CN112280811B - Method for high-yield production of short-chain fatty acid by utilizing microbial symbiotic fermentation technology - Google Patents
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Abstract
The invention relates to a method for high-yield production of short-chain fatty acid by utilizing a microbial symbiotic fermentation technology. The invention utilizes the microbial symbiotic fermentation technology to produce short-chain fatty acid with high yield, takes the jerusalem artichoke and the seaweed which are high in yield and easy to obtain as raw materials, namely takes food-grade materials as fermentation substrates, utilizes the multi-bacterium symbiotic fermentation technology to produce the short-chain fatty acid, and overcomes the defect of high cost of the existing short-chain fatty acid preparation compared with the process for preparing the short-chain fatty acid by utilizing carbohydrate compositions and pure cellulose in the prior art.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for high-yield production of short-chain fatty acid by utilizing a microbial symbiotic fermentation technology.
Background
Short-chain fatty acids (SCFAs), also called Volatile Fatty Acids (VFA), are organic carboxylic acids with 1-6 carbon atoms, and are end products generated by bacterial fermentation after carbohydrates (oligosaccharides, non-starch polysaccharides, resistant starch and the like) which cannot be digested and absorbed by the small intestine enter the large intestine, and mainly comprise acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid, wherein 90-95% of SCFAs are acetic acid, propionic acid and butyric acid.
Short chain fatty acids are intestinal microbial fermentation products, the effects of which on body health have been extensively studied: SCFAs can improve the digestive absorption capacity of the intestinal tract, promote the development of the intestinal tract and maintain the barrier function of the intestinal tract; the intestinal tract is not only crucial to the digestion and absorption of nutrient substances, but also is a huge immune organ of an organism, and SCFAs regulate the immune function of the intestinal tract through various ways; the SCFAs can regulate the structure of intestinal microbial flora, and the stability of the intestinal flora has important significance for maintaining normal physiological level of organisms; and a large number of researches show that the short-chain fatty acid can also regulate the metabolic health of an organism.
Chinese patent document CN108384816A (application No. CN201810125553.8) discloses short-chain fatty acids and a method for producing short-chain fatty acids by anaerobic fermentation of sludge, which mainly comprises adding a methane inhibitor into the sludge at the later stage of anaerobic fermentation, stirring and mixing, and applying a magnetic field around a reactor to produce short-chain fatty acids. Chinese patent document CN104498541A (application No. CN201410841735.7) discloses a method for producing short-chain volatile fatty acids by using kitchen waste and short-chain volatile fatty acids, mainly using kitchen waste as fermentation substrate, using sludge as inoculum, stirring and mixing uniformly to obtain fermentation substrate; adding alkyl polyglycoside into the fermentation substrate, and carrying out anaerobic fermentation under the stirring condition to produce short-chain volatile fatty acid. The short-chain fatty acid prepared by the method is a chemical material, is mainly used as an external carbon source for biological nitrogen and phosphorus removal, improves the nitrogen and phosphorus removal efficiency, and is difficult to apply in the fields of food and medicine. The short-chain fatty acid is taken as a metabolite of intestinal microorganisms, has important significance for maintaining the health of organisms, has wide application prospect in the fields of food and medicine, and needs further research on a preparation scheme, a production process and the like of high-yield short-chain fatty acid on the basis of comprehensively considering the yield and the safety of the short-chain fatty acid.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for high-yield production of short-chain fatty acid by utilizing a microbial symbiotic fermentation technology, the method is scientific and reasonable, has strong operability, is easy to realize large-scale production, and has high short-chain fatty acid yield, and the prepared short-chain fatty acid can be applied to the health fields of food, medical treatment and the like.
The technical scheme of the invention is as follows:
a method for producing short chain fatty acid with high yield by utilizing a microbial symbiotic fermentation technology comprises the following steps:
(1) preparing a fermentation slurry culture medium: preparing a fermentation broth culture medium by using jerusalem artichoke pulp, seaweed powder and sodium glutamate as raw materials, dispersing the jerusalem artichoke pulp, the seaweed powder and the sodium glutamate in water, homogenizing and mixing, adjusting the pH to 6-8, and sterilizing at high temperature to obtain the fermentation broth culture medium;
(2) inoculation: activating yeast, bifidobacterium and ruminococcus, inoculating the activated yeast, bifidobacterium and ruminococcus into the fermentation slurry culture medium prepared in the step (1), and performing anaerobic fermentation in a fermentation tank;
(3) fermentation: maintaining the fermentation temperature at 26-29 deg.C for the first fermentation for 0.5-2 h; and after the first fermentation is finished, raising the fermentation temperature to 35-39 ℃ for second fermentation for 4-6h, and after the fermentation is finished, cooling to 8-11 ℃ to obtain the fermentation liquor containing the short-chain fatty acids.
According to the invention, the jerusalem artichoke pulp in the step (1) is preferably obtained by washing, airing and pulping fresh jerusalem artichoke.
According to the invention, the seaweed powder in the step (1) is preferably obtained by washing and drying seaweed, cutting the seaweed into blocks, drying the blocks until the water content is below 10%, grinding the blocks into powder of 50-100 meshes, and sieving the powder.
According to the invention, the seaweed in the step (1) is preferably one or more of kelp, undaria pinnatifida, laver, gulfweed and coral seaweed which are optionally mixed.
According to the invention, the fermentation slurry culture medium in the step (1) comprises the following components in percentage by mass: 10-40% of jerusalem artichoke pulp, 2-10% of seaweed powder, 0.1-1% of sodium glutamate and the balance of water.
According to the invention, the homogenization in step (1) is preferably carried out at a pressure of 15-30MPa for 3-10 min.
Preferably, the high temperature sterilization in step (1) is maintained at 113-117 ℃ for 5-20 min.
Preferably, the yeast in step (2) is brewer's yeast or baker's yeast.
Preferably, in step (2), the bifidobacterium is one or a mixture of more than two of bifidobacterium longum, bifidobacterium animalis, bifidobacterium adolescentis and bifidobacterium breve.
Preferably, in step (2), the ruminococcus is ruminococcus braakii.
According to the invention, the inoculation amount of the symbiotic fermentation strain in the step (2) is 5-15% of the volume of the fermentation slurry culture medium, wherein the volume ratio of yeast, bifidobacterium and ruminococcus activating bacteria liquid in the inoculated bacteria liquid is 1 (1-2) to (2-10).
In the present invention, yeasts, bifidobacteria and ruminococcus are all the existing strains, and are commercially available, and the mode of activating and culturing the yeasts, the bifidobacteria and the ruminococcus so that the number and the activity thereof meet the inoculation requirement is not particularly limited, and the specification of the strains or the common mode in the prior art can be adopted.
Has the advantages that:
1. the invention takes jerusalem artichoke pulp, seaweed powder and sodium glutamate as fermentation substrates, wherein jerusalem artichoke tubers are rich in inulin, crude fiber and various minerals and vitamins, wherein the inulin and the crude fiber can be used as carbohydrate substrates in the fermentation process, and the minerals and the vitamins provide necessary nutrient substances for the growth and the propagation of microorganisms; the seaweed is rich in seaweed polysaccharide and dietary fiber, can provide a carbon source for microbial fermentation, and in addition, the seaweed is rich in inorganic elements, so that a good production environment is provided for a fermentation strain, and the influence on the yield of SCFAs due to single nutrient substances is avoided; during the fermentation process, sodium glutamate can be utilized by Glutamic Acid Decarboxylase (GAD) of microorganisms to carry out decarboxylation, SCFAs generated by fermentation and the decarboxylation of glutamic acid jointly adjust the pH value of fermentation liquor, so that the metabolism of the microorganisms is better promoted to produce short-chain fatty acids, and experiments show that the sodium glutamate can obviously improve the yield of acetic acid, propionic acid and butyric acid. The yield of acetic acid can reach 190-215 mmol/L, the yield of propionic acid can reach 60-75 mmol/L, the yield of butyric acid can reach 75-85 mmol/L, and the yield of propionic acid is remarkably increased.
2. The invention utilizes the microbial symbiotic fermentation technology to produce short-chain fatty acid with high yield, takes saccharomycetes, bifidobacteria and ruminococcus as symbiotic fermentation strains, directly influences the growth and the reproduction of the strains and the material metabolism due to different inoculation modes, further influences the fermentation process and the yield of SCFAs (microbial symbiotic fermentation), and strictly controls the inoculation ratio according to the symbiotic relationship of the saccharomycetes, the bifidobacteria and the ruminococcus in a fermentation slurry culture medium; considering that the yeast is facultative anaerobe, the bifidobacterium and the ruminococcus are anaerobe, the fermentation process is carried out step by step, firstly, yeast enrichment fermentation is carried out, the yeast is used for deoxidizing the raw material, and the growth retardation of the bifidobacterium is effectively improved; the first fermentation produces a small amount of ethanol, has an induction activation effect on the ruminococcus, promotes the utilization of the ruminococcus to raw materials in the second fermentation, improves the fermentation efficiency, and further greatly improves the SCFAs yield.
3. The pH of the fermentation slurry culture medium is one of the most important abiotic influence factors in the symbiotic fermentation process, which not only influences the activity of symbiotic fermentation microorganisms, but also has great influence on the SCFAs production rate. The pH value of the fermentation slurry culture medium is strictly controlled to be 6-8, in addition, in the fermentation process, the added sodium glutamate is utilized by Glutamic Acid Decarboxylase (GAD) of microorganisms to carry out decarboxylation, the pH value of the fermentation liquor is adjusted by the SCFAs generated by fermentation and the glutamic acid decarboxylation, and the SCFAs yield is improved together.
4. The invention utilizes the microbial symbiotic fermentation technology to produce short-chain fatty acid with high yield, takes the jerusalem artichoke and the seaweed which are high in yield and easy to obtain as raw materials, namely takes food-grade materials as fermentation substrates, utilizes the multi-bacterium symbiotic fermentation technology to produce the short-chain fatty acid, and overcomes the defect of high cost of the existing short-chain fatty acid preparation compared with the process for preparing the short-chain fatty acid by utilizing carbohydrate compositions and pure cellulose in the prior art.
Drawings
FIG. 1 shows the SCFAs content in different fermentation broths; in the figure, P < 0.01, which is very different from acetic acid in example 2; p < 0.05, significantly different from acetic acid in example 2; # indicates P < 0.01, which is very significant compared to propionic acid in example 2; # denotes P < 0.05, which is significantly different from propionic acid in example 2; Δ means that P < 0.01 was significantly different from butyric acid in example 2, and Δ means that P < 0.05 was significantly different from butyric acid in example 2.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
In the examples, yeasts, bifidobacteria and ruminococcus are all the existing strains and are commercially available, the mode of activating and culturing the yeasts, the bifidobacteria and the ruminococcus so that the number and the activity of the yeasts, the bifidobacteria and the ruminococcus meet the inoculation requirement is not particularly limited, and the specification of the strains or the common mode in the prior art can be adopted.
The brewers yeast (bio-52035), bifidobacterium adolescentis (bio-67246) and ruminococcus braaki (bio-80903) in the examples were purchased from Baiohobrevir biotech, Beijing.
Example 1
A method for producing short chain fatty acid with high yield by utilizing a microbial symbiotic fermentation technology comprises the following steps:
1) pretreating the jerusalem artichoke: cleaning 50kg of fresh jerusalem artichoke, naturally drying the surface water, putting the jerusalem artichoke into a pulping machine for pulping, and pulping for 10min at 20Hz to obtain jerusalem artichoke pulp;
2) pretreatment of seaweed: cleaning 50kg of herba Zosterae Marinae, naturally drying, cutting into blocks with length of 2-5cm and width of 2-5cm, drying in air blast drying oven (70 deg.C) until water content is 10%, grinding in a grinder to 60 mesh, and sieving to obtain Sargassum powder;
3) preparing a fermentation slurry culture medium: dispersing 10kg of Jerusalem artichoke slurry prepared in step 1), 2kg of seaweed powder prepared in step 2) and 0.1kg of sodium glutamate in 87.9kg of water, mixing, homogenizing under 20MPa for 8min, adjusting pH to 6, sterilizing at 115 deg.C for 15min, and cooling to obtain fermentation slurry culture medium;
4) inoculation: sequentially inoculating activated beer yeast (bio-52035), bifidobacterium adolescentis (bio-67246) and ruminococcus brucei (bio-80903) into the fermentation slurry culture medium prepared in the step 3), inoculating the total amount of the yeast, the bifidobacterium and the ruminococcus activating bacterial liquid in the inoculated bacterial liquid at a volume ratio of 1:1:2, and performing anaerobic fermentation in a fermentation tank;
5) fermentation: keeping the fermentation temperature at 28 ℃ for the first fermentation for 2 h; and after the first fermentation is finished, raising the fermentation temperature to 37 ℃ for second fermentation for 4 hours, and after the fermentation is finished, cooling to 8-11 ℃ to obtain the fermentation liquor containing the short-chain fatty acids.
Example 2
A method for producing short chain fatty acid with high yield by utilizing a microbial symbiotic fermentation technology comprises the following steps:
1) pretreating the jerusalem artichoke: cleaning 50kg of fresh jerusalem artichoke, naturally drying the surface water, putting the jerusalem artichoke into a pulping machine for pulping, and pulping for 10min at 20Hz to obtain jerusalem artichoke pulp;
2) pretreatment of seaweed: cleaning 50kg of herba Zosterae Marinae, naturally drying, cutting into blocks with length of 2-5cm and width of 2-5cm, drying in air blast drying oven (70 deg.C) until water content is 10%, grinding in a grinder to 60 mesh, and sieving to obtain Sargassum powder;
3) preparing a fermentation slurry culture medium: dispersing 20kg of Jerusalem artichoke slurry prepared in step 1), 5kg of seaweed powder prepared in step 2) and 0.5kg of sodium glutamate in 74.5kg of water, mixing, homogenizing under 20MPa for 8min, adjusting pH to 7, sterilizing at 115 deg.C for 15min, and cooling to obtain fermentation slurry culture medium;
4) inoculation: sequentially inoculating activated beer yeast (bio-52035), bifidobacterium adolescentis (bio-67246) and ruminococcus brucei (bio-80903) into the fermentation slurry culture medium prepared in the step 3), inoculating the total amount of the yeast, the bifidobacterium and the ruminococcus activating bacterial liquid in the inoculated bacterial liquid at a volume ratio of 1:1:5 to perform anaerobic fermentation in a fermentation tank, wherein the total amount of the yeast, the bifidobacterium adolescentis and the ruminococcus brucei activating bacterial liquid is 10% (v/v) of the fermentation slurry culture medium;
5) fermentation: keeping the fermentation temperature at 28 ℃ for the first fermentation, wherein the fermentation time is 1 h; and after the first fermentation is finished, raising the fermentation temperature to 37 ℃ for second fermentation for 5 hours, and after the fermentation is finished, cooling to 8-11 ℃ to obtain the fermentation liquor containing the short-chain fatty acids.
Example 3
A method for producing short chain fatty acid with high yield by utilizing a microbial symbiotic fermentation technology comprises the following steps:
1) pretreating the jerusalem artichoke: cleaning 50kg of fresh jerusalem artichoke, naturally drying the surface water, putting the jerusalem artichoke into a pulping machine for pulping, and pulping for 10min at 20Hz to obtain jerusalem artichoke pulp;
2) pretreatment of seaweed: cleaning 50kg of herba Zosterae Marinae, naturally drying, cutting into blocks with length of 2-5cm and width of 2-5cm, drying in air blast drying oven (70 deg.C) until water content is 10%, grinding in a grinder to 60 mesh, and sieving to obtain Sargassum powder;
3) preparing a fermentation slurry culture medium: dispersing 40kg of Jerusalem artichoke slurry prepared in step 1), 10kg of seaweed powder prepared in step 2) and 1kg of sodium glutamate in 49kg of water, mixing, homogenizing under 20MPa for 8min, adjusting pH to 8, sterilizing at 115 ℃ for 15min, and cooling to obtain a fermentation slurry culture medium;
4) inoculation: sequentially inoculating activated beer yeast (bio-52035), bifidobacterium adolescentis (bio-67246) and ruminococcus brucei (bio-80903) into the fermentation slurry culture medium prepared in the step 3), inoculating the total amount of the yeast, the bifidobacterium and the ruminococcus activating bacterial liquid in the inoculated bacterial liquid at a volume ratio of 1:2:10 to perform anaerobic fermentation in a fermentation tank, wherein the total amount of the yeast, the bifidobacterium and the ruminococcus activating bacterial liquid is 5% (v/v) of the fermentation slurry culture medium;
5) fermentation: keeping the fermentation temperature at 28 ℃ for the first fermentation for 2 h; and after the first fermentation is finished, raising the fermentation temperature to 37 ℃ for second fermentation for 6h, and after the fermentation is finished, cooling to 8-11 ℃ to obtain the fermentation liquor containing the short-chain fatty acids.
Comparative example 1
A short chain fatty acid was prepared as described in example 2, except that in step 3), pH was adjusted to 5 and the other treatments were the same as in example 2.
Comparative example 2
A short chain fatty acid was prepared as described in example 2, except that in step 3), pH was adjusted to 9 and the other treatments were the same as in example 2.
Comparative example 3
Short-chain fatty acids were prepared as described in example 2, except that no sodium glutamate was added in the preparation of the fermentation broth in step 3), and the other treatments were the same as in example 2.
Comparative example 4
Short-chain fatty acids were prepared as described in example 2, except that in step 4), the inoculated microorganism was ruminococcus brucei (bio-80903), and the other treatment was the same as in example 2.
Comparative example 5
Short-chain fatty acids were prepared as described in example 2, except that in step 5), fermentation was carried out in the same manner as in example 2, except that instead of stepwise fermentation, the temperature of the fermentor was directly maintained at 37 ℃ for 6.5 hours.
Examples of the experiments
The fermentation liquor obtained after the fermentation of the examples 1-3 and the comparative examples 1-5 is subjected to SCFAs detection, and the steps are as follows:
after fermentation, the fermentation liquor is centrifuged for 20min at 6500rpm and 4 ℃, supernatant is filtered by a 0.22-micron filter membrane, filtrate is diluted by 10 times by sterile water, short-chain fatty acid in the supernatant is detected by an Agilent Technologies 7890A gas chromatograph, and the chromatographic conditions are as follows: a chromatographic column: DA-FFAP (30m × 0.25mm, 0.25 μm); temperature rising procedure: the initial column temperature is 100 deg.C, maintained for 1min, increased to 130 deg.C at a rate of 3 deg.C/min, maintained for 1min, increased to 200 deg.C at a rate of 20 deg.C/min, and maintained at 200 deg.C for 10 min. FID and injection port temperature: respectively at 230 ℃ and 230 ℃. Carrier gas: nitrogen gas. Gas volume flow rate: hydrogen, air and nitrogen were mixed at flow ratios of 30, 300, 20mL/min, respectively. Sample introduction: automatically injecting samples, wherein the sample injection amount is 1 mu L; run time 25min, results are shown in FIG. 1.
As can be seen from FIG. 1, the SCFAs content in the fermentation broth after the fermentation in examples 1-3 is not significantly different (P > 0.05), wherein the acetic acid, propionic acid and butyric acid content in the fermentation broth in example 1 are 211.32mmol/L, 64.68mmol/L and 76.70mmol/L respectively, the acetic acid, propionic acid and butyric acid content in the fermentation broth in example 2 are 195.04mmol/L, 73.76mmol/L and 76.86mmol/L respectively, and the acetic acid, propionic acid and butyric acid content in the fermentation broth in example 3 are 193.84mmol/L, 69.35mmol/L and 80.48mmol/L respectively; compared with the example 2, the contents of acetic acid, propionic acid and butyric acid in the fermentation liquid after the fermentation of the comparative examples 1 and 2 are very different (P is less than 0.01); in comparative example 3, acetic acid and butyric acid have significant difference (P < 0.05), and propionic acid has very significant difference (P < 0.01); in comparative example 4, acetic acid has a significant difference (P < 0.05), propionic acid and butyric acid have a very significant difference (P < 0.01); in comparative example 5, acetic acid and propionic acid have a very significant difference (P < 0.01), and butyric acid has a significant difference (P < 0.05); neither pentanoic acid nor hexanoic acid were significantly different in each comparative example (P > 0.05). The result shows that the production method of the invention is obtained by adjusting and optimizing the fermentation culture medium, the symbiotic fermentation strain and the fermentation method, the short-chain fatty acid can be produced at high yield by utilizing the microbial symbiotic fermentation technology of the invention, and the invention has wide application prospect in the fields of food and medicine.
Claims (6)
1. A method for producing short-chain fatty acid with high yield by utilizing a microbial symbiotic fermentation technology is characterized by comprising the following steps:
(1) preparing a fermentation slurry culture medium: preparing a fermentation broth culture medium by using jerusalem artichoke pulp, seaweed powder and sodium glutamate as raw materials, dispersing the jerusalem artichoke pulp, the seaweed powder and the sodium glutamate in water, homogenizing and mixing, adjusting the pH to 6-8, and sterilizing at high temperature to obtain the fermentation broth culture medium;
(2) inoculation: activating yeast, bifidobacterium and ruminococcus, inoculating the activated yeast, bifidobacterium and ruminococcus into the fermentation slurry culture medium prepared in the step (1), and performing anaerobic fermentation in a fermentation tank;
the yeast is beer yeast, the bifidobacterium is bifidobacterium adolescentis, and the ruminococcus is ruminococcus braakii;
the inoculation amount of the symbiotic fermentation strain is 5-15% of the volume of the fermentation slurry culture medium, wherein the volume ratio of yeast, bifidobacterium and rumen coccus activating bacteria liquid in the inoculated bacteria liquid is 1 (1-2) to 2-10;
(3) fermentation: maintaining the fermentation temperature at 26-29 deg.C for the first fermentation for 0.5-2 h; and after the first fermentation is finished, raising the fermentation temperature to 35-39 ℃ for second fermentation for 4-6h, and after the fermentation is finished, cooling to 8-11 ℃ to obtain the fermentation liquor containing the short-chain fatty acids.
2. The method according to claim 1, wherein the jerusalem artichoke pulp in step (1) is obtained by washing fresh jerusalem artichoke, air-drying, and pulping.
3. The method according to claim 1, wherein the seaweed powder in step (1) is obtained by washing and air-drying seaweed, cutting into pieces, drying until the water content is less than 10%, grinding into powder of 50-100 meshes, and sieving.
4. The method according to claim 1, wherein the seaweed in step (1) is one or more of kelp, wakame seaweed, laver, gulfweed and coral seaweed.
5. The method of claim 1, wherein the fermentation broth medium in step (1) comprises the following components in percentage by mass: 10-40% of jerusalem artichoke pulp, 2-10% of seaweed powder, 0.1-1% of sodium glutamate and the balance of water.
6. The method according to claim 1, wherein the homogenizing in step (1) is homogenizing at a pressure of 15-30MPa for 3-10 min;
the high-temperature sterilization is carried out at the temperature of 113-117 ℃ for 5-20 min.
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