Note: Descriptions are shown in the official language in which they were submitted.
88183992
Title
A process for the production of caviar or a caviar-like product from live,
mature eggs of
fish or crustaceans, and such products
Description
The invention relates to a method for the production of caviar or a caviar-
like product
from live, mature eggs of fish or crustaceans, said live, mature eggs being in
a
fertilizable but unfertilized state and having a natural potassium content in
the egg
plasma, by treating the live, mature eggs in a saline solution which does not
damage
them and then in at least one solution containing water and at least one
cationic
component dissolved therein which causes stabilization of the egg envelope of
the live,
mature eggs, and to caviar or a caviar-like product.
The prepared, unfertilized eggs, especially from fish, are regarded as a
delicacy and are
increasingly consumed. The term "roe" is used to describe (in layman's terms)
eggs at
any stage of maturity, i.e. from immature to mature, whereas the degree of egg
development is not clearly defined. "Spawn" refers to live, mature eggs laid
by a female
fish, lobster or other aquatic animal in water to be fertilized. Ovulated eggs
are mature,
fertile, live eggs which are released from the follicle shells in the ovaries
and released into
the body cavity. From there, they are then spawned or stripped. Caviar may be
produced
only from roe of female fish of the various sturgeon species in accordance
with FAO
Codex Alimentarius. In addition to the wild sturgeons, sturgeons bred in
freshwater
aquaculture facilities are now also used for caviar production. Sturgeon spawn
occurs
only in fresh water except for a few exceptions. The best-known sturgeon
species
(Acipenseridae) include A. baerii, A. guldenstaedtii, Huso (also known as
Beluga
sturgeon), A. transmontanus, A. ruthenus and its albino. However, the hybrids
between A.
schrenckii (female) and A. dauricus (male) and the American "Paddlefish"
(Polydon
spatula) closely related to the sturgeons are also to be named. Various types
of caviar
are known on the market, such as Sevruga, Osietra and Beluga. The white caviar
(also
known as "golden caviar") is obtained from albino sturgeon. The species A.
ruthenus
albino is occasionally used in aquaculture facilities to produce the so-called
"Tsar caviar".
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However, this is not the "real Tsar caviar" which originates from the albino
of the Huso
huso and is very rare.
Currently, caviar-like products produced from approximately 38 further fish
species that
do not belong to the sturgeon species are produced and marketed, see, for
example, the
publication by P Bronzi et al.: "Present and future sturgeon and caviar
production and
marketing: A global market overview" (Journal of Applied Ichthyology 2014, 30
SI, 6,
1536-1546). These include tuna, lumpfish, salmon, trout, herring, cod, carp,
whitefish and
capelin, the immature roe of which is used to make caviar-like products (also
known as
'caviar substitutes' or 'false caviar). The roe from lobster, large crayfish
and other
crustaceans can also be processed into caviar-like products. The method of the
invention
and the products that can be produced refer to these fish and crustaceans (as
well as
other suitable but not mentioned fish and crustaceans). Unless caviar from
sturgeon is
explicitly mentioned, caviar as well as caviar-like products from fish other
than sturgeon
and from crustaceans, in particular lobsters and crayfish, are regularly meant
and
comprised in the following.
Caviar and caviar-like products are valuable foods. Caviar is rich in protein
with a high
content of essential amino acids and fat. Caviar contains the vitamins D, E,
B12 and
niacin, the minerals sodium, potassium, magnesium and calcium as well as the
trace
elements phosphorus, fluorine, iodine and zinc. Moreover, it has a high
content of
valuable cholesterol (HDL). Caviar and caviar-like products can be used both
as food
and as a substance in the cosmetic industry or in other industries which work
with
such valuable substances. The size and firmness of the eggs are highly
dependent on
the type of fish or crustacean in question as well as on maturity and thus on
the time
of harvest.
There are currently some caviar products on the market made from mature,
ovulated eggs
from sturgeon. Nowadays, however, caviar, which is immature roe taken together
with the
ovaries from killed sturgeons, is still mainly offered. This is the
conventional caviar
extraction method. In the case of caviar from aquaculture sturgeons, it was
initially
assumed that here too - as in the case of wild caviar in the past - the
immature eggs had
sufficient strength without further treatment against the invasive washing
steps to remove
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the remains of the gonad tissue and for the repackaging. However, experience
gained over
the past 20 years in the extraction of caviar from aquaculture sturgeon has
shown that this
immature roe from dead aquaculture sturgeon is too soft and can only be
further processed
by the use of borax or other preservatives or pasteurization into a product
suitable for
repackaging and keeping for more than 2 to 3 months.
The killing of females to extract caviar from wild catches, combined with
drastic
overfishing, industrial, agricultural and domestic waste water pollution of
the waters and
the construction of weirs and dams blocking migration routes to freshwater
spawning
grounds, has led to a massive threat to the wild populations of approximately
27
different sturgeon species. In many regions poaching and illegal black trade
still prevail
despite the protection of sturgeons by the Washington Convention on
International
Trade in Endangered Species of Wild Fauna and Flora (CITES). Cost-intensive
repopulating programs have been initiated worldwide which, however, according
to
reports by the World Sturgeon Conservation Society, are unfortunately showing
little
success above all in China, but also in Iran and Russia. Only the measures for
preserving and repopulating the stocks in the USA and Canada are showing
initial
success. In addition to the permitted capture of spawning animals, various
species of
sturgeon from aquaculture are also released into the wild with varying degrees
of
success as part of conservation programs to save stocks threatened with
extinction.
Crayfish and noble crayfish populations are also severely threatened by
environmental
pollution and imported diseases such as the crayfish plague. Decisive progress
in
crayfish farming in aquaculture and extensive stocking measures play an
important role
in the conservation of indigenous stocks. In aquaculture, the female animals
are kept
alive for breeding and the eggs are obtained by stripping. However, in
aquaculture
caviar production, female sturgeons are usually simply killed in the
conventional way
because more gentle methods are not mastered. This completely disregards the
fact
that they show a considerably improved reproductive performance with
increasing age.
The "Cesarean section method", which is also partially practiced in Russia, is
by no
means one of the gentle methods, as it is associated with a high mortality
rate of the
sturgeons treated in this way.
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State of the art
From RU 2 232 523 C2 a method for producing granular caviar from ovulated
sturgeon roe is known. The harvested ovulated eggs are initially treated in a
hot,
1.5 percent to 2 percent aqueous solution of a preservative to prepare them
for
subsequent pasteurization at temperatures of 65 C to 70 C. Apart from the fact
that
each heating process has a significant effect on the taste of the roe, the use
of
ovulated eggs, which have a very soft and sticky egg envelope, does not
guarantee
that they will withstand subsequent treatments with preservatives without
bursting.
However, even a small fraction of burst eggs significantly deteriorates the
quality of
the caviar, since the burst eggs are difficult to remove. Pasteurization
results in
denaturation of the valuable proteins and gives the caviar a mealy flavor.
In connection with the extraction of ovulated eggs from sturgeon, for example,
from the
publication by M.Szczepkowski et al.: "A simple method for collecting sturgeon
eggs
using a catheter" (Arch. Pole. Fich. (2011) 19:123-128) the use a catheter for
this
purpose is known. As a result, the eggs can easily be removed or sucked off by
a
vacuum. It is also known to simply massage the eggs out of the abdominal
cavity of the
sturgeon. This method is referred to as "stripping" and is the most gentle
harvesting
method.
The closest prior art to the invention is disclosed in WO 2007/045233 Al.
Described is
a method for producing caviar or caviar-like products from mature ovulated but
unfertilized eggs of aquatic animals, preferably fish, by means of exogenous
treatment
of the mature eggs in a solution, wherein an endogenous, morphological change
of the
acellular egg envelope, which separates the egg cell (egg plasma with
surrounding egg
envelope) from the environment is brought about with structural stabilization.
The
solution used contains water and at least one cationic component (calcium
cations
Ca) which is dissolved in water at a predetermined concentration and induces
structural stabilization upon contact with the eggs. Calcium is a cellular
signal
transduction molecule which induces a calcium wave in the egg cell in its egg
plasma,
which in turn leads to a cortical reaction and to the discharge and activation
of
ovoperoxidase. This enzyme ensures irreversible structural cross-linking of
protein
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strands in the extracellular casing by the incorporation of tyrosine molecules
into the
zona radiata intema and the zona radiata externa. The induced process in the
live egg
having metabolism thus leads to the desired structural stabilization of the
egg envelope.
Such stabilization cannot be brought about in immature eggs because the
corresponding receptors and enzyme cascades have not yet matured. In the case
of
killed eggs, the process cannot be initiated at all, since metabolism no
longer takes
place. Living, mature eggs immediately form a sticky layer due to the ovarian
fluid when
they come into contact with water so that they can stick to stones and plants
in the
spawning area. The eggs are therefore rinsed in a non-living ("physiological")
saline
solution prior to treatment in order to remove the ovarian liquid.
Furthermore, the live,
mature eggs have a natural potassium content in the egg plasma. For example,
no
harmful doses of potassium (e.g. to trigger ovulation) were added to them from
outside
before harvesting.
The reaction chain described is referred to in the literature as the "second
reaction".
This is a slow metabolic reaction which, after the fusion of a first sperm
with the egg,
creates a permanent physical-mechanical structure to protect the egg against
other
sperm aggregated outside the egg (polyspermy), but above all against
environmental
toxins, microbes and mechanical damage to the emerging embryo. In the known
method, this second reaction is initiated without fertilization by a sperm
having taken
place. The structural stabilization achieved provides a "plop effect" and
explosive
discharge of the liquid egg plasma upon consumption of the product. The
preference
for a specific strength of the plop effect is highly dependent on the use of
the caviar
and on the consumer.
Furthermore, it is known from EP 2 622 226 B1 to preserve the immature roe of
fish
reared in aquaculture in a composition of the flavonoid and antioxidant
Taxifolin
(Dihydroquercetin) and an organic salt, in particular potassium citrate.
However, the high
concentrations of the composition used lead to blatant changes in the
intracellular ion
environment, which initiate the programmed cell death (apoptosis). The roe
treated in this
way is therefore killed immediately. The publications SU 1662469 Al and
RU 2 048 111 Cl also show methods for the preservation of sturgeon eggs in
which
potassium compounds are used in such enormously high concentrations that
apoptosis
Date Recue/Date Received 2022-07-14
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is immediately triggered in the eggs and they die off. The same also applies
to the
EP 2 868 207 B1 corresponding to the above-mentioned RU 2 232 523 C2, wherein
an
additional denaturation by heating takes place.
The publication by G. E. Bledsoe et al.: "Caviars and Fish Roe products"
(critical
Reviews in Food Science and Nutrition, Vol. 43, No. 3, May 1, 2003, pages 317
to 356)
discloses the use of potassium nitrate in the context of the application of
nitrate as a
general preservative for eggs of crabs, sturgeons and other fish. However,
this takes
place ¨ as is customary in all preservation processes ¨ at such high
concentrations that
a cell-toxic effect occurs which significantly disrupts the osmotic balance
and
immediately kills the treated eggs. Furthermore, only immature eggs in an
early stage of
development are preserved by slaughtered fish, which on the one hand have to
be
rubbed mechanically out of the gonads, accepting possible damage, and on the
other
hand do not yet have any fully developed structures in the mature egg
envelope, so that
they cannot be used in the invention.
DE 2 416 685 A discloses a method for the improved preservation of the red
color
when preserving salmon roe or salmon by adding a food additive in the form of
citrate
which is permitted under food law. After completion of the method, the latter
remains
detectable in the final product and changes its composition. The high
concentrations
used (5 to 10 weight percent) kill the live eggs immediately upon contact.
Only
because immature roe is used can it be rinsed with water. As already
mentioned,
mature roe would form a sticky gel layer. Freezing immature eggs both before
and
after preservation always leads to freezing damage to the egg envelope and to
hardening water loss, because the undeveloped and unstabilized egg envelope
cannot protect the egg. JP S63 ¨ 36 763 A discloses a method for reducing
sodium
chloride in the preservation of fish roe in order to reduce the salty taste.
The sodium
chloride is also substituted by various potassium compounds. However, this
occurs
again in such high concentrations that the eggs, which are immature eggs, are
killed
and are no longer able to perform metabolic work. JP 2001 ¨ 299 285 A
discloses a
method for treating roe frozen in an immature state in order to improve
texture. The
eggs are rinsed at 5 C for up to 24 h and using potassium-containing
chemicals. Such
a long treatment duration interrupts every metabolic process. Since the roe
was frozen
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without protection against freezing, the immature eggs no longer have any
metabolic
activity and are also not fertile. They cannot therefore be used for the
method of the
present invention either. The method of the invention is not, however,
concerned with
subsequent and killing preservation, decolorization or freezing, but with the
original
production of caviar and caviar-like products from untreated mature live eggs.
Preservation or freezing after treatment of the live eggs is only an optional
additional
step in the invention. Decolorization is completely dispensed with, since it
is not
necessary.
From the publication by Huang Hui et al.: "Effect of Synthetic Preservatives
on
Volatile Flavor Compounds in Caviar of Sturgeon" (Huso dauricus x A.
schrenckii)([J]
FOOD SCIENCE, 2015, 36(12): 97-103), it is known that it is preferable to
prevent
loss of taste during the cool storage of caviar by synthetic preservation with
the
preservatives potassium sorbate (E202, sorbic acid) and ascorbate (vitamin C).
A
constant 0.5 per mil potassium sorbate was used in various test groups.
Immature
eggs were used which are to obtain a more intense taste as a result of the
preservative treatment. Potassium sorbate is considered to inhibit mold growth
and
fermentation but can also impair the taste of a product.
The publication by L. Dufresne et al.: "Kinetics of actin assembly attending
fertilization
or artificial activation of sea urchin eggs" (Experimental Cell Research,
Elsevier,
Amsterdam, NL, vol. 172, No.1, September 1987, pages 32 to 42) deals with the
artificial activation of eggs of sea urchins. Although the present invention
does not deal
with the treatment of sea urchin eggs, because they do not show a suitable
structure
(only an egg envelope with two layers), this publication will be briefly
discussed here.
On the one hand, sea urchin eggs are used which were obtained by injecting a
solution
of 0.5 M KCI with an extremely high potassium chloride concentration into the
abdominal cavity of the sea urchin. As a result, the egg has already been
strongly
influenced in its electrical polarization state and its natural potassium
content in the egg
plasma was evidently altered. Furthermore, all the sea urchin eggs come into
contact
with water before the treatment and form a gel layer which has to be
subsequently
mechanically removed. This fundamentally changes the morphological and
physiological properties of the acellular egg envelope as well. The sea urchin
eggs
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cannot be used in the invention not only because of their fundamentally
different
structure, but also because of the massive metabolic interventions with
potassium
chloride during their harvest. Furthermore, calcium-free seawater is not
deionized, it
contains, among other things, more than 10 g of sodium, 0.43 g of potassium
and 1.3 g
of magnesium and 20 g of chlorine per liter. Thus, rinsing the eggs in calcium-
free water
does not correspond to rinsing in a saline solution that does not harm the
living, mature
eggs.
The structure of the membrane of eggs of fish and crustaceans follows a basic
pattern
and is described for the method in accordance with the terminology of the
publication
by Siddique et al.: "A review of the structure of sturgeon egg envelopes and
of the
associated terminology", (J. Appl. Ichthyol. (2014), 1-10). The following is a
brief
description of how to establish a conceptual concordance.
In maturing eggs (oocytes, egg cells) in the ovaries of the animal, the
follicle, consisting
of granulosa cells (also known as follicular cells, follicular membrane,
follicular
epithelium) and thecal cells (also known theca interna and externa), surrounds
the egg
to supply it with signaling substances and nutrients. Between the granulosa
cells and
the thecal cells there is also the base lamina (in science also called
perifollicular
membrane, membrane, basal lamina). The egg plasma (oocyte plasma, olemna,
cytoplasm, inner egg) is surrounded by the egg envelope (oocyte membrane,
plasma
membrane PM, cellular egg envelope). During ovulation, the egg is removed from
the
follicular cells and released into the abdominal cavity of the fish. The
ovulated egg only
retains its acellular egg envelope (also known in science as extracellular
matrix or
extracellular membrane), which was formed during egg maturation and is
structurally
formed from the outside to the inside from:
= Alveolar layer AL (also referred to in science as gel coat, adhesive
layer, gel
shell, (second external) gelatinous shell, layer 3, chorion (2)), outermost
layer of the
acellular egg envelope
= Zona radiata externa ZRE (in science also referred to as external
vitelline
sheath (zona radiata = vitelline sheath), outer vitelline zone, external
vitelline
membrane, layer 2, chorion layer 2, zona pellucida externa at., zona radiata
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externa lat., layer 1B of sheath, second sheath), outer part of vitelline
sheath, lies
directly under the alveolar layer
= Epilayer EP (in science also referred to as epilayer 1, layer 4, outer
layer of the
first membrane), separates the ZRE from the ZRA, not present in all types of
eggs
= Zona radiata interna ZRI (in science also referred to as internal
vitelline sheath,
inner vitelline zone, internal vitelline membrane, chorion layer 1, zona
pellucida
Interne lat., zona radiata Interne lat., layer 1A of the sheath, inner layer
of the first
sheath), inner part of the vitelline membrane, closely connected to the ZRA
and
= perivitelline gap (in science also referred to as extra oocyte matrix,
gap with the
microvilli protuberances of the egg plasma), narrow space between the ZRI and
the
egg envelope, into which the egg plasma inserts numerous microvilli (MV).
Live ovulated egg cells are electrically excitable by ion channels located in
their plasma
membrane. Changes in the electrical properties of the plasma membrane
constitute,
among other things, a prerequisite for egg activation and have an effect on
the egg
envelope. Pioneering work on marine invertebrates demonstrated ion currents of
potassium
cations through the egg envelope, which cause a temporary change of potential
across the
membrane (fertilization potential FP). This potential is generated by the
activation of a
transient voltage-dependent inward current into the egg interior.
Depolarization of the
membrane potential (RP) was shown to result from ion flow through the egg
envelope (ion
current (fertilization current FC)). This current flows through the openings
of non-specific
and highly conductive ion channels, which can be activated by sperm or
artificial chemical
or mechanical effects. The hypothetical models for the role of different ion
channels and the
relevant ions to date show species-specific differences.
In egg cells, potassium plays a central role in nature, compare the
publication by
E. Tosti et al: "Electrical events during gamete maturation and fertilization
in animals
and humans" (2004 Human Reproduction Update, vol. 10, no. pp 53-65). Potassium
K+
is the cation which decisively determines the rest potential of the egg cell.
The
potassium + gradient and the permeability of the egg for ions are regulated by
transport
proteins and ion channels. The natural intracellular potassium cation
concentrations are
50 mmo1/1 in mature, unfertilized eggs of the sturgeon A. baerii according to
studies at
the Alfred Wegener Institute. Extracellular calcium, on the other hand, does
not affect
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the rest potential/fertilization potential of the egg and is also not involved
in the first fast
electrical block (see below) itself. The ion composition in the interior of
the egg cell is
different from the ion composition in the surrounding environment. This
separation
between cell interior and external medium must be maintained for metabolic
activity and
thus for cell survival. The different distribution of electric charges inside
and outside the
cell forms an electrical gradient across the egg envelope, which can be
measured as a
potential difference (rest potential).
Problem and Solution
Based on the prior art closest to the invention according to WO 2007/045233
Al, which
is also used for the production of caviar and caviar-like products, the
problem for the
present invention is to further develop the process described therein on the
basis of live,
unfertilized, mature eggs of fish or crustaceans in such a way that a
modification of the
sensory properties with respect to texture, taste, transport, storage and deep-
freezing of
the mature eggs can be achieved. Thereby, however, the aforementioned
advantages
of caviars or caviar-like products producible by the process or otherwise,
which are also
provided by the invention, are to be maintained.
The present invention provides a solution for this task. Thus, in one aspect,
the present
invention provides a method for producing caviar or a caviar-like product from
live,
mature, ovulated eggs from fish or crustaceans which have three or more layers
in the
egg envelope, wherein the live, mature, ovulated eggs are in a fertile but
unfertilised
state and have a natural potassium content in the egg plasma, the method
comprising:
treatment of the live, mature, ovulated eggs with a 0.6 percent to 1.0 percent
saline
solution to remove ovulation fluid and subsequently, in a potassium exposure
step, with
a potassium-containing solution which acts to stabilise the egg envelope of
the live,
mature, ovulated eggs, wherein in the potassium exposure step, the potassium-
containing solution comprises potassium dissolved in deionised water in a
concentration of potassium cations of between 0.1 mmo1/1 and 3.0 mmo1/1,
wherein
prior to the addition of a potassium donor for the formation of the potassium
cations, the
deionised water is at a temperature in the natural spawning temperature range
for the
fish and crustaceans of between 1 C and 29 C, and wherein the live, mature,
ovulated
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eggs are treated in the potassium-containing solution for a potassium exposure
time of
between 5 min and 30 min,
and finally, dipping of the live, mature, ovulated eggs in a 0.6 percent to
1.0 percent
saline solution.
The inventive method for the production of caviar or a caviar-like product
based on live,
mature eggs of fish or crustaceans is characterized according to the invention
in that, in
a potassium exposure step, potassium is dissolved in the water as the cationic
component in a concentration which does not damage the live, mature eggs and
does
not change their natural potassium content, the water being deionized prior to
the
addition of a potassium re-donor for the formation of the cationic component
and being
at a temperature which is not harmful to the living, mature eggs, and in that
the living,
mature eggs are treated in the solution for a potassium exposure time until a
desired
elastic stabilization of the egg envelope is achieved.
In the process of the invention, live, mature eggs are used which can be
obtained
naturally without damaging the fish or the crustacean. The live, mature eggs
are fertile,
but unfertilized. The ovarian fluid was removed by a previous rinsing with a
saline
solution that does not harm the live, mature eggs, so that no sticky gel layer
can form on
the outer envelope. In addition, the eggs have a natural, artificially
unaltered potassium
content. Only cell-physiologically effective concentrations of potassium
cations are
used. The live eggs, which in fish and crustaceans in the egg envelope have
more than
two layers, i.e. three or more layers, are electrically activated. The
starting product are
live, mature, fertile, but unfertilized eggs with a fully functional
metabolism, so that even
the lowest concentrations of potassium cations, which cause no damage and
leave no
traces in the egg, can trigger transport processes through the egg envelope
and
metabolic processes in the egg plasma. The caviar or caviar-like products
produced in
this way exhibit a new texture with an advantageous stabilizing elasticity
through a new
hyaline, acellular layer (elastic stabilization layer) in the egg envelope,
which becomes
somewhat softer at room temperature without limiting the stability of the
caviar. The
desired degree of elastic stabilization can easily be determined by self-
testing (degree
of elasticity of the eggs). The taste is pleasantly fresh and spicy, without
being "fishy".
The purity of the eggs used, even without the addition of preservatives such
as borax,
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which is already banned in many countries, results in a long shelf life (9 to
12 months)
at standard temperatures between -2 C and -4 C. The eggs treated with this
method
can also be initially frozen without any loss of quality, resulting in
enormous advantages
in terms of storage and transport, see below.
By bringing the live, mature eggs into contact with the potassium cations (K+)
in
physiological, i.e. non-damaging, concentration according to the invention,
the eggs are
changed within the framework of electrical events and the so-called "first
reaction" is
triggered, which leads in the shortest time (seconds to minutes) to the
electrically
induced removal of the stickiness upon contact with water and, in the further
course of
treatment, to the formation of a new, elastic stabilizing layer within the egg
envelope.
This new stabilizing layer gives the egg envelope elasticity, so that the
invention already
produces a caviar or caviar-like product of the highest quality at this stage,
which can
also be subjected to optional processing steps, in particular preservation and
deep-
freezing (at -18 C) without any loss in quality.
An egg activation comprises and passes through a whole series of cell-
biological
cascades. The "second (slow) reaction" (slow block) with a cortical reaction
used in
WO 2007/045233 Al is followed by calcium-dependent enzyme activation for the
amplification and ultimately massive structural alteration of the egg envelope
by
irreversibly tyrosine-linked protein strands in the zona radiata intema and
the zona
radiata externa. In nature, this prepares the first cell division for
embryonic
development. By contrast, the "first (fast) reaction" (fast block, electrical
block, fast
electrical block) used in certain embodiments of the present invention with
subsequent
depolarization/hyperpolarization and its stabilization of different duration
depending on
the animal species is at the beginning of all cell-biological cascades. Both
processes
differ significantly on account of the substances used, namely A) potassium
cations for
rapid electrical blocking with depolarization of the egg envelope and the thus
triggered
egg activation and formation of a single, additional new zone in the egg
envelope
(formation of an elastic structuring layer) and B) calcium cations for slow
mechanical
blocking with an enzymatically controlled morphological conversion in the
existing layers
of the egg envelope (formation of a structural stabilization layer).
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The first electrical event is rapid depolarization or even hyperpolarization
within
milliseconds and is intended to prevent further sperm from adhering to the egg
after
fertilization in nature. Rapid hyperpolarization is detached from a steady
hyperpolarization within the following up to 60 min (in some types of aquatic
animals
such as lobsters, even up to 5 hours). Although the sperm remaining in the
vicinity of
the egg can still undergo attachment after the fast electrical block and
remain stuck in
the vitelline coat of the egg envelope (soft coat), it cannot penetrate
through the plasma
membrane of the egg for actual fertilization, as could be shown on mollusks.
If
potassium cation exposure continues in accordance with certain method
embodiments
according to the invention, the formation of a completely new, hyaline
(translucent,
glassy, gel-like) zone (elastic stabilizing layer) is observed in the live,
fertile, but
unfertilized and mature egg, which is unknown in the literature to date, which
is GAG-
positive (increased occurrence of glucosaminoglycans) and eosinophil (dyeable
with the
red, acidic diagnostic dye eosin) for the visualization of cell organelles,
plasma proteins,
connective tissue and its precursors), and in which sperm would get stuck. The
formation of this elastic stabilizing layer is the first to take place within
the continuous
hyperpolarization of 10 s and more in partial areas over the egg envelope and,
after
completion, is located between the zona radiata externa and the alveolar layer
in live
eggs with a structural design similar to that of fish and crustaceans (at
least two layers
in the egg envelope). The cause for the formation of the new elastic
stabilization layer is
seen in the continuous depolarization of the egg envelope by the supply of
potassium
cations in physiological concentration according to invention.
According to EU legislation of the member states on food supplements, only the
potassium compounds listed therein, such as potassium bicarbonate (potassium
hydrogen carbonate, KHCO3) (CAS no. 298-14-6), potassium carbonate (K2CO3)
(CAS
no. 584-08-7), potassium citrate (CAS no. 6100-05-6), potassium hydroxide
(KOH)
(CAS no. 1310-58-3), potassium chloride (KCI) (CAS no. 7447-40-7), K,
potassium
iodide (KI) (CAS no. 7681-11-0), potassium iodate (KI03) (CAS no. 7758-05-6),
may be
used for nutritional purposes. Similarly, the proposal for a Regulation of the
European
Parliament and of the Council of 11/10/2003 (COM (2003) 671 final) allows
these
compounds to be added to food. Certain potassium compounds, such as potassium
citrate (E 332), potassium lactate (E 326), potassium orthophosphates (E 340),
may
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also be added to foods for technological purposes. The method of treatment of
live,
mature eggs of fish or crustaceans with potassium cations in concentrations
compatible
with cells (physiological concentration, i.e. not harmful to the egg) and
without the
formation of residues, according to the invention, allows caviar or caviar-
like products to
be produced which meet all national and international quality requirements by
authorities, distributors and consumers. The studies on intracellular ion
concentrations
using optical emission spectroscopy (OES) in the cytoplasm of eggs at the
Alfred
Wegener Institute, in which potassium was used as a new substance for the
continuous
depolarization of the outer egg envelope, show no changes in concentration in
the egg
plasma after treatment, even with different concentrations and duration of
treatment.
The potassium cations used in certain method embodiments according to the
invention
thus clearly apply as a processing aid. A processing aid is used in the
industrial
processing and production of foodstuffs. The processing aids are food
additives which
are added in order to facilitate technical processes such as cutting and
filtering. In the
end product, however, the processing aids must not be present at all or only
in
unavoidable (small) residues. In contrast to changing food additives which
also have to
be declared on the packaging, the processing aids must no longer have any
effect in the
end product, which is of particular advantage. Their use must be technically
unavoidable, technologically ineffective, harmless to health and odorless and
tasteless.
Since the substances are no longer present or active in the treated foods,
their use
does not have to be labeled. This also applies to residues, reaction products
or residual
contents.
It is preferred and advantageous if at least one potassium salt, preferably
the salt of
citric acid (potassium citrate E332) and/or the salt of hydrochloric acid
(potassium
chloride E508) and/or the salt of sorbic acid (potassium sorbate E202) is
dissolved in
the water as potassium donor for the formation of the cationic component.
Potassium
donor is taken to mean a compound having potassium, which after its
dissolution in
water supplies the potassium cations, wherein its concentration is determined
by the
concentration of the particular potassium compound and its structural formula
in the
water. The potassium salts mentioned are even all approved as food additives
with E
numbers, although in certain embodiments of the invention they are used only
as
processing aids which no longer occur in the end product and are not subject
to
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declaration. An advantageous and preferred concentration of the potassium
cations in
one solution with previously deionized water is between 0.1 mmo1/1 and 3.0
mmo1/1,
preferably 0.1 mmo1/1, 0.5 mmo1/1, 0.65 mmo1/1, 1.6 mmol/lor 2.0 mmo1/1,
particularly
preferably 1.0 mmol/lor 1.5 mmo1/1 in accordance with an embodiment. All
margin and
intermediate values (integer and non-integer) should also always be included
in all
ranges (also other parameters) made within the scope of this invention. In
order to
produce the potassium cation concentrations mentioned in the water, it must be
deionized. However, since water molecules also decompose constantly in water,
it is
understood that only a degree of deionization can be achieved by technical
means
(electrical conductivity in water between 1 pS and 15 pS at 25 C as a measure
of the
achievable deionization).
Furthermore, the potassium exposure time in the potassium exposure step is
preferably
and advantageously between 5 min and 30 min, preferably at 10 min, 12 min, 15
min,
20 min or 25 min. Other potassium exposure times in this range are also easily
selectable. Exposure times of up to 50 min or more may even occur in the
treatment of
mature lobster eggs (crustacean). The formation of the new elastic
stabilization layer,
which seals the egg envelope, starts already a few seconds (up to 10 s) after
the start of
the treatment. However, since the eggs do not all react simultaneously with a
continuous depolarization and formation of the stabilization layer in partial
regions of the
surface of the egg envelope, an extended treatment time of up to 10 min is
recommended to achieve continuous depolarization in all treated eggs. As the
treatment
time increases, it migrates and is localized between the zona radiata externa
and the
alveolar layer in the entire egg envelope around the egg. As a result, the
live, mature
eggs are elastically stabilized by the invention in such a way that they can
be salted,
repackaged and deep-frozen without problems.
As a further optional modification, certain embodiments of the method
according to the
invention also provide for a calcium exposure step, which can be carried out
after the
potassium exposure step or before it. The respective changes to the egg
envelope
occur independently of each other in both sequences in their described
characteristics.
In the calcium exposure step, calcium is preferably and advantageously
dissolved in
another solution with water as a cationic component in a concentration which
does not
Date Recue/Date Received 2022-07-14
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damage the live, mature eggs (i.e. physiological), wherein the water is
deionized before
the addition of a calcium donor for the formation of the cationic component.
The live,
mature eggs are treated in the calcium exposure step until a desired
structural
stabilization of the egg envelope is achieved. The desired degree of
structural
stabilization can be readily determined by self-testing (degree of plop effect
of the
eggs). In the calcium exposure step, at least one calcium salt, preferably
calcium citrate,
calcium chloride and/or calcium sorbate, is advantageously and preferably used
as
calcium donor (calcium supplier, definition see potassium donor). Calcium
salts are
authorized in the European Union as food additives under the numbers E333 and
E509
without a quantitative limit and E203 with a quantitative limit. In German,
the spelling
forms "Kalium (potassium) and "Calcium" (not Kalzium) were chosen to better
distinguish the use of the two ion types.
Calcium is already physiologically present in the egg cell and an essential
component in
the cell metabolism. It is known from WO 2007/045233 Al, which has already
been
referred to above, that calcium chloride is used to structurally strengthen
the egg
envelope by irreversibly cross-linking proteins through the incorporation of
tyrosine
molecules. In addition to the improved and adjustable elasticity of the egg
envelope,
which is basically achieved with the invention, it can still be mechanically
solidified
structurally by the calcium exposure step. Thus, an optimal, stabilizing
combination can
be achieved for certain caviar types and caviar substitutes. This is
particularly
advantageous for very large unstable eggs (larger than 3.2 mm in diameter,
e.g. eggs
from the Beluga sturgeon or the white sturgeon) or for those which are
particularly soft
when they are mature (maximum force less than or equal to 0.3 N until they
burst in the
hardness test, e.g. eggs from the Sterlet sturgeon). The application of both
treatment
steps results in a high-quality caviar or caviar-like product for eggs that
are problematic
(size, softness).
The concentration of the calcium cations in the other solution is
advantageously
between 0.1 mmo1/1 and 3.0 mmo1/1, preferably 0.1 mmo1/1, 0.5 mmo1/1, 0.8
mmo1/1,
1.0 mmo1/1, 1.5 mmo1/1, 1.6 mmol/lor 2.0 mmo1/1. The calcium exposure time is
preferably between 9 min and 30 min, preferably 10 min, 12.5 min, 15 min, 16
min,
20 min or 25 min. The choice of treatment duration should take account of the
fact that
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the strength of the egg envelope increases steadily with increasing calcium
exposure
time until a limit value is reached. In nature, fertilized eggs from fish
attain a strongly
hardened egg envelope after approx. 60 minutes, which is no longer suitable
for
consumption. This may take up to 24 hours for the lobster.
An important process parameter in certain embodiments of the method according
to the
invention is the temperature of the solutions in which the mature eggs are
treated. This
is said to be physiological, which means that it does not hinder the natural
processes in
the live eggs. In certain embodiments, the temperature of the solutions is
always in the
range of the natural spawning temperature of fish or crustaceans. This ensures
that the
electrical activation of the egg envelope caused in the potassium exposure
step is
carried out reliably with depolarization starting at rest potential. In
unnatural spawning
temperatures, for example in fish or crustaceans from the polar regions above
15 C, no
electrical egg activation occurs, and the live, mature eggs cannot be
electrically or
enzymatically stabilized. They become atretic. The same applies to the eggs of
fish or
crustaceans from the mixed and tropical zones. The basic rule is that at
temperatures
above 35 C, the degeneration of the solution results in a severe loss of
quality in eggs.
In order to adapt the temperatures of the solutions to the natural habitats,
the present
invention broadly divides the life zones of fish and crustaceans, the eggs of
which can
be used, during the periods of natural reproduction into three climate zones:
polar zones
(at the poles), temperate zones (between the polar zones and the tropical
zone),
tropical zone (around the equator). The present invention prefers and
advantageously
exploits the fact that the temperature of one solution (potassium exposure)
and/or of the
other solution (calcium exposure) is taken from a polar temperature range
between 1 C
and 15 C, preferably between 5 C and 12 C, especially preferred 10 C, a
moderate
temperature range between 10 and 20 C, preferably 15 C, especially preferred
12 C, or
a tropical temperature range between 20 C and 29 C, preferably 27 C,
especially
preferred 21 C. The invention preferably avoids temperatures resulting in a
change -
degeneration, cell death - of the eggs, as is the case, for example, with
pasteurization
by heating to temperatures above 40 C. The invention preferably avoids this at
any
point in the process.
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Since the cation concentrations used in certain embodiments of the method
according
to the invention trigger animal-specific physiological reactions of electrical
(potassium
exposure) and metabolic (calcium exposure) nature and thus influence
processing
resulting in a stable edible end product, it must always be assumed that
deionized water
is present in the solution in order to achieve an exact concentration of
electrically
(potassium) or metabolic (calcium) active cations (positively charged). It is
therefore
technically achievable and thus preferred and advantageous if the deionized
water at
25 C has an electrical conductivity between 1 pS/cm and 15 pS/cm, preferably
pS/cm or less, particularly preferred 1 pS/cm. Drinking water and well water,
depending on the regional source, consist of a highly different composition of
different
ions, which may even have antagonistic effects on cell metabolism under
certain
circumstances. When the temperature is 25 C, the electrical conductivity of
pure water,
for example, is 0.055 pS/cm, deionized water 1 pS/cm, rainwater 50 pS/cm or
drinking
water 500 pS/cm. In order to be able to obtain reproducible results, it is
important to
know the deionized water in its electrical conductivity.
Since live cells in the form of activatable mature oocytes are treated with
certain
embodiments of the method according to the invention, it is also important,
among other
things, for the solutions to be adapted to the metabolism of the cells so that
the
metabolic processes induced in the method can also take place. It is therefore
advantageous and preferred if the one and/or the other solution has a pH
(physiological,
not detrimental to the living organism) of between 6.8 and 8.0, preferably
between
7.0 and 7.9, particularly preferred 7.2 or 7.4 or 7.5. In particular, the pH
adjusted in the
solution is relevant for the slow metabolic reaction in the calcium exposure
step. Since
enzymatic processes in the cell are highly regulated by the pH, the
intracellular pH in
the potassium exposure step (electrical process) was also examined. However,
the pH
in the egg plasma of the eggs treated with the various potassium-based
substances at
different concentrations and durations remains substantially unchanged in the
pH
optimum between 7 and 8 and shows the expected individual differences in the
case of
individual fish and crustaceans.
In embodiments of the invention, the different exposure steps are used to
stabilize the
endogenous egg envelope of the live, mature eggs (elastically and optionally
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structurally). Thus, the caviar or the caviar-like product is already ready
for further
processing, such as salting and packaging. During ovulation, the live, mature,
ovulated
eggs are pushed out of the follicular cells so that no more tissue residues of
blood
vessels or follicular cell residues adhere, to which bacteria or fungi could
colonize.
Harvested ovulated eggs therefore have a fine purity and thus the best
conditions for
long shelf life. This is reliably ensured if, following the last exposure step
carried out for
preservation and flavor intensification, a mild salting is carried out with
2.0% to 3.8%,
preferably 3.5%, sodium chloride in relation to a quantity of caviar or caviar-
like product.
The sodium chloride should preferably be free of potassium and calcium donors,
as are
contained, for example, in trickle aids, since this prevents uncontrolled
changes in the
egg envelope due to salting. Caviar from sturgeon eggs is dry salted with
simple
common salt (sodium chloride NaCI), wet salting is often carried out when roe
from
other fish species is processed into caviar-like products such as salmon and
trout
caviar. Salting in the specified area, which can optionally be carried out
with the
invention, is a very light salting process, also known as "malossol", which is
a clear sign
of high quality. Pasteurization or heating to a temperature of 60 C and above
is
preferably dropped entirely for the caviar or caviar-like product prepared
according to
the method of the invention, as this is not necessary and would only harm the
quality of
the product and its sensory properties. As a result of malossol salting, the
caviar or
caviar-like products produced using the method in question have a minimum
shelf-life of
at least 9 to 12 months if stored at -2 C. It does not freeze in the process
due to light
salting.
A further improvement in the quality of the produced caviar or caviar-like
product results
from certain embodiments of the invention if, in accordance with a further
modification,
storage of the caviar or caviar-like product in airtight glass containers for
several
months, preferably one to three months, is carried out preferentially and
advantageously
following preservation and intensification of flavor. The caviar "matures"
through storage
and, depending on the degree of maturity, gains in taste intensity. However,
this
maturation is to be seen in the sense of a further development of taste (as
with cheese,
for example) and has nothing to do with the "degree of maturity" of the mature
eggs
used in the invention in the sense of biological development. Here, maturity
refers to the
possibility of fertilization and thus to the development state of the live
eggs. When the
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caviar matures in relation to taste, it is stored in glass containers, which
provide
sufficient space for the caviar to mature, since it is not pressed (as in the
case of
packaging in metal snap-on lid cans) and thus retains its taste-intensive
oils. The caviar
thus packaged in glass according to certain embodiments of the invention is
not to be
confused with pasteurized caviar, which is also frequently packaged in glass.
Furthermore, storage in environmentally friendly glass containers avoids the
often
criticized metallic taste of caviar conventionally packaged in metal
containers.
In accordance with the next modification of the process, it is preferable and
advantageous to carry out freezing of the caviar or caviar-like product in a
temperature
range between -20 C and -15 C, preferably at -18 C, following preservation and
intensification of flavor or storage and maturation of flavor. Ideally, the
caviar matures in
taste for human consumption to the desired degree of maturity of the
respective
customer and is either freshly frozen after 14 days subsequent to production
or after a
maximum of 3-4 months of maturing. The caviar is either frozen in 500 g glass
containers before the repackaging or after the repackaging for the end
customer in 30 g,
50 g, 125 g, 250 g or 500 g (possibly up to 1000 g) glasses which can be
vacuumed.
Caviar obtained under conventional slaughter cannot be frozen. Although
pasteurized or
heated caviar can be frozen, it exhibits extreme quality losses due to heat
treatment.
The possibility of freezing the caviar or caviar-like product according to the
invention
enables optimal caviar marketing that meets the current demands of convenience
food.
Marketing has so far reached its limits due to the specific transport and
storage
temperatures of -2 C to -4 C, which must be strictly adhered to, as these are
not
maintained by most suppliers. Therefore, conventionally obtained caviar is
treated or
pasteurized with harmful preservatives, such as borax, in order to preserve it
for at least
a period of 12 months and longer. However, the caviar produced with
embodiments of
the present invention can simply be frozen and thus stored and kept fresh for
a longer
period of time. Experiments have shown that caviar thawed slowly in the
refrigerator at
+4 C to +7 C does not lose taste or texture.
In certain embodiments of the invention, the eggs are treated in a solution
bath (an
aqueous solution, a solution with water). The eggs are added and left in the
solution
bath until ¨ depending on the type of egg used ¨ the desired degree of
stabilization
Date Recue/Date Received 2022-07-14
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(elastic and possibly structural) has been achieved. The eggs are then simply
removed
from the bath. In order to reliably prevent an undesired further stabilization
after removal
by the cations in the still adhering solution, in certain embodiments of the
invention it is
preferable and advantageous, in accordance with a next modification of the
method, if
the removal of the respectively introduced cations from the mature eggs is
carried out
after achieving the desired elastic (and optionally structural) stabilization,
to dip (briefly
immerse) the live, mature eggs in a saline solution (physiological saline
solution) that
does not harm them. This rinses off the cations and immediately disrupts the
stabilization processes they cause. The (desired) degree of stabilization of
the egg
envelope achieved so far is reliably preserved as the final state.
The live, mature eggs used in certain embodiments of the invention are fertile
but not
fertilized. They are generally not wetted by water and have a natural
potassium content
in the egg plasma. Such live eggs can either be released from the gonads into
the
abdominal cavity of the fish and harvested from there via the genital opening.
This can
be done, for example, by natural spawning, by stripping (massage of the
abdominal
cavity from the outside) or by using a catheter through which the eggs are
drained or
sucked out of the abdominal cavity. Eggs released from the gonads into the
abdominal
cavity are referred to as ovulated eggs (maturity level 5), which are still
surrounded by a
slimy ovulation fluid. In order to avoid the formation of a sticky layer on
the eggs upon
contact with water, the ovulation fluid is rinsed off with physiological
saline solution
before starting the treatment. Ovulated eggs can be obtained from live
animals, which is
particularly sustainable. However, live, mature eggs of maturity levels 3 or 4
can also be
used for the invention, which are taken from the dead animal in the gonads and
then
isolated. A good overview of the different maturity levels of cod is given in
the
publication by I. G. Katsiadaki et. al.: "Assessment of quality of cod roes
and
relationship between quality and maturity stage" (J. Sci Food Agric 79:1249-
1259
(1999)) can be found there in particular in Table 1. A numerical definition of
the degree
of maturity is possible with the help of the so-called "polarization index".
This is
calculated from the ratio of the distance between cell nucleus and cell
membrane to the
diameter of the ice between the animal and vegetative pole (large half-axis).
In
accordance with the next embodiment of the invention, it is therefore
preferred and
advantageous to treat live, mature eggs of fish or crustaceans with a
polarization index
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PI of 0.05 _5 PI 5 0.15, preferably 0.05 5 PI 5 0.12. Eggs with this PI are
particularly
suitable for harvesting for treatment according to the invention. Further
information on
the polarization index PI of eggs can be found, for example, in the sturgeon
breeding
guidelines (publication FAO Ankara 2011 Fisheries and Aquaculture Technical
Paper
570 "Sturgeon Hatchery Practises and Management for Release - Guidelines").
The method of the invention can be used to treat the live, mature eggs of fish
and
crustaceans (scientific name Crustacea) whose eggs have the basic structure
required
for the invention (more than two layers in the egg envelope) and which are
suitable for
consumption in the form of caviar or caviar-like products. It is preferable
and
advantageous to treat live, mature eggs from fish or crustaceans caught in the
wild or
from aquaculture, which have been ovulated and obtained by stripping or other
targeted
harvesting, such as catheterization. In doing so, for example, animals
intended for
restocking in the wild, such as from a restocking project, can also be
harvested. The
proceeds from the sale of caviar and caviar-like products can then be returned
to the
stocking measures. It is particularly preferred and advantageous if, in
certain
embodiments of the invention, live, mature eggs of recent and natural living
bony fish,
preferably of live sturgeon species, are treated. Embodiments of the invention
can then
be used to produce (genuine) caviar of the highest quality. Other caviar-like
products
from lobsters or other crustaceans, e.g. crayfish, can also be produced to the
highest
quality with embodiments of the method according to the invention.
Furthermore, very
large (above 3.2 mm diameter) or soft, unstable eggs (texture in the hardness
test
below 0.3 N, above which the eggs burst) can be treated preferentially and
advantageously, since embodiments of the method according to the invention can
also
optionally comprise two exposure steps with both elastic (electrically
stimulated) and
structural (enzymatically stimulated) stabilization of the egg envelope.
Finally, embodiments of the invention also include different products from
live, mature
eggs of fish or crustaceans, which can be produced with the inventive method
but also
with other methods. The products are characterized in that an elastic
stabilization layer
in the form of an eosinophilic, hyaline layer with incorporated
glucosaminoglycans is
additionally formed in the egg envelope. In this case, however, the live egg
is
unfertilized, which is why the elastic stabilization layer does not occur in
nature. In
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certain embodiments of the invention, the elastic stabilization layer lies
between the
zona radiata intema and the alveolar layer, preferably between the zona
radiata externa
and the alveolar layer. It can therefore occur only in the case of live eggs
with a more
than two-layer structure of the egg envelope. Sea urchins, for example, only
show
exactly two layers within the egg envelope. The new stabilization layer is
transparent,
gel-like and elastic and can be histologically dyed red with eosin and blue
with alcian.
During production using certain embodiments of the invention, its
characteristics are
influenced by the potassium cation concentration used in the potassium
exposure step
and its position is influenced by the potassium exposure time.
To remove the ovarian fluid, the live, mature eggs are treated with a saline
solution that
does not harm the eggs prior to treatment. This is preferably and
advantageously a
physiological saline solution. Furthermore, it is advantageous and preferred
if the saline
solution is formed as 0.6 percent to 1.0 percent saline solution, particularly
preferably as
0.9 percent saline solution. For example, to prepare a 0.9 percent saline
solution, 9 g of
sodium chloride (NaCI) are dissolved per 1 liter of water used. This
concentration
corresponds to the natural occurrence in the human organism, it is therefore
called
"physiological" saline solution.
Certain embodiments of the invention further relate to caviar or a caviar-like
product of
unfertilized, mature eggs of aquatic animals, characterized in that an
irreversible cross-
linking of protein strands by incorporated tyrosine molecules is additionally
formed in the
egg envelope. This additional irreversible cross-linking is located in the
zona radiata
interna and the zona radiata extema of the live eggs of fish or crustaceans.
Irreversible
cross-linking leads to additional structural stabilization of the egg
envelope. Together
with the existing elastic stabilization, it can also be used to treat
particularly large or soft
eggs. The caviar or the caviar-like product can be prepared according to
embodiments
of the invention, wherein the structural degree of stabilization in the egg
envelope then
depends on the calcium exposure time and the calcium cations concentration in
the
calcium exposure step. Other methods of making caviar or a caviar-like product
with the
same nature of irreversible protein cross-linking in the egg envelope are also
applicable.
Further embodiments of the methods and products of the invention can be found
in the
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following specific description part relating to the exemplary embodiments, but
in no way
limit the scope of the present invention to such exemplary embodiments.
Examples
In the following, the method for the production of caviar or a caviar-like
product from
live, mature eggs of aquatic animals and such products according to the
invention and
their advantageous modifications for further understanding of the invention
are further
explained by means of embodiment examples and figures. Thereby shows the
Figs. 1A, B, C SEM images for comparing obtained live eggs in the immature
and
in the mature state (prior art),
Fig. 2 an initial table of measurements of egg envelope thickness
during
treatment of live, mature eggs from Siberian sturgeon,
FIG. 3 a second table of measurements of the egg envelope
thickness during
the treatment of live, mature eggs from the Beluga sturgeon,
Figs. 4A, B, C, D SEM images of the treatment series for forming the
stabilizing layer
of differently treated live, mature eggs of sturgeon with potassium
cation treatment compared to the double treatment with potassium
and calcium cations as well as calcium cations alone,
Figs. 5A, B, C, D TEM images of the structure of the layers of the egg
envelope with
the formation of the new stabilization layer (SS) between the zona
radiata extema (ZRE) and the alveolar layer (AL),
Figs. 6A, B, C, D TEM images of the cortical granules of untreated and
differently (only
potassium cations, double treatment with potassium and calcium
cations, only calcium cations) treated mature sturgeon eggs,
Figs. 7A, B, C, D light microscopic images of untreated mature eggs of the
Siberian
sturgeon and of live, mature eggs from the Siberian sturgeon
treated with potassium ions,
Figs. 8A, B, C, D light microscopic images of live, mature eggs of the
Siberian
sturgeon treated with calcium cations and of live, mature eggs
treated with potassium cations and with calcium cations, and
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Figs. 9A, B SEM and light microscopic images of the structure of the
egg
envelope of the Beluga sturgeon after treatment of the live, mature
eggs with potassium and calcium cations.
It is known from studies of the relationship between the weight and/or age of
sturgeons and the size of the caviar grain or the quantity of caviar harvested
that the
ovoid diameter and thus the quality of the caviar increases with the weight
and/or
age of the sturgeon. Furthermore, the amount of caviar harvested increases
with the
increasing weight and age of the sturgeon and thus its economic success.
Sturgeons do not become sexually mature in their natural environment until the
age
of 12 to 26 years, depending on the species. Most sturgeons spend the growth
phase until their first reproduction in the sea or in estuaries and then
migrate into the
rivers to find their spawning grounds on stony ground in fresh water. But
also, in
aquaculture, sturgeons need approximately 5 to 16 years, depending on the
sturgeon species, to reach the first sexual maturity and thus the first caviar
harvest.
In aquaculture, the repeated harvesting of caviar from live females over many
years
presupposes animal-friendly keeping of the fish with optimal feeding and low
stocking density and is always economically and ecologically sensible due to
its late
sexual maturity and long lifespan. A production of caviar in the economically
interesting ton range is easily possible in a coordinated workflow from
harvest and
treatment of the stripped eggs to caviar. Suitable upscaling measures can
achieve a
daily production of 80 kg and more depending on the age of the fish and the
amount
of caviar associated with it.
Certain embodiments of the invention use live, mature eggs that have
previously
been cleaned with physiological saline solution. These are ovulated eggs that
have
previously been squeezed out of the gonad by fine muscle fibers of the
follicular
cells due to their stage of maturity (stage of ovulation readiness and
fertility and), a
process known as ovulation. The ovulated eggs are released into the fallopian
tubes and the abdominal cavity of the fish and without cell residues and other
residues. They can then be removed by massaging the abdomen without affecting
the life of the fish. The completely clean surface of the eggs does not allow
any
niches or wrinkles for bacterial or fungal infestation, which results in a
long shelf
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life of the caviar or caviar-like product. It is not necessary to use
conservation
methods such as borax, which is harmful to human health. Figs. 1A, B, C show
prior art scanning electron microscope (SEM) images of a live egg and the
ovulation process. Fig. 1A shows an immature oocyte with follicular cells such
as
those found in conventional caviar from the killed sturgeon. Fig. 1B shows in
situ
ovulation and release of the mature egg cell from the surrounding follicular
cell.
Fig. 1C then shows a live, mature sturgeon egg which is obviously completely
smooth and clean.
In an exemplary embodiment with live, mature eggs after ovulation, a possible
method workflow according to an embodiment of the invention is explained in
more
detail below with some optional additional steps:
= Stripping of live female sturgeon from live eggs at maturity stage V
after
dissolution of the germinal vesicles,
= Immediate transport of the striped live eggs together with the ovarian
fluid to a
caviar laboratory (waiting times are largely avoided, unavoidable waiting
times are
bridged on ice and under exclusion of oxygen by covering the ovarian fluid
with an
air-impermeable plastic film),
= Immediate thorough rinsing of the live eggs in 0.9 percent physiological
saline
solution until the ovarian fluid is completely removed,
= Performing of the potassium exposure step:
= Preparing a 0.1 to 2 millimolar potassium cation solution from potassium
citrate in deionized water having a conductivity of 10 pS/cm (at 25 C) and a
temperature in the polar temperature range of 10 C,
= Introducing the live, mature eggs into the solution for a potassium
exposure
time of 10 min and
= Removing the treated eggs from the solution; and
= Brief dipping of the treated eggs into a 0.9 percent physiological saline
solution.
Other extraction methods for the mature eggs are also possible. Even with the
use of
live, mature eggs taken from a previously killed animal, blood and fat must be
rinsed off
or the eggs must even be rubbed out of the gonads, which is achieved by the
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pretreatment with a preferably physiological saline solution. By dipping, the
elasticity
and the diameter of the stabilization layer can be additionally controlled
(that is to say in
addition to the selection of the duration of the exposure time). Due to the
electrical
influence of the introduced potassium cations, the described treatment effects
the
forming of a hyaline, elastic stabilization layer between the zona radiata
extema and the
alveolar layer in the egg envelope. In eggs of normal condition and softness,
treatment
with the potassium exposure step is sufficient. However, when particularly
large, soft or
sensitive eggs of some sturgeon species, for example Huso huso, Acipenser
transmontanus or Acipenser ruthenus are used, a calcium exposure step can also
be
connected (or put in front):
= Additional performing of the calcium exposure step:
= Preparing another 0.5 to 2 millimolar calcium cations solution of calcium
chloride in deionized water having a conductivity of 10 pS/cm (at 25 C.) and
a temperature in the polar temperature range of 10 C,
= Introducing the live, mature eggs into the other solution for a calcium
exposure time of 12 min, and
= Removing the treated eggs from the other solution, and
= Brief dipping of the treated eggs into a 0.9 percent physiological saline
solution.
In addition to the elastic stabilization layer from the potassium exposure
step, this also
forms a structural protein cross-linking of the egg envelope which is located
in fish and
crustaceans in the already present zones radiata interna and radiata extema of
the egg
envelope. This additional structural protein cross-linking of the egg envelope
by tyrosine
residues gives in particular large and soft or sensitive eggs plastic firmness
¨ in addition
to the elasticity from the potassium exposure step. Optional dipping is also
used here
for additional controllability. The resulting product is (genuine) caviar from
live, mature
eggs, which can then be further processed as follows:
= Mixing the caviar with dry (I(+- and Ca-free trickle aids) sodium
chloride NaCI
(3.5 g/100 g caviar, 3.5%), which corresponds to a malossol salting for
preservation,
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= Filling the lightly salted caviar in glass containers, preferably 500 g
maturing
glasses, and air-tightly vacuum-sealing the containers with screw caps and
labeling,
= Storing of the glass containers at -2 C for 2 to 4 months for further
maturing of
the caviar and optionally
e Freezing of fresh caviar or caviar matured according to customer
requirements in
glass containers at -18 C.
The live, mature eggs treated in the potassium exposure step form a completely
new
zone due to the treatment: the stabilization layer SS, which is elastic and
hyaline (gel-
like). The stabilization layer SS can be easily colored for detection. It is
located
between the alveolar layer AL and the zona radiata externa ZRE and has so far
not
been described in the literature. Refer to descriptive Introduction with the
corresponding Siddique glossary for the structural design of the egg of fish
and
crustaceans.
The table in Fig. 2 is based on measurements of the diameter (in pm) of the
extracellular egg envelope on mature eggs of the Siberian sturgeon on the
basis of
cryosections of constant layer thickness (10 pm) using computer-controlled
image
analysis (Zeiss) under the influence of different treatments to stabilize the
egg
envelope. The table shows the formation of a new stabilization layer SS and
the
diameter of existing layers (ZRI, ZRE, AL) of the egg envelope under treatment
with different concentration additions in mmo1/1 of potassium cations K+ alone
(from
potassium citrate) and in combination with calcium cations Ca ++ (from calcium
chloride) during the various exposure steps according to the invention. Live,
mature eggs from the Siberian sturgeon Acipenser baerii were treated. In
addition
to the acronyms already explained in the foregoing and in the case of
Siddique,
Nly means mean value, and Std means standard deviation. The values given are
those for the new elastic stabilization layer SS. Furthermore, reference is
made to
the state of the art in accordance with the above-mentioned WO 2007/045233 Al.
The quality controls after the treatments showed that only at concentrations
of
1 mmo1/1 and 1.5 mmo1/1 potassium cations a thickness of the egg envelope of
at least
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12 pm is achieved and an intermediate product is formed which has lost its
stickiness
and is stable enough for the further processing of caviar. Furthermore, it was
shown
that a treatment duration of preferably 10 minutes is reasonable so that all
live eggs in
the solution react metabolically. A treatment amount of 2.5 kg of caviar (in
approx. 251
solution) in a treatment unit could be achieved. The sensory examination of
the caviar
after the treatment with potassium cations according to the invention showed
that the
elastic texture of the caviar of the Siberian sturgeon did not show any
differences in
concentration variations between 1 mmo1/1 and 1.5 mmo1/1. On the other hand,
the
eggs treated in the one solution with lower potassium cations concentrations
are
different in texture and only a few eggs are stable, while the untreated eggs
are very
soft and burst. In accordance with the sensory tests carried out, treatment
with two
exposure steps (potassium and calcium cations) leads to a solid, pearly
product, also
referred to as "super plop" in the case of eggs of the Siberian sturgeon.
The table in Fig. 3 shows the presence and diameters (in pm) of layers of
extracellular
egg envelope with different treatments in accordance with the invention of
live large
eggs from the Beluga sturgeon Huso huso. During the treatment of this caviar
with
potassium cations (from potassium citrate), the formation of the new
eosinophilic
stabilization layer SS in the extracellular egg envelope was also observed,
which is also
located between the ZRE and the AL. The sensory tests carried out on the large
eggs
of the beluga sturgeon showed that double treatment with potassium and calcium
cations leads to optimal results with regard to the texture of the fragile
live, mature eggs.
Figs. 4A, B, C, D show SEM images of the change in the structure of the
extracellular egg envelope of live, mature eggs, by way of example of the
Siberian
sturgeon under various treatments. Two magnifications are shown: left 6000x
and
right (cutouts) 12000x. The treatments were always carried out on the live
egg,
which was shock frozen in hexane at -80 C in order to produce histological
cryosections to maintain its native state.
Fig. 4A In untreated mature eggs, the zones of the egg envelope show no clear
separation from one another (prior art).
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Fig. 4B Under the influence of 0.5 mmo1/1 potassium, the new stabilization
layer
SS is already formed between the zona radiata externa ZRE and the alveolar
layer
AL, while the zona radiata interna ZRI and zona radiata externa ZRE show an
unchanged loose protein structure as in untreated eggs.
Fig. 4C The successive double treatment of the eggs with potassium and
calcium cations shows both characteristic morphological features in the SEM,
namely the stabilization layer SS by the potassium treatment AND the twisting
and
cross-linking of the protein strands in the zona radiata interna ZRI and the
zona
radiata externa ZRE, which is characteristic for a calcium treatment, compare
Fig. 4D.
Fig. 4D Calcium treatment alone leads to strong twisting and cross-linking of
the
loose protein strands in the zona radiata interna ZRI and the zona radiata
externa
ZRE (prior art), compare Fig. 4A and Fig. 4B without calcium treatment.
Figs. 5A, B, C, D show transmission electron microscope images (TEM, 3000-fold
magnification) of the multi-layered structure of the egg envelope of mature
sturgeon
eggs during treatment with 1.0 mmo1/1 potassium cations. Fig. 5A shows the
zona
radiata interna ZRI with loose, cloudy fibrils separated from the zona radiata
extema
ZRE by the epilayer EP (phenomenon can only be identified ultra-structurally).
The
zona radiate externa ZRE is characterized by a filamentous network of
elongated
fibrils. Fig. 5B shows the ultra-structural formation of the new stabilization
layer SS
with a fine-granular structure directly between the zona radiata externa ZRE
and ¨ in
accordance with Fig. 5C ¨ the alveolar layer AL, which is permeated to the
periphery
of the egg envelope ¨ in accordance with Fig. 5D ¨ by small ductuli (small
ducts,
channels). The ultra-structural analyzes confirm that treatment with potassium
cations
(1.0 mmo1/1) in accordance with certain embodiments of the invention leads to
the
formation of a previously unknown new stabilization layer SS with amorphous
structure and positioning in fish and crustaceans between the zona radiata
extema
ZRE and the alveolar layer AL.
Figs. 6A, B, C, D show TEM images (3000-fold magnification) of the cortical
granules
CG in the peripheral egg plasma within the plasma membrane of the mature eggs,
wherein Fig. 6A shows an untreated egg (prior art), Fig. 6B shows an egg
treated with
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potassium cations, Fig. 6C shows an egg treated with potassium and calcium
cations
and Fig. 6D shows an egg treated only with calcium cations (prior art).
Cortical granules are secretory organelles (structurally delimitable regions)
found in
eggs and closely associated with the fertilization event. Cortical granules
contain
enzymes such as peroxidase and structural elements for tyrosine cross-linking
of the
zona radiata interna ZRI and the zona radiata externa ZRE. As analyzed by TEM
under the influence of the various treatments, the cortical response and the
discharge of its contents are triggered by treatment with calcium cations. An
identical
process also occurs in natural fertilization by the sperm-induced calcium wave
in the
egg plasma membrane. In the untreated egg (Fig. 6A), the cortical granules CG
with
their enzyme endowment are clearly recognizable as large, round vesicles
(bubbles)
in the peripheral egg plasma, which also contain structural elements.
Likewise, the
cortical granules CG are still present unchanged during potassium treatment
alone
in accordance with the invention (Fig. 6B). However, strong vesicular
transport can
be observed at the egg envelope from the egg plasma into the acellular egg
envelope. In Fig. 6A and Fig. 6B the yolk is marked with D. In a double
treatment
with potassium and calcium cations (Fig. 6C), both the discharge (arrows) of
the
contents of the cortical granules CG and the formation of the new
stabilization layer
SS occur. Treatment with calcium cations alone (Fig. 6D) only leads to a
cortical
response, wherein the contents are discharged into the acellular egg envelope
and
the enzymatic cross-linking of the zona radiata intema ZRI and zona radiata
externa
ZRE is initiated by tyrosine residues. Empty vacuoles V remain in the egg
plasma.
For the diagnostic screening of cryosections for potassium effect in the
invention, in
Fig. 7A and Fig. 7B, light microscopical images (400-fold magnification) of
untreated
live eggs of the Siberian sturgeon Acipenser baerii from the prior art are
shown. The
left photograph in accordance with Fig. 7A shows HE staining (hematoxylin-
eosin
staining), the right photo in accordance with Fig. 7B shows alcian blue
staining. This
test stains glucosaminoglycans GAG, hyaluron and fibrin. It can be seen that
the
alcian blue is missing in the various layers of the egg envelope but is
distinctly present
in the ooplasm OP. The individual layers are characterized according to the
above
embodiments and are marked in their thicknesses by double arrows.
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Fig. 7C and Fig. 7D, on the other hand, show photographs of cryosections of
live,
mature eggs in the ovulated state of the Siberian sturgeon Acipenser baerii
treated
with the potassium exposure step method of the present invention. The eggs
were
treated with a concentration of 1.5 mmo1/1 potassium cations (from potassium
citrate). The occurrence of the new stabilization layer SS between the zona
radiata
externa ZRE and the alveolar layer AL in the extracellular egg envelope is
noticeable. The left photo in accordance with Fig. 7C shows that this new
stabilizing layer SS is particularly eosinophilic after staining with eosin.
The right
photo in accordance with Fig. 7D shows that this new stabilization layer SS is
particularly rich in GAG after staining with alcian blue. The particularly
advantageous elasticity of the new stabilization layer SS can be derived
therefrom.
For diagnostic screening of cryosections for calcium effect alone and double
treatment
effect by potassium and calcium cations, Fig. 8A and Fig. 8B show photos from
the
prior art of cryosections of mature eggs of the Siberian sturgeon Acipenser
baeiii
treated with the method in accordance with WO 2007/045233 Al, magnified 400-
fold.
The left photograph in accordance with Fig. 8A shows HE staining (hematoxylin-
eosin
staining), the right photo in accordance with Fig. 8B shows alcian blue
staining. The
ovulated eggs of the Siberian sturgeon were treated. The left photo in
accordance with
Fig. 8A with the HE staining reveals the protein strands cross-linked by
tyrosine
molecules in the zona radiata intema ZRI and zona radiata externa ZRE. A
distinct
separation between the two zones can be seen. The cross-linking results in
structural
stabilization of the egg envelope. In the right photo in accordance with Fig.
8B with the
alcian blue staining, it can be seen that the zona radiata intema ZRI and the
zona
radiata externa ZRE are stained only very weakly, which has resulted in little
GAG and
a lower elasticity, whereas in the ooplasm OP a strong staining indicates very
much
GAG.
In Fig. 8C and Fig. 8D, photos of cryosections of live ovulated eggs processed
with
the method of the invention are shown, which have been treated in the
additional
calcium exposure step with the method in accordance with
WO 2007/046233 Al. The mature ovulated eggs of the Siberian sturgeon Acipenser
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baerii were treated with 1.5 mmo1/1 potassium cations (from potassium citrate)
in the
potassium exposure step and with 1.6 mmol/lcalcium cations (from calcium
chloride) in the calcium exposure step. In addition to solidifying cross-
linking of the
egg envelope shown in the photos in accordance with Fig. 8A and Fig. 8B, the
new
hyaline stabilization layer SS with its stabilization function of the egg
envelope can
also now be seen in the photos in accordance with Fig. 8C and Fig. 8D. The
treated
live, mature eggs of the Siberian sturgeon are thus stabilized both
elastically (by
GAGs) and structurally (by protein cross-linking) and form a perfect caviar.
Fig. 9A shows a characteristic SEM image (magnification 12000-fold) of the egg
envelope of live, mature ovulated eggs from the Beluga sturgeon Huso huso
after
double treatment according to the invention. Fig. 9B shows a light microscope
image (400-fold magnification) of the egg envelope of live, mature, ovulated
eggs
from the Beluga sturgeon Huso huso after double treatment according to the
invention. Both images show the new stabilization layer SS and the cross-
linking of
the zona radiata interna ZRI and zona radiata externa ZRE. Furthermore, it is
noticeable that the eggs of the Huso huso have an extremely distinctive
alveolar
layer AL with large vacuoles V. The screening of the cryosections with H&E
staining confirms the formation of the new elastic stabilization layer SS with
one
potassium cation exposure step and the additional protein cross-linking in the
egg
envelope with two exposure steps with potassium and calcium cations.
List of reference signs
AL Alveolar layer
Ca ++ Calcium cations
CG Cortical granules
CYT Cytoplasm (OP)
D Yolk
EP Epilayer
K+ Potassium cations
Mv Mean value
OP Oocyte plasma (egg plasma)
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pAL Alveolar layer (toward periphery of the egg envelope)
PO Plasma membrane of oocytes (egg cell)
SS Stabilization layer
Std Standard deviation
V Vacuole
ZRI Zona radiata interna
ZRE Zona radiata extema
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