Note: Descriptions are shown in the official language in which they were submitted.
~Z~l~
This invention relates to processes for converting dairy
whey fractions into commercially useful products, to the novel products
thus produced, and to methods of using them; More particu]arly, this
invention relates to a process for treating substantially deproteinized
dairy whey lactose permeates (WLP) with a base to produce a lactose-
rich aqueous solute fraction which is capable of supporting good growth
of a wide variety of microorganisms and a microcrystalline cloud fraction
which can be converted to a dry, free-flowing, odorless and tasteless
composition which has emulsifying and suspending properties which render
it useEul for a wide variety of applications in the food, pharmaceutical,
cosmetics, and other industries.
As noted by Alan G. Lane in J. Appl. Chem. Biotechnol. 27:
165-169 (1977), the disposal of whey resulting from the manufacture
of cheese and casein presents environmental and economic problems of
enormous magnitude, with the annual production of whey in the United
States estimated to have a pollution strength equivalent to the sewage
from 10 million people. While some whey is used as an animal feed
(e.g. see U.S. Patents 3,343,962 and 3,497,359 to Herbert R. Peer and
U.S. Patent 4,320,15() to Paul R. Austin et al.), most has l>eerl reg,lrcled
as waste and disposed of by traditional methocls. While recent developments
in u]trafiltration (UF) technology have made it possible to recover
proteins from whey economically, disposal of the remaining deproteinized
whey lactose permeate presents serious difficulties since it contains
most of the lactose (45 g/l) and thus most of the pollutional strength
(biological and chemical oxygen demand) from the original whey.
In one approach to this problem, fermentation techniques
have been developed for converting the lactose into food yeasts, e.g.
Kluyvcromyces fragilis, thereby attempting to overcome the limited
8 1 ;~
market for lactose itself. Such processes have generally involved
fermentation of the whey or the whey lactose permeate, first without
prior concentration and later by dialysis culture techniques such as
reported by ~ane. While offering the potential for removing up to
90 percent of the lactose present in the whey lactose permeate, such
methods suffer the disadvantage of yielding a single product of limited
utility.
The dialysis continuous fermentation of deproteinized whey
has been applied to the production of Lactobacillus cells, e.g. as
reported by R.W. Steiber et al. in J. Dairy Sci. 63: 722-730 (1980).
Using deproteinized whey as the substrate, the fermentor contents are
maintained at a constant pH of 5.5 by the addition of ammonia and dialyzed
through a semipermeable membrane against water; cell production was
double that of ordinary continuous fermentation.
Both sweet whey permeate and acid whey permeate have been
used as a feedstock in ethanol production using ~-galactosidase and
Saccharomyces cerevisiae, e.g. as reported by Barbel Hahn-Hagerdal
in Applied Biochemistry and Biotechnology 7: 43-45 (1982). Although
more than 50 percent of the lactose was converted to ethano], thc eluate
contained less than 2 percent ethanol yield bascd 011 thc welght/unit
volume of whey permeate feedstock.
The use of whole whey as a bacteriological culture medium
has been reported by Emel Celikkol in Mikrobiyol. Bul. 9(4): 273-279
(1975) and in U.S.S.R. Patent 819,166. As summarizèd in Chem. Abs.
84: 72629u and 95: 59904n respectively, the former process uses untreated
whole whey, while the latter process removes lactose from the initial
whey and hydrolyzes the proteins therein. For reasons which have heretofore
not been fully appreciated by the prior art, neither of these methods
has gaincd widespread use for either industrial or clinical grades
- 2 --
Z~
of culture media.
Whey colloidal precipitates have found use as clouding, stabilizing,
- emulsifying, thickening, and gelling additives (depending in general
on the concentration in which the precipitate is employed) to food
grade compositions, e.g. as described by U.S. Patents 4,143,174 and
4,209,503 to Syed M.M. Shah et al. Shah et al. do not describe useful
applications for the supernatant liquid which is separated from the
colloidal precipitate.
A variety of solids can be obtained from dairy whey permeates,
depending on the temperature, pH, and other conditions under which
they are formed, e.g. see Eustache U.S. Patents 4,042,575 and 4,042,576.
Pederson describes in Patent No. 4,202,909 a method for recovering
lactose from whey permeates by forming a precipitate upon heating to
180-200 F, and separating the supernatant liquid therefrom. Other
than the recovery of lactose, Pederson does not disclose any industrial
or commercial uses for the precipitate or for the supernatant liquid.
U.S. Patent 4,o36,999 to Donald A. Grindstaff describes pretreatment
of raw acid cheese whey by adjusting the pH to above 6.5 and separating
insoluble solids therefrom. Separated solids are t:reated by adding
calcium ion, heating and drying to form a product which is useful as
a nonfat dried milk substitute in bakery products. It has now been
found that a partic:ular combination of temperature and pH employed
in accorclance with the present invention gives a unique combination
of useful co-products, and that the solubility properties of the precipitate
can be varied depending on the extent of water removed therefrom.
It is a general object of one aspect of the present invention
to provide a simple and inexpensive method for converting deproteinized
dairy whey permeates into industrially useful products.
-- 3 --
It is an overall object of another aspect of the present
invention to provide a method for converting deproteinized lactose
rich dairy whey fractions into at least one fraction containing a micro-
crystalline cloud material (i.e. formed of microScopic crystals not
observable by the naked eye) and at least one lactose-rich aqueous
solute fraction, each of which has further use in a variety of industrial,
commercial and clinical applications.
It is a principal object of still another aspect of the present
invention to provide a lactose rich product derived from lactose rich
dairy whey fractions, which product is useful for formulating industrial
fermentation media, clinical diagnostic culture media, and other growth
media for culturing of microorganisms.
An object of yet another aspect of the present invention
is to provide improvements in processes for culturing microorganisms
employing these media.
An object of a second principal aspece of the present invention
is to provide a microcrystalline cloud material useful as an emulsifying,
suspending, and/or gelling agent.
An objcct of yet al-other aspc?ct of the presc~nt invention
is to provide improved methods for emulsifying and suspending a wide
variety c,f compositions empl-)yil-g these agents.
An obje(:~- ot a mc~r- part~ lar asper~ of tlle presenr invention
is 1() provide impr-ved toccl gra(le ad(litiv(s t(-r use in iol-(ls~ pl~arm.J(:eut-ical
rarliers, ccsmeti~ b;~sl~s, dell~itri~e b.lses, allll th(~ like.
- 4
1~Z0~81Z
By one aspect of the present invention a process is provided for
preparing a dry powder microbiological culture medium which can be recon-
stituted with water and autoclaved to form a clear, pH neutral culture ~edium
capable of supporting the growth of microorganisms under suitable growth con-
dditions, the process comprising: (a) raising the pH of a dairy whey lactose
permeate having a pH below 7 to a selected pH between pH 8 and pH 10 at which
essentially all of the dissolved solids,which would become insoluble when the
permeate is autoclaved for 10-20 minutes at 121 degrees C and 15 psi, preci-
pitate as a microcrystalline solids; (b) separating the supernatant from the
resulling precipitate to form a microcrystalline solid phase consisting
essenl:ially of the previously dissolved solids; (c) removing components hav-
ing a molecular weight above 100 kdal from the supernatant to form a lactose-
rich supernatant which is essentially free of the dissolved solids; and
(d) drying the resultant lactose-rich supernatant to form the dry powder
microbiological culture medium.
The pH preferably is raised to pH 9.
In the above process, step (c) comprises the step of ultrafiltra-
tion across a filter which retains components having a molecular weight above
100 kdal but preferably comprises the step of ultrafiltration across a filter
which retains components having a molecular weight above 20 kdal.
The process further may include lowering the pH of the separated
solute phase to 6.8-7.1, e.g. by the addition of a nontoxic Lewis acid to the
solute phase, or by autoclaving the solute phase to form a sterile micro-
biological culture medium, or without the addition of extraneous acid to the
solute phase.
The process may further additionally include spray drying the
separated solute phase to a moisture content of less than 10 percent by weight.
5 --
:
~Z~Z81'~
By another aspect of this invention ! a dry powder microbial cul-
ture medium is provided which can be reconstituted with water and autoclaved
to form a clear pH neutral culture medium capable of supporting the growth
of microorganisms under suitable growth conditions, the culture medium
being substantially free of components which could be retained by a filter
having a pore size which passes components having a molecular weight below
100 }cdal and which is essentially free of dissolved solids which would be~
come insoluble when a dairy lactose permeate is autoclaved for 10~20 minutes
at 121C and 15 psi.
The medium is preferably substantially free of con~ponents which
would be retained by a filter having a pore siæe which passes c nr~ntS
having a lecular weight below 20 kdal. In dry powder form, the product
would have a moisture content of less than 10 percent by weight.
The medium may further include a growth promoting amount of
extraneous nontoxic assim;lahle carbon atoms, preferably glucose. It may
also include a growth promoting amount of extraneous nontoxic assimilable
nitrogen atoms, preferably a yeast extract, hydrolyzed casein, or mixtures
thereof. Furthermore, it may further include an added gelling agent in an
amount capable of forming a gel when the dry powder is admixed with water.
The medium may further contain hydrolyzed casein, yeast extract,
cyste:ine HCl, and glucose added in amounts effective to produce a microbiolo-
gical culture medium suitable for the cultivation of both aerobic and anaero-
bic bacteria. Alternatively, it may further contain hydrolyzed casein, yeast
extract, and glucose added in amounts effective to produce a general purpose
microbiological growth medium or it may further contain hydrolyzed casein,
yeast extract, glucose, and a colorimetric oxidation-reduction indicator
added in amounts effective to produce a culture medium suitable for the
cultivatiorl of anaerobic bacteria.
.~ ~ - 6 -
~ZIl~Z81;~
The present invention, in another aspect, provides a sterile
microbiological culture medium consisting essentially of an aqueous solution
of the dry powder culture medium as ~escribed above. This culture medium may
be sterilized by autoclaving, and may have a solids content of 3.5% (wt/vol).
Such culture medium may have a total glucose content of 0.5% and
furthe:r containing additives of 0.5~ hydrolyzed casein and 0.05% yeast extract
to produce a general purpose microbiological growth medium~ Alternatively it
may have a total glucose content of 0.4% and further containing additives of
a nontoxic gelling agent in an amount effective to reduce oxygen diffusion,
0.5% hydrolyzed casein, and 0.05% yeast extract to produce a microbiological
culture medium suitable for the cultivation of both aerobic and anaerobic
bacteria, or iS may have a total glucose content of 0.5% and further contain-
ing additives of 0.5% hydrolyzed casein, 1% yeast extract! 0.2% cysteine HCl~
0.05% hemin, 0.1% vitamin K3, and an effective amount of a colorimetric
oxidation-reduction indicator and having a pH of 7.8 and an oxidation-reduc-
tion potential of -150 mV or less to produce a culture medium suitable for the
cultivation of anaerobic bacteria.
Thus, the present invention, in one aspect, provides a lactose-rich
aqueous solute phase, useful as a microbiological culture medium, which does
not form a precipitate upon autoclaving; and a microcrystalline cloud precipi-
tate which is useful as a food grade additive to cause clouding, stabilization,
P~
~2g~Z~
emulsification, and thickening of food, pharmaceutical, cosmetic, and
other compositions.
The present invention in another of its aspects is directed
to a process for treating lactose rich deproteinized dairy whey fractions
to form at least one product comprising microcrystalline cloud material
and at least one fraction comprising a lactose rich aqueous solute
phase. Each of these end products may be further processed according
to the present invention to produce useful compositions or to provide
materials which are useful in industrial and commercial processes.
In particular, the solute phase according to the present invention
is useful as a microbiological culture medium for clinical and industrial
uses, including aerobic and anaerobic fermentation processes. The
microcrystalline cloud material formed according to the present invention
can be utilized as an emulsifying and/or gelling agent, which can be
particularly useful for emulsifying or gelling proteins useful as food
additives, pharmaceutical compositions, cosmetics, and the like.
Generally, whole whey is presently commercially processed
by ultrafiltration in order to collect the protein rich retentate.
The lactose rich permeate Erom th(~ ultratiltr.lt~e step has hec~ll further
treated to rc(:over the lactc)sc~ .IIlCI/Ol' la~ti~ ~ri(l, or the pcrmeate
may be dried ancl useù dS a tert-ilizer. Tlle presenr inventioll in otller
aspects is directecl t- treLItment of the la(:t~ ricll pcrmearc ro torm
otllcr usciul products.
-- 8
~,
In the accompanying drawings:
Figures I and 2 are flow diagrams of a presently preferred
process and practical applications according to an aspect of the present
invention.
Figures 3-5 are graphs showing the effect of pH on the zeta
potential of illustrative cloud fractions of the present invention
determined according to the process of Example 15; those areas in
which the zeta potential is at lcast about 5mv (either ~- or -) represent
generalLy satisfac~:ory pH ranges for the formcltion of stable emulsions
()
~Z~2~1~
or suspensions.
- Figure 6 is a scanning electron micrograph (SEM~ of a commercial
preparation of spray dried industrial grade culture medium prepared
according to the process of Example 10;~
Figure 7 is an SEM of the microcrystalline cloud fraction
obtained as a precipitate concurrently with the solute phase from which
the culture medium shown in Figure 6 was produced.
Figure 8 is an SEM of a microcrystalline cloud fraction similarly
obtained from a different source of deproteinized dairy whey lactose
permeate; and
Figure 9 is an SEM of a microcrystalline cloud fraction similarly
obtained from yet another source of whey lactose permeate which contained
a high protein level due to membrane leakage.
Figure 1 shows a general process according to an aspect of
the present invention, wherein whole whey is subjected to ultrafiltration
to produce a lactose rich dairy whey permeate. The solids concentration
of the permeate is adjusted to an appropriate concentration and the
pH is adjusted to 8 - 10. The adjustment of the pH results in the
-- 10 --
lZq~
formation of a cloud which is separated fr~n the supernatant by
centrifugati~n and/or ultrafiltration. The solute fraction may be
optionally spray dried for later use, or used as is for further
processing as a microbiological culture medium. The cloud fraction
may be used as is, concentrated to a paste or dried for use as an
enlulsifier, for emulsifying aqueous or oily liquids or in emulsifying
or gelling proteins. The resultant emulsions or gels may be combined
with other ingredients appropriate for the desired end use.
Referring to Figure 2, the solute fraction may be supplemented
lo with an appropriate nutrient, then sterilized by autoclaving or
filtration. Alternatively, the unsterilized supplemented solute
frdction may be optionally spray dried for storage until further use,
then sterilized prior to use by autoclaving or filtration. The
sterilized solute fraction, either unsupplemented or supple~ented with
additional nutrients, can then be utilized as a liquid or a solid
culture medium. If a solid culture medium is desired, a gelling agent
is added to form a solid culture medium and the medium is contacted
with an appropriate microorganism, the microorganism allowed to grow
and the microorganism and/or the desired biological product is
isolated. if used as a liquid culture medium, the supplemented or
unsupplemented solute fraction may be utilized in either batch or
continuous processes. In a typical batch process the liquid solute
fraction is contacted with the microorganism under suitable growth
conditions, and transferred successively to larger tanks (staging).
The desired microorganism or biological products from the
microorganisnl are isolated. Alternatively, the liquid solute fraction
can be used in a continuous process wherein the microorganism is
contacted with the medium and allowed to grow to a desired cell
density. The nutrient containing medium is continuously flowed into
the culture while simultaneously withdrawing spent nutrient. The
spent nutrient is collected and the desired biological products
re,~oved therefrom.
Suitable deproteinized lactose whey permeates (WLP) which can be
used as starting materials are commercially available or can be
.,
lZ~Z8~6~
prepared by techniques known to those ski~led in the art from either
sweet or sour (acid) dairy whey derived from hard cheeses, e.y. Swiss
or Mozarella" or from soft cheeses, e.g. cottage cheese. Commercially
available starting materials which have been successfully employed
herein include Foremost-McKesson, Inc. lactose permeate prepare~
according to the methods described in U.S. Patent 3,615,664 to Leo H.
Francis (Figures 6, 8, and 9) and Express Food Company's deproteinized
whey syrup solids (Figure 7).
The lactose rich dairy whey permeates suitable as starting
materials according t:o aspects of the present invention are generally
deproteinized, e.g. by ultrafiltration or other membrane separation
techniques. The percentage solids content therein may vary, depending
upon prior processing. The particular type of ultrafiltration
equipment and membrane employed in prep~ring the WLP starting material
does not appear to be critical, since comparable results have been
obtained from WLP preparations filtered with commercially available
Abcor (cellulosic and noncellulosic tubular membranes), DDS (De Danske
Sukkerfabrikker, polysulfone and cellulosic flat sheet membranes),
Dorr-Oliver (polysulfone and cellulosic bonded plate m~mbranes), and
Ladish (polysulfone and cellulosic spiral wound membranes)
ultrafiltration equipment. Since membranes generally have a molecular
weight cutoff of 17 - 20 kdal (kilodaltons) for the primary
permeate, it is important that the membranes employed do not have
pinhole defects which result in protein leaks, as the quality of the
final products is impaired with such- materials. Conventional
operating conditions for such ultrafiltration membranes are p~ O - 14,
temperatures of 38 - 80 C, and pressures of 60 - 145 psi.
The WLP starting material, either in spray dried form or obtained
in a liquid stream at a concentration of 5-40 percent total solids
(wt/vol), is either dissolved in or diluted with water or evaporated
to a solids content of 2-20 percent, preferably ~, 18 percent
solids. Concentrations much below this range may yield a liquid phase
which has an inadequate nutrient content for use as a culture medium
product (although acid WLP appears to have a higher content of
assimilable nitrogen sources than sweet WLP),' while concentrations
much above this range may fail to stay in solution during processing.
Excessively high concentrations above 20 -- 25 percent 501 ids also
impede the removal of the WLP components which precipitate upon
autoclaving.
These components, which are collectively referred to herein as a
microcrystalline cloud fraction, are precipitated from the WLP
solution by raising the pH thereof to precipitate the cloud material.
This is generally accomplished by the addition of sufficient non-toxic
Lewis base, preferably an inorganic base, e.g. an alkali metal
hydroxide anld especially a~monium hydroxide (which is preferably
generated in situ by bubbling ammonia gas through the diluted WLP,
forming the relatively nontoxic ammonium ion) to raise the pH of the
diluted WLP to 8-10, preferably to pH 9. The material
which is used to adjust the pH of the permeate does not appear to be
critical provided that it is not, or does not form, materials which
are t~xic. The products according to aspects of the present invention may be
utilized in food products, pharmaceuticals, cosmetics and as media for
growing microorganisms; therefore the pH adjusting agent for such
2') applications will be limited to those materials which are nontoxic to
animals and microorganisms. Upon adjustment of the pH as described
above, a microcrystalline cloud precipitate is formed. The
temperature at which the precipitation step is carried out is not
particularly critical. Conveniently, temperatures in the range of 20
- 50 C may be utilized.
This increase in the pH of diluted WLP results in precipitation of
the microcrystalline cloud fraction, with optimal yield usually
obtained at ab~ pH 9. The optimum pH for removing all of the cloud
fraction can be determined by autoclaving aliquots of the WLP solution
after raising the pH thereof to selected values within the 8-10 range
and separating the thus produced cloud fraction. If too low or too
high a pH is cmployed, cloudy and/or dark colored solutions are
obtained upon subsequent autoclaving the solute fraction.
- 13 -
~Z81~
The precipitate is physically separated from the culture medium,
e.g. by centrifugation at 11,7509 and filtration across a 0.45~ pore
size membrane or by ultrafiltration across a 20 - 100 kdal molecular
weight exclusion ~embrane, generally 10 - 50 kdal and preferably 20-30
kdal, and saved for use as an emulsifying or suspending agent as
discussed below. Centrifugation alone without subsequent filtration
is generally unsatisfactory, since the clear supernatant frequently
turns cloudy upon subsequent autoclaving, thereby limiting its
applicability as a culture medium. Ultrafiltration across a smaller
pore size membrane, e.g. 10 kdal, is unsatisfactory since the
resultant culture medium results in poor growth compared to one which
has been filtered across a 20 kdal or larger pore size membrane. The
cloud fraction may be dried and utilized as an emulsifying or gelling
agent in various applications as described below.
The solute fraction may be sterilized in an autoclave or subjected
to sterile filtration (preferably in the pH range of 6.8 - 7.1)
and used as a culture medium for growth of microorganisms. The solute
fraction contains useful quantities of assimilable carbon, nitrogen,
phosphorous and other nutrients, including the sugar sources lactose,
sucrose, galactose and glucose. The principal carbon source is the
lactose present in the starting WLP. Of the available sugar in a 3.5
percent solids unsupplemented media after autoclaving at 121C/15 psi,
a typical co~lposition is: 53.0 percent 3-lactose (11.8 mg/ml); 44.8
percent a-lactose + sucrose (9.97 mglml); 1.2 percent galactose (0.27
mg/ml); and 1.0 percent glucose (0.23 mg/ml~.
It has been found that ~LP, although essentially protein free,
generally contains adequate amounts of metabolizable nitrogen in the
form of free amino acids and low molecular weight polypeptides. Thus,
in accordance with aspects of the present invention, there is no need to
hydrolyze separated proteins to increase the assimilable nitrogen
content as described in U.S.S.R. Patent 819,166; in any event, most
proteins have already been removed during the ultrafiltration process
and are not available as a component of the WLP starting material. If
supplementdtion of nitrogen sources is desired, it can be achieved by
- 14 _
lZ~2~1~
the addition of conventional nitrogen -.sources~ including yeast
extracts and peptones of commonly available animal and vegetable
proteins, such as casein and soy. Other sugar sources e.g.,
glucose, and buffering agents, cofactors, and the like, may be added
as necessary to support growth of the microorganism of choice. Thos~
of` ordinary skill in the art of microorganism culture can readily
determine any necessary nutrient supplements, buffering agents (i.e.,
to an optimal p~ range in which the organism is viable), and the like
which are necessary to grow a particular desired microorganism.
The solute fraction of aspects of the present invention is useful both in
industrial scale processes and as a starting material tor tne
prepardtion of various microbiological culture media which are useful
in clinical diagnostic testing methods. For use ~ith microorganisms
which do not normally metabolize lactose, or for use in clinical
screening applications where such organisms may be encountered, the
metabolizable carbon content of the medium can be enhanced by the
addition of glucose7 generally to a total concentration of 3l 0.5
mg/ml .
The solute fraction may be utilized to prepare a solid or liquid
20 clinical grade culture medium, a liquid or solid aerobic culture
medium, a li~uid or solid anaerobic culture medium, a general
industrial fermentation medium, a fermentation medium for the
production of antibiotics, a culture medium for the preparation of
cheese, and the like.
An exemplary useful general purpose aerobic medium in accordance
with an aspect of the present invention comprises an aqueous co~position
supplemented with yeast extract, amino acids and glucose in the
following proportions:
clarified solute fraction at 3.5 percent solids
yeast extract (Amber 510) 0.05 percent
amino acid mix (U.S. Biochemicals) 0.5 percent
glucose (USP grade) 0.05 percent
The above supplemented culture medium has a protein analysis
(Lowry protein content) lower than conventional nutrient broths, as
_ 15 -
shown belo~ in Table 1, therefore it is unexpected that thesupplemented medium according to the present invention would be useful
for supporting the growth of microorganisms. A typical amino acid
analysis is shown in Table 2. The data shown in Tables 1 - 4 are
representative of the general purpose microbiological culture medium
of Example L which has been supplemen$ed with 0.5 percent casamino
acids, Q.05 percent yeast extract, and 0.05 percent glucose.
TABLE 1
LOWRY PROTEIN ANAL SIS
Medium mg/ml Protein
BBL Nutrient Broth 4.8
Difco Penassay ~roth 3.8
WLP Mediwm, supplemented 1.2
Skim Milk 30.0
It may be seen from the following amino acid analysis of the above
described supplemented solute fraction that it contains an adequate
range of amino acids to support microorganism growth. However, should
a particular application require an unusual amount of a particular
amino acid, such as required for growing microorganisms which are
deficient in the genetic mechanisms for producing a given amino acid,
the medium can be supplemented accordingly.
_ 16 -
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TABL 2 - - -
TYPICAL AMINO ACID ANALYSIS OF
SUPPLEMENTED WLP MEDIUM
Amino Acld Approximate ~ moles/ml
Alanine 3.89
Arginine 1.05
Aspartic acid 2.63
Glutamic acid 9.4
Glycine 1.91
]0 Histidine 0.93
Isoleucine . 1.88
Leucine 3.38
Lysine 3.09
Methionine 1.08
Phenylalanine 1.46
Serine . 4.56
Threonine 1.90
Valine 3.45
The supplemented WLP medium exhibits good buffering capacity to
both acid and base addition, as shown in Tables 3 and 4. This is an
unexpected and advantageous property since m~st microorganisms will
survive only within a limited pH range and the present supplemented
solute fraction exhibits buffering capacity comparable to that of
conventional nutrient broths without the addition of buffering agents.
TABLE 3
ACID B_FFERING CAPACITY
pH after successive 0.1 ml a~ditions
of lN HCl to 25 ml broth
Medium 0 1 2 . 3 4 5
Difco Penassay 6.92 6.64 6.345.965.28 4.37
Broth
Wl.P Medium, 6.82 5.59 4.664.143.73 3.32
(supplemented)
BBL Nutrient Broth 6.80 4.383.583.04 2.60 2.31
- 1 7
TABLE 4
B SE BUFFERING CAPACITY
pH after successive 0.1 ml additions
of lN NaOH to 25 ml broth
Medium 0 1 2 3 4 5
_
Difco Penassay 6.93 6.93 6.96 6.99 7.02 7.05
Broth
WLP Medium, 6.80 6.93 7.04 7.17 7.31 7.45
(supplemented)
BBL Nutrient Broth 6.74 6.99 7.19 7.37 7.55 7.71
The supplemented WLP medium can be prepared either in liquid form
or spray-dried, preferably to a moicture content of less than 10
percent by weight, e.g. about 6 percent by weight, for greater storage
stability. When preparing a liquid broth, any desired supplements can
be added prior to autoclaving at 121 C for 15-20 minutes. In this
manner, various types of culture media can be readily prepared from
the basic unsupplemented WLP solute fraction. Presently preferred
media are:
1) a general purpose growth medium of solute phase preferably
supplemented with ~ L 0.25 - 0.5 percent casamino acids, 0.05
percent yeast extract and 0.05 percent glucose, which compares
favorably with widely used general nutrient broths, e.g. Difco
Penassay broth, Oxoid Lablemco broth and Nutrient Broth No. 2, and 8BL
Nutrient broth;
2) a primary isolation medium ~PIM) for the cultivation of both
aerobic and anaerobic microorganisms from primary clinical specimens.
This material is frequently supplemented with 0.25 - 0.5 percent
casamino acids, 0.5 percent yeast extract, 0.4 - 0.5 percent glucose,
0.1 percent agar or other gelling agent to reduce oxygen diffusion and
0.05 percent cysteine HCl as a reducing agent. When boiled before use
to reduce the oxygen content, the resulting clinical grade medium
compdres favorably with widely used thioglycollate broth; and
_ 18 -
l~Z~l~
3) a pre-reduced, sterile, anaerobically prepared medium for the
cultivation of facultative and obligate anaerobic microorganisms,
which is preferably supplemented with 0.25 - O.S percent casamino
acids, 1 percent yeast extract, 0.5 percent glucose, and 0.001 percent
resazurin as an oxidation-reduction indicator. The latter medium is
boiled under a nitrogen atmosphere for approximately 10 minutes and
then supplemented with 0.2 percent cysteine HCl, 0.50 mg/ml hemin, 1
mQ/ml vitamin K3, and adjusted to pH 7.8 with ammonium hydroxide
prior to being stored under a nitrogen atmosphere. To prepare tubes
lo of pre-reduced agar medium, agar is first added to the tubes to give
the final concentration desired, and pre-reduced broth medium added to
the agar in the- tubes. After autoclaving at 121 C/15 psi for 20
minutes, the remaining solid agar was dissolved by inverting the tubes
several times. This medium has an oxidation-reduction potential of
-1SOmV or lower, and the colorimetric redox indicator turns pink upon
oxidation of the medium; and
4) An industrial fermentation medium, in either liquid or solid
form, e.g. containing solute fraction diluted to 3.5 percent solids
and supplemented with 0.25 percent Amber 510 brewer's yeast
extract. For the solid medium, any conventional gelling agent can be
added, e.g. 1.5 percent agar. A typical analysis of such an
industrial fermentation medium is as follows.
. .
19 -
~2l~
Typical Analysis: V tamins (mg/100 gm)
Proteins Kjeldahl B1 0.30
(percent N x 6.32)12.10 B2 16.60
Protein, Lowry 3.5 Niacin 21.70
percent fat <1.0 Trace Minerals: (mg/100 gm)
percent ash <1.0 Aluminum <0.906
Barium 0.121
percent carbohydrate81.5 Boron 0.242
Calcium 26.21
percent moisture 6.5 Chromium <0.121
Copper <0.181
Bulk density, gm/cc 0.63 Iron 0.181
Magnesium 34.97
Solubility in H~0, Manganese 0.060
gms/100 ml 30 C 24.5 Phosphorus 341.56
Sodium 580.14
Sugar Profile (percent): Strontium O.J85
Gal~actosë 0.8 Zinc 0.604
Glucose 0.7
Lactose 81.5 Microbiological~
Sucrose trace
CFU 220/gm
Coliform negative
Particle Size:
Amino Acid Profile:(mg/100 g)
~ 85 percent passes Tyler 270 screen
,20 Arginine 160
Cystine 30 pH after autoclaving:
Glutamic acid 380
Glycine 230 6.5 (3 percent total solids)
Histidine 100
Isoleucine 190
Leucine 270
Lysine 270
Methionine 90
Ph~nylalanine 180
Threonine 150
Tryptophan 40
Tyrosine 170
Valine 180
2 0 -
l~)Z8~
The above described industrial fermentation formulation supports
growth with the following industrially important organisms:
Streptomyces griseus - produces streptomycin (detectable
levels within 24 hrs.) and pronase
Penicillium notatum - produces penicillin (detectable
levels within 24 hrs.
Saccaromyces cerevisiae - produces ethanol
Aspergillus niger - produces citric acid
5) Industrial fermentation media with increased glucose content.
One such medium consists of solute fraction diluted to 2.0 percent solids!
and supplemented with 0.25 percent a yeast extract, known by the Trade Mark
AMBER 510 from Amber Laboratories Inc. and 1.0 percent dextrose. Another
such medium consists of solute fraction that is passed through an im~.obilized
lactose reactor, diluted to 3.0 percent solids, and supplemented with 0 25
percent AMBER 510 yeast extract. The residence time of the solute fraction in
the reactor is used in conjunction with the pH and temperature of the reaction
to control the final dextrose concentration of this medium.
It may be seen therefore that the solute fraction may be utilized,
either in the supplemented or unsupplemented form, to produce antibiotics,
such as; streptomycin and penicillin. Furthcrmorc, the solute fraction accord
ing to aspects of the present invention may be used as a starter culture
growth medium, e.g. in the biological production of hard and soft cheeses.
While relatively unimportant for use in certain industrial fermen-
tation processes, the optical clarity of a broth culture medium is highly
important in clinical applications. For this purpose, it is advisable to
screen samples of supplements intended to be used, as in some instances it has
been found that certain samplcs will not yield the desired clear product. At
high yeast extract concentrations of around 1 percent, water soluble autolyzed
.,
g;~281;~
yeast extract, known by the Trade Mark AMBEREX 510 obtained from Amber Labora-
tories, Inc., and Nestle yeast extracts obtained from the BBL Microbiology
Division of Becton, Dickinson and Co. have proved satisfactory. Amino acid
supple:ments from Difco
- 2l a -
Laboratories, Inc., U.S. Biochemical Cor~., and Marcor Development
Corp. are likewise satisfaotory for use in the present process.
For use as a liquid culture media, the solute fraction obtained by -
the process of as~ects of this invention can be sterilized by conventionalmethods such as sterile filtration or autoclaving. Once autoclaved,
the sterile media should not be re-autoclaved, as this causes a
material reduction in microbial growth potential. If sterile
filtration alone is employed, generally through a 0.22~ filter, it i5
necessary to reduce the pH of the broth to ~ 6.8 - 7.1 by the
addition of a suitable nontoxic acid, e.g. HCl. This can be
accomplished either before or after filtration, but in any event must
be done prior to use. Sterilization of liquid culture media by
autoclaving has been found to inherently reduce the pH thereof from
about pH 9 to the desired range, and for that reason an initial pH
adjustment to pH 9 together with autoclaving is presently preferred.
The reason for this is not fully known, but may be the result of
polypeptides or other organic buffering constituents of the medium
being degraded by the heat of autoclaving.
In addition to its use as a brothj the basic unsupplemented solute
?0 phase of aspects of the present invention can be ~ade up into solid or semisolid
plates or slant tubes by the addition of a gelling agent, e.g. ag~r
agar, Carrageenan, pectin, silicone gel, guar gum, locust bean gum,
various gella~le polysaccharides, etc. according to known techniques.
These gelling agents can be used with or without other additives such
as defibrinated sheep or horse blood, proteins, litmus, etc. to form
culture media suitable for use as blood agar, protease assay agar,
litmus agar, etc. For example, the liquid medium is easily prepared
in the form of pour plates by the addition of 1.5 percent (wt/vol)
agar. In general, the unsupplemented solute phase culture medium of
the present invention can be modified as desired by the addition of a
wide variety of supplements depending on its ultimate intended use,
e.g. see the Media section at pages 601-656 of the American Type
Culture Collection Catalogue of Strains 1, 15th Edition (1982).
_ 22 --
lZ~Z~
Alternatively, the solute fraction can-be spray dried to a powder
in order to increase shelf life and save transportation costs.
Because the solute fraction must be in a concentrated form for spray
drying, the use of WLP starting materials in concentrations greater
than the 3.5 percent generally employed for liquid media is preferred,
and concentrations as high as 20 percent have proven satisfactory. As
the solids content of the WLP starting material approaches 30 percent,
it has been found that some of the solid material may remain in
suspension and not be precipitated by pH adjustment. Spray drying of
media containing supplements e.g. 0.05 percent yeast extract and/or
0.25 - 0.5 percent casamino acids is readily accomplished. Spray
drying of unsupplen~nted solute fraction generally requires drier air
to compensate for the lack of seed particles in the supplements, which
dry rapidly and form a nucleus upon which the rest of the materials
can dry. Use of a portable, general-purpose spray drier e.g. that
manufactured by Niro Atomizer, Inc. is quite satisfactory with a
temperature o~ 200C and an outlet stack temperature of about
80 C. Using such conditions, the mDisture content in the basic
supplemented medium is reduced to 6 percent.
It will be appreciated that the culture media of the present
invention can be employed in the fermentative production of
antibiotics, enzymes, organic acids, alcohols, and ketones and can
also be used as a starter culture growth medium, e.g. in the
biological production of hard and soft cheeses e.g. American,
Swiss, Italian, cheddar, Mozarella, and cottage cheeses. These WLP
media are distinctly different from whole whey-based cheese starter
cultures as illustrated, inter alia, by G.W. Reinbold et al. U.S.
_ _ . _ _ _
Patent 3,998,700; D.L. Andersen et al. U.S. Patent 4,020,185; R.S.
Porubcan et al. U.S. Patent 4,115,199; and W.E. Sandine et al. U.S.
Patent 4,282,255.
The microcrystalline cloud fraction which is precipitated at an
alkaline pH, preferably at pH 9, and separated frorn the culture
n~dium by centrifugation or ultrafiltration across a 20 - 100 kdal
membrane is generally harvested as an aqueous pellet material which
- 23 -
has the consistency of shortening at 4 C and becomes more free-flowing
upon warming to room temperature. When dried, this precipitate is a
tasteless, odorless, chalky ~hite free-flowing powder; typically,
about 15 percent of the input WLP solids which are processed are
recovered as this dried precipitate powder.
This precipitate is different in nature from whey permeate
precipitates reported by other investigators. Unlike the
superficially similar materials reported by Shah et al. in U.S.
Patents 4,143,174 and 4,209,503, the microcrystalline cloud fraction
lo of the present invention is insoluble in petroleum ether, as shown in
Table S. The physical characteristics of the microcrystalline cloud
fr~ction of aspects of this invention are critically dependant upon the form in
which the precipitate is recovered. When recovered as a concentrated
1i4uid, it forms a gel in water and is immiscible in petroleum ether.
~nen further concentrated into a paste form, the microcrystalline
cloud fraction becomes insoluble in both water and petroleum ether.
Once dried, e.g. to b 6 percent moisture, the microcrystalline
cloud fraction is only transiently suspendable in water but is still
insoluble in petroleum ether.
TABLE 5
St)LUBlLlTY OF CLOUD FRACTION
__ _ _ _ _
Solvent Solubilit~y
Concentrated Cloud Fraction Pellet
Ethy-r acetatë insoluble, nondispersing paste
Benzene insoluble, nondispersing paste
Toluene insoluble, nondispersing paste
Chloroform insoluble, nondispersing paste
Petroleum ether insoluble, nondispersing paste
Methanol very cloudy suspension
Ethanol very cloudy suspension
Propanol very cloudy suspension
Butanol slight suspension
lN HCl cloudy suspension
lN NaOH cloudy suspension
- 24 --
i2~2~
Dry Cloud Fraction~
Water, 5 percent solids slight suspension
Water, 10 percent solids slight suspension
Water, 20 percent solids slight suspension
Petroleum ether, 5 percent solids insoluble particles
Petroleum ether, 10 percent solids insoluble particles
Petroleum ether, 20 percent solids insoluble particles
When chemically analyzed by ICP analysis, the microcrystalline
cloup fraction of this invention is also demonstrably different in
nature from both unprocessed spray dried WLP and the precipitate that
forms when spray-dried WLP is resuspended to 20 percent concentration
(wt/vol) and cooled at 4 C- for 72 hours~ as shown in Table 6. The
data shown are from the same starting material sample, which had a
maximum water solubility at room temperature of about 20 percent, a
normal pH at that concentration of 5.5 to 6.0, and contained less than
1 mg/100 9. of carbohydrates and essentially no protein or fat. Data
were obtained by ICP analysis according to Industrially Coupled
Plasma-Atomic Emission Spectroscopy Method 3.005 of the American
Organization of Analytical Chemists and compared to a sample prepared
according to the process of Pederson, U.S. Patent 4,202,909 which
involves heating to only 140 to 150 F. All samples were prepared by
resuspending 20 percent (wt/vol) spray-dried WLP in water prior to
individual processing.
-25 -
12~2~
TABL~ 6 ~ -- s
ICP ANALYSIS OF CLOUD FRACTION
mg/rOOg So~ids (Dry Bas ~
Cold Pederson
Alkaline pHPrecipitate Precipitate
Spray Dried WLP Precipitate 4 C, 72 hrs. 78 C.
Calcium 348.-438 5,392. 15,800. 7,934.
Iron` 0.11-.12 6.65 6.59 4.77
Phosphorous 488.-491 782.8 11,448. 4,075.
Magnesium 150. 5,733. 2,370. 548.2
Zinc 0.06 .08 0.79 3.42 1.39
Copper 0.025 0.75 0.79 0.37
Sodium 774.-863 461.6 718. 600.9
Chromium 0.036-.0370.48 l.57 0.50
Aluminum 1.1-1.3 52.96 15.66 6.50
Barium 0.025-.0280.82 0.57 D.57
Strontium 0.11-.22 1.54 6.6~ 3.74
Boron 0.06-.07 3.70 0.631 0.38
Manganese 0.005-.Oll0.21 0.21 0.18
20 By determining the zeta potential as a function of pH for the
microcrystalline cloud fractions prepared from various sources of
starting materials in accordance with the present invention, suitable
pH ranges can be determined in which stable emulsions or colloids can
be formed. Since the zero-point-of-charge corresponds to the pH in
which the materials in suspension are least stable (not unlike the
isoelectric point or pI for proteins), pH values which give a zeta
potential of at least 5mv are generally preferred, with the
gredter deviation from the zero-point-of-charge generally resulting in
the gredtest stability. However, in the acid range, such high acidity
- 26 -
~28i~
may result in degradation of polypeptide components present in the microcry-
stalline cloud fraction.
Taking into account these unique solubility properties, an air dried
microcrystalline cloud fraction of aspects of the present invention can be
employed in a wide variety of industrial applications, e.g. as a food grade
emulsifier or suspending agent for pharmaceutical, cosmetic, and food materials
using techniques known in the art.
Without further elaboration, it is believed that one skilled in the
art can, using the preceding description, utilize the present invention to its
fullest extent. I'he following preferred specific embodiments are, therefore,
to be construed as merely illustrative and not limitative of the rr~ ; nd~r of
the disclosure in any way whatsoever. In the following Examples, the tempera-
tures are set forth uncorrected in degrees Celsius; unless otherwise indicated,
all parts and percentages are by weight.
EXAMPLE 1
Preparation of Solute Fraction and Cloud Fraction
7 g of WL2 (obtained from Express Foods Co. and similar to products
commercially available from Foremost McKesson, Inc. and other sources) was
made up to 200 m].. with deionized water (3.5 perccnt solids content, wt/vol).
The mi~ture was c;tirred for a few minutes to mix well, as some solids tend to
fall out of solution if the mixture is not stirred. The pH was increased from
an initial pH of 6.09 to 8.99 by the addition of 2.15 ml of 5.5 N NH40H while
stirring, and centrifuged for 10 minutes at 8500 rpm (11,800g) in a centrifuge
known by the Trade Mark SORVATr RC-5B using a GSA rotor refrigerated at 4~C.
1.26 g of a solft, white microcrystalline cloud fraction pellet were obtained
per 10() ml of starting solution. The supernatant was poured through a 0.45 ~,
- 27 -
Z~
115 ml filtration unit known by the Trade Mark N~LGENE, yielding 200 ml of
clear material having a pH of 9.04. After autoclaving at 121C/15 psi for
20 minutes, a dull organge and crystal clear unsupplemented culture medium
was obtained, having a final pH of 7.07.
- 27 a -
As a control, the above process was repeated using whole whey as
the starting material. The initial pH was 6.26, and 2.4 ml of NH40H
were added to bring the pH up to 8.98. Following centrifugation, 1.08
g of a hard, tan pellet were obtained per 1nO ml of starting
material The supernate was not clear, but had fluffy material
floating throughout it. Only t 25 ml of the supernate could be
passed through the filter unit until it clogged and the filter had to
be changed. Following filtratic)n, the supernate was still cloudy and
had a pH of 8.98. After autoclaving, a dull orange, cloudy liquid was
obtained having a pH of 7.08.
EXAMPLE 2
Preparation of Supplemented Culture Medium
__ _ _ _~_
From Acid (Sour) Dairy Whéy Solute Fraction
Following the procedure of Example 1, a microbiological culture
medium was prepared from acid whey having an initial pH of 4.45 which
was obtained from cottage cheese production at the Giant Food, Inc.
dairy plant at Lanham, Maryland. The whole acid whey was
ultrafiltered thru a 30 kdal Dorr-Oliver filter unit, yielding a
primary retentate and a primary permeate. The primary permeate was
adjusted to pH 9 with NH40H and the ultrafiltration process was
repeated, yielding a secondary microcrystalline cloud fraction and a
secondary permeate. The secondary permeate was supplemented with 0.25
percent casamino acids, 0.05 percent yeast extract, and 0.05 percent
glucose prior to autoclaving for 20 minutes at 121 C/15 psi. The
resulting autoclavecl culture medium was clear and golden in color,
with a pH of 8.15.
EXAMPLE 3
PreciQitation with Other Bases
The procedure of Example 1 was followed, except KOH was used to
adjust the pH. From an initial pH of 6.09, 0.2 ml of 6N KOH and 0.2
ml of lN KOH were added to bring the pH to 8.92. 1.66 9 of cloud
fraction were obtained as a soft, white pellet per 100 ml of starting
material. Following filtration, the supernate was clear and had a pH
- 28 -
Z~l~
of 8.78. A~ter autoclaving, the liquid was golden colored and veryslightly cloudy, with a final pH of 6.25.
When NaOH was substituted for the N~40H in the procedure of
Example 1, the initial pH of 6.08 was raised to pH 8.90 by the
addition of 0.4~ ml of 3N NaOH. Centrifugation yielded 1.62 9 per 100
ml of starting material of microcrystalline cloud fraction as a soft
white pellet. After ultrafiltration across a 0.45~ membrane, the
supernate was clear and had a pH of 8.75. After autoclaving, a golden
colored~ slightly cloudy liquid was obtained having a final pH of
lo 6.25.
EXAMPLE 4
_omparative Growth Characteristics
The autoclaved clear culture media from Examples 1 and 3 were
evaluated for their ability to support the growth of common laboratory
culture strains, Bacillus subtilis 6051a, Enterobacter aerogenes
E13048, and _scherichia coli HS. Tubes of culture media, both
unsupplemented and supplemented with 1 percent BBL yeast extract, 0.5
percent Difco casamino acids, and 0.5 percent sucrose (Sigma Ohemical
Co.), were inoculated and incubated at 35 C for 5 hrs, after which
optical density readings were made at 660 nm. Difco Penassay broth
and BBL Nutrient broth were used as controls. The results in this and
the following experiments were scored according to the following
scale, which roughly correlates to half-log differences in measured
optical density: -
++++ Excellent growth; O.D. 0.3-1.0
~+~ Good growth; O.D. 0.1-0.3
++ Moderate growth; O.D. 0.03-0.1
+ Some growth; O.D. 0.005-0.03
No growth; O.D. 0-0.005.
The results dr e shown in Table 7.
- 29 -
Z~
TABLE 7 ~ ~
Preliminary Growth Screening
Culture Medium B. subtilis E. aer~enes E. coli
_ . . _
Difco Penassay broth +~++ ++++ ++++
BBL Nutrient broth ++++ ++++ ++++
3.5 percent WLP* 'NaOH) ++ +++ +++
3.5 percent WLP* NaOH) + supp ++++ ++++ ++++
3.5 percent WLP* KOH) ++ +++ +++
3.5 percent WLP* KOH) + supp ++++ ++++ ++++
lo 3.5 percent WLP* NH40H) +++ +++ +++
3.5 percent WLP* NH40H)+supp ++++ ++++ ++++
* as WLP sol ds
EXAMPLE 5
Evaluation of pH Importance
_ __ _
In order to evaluate the importance of the pH employed for
precipitation of the microcrystalline cloud fraction, a series of
culture media supplemented as in Example 2 were prepared in which the
initial pH was adjusted to between 4 and 11 using HCl or NH40H as
required. With the exception of the initial pH, the media were
.20 prepared as in Example 1 and the supplement added prior to
autoclaving, at which time all of the samples appeared similar and
filtered easily. The differences in the products obtained following
autoclaving are shown in Table 8.
- 30 -
1~6)Z81;~
TABLE 8
Process pH Appearance After Autoclaving pH After Autoclavin~ -
4 with HCl clear, light green 4.5
5 with HCl clear, light green 5.5
6 no addition slightly opaque 6.0
7 with NH40H very cloudy, light yellow 6.1
8 with NH40H very cloudy, golden 6.4
9 with NH40H clear, root beer color . 7.1
10 with NH40H very dark brown 8.8
11 with NH40H like liquid chocolate 9.7
EXAMPLE 6
Representative Growth Cur~es
Following the procedure of Example 4, whey permeate culture medium
produced according to the procedure of Example 1 and supplemented with
0.25 percent casamino acids and 0.05 percent yeast extract, both with
and without 0.05 percent glucose, was compared with Difco Penassay
broth and BBL nutrient broth for its ability to support the growth of
a representative variety oF clinically important microorganisms. The
results are presented in Table 9 and show that the solute phase
culture media of aspects of this invention compare favorably with two current
widely accepted industry standards.
- 31 -
lZ~Z~
TABLE 9
REPRESENTATIYE GROWTHS IN LIQUID MEDIA
Whey Permeate Whey Permeate DIFCO BBL
Media (suppl.) Media (suppl.) Penassay Nutrient
Microorgan m w/out glucose w/glucose Broth Broth
Bacillus
subtilis 6051a ++++ not done ++++ ++++
Escherichia
coli HS l++++ not done ++++ t+++
Enterobacter
_erogenes E13048 ++++ not done ++++ ++++
Streptococcus
faecalis E19433 ++++ ++++, ++++ ++
Staphylococcus
aureus 6538P ++++ ++++ ++++ ++++
Proteus
mirabilis 259:33 ++++ +++~ ++++ ++++
- Klebsiella
pneumoniae 23357 ++++ . ++++ ++++ ++++
Pseudomonas
fluorescens 15453 ++++ ++++ ++++ ++++
Salmonella
typhimurium LT2 ++++ ++++ ++++ ++++
Shigella
sonnei ++++ ++++ ++++ ++++
___
Salmonella
typhimurium 211a +++ ++++ ++++ +++
_ _
- 32 -
l~g)ZI~
EXAMPLE 7 --~
Effect of Autocla g on Growth
ln order to evaluate the importance of achieving a neutral pH in
the final product through the autoclaving process, a filter sterilized
glucose supplemented medium control was prepared otherwise
corresponding to the culture medium used in Example 6 except that the
final pH was adjusted to pH 7 by the addition of HCl rather than as a
resùlt of the autoclaving treatment. The results are shown in Table
10.
_ 33 -
t
lZOZ~
TABLE 10
REPRESENTATIVE GROWTHS IN LIQUID MEDIA
Autoclaved Filter Sterilized DIFCO BBL
Whey Permeate Whey Permeate Penassay Nutrient
Microorganism Media (supp) Media (supp) Broth Broth
Bacillus
subtilis 6051a ++++ ++++ ++++ ++++
__ _ _
Escherichia
col~ HS ++++ ++++ +++~ +~++
lo Enterobaster
aerogenes E13048 ++++ -~+++ ++++ ++++
Streptococcus
_ caTls E19433 ++++ ++++ ++++ +++
Staphylococcus
aureus 6-~38P ++~ +++ +++ +++
Proteus
mirab ~rTs 25933 ++++ +++~ ++++ ++++
Klebsiella
pneumoniae 23357 ++++ ++++ . ++++ ++++
Pseudo~o-as
fr ore~c~ns 15453 ++++ ++++ ++++ ++++
typhinlr um LT2 ++++ ++++ ++++ ++++
Shigella
sonnei ++++ ++++ ++++ +++
Salmonella
typhimurium 21a + +++ ++++
.__
It can be seen from the last entry on the above table that there
are apparently some nutrients required for the growth of Salmonella
typhimurium which are changed by the autoclaving treatment and become
,_ _ _ _ __
not as readily metaboli~able as in the sterile filtered medium.
Nonetheless, both the autoclaved and sterile filtered media were
superior to the nutrient broth control.
- 34 _
;~2~2~
EXAMPLE 8
- Preparation of Anaerobic Culture Medium
Following the procedure of L.V. Holdeman et al. (Ed.) in Anaerobe
Laboratory Manual, qth Edition (1977), a pre-reduced anaerobic cultur¢
medium was prepared by weighing out the dry ingredients 0.5 percent
casamino acids, 1 percent yeast extract, and 0~5 percent dextrose
immediately before use, adding water and resazurin, and heating under
a nitrogen atmosphere. The solution was gently boiled until the
resazurin turned from blue to pink to colorless in 5-10 minutes.
After cooling in an ice bath under a nitrogen atmosphere, the cysteine
was added. This was done after partial reduction of the medium by
boiling in order to prevent oxidation of the cysteine, since oxidized
cysteine can be toxic for some fastidious anaerobes. The pH was
adjusted to 7.8 with NH40H as measured by test paper while bubbling
nitrogen through the liquid, which was then dispensed into tubes which
had been flushed with nitrogen. To prepare tubes of pre-reduced agar
medium, agar was first added to the tubes to give the final
concentration desired, and pre-reduced broth medium added to the agar
in the tubes. After autoclaving at 121 C/15 psi for 20 minutes, the
2~ remaining solid agar was dissolved by inverting the tubes several
times.
EXAMPLE 9
Representative Growths in Anaerobic Culture Medium
Liquid anaerobic culture media were tested against three strains
of Bacteroides for its ability to support anaerobe growth. Test
samples containing 0.05 percent yeast extract plus 0.05 percent
glucose (Medillm 1), and 1.0 percent yeast extract plus 0.5 percent
glucose (Medium 2, from Example 8) were inoculated with the anaerobic
microorganisms. Difco brain heart infusion broth (BHI) was used as
one control medium; a medium containing 1 percent tryptone, 2 percent
yeast extract, and 2 percent gluccse (TYG) served as a second
- 35 -
~2~2~
control. Optical density rPadings were made during the first eight
hours of incubation. The results are summarized in the following
Table:
TA,BLE I 1
ANAEROBIC GROWTH SCREENING
Mirrnnrg;~ni RHI krnth T`~l~ Rrnth ~IPrl jl 1 MPrlil ?
r t P r i nrlP c
r. i fn-- i c v.~?? t~ t +tt t tt~
RAr tPr i nrlPc
frAgi 1 ic
ATP.I' ?~i?R!~i tt+ I t+~ ~t~ t~t~
RAr tPr i nrlPc
~rArJil ic 47Si-l tt~ t~ tt~t
EX~MPLE I ~
PrPp~r2tinn nf R~cir RllltllrP MPr~ fnr
Irrl~lCtr; A1 FPr- -nt~t i nnc
~ .5 percent WLP (wt./vol; commercially availa~hle frc~
Foremost-McKesson, Inc. or Express Foods Co.) was adjusted to pH 9 with
NH40H ancl ultrafiltered through a 30 ~dal Dorr-Oliver filter unit.
The permeate was supplemented with 0.25 percent Amber BYF10d Yeast
extract prior to autoclauing for 2a minutes at 121C/15 psi. The
resulting autoclaved culture medium was only slightly cloudY, golden in
color, and had a pH of 6.71. If a clear medium is desired, a yeast
extract that is readily soluble, such as Ambere~ 510 Yeast extract maY
be substituted for ~mber BYF100.
EX~1PLE I I
Tnl' ctr i Al FPr---ntAt i nn PrnrPcc
Ft~rilll~c rPrPllc cllhc. thllrinrJipncic~ UAr. PPrl inPr o~tained from
Mr. Howard T. Dulmage of the U.S. Department of ~griculture Ootton
Research Institute, Brown-cville~ Texas was selected to e~emplifY the
-36-
``jl,
capability of the culture medium of the presenr invention to support
an industt-ial fermentation process using the methodology described by
Dulm3ge et al. in 3. Invert. Pathol. 22: 273 - 277 (1973). This -
organism produces a 6-endotoxin and is used as a biological
insecticide in the control of lepidopteran pests, e.g. as the worrn
~iller available under the trademark DIPEL 4L from Abbott
Labordtories, Chicago, Ill. The development of parasporal crystals
and spores of Bacil_us_ thurin~iensis was monitored under phase
contrast microscopy following the procedure of L. A. Bulla et al.
described in Applied Microbiology 18 (4): 490 - 495 (1969).
- Heat shoc~ing at 70 C was used to compare the degree of
sporulation in the rnodified culture medium of Example 10 containing
0.25 percent Amber BYF100 compared with the GYS medium described by
Bulla et al. and the B4, B4b, and B8b media described by Dulmage et
al. Heat resistance was used as a measure of completed spore
formation .
After 24 hr, sporulation approached its maXimum level with the
culture medium of aspects of this invention, whereas sporulation with the GYS
medium did not approach maximum levels until 48 hr; in addition, the
maximum sporulation level obtained was 100 fold higher than with GYS.
Similar e~periments comparing the cultllre medium of tnis invention
\~ith B4, B4b, and B~b media showed sporulation after 24 hollrs from 10
to 100 fold higher with the former; in addition, the maximum
sporulation level obtained was from 5 to 10 fold higher.
EXAMPLE 12
Industrial Fermentation Medium
The basic culture n~dium of Example 1 was supplernented with 0.25
percent Am'.~el- BFY 1~ yeast extract befnre autnclaving. Aliquots of
tl)e resllltincl medium were inoculated with several organisms of
industrial interest. Colony morphologies and dry cell weight yields
w~re recorded and dre shnwn in Tahle 12. This experiment demonstrates
that t:he culture medium of aspects of this invention can be used in industrial
fern~ntation processcs.
TABLE 12
COLoNY GR~wTH CHARACTERISTICS
tr~i~ r.n1nny Mnrrhnlnr~y ~ry r~ll t-i~ht yiPl~*
AcrPrgillllc nigPr 5i ngle, large 0.76g/10~ ml
hyphal mat
PPnirillil nnt~tll di sperse, bead- Q.47g~100 ml
li~e growth
~tnPrt~ yrP~ 3riCPII~ well dispersed ~.21g/1~ ml
r~rrh~r~ yrlPc rPrPu~ P well dispersed 0.287c~'l00 ml
* 5 days after 1/10 vol. inoculation and incubation at 80C
with shaKing.
EXAMPLE 13
Antihintirc Prnrlllrtinn
The same ~asic culture medium, unsupplemented, was used to
demonstrate the production of anti~iotic~ ~y two commonly used
industrial microorganisms. The results, which are reported in Table
13, demonstrate that, while not yet optimi~ed~ drug production did
occur in useful quantities.
T~BLE 13
ANTIBIOTICS PRODUCTI~N
~$~ain Antihintir Anitihintir llnitc/rl*
PPnirillil~ nnt~tll penicillin ~.00~4 Units/ml
~trPrtr rr~-; gric~ streptomycin 0.00735 Units/ml
* One day after 1/10 volume inoculation and incubation at
25~-30C with agitation
-38-
EXAMPLE 14 '~ '
Preparation of a Nutrient Supplemented
Medium from Solute Fraction
Solute fraction prepared as in Example 1 was supplemented with 0.5
percent casamino acids, 0.05 percent yeast extract, and 0.05 percent
glucose. Tubes of broth were inoculated with various microorganisms
aild growth was observed either by plate count as reported in Table 14
or visual7y as reported in Table 15.
TABLE 14
lo PLATE COUNT OBSERVATIONS OF GROWTH
Colony Counts per ml. after 24 hrs at 37 C
Supplemented Solute Control Media
nrganism Tested(BHI, PABA, AGAR)
r.. meninqitidis 80 250
''.~''~i'nfTu'enzae 60 0
. ovitis 1250 1800
- 39 -
-
T.qBLE I 5
~I SUAL OBSERWITI QNS OF ~ROWTH
~rr~th ithin ~4 hrc At ~0~ ~rr th ~ ithin 4~ hrc At ~RO~
Alr~l igPnPc ~aPrAl ic Ar inPtnhartP~ rAlrnarPt irnc.
~r i 1 1 "c rPrPIlc l':nrynPh~rtPr i 11 Cp .
P. r r~atPr i I Mi rrnrnrrllc cr,
P. c~lhtilic Mirrnrnrrllc lycnrlPiktirllc
P. thyrinrJiPnciS PlAnnrrArrllc cr.
~itrnhArtpr ~rPnnr~ arrinA c~p,
FntPrnh~rtpr AprnrJpnpc ~ArrinA llrPap
FcrhPr i rh i a rnli
~i rrnrnrrllC I lltPllC
PrntPIlc ull 1 ~r~Ar i C
PcPll~ --AC. AprnrJinncA Fun~i grew
P Plnn~Ata after ?-3 days
~hn~ncpirillll rllhrll
.r;alr--Plla typhir~lrill Ac~pPrgillllc nir~Pr
~PrrAt jA r~rrPcrpns ~nr-Atn~yrpc Ctr---itis
~tAphylrlrnrrllc AnrPlls PPnirillill ep,
~itrPptnrnrrllc faPrAl ic
~trPp trrnrrllc 1 Ar t i s
EX~MPLE 15
~lle~pn~inrJ an~ r-llcifyinrJ Prr~Prtipc rJ~ Cl~ Frartinnc
The stability of colloids comprising three microcrystalline cloud
fraction samples was measured by determining the zeta potential. Each
sample was diluted in deionized water to a 8.1~ percent sUcpencion~
and the electrophoretic mo~ility was determined using a 2eta-Meter
(Zeta-Meter, New ~orK, NY). With this instrument, a suspension of the
sample is decanted into an electrophoretic cell and a potential applied
across a pair of electrodes inserted into the cell. The average time
for a particle to move horizontally between two lines of a grid is
observed thru a microscope and recorded. This time is then translated
into the zeta potential using standard conversion charts.
The first sample suspension, air dried Express Foods
microcrystalline cloud fraction, was only moderatelY stable, with the
sol id dispersing slowlY over a period of 15 minutes and some larger
partlcles settling quicKly to the bottom of the container when
~4~ ~
,
~tirring was stopped. The second sample suspension, E%pre~s Foods
microcrystalline cloud fraction wet pellet, was extremely stable, while
the third sample, FGA-I microcrystalline cloud fraction~ also appeared
extren.ely stable but was stirred for ~4hr prior to measurement o~ its
~eta potential.
The effect of pH on the 2eta potential was determined for each of
the three materials. The zeta potential for each sample was negative
in the neutral pH range, and became more negative with increasing
~asicity, and positive with increasing acidity. There was 50me
evider,ce for dissolution in the acid pH range. The
zero-point-of-charge, i.e. the pH at which the ~eta potential of the
sur4ace of the particle reached zero, was as follows: ~ample I - 4.2;
Sample 2 = 2.4; Sample 3 = 4.5. The results of plotting pH vs. ~eta
potential are shown in Figures 3 - 5.
EX~MPLE 16
P~rtirlP ~i7P r~ictrihlltinn
Four samples were examined and photographed by scanning electron
microscopy (SEM) to determine particle si_e. The scanning electron
micrographs are shown in Figure 6 through 9; the distance between
solid white squares on the lower border of each photograph is 10~um.
Figure 6 represents the basic culture medium for industrial
fermentations prepared as descri~ed in Example 19. Figure 7
represents microcrystalline cloud fraction from whey lactose permeate
~Express Foods Co.~ generated as described in Example 1, separated
from the culture medium bY ultrafiltration, and subsequently
spray-dried. Figure 8 represents microcrystalline cloud fraction
produced from whey lactose permeate (Foremost-McKesson, Inc.)
generated bY Mozarella cheese manufacture. The microcrystalline cloud
fraction was generated as described in Example 1, separated from the
culture medium by ultrafiltration, and subsequentlY spray-dried.
Figure 9 represents microcrystalline cloud fraction produced from whey
lactose permeate (Foremost-McKesson, Inc.~ generated by Swiss cheese
-41-
manufacture. The microcrystalline cloud fraction was again generated
as described in Example 1, separated from the culture medium hy
ultrafiltration and subsequentlY spray-dried.
~MPLE 1 7
~nlllhil ity in L'~tPr ;.nri PPtrnlPI ~thPr
The solubility characteri~tics of the microcrystalline cloud
fractions of Example 16 (Figures 7 - 9) in water, petroleum ether, IN
HCI, and IN NaOH were examined. ~.5 g, I.~ g, and 2.~ g of cloud
material were added to IB m1 aliquots of each so1~ent. The solutions
were shaXen ~igorously and allowed to stand. The re5ulting solubilitY
profiles appear in Table 16.
-42-
1~(3Z~
TABIE 16
SOLUBILITY CHARACTERISTICS IN ~ATER9
PETRGLEUM ETHER AND ACIDIALKALI
Spray Spray Spray
Co~npound Dried E.F. Dried FGA-1Dried FGA-2
Wd ter, 5 Transient Cloudy, Cloudy,
percent so~idssuspension, partial partial
insoluble suspensionsuspension
Water, 10 Transient Cloudy, Cloudy,
10 percent solidssuspension, partial partial
insoluble suspensionsuspension
Water, 20 Transient Cloudy, Cloudy,
percent solidssuspension, partial partial
insoluble suspensionsuspension
Petroleum Insoluble Insolub.leInsoluble
ether, 5 (film) (film) (film)
percent solids
Petroleum Insoluble Insoluble Insoluble
ether, 10 (film) (film) (film)
20 percent solids
Petroleum Insoluble Insoluble Insoluble
ether, 20 (film) (film) (film)
percent solids
lN HC1, 5 Transient Cloudy,Cloudy, almost
per cent solids suspension, partial complete,
insoluble suspensionsuspension
IN HCl, 20 Cloudy, partial Cloudy, partial Cloudy, partial
percent solidssusp. suspensionsuspension
(significant (floating
amount ofmaterial)
stable foam)
_ 43 -
TABLE 16 (CONTINUED)
SOLUBILITY CHARACTERISTICS IN WATER,
PETROLEUM ETHER ANC ACIDlALKhLI
Spray Spray Spray
Compound Dried E.F. Dried FGA-1 Dried FGA-2
_ _ _ _
lN NaOH, 5 Partially Partially
Partially
percent solids soluble, soluble,
soluble,
supernate supernate supernate
clean yellow clear orange clear yellow
(floating (floating
material) ~ material)
lN NaOH, 20 Partially Stable, dark
Partially
percent solids soluble, orange foam
soluble,
supernate clear orange
clear orange supernate
(significant
amount of
floating
material)
lZ6)Z81~
EXAMPLE 1B-~
Solubility in Or~anic Liquids
_ __ _ _ _
The solubilities of the microcrystalline cloud fraction materials
of Example 10 (Figures 7 - 9) were characterized further in a variety
of organic solvents. In general, 0.5 9 of cloud material was added to
5ml of each solvent. The solutions were shaken vigorously and allowed
to stand. In the case of glycerol, 59 were added to 5~m1 and the
solution WdS stirred mechanically. The resulting solubility profiles
ap?ear in Table 17.
TABLE 17
SOLUBILITY CHARACTERISTICS IN ~RGANIC LIQUIDS
Solubility of Cloud Fractions at 10 percent wt/vol.
Dielectric Spray Spray Spray
Constant Dried Dried Dried
SolYent a 25 C E.F. FGA-l FGA-2 FGA-2 Wet
Glycerol 42.5 2 2 2
Methanol 32.6 7 7 7 7
Ethanol 24.3 4 7 7 7
Acetone 20.7 7 3 3 2
-20 2-Propanol 20.1 7 2 7 8
n-Butanol 17.1 7 7 7 7
Ethyl
acetate 6.0 5 5 5
Chloroform 4.8 (20 C) 5 5 5 9
Ethyl ether 4.3 5 5 5 9
Toluene 2.4 7 6 5 9
Benzene 2.3 7 6 6 9
Hexanes,
practical 1.9 (20C) 7 7 7 9
1 = Cloudy, total suspension
2 = Cloudy, partial suspension
3 = Cloudy, partial suspension with floating material
4 = Cloudy, slight suspension
5 = Partial, particulate suspension
6 , Parlial, partic~late suspension with floating material
7 - Transient suspension, insoluble
8 _ Trailsient partial suspension, insoluble
9 = Insolilble
- 45 -
1~0;~
EX~1PLE I 5'
r- llci~ tinn n~ ~P~t~hlP nil
A solution of microcrystalline cloud fractions from Example IS
~Figures 7 and 8) was prepared c,y sha~ing 2~ parts of the moist
precipitate in I~ parts of water. Thirty parts of 5 percent vinegar
were added tc this solution ànd the resultant mixture was stirred,
thicKening noticeablY. Fifty parts of sucrose were then added with
stirring, causing further thic~enir,~. Thereafter, Ia0 parts of liquid
vegetaDle oil ~peanut oil) were added and the mixture was homogeni2ed
in a Waring Dlender at high speed for ~ minutes The resultant
emulsion layer was sta~le for at least 4 hours and had the visCositY of
a mayonndise mixture.
~ 5 a control, the a~ove process was repeated without the adclition
of microcrystalline cloud fraction. This process showed no thic~ening
of the mixture following the addition of vinegar and sucrose. The oil
and water 1ayers formed no emulsion, and separation into two distinct
layers was complete only two minutes after attempted homogenization.
EX~MPLE 20
r-ll5ifi~atinn nf nransp Plllp ~'~ch
Following the procedure of the preceding example, I~ ml of orange
pulp wash ~the H~O soluDle fraction of citrus pulp and ruptured juice
~esicles) Wc15 added to 1~ ml of distilled water containing ~9 of
microcrystalline cloud fraction of Example I6. Immediately after
mixing, all of the material was in a single emulsion layer and remained
so for at least tWD hours. ~pproximatelY 18 hours later, a small
portion of liquid had formed a lower~ clearing laYer under the
emulsion. With the microcrystalline cloud fraction sample of Figure 8,
the emulsion layer had 501 idified.
~ s a control, the abo~e process was repeated without the addition
of mitrocrYstalline cloud fraction. The lower, clearing layer began to
form after less than ~ minutes, and the remaining emulsion layer was
not thic~ as in the samples containing the cloud fraction.
--46--
~'
~oz~
EXf~MPLE 21
F-- 1 ~i f i ~ A t i rln nf HP YA n~c.
This example illustrates the ability of the microcrystalline cloud
fractior, of this inYentiOn to emulsify non-polar hydrocarbons.
Foll3wing the procedure of the preceding Examples, I0 ml of technical
hexanes wa~ added to 10 ml of distilled water containing 2 g of
microcrystalline c~oud fraction from the two different sources.
In,mediately after mixing, an upper foam layer extended to the top of
the test tube, and the foam was still at this height after 37 minutes.
~pproximately I8.5 hrs. later, the foams in both tu~es had ~ecome
gelatinous.
~ s a control, the a~ove process was repeated without the addition
of microcrystalline cloud fraction. The technical hexanes and water
separated completely into two different, clear phases immediately after
vortex mixing was ended.
EXAMPLE 22
r- l~i f i r ~ t i nn r f ~r ~ nil
Using South ~a~ota intermediate grade crude oil containing I.6
percent sulfur, 10 ml of the oil sample were added to 10 ml distilled
water containing 2 g of both of the microcrystalline cloud fraction of
the preceding examples. Samples containing microcrYstalline cloud
fraction formed two sta~le phases. The upper phase ~ecame gelatinous,
with the Figure 8 microcrystalline cloud fraction sample gelling at
about ~0 minutes, and the Figure 7 sample gelling less dramatically
after a~out 18 hours. The Figure 8 sample exhi~ited a relatively poor
capacity to coat a plastic tube compared to the Figure 7 and control
samples.
As a control, the above process was repeated without the addition
of microcrystalline cloud fraction. The oil and water formed a single
liquid phase and formed no gel upon standing.
-47-
EX~MPLE 2~
F- lci~irAtin~ nf ~PntnnitP
This example illustrates the use of the microcrystalline cloud
fraction to emulsify particulate inorganic solids. Following the
procedures of the preceding examples, ~.29 bentonite was added to 20 ml
distilled water containing 2g of dry microcrYstalline cloud fraction
o~tained acrording to Example 1~. With the Figure 8 microcrystalline
cloud fraction, an upper foam and a lower, frothy layer were formed.
The frothy layer was stable for at lea~t 18 hours.
~ s a control, the a~o~e process was repeated without the
addition of microcrystalline cloud fraction. Im~ediately after mixing,
a single, frothy layer was obtained which, within approximatelY 0.5 hr,
also showed the presence of a lGwer, clear layer.
EX~MPLE 24
r- lci~irAtinn nf PrntPin ~y ~lnll~ Fr~,tin~c
This example illustrates the capacity of microcrystalline
cloud fraction to emulsify and gel protein. 100 ml aqueous solutions
were prepared using microcrystalline cloud fraction generated from WLP
commercially available from Express Foocds Co. and Sa~orpro 75 whey
protein concentrate which is also commerciallY a~aila~le from Express
Foods Co. Sanlples were whipped in a Waring t~lender at high ~peed for
minutes. Foam height and the viscosity of the resulting emulsions were
docunented, demonstrating that the addition of either 10 or 20 percent
microcrystalline cloud fraction increased both the foam height and
~iscosity of 10 percent whey protein concentrate solutions. The
results are shown in Table 18.
-48-
T~BLE I8
_MULSIFIC~TIoN OF P~OTEIN B~ CLOUD FRACTION
Foam height ~iscositY (Seconds for
I00 ml aqueous solution when 1~0ml liq. 5nl1 to drop from pipet;
whipped at high speed settled out in relative to a value
for 3 minutes 200ml ~ea~er of 3 for H20
I0 percent WLP I.8 cm ~ sec.
I0 percent WlP,
I0 percent cloud
fraction 2.5 cm 5 sec.
10 percent WLP,
20 percent cloud
fraction 3.5 cm 5.8 sec.
EXQMPLE 25
I~P 11 i n~ n~ PrntP i n l~y rl nllrl Fra~ t i nn
In addition to emulsifying protein, microcrystalline cloud fraction
also gels protein at concentrations lrJwer than that at which gelling
would normally occur. Using the same materials descri~ed abo~e, 10 ml
aqueous solutions were prepared, ~ortexed, and incu~ated at 80C for 20
minutes. With the addition of 20 percent microcrystalline cloud
fraction, I0 percent whey protein concentrate solidifies at ~0C.
Without the addition of microcrystalline cloud fraction no
solidification of the 10 percent protein solution occurs, as shown in
Ta~le I9:
-49-
TABL~
BELLlN~i OF PROTEIN SOLUTI l:lNS
~rlU~nllc !~nllltinn R~Or: ~nr ~ rinlltPc
10 percent cloud fraction slight suspension w~large pellet sediment
~0 percent cloud fraction 51 ight suspension w~large pellet sediment
10 percent WLP cloudy suspension, thic~ coating
20 percent WLP solid pellet
I0 percent cloud fraction ~
10 percent WLP mil~y suspension w~pellet
I0 percent cloud fraction ~
20 percent WLP solid pellet
20 percent cloud fraction
I0 percent WLP I:I solid pellet and thick coatinc~
20 percent cloud fraction ~
20 percent WLP solid pellet
XAMPLE 26
Preparation of Industrial Fermentation
M~rli A ~ 'i th InrrPacPrl 1~1 llrncP l;nntPnt
Permeate i5 prepared as in Example 1~, with the exception that 20
percent ~wt.~vol.) whey lactose permeate is used as the starting
material. The resulting permeate is spray-dried and used to prepare
basic culture media for industrial 4ermentations that are increased in
glucose content relative to that of Example 10. One such culture
medium is produced by preparing a 2.0 percent solids solution of
spray-dried permeate and supplementing with ~.25 percent ~mber 510
yeast extract and I 0 percent dextro6e prior to autoclauing for 20
minutes at I2IG/IS p5i. The resulting autocla~ed culture medium is
clear, golden in color, and has a pH of 6.5. Another such culture
medium i5 produced ~y first preparing a IS percent 501 ids solution of
spray-dried permeate, the solution ha~ing a pH of 6.5.
This solution is passed through an immobili2ed en2Yme reactor
of the type described ~y A. G. Hausser et al. in RintPrhnnlngy ~n~
Rinphycirc ~U pages 525-53~ (I983) at a rate of 6 ml/min at a
temperature of 378, resulting in the conversion of 47 percent of the
_5~ _
1' ~;
l~X~
permeate lactose to glucose and galactose by immobilized acid lactase
enzyme. The en~ymatic conversion was carried out without pH
adjustment of the permeate and was, therefore, at a pH that was
non-optimal for the acid lactase enzyme. This use of a non-optimal pH
resulted in a 47 percent con~ersion, which was desirable for this
example since it resulted in an approximately I percent glucose
cor,centration when the permeate solids were adjusted to ~ percent. The
resulting medium was then comparable to the 1 percent
glucose-supplemented medium. Adjusted to a solids le~el of B~a
percent, this lactase treated permeate contains 1.24 percent glucose.
3.0 percent lactase-treated permeate, supplemented with ~.25 percent
~mber 5l0 )~east extract and autoclaved for 20 minutes at l2lC~I5 psi,
gives a clear, golden culture medium with a final pH of 6.5.
The glucose supplemented and lactase-treated basil culture
media of this example were tested against se~eral microorganisms for
their growth support characteristics relati~e to the basic industrial
culture medium descri~ed in Example l0. The results appear in Table 20
below.
TABLE 20
REPRESENTATI~E GRoWTH IN INDUSTRlAL FERMENTATIoN
MEDI~ WITH INCREASEl? GLUCOSE CONTENT
MediumMicroorganism
B.allrPIlc ~.~aPralic ~.cllhtilic F.rnli P.~lllnrPcrPng
Basic culture medium
for industri al fer-
mentation. tt +ttt tttt tttt tttt
Gl ucose supplemented
basic culture medium
for industrial fer-
mentation. tt tttt ttt~ tttt tttt
Lactose-treated basit
culture medium for
industrial fermen-
tation. ttt t~tt tttt tttt ttt~
-51-
l~oz~
Ir,dustrial culture media with a wide range of glucose to lactose
ratios can ~e prepared by varying the extent of either the clextrose
supplementation, or the lactose hyclrolysis described abo~e.
Spæcifically, if a high level of lactose hydrolysis i~ desired a
neutral lactose en2yme is immobili~ed using a neutral buffer system and
no further pH adjustment is made before permeate j5 passed through the
reactor. Further, media with di~fering glucose to lactose ratios can
also be prepared by dry blending the appropriate amounts of permeate
solids with dextrose, or permeate solids with lactose-treated permeate
solids.
EX~MPLE 27
rhPP~P r;tartPr l:--l t~lrP MP~
Following the procedure of Example 2 but adjusting the primary
permeate to pH 8.5-~.0 and supplementing the secondarY permeate with
~.25 percent ~mber 51~ or ~mber 10a3 yeast extract gives an essentially
neutral clear golden culture medium which is suitable for growing
commercial cheese starter cultures available from Chris Hansen
Laboratories, Milwau~ee, Wisconsin and for growing cultures of
~trPptnrnrrll~ rrr-nri~ (ATCC 19257), ~trPrtnrnrr"c lartic (ATCC 1~435~,
and ~trPrtnrnrrl,~ ~iarPtylarti~ (ATCC 15346).
Culture growth on these media, as measured by viable plate count,
equals growth produced bY currently available cheese starter culture
media when temperatures and agitation are controlled identically and no
external pH control is used. If, however, pH is controlled by the
addition of a base to maintain the culture broth in the range of pH 6.~
to 6.5~ the cell density reaches 5 to l~ times that obtained in the
currently available commercial media. Furthermore, these growth levels
can be obtained reproducably in 8 hrs with appropriate inoculum as
opposed to 16-2~ hrs typically required in commercial media such as
Nordica, In-Sure and Phase 4, which include internal phosphate
buffering.
_c,~_
`~
81;~
The preceding examples can be repeated with simila.r success by
substituting the generically or specificallY descri~ed reactants and/or
operating conditions of this invention for those specificallY used in
the examples. F~om the foregoing description, one skilled in the art
to which thi~ in~/ention pertains can easily ascertain the essential
characteristics thereof and, without departing from the spirit and
~cope of the present invention, can ma~e various changes and
modifications to adapt it to various usages and conditions.
n ~ t r i ~ 1 ~ p l i r I h i 1 i t y
~ 5 can be seen from the present specification and examples, the
present invention is industrially useful in providing a plurality of
commercially useful products from lactose rich dairy whey permeate
which has heretofore normally been considered a waste material. ~ne
principal product comprises microbiological culture media which are
capable of supporting good growth of a wide variety of microorganisms;
a second product comprises a food grade emulsifying or stabilizing
agent which is capa~le of emulsifying or stabilizing a wide variety of
products~
-5~-
"~.
