CA1202812A - Conversion of clarified dairy whey lactose permeates to culture media and other commercially useful products - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A process is provided for preparing a dry powder microbiological culture medium which can be reconstituted with water and autoclaved to form a clear, pH neutral culture medium capable of supporting the growth of micro-organisms under suitable growth conditions. The process comprises (a) rais-ing 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 min-utes at 121 degrees C and 15 psi precipitate as microcrystalline solids;
(b) separating the supernatant from the resulting precipitate to form a microcrystalline solid phase consisting essentially of the previously dis-solved solids; (c) removing components having 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 lac-tose-rich supernatant to form the dry powder microbiological culture medium.
This dry powder microbiological culture medium is useful itself and as a basic formulation for preparing a wide variety of microbiological culture media. The precipitate formed is useful as a food grade additive to cause clouding, stabilization, emulsification, and thickening of food, pharmaceuti-cal, cosmetic, and other compositions.

Description

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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

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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.

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.

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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.

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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.

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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~

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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.

~, 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 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.

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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.

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.

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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.

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 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.

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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 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 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.

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 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.

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 ~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.

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 ~, 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.

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.

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

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.

_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.

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 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.

1~6)Z81;~

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 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.

lZ~Z~

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 +++ ++++ ++++ +++
_ _ 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~

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~

- 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.

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 ~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 ``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.

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.

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.

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 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.

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 -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 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.

1~(3Z~

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.

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 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.

~' ~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.

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.

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.

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.

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:

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 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.

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~

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~-"~.

Claims (26)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a dry powder microbiological culture medium 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, said 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 auto-claved for 10-20 minutes at 121 degrees C and 15 psi precipitate as microcry-stalline solids;
b) separating the supernatant from the resulting precipitate to form a microcrystalline solid phase consisting essentially of said previously dissolved solids;
c) removing components having a molecular weight above 100 kdal from the supernatant to form a lactose-rich supernatant which is essentially free of said dissolved solids; and d) drying the resultant lactose-rich supernatant to form said dry powder microbiological culture medium.

2. A process according to claim 1, wherein the pH is raised to pH 9.

3. The process of claim I wherein step c) comprises the step of ultrafiltration across a filter which retains components having d molecular weight above 100 kdal.

4. The process of claim 3 wherein step c) comprises the step of ultrafiltration across a filter which retains components having a molecular weight above 20 kdal.

5. A process according to claim 1, further comprising lowering the pH of the superated solute phase to 6.8-7.1%.

6. A process according to claim 5, wherein the pH is lowered by the addition of a nontoxic Lewis acid to the solute phase.

7. A process according to claim 6, wherein the pH is lowered by autoclaving the solute phase to form a sterile microbiological culture medium.

8. A process according to claim 6, wherein the pH is lowered with-out the addition of extraneous acid to the solute phase.

9. A process according to claim 1, further comprising spray drying the separated solute phase to a moisture content of less than 10 percent by weight.

10. A dry powder microbiological culture medium which can be recon-stituted with water and autoclaved to form a clear pH neutral culture medium capable of supporting the growth of microorganisms under suitable growth conditions, said culture medium being substantially free of components which could be retained by a filter having a pore size which passes components hav-ing a molecular weight below 100 kdal and which is essentially free of dissolved solids which would become insoluble when a dairy lactose permeate is autoclaved for 10-20 minutes at 121°C and 15 psi, whenever prepared by the process of claim 1 or by its obvious chemical equivalents.

11. The dry powdered microbiological culture medium according to claim 10, which is substantially free of components which would be retained by a filter having a pore size which passes components having a molecular weight below 20 kdal.

12. The dry powder microbiological culture medium according to claim 10 having a moisture content of less than 10 percent by weight.

13. A dry powder microbiological culture medium according to claim 10 further containing an added source of nontoxic assimilable carbon atoms.

14. A dry powder microbiological culture medium according to claim 11 wherein said source is glucose.

15. A dry powder microbiological culture medium according to claim 10 further containing an added source of nontoxic assimilable nitrogen atoms.

16. A dry powder microbiological culture medium according to claim 15 wherein said source is a yeast extract, hydrolyzed casein, or mixtures thereof.

17. A dry powder microbiological culture medium according to claim 10 further containing an added gelling agent in an amount capable of forming a gel when said dry powder is admixed with water.

18. A dry powder microbiological culture medium according to claim 17 further containing hydrolyzed casein, yeast extract, cysteine HC1, and ?
glucose added in amounts effective to produce a microbiological culture medium suitable for the cultivation of both aerobic and anaerobic bacteria.

19. A dry powder microbiological culture medium according to claim 10 further containing hydrolyzed casein, yeast extract, and glucose added in amounts effective to produce a general purpose microbiological growth medium.

20. A dry powder microbiological culture medium according to claim 10 further containing hydrolyzed casein, yeast extract, glucose, and a colori-metric oxidation-reduction indicator added in amounts effective to produce a culture medium suitable for the cultivation of anaerobic bacteria.

21. A sterile microbiological culture medium consisting essen-tially of an aqueous solution of the dry powder culture medium according to claim 10.

22. A sterile microbiological culture medium according to claim 21 which has been sterilized by autoclaving.

23. A sterile microbiological culture medium according to claim 21 having a solids content of 3.5% (wt/vol).

24. A sterile microbiological culture medium according to claim 21 having a total glucose content of 0.5% and further containing additives of 0.5% hydrolyzed casein and 0.05% yeast extract to produce a general purpose microbiological growth medium.

25. A sterile microbiological culture medium according to claim 21 having a total glucose content of 0.4% and further containing additives of a montoxic 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.

26. A sterile microbiological culture medium according to claim 21 having a total glucose content of 0.5% and further containing additives of 0.5% hydrolyzed casein, 1% yeast extract, 0.2% cysteine HC1, 0.05% hemin, 0.1% vitamin K3, and an effective amount of a colorimetric oxidation-reduc-tion indicator and having a pH of 7.8 and an oxidation-reduction potential of -150 mV or less to produce a culture medium suitable for the cultivation of anaerobic bacteria.