud, but because lactic acid bacteria generally grow rapidly, it is easy to cause a large amount of lactic acid accumulation during culture, inhibiting the growth of most microorganisms including caproic acid-producing bacteria, which is not conducive to the stability and diversity of the cellar mud microecology.

[0005] Therefore, if a functional bacterium for co-cultivation of caproic acid-producing bacteria can be provided to achieve synergistic growth and increase the rate of caproic acid production, it is expected to be used for the rapid regulation of cellar mud microecology and promote the aging of new cellars. Summary of the invention

[0006] The purpose of the present invention is to provide a co-cultured caproic acid-producing microbial agent and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a composite microbial agent, which includes caproic acid bacteria and combined lactobacillus.

[0008] Preferably, the caproic acid bacteria is Ruminococcaceae bacterium, and the deposit number is GDMCC No: 60133.

[0009] Preferably, the combined lactobacillus is Ligilactobacillus acidipiscis, CICC 10822.

[0010] Preferably, based on the number of viable bacteria, the ratio of caproic acid bacteria to combined lactobacillus is 10:3 to 30:1.

[0011] Accordingly, the preparation method of the bacterial agent comprises the following steps:

[0012] (1) Combined lactobacillus culture to OD 600 The combined lactobacillus seed solution is 0.8 to 1.0;

[0013] (2) Cultivate the caproic acid bacteria to OD 600 The hexanoic acid bacteria seed solution is 1.2 to 1.5;

[0014] (3) The caproic acid bacteria seed liquid and the combined lactobacillus seed liquid are subjected to combined anaerobic fermentation to obtain a liquid composite microbial agent.

[0015] Correspondingly, the method of producing caproic acid by co-culturing the bacterial agent is to inoculate the bacterial agent into a matrix that can provide growth for caproic acid bacteria and combined lactobacillus, control the culture temperature to 25-35°C, control the culture pH to 5-9, and perform combined anaerobic fermentation culture.

[0016] Correspondingly, the method for improving / optimizing the cellar environment and quickly preparing high-quality cellar mud by using the bacterial agent is as follows: the mass ratio of bacterial agent: water: loess = 0.3:0.16:1, the mixture is evenly stirred, loaded into a fermentation tank, sealed, and cultured at 30-35° C. for 30-60 days.

[0017] Preferably, water is replaced with a cellar mud nutrient solution, wherein the cellar mud nutrient solution comprises glucose, peptone, beef extract, yeast powder, soluble starch, cysteine ​​hydrochloride, sodium chloride, sodium acetate and yellow water.

[0018] Correspondingly, the cellar mud is used in winemaking.

[0019] Preferably, the cellar mud is applied to the cellar wall of the cellar.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a new composite microbial agent that can produce caproic acid after co-cultivation. Compared with the simple cultivation of caproic acid bacteria, the co-cultivation can significantly increase the caproic acid production. Compared with the simple cultivation of lactic acid bacteria, the co-cultivation can reduce the accumulation of lactic acid and help convert lactic acid into caproic acid.

[0022] The present invention can achieve the situation that the growth of caproic acid bacteria and the production of caproic acid are not affected by the rapid and large-scale accumulation of lactic acid without the need to add calcium carbonate or other pH buffers by selecting specific lactic acid bacteria and caproic acid bacteria, and controlling the conditions of co-cultivation. In the co-cultivation process, the combined lactobacillus first uses carbon sources such as glucose and fructose as substrates to ferment and produce lactic acid, and the caproic acid bacteria further synthesize caproic acid using lactic acid as a substrate. In this process, it is not necessary to extract the intermediate lactic acid, and the process link of adjusting pH in the two-stage fermentation and the cost of lactic acid purification can be exempted, thereby reducing the production cost of biosynthetic lactic acid, and the process is simple and feasible.

[0023] In addition to being used to prepare caproic acid or products containing caproic acid, the composite microbial agent can also be used to make or optimize cellar mud, help quickly remove miscellaneous bacteria in loess, help beneficial bacteria quickly occupy a dominant ecological niche, and promote aging of new cellars.

[0024] The composite microbial agent has the advantages of simple preparation process, short culture cycle, low cost and energy consumption, good stability, significant benefits and broad market application prospects. DETAILED DESCRIPTION

[0025] 1. The present invention provides a composite microbial agent, which includes caproic acid bacteria and combined lactobacillus. The agent can be in the form of common agents in the art, such as liquid or freeze-dried powder. The preferred scheme is: in terms of viable cell count, in the agent, caproic acid bacteria: combined lactobacillus = 10:3 to 30:1; more preferably: caproic acid bacteria: combined lactobacillus = 10:3.

[0026] Preferably, the caproic acid bacteria is caproic acid bacteria (Ruminococcaceae bacterium) CPB6, which is the rumen bacteria in patent 201710092267.1, GDMCC No: 60133 (through long-term culture, the performance of rumen bacteria CPB6 is slightly degraded compared with the performance when it was just preserved); the combined lactobacillus is acid fish combined lactobacillus (Ligilactobacillus acidipiscis) H2, CICC 10822.

[0027] Preferably, in the bacterial agent, the live bacterial concentrations of caproic acid bacteria and combined lactobacillus are both ≥10 8 CFU / mL or ≥10 8 CFU / g.

[0028] Second, the present invention also provides a method for preparing the bacterial agent, which specifically comprises the following steps:

[0029] 1. Inoculate freeze-dried caproic acid bacteria and combined lactobacillus into fresh YRCM and RM culture medium respectively, and culture them at 30-35°C for 24-48h for activation to obtain caproic acid bacteria suspension and combined lactobacillus suspension.

[0030] 2. Inoculate the combined Lactobacillus suspension into RM medium at a 10% (v / v) inoculation rate and culture at 30-37°C for 24 h to obtain the OD 600 The combined lactobacillus seed solution is 0.8 to 1.0.

[0031] RM medium (L): 5 g glucose, 10 g peptone, 10 g beef extract, 3 g yeast powder, 1 g soluble starch, 0.5 g cysteine ​​hydrochloride, 5 g sodium chloride, and 3 g sodium acetate.

[0032] 3. Inoculate the caproic acid bacteria suspension into YRCM medium at a 10% (v / v) inoculation rate and culture anaerobically at 30-35°C for 24 h to obtain the OD 600 It is a seed suspension of 1.2 to 1.5.

[0033] YRCM medium (L): 5 g glucose, 10 g peptone, 10 g beef extract, 3 g yeast powder, 1 g soluble starch, 0.5 g cysteine ​​hydrochloride, 5 g sodium chloride, 3 g sodium acetate, 100 mL yellow water.

[0034] 4. Inoculate the caproic acid bacteria seed liquid and the combined lactobacillus seed liquid into the fermentation medium RM at a 5% inoculation amount and the required ratio of caproic acid bacteria and combined lactobacillus in the bacterial agent for anaerobic fermentation for 3 to 5 days to obtain the liquid composite microbial agent.

[0035] Fermentation medium RM (L): glucose 10 g, yeast powder 3 g, sodium acetate 5 g, peptone 5 g, beef powder 5 g, NH4Cl1.0 g, K2HPO4 0.45 g, MgSO4×7H2O 0.1 g, resazurin (0.05% w / v) 0.5 mL, L-cysteine ​​hydrochloride 0.5 g, trace elements 1 mL.

[0036] The liquid composite microbial agent can also be further concentrated to form composite microbial sludge as needed, and an active protective agent is added to the composite microbial sludge, mixed to obtain a mixed suspension; the mixed suspension is pre-frozen, and the pre-frozen bacteria are placed in a vacuum freeze-drying device for freeze-drying to obtain a composite microbial agent dry powder.

[0037] An optional solution is that the active protective agent components (w / w) are: 5% peptone, 3% sodium chloride, 4.5% glucose, 3.8% beef extract powder, 2.5% glycerol, 2% corn flour, 2.5% sodium alginate, 0.6% cysteine ​​salt and 0.9% dipotassium hydrogen phosphate, and the balance is water.

[0038] 3. The present invention provides a method for producing caproic acid by using the composite microbial agent. The method comprises: inoculating the composite microbial agent into a matrix for the growth of caproic acid bacteria and combined lactobacillus, controlling the culture temperature to 25-35°C (preferably 30°C), controlling the culture pH to 5-9 (preferably pH 5.5), and conducting combined anaerobic fermentation culture. Preferably, the carbon source is glucose or fructose, and the preferred nitrogen source is peptone.

[0039] Fourth, the present invention also provides a method for improving / optimizing the cellar environment and rapidly preparing high-quality cellar mud using the composite microbial agent. The method comprises the following steps:

[0040] 1. Pretreatment of loess: Select neutral loess, beat it into fine powder, pass it through a 100-mesh sieve, and air-dry it naturally.

[0041] 2. Prepare pit mud nutrient solution and mix it with water for later use: 15g glucose, 10g peptone, 10g beef extract, 3g yeast powder, 1g soluble starch, 0.5g cysteine ​​hydrochloride, 5g sodium chloride, 3g sodium acetate, 100mL yellow water, and make up to 1000mL with water.

[0042] 3. Mix liquid microbial agent: pit mud nutrient solution: loess in a mass ratio of (0.1-0.3):(0.16-0.36):1, stir evenly, put into a fermentation tank, seal, and culture at 30-35°C for 30-60 days. During this period, regularly add pit mud nutrient solution to maintain the water content of the pit mud at 45-50%.

[0043] The prepared cellar mud can be directly pasted on the cellar walls around the cellar for fermenting strong-flavor liquor.

[0044] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0045] Example 1: Optimization of conditions for co-cultivation of caproic acid bacteria and combined lactobacillus to produce caproic acid

[0046] 1. Obtain microbial seed solution

[0047] The combined Lactobacillus was inoculated into RM medium at a rate of 10% (v / v) and cultured at 35°C for 24 h to obtain the OD 600 The total number of combined lactobacillus colonies in the seed solution was 1.74×10 8 CFU / mL.

[0048] The caproic acid bacteria were inoculated into YRCM medium at a rate of 10% (v / v) and cultured anaerobically at 35°C for 24 h to obtain the OD 600 The seed solution is 1.0 to 1.2. The number of inoculated strains of caproic acid bacteria in the seed solution is 2.7×10 9 CFU / mL.

[0049] 2. Effect of carbon source type on caproic acid production in co-culture

[0050] The combined Lactobacillus seed liquid and the caproic acid bacteria seed liquid were inoculated into the fermentation medium RM at a volume ratio of 1:1 and a 5% inoculation amount, and anaerobically cultured at 30° C. in an anaerobic fermentation tank for 5 days.

[0051] Fermentation medium RM (L): glucose 10 g, yeast powder 3 g, sodium acetate 5 g, peptone 5 g, beef powder 5 g, NH4Cl1.0 g, K2HPO4 0.45 g, MgSO4×7H2O 0.1 g, resazurin (0.05% w / v) 0.5 mL, L-cysteine ​​hydrochloride 0.5 g, trace element solution 1 mL, pH = 6.0.

[0052] Trace element solution (L): FeCl2·4H2O 1.5g; CoCl2·6H2O 0.19g; MnCl2·4H2O0.1g; ZnCl2 0.07g; NaMO4·2H2O 0.036g; NiCl2·6H2O 0.024g; H3BO3 0.006g; CuCl2·2H2O 0.002g; HCl (25% w / w) 10mL.

[0053] The glucose in the fermentation medium RM was replaced with D-fructose, sucrose, lactose, maltose, mannitol and soluble starch in equal amounts, and the other culture conditions were exactly the same; at the same time, 5% pure caproic acid bacteria seed solution was inoculated as a control, and the other conditions were the same to obtain the fermentation broth after anaerobic fermentation in each group. The caproic acid content in the fermentation broth of each group was determined by high performance liquid chromatography (HPLC), and 3 replicates were set for each group, and the results were averaged (the subsequent data were all processed in the same way and will not be repeated), as shown in Table 1.

[0054] Table 1 Comparison of the effects of carbon sources on caproic acid production

[0055]

[0056] The results showed that the use of glucose and D-fructose as carbon sources can significantly improve the ability of co-culture to produce caproic acid. Among them, when the caproic acid bacteria are fermented alone, it is difficult to use D-fructose to produce caproic acid, but after co-culture with Lactobacillus, the caproic acid production is significantly improved.

[0057] 3. Effect of glucose concentration on caproic acid production in co-culture

[0058] According to the method and culture conditions of step 2, the glucose concentration in the fermentation medium RM was adjusted, and the caproic acid content in each group of fermentation broth was measured after co-cultivation. The results are shown in Table 2.

[0059] Table 2 Comparison of the effect of glucose concentration on caproic acid production

[0060] Glucose concentration Caproic acid content 5g / L 1.74g / L 10g / L 3.35g / L 15g / L 4.54g / L 20g / L 4.67g / L 25g / L 4.10g / L 30g / L 4.64g / L

[0061] 4. Effect of pH on hexanoic acid production in co-culture

[0062] According to the method and culture conditions of step 2, fermentation was carried out using fermentation medium RM, the pH of the basic RM medium was adjusted, and the caproic acid content in each group of fermentation broth was measured after co-cultivation. The results are shown in Table 3.

[0063] Table 3 Comparison of the effect of pH on caproic acid production

[0064] pH Caproic acid content 4.5 0 5.5 3.59g / L 6.5 3.46g / L 7.5 3.32g / L 8.5 3.02g / L

[0065] 5. Effect of microbial ratio on caproic acid production in co-culture

[0066] According to the method and culture conditions of step 2, the inoculation ratio of combined lactobacillus and caproic acid bacteria seed solution was adjusted, and the caproic acid content in each group of fermentation broth was measured after co-cultivation. The results are shown in Table 4. The caproic acid yields of different inoculation ratios were not much different, but were significantly higher than CPB6 single strain fermentation.

[0067] Table 4 Comparison of the effect of inoculation ratio on caproic acid production

[0068] Combined Lactobacillus: Caproic acid bacteria Caproic acid content 5:1 3.41g / L 3:1 3.53g / L 1:1 3.35g / L 1:3 3.15g / L 0:1 2.57g / L

[0069] 6. Effect of microbial inoculation order on hexanoic acid production in co-culture

[0070] According to the method and culture conditions of step 2, synchronous co-culture and co-culture according to different inoculation orders are performed respectively. The specific methods include: (1) synchronous co-culture: according to the method of step 2, the combined bacteria Lactobacillus and caproic acid bacteria are inoculated at the same time for mixed fermentation; (2) sequential mixed fermentation 1: first inoculate 2.5% caproic acid bacteria seed liquid, and then inoculate 2.5% combined bacteria Lactobacillus seed liquid 12 hours later; (3) sequential mixed fermentation 2: first inoculate 2.5% combined bacteria Lactobacillus seed liquid, and then inoculate 2.5% caproic acid bacteria seed liquid 12 hours later.

[0071] After the co-culture, the contents of caproic acid in the fermentation broth obtained by the three methods were 3.35 g / L, 3.09 g / L and 0.98 g / L, respectively. Therefore, the simultaneous inoculation and co-culture of Lactobacillus and caproic acid bacteria has the strongest ability to produce caproic acid.

[0072] In addition, if general lactic acid bacteria (such as lactic acid bacteria RJ25 separated and preserved by the laboratory) are used to co-cultivate with caproic acid bacteria, lactic acid bacteria grow faster, easily metabolize quickly and accumulate a large amount of acid, inhibit the growth of caproic acid bacteria, thereby affecting the generation of caproic acid, it is necessary to add a large amount of pH buffers such as calcium carbonate to regulate pH. And using the combined lactobacillus provided by the present invention, when co-cultivating with caproic acid bacteria under specific conditions, the two can grow in coordination, without the need to additionally add pH buffers such as calcium carbonate (of course, adding an appropriate amount of calcium carbonate in the culture will not have a negative impact).

[0073] Example 2: Preparation / Optimization of Pit Mud by Co-cultivation of Caproic Acid Bacteria and Combined Lactobacillus

[0074] 1. Prepare the bacterial agent: prepare the caproic acid bacteria seed liquid and the combined lactobacillus seed liquid in the manner of Example 1. Inoculate the caproic acid bacteria seed liquid and the combined lactobacillus seed liquid (the volume ratio of the two is 1:1) into the fermentation medium RM at an inoculation amount of 5% (v / v), and culture anaerobically at 30° C. in an anaerobic fermentation tank for 5 days to obtain a composite microbial agent. At the same time, under the same inoculation and culture conditions, the caproic acid bacteria seed liquid and the combined lactobacillus seed liquid are inoculated into the fermentation medium RM respectively to prepare pure caproic acid bacteria agent and pure combined lactobacillus agent.

[0075] 2. Preparation of cellar mud: Select neutral loess, beat it into fine powder, pass it through a 100-mesh sieve, and air-dry it naturally.

[0076] Prepare pit mud nutrient solution: 15g glucose, 10g peptone, 10g beef extract, 3g yeast powder, 1g soluble starch, 0.5g cysteine ​​hydrochloride, 5g sodium chloride, 3g sodium acetate, and 100mL yellow water.

[0077] According to the mass ratio of bacterial agent: pit mud nutrient solution: water: loess = 3:1.6:1:10, the composite microbial agent, pure caproic acid bacteria agent and pure combined lactobacillus agent prepared in step 1 are respectively mixed with the pit mud nutrient solution and loess to make artificial pit mud with a moisture content of 50-55%, and then respectively loaded into fermentation tanks, sealed, and cultured at 30°C for 60 days. At the same time, a control group is set up, and the microbial agent is replaced with an equal amount of sterilized living cell-free bacterial solution (121°C, sterilized for 20 minutes), and other methods are the same.

[0078] 3. After the cultivation, the organic acid content in the pit mud of each group was determined. The results are shown in Table 5.

[0079] Table 5 Comparison table of organic acid content in each group of pit mud (unit: mg / kg dry pit mud)

[0080] Group Acetic acid Butyric acid Hexanoic acid Control group 3190.57 8676.12 363.27 Pure combined lactobacillus group 2448.04 5350.00 314.07 Pure caproic acid bacteria group 4255.86 7963.07 746.88 Complex microbiome 2904.52 6625.84 1020.74

[0081] The results showed that after 60 days of cultivation under the same conditions, the caproic acid content in the pit mud prepared by the composite microbial agent prepared by the present invention increased significantly compared with the control group or other microorganisms, indicating that the composite microbial agent provided by the present invention can effectively promote the production of caproic acid in the pit mud that is beneficial to the quality of liquor, thereby quickly improving the quality of the pit mud.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent comprises caproic acid bacteria and combined lactobacillus.

2. The bacterial agent according to claim 1, characterized in that: The caproic acid bacteria is Ruminococcaceaebacterium, and the deposit number is GDMCC No:60133.

3. The bacterial agent according to claim 1 or 2, characterized in that: The combined lactobacillus is Ligilactobacillus acidipiscis, CICC 10822.

4. The bacterial agent according to claim 1, characterized in that: According to the number of live bacteria, the ratio of caproic acid bacteria to combined lactobacillus is 10:3 to 30:

1.

5. The method for preparing the bacterial agent according to any one of claims 1 to 4, characterized in that: The steps include: (1) Combined lactobacillus culture to OD 600 The combined lactobacillus seed solution is 0.8 to 1.0; (2) Cultivate the caproic acid bacteria to OD 600 The hexanoic acid bacteria seed solution is 1.2 to 1.5; (3) The caproic acid bacteria seed liquid and the combined lactobacillus seed liquid are subjected to combined anaerobic fermentation to obtain a liquid composite microbial agent.

6. A method for producing caproic acid by co-cultivation of the bacterial agent according to any one of claims 1 to 4, characterized in that: The bacterial agent is inoculated into a matrix that can be used for the growth of caproic acid bacteria and combined lactobacillus, the culture temperature is controlled at 25-35° C., the culture pH is controlled at 5-9, and combined anaerobic fermentation culture is performed.

7. A method for improving / optimizing the cellar environment and rapidly preparing high-quality cellar mud using the microbial agent described in any one of claims 1 to 4, characterized in that: The mass ratio of bacterial agent: water: loess is 0.3:0.16:

1. Mix and stir evenly, put into a fermentation tank, seal it, and culture it at 30-35°C for 30-60 days.

8. The method according to claim 7, characterized in that: The water is replaced with a pit mud nutrient solution, which includes glucose, peptone, beef extract, yeast powder, soluble starch, cysteine ​​hydrochloride, sodium chloride, sodium acetate and yellow water.

9. Use of the cellar mud prepared by the method according to claim 7 or 8 in winemaking.

10. The use according to claim 9, characterized in that: The cellar mud is spread on the cellar wall of the cellar.

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