Source: http://www.google.com/patents/US6610519?dq=6289460
Timestamp: 2016-02-07 18:51:51
Document Index: 129361081

Matched Legal Cases: ['application No. 60', 'Art 46267', 'Art 22184', 'Art 6267', 'Art 22184', 'Art 8418', 'Art 6580', 'Art 4873', 'Art 6267', 'Art 1182', 'arts 21']

Patent US6610519 - Solid phytase composition stabilized with lactic acid provided by corn steep ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsA solid phytase composition having a phytase activity of above 20 FYT/g is prepared containing a lactic acid source such as corn steep liquor to stabilize the phytase. A starch source, disaccharide, filler, carrier, vitamins and/or minerals may be present in the composition. The composition can be prepared...http://www.google.com/patents/US6610519?utm_source=gb-gplus-sharePatent US6610519 - Solid phytase composition stabilized with lactic acid provided by corn steep liquorAdvanced Patent SearchPublication numberUS6610519 B1Publication typeGrantApplication numberUS 09/410,503Publication dateAug 26, 2003Filing dateOct 1, 1999Priority dateOct 2, 1998Fee statusPaidAlso published asUS8846361, US20040106182, US20090169681Publication number09410503, 410503, US 6610519 B1, US 6610519B1, US-B1-6610519, US6610519 B1, US6610519B1InventorsLotte Rugholm Henriksen, Erik MarcussenOriginal AssigneeNovozymes A/SExport CitationBiBTeX, EndNote, RefManPatent Citations (12), Non-Patent Citations (1), Referenced by (22), Classifications (18), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetSolid phytase composition stabilized with lactic acid provided by corn steep liquor
US 6610519 B1Abstract
A solid phytase composition having a phytase activity of above 20 FYT/g is prepared containing a lactic acid source such as corn steep liquor to stabilize the phytase. A starch source, disaccharide, filler, carrier, vitamins and/or minerals may be present in the composition. The composition can be prepared by spray drying or granulation. Granulates are preferred for using the composition to animal feed where the phytase eliminates the anti-nutritional effects of phytic acid.
What is claimed is: 1. A solid phytase composition consisting essentially of:
(a) an enzyme having a phytase activity of above 20 FYT/g of the composition, and (b) corn steep liquor in an amount of 0.01-15% by weight to provide lactic acid in an amount sufficient to stabilize the enzyme. 2. The composition of claim 1, wherein the lactic acid is present in an amount of 0.01-10%.
3. The composition of claim 1, having a chromatogram determined by HPLC, which has one or more of peaks 1-10.
4. The composition of claim 1, further consisting essentially of a starch material.
5. The composition of claim 1, further consisting essentially of a disaccharide.
6. The composition of claim 1, further consisting essentially of a carrier material.
7. The composition of claim 1, further consisting essentially of a filler material.
8. The composition of claim 1, further consisting essentially of one or more vitamins, one or more minerals or a mixture of both.
9. The solid phytase composition of claim 1, wherein the enzyme has a phytase activity of at least 25 FYT/g of the composition.
10. The solid phytase composition of claim 9, wherein the enzyme has a phytase activity of at least 50 FYT/g of the composition.
11. The solid phytase composition of claim 10, wherein the enzyme has a phytase activity of at least 100 FYT/g of the composition.
12. The solid phytase composition of claim 4, wherein the enzyme has a phytase activity of at least 250 FYT/g of the composition.
13. The solid phytase composition of claim 12, wherein the enzyme has a phytase activity of at least 500 FYT/g of the composition.
14. The solid phytase composition of claim 13, wherein the enzyme has a phytase activity of at least 750 FYT/g of the composition.
15. The solid phytase composition of claim 14, wherein the enzyme has a phytase activity of at least 1000 FYT/g of the composition.
This application claims priority from U.S. provisional application No. 60/103,522 filed Oct. 8, 1998 and Danish application no. PA 1998 01251 filed Oct. 2, 1998, the contents of which are fully incorporated by reference.
The addition of phytase to animal feed to eliminate the anti-nutritional effects of phytic acid is well-described, see e.g. WO 98/28408 and WO 98/28409.
According to e.g. the two above first-cited WO-references, when supplementary phytase has been added to feed, the phytase activity in the feed is in the range of 0.01-20 units/g.
In a preferred embodiment, one unit of phytase activity is defined as the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: A pH which is within the range of +/−1 pH unit from the optimum pH of the actual enzyme; a temperature which is within the range of +/−20� C. from the optimum temperature of the actual enzyme; using as a substrate phytic acid or any salt thereof in a suitable concentration.
Preferably, the temperature is within the range of +/−10� C. from the optimum temperature; more preferably the temperature is the optimum temperature.
In another preferred embodiment, the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37� C.; substrate: sodium phytate (C6H6O24P6Na12)in a concentration of 0.0050 mole/l.
Phytases have been derived from plants as well as from microorganisms. Amongst the microorganisms, phytase producing bacteria as well as phytase producing fungi are known. From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinson et al, Biochemistry, 1 (1962), 166-171). An alkaline phytase from lilly pollen has been described by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.
Amongst the bacteria, phytases have been described which are derived from Bacillus subtilis (Paver and Jagannathan, 1982, Journal of Bacteriology 151:1102-1108) and Pseudomonas (Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220). Still further, a phytase from E. coli has been purified and characterized by Greiner et al, Arch. Biochem. Biophys., 303, 107-113, 1993).
Phytase producing yeasts are also described, such as Saccharomyces cerevisiae (Nayini et al, 1984, Lebensmittel Wissenschaft und Technologie 17:24-26. However, this enzyme is probably a myo-inositol monophosphatase (Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303). AU-A-24840/95 describes the cloning and expression of a phytase of the yeast Schwanniomyces occidentalis. There are several descriptions of phytase producing filamentous fungi, primarily belonging to the fungal phylum of Ascomycota (ascomycetes). In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., 1993, Gene 133:55-62). EP 0 420 358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger). EP 0 684 313 describes the cloning and expression of phytases of the ascomycetes Myceliophthora thermophila and Aspergillus terreus. Phytases derived from fungi of the phylum Basidiomycota are disclosed in WO 98/28409 and WO 98/28408.
Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 0897010, EP 0897985, PCT/DK99/00153 and PCT/DK99/00154. The phytases disclosed in either of these four patent applications can also be used in the compositions of the present invention.
A solid or dry composition is a particulate material comprising, preferably consisting essentially of, or consisting of, freely flowing particles of a size ranging from (μm) 0.01, or from 1.0, or preferably from around 1-to 1000, or to 1200, or to 1500, or even up to 2000 (μm).
Preferably, a solid or dry phytase composition is such composition which can be prepared from liquid phytase concentrates e.g by spray drying, spray cooling (prilling), or any type of granulation.
For many uses, however, including the use in animal feed, granulates are usually preferred for a number of reasons. One reason being that they may readily be mixed with feed components, or more preferably, form a component of a pre-mix which contains other desired feed additives such as vitamins and minerals.
Agglomeration granulates and agglomerated powders may be prepared using agglomeration technique in a high shear mixer (e.g. L�dige) during which one or more filler materials and the enzyme are co-agglomerated to form granules.
Typical filler materials are salts such as di-sodium sulphate and calcium-lignosulphonate. Other fillers are silica, gypsum, kaolin, talc, magnesium aluminium silicate and cellulose fibres. Optionally, binders such as dextrins are also included in agglomeration granulates.
Typical carrier materials may consist of particulate cores having a suitable particle size. The carrier may be water soluble or water insoluble, e.g. starch, e.g. in the form of cassava or wheat; or a sugar (such as sucrose or lactose), or a salt (such as sodium chloride or sodium sulphate).
WO 97/39116 discloses preferred processes for making solid compositions of the invention in the form of enzyme-containing granules or an enzyme-containing granulate, see in particular the sections of the detailed description therein headed cores, binders, fillers, plasticizers, fibrous materials, superabsorbents, coating layers, enzymes, other adjunct ingredients (these sections being hereby incorporated by reference herein). However, WO 97/39116 does not disclose the inclusion in the solid composition of a lactic acid source. Preferred methods of preparing phytase granulates are referred to in Example 3.
Preferred solid compositions of the invention are enzyme compositions. The preferred compositions are concentrated, viz. of an activity of above 20 units/g. Thus, the concept of solid enzyme composition comprises in particular, but are not limited to, spray-dried enzyme preparations, enzyme granulates, e.g. agglomeration granulates and absorption granulates, coated as well as un-coated, and enzyme-containing pre-mixes for animal feed. Phytase is a preferred enzyme.
Liquid enzyme (phytase) concentrates can e.g. be prepared as follows: The enzyme source, typically a phytase-containing fermentation broth, is subjected to a primary separation step (e.g. using a decanter, a centrifuge, or a filter press), followed by a second polish filtration and/or germ filtration step. Finally the liquid is concentrated, e.g. using ultra filtration, followed by a germ filtration. A typical dry matter content is in the range of 10-30%, preferably 15-25%, more preferably 17-22%.
In the present context, “a” generally means “one or more” or “at least one.” This applies i.a. for the following mandatory or optional components of the compositions of the invention: Phytase, lactic acid source, CSL, starch, disaccharide, filler, carrier.
In the present context a “lactic acid source” or a “lactic acid preparation” is any composition which comprises the compound lactic acid or any lactates, i.e. any salts thereof (lactic acid is 2-hydroxy propanic acid). Likewise, “lactic acid” as used herein includes any lactates. These expressions are used interchangeably for the lactic acid source, resp. the lactic acid, as is, and for the dry matter part thereof.
The solid enzyme compositions of the invention preferably comprise up to 20, preferably up to 15, more preferably up to 10, still more preferably up to 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75 or 0.5% lactic acid. The content of lactic acid is preferably above 0.001, preferably above 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0,28 or above 0.3%. Preferred ranges of content of lactic acid are 0.01-10%, 0.02-9%, 0.03-8%, 0.04-7%, 0.05-6%, 0.06-7% 0.07-6%, 0.08-5%, 0.09-4%, or 0.1-3%.
The concept of a starch source includes any natural or synthetic polysaccharides comprising glucose units interconnected by alpha-1,4- or alpha-1,6-linkages. Purity preferably above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. A preferred starch source is Wheat Starch, which is a commercially available product. The expression “starch source” includes the starches and modified starches described in the section headed “Cores” of WO 97/39116, cited above.
In another preferred embodiment, the solid composition of the invention additionally comprises a disaccharide, preferably in an amount of 0.01-15%, even more preferred 0.1-10%, still more preferred 1-5%.
The concept of disaccharides includes any natural or synthetic disaccharides, whatever the monomers, and whatever the linkage type. Examples of such disacharides are maltose, lactose, cellobiose, sucrose, trehalose (non-limiting list). Preferably, the disaccharides are of a purity of above 10, 20, 30, 40, 50, 60, 70, 80 or even 90%. Preferred disaccharides are lactose and trehalose (alpha-D-glucose alpha-D-glucopyranoside, alpha-1,1 linkage).
In the process of the invention, all steps, e.g. those indicated in claim 14, can be performed simultaneously or sequenially. E.g. steps (i) and (ii) sequentially or preferably simultaneously (mixing the lactic acid source and the phytase before spraying it onto the carrier); steps (iii) and (iv) simultaneously or sequentially, preferably simultaneously, in the same apparatus; applies also to “together with” of claim 12.
10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5) are added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate is preheated) and incubated for 30 minutes at 37� C. The reaction is stopped by adding 250 μl 10% TCA and free phosphate is measured by adding 500 μl 7.3 g FeSO4 in 100 ml molybdate reagent (2.5 g (NH4)6Mo7O24.4H2O in 8 ml H2SO4 diluted to 250 ml). The absorbance at 750 nm is measured on 200 μl samples in 96 well microtiter plates. Substrate and enzyme blanks are included. A phosphate standard curve is also included (0-2 mM phosphate). 1 FYT equals the amount of enzyme that releases 1 μmol phosphate/min at the given conditions.
One FTU is defined a s the amount of enzyme, which at standard conditions (37� C., pH 5,5; reaction time 60 minutes and start concentration of phytic acid 5 mM) releases phosphate equivalent to 1 μmol phosphate per minute.
0,22 M Acetate Buffer With Tween 20 pH 5,5
30 g sodium acetate trihydrate (MW=136,08 g/mol) e.g. Merck Art 46267 per liter and 0,1 g Tween 20 e.g. Merck Art 22184 pr. liter are weighed out.
Tween 20 is added, and pH adjusted to 5,50�0,05 with acetic acid.
0,22 M acetate buffer with Tween 20, EDTA, PO4 3− og BSA.
30 g sodium acetate trihydrate e.g. Merck Art 6267 per liter.
0,1 g Tween 20 e.g. Merck Art 22184 per liter.
30 g EDTA f.eks. Merck Art 8418 pr. liter.
20 g Na2HPO4,2H2O e.g. Merck Art 6580 per liter.
0,5 g BSA (Bovine Serum Albumine, e.g. Sigma Art A-9647 per liter.
The ingredients are dissolved in demineralized water, and pH is adjusted to 5,50�0,05 with acetic acid.
50 mm PO4 3− Stock Solution
0,681 g KH2PO4 (MW=136,09 g/mol) e.g. Merck Art 4873 is weighed out and dissolved in 100 ml 0,22 M sodium acetate with Tween, pH 5,5.
0,22 M Acetate Buffer pH 5,5 Without Tween
150 g sodium acetate trihydrate (MW=136,08) e.g. Merck Art 6267 is weighed out and dissolved in demineralized water, and pH is adjusted with acetic acid to 5,50�0,05.
If the water content is e.g. 8,4% the following is obtained: 0.005��mol/l � 923.8 g/mol ( 1 � 0.084 ) = 5.04   g/l Phytic acid (Na-salt) (MW=923,8 g/mol) e.g. Sigma P-8810 is weighed out and dissolved in 0,22 M acetate buffer (without Tween). Addition of (diluted) acetic acid increases the dissolution speed.
pH is adjusted to 5,50�0,05 with acetic acid.
Add 0,22 M acetate buffer to total volume.
100 g ammonium heptamolybdate tetrahydrate (NH4) 6Mo7O24,4H2O e.g. Merck Art 1182 is dissolved in demineralized water. 10 ml 25% NH3is added.
Bought from fra Bie & Berntsen.
1 part vanadate solution (0,24% ammonium vanadate) +1 part molybdate solution are mixed. 2 parts 21,7% nitric acid solution are added.
Choose a solution volume which allows addition of buffer corresponding to 10 times the sample weight, e.g. 100 g is dissolved in 1000 ml 0,22 M acetate buffer with Tween, see enclosure 1. Round up to nearest solution volume.
Magnets are placed in the beakers and 0,22 M acetate buffer with Tween is added.
If the samples have been weighed (50-100 g) all of the sample is placed in tared beakers. The sample weight is noted. If a sample is too big to handle, it is split into parts of approx. 100 g.
Magnets are placed in the beakers and 0,22 M acetate buffer with Tween, EDTA og PO4 3− is added.
Extracts of premix are diluted to approx. 1,5 FTU/g (A415 (main sample)<1,0).
0,22 M acetate buffer with Tween 20 is used for the dilution.
2�100 ml of the supernatant from the extracted and centrifuged samples are placed in marked glass test tubes and a magnet is placed in each tube.
When all samples are ready they are placed on a water bath with stirring. Temperature: 37� C.
3,0 ml substrate is added.
The samples are taken off the water bath and 2,0 ml stop solution is added (exactly 60 minutes after addition of substrate).
2,0 ml stop solution is added to the samples.
3,0 ml substrate is added to the samples.
2�100 ml are taken from each of the 8 standards and also 4�100 ml 0,22 M acetate buffer (reagent blind).
FTU/g=μmol PO4 3−/(min * g(sample))
100 μl is taken from the extracted and centrifuged sample.
PO4 3− standard curve is linear.
From the regression curve for the PO4 3− standard the actual concentration of the sample is found (concentration in mM):
[PO4 3−]=(x−b)/ax=A 415 a=slope b=intercept with y-axis μmol PO4 3−/min={[PO4 3−](mM)�Vol (liter)�1000 μmol/mmol}/t t=incubation time in minutes.
Vol=sample volume in liter=0,0001 liter
1000=conversion factor from mmol to μmol
FTU/g pr�ve={(x−b)�Vol�1000�F p }/{a�t�C} C=gram sample weighed out
Example: 0,100 ml taken from 1000 ml →Fp=1000/0,100=10000.
Vol=0,0001 l
FTU/g pr�ve={(x−b)�0,0001�1000�10000}/{a�60�C} EXAMPLE 2
The phytase derived from Peniophora lycii is expressed in Aspergillus oryzae, fermented and purified, essentially as described in WO 98/28408. The resulting liquid phytase concentrate is a UF (ultra filtration) concentrate of a dry matter content of 18%. pH is adjusted to 5.
is mixed in a L�dige mixer FM 50 and sprayed with 3.15 kg of a spraying liquid consisting of 1.68 kg of water, 0.625 kg of Corn Steep Liquor (Concentrated Corn Steep Liquor (CCSL) supplied by Amylum N.V. with a dry matter content of 48%) and 0.84 kg of Phytase concentrate (18% dry matter content) prepared as described in Example 2. During and after spraying the moist mixture is exposed to a compacting and granulation influence from the multiple set of knives, as described in Example 1 of U.S. Pat. No. 4,106,991.
The percentage of CSL in this as yet un-coated raw granulate is calculated as follows: 0.625�0.48/(14.68+0.625�0.48+0.84�0.18)=0.300/(14.68+0.30+0.672)=0.300/15.652=1.917%˜2%.
10.5%>1100 μm (micro meter)
92.0%>300 μm
8.0%<300 μm
The granulate is finally sifted to get a product with the particle range 300 μm to 1100 μm, and 6 kg of granulate is coated at 80� C. with
9% fully hydrogenated palm oil, followed by 22.5% of kaolin, Speswhite (dry matter content in 100 g coating material: 22.5 g+9 g=31.5 g), in a manner as described in U.S. Pat. No. 4,106,991, Example 22.
The content of CSL in the resulting final product, the coated granulate, is reduced as compared to the CSL content of the raw granulate as follows: 1.917%/1.315=1.458% ˜1.5%.
The granulate is sifted to obtain a product with the particle range 300 μm to 1200 μm.
The premix ENGA 1-02/Nordkorn. Product. No: 015384 Artikel Nr. 8259.(25 kg drums) is mixed in a L�dige mixer to ensure an even distribution of the premix components and filled into plastics bags with ≈3 kg premix in each bag.
5000000IE
1000000IE
Ca-D-Panthothenate
50 g�1 g of premix is added to each vial and the vials are closed with a screw-on lid. The premix is added using an adjustable cylindrical “scoop” adjusted to give a volume corresponding to 50 g. The vials are mixed by hand until the granulates are evenly distributed in the premix.
The 0 week samples (closed vials), defining for each granulate the level of 100% activity, are frozen immediately after completion of the sample preparation. The samples which are to be stored at 30� C. are re-opened. The open vials are placed in plastic boxes containing 1 liter of glycerol adjusted with water to 43% rH (62% refractometer dry matter measured on a sugar scale) corresponding to ≈10% water in the samples. The lids of the plastic boxes are sealed with strong tape. This means that the water activity is 0,43 during the whole test period of 13 weeks.
The samples are defrosted in a refrigerator (5� C.) night prior to analysis.
The 0 seek samples stored at −18� C. and the corresponding samples stored at 30� C. are analysed the same day in order to eliminate day-to-day and person-to-person variation.
Percentage residual
activity following 13
Granulate code
weeks storage at 30� C.
Control granulate
1.5% CSL
1.5% CSL + 3.8% WS
The samples of granulates in feed are prepared at Biotekn-ologisk Institut, Kolding, Denmark.
The granulate batches are mixed into feed and the mixture is pelletized at 65 � C.
The 0 week samples, defining for each granulate the level of 100% activity, are closed with screw on lids and kept at −18� C.
The samples which are to be stored at 30� C. are not closed. The open vials are placed in plastic boxes containing 1 liter of glycerol adjusted with water to 43% rH (62% refractometer dry matter measured on a sugar scale) corresponding to ≈10% water in the samples. The lids of the plastic boxes are sealed with strong tape. This means that the water activity is 0,43 during the whole test period of 13 weeks.
After completion of the storage period the samples are removed from the glycerol boxes, closed with screw on lids and frozen. The samples are defrosted in a refrigerator (5� C.) over-night prior to analysis.
Samples for the homogeneity test are kept refrigerated at �5� C. until analysis.
The mash feed, the feed heated to 65� C., and the feed pellets without added enzyme all contain ≈0.5 FTU/g feed as expected.
5 samples of the mash feed with enzyme added and heated to 65� C. are analysed for homogeneity. The relative standard deviation is 2% to 11%. In conclusion, the homogeneity is acceptable.
The storage stability is measured after 13 weeks. The 0 week samples stored at −18� C. and the corresponding samples stored at 30� C. are analysed the same day in order to eliminate day-to-day and person-to-person variation.
weeks storage at
In a first granulation experiment, the disaccharide lactose was added in an amount of 2% together with 3% of the, lactic acid source Corn Steep Liquor (CSL).
In a second granulation experiment, 3% Wheat Starch (WS)was applied—in addition to the two components of the first experiment.
The storage stability of the resulting phytase granulate composition per se is examined using the following rather strict conditions: 4 weeks, 40� C. and a relative humidity of 60%.
CSL + lactose
CSL + WS +
A liquid phytase concentrate and a solid composition—i.e. a granulate—was prepared according to the teachings of Examples 2 and 3, using a so-called consensus phytase as described in EP 0897010.
Granulation experiments were conducted essentially as described in Example 6. However, for storage stability samples are also stored at 30� C. The results are shown in Table 4 below.
40� C., 60% RH
15 samples from various batches of CSL from various suppliers (Roquette Freres, 4 Rue Patou, F-59022 Lille Cedex, France; Staral s.a., Z.I.ET Portuaire, B.P. 32, F-67390 Marckol-sheim, France; and Cerestar Scandinavia A/S, Skovlytoften 33, DK-2840 Holte, Denmark) are tested as described below.
Weigh 5.0 g CSL into a 100 ml flask. Add 40 ml MQ-water (demineralized water filtered through a Milli-q filter) and incubate at 70� C. for 15 minutes while shaking at 200 rpm. Add 12 ml of Carrez-I-solution (Potassium-hexacyanoferrat(II)-trihydrate) and shake. Add 12 ml Carrez-II-solution (Zinc sulphatehepta hydrate) and shake. Add 20 ml 0.5N NaOH and shake. Let cool and add MQ-water ad 100 ml, shake. 10 ml of this preparation is transferred to a vial and centrifuged for 10 minutes at 4000 rpm. The supernatant is filtered at a 0.5 μm filter for HPLC analysis. Each sample is analyzed twice, include sample blinds (MQ-water and Carrez-solutions).
Supelcosil LC-18-DB, No. 088877AE
Shimadzu SPDM6A-diodearray from 220 nm to 350 nm.
For analyzing data, use peak areas resulting from inte-
gration at 260 nm.
HP 1080 gradient pump
A)MQ-water
0 min A
32.807 7445
5.191 10331
11.014 14124
29.306 15322
6.132 18831
8.003 26373
37.388 29926
28.075 30607
38.739 42971
40.0810 43427
In the % SD column of Table 5, characteristic peaks are indicated by way of a superscript number (1,2,3, . . . , 9,10). In what follows, these peaks will be referred to as peak-1, peak-2, peak-3, . . . , peak-9, peak-10, respectively. The whole group of ten peaks is referred to as peaks 1-10. Sub-groups are referred to by analogy, e.g. peaks 1-5 for the five peaks numbered 1 to 5, peaks 1,3,5 for peak-1, peak-3 and peak-5 etc. Thus, the presence of one or more of these peaks in a sample is indicative of the presence of CSL. In preferred embodiments, the presence of one, two, three, four, five, six, seven, eight, nine or all ten of these peaks is indicative of the presence of CSL. In more preferred embodiments, the presence of five, seven, eight or ten peaks is indicative of the presence of CSL. The presence of five peaks is most preferred.
The above qualitative method can be made quantitative by comparing with a batch denominated by Roquette Freres to be a standard batch. A particularly preferred standard CSL batch from Roquette Freres is SOLULYS�L 48 L CAS No. 66071-94-1, EINECS: 266-113-4.
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