U.S. patent application number 10/618477 was filed with the patent office on 2004-06-03 for solid phytase compositions.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Henriksen, Lotte Rugholm, Marcussen, Erik.
Application Number | 20040106182 10/618477 |
Document ID | / |
Family ID | 27761041 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040106182 |
Kind Code |
A1 |
Henriksen, Lotte Rugholm ;
et al. |
June 3, 2004 |
Solid phytase compositions
Abstract
The present invention relates to solid enzyme, in particular
phytase, compositions stabilized with a lactic acid source such as
Corn Steep Liquor (CSL), and methods of producing the same.
Preferred compositions additionally comprise starch and
disaccharides such as lactose or trehalose.
Inventors: |
Henriksen, Lotte Rugholm;
(Vanlose, DK) ; Marcussen, Erik; (Ballerup,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27761041 |
Appl. No.: |
10/618477 |
Filed: |
July 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10618477 |
Jul 11, 2003 |
|
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09410503 |
Oct 1, 1999 |
|
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6610519 |
|
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60103522 |
Oct 8, 1998 |
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Current U.S.
Class: |
435/196 ;
424/94.6 |
Current CPC
Class: |
C12N 9/96 20130101; Y10S
426/807 20130101; C12N 9/16 20130101; C12N 9/98 20130101 |
Class at
Publication: |
435/196 ;
424/094.6 |
International
Class: |
A61K 038/46; C12N
009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 1998 |
DK |
PA 1998 01251 |
Claims
1. A solid phytase composition comprising: (a) an enzyme having
phytase activity, and (b) a lactic acid source, wherein the phytase
activity of the composition is above 20 units/g.
2. The composition of claim 1, wherein the lactic acid source
comprises at least 10% lactic acid.
3. The composition of claim 1, which comprises lactic acid in an
amount of 0.01-10%.
4. The composition of claim 1, wherein the lactic acid source is
Corn Steep Liquor (CSL).
5. The composition of claim 4, the chromatogram of which, when
analyzed by HPLC according to Example 8 herein, reveals the
presence of one or more of peaks 1-10.
6. The composition of claim 4, comprising CSL in an amount of
0.01-15%.
7. The composition of claim 1, further comprising a starch
source.
8. The composition of claim 1, further comprising a
disaccharide.
9. The composition of claim 1, additionally comprising a carrier
material.
10. The composition of claim 1, additionally comprising a filler
material.
11. The composition of claim 1, additionally comprising one or more
vitamins, one or more minerals or a mixture of both.
12. A process for preparing a solid phytase composition which
comprises drying a lactic acid source together with an enzyme
having phytase activity, wherein the phytase activity of the solid
composition is above 20 units/g.
13. The process of claim 12, wherein the lactic acid source is Corn
Steep Liquor (CSL).
14. The process of claim 12, wherein the solid phytase composition
is a phytase granulate composition, and which process comprises:
(a) spraying the enzyme having phytase activity onto a carrier; (b)
spraying the lactic acid source onto the carrier; (c) mixing; and
(d) drying.
15. The process of claim 12, wherein the drying is a
spray-drying.
16. The process of claim 12, further comprising the addition of one
or more starch sources.
17. The process of claim 12, further comprising the addition of one
or more disaccharides.
18. A solid composition having a phytase activity above 20 units/g
and being obtainable by the process of claim 12.
19. A solid phytase composition consisting essentially of: (a) an
enzyme having a phytase activity of above 20 FYT/g of the
composition, and (b) a lactic acid source in an amount of 0.01-15%
by weight to provide lactic acid in an amount sufficient to
stabilize the enzyme.
20. The composition of claim 19, wherein the lactic acid is present
in an amount of 0.01-10%.
21. The composition of claim 19, having a chromatogram determined
by HPLC, which has one or more of peaks 1-10.
22. The composition of claim 19, further consisting essentially of
a starch material.
23. The composition of claim 19, further consisting essentially of
a disaccharide.
24. The composition of claim 19, further consisting essentially of
a carrier material.
25. The composition of claim 19, further consisting essentially of
a filler material.
26. The composition of claim 19, further consisting essentially of
one or more vitamins, one or more minerals or a mixture of
both.
27. The solid phytase composition of claim 19, wherein the enzyme
has a phytase activity of at least 25 FYT/g of the composition.
28. The solid phytase composition of claim 27, wherein the enzyme
has a phytase activity of at least 50 FYT/g of the composition.
29. The solid phytase composition of claim 28, wherein the enzyme
has a phytase activity of at least 100 FYT/g of the
composition.
30. The solid phytase composition of claim 29, wherein the enzyme
has a phytase activity of at least 250 FYT/g of the
composition.
31. The solid phytase composition of claim 30, wherein the enzyme
has a phytase activity of at least 500 FYT/g of the
composition.
32. The solid phytase composition of claim 31, wherein the enzyme
has a phytase activity of at least 750 FYT/g of the
composition.
33. The solid phytase composition of claim 32, wherein the enzyme
has a phytase activity of at least 1000 FYT/9 of the composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 09/410,503
filed Oct. 1, 1999 which claims priority or the benefit under 35
U.S.C. 119 of Danish application no. PA 1998 01251 filed Oct. 2,
1998 and U.S. provisional application No. 60/103,522 filed Oct. 8,
1998, the contents of which are fully incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to solid phytase compositions
which have been stabilized with a lactic acid source such as Corn
Steep Liquor (CSL), and methods of producing the same.
[0004] 2. Description of Related Art
[0005] 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.
[0006] The stabilization of liquid phytase formulations with urea,
glycerol or sorbitol is disclosed in WO 93/16175.
[0007] Salt-stabilized solid phytase compositions are disclosed in
EP 0 758 018 A1.
[0008] Plant seeds, cereal grains and legumes are usual components
of animal feed. Some of those seeds contain phytic acid, and often
also endogenous phytase enzymes.
[0009] According to investigations performed by the applicant,
endogenous phytase activity in animal feed is at a very low level
of around 0.5 units/g.
[0010] 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.
SUMMARY OF THE INVENTION
[0011] The present invention relates to solid phytase compositions
which comprise (a) an enzyme having phytase activity; and (b) a
lactic acid source, wherein the phytase activity of the composition
is above 20 units/g.
DETAILED DESCRIPTION OF THE INVENTION
[0012] In the present context, the expression "enzyme (or
polypeptide) having phytase activity" or "phytase" includes any
enzyme capable of effecting the liberation of inorganic phosphate
from phytic acid or from any salt thereof (phytates).
[0013] Phytic acid is myo-inositol 1,2,3,4,5,6-hexakis dihydrogen
phosphate (or for short myo-inositol hexakisphosphate). In what
follows, unless otherwise indicated, the terms "phytic acid" and
"phytate," are used synonymously or at random.
[0014] In the present context, the term "units" means units of
enzyme, in particular phytase, activity. Any method for determining
phytase activity can be used.
[0015] 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.degree. C. from the optimum temperature of the actual enzyme;
using as a substrate phytic acid or any salt thereof in a suitable
concentration.
[0016] Preferably, the substrate is dodeca-sodium phytate in a
concentration of 0.005 mole/l.
[0017] Preferably, the pH is within the range of +/-0.5 pH unit
from the optimum pH; more preferably the pH is the optimum pH.
[0018] Preferably, the temperature is within the range of
+/-10.degree. C. from the optimum temperature; more preferably the
temperature is the optimum temperature.
[0019] Preferably, the optimum pH and optimum temperature refers to
the use of sodium phytate as a substrate.
[0020] 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.degree. C.;
substrate: sodium phytate (C.sub.6H.sub.6O.sub.24P.sub.6Na.sub.12)
in a concentration of 0.0050 mole/l.
[0021] In a further preferred embodiment, the phytase activity is
measured using the FTU assay.
[0022] The FYT- and FTU-assays are described in more detail in the
experimental part.
[0023] In preferred embodiments, the phytase activity of the solid
composition of the invention is above 25, 50, 100, 250, 500, 750 or
even above 1000 units/g.
[0024] Optionally, the phytase activity of the solid composition is
below 100,000 units/g, more preferably below 75,000 units/g, even
more preferably below 50,000 units/g, or below 40,000 units/g, or
below 25,000 units/g, or even below 10,000 units/g, mostly
preferred below 5,000 units/g.
[0025] Preferred ranges of phytase activity are
25-100,000,25-75,000, 35-50,000, or 50-40,000 units/g; more
preferably 100-25.000 units/g; even more preferably 500-10.000
units/g; mostly preferred 1000-5000 units/g.
[0026] In the present context, any enzyme having phytase activity
can be used.
[0027] 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.
[0028] 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).
[0029] 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.
[0030] 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.
[0031] Phytases derived from fungi of the phylum Basidiomycota are
disclosed in WO 98/28409 and WO 98/28408.
[0032] 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.
[0033] 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 (.mu.m) 0.01, or
from 1.0, or preferably from around 1 to 1000, or to 1200, or to
1500, or even up to 2000 (.mu.m).
[0034] 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.
[0035] For spray drying, no further components need to be added to
the liquid phytase concentrate.
[0036] For spray cooling, a meltable component--such as palm oil
(and/or another meltable vegetable oil or fat), hydrogenated palm
oil (and/or another hydrogenated vegetable oil), tallow,
hydrogenated tallow or a wax functions as a matrix. The phytase and
other ingredients, if any, are introduced into the melted, meltable
component, and the melt is then allowed to solidify under
particle-forming conditions, typically in a spray drying tower.
[0037] For many uses, however, including the use in animal feed,
granulates are usually preferred for a number of reasons. One
reason is 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.
[0038] The particle size of the enzyme granulates preferably is
compatible with that of the other components of the mixture. This
provides a safe and convenient mean of incorporating enzymes into,
e.g., animal feed.
[0039] The size of a particle may be regarded as the greatest
linear dimension of the particle; thus, in the case of, e.g., a
substantially spherical particle (such as a substantially spherical
granulate particle), the particle size in question will be the
diameter of the particle.
[0040] Agglomeration granulates and agglomerated powders may be
prepared using agglomeration technique in a high shear mixer (e.g.
Lodige) during which one or more filler materials and the enzyme
are co-agglomerated to form granules.
[0041] Absorption granulates are prepared by having cores of one or
more carrier materials to absorb/be coated by the enzyme.
[0042] 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.
[0043] 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 cassaya or
wheat; or a sugar (such as sucrose or lactose), or a salt (such as
sodium chloride or sodium sulphate).
[0044] Optionally, the granulates are coated with a coating
mixture. Such mixture comprises coating agents, preferably
hydrophobic coating agents, such as hydrogenated palm oil and beef
tallow, and if desired other additives, such as calcium carbonate
or kaolin.
[0045] 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.
[0046] Preferred methods of preparing phytase granulates are
referred to in Example 3.
[0047] 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.
[0048] 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%.
[0049] 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.
[0050] Unless otherwise indicated, all percentage indications are
weight/weight, by reference to dry matter content. Preferably,
"units/g" also refers to dry matter content. Dry matter content can
be determined by any method known in the art, such as refractometer
or drying in an oven to release humidity.
[0051] Unless otherwise indicated, the expression "above" generally
means ">", whereas the expressions "up to" or "below" mean
".ltoreq.".
[0052] 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.
[0053] A non-limiting list of lactic acid sources is the following:
Lactic acid and lactates as relatively pure chemical compounds
(purity of, say, above 70%, 80%, 90%); lactic acid and lactates as
more impure substances (purity of, say, above 5%, 10%, 15%, 20%,
25%, 30%, 40%, 50%); any natural or synthetical composition which
comprises lactic acid in an amount of above 5%, preferably above
10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 80%, 90%.
[0054] 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%.
[0055] Any assay for lactic acid can be used. Preferred lactic acid
assays are from SIGMA: (1) Assay kit catalogue no. 735-10
(enzymatical assay, lactate degraded to pyruvate and hydrogen
peroxide in the presence of oxidase); or (2) Assay kit catalogue
no. 826-A and 826-B (ultraviolet, endpoint, lactate converted into
pyruvic acid in the presence of lactate dehydrogenase and NAD).
[0056] A preferred lactic acid source is Corn Steep Liquor or CSL.
CSL is a commercially available product, see for instance Merck
Index, 1996, 4.sup.th edition, Index no. 2598. It is a viscous
yellowish or dense brown liquid obtained by concentration of corn
steep water. The dry matter content is usually 45-55%, preferably
48-52%. The pH is in the range of 3-5, preferably 3.5-4.5. The
protein content (Dry Matter) is typically 30-50%, preferably
35-45%. The acidity (as lactic acid) is typically 10-30% (Dry
Matter), preferably 12-25%.
[0057] "CSL" as used herein refers to the product as such, or to
its dry matter part.
[0058] In a preferred embodiment, the solid composition of the
invention comprises 0.01-15%, preferably 0.1-10%, more preferably
1-5% of CSL.
[0059] For analyzing CSL-content, any method can be used. A
preferred HPLC method for fingerprinting and quantifying CSL is
indicated in Example 8. Another preferred method is Head Space Gas
Chromatograph (HS-GC), preferably combined with mass spectrometry
(MS).
[0060] In a preferred embodiment, the solid composition of the
invention additionally comprises a starch source, typically in an
amount of 0.1-20%, more preferably 0.2-10%, still more preferably
1-5%.
[0061] 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 is 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.
[0062] 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%, even more
preferrably 1-5%.
[0063] 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).
[0064] In the process of the invention, all steps, e.g. those
indicated in claim 14, can be performed simultaneously or
sequentially. 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.
[0065] Further preferred embodiments of the invention are the
following:
[0066] A solid composition which comprises at least one enzyme
having phytase activity, and Corn Steep Liquor (CSL), wherein the
phytase activity of the composition is in the range of 20-50.000
units/g. A preferred amount of CSL is within the range of 0.01-15%
(dry matter content and w/w). Preferably, the composition
additionally comprises Wheat Starch (WS), preferably in an amount
within the range of 0.01-20% (dry matter content and w/w);
[0067] A process for preparing a granulate composition having a
phytase activity in the range of 20-50.000 units/g, which method
comprises the steps of (i) spraying a liquid phytase concentrate
onto a carrier; (ii) spraying CSL onto the carrier; (iii) mixing;
and (iv) drying;
[0068] A process for preparing a spray dried solid composition
having a phytase activity in the range of 20-50.000 units/g, which
method comprises the step of adding CSL to a liquid phytase
concentrate before spray drying it.
[0069] The activities of the solid phytase compositions prepared in
the present examples are in the range of 1000-3000 FTU/g before
storage.
EXAMPLE 1
[0070] FYT Assay
[0071] 10 .mu.l diluted enzyme samples (diluted in 0.1 M sodium
acetate, 0.01% Tween 20, pH 5.5) are added into 250 .mu.l 5 mM
sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween 20, pH
5.5 (pH adjusted after dissolving the sodium phytate; the substrate
is preheated) and incubated for 30 minutes at 37.degree. C. The
reaction is stopped by adding 250 .mu.l 10% TCA and free phosphate
is measured by adding 500 .mu.l 7.3 g FeSO4 in 100 ml molybdate
reagent (2.5 g (NH.sub.4).sub.6Mo.sub.7O.sub.240.4H.sub.2O in 8 ml
H.sub.2SO.sub.4 diluted to 250 ml). The absorbance at 750 nm is
measured on 200 Al 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 .mu.mol phosphate/min at the given conditions.
[0072] FTU Assay
[0073] One FTU is defined as the amount of enzyme, which at
standard conditions (37.degree. C., pH 5.5; reaction time 60
minutes and start concentration of phytic acid 5 mM) releases
phosphate equivalent to 1 .mu.mol phosphate per minute.
[0074] 1 FTU=1 FYT
[0075] The FTU assay is preferred for phytase activity measurements
on animal feed premixes and the like complex compositions.
[0076] Reagents/Substrates
[0077] Extraction Buffer for Feed etc.
[0078] This buffer is also used for preparation of
PO.sub.4-standards and further dilution of premix samples.
[0079] 0.22 M Acetate Buffer with Tween 20 pH 5.5
[0080] 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.
[0081] The sodium acetate is dissolved in demineralized water.
[0082] Tween 20 is added, and pH adjusted to 5.50+0.05 with acetic
acid.
[0083] Add demineralized water to total volume.
[0084] Extraction Buffer for Premix
[0085] 0.22 M acetate buffer with Tween 20, EDTA, PO.sub.4.sup.3-
og BSA.
[0086] 30 g sodium acetate trihydrate e.g. Merck Art 6267 per
liter.
[0087] 0.1, g Tween 20 e.g. Merck Art 22184 per liter.
[0088] 30 g EDTA f.eks. Merck Art 8418 per liter.
[0089] 20 g Na.sub.2HPO.sub.4, 2H.sub.2O e.g. Merck Art 6580 per
liter.
[0090] 0.5 g BSA (Bovine Serum Albumine, e.g. Sigma Art A-9647 per
liter.
[0091] The ingredients are dissolved in demineralized water, and pH
is adjusted to 5.50.+-.0.05 with acetic acid.
[0092] Add demineralized water to total volume.
[0093] BSA is not stable, and must therefore be added the same day
the buffer is used.
[0094] 50 mM PO.sub.4.sup.3- Stock Solution
[0095] 0.681 g KH.sub.2PO.sub.4 (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.
[0096] Storage stability: 1 week in refrigerator.
[0097] 0.22 M Acetate Buffer pH 5.5 without Tween
[0098] This buffer is used for production of phytic acid
substrate).
[0099] 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.
[0100] Add demineralized water to 5000 ml.
[0101] Storage stability: 1 week at room temperature.
[0102] Phytic Acid Substrate; 5 mM Phytic Acid
[0103] The volume of phytic acid is calculated with allowance for
the water content of the used batch.
[0104] If the water content is e.g. 8.4% the following is
obtained:
(0.005 mol/l.times.923.8 g/mol)/(1/0.084)=5.04 g/l
[0105] 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.
[0106] pH is adjusted to 5.50.+-.0.05 with acetic acid.
[0107] Add 0.22 M acetate buffer to total volume.
[0108] 21.7% Nitric Acid Solution
[0109] For stop solution.
[0110] 1 part concentrated (65%) nitric acid is mixed into 2 parts
demineralized water.
[0111] Molybdate Reagent
[0112] For stop solution.
[0113] 100 g ammonium heptamolybdate tetrahydrate
(NH.sub.4).sub.6Mo.sub.7- O.sub.24, 4H.sub.2O e.g. Merck Art 1182
is dissolved in demineralized water. 10 ml 25% NH.sub.3 is
added.
[0114] Add demineralized water to 1 liter.
[0115] 0.24% Ammonium Vanadate
[0116] Bought from Bie & Berntsen.
[0117] Molybdate/Vanadate Stop Solution
[0118] 1 part vanadate solution (0.24% ammonium vanadate)+1 part
molybdate solution are mixed. 2 parts 21.7% nitric acid solution
are added.
[0119] The solution is prepared not more than 2 hours before use,
and the bottle is wrapped in tin foil.
[0120] Samples
[0121] Frozen samples are defrosted in a refrigerator
overnight.
[0122] Sample size for feed samples: At least 70 g, preferably 100
g.
[0123] Feed Samples
[0124] 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.
[0125] If the sample size is approx. 100 g all the sample is ground
in a coffee grinder and subsequently placed in tared beakers. The
sample weight is noted. It is not necessary to grind not-pelleted
samples. If a sample is too big to handle, it is sample split into
parts of approx. 100 g.
[0126] Magnets are placed in the beakers and 0.22 M acetate buffer
with Tween is added.
[0127] The samples are extracted for 90 minutes.
[0128] After extraction the samples rest for 30 minutes to allow
for the feed to sediment. A 5 ml sample is withdrawn with a
pipette. The sample is taken 2-5 cm under the surface of the
solution and placed in a centrifuge glass, which is covered by a
lid.
[0129] The samples are centrifuged for 10 minutes at 4000 rpm.
[0130] Premix Samples
[0131] Choose a solution volume which allows addition of buffer
corresponding to 10 times the sample weight. Round up to nearest
solution volume.
[0132] 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.
[0133] Magnets are placed in the beakers and 0.22 M acetate buffer
with Tween, EDTA and P.sub.4.sup.3- is added.
[0134] The samples are extracted for 60 minutes.
[0135] After extraction the samples rest for 30 minutes to allow
for the premix to sediment. A 5 ml sample is withdrawn with a
pipette. The sample is taken 2-5 cm under the surface of the
solution and placed in a centrifuge glass, which is covered by a
lid.
[0136] The samples are centrifuged for 10 minutes at 4000 rpm.
[0137] Analysis
[0138] Extracts of feed samples are analyzed directly.
[0139] Extracts of premix are diluted to approx. 1.5 FTU/g
(A.sub.415 (main sample)<1.0).
[0140] 0.22 M acetate buffer with Tween 20 is used for the
dilution.
[0141] Main Samples
[0142] 2.times.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.
[0143] When all samples are ready they are placed on a water bath
with stirring. Temperature: 37.degree. C.
[0144] 3.0 ml substrate is added.
[0145] Incubation for exactly 60 minutes after addition of
substrate.
[0146] The samples are taken off the water bath and 2.0 ml stop
solution is added (exactly 60 minutes after addition of
substrate).
[0147] The samples are stirred for 1 minute or longer.
[0148] Feed samples are centrifuged for 10 minutes at 4000 rpm (It
is not necessary to centrifuge premix samples).
[0149] Blind Samples
[0150] 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.
[0151] 2.0 ml stop solution is added to the samples.
[0152] 3.0 ml substrate is added to the samples.
[0153] The samples are incubated for 60 minutes at room
temperature.
[0154] The feed samples are centrifuged for 10 minutes at 4000 rpm
(it is not necessary to centrifuge premix samples).
[0155] Standards
[0156] 2.times.100 ml are taken from each of the 8 standards and
also 4.times.100 ml 0.22 M acetate buffer (reagent blind).
[0157] A.sub.415 is measured on all samples.
[0158] Calculation
[0159] FTU/g=.mu.mol PO.sub.4.sup.3-/(min*g (sample))
[0160] C g sample is weighed out (after grinding).
[0161] 100 .mu.l is taken from the extracted and centrifuged
sample.
[0162] PO.sub.4.sup.3- standard curve is linear.
[0163] From the regression curve for the PO.sub.4.sup.3- standard
the actual concentration of the sample is found (concentration in
mM):
[0164] [PO.sub.4.sup.3-]=(x-b)/a x=A.sub.415 a=slope b=intercept
with y-axis
[0165] .mu.mol PO.sub.4.sup.3-/min={[PO.sub.4.sup.3-]
(mM).times.Vol (liter).times.1000 .mu.mol/mmol}/t
[0166] t=incubation time in minutes.
[0167] Vol=sample volume in liter=0.0001 liter
[0168] 1000=conversion factor from mmol to .mu.mol
[0169] FTU/g.sub.pr.o
slashed.ve={(x-b).times.Vol.times.1000.times.F.sub.p-
}/{a.times.t.times.C}
[0170] C=gram sample weighed out
[0171] F.sub.p=Relation between the sample taken out and the total
sample (after extraction).
[0172] Example: 0.100 ml taken from 1000
ml.fwdarw.F.sub.p=1000/0.100=1000- 0.
[0173] Reduced expression with insertion of the following
values:
[0174] t=60
[0175] Vol=0.0001 l
[0176] F=10000
[0177] FTU/g.sub.pr.o
slashed.ve={(x-b).times.0.0001.times.1000.times.1000-
0}/{a.times.60.times.C}
EXAMPLE 2
[0178] Preparation of a Concentrated Liquid Phytase Preparation
[0179] The phytase derived from Peniophora lycli 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.
EXAMPLE 3
[0180] Preparation of Phytase Granulates
[0181] A coated phytase granulate with 1.5% CSL is prepared as
follows:
[0182] 14.68 kg of a powder composition with the formulation
[0183] 0.75 kg kaolin, Speswhite, English China Clay
[0184] 1.80 kg of fibrous cellulose, Arbocel BC 200
[0185] 11.23 kg finely ground sodium sulphate
[0186] 0.90 kg Carbohydrate binder, Tackidex G155 from Roquette
[0187] is mixed in a Lodige 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.
[0188] The percentage of CSL in this as yet un-coated raw granulate
is calculated as follows:
0.625.times.0.48/(14.68+0.625.times.0.48+0.84.time-
s.0.18)=0.300/(14.68+0.30+0.672)=0.300/15.652=1.917%-2%.
[0189] The granulate is dried in a fluid bed to a water content
below 3%, resulting in a light coloured granulate with the
following particle distribution:
[0190] 10.5%>1100 .mu.m (micro meter)
[0191] 92.0%>300 .mu.m
[0192] 8.0%<300 .mu.m
[0193] The granulate is finally sifted to get a product with the
particle range 300 .mu.m to 1100 .mu.m, and 6 kg of granulate is
coated at 80.degree. 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.
[0194] 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%.
[0195] The granulate is sifted to obtain a product with the
particle range 300 .mu.m to 1200 .mu.m.
[0196] The control granulate used below is prepared as described
above, except for no CSL being added.
[0197] Granulates additionally comprising wheat starch and lactose
or trehalose are prepared in a corresponding manner.
EXAMPLE 4
[0198] Storage Stability of Phytase Granulates in Premix
[0199] The phytase granulates indicated in Table 1 below are
prepared according to Example 3. "Control" indicates a phytase
granulate prepared according to the method of Example 3, but with
no addition of CSL.
[0200] The granulates are weighed directly into each vial. The
exact weight of the granulate is recorded. The vials are covered
with a clean towel and left at room temperature overnight.
[0201] The premix ENGA 1-02/Nordkorn. Product. No: 015384 Artikel
Nr. 8259 (25 kg drums) is mixed in a Lodige mixer to ensure an even
distribution of the premix components and filled into plastic bags
with .apprxeq.3 kg premix in each bag.
[0202] The composition of the premix is as follows (per kilo):
1 5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E
1000 mg Vitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg
Vitamin B6 7666 mcg Vitamin B12 12333 mg Niacin 33333 mcg Biotin
300 mg Folic Acid 3000 mg Ca-D-Panthothenate 1666 mg Cu 16666 mg Fe
16666 mg Zn 23333 mg Mn 133 mg Co 66 mg I 66 mg Se 5.8 % Calcium 25
% Sodium
[0203] 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.
[0204] 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.degree. C. are re-opened. The open vials are placed in
plastic boxes containing 1 litre of glycerol adjusted with water to
43% rH (62% refractometer dry matter measured on a sugar scale)
corresponding to .apprxeq.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.
[0205] After completion of the storage period the samples are
removed from the glycerol boxes, closed with screw on lids and
frozen.
[0206] The samples are defrosted in a refrigerator (5.degree. C.)
overnight prior to analysis.
[0207] The 0 week samples stored at -18.degree. C. and the
corresponding samples stored at 30.degree. C. are analysed the same
day in order to eliminate day-to-day and person-to-person
variation.
[0208] The results are shown in Table 1 below; CSL=Corn Steep
Liquor and WS=Wheat Starch.
[0209] Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL
plus 5% WS showed similar performance.
2TABLE 1 Percentage residual activity following 13 Granulate code
Granulate weeks storage at 30.degree. C. 1 Control granulate 61% 2
Control granulate 64% 3 1.5% CSL 81% 4 1.5% CSL 86% 5 1.5% CSL 84%
6 1.5% CSL 85% 7 1.5% CSL + 3.8% WS 84% 8 1.5% CSL + 3.8% WS 90% 9
1.5% CSL + 3.8% WS 84%
EXAMPLE 5
[0210] Storage Stability of Phytase Granulates in Feed
[0211] The phytase granulates indicated in Table 2 below are
prepared according to Example 3. "Control" indicates a phytase
granulate prepared according to the usual standard method of
Example 3, except for neither WS nor CSL nor disaccharides being
added.
[0212] The samples of granulates in feed are prepared at
Bioteknologisk Institut, Kolding, Denmark.
[0213] The composition of the feed is as follows:
[0214] 74.0% wheat
[0215] 20.7% roasted soy cake
[0216] 5.0% soy oil
[0217] 0.3% Premix Enga 1-02/Nordkorn
[0218] The feed is dried to a water content of .ltoreq.10% water
before addition of the phytase granulates.
[0219] The granulate batches are mixed into feed and the mixture is
pelletized at 65.degree. C.
[0220] The feed pellets are sample split and filled into 100 ml
sample vials.
[0221] The 0 week samples, defining for each granulate the level of
100% activity, are closed with screw on lids and kept at
-18.degree. C.
[0222] The samples which are to be stored at 30.degree. C. are not
closed. The open vials are placed in plastic boxes containing 1
litre 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.
[0223] 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.degree. C.)
overnight prior to analysis.
[0224] Samples for the homogeneity test are kept refrigerated at
+5.degree. C. until analysis.
[0225] The mash feed, the feed heated to 65.degree. C., and the
feed pellets without added enzyme all contained .apprxeq.0.5 FTU/g
feed as expected.
[0226] 5 samples of the mash feed with enzyme added and heated to
65.degree. C. are analysed for homogeneity. The relative standard
deviation is 2% to 11%. In conclusion, the homogeneity is
acceptable.
[0227] 5 samples of the feed pellets are analysed for homogeneity.
The relative standard deviation is 2% to 10%. In conclusion, the
homogeneity is acceptable.
[0228] The storage stability is measured after 13 weeks. The 0 week
samples stored at -18.degree. C. and the corresponding samples
stored at 30.degree. C. are analysed the same day in order to
eliminate day-to-day and person-to-person variation.
[0229] The results of the phytase residual activity measurements
are shown in Table 2 below (endogenous activity has been subtracted
from the total activity before calculating the residual activity);
CSL=Corn Steep Liquor and WS=Wheat Starch.
[0230] Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL
plus 5% WS showed similar performance.
3TABLE 2 Percentage residual activity following 13 Granulate code
Granulate weeks storage at 30.degree. C. 1 Control granulate 53% 2
Control granulate 55% 3 1.5% CSL 74% 4 1.5% CSL 89% 5 1.5% CSL 82%
6 1.5% CSL 73% 7 1.5% CSL + 3.8% WS 82% 8 1.5% CSL + 3.8% WS 91% 9
1.5% CSL + 3.8% WS 89%
EXAMPLE 6
[0231] Phytase Granulates per se; Granulation Yield and Storage
Stability
[0232] The liquid concentrate of Example 2 was used to prepare
experimental solid phytase compositions according to the method of
Example 3.
[0233] 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).
[0234] In a second granulation experiment, 3% Wheat Starch (WS) was
applied--in addition to the two components of the first
experiment.
[0235] In a third granulation experiment, the disaccharide
trehalose, in an amount of 2%, as well as 3% WS, was added together
with 3% of the lactic acid source CSL.
[0236] The effect on granulation yield and storage stability of the
resulting granulates per se is examined.
[0237] Granulation yield is calculated as phytase units remaining
in the product leaving the granulation unit, relative to phytase
units of the liquid concentrate entering the unit.
[0238] The storage stability of the resulting phytase granulate
composition per se is examined using the following rather strict
conditions: 4 weeks, 40.degree. C. and a relative humidity of
60%.
[0239] The results are shown in Table 3 below.
4 TABLE 3 Granulation yield Storage stability Experiment Batch 22
Batch 24 Batch 22 Batch 24 Control 75% 70% 43% 47% CSL + lactose
80% 67% 47% 50% CSL + WS + lactose 81% 79% 56% 54% CSL + WS +
trehalose 85% 82% 63% 62%
EXAMPLE 7
[0240] Storage Stability of a Granulate Composition of Another
Phytase
[0241] 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.
[0242] Granulation experiments were conducted essentially as
described in Example 6. However, for storage stability samples are
also stored at 30.degree. C. The results are shown in Table 4
below.
5 TABLE 4 Storage stability Granulation 30.degree. C. 40.degree.
C., 60% RH Experiment yield 8 weeks 17 weeks 4 weeks Control 82%
85% 85% 40% CSL + WS + 92% 97% 95% 48% lactose
EXAMPLE 8
[0243] Characterization of CSL using High Performance Liquid
Chromatography (HPLC)
[0244] 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
Marckolsheim, France; and Cerestar Scandinavia A/S, Skovlytoften
33, DK-2840 Holte, Denmark) are tested as described below.
[0245] Carrez-Precipitation
[0246] 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.degree. C. for 15 minutes while shaking at 200 rpm.
Add 12 ml of Carrez-1-solution (Potassium-hexacyanoferrat(II)-tri
hydrate) and shake. Add 12 ml Carrez-II-solution (Zinc
sulphate-hepta 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 .mu.m filter for
HPLC analysis. Each sample is analyzed twice, include sample blinds
(MQ-water and Carrez-solutions).
[0247] Chromatography Parameters
[0248] Column: Supelcosil LC-18-DB, No. 088877AE
[0249] Detector: Shimadzu SPDM6A-diodearray from 220 nm to 350
nm.
[0250] Data analysis: For analyzing data, use peak areas resulting
from integration at 260 nm.
6 Pump: HP 1080 gradient pump Eluents: A) MQ-water B) 30% MeOH C)
60% MeOH D) 90% MeOH Gradients: 0 min A 15 min A 35 min B 50 min C
60 min C 65 min D 70 min D 75 min C 80 min B 85 min A 90 min A
[0251] The results of variable statistics on 15 samples of CSL
analyzed by HPLC are shown in Table 5 below:
7TABLE 5 Peak Standard retention Deviation time Min Max Mean (SD) %
SD 6791 22154 114227 79192 25978 32.80.sup.7 7445 1934348 2326644
2135867 110911 5.19.sup.1 10331 4746 132048 44503 52860 118.78
11844 0 3020 201 780 387.30 12440 124723 183826 155946 17166
11.01.sup.4 14124 79613 186731 119578 35032 29.30.sup.6 15322 0
27204 13205 11537 87.37 16187 314623 380627 346898 21271 6.13.sup.2
18831 18494 148325 130989 10481 8.00.sup.3 26373 0 8482 2288 3454
150.99 26833 0 59860 25622 19252 75.14 27672 0 46259 24388 13200
54.12 28053 0 11383 3085 3169 102.71 28762 5643 60078 31762 20966
66.01 29491 3657 14650 8970 3353 37.38.sup.8 29926 40184 89538
52754 14807 28.07.sup.5 30607 0 44749 14732 15523 105.37 30951 0
19732 2423 5786 238.80 31825 0 16090 2624 4909 187.06 32454 0 15725
1288 4050 314.36 32636 0 28268 7516 9534 126.85 33068 0 36398 4570
9707 212.42 33394 0 96671 30197 33359 110.47 33646 0 24856 4042
7600 188.02 34108 0 11826 2144 3922 182.94 34464 0 29248 9794 8990
91.79 35309 0 14392 8793 6491 73.82 36826 0 29619 9667 10423 107.82
42457 0 45570 32023 12404 38.73.sup.9 42971 29905 102074 45303
18156 40.08.sup.10 43427 0 49318 6293 14306 227.33 43812 0 13837
5851 4924 84.16 45519 0 12487 3570 4815 134.87 46032 0 11850 4728
4756 100.59 46654 0 36357 23766 11561 48.65 47034 0 37769 17540
11212 63.92 47268 0 20271 5414 6969 128.72 47784 0 5218 1569 2091
133.25 48494 0 5858 1872 1595 85.20 48859 0 10935 2923 3748 128.25
49180 0 24091 11016 8573 77.82 49467 0 23885 10146 8372 82.52 49905
0 22011 4205 8506 202.31
[0252] 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.
[0253] For samples of an unknown content of CSL, suitable dilutions
are found using simple trial-and-error techniques.
[0254] 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.RTM. L 48 L CAS No. 66071-94-1, EINECS:
266-113-4.
* * * * *