CN111944730A - Lactobacillus paracasei capable of efficiently utilizing jerusalem artichoke powder and application thereof - Google Patents
Lactobacillus paracasei capable of efficiently utilizing jerusalem artichoke powder and application thereof Download PDFInfo
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Abstract
The invention discloses lactobacillus paracasei capable of efficiently utilizing jerusalem artichoke powder and application thereof, wherein the preservation number of the lactobacillus paracasei is CCTCC NO: m2020386. The strain grows and produces acid quickly, and can well utilize glucose, fructose, mannose, sucrose, oligomannose, glucose, fructose, a mixture of sucrose and fructo-oligosaccharide and inulin to produce L-lactic acid. The strain has the capability of producing inulase, can produce lactic acid by using short-chain or long-chain inulin polysaccharide, can produce high-concentration L-lactic acid by using Jerusalem artichoke powder raw material (mainly comprising inulin polysaccharide) with high-concentration substrate under the conditions of no sterilization of culture medium, no addition of nitrogen source, only addition of ionized water and light calcium carbonate, and has the number of lactic acid bacteria reaching 230 multiplied by 108CFU/ml above.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus paracasei capable of efficiently utilizing jerusalem artichoke powder and application thereof.
Background
Lactic acid is an indispensable chemical in the industrial field. The polylactic acid polymerized by using lactic acid as a precursor is a novel biodegradable and renewable bio-based material and is an environment-friendly material which has the most potential to replace the traditional plastics at present. In recent years, due to the problem of grain safety caused by lactic acid fermentation with traditional grains as substrates and the problem of high cost of refined sugar substrates in the traditional process, cheap non-grain crops are sought as lactic acid fermentation substrates, the problem of limited raw material sources and high cost becomes one of the hotspots of the current lactic acid production research, the fermentation process is improved on the basis, and the improvement of the yield and the efficiency of the lactic acid production becomes a main target. The jerusalem artichoke is a non-grain ecological and economic crop, has strong adaptability, can absorb and store saline-alkali ions of soil, improves the saline-alkali property of the soil, has wide planting range in northwest of China, and becomes a good fermentation substrate source because the rhizome of the jerusalem artichoke is rich in inulin.
Lactic acid bacteria are a group of facultative anaerobic or microaerophilic bacteria that are capable of producing lactic acid efficiently via the glycolytic pathway under anaerobic conditions (homolactic fermentation) or the hexosephosphate pathway under aerobic conditions (heterolactic fermentation). Most of lactic acid bacteria are well-known safe strains, not only harmless to the human body, but also beneficial, especially Lactobacillus plantarum (Lactobacillus plantarum) and Lactobacillus casei (Lactobacillus casei) are very widely and have a long history of use in the food industry. Furthermore, the biomass of the beneficial lactic acid bacteria after fermentation is a beneficial by-product of the fermentation process (de Lima et al 2016). However, most lactic acid bacteria have the characteristics of low fermentation temperature, high risk of bacterial contamination and high nutritional requirement, and limit the application of the lactic acid bacteria in industrial scale.
In the traditional lactic acid fermentation, refined sugar or grain crops rich in starch are used as substrates, then people gradually aim at renewable and low-cost lignocellulose raw materials, and lignocellulose biomass has become a research hotspot of lactic acid production raw materials in recent years. Compared with refined sugar and starch from grain crops, Jerusalem artichoke is an ecological crop without occupying cultivated land and has low price. Compared with lignocellulose raw materials, the biological refining method using the jerusalem artichoke as the substrate has the obvious advantages that: (1) the pretreatment of the lignocellulose raw material needs a complex process and high energy consumption, while the pretreatment process of the jerusalem artichoke raw material is simple, and only simple peeling and crushing are needed to be carried out on the jerusalem artichoke tubers, and the jerusalem artichoke tubers are extracted by hot water to obtain jerusalem artichoke juice for fermentation if necessary; (2) obtaining fermentable sugars from lignocellulosic materials requires the action of various enzyme systems, while inulin polysaccharides in jerusalem artichoke tubers require only inulinase to be hydrolyzed into fermentable sugars; (3) the process of the multi-enzyme system common enzymolysis of the wood fiber raw material is complex, the enzymolysis efficiency is generally low, the cost of the enzyme greatly increases the fermentation cost, and compared with the inulin enzymolysis process, the process is more efficient and convenient, and the cost of the inulase is relatively low.
The jerusalem artichoke is used as a high-quality and environment-friendly biorefinery raw material, has great advantages in the production of lactic acid compared with starch and wood fiber raw materials, and has wide application prospect, however, few research reports about the fermentation production of lactic acid by the jerusalem artichoke raw material exist at present. Although there has been some progress in the simultaneous saccharification and fermentation of lactic acid using jerusalem artichoke-derived substrates, research in the last decade has shown that some of the problems in the production process still need to be optimized: (1) most strains cannot directly utilize inulin, exogenous inulase is needed for producing lactic acid by taking a jerusalem artichoke matrix as a raw material, and the production cost of the lactic acid is increased; (2) the optical purity of the obtained L-lactic acid is not more than 95 percent and does not reach the industrial standard grade of synthesizing polylactic acid precursor L-lactic acid; (3) the fermentation substrate requires extraction from hot water extracts of jerusalem artichoke or direct purchase of commercial inulin, resulting in increased costs for industrial applications.
Disclosure of Invention
The invention aims to provide Lactobacillus paracasei capable of efficiently utilizing jerusalem artichoke powder, which is Lactobacillus paracasei (Lactobacillus paracasei) F204 with a preservation number of CCTCC NO: m2020386.
Another object of the present invention is the use of Lactobacillus paracasei F204 for the preparation of L-lactic acid.
In order to achieve the purpose, the invention adopts the following technical measures:
the applicant obtains a lactobacillus capable of efficiently utilizing jerusalem artichoke powder by screening laboratory lactobacillus resources, the lactobacillus is identified as lactobacillus paracasei through a 16S rDNA gene sequence and a whole genome sequence, the strain is delivered to a China center for type culture collection for preservation in 7-31 months in 2020, and the strain is classified and named as follows: lactobacillus paracasei F204 with the preservation number of CCTCC NO: m2020386, address: wuhan university in Wuhan, China.
The Lactobacillus paracasei F204(Lactobacillus paracasei F204) is a gram-positive bacillus, has no spore, no flagellum and is facultative anaerobic. The colony on MRS solid medium is milky white, has smooth and moist surface, round bulge and neat edge, and the diameter is generally 1.0-2.0mm (figure 1). The strain was simply stained to be rod-shaped under an electron microscope (FIG. 2). The L-lactic acid is produced by homofermentation of glucose, fructose, mannose, sucrose, manno-oligosaccharide and inulin. The optimal growth pH value is 5.5 in a pure sugar culture medium, and the optimal growth temperature is 34 ℃.
The application of Lactobacillus paracasei (Lactobacillus paracasei) F204 in preparing L-lactic acid comprises the steps of producing the L-lactic acid by fermenting the strain;
in the above applications, preferably, the carbon source to be transformed in the fermentation process includes: fructose; glucose; mannose; sucrose; mannose oligomer; a mixture of glucose, fructose, sucrose and fructo-oligosaccharides; and inulin.
In the above application, preferably, the jerusalem artichoke powder is used for producing the L-lactic acid by fermentation, and the culture medium in the fermentation process is as follows:
fermentation medium of jerusalem artichoke powder: jerusalem artichoke powder 215-250g/L, light CaCO380-100 g/L,MgSO4·7H2O 2-3g/L，K2HPO4 2-3g/L，MnSO4·H2O 0.05-0.10g/L，FeSO4·7H2O0.01-0.02 g/L, natural pH, no sterilization. In the above-mentioned applications, it is preferable that the high concentration of L-lactic acid is prepared using a high concentration of sugar, which isThe culture medium in the fermentation process is as follows:
160g/L of glucose or fructose or inulin 150-4·7H2O 2-3g/L，K2HPO4 2-3g/L，MnSO4·H2O 0.05-0.10g/L，FeSO4·7H2O0.01-0.02 g/L, adjusting initial pH to 5.8-6.0, and sterilizing the culture medium.
In the above-mentioned application, preferably, the fermentation method comprises: the culture medium is heated to 37-40 ℃, 10-12% of seed liquid is directly inoculated for anaerobic fermentation, and the stirring speed is 150-.
Compared with the prior art, the invention has the following advantages:
the Lactobacillus paracasei (Lactobacillus paracasei) F204 provided by the invention has the capability of rapidly producing L-lactic acid by using monosaccharide and oligosaccharide and producing the L-lactic acid with high product concentration by using glucose, fructose and inulin with high substrate concentration. In addition, the more unique characteristic is that the lactobacillus paracasei F204 expresses inulinase, and short-chain and long-chain inulin and jerusalem artichoke powder raw materials can be directly utilized to produce the L-lactic acid. Inulin with a polymerization degree of at least up to 23 can be used, whereas inulin is less available for FFn types with lower polymerization degrees. Especially, the Jerusalem artichoke powder can be directly used for producing the L-lactic acid by fermentation under the conditions of not adding a nitrogen source, only adding ionized water and light calcium carbonate, and the optical purity of the L-lactic acid produced by the fermentation of the strain is more than 98 percent.
Drawings
FIG. 1 is a morphological diagram of a Lactobacillus paracasei F204 colony;
FIG. 2 is a simple staining microscopic morphology of Lactobacillus paracasei F204;
FIG. 3 shows the results of the production of lactic acid by Lactobacillus paracasei F204 using different pure sugars;
fru: fructose; glu: glucose; man: mannose; and Sur: sucrose; MOS: mannose oligomer; FOS: glucose, fructose, a mixture of sucrose and fructo-oligosaccharides (fructo-oligosaccharides > 90%, the content of glucose, fructose and sucrose < 10%), the degree of polymerization of fructo-oligosaccharides is 2-10; p95: FFn type inulin with polymerization degree of 4-5; CLR: GFn type inulin, the polymerization degree is 7-9; TEX: GFn-type inulin, with a degree of polymerization > 23; HP: GFn type inulin, polymerization degree 21-26; HD: GFn type inulin with polymerization degree of 2-60.
FIG. 4 shows the results of simultaneous saccharification and lactic acid fermentation of Lactobacillus paracasei F204 in 5L and 50L anaerobic fermentors;
wherein (A)5L anaerobic fermentation tank experiment; (B)50L anaerobic fermenter experiment.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available. The lactic acid is L-lactic acid.
Example 1: screening of strains
1) Among lactobacillus resources in the laboratory of the applicant, 8 lactobacillus strains with high inulin polysaccharide utilization rate are connected to an MRS liquid culture medium activating strain from a glycerol tube, and then are switched to an MRS liquid culture medium for standing culture for 18h to serve as seed liquid. Respectively inoculating the seed solution into strain screening culture medium, taking corresponding culture medium without adding Jerusalem artichoke powder as blank control, inoculating with an amount of 1% (v/v), standing at 37 deg.C for culturing, and adjusting pH to 5.5-6 with 10mol/L NaOH every 6h (determined by pH test paper, and the process is aseptic operation). And (4) after the pH value of the fermentation solution is not changed any more, considering that the acid production is finished. And collecting fermentation liquor at the fermentation end point of each experimental group, and measuring the contents of lactic acid, acetic acid, glucose, fructose and sucrose in the fermentation liquor. As shown in Table 1, the yield of lactic acid produced by Lactobacillus paracasei F204 and the conversion rate of the Jerusalem artichoke powder are the highest, and the yields are respectively 32.65g/L and 0.65g/g of substrate raw material.
The strain screening culture medium comprises the following components: 50g/L of jerusalem artichoke powder, 10g/L of yeast extract and MgSO4·7H2O 2g/L，K2HPO4 2g/L，MnSO4·H2O 0.05g/L，FeSO4·7H2O 0.01g/L；pH 6.5。
TABLE 1 results of lactic acid and acetic acid production by different lactic acid bacteria directly using Jerusalem artichoke powder
Identification and preservation of Lactobacillus paracasei F204
The selected target strain F204 was simply stained with crystal violet and observed by an optical microscope. The strain is found to be gram-positive bacillus, non-spore, non-flagellum and facultative anaerobic. The colony on MRS solid medium is milky white, smooth and moist surface, round, raised, and regular edge, and the diameter is generally 1.0-2.0mm (FIG. 1). The strain was simply stained to be rod-shaped under an electron microscope (FIG. 2). The optimum growth pH is 6.0-6.5; the optimum growth temperature is 34 ℃.
And (3) carrying out genome sequencing on lactobacillus paracasei F204 to obtain the full-length 16S rDNA, and comparing the rDNA with an NCBI database to determine the strain of lactobacillus paracasei. The strain is delivered to China Center for Type Culture Collection (CCTCC) for collection at 31/7/2020, and is classified and named as follows: lactobacillus paracasei F204 with the preservation number of CCTCC NO: m2020386, address: wuhan university in Wuhan, China.
Example 2:
the lactobacillus paracasei F204 can efficiently utilize different carbon sources to produce lactic acid:
preparing a pure sugar fermentation culture medium: 50g/L carbon source to be transformed, 5g/L yeast extract, MgSO4·7H2O 2g/L，K2HPO4 2g/L，MnSO4·H2O 0.05g/L，FeSO4·7H2O is 0.01 g/L. The initial pH was 6.5.
The carbon source to be transformed is: fructose (Fru); glucose (Glu); mannose (Man); sucrose (Sur); mannose Oligomer (MOS); glucose, fructose, a mixture of sucrose and fructo-oligosaccharides (FOS, comprising 5% glucose, fructose and sucrose, 95% fructo-oligosaccharides), the degree of polymerization of fructo-oligosaccharides being 2-10; FFn-type inulin (P95), having a degree of polymerization of 4 to 5; GFn-type inulin (CLR), having a degree of polymerization of 7 to 9; GFn-type inulin (TEX), with a degree of polymerization > 23; GFn-type inulin (HP), having a degree of polymerization of 21 to 26; GFn-type inulin (HD), polymerization degree of 2-60.
The corresponding medium without carbon source was used as a blank.
Inoculating fresh seed liquid of lactobacillus paracasei F204 according to the inoculation amount (v/v) of 1 percent, standing in a triangular flask and culturing at 37 ℃, and adjusting the pH to be between 5.5 and 6 by using 10mol/L NaOH every 6 hours. And (4) after the pH value of the fermentation solution is not changed any more, determining that acid production is finished, and measuring the concentration of lactic acid and the concentration of acetic acid by using a high performance liquid chromatography.
The conversion rate of the jerusalem artichoke powder by the lactic acid is calculated as follows: lactic acid concentration/carbon source substrate concentration;
the conversion of lactic acid to pure sugar substrate was calculated as follows: lactic acid concentration/pure sugar substrate concentration × 100%
The results of the experiment are shown in figure 3,
the lactobacillus paracasei F204 provided by the invention can well utilize a mixture of glucose, fructose, mannose, sucrose, oligomannose, glucose, fructose, sucrose and fructo-oligosaccharide (comprising 5% of glucose, fructose and sucrose and 95% of fructo-oligosaccharide) to produce lactic acid, and the conversion rates are 83.29%, 88.72%, 89.70%, 95.93%, 88.56% and 76.37% respectively; the inulin utilization rate for the polymerization degree of 7-9 was 94.98%, the inulin utilization rate for the polymerization degree of 21-26 was 83.38%, the inulin utilization rate for the polymerization degree of >23 was 85.88%, and the inulin utilization rate for the polymerization degree of 2-60 was 82.68%.
After fermentation, a fermentation medium containing different carbon sources (the different carbon sources are fructose (Fru), glucose (Glu), mannose (Man), sucrose (Sur), oligomannose (MOS), a mixture (FOS) of glucose, fructose, sucrose and fructo-oligosaccharide, wherein the polymerization degree of the fructo-oligosaccharide is 2-10, FFn-type inulin (P95) has the polymerization degree of 4-5, GFn-type inulin (CLR) has the polymerization degree of 7-9, GFn-type inulin (TEX) has the polymerization degree of more than 23, and GFn-type inulin (HP) has the polymerization degree of 21-26); GFn type inulin (HD), polymerization degree 2-60, lactic acid concentration in the fermentation liquid is as follows in sequence: 41.64g/L, 44.36g/L, 44.85g/L, 47.96g/L, 44.28g/L, 38.18g/L, 3.08g/L, 47.49g/L, 42.94g/L, 41.69g/L and 41.34 g/L. The conversion rate of lactic acid to pure sugar substrate was 83.29%, 88.72%, 89.70%, 95.93%, 88.56%, 76.37%, 6.16%, 94.98%, 85.88%, 83.38%, 82.68%.
Example 3:
production of high concentration lactic acid using high concentration substrate glucose, fructose and inulin
Preparation of lactobacillus paracasei F204 seed solution: MRS liquid culture medium, pH 6.5-6.8. Sterilizing at 115 deg.C under high temperature and high pressure for 20min, cooling to 37 deg.C, aseptically inoculating 1% bacteria solution (v/v, 10%) activated from glycerol tube7-108CFU/mL). Standing and culturing for 18h to obtain seed liquid.
Fermentation medium: 150g/L of glucose or fructose or inulin, 15g/L of yeast extract, 80g/L of light calcium carbonate and MgSO4·7H2O 2g/L，K2HPO4 2g/L，MnSO4·H2O 0.05g/L，FeSO4·7H2O0.01 g/L, adjusting the initial pH to 5.8, sterilizing the culture medium at 115 ℃ for 20min under high temperature and high pressure, and cooling to 40 ℃.
The fermentation process comprises the following steps: the culture medium was warmed to 40 ℃ and inoculated directly with 10% seed solution (25X 10)8CFU/mL) was fermented anaerobically with a stirring rate of 150 r/min. High-concentration lactic acid can be obtained by anaerobic fermentation (wherein the fermentation period with glucose as a substrate is 30h, and the fermentation period with fructose and inulin as substrates is 33 h.) and the content of L-lactic acid in the culture solution is detected.
The results were: 141.20g/L of L-lactic acid is produced by using 150g/L of glucose; 149.45g/L of L-lactic acid is produced by using 150g/L of fructose; 145.41g/L of L-lactic acid was produced using 150g/L of inulin. Meanwhile, the number of viable bacteria in the fermentation time of 30-33h reaches 200-400 multiplied by 108CFU/ml。
Example 4:
lactobacillus paracasei F204 directly utilizes jerusalem artichoke powder as raw material to produce high-concentration L-lactic acid
Fermentation medium of jerusalem artichoke powder: jerusalem artichoke powder 215g/L, light CaCO380 g/L,MgSO4·7H2O 2g/L，K2HPO42g/L，MnSO4·H2O 0.05g/L，FeSO4·7H20.01g/L of O, and the mixture is charged into a 5L or 50L anaerobic fermentation tank (the liquid charge amount is 3L or 30L) after being uniformly mixed, and is added at one time. Natural pH (about 5.8), no sterilization, ready for use.
The jerusalem artichoke powder is prepared by air drying and crushing non-peeled jerusalem artichoke tubers, and sieving with a 60-mesh sieve.
The fermentation process comprises the following steps: the culture medium was warmed to 40 ℃ and inoculated directly with 10% seed solution (25X 10)8CFU/mL) was fermented anaerobically with a stirring rate of 150 r/min. High-concentration lactic acid can be obtained by anaerobic fermentation for 39h (5L) or 42h (50L).
In the 5-L and 50-L anaerobic fermentors, the lactic acid yields were 144.08g/L and 139.57g/L, respectively, the yields were 0.67g/g and 0.65g/g, respectively, the average productivities were 4.37g/L/h and 3.32g/L/h, respectively, the conversion efficiency was 99% of the theoretical yield, and the optical purity of the produced L-lactic acid was 99% (FIG. 4).
After the fermentation is finished (the fermentation period is 39h and 42h respectively), the viable count of the lactobacillus paracasei F204 reaches 230 multiplied by 108CFU/ml。
Claims (6)
1. Separated lactobacillus paracasei (L.paracasei)paracasei Lactobacillus) The preservation number of the lactobacillus paracasei is CCTCC NO: m2020386.
2. Use of lactobacillus paracasei according to claim 1 for the fermentative production of L-lactic acid.
3. The use of claim 2, wherein the carbon source to be transformed in the fermentation process comprises: fructose; glucose; mannose; sucrose; mannose oligomer; a mixture of glucose, fructose, sucrose and fructo-oligosaccharides; and inulin.
4. The use according to claim 2, wherein the culture medium during the use is:
fermentation medium of jerusalem artichoke powder: jerusalem artichoke powder 215-250g/L, light CaCO380-100 g/L, MgSO4∙7H2O 2-3 g/L，K2HPO4 2-3 g/L，MnSO4∙H2O 0.05-0.10 g/L，FeSO4∙7H2O0.01-0.02 g/L, natural pH, no sterilization.
5. The use according to claim 2, wherein the culture medium during the use is:
160g/L of glucose or fructose or inulin 150-4∙7H2O 2-3 g/L，K2HPO4 2-3 g/L，MnSO4∙H2O 0.05-0.10 g/L，FeSO4∙7H2O0.01-0.02 g/L, adjusting initial pH to 5.8-6.0, and sterilizing the culture medium.
6. The use according to claim 4 or 5, wherein the fermentation process comprises: the culture medium is heated to 37-40 ℃, 10-12% of seed liquid is directly inoculated for anaerobic fermentation, and the stirring speed is 150-.
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| EP4070670A1 (en) * | 2021-04-08 | 2022-10-12 | University College Cork-National University of Ireland Cork | Lacticaseibacillus paracasei em025-11 and uses thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4070670A1 (en) * | 2021-04-08 | 2022-10-12 | University College Cork-National University of Ireland Cork | Lacticaseibacillus paracasei em025-11 and uses thereof |
| WO2022214700A1 (en) * | 2021-04-08 | 2022-10-13 | University College Cork - National University Of Ireland, Cork | Lacticaseibacillus paracasei em025-11 and uses thereof |
| CN113832072A (en) * | 2021-10-20 | 2021-12-24 | 江南大学 | Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof |
| CN113832072B (en) * | 2021-10-20 | 2023-10-27 | 江南大学 | Lactococcus lactis subspecies lactate with inulin utilization capacity and application thereof |
| CN114317366A (en) * | 2022-01-04 | 2022-04-12 | 澳优乳业（中国）有限公司 | Bacterial strain and application thereof |
| CN114317366B (en) * | 2022-01-04 | 2023-07-07 | 澳优乳业（中国）有限公司 | Bacterial strain and application thereof |
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