EP0119254A1 - Umwandlung geklärter laktosepermeaten aus molke in kulturmedien und sonstige kommerziell nutzbare erzeugnisse - Google Patents

Umwandlung geklärter laktosepermeaten aus molke in kulturmedien und sonstige kommerziell nutzbare erzeugnisse

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Publication number
EP0119254A1
EP0119254A1 EP83903238A EP83903238A EP0119254A1 EP 0119254 A1 EP0119254 A1 EP 0119254A1 EP 83903238 A EP83903238 A EP 83903238A EP 83903238 A EP83903238 A EP 83903238A EP 0119254 A1 EP0119254 A1 EP 0119254A1
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EP
European Patent Office
Prior art keywords
percent
culture medium
microbiological culture
process according
microcrystalline
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP83903238A
Other languages
English (en)
French (fr)
Other versions
EP0119254A4 (de
Inventor
Kathleen M. Keggins
Ann C. Davis
Edward M. Sybert
Thomas D. Mays
Robert Austin Milch
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Igi Biotechnology Inc
Original Assignee
Igi Biotechnology Inc
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Filing date
Publication date
Priority claimed from US06/418,067 external-priority patent/US4544637A/en
Application filed by Igi Biotechnology Inc filed Critical Igi Biotechnology Inc
Publication of EP0119254A1 publication Critical patent/EP0119254A1/de
Publication of EP0119254A4 publication Critical patent/EP0119254A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K5/00—Lactose
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00—Whey; Whey preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Definitions

  • 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.
  • WLP substantially deproteinized dairy whey lactose permeates
  • 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).
  • 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.
  • Whey colloidal precipitates have found use as clouding, stabilizing, emulsifying, thickening, and gelling additives (depending in general on the concentration in which the precipitate is employed) to food grade compositions, e.g. as described by U.S. Patents 4,143,174 and 4,209,503 to Syed M.M. Shah et al . Shah et al . do not describe useful applications for the supernatant liquid which is separated from the colloidal precipitate.
  • a variety of solids can be obtained from dairy whey permeates, depending on the temperature, pH, and other conditions under which they are formed, e.g. see Eustache U.S. Patents 4,042,575 and 4,042,576.
  • Pederson describes in Patent No. 4,202,909 a method for recovering lactose from whey permeates by forming a precipitate upon heating to 180-200 ⁇ F, and separating the supernatant liquid therefrom. Other than the recovery of lactose, Pederson does not disclose any industrial or commercial uses for the precipitate or for the supernatant liquid.
  • 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.
  • 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.
  • OMPI 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.
  • FIGS. 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.
  • 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.
  • 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.
  • 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.
  • 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
  • OMPI 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.
  • the solute fraction may be supplemented with an appropriate nutrient, then sterilized by autoclaving or filtration.
  • 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.
  • the supplemented or unsupplemented solute fraction may be utilized in either batch or continuous processes.
  • 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.
  • 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 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.
  • 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 ⁇ C, and pressures of about 60 - 145 psi.
  • the WLP starting material is 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.
  • 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.
  • 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.
  • 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.
  • the precipitate is physically separated from the culture medium, e.g. by centrifugat on at ll,750g and filtration across a 0.45 ⁇ 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.
  • 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.
  • 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).
  • WLP although essentially protein free, generally contains adequate amounts of metabolizable nitrogen in the form of free amino acids and low molecular weight polypeptides.
  • 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.
  • 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.
  • buffering agents i.e., to an optimal pH range in which the organism is viable
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any desired supplements can be added prior to autoclaving at 121°C for 15-20 minutes.
  • various types of culture media can be readily prepared from the basic unsupplemented WLP solute fraction.
  • Presently preferred media are:
  • 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;
  • PIM primary isolation medium
  • 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.
  • 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
  • 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.
  • any conventional gelling agent can be added, e.g. about 1.5 percent agar.
  • Lactose 81.5 Microbiological
  • Industrial fermentation media with increased glucose content consist 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.
  • solute fraction may be utilized, either in the supplemented or unsupplemented form, to produce antibiotics, such as streptomycin and penicillin.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • the liquid medium is easily prepared in the form of pour plates by the addition of 1.5 percent (wt/vol) agar.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • OMPI 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.
  • 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.
  • 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.
  • 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.
  • acid range such high acidity
  • OMPI . W1PO may result in degradation of polypeptide components present in the microcrystalline cloud fraction.
  • 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.
  • 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.
  • WLP obtained from Express Foods Co. and similar to products commercially available from Foremost McKesson, Inc. and other sources
  • 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 0.45 ⁇ , 115 ml Nalgene filtration unit, yielding 200 ml of clear material having a pH of 9.04.
  • EXAMPLE 2 Preparation of Supplemented Culture Medium From Acid (Sour) Dairy Whey Solute Fraction
  • 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
  • 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:
  • 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.
  • OMPI EXAMPLE 7 Effect of Autoclaving on Growth
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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
  • PeniciIlium notatum disperse bead ⁇ 0.47g/100 ml like growth
  • 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.
  • 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.
  • 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.
  • 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
  • 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 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 lOO ⁇ m.
  • 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.
  • 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.
  • Petroleum Insoluble Insoluble Insoluble ether 10 (film) (film) (film) percent solids
  • Petroleum Insoluble Insoluble Insoluble ether 20 (film) (film) (film) percent solids
  • 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.
  • This example illustrates the ability of the microcrystalline cloud fraction of this invention to emulsify non-polar hydrocarbons.
  • 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.
  • an upper foam layer extended to the top of the test tube, and the foam was still at this height after 37 minutes.
  • the foams in both tubes had become gelatinous.
  • This example illustrates the use of the microcrystalline cloud fraction to emulsify particulate inorganic solids.
  • 0.2g bentonite was added to 20 ml distilled water containing 2g of dry microcrystalline cloud fraction obtained according to Example 16.
  • an upper foam and a lower, frothy layer were formed.
  • the frothy layer was stable for at least 18 hours.
  • 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.
  • microcrystalline cloud fraction also gels protein at concentrations lower than that at which gelling would normally occur.
  • 10 ml aqueous solutions were prepared, vortexed, and incubated at 80°C for 20 minutes.
  • 10 percent whey protein concentrate solidifies at 80"C.
  • no solidification of the 10 percent protein solution occurs, as shown in Table 19:
  • 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 ⁇ 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.
  • 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.
  • 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 2 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 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.
  • 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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EP19830903238 1982-09-14 1983-09-06 Umwandlung geklärter laktosepermeaten aus molke in kulturmedien und sonstige kommerziell nutzbare erzeugnisse. Withdrawn EP0119254A4 (de)

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US418067 1982-09-14
US06/418,067 US4544637A (en) 1982-09-14 1982-09-14 Culture media from clarified diary whey lactose permeates
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US4698303A (en) * 1985-02-15 1987-10-06 Engenics, Inc. Production of lactic acid by continuous fermentation using an inexpensive raw material and a simplified method of lactic acid purification
NZ216353A (en) * 1985-06-05 1988-05-30 Univ Kentucky Res Found Manufacture of lac + fungi
US4771001A (en) * 1986-03-27 1988-09-13 Neurex Corp. Production of lactic acid by continuous fermentation using an inexpensive raw material and a simplified method of lactic acid purification

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FR1443098A (fr) * 1965-01-20 1966-06-24 Perfectionnements à la fabrication des fromages
FR2274231A1 (fr) * 1974-06-13 1976-01-09 Baignes Ste Radegonde Laiterie Procede d'epuration et de valorisation des sous-produits laitiers
US3963580A (en) * 1975-02-10 1976-06-15 Microlife Technics, Inc. Method for determining the suitability of milk for bacterial fermentation activity
DE2631655A1 (de) * 1976-07-14 1978-01-19 Franz Xaver Julius Prof Roiner Verfahren zum herstellen eines kulturenkonzentrats fuer saure lebensmittel

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US3930039A (en) * 1971-07-30 1975-12-30 Molkerei J A Meggle Milchindus Method of preparing a protein concentrate from whey
CH556143A (fr) * 1972-09-11 1974-11-29 Nestle Sa Procede de preparation d'une fraction soluble des proteines du petit lait.
US4036999A (en) * 1974-08-30 1977-07-19 Stauffer Chemical Company Treatment of whey
FR2288477A1 (fr) * 1974-10-22 1976-05-21 Manche Union Coop Agr Laiti Procede de production d'acide sialique et de glycoproteines a partir de lactoserum de fromagerie
FR2288473A1 (fr) * 1974-10-22 1976-05-21 Manche Union Coop Agr Laiti Procede de traitement du lactoserum de fromagerie, notamment en vue de l'extraction de glycoproteides et d'acide sialique
US4143174A (en) * 1975-07-24 1979-03-06 Beatrice Foods Co. Food composition containing whey colloidal precipitate
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US4402986A (en) * 1981-07-23 1983-09-06 Stauffer Chemical Company Bulk starter media

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FR1443098A (fr) * 1965-01-20 1966-06-24 Perfectionnements à la fabrication des fromages
FR2274231A1 (fr) * 1974-06-13 1976-01-09 Baignes Ste Radegonde Laiterie Procede d'epuration et de valorisation des sous-produits laitiers
US3963580A (en) * 1975-02-10 1976-06-15 Microlife Technics, Inc. Method for determining the suitability of milk for bacterial fermentation activity
DE2631655A1 (de) * 1976-07-14 1978-01-19 Franz Xaver Julius Prof Roiner Verfahren zum herstellen eines kulturenkonzentrats fuer saure lebensmittel

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CHEMICAL ABSTRACTS, vol. 85, no. 1, 5th July 1976, page 326, abstract 3993s, Columbus, Ohio, US; M.S. FODA: "A novel approach for whey utilization in production of native and microbial proteins", & MILCHWISSENSCHAFT 1976, 31(3), 138-41 *
CHEMICAL ABSTRACTS, vol. 90, 26th March 1979, page 433, abstract 101921q, Columbus, Ohio, US; A. GIEC et al.: "Ultrafiltration of whey for protein concentrates and yeast/propionic acid bacterial biomass production", & ACTA ALIMENT. POL. 1978, 4(3), 247-54 *
FOOD SCIENCE TECHNOLOGY ABSTRACTS, vol. 83, no. 1, ref. 83004116; S. SOBACK: "Growth of some lactic acid bacteria in milk containing sulfadiazine", & ACTA VETERINARIA SCANDINAVICA, vol. 22, no. 3/4, pages 493-500, 1981 *
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See also references of WO8401104A1 *

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