CONVERSION OF CLARIFIED DAIRY WHEY LACTOSE PERMEATES TO CULTURE MEDIA AND OTHER COMMERCIALLY USEFUL PRODUCTS

DESCRIPTION OF THE INVENTION

Cross-Reference to Related Application This application is a Continuation-in-Part of copending, commonly assigned U.S. Patent Applications Ser. No. 06/418,067 filed September 14, 1982 and Ser. No. 06/471,570 filed March 2, 1983, the contents of which are incorporated by reference herein.

Technical Field of the Invention 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 particularly, 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 useful for a wide variety of applications in the food, pharmaceutical, cosmetics, and other industries.

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Background Art

As noted by Alan S. 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 4,320,150 to Paul R. Austin et al.), most has been regarded as waste and disposed of by traditional methods. While recent developments in ultrafiltration (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 (about 45 g/1) 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. Kluyveromyces fragil s, thereby attempting to overcome the limited 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 Lane. While offering the potential for removing up to 90 percent of the lactose present in the whey lactose permeate, such methods suffer the disadavatage 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 fer entor 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 App ied Biochemistry and Biotechnology 7_: 43-45 (1982). Although more than 50 percent of the lactose was converted to ethanol, the eluate contained less than 2 percent ethanol yield based on the weight/unit volume of whey permeate feedstock.

The use of whole whey as a bacteriological culture medium has been reported by Emel Celikkol in Mikrobiyol. Bui. 9_(4): 273-279 (1975) and in U.S.S.R. Patent 819,166. As summarized in Che . 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 gained widespread use for either industrial or clinical grades 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 ^β 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 4,036,999 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 treated 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 particular combination of temperature and pH employed in accordance 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.

Disclosure of the Invention

It is a general object 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 the present invention to provide a method for converting deproteinized lactose rich dairy whey fractions into at least one fraction containing a icrocrystalline 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 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.

A further object of the present invention is to provide improvements in processes for culturing microorganisms employing these media.

A second principal object of the present invention is to provide a microcrystalline cloud material useful as an emulsifying, suspending, and/or gelling agent.

Yet another object of the present invention is to provide improved methods for emulsifying and suspending a wide variety of compositions employing these agents.

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A more particular object of the present invention is to provide improved food grade additives for use in foods, pharmaceutical carriers, comsetic bases, dentifrice bases, and the like.

'Upon study of the specification and appended claims, further objects, features and advantages of the present invention will become more fully apparent to those skilled in the art to which this invention pertains.

Brief Description of the Drawings

Figures 1 and 2 are flow diagrams of a presently preferred process and practical applications according to 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 least about 5mv (either + or -) represent generally satisfactory pH ranges for the formation of stable emulsions 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 of the present invention;

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.

Best Mode for Carrying Out the Invention Briefly, the above and other objects, features and advantages of the present invention are attained in one aspect thereof by providing a method for separating dissolved solids from deproteinized dairy whey

lactose permeate materials which would otherwise form a precipitate upon autoclaving the permeate to form a) a lactose-rich aqueous solute phase, useful as a microbiological culture medium, which does not form a tnrecipitate upon autoclaving; and b) a microcrystalline cloud precipitate which is useful as a food grade additive to cause clouding, stabil zation, emulsification, and thickening of food, pharmaceutical, cosmetic, and other compositions.

The present invention is directed to a method 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 from the ultrafiltration step has been further treated to recover the lactose and/or lactic acid, or the permeate may be dried and used as a fertilizer. The present invention is directed to treatment of the lactose rich permeate to form other useful products.

Figure 1 shows a general process according to 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 about 8 - 10. The adjustment of the pH results in the

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formation of a cloud which is separated from the supernatant by centrifugation 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 emulsifier, 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 with an appropriate nutrient, then sterilized by autoclaving or filtration. Alternatively, the unsterilized supplemented solute fraction may be optionally spray dried for storage until further use, then sterilized prior to use by autoclaving or filtration. The sterilized solute fraction, either unsupple ented or supplemented 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 microorganism 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 removed therefrom.

Suitable deproteinized lactose whey permeates (WLP) which can be used as starting materials are commercially available or can be

prepared by techniques known to those skilled in the art from either sweet or sour (acid) dairy whey derived from hard cheeses, e.g. 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 prepared 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 to 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 preparing 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), DOS (De Danske Sukkerfabrikker, polysulfone and cellulosic flat sheet membranes), Dorr-Oliver (polysulfone and cellulosic bonded plate membranes), and Ladish (polysulfone and cellulosic spiral wound membranes) ultrafiltration equipment. Since membranes generally have a molecular weight cutoff of about 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 pH 0 - 14, temperatures of about 38 - 80 ^β C, and pressures of about 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 about 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 solids 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 and especially ammonium 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 about 8-10, preferably to about 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 toxic. The products according to the present invention may be utilized in food products, pharmaceuticals, cosmetics and as media for growing microorganisms; therefore the pH adjusting agent for such 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 about 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 employed, 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 centrifugat on at ll,750g and filtration across a 0.45μ pore size membrane or by ultrafiltration across a 20 - 100 kdal molecular weight exclusion membrane, 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 about 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 121°C/15 psi , a typical composition is: 53.0 percent s-lactose (11.8 mg/ l); 44.8 percent o-lactose + sucrose (9.97 mg/ml); 1.2 percent galactose (0.27 mg/ml); and 1.0 percent glucose (0.23 mg/ml).

It has been found that WLP, 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 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 supplementation of nitrogen sources is desired, it can be achieved by

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 such as glucose, and buffering agents, cofactors, and the like, may be added as necessary to support growth of the microorganism of choice. Those of ordinary skill in the art of microorganism culture can readily determine any necessary nutrient supplements, buffering agents (i.e., to an optimal pH range in which the organism is viable), and the like which are necessary to grow a particular desired microorganism.

The solute fraction of the present invention is useful both in industrial scale processes and as a starting material for the preparation of various microbiological culture media which are useful in clinical diagnostic testing methods. For use with 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 glucose, generally to a total concentration of about 0.5 mg/ml .

The solute fraction may be utilized to prepare a solid or liquid clinical grade culture medium, a liquid or solid aerobic culture medium, a liquid 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 the present invention comprises an aqueous composition 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

shown below in Table 1, therefore it is unexpected that the supplemented 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 1 which has been supplemented with 0.5 percent casamino acids, 0.05 percent yeast extract, and 0.05 percent glucose.

TABLE 1

LOWRΪ ' PROTEIN ANALYSIS

Medium mg/ml Protein

BBL Nutrient Broth • 4.8 Difco Penassay Broth 3.8 WLP Medium, supplementteedd 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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TABLE 2

TYPICAL AMINO ACID ANALYSIS OF

SUPPLEMENTED WLP MEDIUM

Amino Acid Approximate u moles/ml

Alanine 3.89

Arginine 1.05

Aspartic acid 2.63

Gluta ic acid 9.45

Glycine 1.91

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 most 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 BUFFERING CAPACITY pH after successive 0.1 ml additions of IN HC1 to 25 ml broth

Med i urn 0

Difco Penassay 6.92 6.64 6.34 5.96 5.28 4.37 Broth

WLP Medium, 6.82 5.59 4.66 4.14 3.73 3.32 (supplemented) BBL Nutrient Broth 6.80 4.38 3.58 3.04 2.60 2.31

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TABLE 4 BASE BUFFERING CAPACITY pH after successive 0.1 ml additions of IN NaOH to 25 ml broth

Medium 2 I 1 2 1 I

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 moisture 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 about 0.25 - 0.5 percent casa ino 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 BBL 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 compares favorably with widely used thioglycollate broth; and

3) a pre-reduced, sterile, anaerobically prepared medium for the cultivation of facultative and obligate anaerobic microorganisms, which is preferably supplemented with 0.25 - 0.5 percent casamino actds, 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 he in, 1 mg/ml vitamin K ₃ , and adjusted to pH 7.8 with ammonium hydroxide prior to being stored under a nitrogen atmosphere. To prepare tubes 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 -150mV 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 about 0.25 percent Amber 510 brewer's yeast extract. For the solid medium, any conventional gelling agent can be added, e.g. about 1.5 percent agar. A typical analysis of such an industrial fermentation medium is as follows:

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Typical Analysis: Vitamins (mg/100 gm)

Protein, Kjeldahl Bl 0.30

( _* percent N x 6.32) 12.10 B ₂ 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 carbohydrate 81.5 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 H7O, Manganese 0.060 gms/100 ml 30 C 24.5 Phosphorus 341.56

Sodium 580.14

Sugar Profile (percent) ; Strontium 0.785

Galactose 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

Arginine 160

Cystine 30 pH after autoclaving:

Glutamic acid 380

Glycine 230 6.5 (3 percent total solids)

Histidine 100

Isoleucine 190

Leucine 270

L sine 270

Methionine 90

Phenylalanine 180

Threonine 150

Tryptophan 40

Tyrosine 170

Valine 180

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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

PeniciIlium notatu - 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 Amber 510 yeast extract and 1.0 percent dextrose. Another such medium consists of solute fraction that is passed through an immobilized 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. Furthermore, the solute fraction according to the present invention may be used as a starter culture growth medium, such as in the biological production of hard and soft cheeses.

While relatively unimportant for use in certain industrial fermentation 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 samples will not yield the desired clear product. At high yeast extract concentrations of around 1 percent, Amberex 510 water soluble autolyzed yeast extract obtained from Amber Laboratories, Inc., and Nestle yeast extracts obtained from the BBL Microbiology Division of Becton, Dickinson and Co. have proved satisfactory. Amino acid supplements from Difco

Laboratories, Inc., U.S. Biochemical Corp., and Marcor Development Corp. are likewise satisfactory for use in the present process.

For use as a liquid culture media, the solute fraction obtained by the* process of this invention can be sterilized by conventional methods 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 0.22μ filter, it is necessary to reduce the pH of the broth to about 6.8 - 7.1 by the addition of a suitable nontoxic acid such as 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 broth, the basic unsupplemented solute phase of the present invention can be made up into solid or semisolid plates or slant tubes by the addition of a gelling agent such as agar agar, Carrageenan, pectin, silicone gel, guar gum, locust bean gum, various gellable 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 I, 15th Edition (1982).

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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 dry-ing, 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 such as 0.05 percent yeast extract and/or 0.25 - 0.5 percent casamino acids is readily accomplished. Spray drying of unsupplemented 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 such as that manufactured by Niro Atomizer, Inc. is quite satisfactory with a temperature of about 200°C and an outlet stack temperature of about 80°C. Using such conditions, the moisture content in the basic supplemented medium is reduced to about 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 such as 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 about pH 9, and separated from the culture medium by centrifugation or ultrafiltration across a 20 - 100 kdal membrane is generally harvested as an aqueous pellet material which

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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 white 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 of the present invention is insoluble in petroleum ether, as shown in Table 5. The physical characteristics of the microcrystalline cloud fraction of this invention are critically dependant upon the form in which the precipitate is recovered. When recovered as a concentrated liquid, it forms a gel in water and is immiscible in petroleum ether. When further concentrated into a paste form, the microcrystalline cloud fraction becomes insoluble in both water and petroleum ether. Once dried, e.g. to about 6 percent moisture, the microcrystalline cloud fraction is only transiently suspendable in water but is still insoluble in petroleum ether.

TABLE 5 SOLUBILITY OF CLOUD FRACTION

Solvent Solubility

Concentrated Cloud Fraction Pellet:

Ethyl acetate insoluble, nondispersing paste

Benzene insoluble, nondispersing paste

To!uene insoluble, nondispersing paste

Chlorofor insoluble, nondispersing paste

Petroleum ether insoluble, nondispersing paste

Methanol very cloudy suspension

Ethanol very cloudy suspension

Propanol very cloudy suspension

Butanol slight suspension

IN HCl cloudy suspension

IN NaOH cloudy suspension

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 cloud 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 g. of carbohydrates and essentially no protein or fat. Data were obtained by ICP analysis according to Industrially Coupled PTas a-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.

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TABLE 6 ICP ANALYSIS OF CLOUD FRACTION ^" mg/lOOg Solfds (Dry Basis) ~

Cold Pederson

Alkaline pH Precipitate Preci \

Spray Dried WLP Precipitate 4°C, 72 hrs, 4 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-.037 0.48 1.57 0.50

Aluminum 1.1-1.3 52.96 15.66 6.50

Barium 0.025-.028 0.82 0.57 0.57

Strontium 0.11-.22 1.54 6.60 3.74

Boron 0.06-.07 3.70 0.631 0.38

Manganese 0.005-.011 0.21 0.21 0.18

By determining the zeta potential as a function of pH for the microcrystall ne 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 pl for proteins), pH values which give a zeta potential of at least about 5mv are generally preferred, with the greater deviation from the zero-point-of-charge generally resulting in the greatest stability. However, in the acid range, such high acidity

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may result in degradation of polypeptide components present in the microcrystalline cloud fraction.

Taking into account these unique solubility properties, an air drred microcrystalline cloud fraction 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. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. In the following Examples, the temperatures 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 WLP (obtained from Express Foods Co. and similar to products commercially available from Foremost McKesson, Inc. and other sources) was made up to 200 ml. with deionized water (3.5 percent solids content, wt/vol). The mixture was stirred 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 NH ₄ 0H while stirring, and centrifuged for 10 minutes at 8500 rpm (ll,800g) in a Sorvall RC-5B centrifuge using a GSA rotor refrigerated at 4°C. 1.26 g of a soft, white microcrystalline cloud fraction pellet were obtained per 100 ml of starting solution. The supernatant was poured through a 0.45μ, 115 ml Nalgene filtration unit, yielding 200 ml of clear material having a pH of 9.04. After autoclaving at 121°C/15 psi for 20 minutes, a dull orange and crystal clear unsupplemented culture medium was obtained, having a final pH of 7.07.

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 NH.OH were added to bring the pH up to 8.98. Following centrifugation, 1.08 g "of a hard, tan pellet were obtained per 100 ml of starting material. The supernate was not clear, but had fluffy material floating throughout it. Only about 25 ml of the supernate could be passed through the filter unit until it clogged and the filter had to be changed. Following filtration, 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 Whey 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 Lanha , 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 NH-OH 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 autoclaved culture medium was clear and golden in color, with a pH of 8.15.

EXAMPLE 3 Precipitation 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 IN KOH were added to bring the pH to 8.92. 1.66 g 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

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of 8.78. After autoclaving, the liquid was golden colored and very slightly cloudy, with a final pH of 6.25.

When NaOH was substituted for the NH-OH in the procedure of Example 1, the initial pH of 6.08 was raised to pH 8.90 by the addition of 0.45 ml of 3N NaOH. Centrifugation yielded 1.62 g per 100 ml of starting material of microcrystalline cloud fraction as a soft white pellet. After ultrafiltration across a 0.45μ 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 6.25.

EXAMPLE 4 Comparative 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 Escherichia 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 Chemical 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 are shown in Table 7.

TABLE 7 Pre iminary Growth Screening

Culture Medium B. subtilis E. aerogenes 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 ++++ ++++ ++++

3.5 percent WLP* (NH 0H) +++ +++ +++

3.5 percent WLP* (NH4θH)+supp -H-++ ++++ ++++

* as WLP solids

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 NH.OH as required. With the exception of the initial pH, the media were 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.

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TABLE 8

Process pH Appearance After Autoclaving pH After Autoclaving

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 NH ₄ 0H very cloudy, light yellow 6.1

8 with NH ₄ 0H very cloudy, golden 6.4

9 with NH ₄ 0H clear, root beer color 7.1

10 with NH ₄ 0H er dark brown 8.8

11 with NH ₄ 0H like liquid chocolate 9.7

EXAMPLE 6 Representative Growth Curves 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 this invention compare favorably with two current widely accepted industry standards.

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TABLE 9 REPRESENTATIVE GROWTHS IN LIQUID MEDIA

Whey Permeate Whey Permeate DIFCO BBL Media (suppl.) Media (suppl.) Penassay Nutrient

Microorganism w/out glucose w/glucose Broth Broth

Bacillus subtilis 6051a ++++ not done ++++

Escheri ^' chia coli HS ++++ not done ++++

Enterobacter aerogenes E 3048 ++++ not done ++++

Streptococcus faecalis El$433 ++++ ++++ ++++

Staphylococcus aureus 6538P ++++ ++++ ++++

Proteus mirabilis 25933 ++++ ++++ ++++

Klebsiella pneumomae 23357 ++++ ++++ ++++ ++++

Pseudomonas fluorescens 15453 ++++ ++++ ++++ ++++

Salmonella typhimuπum LT2 ++++ ++++ ++++

Shigella sonnei -H-++ ++++ ++++

Salmonella typhimuriu 21a ++++ +++

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EXAMPLE 7 Effect of Autoclaving on Growth In 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 result of the autoclaving treatment. The results are shown in Table 10.

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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 coli HS ++++ ++++ ++++ ++++

Enterobacter aerogenes El3048 ++++ ++++ ++++ ++++

Streptococcus faecalis El9433 ++++ ++++

Staphylococcus aureus 6538P +++ +++ +++ +++

Proteus mirabil s 25933 ++++ ++++ ++++

Klebsiella pneumoniae 23357 ++++ ++++ ++++

Pseudomonas fluorescens 15453 ++++ ++++ ++++ ++++ typhimurium LT2 ++++ ++++ ++++ ++++

Shigella sonnei ++++ -H-++ ++++ +++

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 metabolizable as in the sterile filtered medium. Nonetheless, both the autoclaved and sterile filtered media were superior to the nutrient broth control.

EXAMPLE 8 Preparation of Anaerobic Culture Medium - Following the procedure of L.V. Holdeman et al. (Ed.) in Anaerobe Laboratory Manual, 4th Edition (1977), a pre-reduced anaerobic culture 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 NH ₄ 0H 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 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 (Medium 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 glucose (TYG) served as a second

control. Optical density readings were made during the first eight hours of incubation. The results are summarized in the following Table:

TABLE 11 ANAEROBIC GROWTH SCREENING

Microorganism BHI Broth TYG Broth Medium 1 Medium 2

Bacteriodes uniformis v622 ++++ ++++ ++++

Bacteriodes fragilis ATCC 25285 ++++ ++++ ++++

Bacteriodes fragilis 479-1 ++++ +++ ++++ ++++

EXAMPLE 10 Preparation of Basic Culture Medium for Industrial Fermentations 3.5 percent WLP (wt./vol; commercially available from Foremost-McKesson, Inc. or Express Foods Co.) was adjusted to pH 9 with NH ₄ 0H and ultrafiltered through a 30 kdal Dorr-Oliver filter unit. The permeate was supplemented with 0.25 percent Amber BYF100 yeast extract prior to autoclaving for 20 minutes at 121°C/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 Amberex 510 yeast extract may be substituted for Amber BYF100.

EXAMPLE 11 Industrial Fermentation Process Bacillus cereus subs, thuringiensis, var. Berliner obtained from Dr. Howard T. DuImage of the U.S. Department of Agriculture Cotton Research Institute, Brownsville, Texas was selected to exemplify the

capability of the culture medium of the present invention to support an industrial fermentation process using the methodology described by Dulmage et al. in J. Invert. Pathol. 22_: 273 - 277 (1973). This organism produces a δ-endotoxin and is used as a biological insecticide in the control of lepidopteran pests, e.g. as the worm killer available under the trademark DIPEL 4L from Abbott Laboratories, Chicago, 111. The development of parasporal crystals and spores of Bacillus thuringiensis 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 shocking at 70°C was used to compare the degree of sporulation in the modified 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 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 experiments comparing the culture medium of this invention with B4, B4b, and B8b media showed sporulation after 24 hours 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 medium of Example 1 was supplemented with 0.25 percent Amber BFY 100 yeast extract before autoclaving. Aliquots of the resulting medium were inoculated with several organisms of industrial interest. Colony morphologies and dry cell weight yields were recorded and are shown in Table 12. This experiment demonstrates that the culture medium of this invention can be used in industrial fermentation processes.

TABLE 12 COLONY GROWTH CHARACTERISTICS

Strain Colony Morphology Dry cell weight yield* Aspergillus niger single, large 0.76g/100 ml hyphal mat

PeniciIlium notatum disperse, bead¬ 0.47g/100 ml like growth

Streptomyces griseus well dispersed 0.21g/100 ml Saccharomyces cerevisiae well dispersed 0.287g/100 ml

* 5 days after 1/10 vol. inoculation and incubation at 30°C with shaking.

EXAMPLE 13 Antibiotics Production The same basic culture medium, unsupplemented, was used to demonstrate the production of antibiotics by two commonly used industrial microorganisms. The results, which are reported in Table 13, demonstrate that, while not yet optimized, drug production did occur in useful quantities.

TABLE 13 ANTIBIOTICS PRODUCTION

Straiπ Antibiotic Anitibiotic units/ml* PeniciIlium notatum penicillin 0.0064 Units/ml Streptomyces griseus streptomycin 0.00735 Units/ml

* One day after 1/10 volume inoculation and incubation at 25-30°C 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 and growth was observed either by plate count as reported in Table 14 or visually as reported in Table 15.

TABLE 14 PLATE COUNT OBSERVATIONS OF GROWTH

Colony Counts per ml. after 24 hrs at 37°C

Supplemented Solute Control Media

Organism Tested (BHI, PABA, AGAR)

N. eningitidis 80 250

H. influenzae 60 0

B. ovitis 1250 1800

TABLE 15 VISUAL OBSERVATIONS OF GROWTH

Grow-th within 24 hrs at 30°C Growth within 48 hrs at 30 C

Alcaligenes faecalis Acinetobacter calcoaceticus Bacillus cereus Corynebacterium spT B. megaterium Micrococcus sp. B. subtilis Micrococcus lysodeikticus B. thyringiensis Planococcus sp. Citrobacter freundii Sarcina sp. Enterobacter aerogenes Sarcina ureae Escherichia cofT Micrococcus luteus Proteus vu garis Pseudo onas aeruginosa Fungi grew P. elongata after 2-3 days Rhodospirillurn rubrum Salmonella typhimurium Aspergillus niger Serratia marcescens Doratomyces stemonitis Staphylococcus aureus PeniciIlium sp. Streptococcus faecalis Streptococcus factis

EXAMPLE 15 Suspending and Emulsifying Properties of Cloud Fractions

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 0.100 percent suspension, and the electrophoretic mobility was determined using a Zeta-Meter (Zeta-Meter, New York, 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 solid dispersing slowly over a period of 15 minutes and some larger particles settling quickly to the bottom of the container when

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stirring was stopped. The second sample suspension, Express Foods microcrystalline cloud fraction wet pellet, was extremely stable, while the third sample, FGA-1 microcrystalline cloud fraction, also appeared extremely stable but was stirred for 24hr prior to measurement of its zeta potential.

The effect of pH on the zeta 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 basicity, and positive with increasing acidity. There was some evidence for dissolution in the acid pH range. The zero-point-of-charge, i.e. the pH at which the zeta potential of the surface of the particle reached zero, was as follows: Sample 1 = 4.2; Sample 2 = 2.4; Sample 3 = 4.5. The results of plotting pH vs. zeta potential are shown in Figures 3 - 5.

EXAMPLE 16 Particle Size Distribution Four samples were examined and photographed by scanning electron microscopy (SEM) to determine particle size. 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 lOOμm. Figure 6 represents the basic culture medium for industrial fermentations prepared as described in Example 10. 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 by ultrafiltration and subsequently spray-dried.

EXAMPLE 17 Solubility in Water and Petroleum Ether The solubility characteristics of the microcrystalline cloud fractions of Example 16 (Figures 7 - 9) in water, petroleum ether, IN HCl, and IN NaOH were examined. 0.5 g, 1.0 g, and 2.0 g of cloud material were added to 10 ml aliquots of each solvent. The solutions were shaken vigorously and allowed to stand. The resulting solubility profiles appear in Table 16.

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TABLE 16

SOLUBILiπ CHARACTERISTICS IN WATER,

PETROLEUM ETHER AND ACID/ALKALI

_* Spray Spray Spray

Compound Dried E.F. Dried FGA-1 Dried FGA-2

Water, 5 Transient Cloudy, Cloudy, percent solids suspension, partial partial insoluble suspension suspension

Water, 10 Transient Cloudy, Cloudy, percent solids suspension, partial partial insoluble suspension suspension

Water, 20 Transient Cloudy, Cloudy, percent solids suspension, partial partial insoluble suspension suspension

Petroleum Insoluble Insoluble Insoluble ether, 5 (film) (film) (film) percent solids

Petroleum Insoluble Insoluble Insoluble ether, 10 (film) (film) (film) percent solids

Petroleum Insoluble Insoluble Insoluble ether, 20 (film) (film) (film) percent solids

IN HCl, 5 Transient Cloudy, Cloudy, almost percent solids suspension, partial complete, insoluble suspension suspension

IN HCl, 20 Cloudy, partial Cloudy, partial Cloudy, partia percent solids susp. suspension suspension

(significant (floating amount of material) stable foam)

TABLE 16 (CONTINUED)

SOLUBILITY CHARACTERISTICS IN WATER,

PETROLEUM ETHER AND ACID/ALKALI

Spray Spray Spray

Compound Dried E.F. Dried FGA-1 Dried FGA-2

IN NaOH, Partially Partially Partially

5 percent soluble, soluble, soluble, solids supernate supernate supernate clear yellow clear orange clear yellow

(floating (floating material) material)

IN NaOH, Partially Stable, dark Partially

20 percent soluble, orange foam soluble, solids supernate clear orange clear orange supernate (significant amount of floating material )

EXAMPLE 18 Solubility in Organic Liquids The solubilities of the microcrystalline cloud fraction materials of Εxample 10 (Figures 7 - 9) were characterized further in a variety of organic solvents. In general, 0.5 g of cloud material was added to 5ml of each solvent. The solutions were shaken vigorously and allowed to stand. In the case of glycerol, 5g were added to 50ml and the solution was stirred mechanically. The resulting solubility profiles appear in Table 17.

TABLE 17 SOLUBILiπ CHARACTERISTICS IN ORGANIC LIQUIDS Solubility of Cloud Fractions at 10 percent wt/vol

Dielectric Spray Spray Spray

Constant Dried Dried Dried

Solvent a 25°C E.F. FGA-1 FGA-2 FGA-2 Wet

Glycerol 42.5 2 2 2 1

Methanol 32.6 7 7 7 7

Ethanol 24.3 4 7 7 7

Acetone 20.7 7 3 3 2

2-Propanol 20.1 7 2 7 8 n-Butanol 17.1 7 7 7 7

Ethyl acetate 6.0 5 5 5 1

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 (20°C) 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 = Partial, particulate suspension with floating material

7 -x Transient suspension, insoluble

8 = Transient partial suspension , insoluble

9 = Insoluble

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^

EXAMPLE 19 Emulsification of Vegetable Oil

A solution of microcrystall ne cloud fractions from Example 16 (Figures 7 and 8) was prepared by shaking 20 parts of the moist precipitate in 100 parts of water. Thirty parts of 5 percent vinegar were added to this solution and the resultant mixture was stirred, thickening noticeably. Fifty parts of sucrose were then added with stirring, causing further thickening. Thereafter, 100 parts of liquid vegetable oil (peanut oil) were added and the mixture was homogenized in a Waring blender at high speed for 2 minutes. The resultant emulsion layer was stable for at least 4 hours and had the viscosity of a mayonnaise mixture.

As a control, the above process was repeated without the addition of microcrystalline cloud fraction. This process showed no thickening of the mixture following the addition of vinegar and sucrose. The oil and water layers formed no emulsion, and separation into two distinct layers was complete only two minutes after attempted homogenization.

EXAMPLE 20 Emulsification of Orange Pulp Wash

Following the procedure of the preceding example, 10 ml of orange pulp wash (the H^O soluble fraction of citrus pulp and ruptured juice vesicles) was added to 10 ml of distilled water containing 2g of microcrystalline cloud fraction of Example 16. Immediately after mixing, all of the material was in a single emulsion layer and remained so for at least two hours. Approximately 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 solidified.

As a control, the above process was repeated without the addition of microcrystalline cloud fraction. The lower, clearing layer began to form after less than 30 minutes, and the remaining emulsion layer was not thick as in the samples containing the cloud fraction.

EXAMPLE 21 Emulsification of Hexanes

This example illustrates the ability of the microcrystalline cloud fraction of this invention to emulsify non-polar hydrocarbons. Following the procedure of the preceding Examples, 10 ml of technical hexanes was added to 10 ml of distilled water containing 2 g of microcrystalline cloud fraction from the two different sources. Immediately 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. Approximately 18.5 hrs. later, the foams in both tubes had become gelatinous.

As a control, the above 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 Emulsification of Crude Oil

Using South Dakota intermediate grade crude oil containing 1.6 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 stable phases. The upper phase became gelatinous, with the Figure 8 microcrystalline cloud fraction sample gelling at about 90 minutes, and the Figure 7 sample gelling less dramatically after about 18 hours. The Figure 8 sample exhibited 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.

EXAMPLE 23 Emulsification of Bentonite

This example illustrates the use of the microcrystalline cloud fraction to emulsify particulate inorganic solids. Following the procedures of the preceding examples, 0.2g bentonite was added to 20 ml distilled water containing 2g of dry microcrystalline cloud fraction obtained according to Example 16. With the Figure 8 microcrystalline cloud fraction, an upper foam and a lower, frothy layer were formed. The frothy layer was stable for at least 18 hours.

As a control, the above process was repeated without the addition of microcrystalline cloud fraction. Immediately after mixing, a single, frothy layer was obtained which, within approximately 0.5 hr, also showed the presence of a lower, clear layer.

EXAMPLE 24 Emulsification of Protein by Cloud Fractions 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 Foods Co. and Savorpro 75 whey protein concentrate which is also commercially available from Express Foods Co. Samples were whipped in a Waring blender at high speed for 3 minutes. Foam height and the viscosity of the resulting emulsions were documented, demonstrating that the addition of either 10 or 20 percent microcrystalline cloud fraction increased both the foam height and viscosity of 10 percent whey protein concentrate solutions. The results are shown in Table 18.

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TABLE 18 EMULSIFICATION OF PROTEIN BY CLOUD FRACTION

Foam height Viscosity (Seconds for

100 _, ml aqueous solution when 100ml liq. 5ml to drop from pipet; whipped at high speed settled out in relative to a value for 3 minutes 200ml beaker of 3 for H ₂ 0

10 percent WLP 1.8 cm 4 sec.

10 percent WLP, 10 percent cloud fraction 2.5 cm 5 sec.

10 percent WLP, 20 percent cloud fraction 3.5 cm 5.8 sec.

EXAMPLE 25 Gelling of Protein by Cloud Fraction In addition to emulsifying protein, microcrystalline cloud fraction also gels protein at concentrations lower than that at which gelling would normally occur. Using the same materials described above, 10 ml aqueous solutions were prepared, vortexed, and incubated at 80°C for 20 minutes. With the addition of 20 percent microcrystalline cloud fraction, 10 percent whey protein concentrate solidifies at 80"C. Without the addition of microcrystalline cloud fraction no solidification of the 10 percent protein solution occurs, as shown in Table 19:

TABLE 19 GELLING OF PROTEIN SOLUTIONS

Aqueous Solution 80°C for 20 minutes

10 percent cloud fraction slight suspension w/large pellet sediment

20 ^* percent cloud fraction slight suspension w/large pellet sediment

10 percent WLP cloudy suspension, thick coating

20 percent WLP solid pellet

10 percent cloud fraction +

10 percent WLP milky suspension w/pellet 10 percent cloud fraction +

20 percent WLP solid pellet

20 percent cloud fraction +

10 percent WLP 1:1 solid pellet and thick coating 20 percent cloud fraction +

20 percent WLP solid pellet

EXAMPLE 26 Preparation of Industrial Fermentation Media with Increased Glucose Content

Permeate is prepared as in Example 10, 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 fermentations 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 0.25 percent Amber 510 yeast extract and 1.0 percent dextrose prior to autoclaving for 20 minutes at 121 ^β C/15 psi. The resulting autoclaved culture medium is clear, golden in color, and has a pH of 6.5. Another such culture medium is produced by first preparing a 15 percent solids solution of spray-dried permeate, the solution having a pH of 6.5.

This solution is passed through an immobilized enzyme reactor of the type described by A. G. Hausser et al . in Biotechnology and Biophysics XXV pages 525-539 (1983) at a rate of 6 ml/min at a temperature of 37°C, resulting in the conversion of 47 percent of the

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permeate lactose to glucose and galactose by immobilized acid lactase enzyme. The enzymatic conversion was carried out without pH adjustment of the permeate and was, therefore, at a pH that was norr-optimal for the acid lactase enzyme. This use of a non-optimal pH resulted in a 47 percent conversion, which was desirable for this example since it resulted in an approximately 1 percent glucose concentration when the permeate solids were adjusted to 3 percent. The resulting medium was then comparable to the 1 percent glucose-supplemented medium. Adjusted to a solids level of 3.0 percent, this lactase treated permeate contains 1.24 percent glucose. 3.0 percent lactase-treated permeate, supplemented with 0.25 percent Amber 510 yeast extract and autoclaved for 20 minutes at 121°C/15 psi, gives a clear, golden culture medium with a final pH of 6.5.

The glucose supplemented and lactase-treated basic culture media of this example were tested against several microorganisms for their growth support characteristics relative to the basic industrial culture medium described in Example 10. The results appear in Table 20 below.

TABLE 20 REPRESENTATIVE GROWTH IN INDUSTRIAL FERMENTATION MEDIA WITH INCREASED GLUCOSE CONTENT Medium Microorganism

S.aureus S.faecalis B.subtilis E.coli P.fluorescens

Basic culture medium for industrial fer¬ mentation. ++ ++++ ++++ ++++ ++++

Glucose supplemented basic culture medium for industrial fer¬ mentation. ++++ ++++ ++++ ++++

Lactose-treated basic culture medium for industrial fermen¬ tation. +++ ++++ ++++ ++++

Industrial culture media with a wide range of glucose to lactose ratios can be prepared by varying the extent of either the dextrose supplementation, or the lactose hydrolysis described above. Specifically, if a high level of lactose hydrolysis is desired a neutral lactose enzyme is immobilized using, a neutral buffer system and no further pH adjustment is made before permeate is passed through the reactor. Further, media with differing 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.

EXAMPLE 27 Cheese Starter Culture Medium

Following the procedure of Example 2 but adjusting the primary permeate to pH 8.5-9.0 and supplementing the secondary permeate with 0.25 percent Amber 510 or Amber 1003 yeast extract gives an essentially neutral clear golden culture medium which is suitable for growing commercial cheese starter cultures available from Chris Hansen Laboratories, Milwaukee, Wisconsin and for growing cultures of Streptococcus cremoris (ATCC 19257), Streptococcus lactis (ATCC 19435), and Streptococcus diacetylactis (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 6.0 to 6.5, the cell density reaches 5 to 10 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 16-20 hrs typically required in commercial media such as Nordica, In-Sure and Phase 4, which include internal phosphate buffering.

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The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those specifically used in the examples. From the foregoing description, one skilled in the art to which this invention pertains can easily ascertain the essential characteristics thereof and, without departing from the spirit and scope of the present invention, can make various changes and modifications to adapt it to various usages and conditions.

Industrial Applicability As 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. One 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 capable of emulsifying or stabilizing a wide variety of products.

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