NZ205569A - Production of clouding agent, stabilising agent, emulsifying agent, thickening agent or microbiological culture medium from dairy whey lactose permeate - Google Patents

Description

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PATENTS ACT 195 3 Number: Da te: iwwv <-an,M: 2.-5-83 &»eci!Ieatioo Filod: Class: ll RX5Cai|o<i R^SiijoO ,[!!!*!* !Q{F. f TKCcr? !!!!!!!!!!!!! ft&Hcation Dots.: P,Q, Mo; J.3Q& COMPLETE SPECIFICATION Insert Title • of Invention.

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CONVERSION OF CLARIFIED DAIRY WHEY LACTOSE PERMEATES TO CULTURE MEDIA AND OTHER COMMERCIALLY USEFUL PRODUCTS I/WE IGI BIOTECHNOLOGY INC., a Maryland corporation of 9110 Red Branch Road, Columbia, Maryland 21045, United States of America hereby declare the invention for which I/we pray that a patenb may be granted to me/us and the method by which it is to be performed, to be particularly described in and by the follov/ing statement:- fThe—f ollowing—page—i-s—numbacd—1 la - Indicate if following page is numbered '1(a)' 2 205569 Tprhniral F i p 1 rl n-f thp Tnupntinn This invention relates to processes -for converting dairy whey -fractions into commercially use-ful 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 o-f supporting good growth o-f a wide variety o-f microorganisms and a macrocrystalline cloud -fraction which can be converted to a dry, -free—f 1 owing, odorless and tasteless composition which has emulsi-fying and suspending properties which render it use-ful for a wide variety of applications in the food, pharmaceutical, cosmetics, and other industries. 205569 3 N.Z. PATENT OFF ICE SEP 1984 RECEIVED As noted by Alan 6. Lane in J. Appl. Chem. Biotechnol . 22: 165-16? (1977), the disposal o-f whey resulting -from the manufacture o-f cheese and casein presents environmental and economic problems o-f 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 4,328,158 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. K1 nyuprnmvrp<^ -fragility 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 98 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 Lact.nhflr.i 1 lus cells, e.g. as reported by R.W. Steiber et al . in J. Dairy Sci. £3: 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. 205569 Both sweet whey permeate and acid whey permeate have been used as a -feedstock in ethanol production using beta-gal ac tosi dase and fiarrharnmvrPQ rprpuig.iap, e.g. as reported by Barbel Hahn-Hagerdal in Applied Biochemistry and Biotechnology 2: 43-45 <1982). Although more than 58 percent of the lactose was converted to ethanol, the eluate contained less than 2 percent ethanol yield based on the weight/unit volume o-f whey permeate -feedstock.

The use o-f whole whey as a bacteriological culture medium has been reported by Emel Celikkol in Mikrobiyol. Bui. 2(4): 273-279 (1975) and in U.S.S.R. Patent 819,166. As summarized in Chem. Abs. 24: 72629u and 25: 59984n 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 hereto-fore not been -fully appreciated by the prior art, neither o-f these methods has gained widespread use -for either industrial or clinical grades of culture media.

Uhey 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 4,289,583 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 4,842,576. 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 l iquid. 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 N.Z. PATENT OFFIC SEP 1984 f- "" r;f; ] V" are treated by adding calcium ion, heating and drying to -form a product which is useful as a non-fat dried milk substitute in bakery products. It has now been -found that a particular combination o-f 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.

It is a general object of the present invention to provide a simple 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 microcrystal 1ine 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 applicat i ons.

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 microorgani sms employing these media.

A second principal object of the present invention is to provide a mi crocrystal1ine 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. and inexpensive method for converting deproteinized dairy whey A more particular object o-f the present invention is to provide improved -food grade additives -for use in -foods, pharmaceutical carriers, cosmetic 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.

Bnie.f Dascr ip.t ion of the Drawings Figures 1 and 2 are flew diagrams of a presently preferred process and practical applications according to the present invention.

Figures 3-5 are graphs shewing 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 format i-on 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 microcrystal1ine 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 microcrystal1ine cloud fraction similarly obtained from a different source of deproteinized dairy whey lactose permeate; and Figure 9 is ah SEM of a microcrystal1ine cloud fraction similarly obtained from yet another source of whey lactose permeate which contained a high protein level due to membrane leakage.

Bast MndP-fon-Camyi ng_Oul the Inupntinn Brie-fly, the above and other objects, features and advantages the present invention are attained in one aspect thereof by providi a method for separating dissolved solids from deproteinized dairy whe. 205569 f # 4 lactose permeate materials, which would otherwise -form a precipitate upon autoclaying the permeate, to form a) a lactose-rich aqueous solute phase, use-ful as a microbiological, culture medium, which does not -form a precipitate upon autoclaving; and b) a microcrystal1ine cloud precipitate which is useful as a food grade additive to cause clouding, stabilization, emu 1sification , 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 microcrystal1ine 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 i$ useful as a microbiological culture medium for clinical and industrial uses, including aerobic and anaerobic fermentation processes. The microcrysta 11 ine 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 commercial1y 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 . j^Xhe.-sol i ds concentr-atj on of the permeate is adjusted to an appropriate concenjJ^^^^fc^nd th|j pH which normally will be below 7 is adjusted to about ^V^IO. Th^x^jus'tment of the pH results in the 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 gej s 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 unsupplemented 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 commercial1y 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 commercial1y available Abcor (cellulosic and nonce 11ulosic tubular membranes), DDS (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 (ki1odaltons) 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°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 2-28 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 205569 N Z. PATENT OFFICE . if SEP 1984 - SCGIVlD assimilable nitrogen sources than sweet WLP), while concentrations much above this range may -fail to stay in solution during processing. Excessively high concentrations above 28 - 25 percent solids also impede the removal o-f the WLP components which precipitate upon autoclaving.

These components, which are collectively referred to herein as a mi crocrystal 1 i ne 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 siiu. 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 macrocrystalline cloud precipitate is formed. The temperature at which the precipitation step is carried out is not particularly critical. Conveniently, temperatures in the range of 28 - 58°C may be utilized.

This increase in the pH of diluted WLP results in precipitation of the microcrystal1ine 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. 11 205569 The precipitate is physically separated from the culture medium," e.g. by centrifugation at 1 1 ,7589 and ■filtration across a 0.4$jj pore size membrane or by ultrafiltration across a 10 - 100 kdal molecular weight exclusion membrane, generally 20 - 50. kdal and preferably 20-30 kdal, and saved for use as an emulsifying or suspending agent as discussed below. Centrifugation alohe without subsequent filtration is generally unsatisfactory, since the clear supernatant frequently turns cloudy upon subsequent autoclaving, thereby limiting its applicability as a culture'median. Ultrafiltration across a membrane of pore size less than 20 kdal, e.g. 10 kdal/ is unsatisfactory when the solute phase is the desired product 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) apd used as a culture medium for growth of microorganisms. The solute fraction contains useful quantities of assimilable carbon, nitrooen', phosphorus and other nutrients, including the sugar sources lactose, sucrose, galactose. and glucose. Ihe. principal carbon source is the lactose present in the starting WLP. Of the available sugar in a 3.5 percent' (wt/vol) solids unsuppl onented media after autoclaving at 121°C/15 psi, a typical composition is: 53.0 percent by weight beta-lactose (11.8 mg/ml); -'44.8 percent by weight alpha-lactose + sucrose (9.97 ng/mL); 1.2 percent ty weight galactose (0.27 mg/ml); and 1.0 percent by weight glucose (0.23 mg/ml).

It has been found that WLP, although essentially proteir. free, generally contains adequate amounts of metabolizable nitrogen in the form of free ajnino acids and Iow molecular weight polypeptides. Thus, in accordance with the present invention, there is no need to hydrolyze separated proteins to increase the assi^Hable nitrogen content as described in U.S.S.R. Patent 817,166; ih'any event, most proteins have 205569 12 already been relieved during the ultrafiltration process and are not available as a component of the WLP starting material. If supplmentation of nitrogen sources is desired, it can be achieved fcy the addition of con/entional nitrogen sources, including yeast extracts and peptones of commonly available animal and vegetahle proteins, such as casein and soy. The source of nitrogen atoms then could be a yeast extract, yeast autolyzatef hydrolyzed casein, soy protein or soy protein lydrolyzate, or a mixture of these. Other sugar sources such as glucose, and buffering agents, cofactors, and the like, m^ be added as necessary to support growth of the microorganism of choice. Those of ordinary skill in the art cf microorganian culture can readily determine an/ necessary nutrient supplements, buffering agents (i.e., to an optimal pH range in which the organisn is viable), and the like which are necessary to grew 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 _wi.fch^ the present invention comprises an aqueous composition „ supplemented with yeast extract, amino acids and glucose in the xmto!lowing proportions: clarified solute fraction at 3.5 percent solids (wt/vol) ^3OCT 19^7 f yeast extract <Amber 510) 0.05 percent by weight amino acid mix (U.S. Biochemicals) 0.5 percent by weight glucose (USP grade) 0.05 percent by weight above supplemented culture medium has a protein analysis (Cowry protein content) lower than conventional nutrient broths, as 205569 13 shown below in Table 1, there-fore it is unexpected that the supplemented medium according to the present invention would be use-ful ■for supporting the growth o-f 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 o-f Example 1 which has been supplemented with 0.5 percent by weight casamino acids, 0.05 percent by weight yeast extract, and 0.05 percent by weight glucose.

TABLE 1 Marii urn 8BL Nutrient Broth Di-fco Penassay Broth WLP Medium, supplemented Sk im Milk mg/ml Prntpin 4.8 3.3 1 .2 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 growi ng mi croorgan i srns which are deficient in the genetic mechanisms for producing a given amino acid, the medium can be supplemented accordingly. 14 205569 TABLE 2 1XB1EAI AMINO- Amino .Ar i rl Alan i ne Argi n i ne Aspartic acid Glutamic acid 61yc i ne Hi st i di ne I soleuc i ne Leuc i ne Lysi ne Me th i on i ne Phenylalan i ne Ser i ne Threon i ne Vali ne 3.8? 1 .05 2.63 9.45 1 .91 0.9 3 1 .83 3.38 3.0? 1 .03 1 .4 6 4.56 1 .90 3.45 The supplemented WLP medium exhibits good bu-f-fering 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 pH after successive 8.1 ml additions of IN HC1 to 25 ml broth Mprliiim II 1 2 3 Difco Penassay Broth 6.92 6.64 6.34 5.96 5.28 4.37 ULP Medi urn, (supp1emen ted) 6.82 5.59 4.66 4.14 3.73 3.32 BBL Nutrient Broth 6.80 4.38 3.58 3.04 2.60 2.31 TABLE 4 BasE_BUE£EeifciG_c&Eani ~n pH after successive 0.1 ml additions of IN NaOH-to 25 ml broth M p rl i 11 m B 1 2 3 4 Di fco Penassay Broth 6.93 6.93 6.96 6.9? 7.02 7.05 ULP Medium 6.80 6.93 7.04 7.17 7.31 7.15 (supp1emented) BBL Nutrient Broth 6.74 6.9? 7.19 7.37 7.55 7.71 The supplemented ULP 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. Uhen preparing a liquid broth, any desired supp\ements can be added prior to autoclaving at 1210C for 15-20 minutes. In this manner, various types of culture media can be readily prepared from the basic unsupp1emented ULP solute fraction. Presently preferred media are : 1) a general purpose gncwth medium cf solute phase preferably supplonented with about 0.25 - 0.5 percent by weight casamino acids, 0.05 percent by weight yeast extract and 0.05 - 0.1 percent by weight 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 mediun (PIM) for the cultivation of both aerobic and araerobic microorganisms from primary clinical specimens. Ihis material is frequently supploriented with 0.25 - 0.5 percent by weight casamiro acids, 0.5 percent by weight yeast extract, 0.4 - 0.5 percent by weight glucose, 0.1 percent by weight agar or other gelling agent to reduce oxygen diffusion and 0.05 percent by weight cysteine HCl as a reducing agent. When boiled before use to reduce the oxygen content, the resulting clinical grade medium ccmpares favorably with widely used thiogly col late broth; and 3) a pre-reduced, sterile, anaerohically prepared mediun for the cultivation of facultative and obligate anaerobic microorganians, which is preferably supplemented with 0.25 - 0.5 percent by weight casamino acids, 1 percent by weight yeast extract, 0.5 percent by weight glucose, and 0.001 percent by weight 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 by weight cysteine HCl, 0.50 mg/ml hemin, 1 mg/ml vitamin K^, 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 o-f -150mV or lower, and the col orimetrie redox indicator turns pink upon oxidation of the medium; and 4) an industrial fermentation mediun, in either liquid or solid form, e.g. containing solute fraction diluted to 3.5 percent (wt/vol) solids and supplemented with about 0.25 percent by weight Mber 510 brewer1 s yeast extract. For the solid medium, ary conventional gelling agent can be added, e.g. about 1.5 percent by weight agar. A typical analysis of such an industrial fermentation medium is as follcws: 205569 17 I. Analysis: (mg/100 om) Protein, Kjeldahl (percent N by weight 12.10 x 6.32) Protein, Lowry 3.5 percent by weight fat <1.0 percent by weight ash <1.9 percent by weight carbohy- 81.5 drate percent by weight moisture <4.5 Bulk density, gm/cc 0.63 Solubi1i ty i n H2O, gms/100 ml 30°C 24.5 Sugan_Eco£il£ (percent): Galac tose 0.8 Glucose 0,7 Lac tose 31.5 Sucrose trace Bl B2 Ni ac i n 0.38 16.60 21.70 Xcacp Miaprals: <mg/100 gm) Alumi num Bar i um Boron Cal c i urn Ch r om i um Copper Iron Magnesi um Manganese Phosphorus Sodi um Stront i um Z i nc Mi r.rrib.i ni ogical: CFU Col i -form <0 .906 8.121 0.242 26. 21 <0.121 <0.131 0.181 34.97 " 0 .060 341.56 580.14 0.785 0 .604 220/gm negat i ve AmiHQ_6cid_Ecafil£:(mg/109 g) Argi ni ne Cyst i ne G1u t am i c acid G1yc i ne Hi st i di ne I soleuc i ne Leuc i ne Lys i ne Me th i on i ne Phenylalan i ne Thr eon i ne Tryp tophan Tyros i ne Vali ne 160 30 330 230 100 198 270 270 90 130 150 40 170 130 85 percent passes Tyler 270 screen pH a-ffprr autflclau ing: 6.5 (3 percent total solids) /« 18 205569 The above described industrial -fermentation formulation supports growth with the following industrially important organisms: £_t££ plfjfTl YT P Qf- i c P 11 c E£niclllium_no±a±um Saccharcrayces cerevisiae Aspangillus-iiigac. produces strep totTiyc i n (detectable levels within 24 hrs.) and pronase produces penicillin (detectable levels wi thin 24 hrs.) produces ethanol produces citric acid ) Industrial fermentation media with increased glucose content. One such mediun consists1 of solute fraction diluted to 2.0 percent sciLids (wt/vol), and suppLonented with 0.25 percent by wei^it ftnber 510 yeast extract and 1.0 percent ky weight dextrose. Another sudi medium consists of salute fraction that is .-passed through an iirmobil ized lactose reactor, diluted to 3.0 percent (wt/vol) solids, and suppioriented with 0.25 percent by weight ftnber 510 yeast extract. The residence time of the solute fraction in the reactor is used in oorrjunction with the pH and temperature of ttie reaction to control, the firal dextrose concentration of this mediun.

It may be seen there-fore that the solute -fraction may be utilized, either in the supplemented or unsupp1emented form, to produce antibiotics, such as streptc-myc i n and penicillin. Furthermore, the solute fraction according to the present invention may be used as a star tip culture growth medium, such as in the biological production o-f Olfc^rd aind soft cheeses.

While relatively unimportant for use in certain industrial Proce5ses» the optical clarity of a broth culture medium highly important in clinical applications. For this purpose, it is £N E f advisable to screen samples of supplements intended to be used, as i n some^i||stances it has been found that certain samples will not yield .j red clear product. At high yeast extract concentrations of around 1 percent by weight, Amber ex 510 water soluble autolyzed yeast extract obtained fran Arriber 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 0.22 jul 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 ? 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 unsuppIemented 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, Carr^ageenan, 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 unsupp1emented 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 681-656 of the American Type Culture Collection Catalogue of Strains I, 15th Edition (1932). 205569 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 cf WLP starting materials in concentrations greater than the 3.5 percent (wt/vol) generally onpLcyed for liquid media is preferred, and concentrations as high as 20 percent (wt/vol) have proven satisfactory. As the sclids content of the WLP starting material approaches 30 percent (wt/vol), it has been found that sane of the solid material may ranain in suspension and not be precipitated fcy pH adjustment. Spr^ drying of media containing supplorients such as 0.05 percent ky weight yeast extract and/or 0.25 - 0.5 percent ty weight casanino 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 2000C 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 by weight. will be appreciated that the culture media of the present N v e n t jjpn can be employed in the fermentative production of i b i o't i c s, enzymes, organic acids, alcohols, and ketones and can also t b^jijsed as a starter culture growth medium, e.g. in the biological l30CT1987rijuction of hard and soft cheeses such as American, Swiss, Italian, ieddar, Mozarella, and cottage cheeses. These UILP media are distinctly different from whole whey-based cheese starter cultures as illustrated, in±£n_alia, by G.W. Reinbold et al. U.S. Patent 3,993,700; D~.L"r^ndersen et al . U.S. Patent 4,020,185; R.S. Porubcan et al . U.S. Patent 4,115,199; andU.E. Sandine et al. U.S. Patent 4,232,255.

The microcrystal1ine 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 205569 21 has the consistency o-f shortening at 4°C and becomes more -free—f 1 owi ng upon warming to room temperature. When dried, this precipitate is a tasteless, odorless, chalky white -free-flowing powder; typically, about 15 percent by weight of the input WLP solids which are processed are recovered W as this dried precipitate pcwder.

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 4,1*43,174 and 4,209,503, the microcrystal1ine cloud fraction of the present invention is insoluble in petroleum ether, as shown in Table 5. The physical characteristics of the microcrystal1ine 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 by weight moisture, the microcrystalline cloud fraction is only transiently suspendable in water but is still insoluble in petroleum ether.

TABLE 5 SBLlJBlU.IX_D£_£LDlJI>_£fiA£Iim Sflluaoi .Sol i,ihi 1 i ty Coneentrafprl r.lnurl Prartinn Ppllpf; Ethyl acetate Benzene Toluene Chi or of or m Petroleum ether Methanol Ethanol Propanol Bu tanol IN HCl IN NaOH insoluble, nondispersing paste insoluble, nondispersing paste insoluble, nondispersing paste insoluble, nondispersing paste insoluble, nondispersing paste very cloudy suspension very cloudy suspension very cloudy suspension sii gh t suspens i on cloudy suspension cloudy suspension /juLwaf 205569 22 Dry CLoud Fraction, Water, 5 percent solids (wt/vciL) Water, 10 percent solids (wt/vol) Water, 20 percent solids (wt/vol) slight suspension sLight suspension slight suspension Petroleum ether, 5 percent solids (wt/vol) insoluhle particles PetraLeun ether, 10 percent solids (wt/vol) insolubLe particles Petroleum ether, 20 percent solids (wt/vol) insoLuhLe particles When chemically analyzed by ICP analysis, the microcrystal1ine cloud fraction o-f this invention is also demonstrably different in nature from both unprocessed -spray dried UILP and the precipitate that forms when spray-dried ULP is resuspended to 20 percent concentration (wt/vol) and cooled at 4CC 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 ICR analysis according to Industrially Coupled P1 asina-A t om i c 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 4,202,90? which involves heating to only 140° to 150°F. All samples were prepared by resuspending 20 percent (wt/vol) spray-dried ULP in water prior to individual processing. 205569 23 TABLE 6 Spray Dcipri U1LE Calcium 348.-438 Iron 8.11-.12 Phosphorous 488.-491 Magnesium 150.

Zinc Copper Sod i um C-hromi um Alum i num Bar i um Stront i um Boron Manganese 6.06-.08 0.025 774.-863 0.036-.037 1.1-1.3 0.025-.028 0 .11-.22 0.06-.07 0.005-.011 Alkaline pH Ecex i p i tatp ,392. 6.65 782.8 ,733. 0.7? 0.75 461 .6 0.48 52.96 0 .82 1 .54 3.70 0 .21 Cold Pederson Precipitate Precipitate 4°R, 7? hr^ 222£~ ' ,800. 6.5? 11,448. 2,378. 3.42 0.7? 718. 7,934. 4.77 4,075. 548.2 1 .39 0.37 600 .9 1 .57 0.50 .66 6.50 0.57 0.57 6.60 3.74 0 .631 0.38 0 .21 0.18 a -function o-f pH By determining the zeta potential as microcrystal1ine 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-poi nt-o-f-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 o-f at least about 5mV are generally preferred, with greater deviation -from the zero-point-o-f-charge generally resultin the greatest stability. However, in the acid range, such high acid N.Z. PATENT OFFICE - ■ SEP 1984 IGCEWED ! may result in degradation o-f polypeptide components present in the microcrystal1ine cloud fraction.

Taking into account these unique solubility properties, an air dried microcrystal1ine 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 7 g of WLP (obtained from Express Foods Co. and similar to products commercial1y 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 6.99 to 8.99 by the addition of 2.15 ml of 5.5 N NH4OH while stirring, and centrifuged for 10 minutes at 8500 rpm <ll,300g) in a Sorval1 RC-5B centrifuge using a GS^i rotor refrigerated at 4°C. 1.26 g of a soft, white microcrystal1ine cloud fraction pellet were obtained per 100 ml of starting solution. The supernatant was poured through a 0.45u, 115 ml Nalgene fi1tration unit, yielding 200 ml of clear material having, a pH of 9.84. After autoclaving at 121°C/15 psi for 28 minutes, a dull orange and crystal clear unsupplemented culture medium was obtained, having a final pH of 7.87.

I N.Z. PATEN l v.' r 10 SEP 1984 y rec^T" 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 o-f NH4OH were added to bring the pH up to 3.98. Following centr i -fugat i on , 1.08 go-fa hard, tan pellet were obtained per 188 ml o-f 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 7.88.

EXAMPLE 2 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 ultrafi1tered thru a 38 kdal Dorr-Oliver filter unit, yielding a primary retentate and a primary permeate. The primary permeate was adjusted to pH 9 with NH4OH and the ultrafiltration process was repeated, yielding a secondary microcrystal1ine cloud fraction and a secondary permeate. The secondary permeate was supplemented with 8.25 percent casamino acids, 8.05 percent yeast extract, and 8.85 percent glucose prior to autoclaving for 28 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 Enaciftllalxon-uitli. Qthar Rasfcs The procedure of Example 1 was followed, except KOH was used to adjust the pH. From an initial pH of 6.89, 8.2 ml of 6N KOH and 8.2 ml of IN KOH were added to bring the pH to 3.92. 1.66 g of cloud fraction were obtained as a soft, white pellet per 188 ml of starting material. Following filtration, the supernate was clear and had a pH 9 p, c r r 6 0 j J u 7 205569 1QSCFV}84 o-f 8.78. After autoclaving, the liquid was golden colored and very slightly cloudy, with a -final pH o-f 6.25 When NaOH was substituted -for the NH^OH in the procedure o-f Example 1, the initial pH of 6.88 was rai sed to pH 8.98 by the addi t i on of 8.45 ml of 3N NaOH. Centrifugation yielded 1.62 g per 188 ml of starting material of microcrystalline cloud fraction as a soft white pellet. After ultrafiltration across a 8.45u membrane, the supernate was clear and had a pH of 8.75. After autoclaving, a golden colored, siight1y c1oudy liquid was obtained having a final pH of 6.25.

The autoclaved clear culture media from Examples 1 and 3 were evaluated for their ability to support the growth of common laboratory culture strains, EarllXus subilJLLs 6851a, Entjernhar tpr aprngenp-s E13843, and Fdrhpr i rhi a mli HS. Tubes of culture media, both unsuppl emented and supplemented with 1 percent BBL yeast extract,-- 8.5 percent Difco casamino acids, and 8.5 percent sucrose (Sigma Chemical Co.), were inoculated and incubated at 35°C for 5 hrs, after which optical density readings were made at 668 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: EXAMPLE 4 + + + + + + + + + + Excellent growth; O.D. 8.3-1.0 Good growth; O.D. 0.1-8.3 Moderate growth; O.D. 8.83-8.1 Some growth; O.D. 8.885-8.83 No growth; O.D. 8-0.885.

The results are shown in Table 7.

N.z. patent OFFICE SEP 1984 205569 r.iil tnpp Mprlinm TABLE 7 Re£ 1 iminary fir\Dui±b-££C££ain^ R. c.juht.i..l-Ls £^—rnl i 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* (NH4OH) + + + + + + + + + 3.5 percent WLP* (NH4QHHSUPP + + + + + + + + + 4 + + * as. WLP sol i ds EXAMPLE 5 In order to evaluate the importance o-f the pH employed -for precipitation o-f the mi crocrystal 1 i ne 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 NH4OH 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 shewn in Table 8. 28 205569 TABLE 8 4 with HCl clear, 1ight green 4.5 with HCl clear, light green .5 6 no addi t i on sii gh 11 y opaque 6/9 7 wi th NH40H very cloudy, light ye 1 lew 6.1 8 with NH4OH very cloudy, golden 6.4 9 wi th NH4OH clear, root beer color 7.1 19 with NH4OH very dark brown 3.8 11 with NH4OH like 1i qu i d chocolate 9.7 EXAMPLE 6 Following the procedure o-f Example 4, whey permeate culture medium produced according to the procedure o-f Example 1 and supplemented with 8.25 percent casamino acids and 0.95 percent yeast extract, both with and without 8.95 percent glucose, was compared with Di-fco Penassay broth and BBL nutrient broth -for its ability to support the growth o-f a representative variety o-f cl i n i cal 1 y important microorganisms. The results are presented in Table 9 and show that the solute phase culture media o-f this invention compare -favorably with two current widely accepted industry standards. 2? 205569 TABLE 9 REPRESENTATIVE GROWTHS IN LIQUID MEDIA m Whey Permeate Whey Permeate DIFCO BBL Media <suppl.) Media <suppl.) Penassay Nutrient ui/nu.t piiim^e ut/plxirq<*p Bcnlh Rrnth aubi i 1 La riflfila + + + + not done >+ + + + + + + + Eschanich ia cnl i-Uft + + + + not done + + + + + + + + + + + + + + + + + + + + not done + + + + + + + + + + + + + + + + + + + + + + + + + + + + Prntpua + + + + + + + + + + + + + + + + Klflisialla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ♦ Salmonella tvph imilP i um I T9 + + + + fih i qp11 a annnpL + + + + + + + + + + + + ++ + + + + + + + + + + + + + + Salmonella. + + + + + + + + + + + + + + 205569 re 1 10 SEP 1984 EXAMPLE 7 Fffprt of AutnrlauiriQ nn firnuiih In order to evaluate the importance o-f 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 shewn in Table 10. 205569 31 TABLE 18 REPRESENTATIVE GROWTHS IN LIQUID MEDIA Autoclaved Filter Sterilized DIFCO BBL M i rrnnpgan i gjn Bar i11 us suhJti lis rifl'Fil a Esrhpr i rh.i.a Fntprnhar tpp BcqIeus Whey Permeate Whey Permeate Mprlia (snpp) + + + + + + + + + + + + ■fapral i s, F19433 + + + + Staphylococcus + + + + + ++ K1 phyi p 1 la pne.umnn i ap...23352 + + + + + + + + + + + + + + ♦ + + + + + + + + + + + + + + + + + + + + + + + + Penassay Nutrient Brnth Rrnth— + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + sonnpi Salmnnp1 la typhimnrinm ?la + + + + + + + + + + + + + + + + + + + + + + It can be seen -from the last entry on the above table that there are apparently some nutrients required -for the growth o-f £almnnpl 1 a typh imur i nm 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. 205569 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 8.5 percent casamino acids, 1 percent yeast extract, and 8.5 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 5-18 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 NH4OH as measured by test paper while bubbling nitrogen through the liquid, which was then dispensed into tubes which had been flushed wi th 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 t?e 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 Liquid anaerobic culture media were tested against three strains of Rartprnidps for its ability to support anaerobe growth. Test samples containing 8.85 percent yeast extract plus 0.85 percent glucose (Medium 1), and 1.8 percent yeast extract plus 8.5 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 9 05569 33 N.Z. PATENT OFFICE SEP 1984 control. Optical density readings were made during the first eight hours of incubation. The results are summarized in the following Table: TABLE 11 ANAEROBIC GRCWTH SCREENING -£H1_£e_o±£i_ _IYil-B£c±li_ Bar tpr i nrlps Bac ter i nrlps icagilis Bar t PxiodES ic.agi 1 i s .47?=! + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Mprtiiim ? + + + + + + + + + + + + EXAMPLE 10 Prpparatinn nf Basic Cul tnr£-H££LLum—£o£ 3.5 percent WLP (wt./vol; commercially available from Foremost-McKesson, Inc. or Express Foods Co.) was adjusted to pH 9 with NH4OH and u1trafi1tered through a 38 kdal Dorr-Oliver filter unit. The permeate was supplemented with 8.25 percent Amber BYF100 yeast extract prior to autoclaving for 28 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 BYF180.

EXAMPLE 11 Industrial Fermentation Prnrp^s Rar ilitis—c£t£us—suhs... th.»c.iiigl£asls^—uar.Bedinet obtai ned from Dr. Howard T. Dulmage of the U.S., Department of Agriculture Cotton Research Institute, Brownsville, Texas was selected to exemplify the 34 205569 capability o-f the culture medium o-f 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 de1ta-endotoxin and is used as a biological insecticide in the control o-f 1 ep i dopter-an pests, e.g. as the worm killer available under the trademark DIPEL 4L -from Abbott Laboratories, Chicago, 111. The development o-f parasporal crystals and spores o-f Ra riling thnr i ngi pn^j g. was monitored under phase contrast microscopy following the procedure o-f L. A. Bulla et al . described in Appl i ed M i cr-obi ol ogy IB <4>: 498 - 495 (1969).

Heat shocking at 78°C was used to compare the degree o-f sporulation in the modified culture medium of Example 16 containing 0.25 percent Amber BYF188 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 108 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 18 to 100 fold higher with the former; in addition, the maximum sporulation level obtained was from 5 to 18 fold higher.

The basic culture medium of Example 1 was supplemented with 0.25 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.

EXAMPLE 12 percent Amber BFY 188 yeast extract before autoclaving. Aliquots of the resulting medium were inoculated with several organisms of industrial interest. Colony morphologies and dry cell weight yields 205569 N 7- PATENT OFFiCE_ SEP 1984 TABLE 12 COLONY GROWTH CHARACTERISTICS Strain tt-noiaturn Sirpptnmxce.s .gnisaus Sac.char0tnyr-es.cpcg.ui si ae Cn 10By Mnr phnlngy single, large hyphal mat disperse, beadlike growth wel1 di spersed well dispersed Dry r p 1 1 hip i gh t y i p 1 rift 0.76g/100 ml 0.47g/108 ml 0,21g/180 ml 0.237g/108 ml * 5 days after 1/10 vol. inoculation and incubation at 30°C wi th shak i ng.

EXAMPLE 13 An t i h i nt i r Prnrlnrtinn 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 Sltaia penicillin streptomyc i n 0 .0864 Un i ts/ml 0.80735 Units/ml.

* One day after 1/18 volume inoculation and incubation at 25-38°Cwith agitation 205569 t: z. PATENT OFF ICE 36 1CSEP1984 EXAMPLE 14 Solute fraction prepared as in Example 1 was supplemented with 8.5 percent casamino acids, 8.05 percent yeast extract, and 0.85 percent glucose. Tubes o-f 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 f:nl nnv r.nnntc. ppr- ml. a-ftpr ?4 hr^ at 3?0f: Organism Tpsifcd Supplemented Solute Control Media < 8HI, PABA, AGAR) N. me.QiagiiidJ-5.

Ln.f I upn7ft£ R. nu i t i s 88 68 1258 250 8 1388 205569 37 N.Z. PATENT OFFiC£ 105EP1984 RECEIVED TABLE 15 VISUAL OBSERVATIONS OF GROWTH Alraligpnp^ -fapralig.

Bacillus, c e.c.aus B*_subillis B_ thyc-ingiansls Cilnoharter -frpnnrlii P. plnngata Rhnrln^p i r i H nm riihrnm Ralmnnplla typhimnrinm SEttalii-manctsctas S i a p h y .1 ococcus_ami£.us Strpptnrnrrns -fapralis BiiQulh-Ltfiibi n 4R hrs at 3fl°C Arinptnhartpr ralrnarpti mjs Mi rrnrnrrng. c.p _ Fungi grew after 2-3 days Aspergillus ninsr Q-oratiaii^cas-stemnn i t is Ppn i r i 1 1 i um =.p .

EXAMPLE 15 The stability o-f colloids comprising three mi crocrystal 1 i ne cloud ■fraction samples was measured by determining the zeta potential. Each sample was diluted in deionized water to a 8.180 percent suspension, and the electrophoretic mobility was determined using a Zeta-Meter (Zeta-Meter, New York, NY). With this instrument, a suspension o-f the sample is decanted into an electrophoretic cell and a potential applied across a pair o-f electrodes inserted into the cell. The average time for a particle to move hor i zon tall y between two lines o-f 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 mi crocrystal 1 i ne cloud -fraction, was onl y moderatel y stable, with the solid dispersing slowly over a period o-f 15 minutes and some larger particles settling quickly to the bottom o-f the container- when 205569 stirring was stopped. The second sample suspension, Express Foods mi crocrystal 1 i ne cloud -fraction wet pellet, was extremely stable, while the third sample, FGA-1 mi crocrystal 1 i ne cloud -fraction, also appeared extremely stable but was stirred -for 24hr prior to measurement o-f its zeta potent i al.

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 Part i r 1 p fii7P D t c.tr i hn t i nn Four samples were examined and photographed by scanning electron microscopy <SEM) to determine particle size. The scanning electron micrographs are shewn in Figure 6 through 9; the distance between solid white squares on the lower border of each photograph is 100um. Figure 6 represents the basic culture medium for industrial fermentations prepared as described in Example 10. Figure 7 represents microcrystal1ine 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 microcrystal1ine cloud fraction produced from whey lactose permeate (Foremost-McKesson, Inc.) generated by Mozarella cheese manufacture. The microcrystal1ine cloud fraction was generated as described in Example 1, separated from the culture medium by ultrafiltration, and subsequently spray-dried. Figure 9 represents microcrystal1ine c':jd fraction produced from whey lactose permeate (Foremost-McKesson, Inc.) generated by Swiss cheese 205569 39 manufacture. The mi crocrystal 1 i ne cloud -fraction was again generated as described in Example 1, separated -from the culture medium by ultra-filtration and subsequently spray-dried.

EXAMPLE 17 The solubility characteristics o-f the microcrystal 1 i ne cloud fractions of Example 16 (Figures 7 - 9) in water, petroleum ether, IN HCl, and IN NaOH were examined. 8.5 g, 1.8 g, and 2.0 g of cloud material were added to 18 ml aliquots of each solvent. The solutions, were shaken vigorously and allowed to stand. The resulting solubility profiles appear in Table 16.

TABLE 16 SOLUBILITY CHARACTERISTICS IN WATER, P-nmpnnnrt Water, 5 percent soli ds Spray Transi ent suspensi on, i nsol ubl e Spray Dr i pd FfiA-i Cloudy, part i al suspensi on Water,10 percent solids Water, 20 percent soli ds Pe troleum ether , 5 percent soli ds Petroleum ether, 10 percent soli ds Petroleum ether, 20 percent soli ds Transi ent suspensi on, i nsoluble Transi ent suspensi on, insoluble Insoluble <fi 1m) Insoluble (•film) I nsol ubl e (film) Cloudy, part i al suspens i on Cloudy, p ar t i a 1 suspensi on Insoluble (f ilm) Insoluble (•film) Insolubl e (film) Cloudy, part i al suspensi on Cloudy, partial suspensi on (si gn i f i cant amount of stable foam) Spray Dc.i.ed FGA-? Cloudy, „par t i al suspensi on Cl oudy, part i al suspens ion Cl oudy, part i al suspensi on Insoluble (film) Insoluble (film) Insoluble (film) Cloudy, almost complete, suspens i on Cloudy, partial suspensi on (floating mater i al) IN HCl, 5 percent soli ds IN HCl, 20 percent soli ds Transient suspensi on, i nsoluble Cloudy, partial susp.

SEP 1984 TABLE 16 ( CONTINUED) SOLUBILITY CHARACTERISTICS IN WATER, Cnmpniinrl IN NaOH, 5 percent soli ds Spray Dr i prt PP.

Part i al1y soluble, supernate c1 ear ye 11ow Spray Driprl FfiA-1 Part ially sol ubl e, supernate clear orange (f1 oat i ng mater i al) Spray Driprl FfiA-? Par t i al 1 y soluble, supernate c 1 ear ye 11ow (■floating mater i al) IN NaOH, 20 percent soli ds Part ially soluble, supernate clear orange Stable, dark orange -foam Partially soluble, clear orange supernate (si gn i -f i cant amount o-f floating mater i al) 205569 12 K.i SEP 1984 RECEIVro EXAMPLE 18 The s-olubilities o-f the microcrystal 1 ine cloud -fraction materials o-f Example 18 (Figures 7-9) were characterized -further in a variety o-f organic solvents. In general, 0.5 g o-f 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 mechanical1y. The resulting solubility profiles appear in Table 17.

TABLE 17 SOLUBILITY CHARACTERISTICS IN ORGANIC LIQUIDS Srnlubi 1 ,i ty. nf r.lniirl Frartinns at 1ft pprrpnt uit/.unl^ Di el ectr- i c Spray Spray Spray Constant Dried Dr i ed Dr- i ed Rnlupnt rf ?5or: F.F.

FPto-1 FfiA-9 G1 ycerol 42.5 2 2 2 1 Methanol 32.6 7 7 7 7 Ethanol 24.3 4 7 7 7 Acetone .7 7 3 3 2 2-Propanol .1 7 2 7 8 n-Butanol 17.1 7 7 7 7 Ethyl acetate 6.0 1 Chloroform 4.8 (20°C) 9 Ethyl ether 4.3 9 Toluene 2.4 7 6 9 Benzene 2.3 7 6 6 9 Hexanes, prac t i cal 1.9 (20OO 7 7 7 9 1 = Cloudy, total suspension 2 = Cloudy, partial suspension 3 = Cloudy, partial suspension with floating material 4 = Cloudy, slight suspension = Partial, particulate suspension 6= Partial, particulate suspension with f1 oating material 7 = Transient suspension, insoluble 8 = Transient partial suspension, insoluble 9 = Insol ubl e 205569 r43- — | 10sepi984 \ L— [7 received EXAMPLE 1? A solution o-f mi crocrystal 1 i ne cloud -fractions -from Example 16 (Figures 7 and . 8) was prepared by shaking 20 parts o-f the moist precipitate in 188 parts o-f 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, 188 parts of liquid vegetable oil (peanut oil) were added arid 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 microcrystal1ine 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 28 Fmnl si f i rat i-on-of-DranQP Pnlp-Uastt Following the procedure of the preceding example, 18 ml of orange pulp wash (the HjO soluble fraction of citrus pulp and ruptured juice vesicles) was added to 18 ml of distilled water containing 2g of microcrystal1ine 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 sma;l 1 portion of liquid had formed a lower, clearing layer under the emulsion. With the microcrystal1ine 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 38 minutes, and the remaining emulsion layer was not thick as in the samples containing the cloud fraction. 205569 EXAMPLE 21 Fmii Isifinalion nf Hexanes This example i11ustrates the ability o-f the mi crocrystal 1 i ne cloud ■fraction o-f this invention "to emulsify non-polar hydrocarbons. Following the procedure of the preceding Examples, 18 ml of technical hexanes was added to 18 ml of distilled water containing 2 g of microcrystal1ine 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 microcrystal1 ine cloud fraction. The technical hexanes and water separated completely into two different, clear phases immediately after vortex mixing was ended.

EXAMPLE 22 Using South Dakota intermediate grade crude oil containing 1.6 percent sulfur, 18 ml of the oil sample were added to 18 ml distilled water containing 2 g of both of the microcrystal1ine cloud fraction of the preceding examples. Samples containing microcrystal1ine cloud fraction formed two stable phases. The upper phase became gelatinous, with the Figure 8 microcrystal!ine cloud fraction sample gelling at about 98 minutes, and the Figure 7 sample gelling less dramatical1y 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 microcrystal1 ine cloud fraction. The oil and water formed a single liquid phase and formed no gel upon standing. 205569 EXAMPLE 23 Fmii 1 q. i f i r a t i nn nf Rpntnnitp This example illustrates the use of the microcrystal]ine cloud ■fraction to emulsify particulate inorganic solids. Following the procedures of the preceding examples, 0,2g bentonite was added to 28 ml distilled water containing 2g of dry microcrystal1ine cloud fraction obtained according to Example 16, With the Figure 3 microcrystal1ine 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 microcrystal1ine cloud fraction. Immediately after mixing, a single, frothy layer was obtained which, within approximately 8.5 hr, also shewed the presence of a lower, clear layer.

EXAMPLE 24 This example illustrates the capacity of microcrystal1ine cloud fraction to emulsify and gel protein. 188 ml aqueous solutions were prepared using microcrystal1ine cloud fraction generated from ULP commerci&11y available from Express Foods Co. and Savorpro 75 whey protein concentrate which is also commercial1y available from Express Foods Co. Samples were whipped in a Waring blender at high speed for 3 mi nutes. Foam height and the viscosity of the resulting emulsi ons were documented, demonstrating that the addition of either 18 or 28 percent microcrystal1ine cloud fraction increased both the foam height and viscosity of 18 percent whey protein concentrate solutions. The results are shown in Table 18.

TABLE 18 EMULSIFICATION OF PROTEIN BY CLOUD FRACTION 109 ml aqueous- solution whipped at high speed ■for 3. mi nutes Foam height when 109ml 1i q settled out in 200ml beaker Viscosity (Seconds for 5ml to drop from pi pet; relative to a value of 3 for H^O .10 percent WLP percent WLP, 10 percent cloud fract i on percent WLP, 20 percent cloud fract i on 1 .8 cm 2.5 cm 3.5 cm 4 sec sec .S sec .

EXAMPLE 25 CbIUqq nl...Pc.ote ia_by nioujd-Enaclion In addition to emulsifying protein, microcrystal1ine cloud fraction also gels protein at concentrations lower than that at which gelling would normally occur. Using the same materials described above, 18 ml aqueous solutions were prepared, vortexed, and incubated at 80°C for 20 minutes. With the addition of 20 percent microcrystal1ine cloud fraction, 10 percent whey protein concentrate solidifies at 30°C. Without the addition of microcrystal1ine cloud fraction no solidification of the 18 percent protein solution occurs, as shown in Table 1?: 205569 4? , '..TBJT OFFICE j 10sep1984 L- i Aqueous Rnlntinn TABLE 19 GELLING OF PROTEIN SOLUTIONS £M£ -f nr ?fl . m i nuias percent cloud -fraction 20 percent cloud fraction percent WLP 20 percent WLP percent cloud fraction + percent WLP 10 percent cloud fraction + 20 percent WLP percent cloud fraction + percent WLP 20 percent cloud fraction + 20 percent WLP slight suspension w/large pellet sediment si ight suspension w/large pellet sediment cloudy suspension, thick coating sol id pellet milky suspension w/pellet sol id pellet 1:1 solid pellet and thick coating solid pellet EXAMPLE 26 Preparation of Industrial Fermentation Permeate is prepared as in Example 16, 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 supp1ementing with 0.25 percent Amber 510 yeast extract and 1.0 percent dextrose prior to autoclaving for 20 minutes at 121°C/15 psi. The resulting autoclaved culture mediurn 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 Rintprhnnlngy.and Bi.flfth.Yii.es—XXL! pages 525-539 (1983) at a rate of 6 ml/mi n at a temperature of 37°C, resulting in the conversion of 47 percent of the 2055 permeate lactose to glucose and galactose by immobilized acid lactase enzyme. The enzymatic conversion was carried out without pH adjustment o-f the permeate and was, there-fore, at a pH that was non-optimal -for the acid lactase enzyme. This use o-f 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 o-f 3,0 percent, this lactase treated permeate contains 1.24 percent glucose. 3.0 percent 1actase-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 o-f 6.5.

The glucose supplemented and 1 ac tase-treated basic culture media o-f 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.

Medi um TABLE 20 REPRESENTATIVE GROWTH IN INDUSTRIAL FERMENTATION MEDIA WITH INCREASED GLUCOSE CONTENT Microorganism S..aureus S^.f aeralis B. c.uht i 1 is P.flunr&scens Basic culture medium for industrial fer-mentat i on.

+ + + + + + + + + + + + + + + + + + Glucose supplemented basic culture medium ■for industrial "fermentation. + + + + + + + + + + + + + + + + + + Lactose-treated basic culture medi um for industrial fermentation. + + + + + + + + + + + + + + + + 4 + + 205569 p.—_—-—-—i atent office i SEP 1984 RECEIVED I Industrial culture media with a wide range o-f glucose to lactose ratios can be prepared by varying the extent o-f 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 soli ds.

EXAMPLE 27 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 518 or Amber 1803 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 St r.e p±ncQf c u S-.r.nemncis (ATCC 19257) , Sirep±£>corcus_Lacl-Ls (ATCC 19435), 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. 205569 N.Z. PATENT OFFICE I 1V/~M-' 1 CT 10SEP1984 The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions o-f this invention -for those specifically used in 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.

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 produc ts. the examples. From the foregoing description, one skilled in the art 51 205569

Claims (1)

1. WHAT WE CLAIM IS: 1: A process for converting dairy whe/ lactose permeate into a microcrystalline cloud fraction and a solute phase, which ccmprises: a) raising the pH c£ a substantially deproteinized dairy whe/ lactose permeate, having a pH below 7, to a pH between 8 and 10 to form a lactose-rich aqueous solute phase and a microcrystalline cloud fraction, which fraction contains substantially all of the dissolved solids f rem said permeate which would form a precipitate upon autoclaving said permeate for 10-20 minutes at 121°C and 15 psi, so that a clear, light colored solute having a pH of substantially 7 can be formed when the alkaline permeate is so autoclaved, said microcrystalline cloud fraction being characterised by being insoluble in petroleum ether after drying to form a pewder; b) separating the microcrystalline cloud fraction -from the solute phase; and c) recovering at least one o-f the microcrystalline cloud -fraction and the solute phase. 2: A process according to claim 1, wherein the pH is raised to substantially pH 9. 3: A process according to claim microcrystalline cloud -fraction is separated -from the ultra-filtration across a 10 - 100 kdal membrane -filter. 4: A process according to claim 1, -further comprising lowering the pH of the separated solute phase to 6.8-7.1. 5: A process according to claim 4, wherein the pH is lowered by the addition c-f a nontoxic acid to the solute phase. 6: A process according to claim 4, wherein the pH is'Tower-ed-by autoclaving the solute phase to form a sterile micro culture med i um. 1, where i n the solute phase by 205569 52 7: A process according to claim 6, wherein the pH is lowered without the addition o-f extraneous acid to the solute phase. 8: A process according to claim 1, -further comprising spray drying the separated solute phase to a moisture content o-f less than 18 percent by we i gh t. 9: A microbiological culture medium capable of supporting the growth of microorganisms under suitable growth conditions, consisting essentially of the solute phase obtained according to the process of claim 1, which is substantially free of components which would be retained by a filter having a pore size which passes components having a molecular .we i gh-t below 100 kdal and which contains substantially all of the components which would be passed by a filter having a pore size which would retain components having a molecular weight above 20 kdal. 10: A microbiological culture medium according to claim 9^ 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 30 kdal. 11: A microbiological culture medium according to claim 9 having a pH of 6.8 - 7.1. 12: A microbiological culture medium according to claim 9 in| a sol ids content of substantially 3.5 percent (wt/vol) . nil Ict 1987 yy I 13: A sterile microbiological culture medium according to 14: A microbiological culture medium according to claim 9 in the form of a free-f1owing powder having a moisture content of less than 10 percent by weight. 53 205569 15; A microbiological culture medium according to claim 9, •further comprising a growth promoting amount of extraneous nontoxic assimilable carbon atoms. 16: A microbiological culture medium according to claim 15, wherein the source of carbon a tans is glucose. 17: A microbiological culture medium according to claim 9, further comprising a growth promoting amount of extraneous nontoxic assimilable nitrogen atoms. 13: A microbiological culture medium according to claim 17, wherein the source of nitrogen., atoms is a yeast extract, yeast autolysate, hydrolyzed casein, soy protein or soy protein hydrolyzate, or a mixture thereof. 19: A microbiological culture medium according to claim 9, further comprising an effective amount of a nontoxic gelling agent. 20: A microbiological culture medium according to claim 9, having nutrient grcwth characteristics of a fermentation medium, characterized fcy having a solids content of substantially 3.5 percent (wt/vol), and further comprising substantially 0.25 percent fcy weight water - soluble ^bre^er1 s yeast extract. 1. v 21: A microbiological culture mediun according to claim 9, having nutrient grcwth characteristics comparable to penassay broth or nutrient °\»hroth, and further comprising 0.25 - 0.5 percent by weight hydrdyzed 'casein, substantially 0.05 percent fcy weight yeast extract, and a total glucose content cf 0.05 - 0.1 percent fcy weight. : A microbiological culture mediun according to claim 9, having nutrient growth characteristics comparable to thiogly col late broth, and further comprising an effective amount cf a nontoxic gelling 54 20S56cI agent to reduce oxygen diffusion therein, 0.25 - 0.5 percent fcy weight hydrolyzed casein, substantially 0.5 peroent fcy weight yeast extract, substantially 0.05 percent fcy weight cysteine HCl, and a total glucose content of substantially 0.5 percent fcy weight. 23: A microbiological culture medium according to claim 9, adapted for the cultivation of anaerobic bacteria, further canprising substantially 0.25 percent fcy weight hydrolyzed casein, substantially 1 percent fcy weight yeast extract, substantially 0.2 percent by weight cysteine HCl, substantially 0.05 peroent (wt/vol) hemin, substantially 0.1 percent (wt/vol) vitamin , a total glucose content of substantially 0.5 percent fcy weight, a pH of substantially 7.8, an oxidation-reduction potential of -150 mV or less,and an effective amount of an oxidation-reduction colorimetric indicator. 24: A microbiological culture medium according to claim 23, wherein said indicator is substantially 0.001 percent fcy weight resazurin. 25: In a bulk microbiological starter mixture comprising viable microorganisns and a suitable nutrient mediun therefor, the improvement wherein said nutrient medium is the microbiological culture medium according to claim 9. 26: An improvement according to claim 25, wherein said microorganisms are cheese-producing microorganisms. 27: In a process for growing a microorganian in vitro under submerged culture nutrient growth conditions in a culture medium containing assimilable carbon, nitrogen, and phosphorus sources, the imprcvenent wherein said culture mediun is the culture medium of claim 9. 28: An improvement according to claim 27, wherein the microorganisn is a bacterium. 205569 55 2?: An improvement according to claim 27, wherein the microorganism is selected -from the group consisting o-f Bar i 1.1 jus, Lactobacillus, KluxuEComxcas, and Sacchac.nmyces species. 39: An improvement according to claim 27, wherein the rn i C r oor g an i sm is RariUtic mrpm c.nhc . thur i ngi pnc,i C,, 31: An improvement according to claim 27, wherein the mi croorgan i sm is selected -from the group consisting o-f As.p.£.cQi 11ns, EenicilJLLum, and Slxsptraaycas species. 32: An improvement according to claim 31, wherein the microorganism is PpniriIlium nntatum- . 33. An improvement according to claim 31, wherein the mi croorgan i sm i s Strpp trimyr£S-Qr.Ls£xis. 34: An improvement according to claim 27, wherein the mi croorgan i sm is obtained -from a clinical isolate. 35: A process according to claim 1, -further comprising drying the separated microcrystalline cloud -fraction to -form a tasteless, odorless, chalky white free—flowing powder. 36: A nontoxic food grade additive consisting essentially of the tasteless, odorless, chalky white free-f1owing powder obtained according to the process of claim 35. 37: In a food, pharmaceutical, cosmetic, or dentifrice composition of matter comprising an effective amount of an added clouding agent, stabilizing agent, emulsifying agent, or thickening agent, the improvement wherein the agent according to claim 36. * 1 o * 205569 56 r* - \^y> 38: In a process for -forming a stable emulsion or suspension o-f a plurality of immiscible materials by adding an emulsifying or stabilizing agent thereto, the improvement wherein said emulsifying or stabilizing agent is the additive of claim 36. 39: An improvement acoording to claim 38, wherein said immiscible materials include an oil and water as the major portion thereof. 40: An improvement according to claim 39, wherein the oil is an edible vegetable oil. 41. A process as claimed in claim 1 substantially as herein described with reference to the accompanying drawings and to the examples herein. 42. A microbiological culture medium as claimed in claim 9 substantially as herein described with reference to the accompanying drawings and to the examples herein. J. D. HARDIE & CO. Patent Attorneys for the Applicant(s).