WO2023055287A1 - Procédé d'extraction et de fermentation - Google Patents

Procédé d'extraction et de fermentation Download PDF

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Publication number
WO2023055287A1
WO2023055287A1 PCT/SG2021/050596 SG2021050596W WO2023055287A1 WO 2023055287 A1 WO2023055287 A1 WO 2023055287A1 SG 2021050596 W SG2021050596 W SG 2021050596W WO 2023055287 A1 WO2023055287 A1 WO 2023055287A1
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Prior art keywords
bran
ferulic acid
fermentation
hours
solid
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PCT/SG2021/050596
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English (en)
Inventor
Kian Hong NG
Siew Choo LIM
Gee Fang BOH
Jia Rong Bernadette LEE
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Wilmar International Limited
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Priority to CN202180099684.3A priority Critical patent/CN117545733A/zh
Priority to PCT/SG2021/050596 priority patent/WO2023055287A1/fr
Publication of WO2023055287A1 publication Critical patent/WO2023055287A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
    • C07C51/44—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation by distillation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the present invention relates generally to the field of biotechnology.
  • the present invention relates to methods of extraction and fermentation.
  • Bran is a major side stream product from flour milling industry. It is commonly used as feed ingredient. However, due to high content of non-starch polysaccharides, its inclusion in feed is limited, especially in monogastric animals. Wheat bran, for example, also contains high amount of ferulic acid and there are studies focusing on exploring ways to extract the phenolic acid from wheat bran. However, there is a dearth of technical exploration of combining both processes for wheat bran treatment. Thus, there is a need for a combined process to better exploit bran obtained as a side product.
  • the present disclosure refers to a method of obtaining ferulic acid from bran followed by subsequent fermentation of the bran, the method comprising: a. combining the bran, water, and an enzyme to obtain a mixture, wherein the enzyme hydrolyses (hemi-)cellulose; b. incubating the mixture of step a in an incubation step; c. separating the mixture of step b into liquid and solid phases; d. isolating the liquid phase obtained in step c and obtaining ferulic acid from the same; e. inoculating the obtained solid phase of step c with a microorganism which ferments bran; f. incubating the inoculated solid phase of step e in a fermentation step; thereby obtaining fermented bran.
  • FIG. 1 shows a schematic diagram summarizing the “Integrated Process” of ferulic acid extraction and fermentation of bran.
  • FIG. 2 shows the experimental setup and outcomes of three processes for treating bran, in this example, wheat bran.
  • the “Integrated Process” includes enzymatic treatment followed by solid-state fermentation using DSM33837.
  • the “Control Process” refers to wheat bran in water (1: 12.5 ratio, w/w) without addition of any exogenous enzymes or DSM33837. “Fermentation” involves only DSM33837 in a solid-state fermentation.
  • FIG. 3 shows the characterization of dried, and modified products of wheat bran. The products were obtained either through “Integrated Process”, “Fermentation” or “Control Process”.
  • FIG. 3a shows live colony-forming units of DSM33837 were recovered in products from both the “Integrated Process” and “Fermentation” but not from the “Control Process”.
  • FIG. 3b shows increased reducing sugar content in both products from the “Integrated Process” and “Fermentation”, but not from the “Control Process”.
  • FIG. 4 shows the experimental setup and outcome of an “Alternative Process” for treating bran, as shown by way of the example of wheat bran.
  • “Alternative Process” includes both enzymatic treatment and Bacillus fermentation in a single step. Quantifiable amounts of ferulic acid could not be detected from using the “Alternative Process”.
  • FIG. 5 shows a schematic diagram summarizing the claimed method applied to the example of ferulic acid extraction and fermentation of wheat bran.
  • Ferulic acid has antioxidant properties, giving it many potential commercial applications in health, cosmetic and food. Extraction of ferulic acid and other phenolic compounds from products such as, but not limited to, bran, is a topic of interest to valorise this lignocellulosic biomass.
  • an enzymatic method was used to extract ferulic acid in an environmentally friendly method.
  • the bran resulting from the methods disclosed herein is solvent-free and can be used in other applications, such as, but not limited to, animal feed ingredients.
  • Extraction of ferulic acid can be performed on various types of biomasses, such as, but not limited to, com stoves, rice bran, wheat bran.
  • the ferulic acid is extracted from bran.
  • the enzyme is obtained from a microorganism.
  • the enzyme can be a synthetic or recombinant enzyme, for example, one that is obtained in the laboratory using genetic methods known in the art. So long as the required enzyme is produced by a microorganism, a person skilled in the art would be able to obtain said enzyme using methods known in the art.
  • such an enzyme can be isolated or obtained from a microorganism of the genus of Aspergillus sp.
  • Ferulic acid esterase also known as feruloyl esterase is an enzyme capable of hydrolysing the ester bond which is present between polysaccharide forming the plant cell wall and phenolic acid. Ferulic acid esterase has been shown to work synergistically with other hemicellulases to breakdown of lignocellulosic biomass. In other words, enzymes that hydrolyse (hemi-)cellulose can be used in the methods and processes disclosed herein.
  • the methods described herein can be performed using one or more enzymes as disclosed herein.
  • the enzymes are, but are not limited to, ferulic acid esterase, xylanase, and combinations thereof.
  • the ferulic acid esterase can be obtained from any one or more of the following exemplary microorganisms: Aspergillus niger, Aspergillus tubingensis, Aspergillus terreus, Aspergillus clavatus,' Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gasseri, and/or Lactobacillus helveticus.
  • the enzyme is Aspergillus niger ferulic acid esterase (AnFAE).
  • Bran refers to the grain husk or the hard outer layers of a grain, such as, but not limited to, cereal grains. It consists of the combined aleurone and pericarp. Along with germ, it is an integral part of whole grains, and is often produced as a by-product of milling in the production of refined grains. Bran makes up about 10% to 15% of the total wheat kernel weight. Specifically, wheat bran is an abundant side product generated during the milling process. In addition, wheat bran contains 10% to 25% starch due to incomplete separation of the endosperm and contains about roughly 35% non-starch carbohydrate mainly, made up of arabinoxylan, a major fibre component in bran.
  • Bran is present in cereal grain, including but not limited to rice, corn (maize), wheat, oats, barley, rye and millet. Bran is not the same as chaff, which is a coarser scaly material surrounding the grain but not forming part of the grain itself, and which is indigestible to humans.
  • meal refers to the coarse-ground, edible part of various grains. Meal is often used in animal feed and can also be characterised as being a coarser blend than flour, whereby flour refers to a powder made by grinding raw grains, roots, beans, nuts, or seeds. It is noted that this does not refer only to the wheat flour commonly used, but refers to the ground product of raw grains, roots, beans, nuts, or seeds. “Meal” also refers to products that remain after oil extraction of grains, brans or oilseeds has been completed. A meal may or may not contain further additives and agents for, for example, enriching the meal, or preventing caking after grinding.
  • defatted refers to the removal of fat from the product in question.
  • defatted meal refers to meal which has a lower fat content than otherwise untreated (non-defatted) meal.
  • the presently disclosed methods can be performed on brans that are naturally enriched in ferulic acids.
  • the bran is bran which contains ferulic acid.
  • the bran is obtained from grains such as, but not limited to, wheat, maize, rice, defatted rice, sorghum, barley, rye and oat.
  • the bran disclosed herein is wheat bran.
  • Arabinoxylan is further associated with varying levels of phenolic acids such as ferulic acid.
  • a method enables the extraction of ferulic acid from bran.
  • the method disclosed herein also allows for subsequent fermentation of the product left over after ferulic acid extraction in the form of a sequential or integrated method or process.
  • non-starch carbohydrate such as arabinoxylan
  • xylan can act as an antinutritional components by trapping energy and nutrients in meal that contains xylan or arabinoxylan.
  • microorganisms disclosed herein function to ferment biomass in order to improve the biochemical and nutritional composition of the biomass. In other words, fermentation of the biomass results in a form of “pre-digestion”, resulting in improved bioavailability of the nutrients and energy contained in the meal and bran treated using the method disclosed herein.
  • microorganisms capable of fermentation capable of fermentation.
  • the microorganism used herein must be capable of fermenting the starting material of the fermentation process.
  • fermentation is performed using microorganisms such as, but not limited to, a microorganism of the genus of Bacillus spp., Lactobacillus spp., Aspergillus spp., Streptococcus spp., and combinations thereof.
  • the microorganism can be, but is not limited to, Bacillus spp. strains DSM33837, DSM33839 and DSM33838, and combinations thereof.
  • the microorganism is DSM33837.
  • the present disclosure describes a method of bran pre-treatment and extraction of ferulic acid in a preceding method.
  • the bran obtained herein can be use as, for example, an animal feed ingredient.
  • a method of obtaining ferulic acid from bran also referred to as the extraction step
  • the method comprising: combining the bran, water, and an enzyme to obtain a mixture, wherein the enzyme hydrolyses (hemi-) cellulose; incubating the mixture in an incubation step; separating the mixture into liquid and solid phases; isolating the obtained liquid phase and obtaining ferulic acid from the same; inoculating the obtained solid phase with a microorganism which ferments bran; incubating the inoculated solid phase in a fermentation step; thereby obtaining fermented bran.
  • the remaining liquid phase is returned to the starting material
  • the incubation step is carried out for at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours, or about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, at least 23 hours, or at least 24 hours, or about 15 minutes, about
  • the duration of the incubation step will depend on, for example, the quality of the enzyme used (and thereby the efficacy of the enzyme). Thus, the more efficient the enzyme, the shorter the incubation duration can be. Such a duration can also depend on the type and quality of the starting material used.
  • bran and water are combined in a ratio of, but not limited to, at least 1:5 (bran to water), or about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 11, about 1: 12, about 1: 13, about 1: 14, about 1: 15, about 1: 16, about 1: 17, about 1: 18, about 1: 19, about
  • the amount of ferulic acid esterase is, but is not limited to, at least O.lOpg per gram of bran, or about O.l lpg, about 0.12pg, about 0.13pg, about 0.14pg, about 0.15pg, about 0.16pg, about 0.17pg, about 0.18pg, about 0.19pg, or about 0.20pg per gram ofbran.
  • the amount of xylanase is, but is not limited to, at least 0.4% per gram of bran, or about 0.5% per gram of bran, about 0.6% per gram of bran, about 0.7% per gram of bran, about 0.8% per gram ofbran, about 0.9% per gram of bran, about 1% per gram of bran, about 1.5% per gram of bran, or about 2% per gram ofbran.
  • the order in which the subprocesses of the claimed process are performed has an impact on the resulting product.
  • the following issues can occur: proteases secreted by bacteria during fermentation can inhibit feruloyl esterase activity, thereby negatively affecting the yield of the method; carry-over of bacteria from fermentation into the liquid phase of ferulic acid extraction can hamper and complicate purification of ferulic acid and the reuse of water, thereby making the resulting method less economical; without being bound by theory, enzyme digestion (in order to release ferulic acid into the liquid phase) requires large amounts of water.
  • the release of ferulic acid may not be efficient; reducing sugars generated by bacterial fermentation will be washed away from the fermented bran if the process was performed simultaneously or in a reversed sequence, thereby diminishing the nutrient content and nutritional value of the resulting fermented bran.
  • the ferulic acid needs to be separated from its (carrier) liquid phase. A person skilled in the art will appreciate that this can be done using methods known in the art. These methods can be, but are not limited to, distillation, vacuum distillation, batch distillation, continuous distillation, solvent extraction, and solidphase extraction.
  • the ferulic acid obtained from the methods disclose herein is obtained at a yield of between 0.4mg ferulic acid per gram bran to 6 mg ferulic acid per gram bran.
  • the yield of ferulic acid is between 0.4mg to 0.8mg, between 0.5mg to Img, between 0.75mg to I.25mg, between 0.8mg to 1.3mg, between 0.9mg to 1.4mg, between 1.3mg to 1.8mg, between 1.5mg to 2mg, between 1.75mg to 2.25mg, between 1.8mg to 2.3mg, between 1.9mg to 2.4mg, between 2.3mg to 2.8mg, between 2.5mg to 3mg, between 2.75mg to 3.25mg, between 2.8mg to 3.3mg, between 2.9mg to 3.4 mg, between 3.3 mg to 3.8 mg, between 3.5 mg to 4 mg, between 3.75 mg to 4.25 mg,
  • separation of a solid phase or component from a liquid phase can be performed using methods such as, but not limited to centrifugation, fdtration, and sedimentation.
  • the fermentation step as disclosed herein is a solid-state fermentation (SSF) step.
  • SSF solid-state fermentation
  • animal feed ingredients include, but are not limited to, animal feed ingredients which contain probiotic bacillus, as a result of the fermentation process of the bran disclosed herein.
  • animal feed ingredients obtained by the process disclosed here have increased reducing sugar content, compared to bran that had not undergone the claimed process.
  • the present disclosure shows an exemplary workflow wherein ferulic acid is first extracted from bran, followed by subsequent solid-state fermentation (SSF) of the residual bran, which can then be further processed to be used in the production of animal feed ingredients. (See, for example, FIG. 1).
  • SSF solid-state fermentation
  • the enzymatic treatment as disclosed herein allows for the extraction of ferulic acid without compromising the growth of the microorganisms required for subsequent fermentation.
  • the water used for ferulic acid extraction containing residual enzymes can be reused for treating subsequent batches of bran. This will also have a positive economic impact on the method disclosed herein, which is then considered to be environmentally friendly and cost-effective compared to known methods.
  • bran contains higher amount of reducing sugar than non-fermented bran, thereby showing and underlining that the bran obtained from the methods disclosed herein has improved bioavailability of the nutrients it contains.
  • Table 1 shows the results of quantification of extracted ferulic acid from the three processes disclosed herein, the “Integrated Process”, the “Control Process” and “Fermentation”. After 16 hours, the liquid fraction is separated from the wheat bran. Ferulic acid from the liquid phase was analysed by HPLC. The amount of ferulic acid was quantified using ferulic acid standard curve. Values that fall below the range covered by the standard curve is considered not quantifiable (N.Q.). The ferulic acid yield from the “Integrated Process” is 1.65 mg/g of wheat bran. Wheat bran treated with the other processes did not result in a quantifiable yield.
  • the residual wheat bran undergoes solid state fermentation with DSM33837 and then dried.
  • the dried solid-state fermentation (SSF) product was rehydrated and analysed for viable microbes, reducing sugar content.
  • a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
  • the term “about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Wheat bran was autoclave sterilized at 121 °C for 15 minutes and dried overnight in an oven.
  • the sterilized wheat bran (19g) was placed in a 1 litre conical flask.
  • 237.5g of water, 2.85pg of recombinant Aspergillus niger ferulic acid esterase (AnFAE) and 95pL (0.5% (v/w)) of commercial xylanase (Megazyme) were added. This meant that the amount of ferulic acid esterase and commercial xylanase used was 0. 15pg and 0.5%, respectively, per gram of wheat bran.
  • the wheat bran to water ratio used in enzymatic hydrolysis was 1: 12.5 (12.5g of water was added to 1g of wheat bran).
  • Control Process 237.5g of water was added to wheat bran.
  • Fermentation 19g of water was added to wheat bran. All flasks were incubated for 16 hours at 37°C. The liquid fraction was recovered, centrifuged, and kept for ferulic acid analysis. The treated wheat bran was subsequently used for solid- state fermentation. Time 0 hour was taken as before enzymatic hydrolysis and time 16 hour was after enzymatic hydrolysis.
  • the concentration of ferulic acid was determined by HPLC (Shimadzu Prominence LC-20AD system equipped with a diode array detector SPD-M2A).
  • a C18 column (5pm x 250mm x 4.6mm) (ODS- HYPERSIL-2, Thermo Fisher) was eluted with a gradient mobile phase consisting of 0.1% acetic acid as eluant A and 100% methanol as eluant B. The flow rate was maintained at 1ml per minute.
  • the column oven was set at 24°C. lOpl of sample, appropriately diluted, were injected into the column for analysis. Peaks were monitored at 340nm.
  • the amount of ferulic acid was quantified using a ferulic acid standard curve.
  • the DNA coding for ferulic acid esterase gene from Aspergillus niger was codon optimized and synthesized. The gene was cloned into a pPIC9K vector using EcoRI/Notl restriction sites and expressed in Pichia pastoris GS115. Protein expression was achieved by growing the cells in buffered minimal methanol -complex medium (BMMY; 0.1M potassium phosphate buffer pH 6.0, 1% yeast extract, 2% peptone, 1.34% YNB without amino acids, 0.4pg/ml biotin, 1% methanol) at 28°C with shaking (200rpm). The cells were fed with 1% methanol every 12 hours for 4 days.
  • buffered minimal methanol -complex medium BMMY; 0.1M potassium phosphate buffer pH 6.0, 1% yeast extract, 2% peptone, 1.34% YNB without amino acids, 0.4pg/ml biotin, 1% methanol
  • the culture was centrifuged at 4,000rpm for 30 minutes to obtain and remove the cell pellet.
  • the supernatant containing the Aspergillus niger ferulic acid esterase was concentrated, and protein concentration was determined via Bradford method, using bovine serum albumin (BSA) as a standard.
  • BSA bovine serum albumin
  • the protein sequence of the synthesized gene is as follows (same sequence as XP_001393337.1 in protein database): ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTIIKGEKIYNAQTDINGWILRDDTSKEIITVFRG TGSDTNLQLDTNYTLTPFDTLPQCNDCEVHGGYYIGWISVQDQVESLVKQQASQYPDYALTVTG HSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSND GIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGVNDAHTTYFGMTSG ACTW (SEQ ID NO: 2)
  • the culture was collected, washed with water, and inoculated at initial OD600 of 0.2 into M9 minimal medium (6.78g/L Na2HPO4*7H20, 3g/L KH2PO4, Ig/L NH4CI, 0.5g/L NaCl) containing 0.5% (w/v) beech wood xylan as sole carbon source.
  • M9 minimal medium (6.78g/L Na2HPO4*7H20, 3g/L KH2PO4, Ig/L NH4CI, 0.5g/L NaCl) containing 0.5% (w/v) beech wood xylan as sole carbon source.
  • the tubes were incubated at 37°C, 220rpm for several days and their enzyme activity was assessed by a plate screen with Azurine-cross-linked xylan (AZCL-xylan) followed by xylanase activity assay performed using the 3, 5 -dinitrosalicylic acid (DNS) method.
  • the phylogeny of isolated strains was further identified by 16S rRNA sequencing. Genomic DNA extraction was performed by the methodology as described by Hoffman et al. (1987; Gene; 57; 267- 272) and used as template in PCR for 16S rRNA partial gene amplification ( ⁇ 500bp) using the following universal primers: WIC1P2336 (5’- CCT ACG GGA GGC AGC AG -3’; SEQ ID NO: 3) and WIC1P2337 (5’- GGA CTA CHV GGG TWT CTA AT -3’; SEQ ID NO: 4). The amplicons were purified and sequenced. Sequence similarity and homology analysis against the GenBank database were carried out using the basic local alignment search tool (BLASTN) on the National Center for Biotechnology Information (NCBI).
  • BLASTN basic local alignment search tool
  • NCBI National Center for Biotechnology Information

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Abstract

La présente invention divulgue des procédés d'obtention d'acide férulique à partir de son suivi d'une fermentation ultérieure du son.
PCT/SG2021/050596 2021-10-01 2021-10-01 Procédé d'extraction et de fermentation WO2023055287A1 (fr)

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