Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation

Definitions

• the subject of the present invention is a new process for the continuous isolation of active proteins and in particular of enzymes from plants or fermentation media, as well as the extraction device.
• Enzymes play an important role in the biogenesis of aromas in fresh foods.
• the synthesis processes which they catalyze give the food its characteristic taste and odor.
• these compounds are often lost or thermally degraded, and the enzymes that synthesize them are inactivated.
• US Pat. No. 4,728,613 describes a process for enriching the enzyme present in one of the 2 phases of an insoluble oil-water mixture.
• the enzyme must still be isolated, which is not described in this patent. This process requiring several steps is therefore slow and tedious.
• it only allows a low yield incompatible with industrial use since it causes part of the activity of the enzyme to be lost at each stage.
• the present invention aims to remedy these problems.
• the active proteins contained in a mixture are precipitated continuously and in a single step, in a suitable organic solvent.
• enzymatic solution extracted from said plant material or from the fermentation medium, in a specific reactor said proteins contained in a solution based on juice of plant material, the conditions in the reactor being adjusted so as to obtain a precipitate of undenatured proteins, said precipitate is then continuously separated.
• a step of maturing the protein precipitate can be applied after the precipitation in the reactor, so as to increase the size of its particles. constitutive. It can be obtained by mixing with a vertical turbine in a continuous reactor or using a static mixer, for example.
• the method according to the invention makes it possible to obtain a protein extract, in particular enzymes, which is not denatured and therefore active.
• a protein extract in particular enzymes, which is not denatured and therefore active.
• the extraction yield as well as the activity of the enzymes are much higher than what can be obtained by conventional methods or batch processes.
• Another object of the invention is a reactor which allows the continuous isolation of numerous active proteins. It allows for example to extract more activity of pectin methylesterase (PME), peroxidase (POX) and a larger quantity of proteins (cf. Table 1). It consists of an angle cell, preferably in the form of a T.
• the reaction conditions are easily adjustable and allow the process to be optimized for each type of protein.
• This reactor also has the advantage of having a simple geometry compared to other reactors, which makes it very easy to handle and to clean.
• the invention relates to the non-denatured protein extract thus obtained and its use for regenerating the aromas and tastes of various food products such as soups and other vegetable-based foods, foods for children, for example.
• This process can also be applicable in the field of biotechnology for "downstream processing", that is to say the separation of an enzyme produced by microorganisms in a bio-fermenter, for example.
• “Fresh aroma or taste” is understood to mean the aroma and taste of fresh tomatoes, that is to say the green, acidic and light notes which are not found in industrial tomato juice, for example.
• active proteins designates the enzymes present in the tomato and partly responsible for the taste and odor which are known to be undenatured. These active proteins are for example enzymes such as peroxidase (POX), acid phosphatase (PA), pectin methylesterase (PME) or alcohol dehydrogenase (ADH).
• POX peroxidase
• PA acid phosphatase
• PME pectin methylesterase
• ADH alcohol dehydrogenase
• fruit and / or vegetables can be used as vegetable material, that is to say any edible plant, whether it is a seed, root, tuber, stem, leaf, flower or fruit, for example.
• vegetable material that is to say any edible plant, whether it is a seed, root, tuber, stem, leaf, flower or fruit, for example.
• plants are used for which it is desired to reinforce the natural fresh taste. We therefore particularly avoid plants whose natural taste can be unpleasant or whose cooked taste is desired, in particular asparagus, peas, soybeans, potatoes, cereals, sea buckthorn, medlars, for example.
• the leaves in particular leek, fennel and cabbage, stems, in particular rhubarb and broccoli, certain roots, in particular carrot, onion, radish, celery and beets, tubers, especially cassava, and fruits, especially tomatoes, zucchini, eggplant, bananas, apples, apricots, melons, watermelons, pears, plums, peaches, cherry, kiwi and mirabelle plum, for example.
• the plant material is prepared in the form of juice and then treated so that the solution contains as many enzymes as possible.
• This initial extraction step consists in dissolving the maximum quantity of enzymes before the actual isolation step in a specific reactor. For this, we can homogenize the plant material and then bring the pH of the homogenate to 5-8.5, and preferably 7.0. Salt can then be added, for example sodium chloride.
• the total salt concentration can be between 0.25-1 M and preferably
• the insoluble parts can then be removed by centrifugation, by example.
• the optimal conditions for each enzyme are presented in Table 2.
• the supernatant thus obtained can be frozen or directly treated in the reactor so as to isolate the active proteins therefrom.
• the extraction yield of the enzymes can be between 50 and 100% for the tomato, for example.
• the solution containing the enzymes to be isolated is thus continuously introduced into a reactor consisting of a cell, two "inlet” branches (enzyme solution and solvent) and an “outlet” branch (for the enzyme isolate in the form of a precipitate), the latter preferably forming an angle of 90 ° relative to the inlets, ie a T-shaped cell. Other angles can be used.
• the solution containing the enzymes to be isolated is mixed with the organic solvent then carried out in the reactor.
• the solvent is preferably chosen from alcohols, and in particular ethanol, or any other derived organic solvent.
• the solvent is injected directly into the cell, through one of the inlet branches of the reactor cell.
• the alcohol is preferably used so that its final concentration is between 40 and 95% by mass and preferably 80%.
• the conditions in the reactor are adjusted so as to obtain a precipitate of undenatured proteins.
• the contact times and the cooling temperature are preferably chosen so that the internal temperature of the mixture remains low, so that the enzymes are not denatured.
• use will be made, for example, of temperatures in the reactor of between -15 ° C. and + 18 ° C. and preferably around 0 ° C.
• the protein precipitate is preferably in contact with the solvent, after passing through the reactor, between 0 to 30 minutes, and preferably 30 seconds.
• the optimal conditions for the isolation of the various enzymes are preferably a final temperature of 0 ° C. in the T-shaped reactor, a final ethanol concentration of 80% and a contact time of the precipitate with the solvent of approximately 30 seconds.
• the precipitate suspension is continuously discharged through the outlet, which preferably forms an angle of 90 ° relative to the inlets of the enzyme solution and the solvent.
• the particle size of the suspension can vary between 1 and 2 microns.
• the precipitate suspension can undergo a maturation step. This step increases the particle size of the suspension.
• the conditions of duration and speed of mixing are preferably adjusted to obtain particles or aggregates of sufficient size.
• the precipitate suspension can either be mixed at a temperature close to 4 ° C in a stirred tank fitted with a vertical propeller at a speed of 100 to 400 rpm (Reynolds number (Re) from 1175 to 4700) for 10 to 60 seconds, for example, and preferably at 300 rpm (about 3500 Re) for 20 seconds, or passed through static mixers at 4 ° C for 30 seconds, for example.
• the precipitate obtained after maturation comprises aggregates whose size can reach 500 microns on average. The precipitate is then continuously separated.
• Continuous separation of the protein precipitate is preferably obtained by simple centrifugation.
• the pellet is recovered and then stored.
• the supernatant can be either removed or treated in a distillation column and the ethanol thus recovered recycled in the process.
• the enzymatic extract thus obtained can then be directly frozen without the addition of water or freeze-dried, for example.
• the reactor is preferably a T-shaped cell in plexiglass, without a mixer, for example.
• the shape of the reactor is such that there are as few dead zones as possible: the inlet flows are preferably at the foot of the mixing volume with an identical diameter of the inlet tubes and preferably around 1, 5 mm in diameter and the outlet flow, perpendicular to the other two, lying upwards.
• the diameter of the outlet tube is preferably 1/3 larger than that of the inlet streams. It can be 2 mm, for example, when the inlet tubes are for example 1.5 mm.
• diameters can vary according to the flow rates to be passed through the reactor, but they must preferably be chosen in order to guarantee a speed of the flow of the source of enzymes at the time of contact of approximately 5 to 20 cm / s, for example and preferably about 11 cm / s, in order to allow good mixing while avoiding possible denaturation of the enzymes.
• This device makes it possible for example to process up to 14 tonnes of tomatoes per day with dimensions of the branches of the reactor of the order of 4 cm for the inlets and about 5.2 cm for the outlet.
• Another subject of the invention relates to the use of the endogenous enzymes or proteins isolated according to the invention for the preparation of cosmetic or food products.
• Said enzymes can thus be used to regenerate their aroma or taste in preparations such as soups, food for children, vegetable juices or purees or cold meats.
• the process is particularly effective for extracting the "active ingredients” from tomatoes, carrots, onions, for example. If, for example, tomatoes are chosen as plant material, the enzymes continuously extracted by the process according to the invention can be used in tomato juice, tomato puree, all frozen and fresh tomato-based products such as than pizza, lasagna, for example.
• tomatoes are washed and then transformed into juice.
• the juice is then treated by a first so-called extraction step so as to dissolve the maximum quantity of enzymes before the isolation step proper in the specific reactor.
• the plant material is homogenized and then the pH of the homogenate is brought to 7.0 by addition of a sodium hydroxide solution.
• NaCl is added so that the final salt concentration is 0.5 M.
• the insoluble parts are then removed by centrifugation.
• the supernatant thus obtained can be frozen or directly treated in the reactor so as to isolate the active proteins therefrom.
• the solution containing the enzymes to be isolated is then introduced via one of the inlet branches of the T-reactor. Ethanol is injected directly into the cell, through the other input branch of the reactor cell.
• the final ethanol concentration is 80%.
• the precipitate of non-denatured proteins obtained is continuously discharged through the branch of the T, placed at 90 ° relative to the inlet branches.
• the temperature of the mixture is approximately 0 ° C.
• the precipitate suspension is then vigorously mixed with a vertical turbine for a few 20 seconds (contact time).
• the precipitate is then continuously separated by centrifugation.
• the pellet is recovered and then stored.
• the supernatant can be either removed or treated in a distillation column and the ethanol thus recovered recycled in the process.
• the enzyme extract is either directly frozen (without adding water) or freeze-dried.
• the enzymes thus isolated by the process according to the invention have a very higher activity yield than that which could be obtained by traditional batch processes for example. This easy to implement process is therefore particularly effective for the isolation of active proteins continuously.
• Table 1, below gives the yields of recovered activities (in%) for pectin methylesterase (PME), peroxidase (POX), alcohol dehydrogenase (ADH) and acid phosphatase (AP).
• the carrots are prepared as described in Example 1.
• the conditions for continuous precipitation are 80% ethanol and a final temperature of 0 ° C.
• the onions are also prepared as in Example 1.
• the conditions for continuous precipitation are 80% ethanol and a final temperature of 0 ° C.
• CSL cysteine sulfoxide lyase
• POX peroxidase
• a batch reactor (discontinuous) and a vertical propeller are used. Ethanol at 94% w / w is added to the tomato extract (80 g, at 4 ° C.) initially present in the reactor, to the desired concentration and then the mixture is continued to stir.
• pectin methylesterase for example, is then measured. For a final concentration of 77%, this value varies from 0 to 20% depending on the temperature of the mixture. In addition, this enzyme is irreversibly denatured if it remains in contact with ethanol for too long. It should also be noted that no activity is measurable in the supernatant.
• the tomato extract is mixed with a PEG 8000 33.3% solution in a batch reactor, cooled to 4 ° C. A slight precipitation appears from a final PEG concentration of 12.35%.
• the solution is then centrifuged at 4 ° C for 10 minutes at 2000g.
• the pellet is recovered and dissolved in water (as for ethanol precipitation) before measuring the enzymatic activities present after precipitation.
• Table 4 Yields of activity recovered for different enzymes present in the tomato by batch batch processes with PEG 8000.
• Table 5 Optimal conditions for the extraction of different tomato enzymes and proteins.
• Table 6 Optimal precipitation conditions for some enzymes. The general optimal conditions: 80% ethanol, 0 ° C, without stirring and a contact time of the precipitate with the solvent of approximately 30 seconds (with high stirring).
• the precipitate suspension obtained at the outlet of the reactor is subjected to stirring at a temperature of 4 ° C. in a stirred tank fitted with a vertical turbine.
• the particle size is measured for mixing speeds of between 100 and 400 rpm (Re of
• Table 7 Median particle size of the precipitate (in ⁇ m) as a function of the speed and the mixing time during the maturation step.
• the time and speed of agitation have a significant effect on the median size of the particles of the precipitate and their aggregation.
• the optimal conditions are a speed of 300 rpm for about 20 s.
• Example 8 comparison of the yields for the T-shaped reactor and a CSTR.
• Table 8 Yields of enzymatic and mass activity for the T-reactor according to the invention and a CSTR.
• the treated and untreated samples are incubated for one hour at 37 ° C.
• the different samples are then tested by a panel, as follows:
• the amount of enzymes added is given in%. For example, if 100 g of tomato paste (which initially corresponds to 600 g of fresh tomatoes) is treated with 10% of enzymes, this means that the quantity of enzymes recovered after precipitation of 60 g of fresh tomatoes was added to the product.

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• Proteomics, Peptides & Aminoacids (AREA)
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• 1999
  • 1999-11-10 CA CA002350215A patent/CA2350215A1/en not_active Abandoned
  • 1999-11-10 EP EP99957301A patent/EP1131339A1/de not_active Withdrawn
  • 1999-11-10 BR BR9915484-6A patent/BR9915484A/pt not_active IP Right Cessation
  • 1999-11-10 AU AU15055/00A patent/AU1505500A/en not_active Abandoned
  • 1999-11-10 JP JP2000583943A patent/JP2002530427A/ja not_active Withdrawn
  • 1999-11-10 WO PCT/EP1999/008699 patent/WO2000031116A1/fr not_active Application Discontinuation
  • 1999-11-10 CN CN99813509A patent/CN1326463A/zh active Pending
• 2001
  • 2001-05-17 US US09/859,315 patent/US20020018831A1/en not_active Abandoned
  • 2001-06-19 ZA ZA200105015A patent/ZA200105015B/en unknown

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