NO880279L - BACTERY LYZING ENZYM PRODUCT FROM STREPTOMYCETS, ITS MANUFACTURING AND USE FOR CONSERVATION OF CHEESE. - Google Patents
BACTERY LYZING ENZYM PRODUCT FROM STREPTOMYCETS, ITS MANUFACTURING AND USE FOR CONSERVATION OF CHEESE.Info
- Publication number
- NO880279L NO880279L NO880279A NO880279A NO880279L NO 880279 L NO880279 L NO 880279L NO 880279 A NO880279 A NO 880279A NO 880279 A NO880279 A NO 880279A NO 880279 L NO880279 L NO 880279L
- Authority
- NO
- Norway
- Prior art keywords
- enzyme product
- cheese
- bacteria
- product
- lysing
- Prior art date
Links
- 238000004519 manufacturing process Methods 0.000 title description 6
- 239000000047 product Substances 0.000 claims description 64
- 102000004190 Enzymes Human genes 0.000 claims description 57
- 108090000790 Enzymes Proteins 0.000 claims description 57
- 235000013351 cheese Nutrition 0.000 claims description 30
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 241000187747 Streptomyces Species 0.000 claims description 4
- 241000187432 Streptomyces coelicolor Species 0.000 claims description 4
- 230000002101 lytic effect Effects 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000012228 culture supernatant Substances 0.000 claims description 3
- 235000011617 hard cheese Nutrition 0.000 claims description 3
- 238000004255 ion exchange chromatography Methods 0.000 claims description 3
- 230000002934 lysing effect Effects 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 description 47
- 235000013336 milk Nutrition 0.000 description 11
- 239000008267 milk Substances 0.000 description 11
- 210000004080 milk Anatomy 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000000243 solution Substances 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 230000005070 ripening Effects 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 241001655322 Streptomycetales Species 0.000 description 4
- 239000008351 acetate buffer Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010011756 Milk Proteins Proteins 0.000 description 2
- 102000014171 Milk Proteins Human genes 0.000 description 2
- 206010042674 Swelling Diseases 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 229920001429 chelating resin Polymers 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000021239 milk protein Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 241000194034 Lactococcus lactis subsp. cremoris Species 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 240000002129 Malva sylvestris Species 0.000 description 1
- 235000006770 Malva sylvestris Nutrition 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 241001153358 Micrococcus luteus NCTC 2665 Species 0.000 description 1
- NUVFQILPDFENMM-UHFFFAOYSA-J O.O.Cl[Ca]Cl.Cl[Ca]Cl Chemical compound O.O.Cl[Ca]Cl.Cl[Ca]Cl NUVFQILPDFENMM-UHFFFAOYSA-J 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 235000014962 Streptococcus cremoris Nutrition 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000002535 acidifier Substances 0.000 description 1
- 229940095602 acidifiers Drugs 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- QXDMQSPYEZFLGF-UHFFFAOYSA-L calcium oxalate Chemical compound [Ca+2].[O-]C(=O)C([O-])=O QXDMQSPYEZFLGF-UHFFFAOYSA-L 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 229940071162 caseinate Drugs 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 235000019879 cocoa butter substitute Nutrition 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000000416 hydrocolloid Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000001806 lysozymelike Effects 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 150000002734 metacrylic acid derivatives Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical class [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 229940108461 rennet Drugs 0.000 description 1
- 108010058314 rennet Proteins 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 235000008983 soft cheese Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- 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
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
- A23C19/0328—Enzymes other than milk clotting enzymes, e.g. lipase, beta-galactosidase
-
- 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
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
- A23C19/063—Addition of, or treatment with, enzymes or cell-free extracts of microorganisms
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
Description
I fransk patent 80 03 321 omtales anvendelsen av lysozymak-tlge produkter fra sekreter av dyr og planter til å hindre senoppblåsning i forskjellige ostetyper. Senoppblåsning ytrer seg i løpet av ostemodningen ved en materialveksling av den tilstedeværende melkesyre under gassdannelse som fører til sterke teksturfeil. Sporene av de anaerobe mikroorganismer som forårsaker denne senblæring, lyserer med de nevnte enzymprodukter med utkimingen. French patent 80 03 321 mentions the use of lysozymak-containing products from secretions of animals and plants to prevent late swelling in different types of cheese. Late swelling manifests itself during cheese ripening by a material exchange of the lactic acid present during gas formation, which leads to strong textural defects. The spores of the anaerobic micro-organisms which cause this late blister, lyse with the mentioned enzyme products with the germination.
Fremstillingen av et baktkerielyserende enzymprodukt fra Streptomyceter er kjent og eksempelvis omtalt i de tyske Offenlegungsschrifter 20 11 935, 20 40 444, 21 46 597 og 34 40 735. Riktignok lar disse fra Streptomyceter fremstilte enzymprodukter seg uten videre anvende til konservering av ost. The production of a bacterial lysing enzyme product from Streptomyceter is known and, for example, described in the German Offenlegungsschrifter 20 11 935, 20 40 444, 21 46 597 and 34 40 735. It is true that these enzyme products produced from Streptomyceter can be used without further ado for the preservation of cheese.
Overraskende har det vist seg at Streptomycet-enzymproduktet, når det eksempelvis fremstilles etter det tyske Offenlegungs-schrift 34 40 735 er er fulgt, med proteaser og bare vanskelig å adskille fra disse. Det er nu funnet at ved hjelp av bakterielyserende proteasefrie enzymprodukt fra Streptomyceter, kan osteprodukter konserveres med bedre resultat enn med tilsvarende produkter fra sekreter av dyr og planter. Ikke bare senblæringen hindres effektivt, men det ble også funnet at vegetative gram-negative kimer, eksempelvis E.coli eller også patogene bakterier, f.eks. Salmonella, Shigella, Campylobactger eller Listeria likeledes hindres i vekst ved enzymprodukter. Dette faktum er av spesiell viktighet med hensyn til konservering av fra råmelk fremstilt ost. I USA opptrådte delvis dødelig forløpende forgiftninger, ved spising av mykost som var kontaminert med Listerai monocyto-genes. Dessuten kan ved hjelp av enzymprodukter fra Strepto-mycetes, den ved hjelp av E.coli frembragte tidlig bæring undertrykkes. Ost lar seg også på denne måte fremstille med lav utgangskontaminasjon, og derved som helhet bedre lag-ringsdyktig. Surprisingly, it has been shown that the Streptomycet enzyme product, when it is produced for example according to the German Offenlegungs-schrift 34 40 735, is accompanied by proteases and is only difficult to separate from these. It has now been found that with the help of bacteria-lysing protease-free enzyme products from Streptomyceters, cheese products can be preserved with better results than with similar products from animal and plant secretions. Not only is late blight effectively prevented, but it was also found that vegetative gram-negative germs, for example E.coli or also pathogenic bacteria, e.g. Salmonella, Shigella, Campylobacter or Listeria are likewise prevented from growing by enzyme products. This fact is of particular importance with regard to the preservation of cheese made from colostrum. In the United States, partially fatal poisonings occurred when eating shellfish contaminated with Listerai monocytogenes. Moreover, with the aid of enzyme products from Strepto-mycetes, the early bearing produced by E.coli can be suppressed. Cheese can also be produced in this way with low initial contamination, and thereby better storability as a whole.
Oppfinnelsen vedrører således: 1.. - Et bakterielyserende enzymprodukt oppnådd ved fermentering av bakterier av.slekten Streptomyces som erkarakterisertved en spesifik lytisk aktitivtet på 20 000 - 60 000 U/mg protein. • The invention thus relates to: 1.. - A bacteria-lysing enzyme product obtained by fermentation of bacteria of the genus Streptomyces which is characterized by a specific lytic activity of 20,000 - 60,000 U/mg protein. •
2. -Fremgangsmåte til fremstilling av det under punkt 1 definerte bakterielyserende enzymprodukt, idet fremgangsmåten erkarakterisert vedat 2. -Procedure for the production of the bacteria-lysing enzyme product defined under point 1, the method being characterized by
a) en bakterie fra slekten Streptomyces kultiveres med et næringsmedium og b) det i kulturoverstående befinnende nevnte enzymprodukt isoleres ved rensning ved hjelp av ioneutvekslerkromatografi. 3. Anvendelse av.bakterielyserende enzymprodukt som definert under punkt 1 - til konservering av osteprodukter. a) a bacterium from the genus Streptomyces is cultivated with a nutrient medium and b) the said enzyme product present in the culture supernatant is isolated by purification using ion exchange chromatography. 3. Application of bacteria-lysing enzyme product as defined under point 1 - for the preservation of cheese products.
I det følgende forklares oppfinnelsen detaljert, spesielt i dens foretrukne utførelsesform. Videre defineres oppfinnelsen i patentkravene. In the following, the invention is explained in detail, especially in its preferred embodiment. Furthermore, the invention is defined in the patent claims.
Det kan prinsippielt ifølge oppfinnelsen finne anvendelse av ethvert lysosymaktivt produkt fra Streptomyceter. I enkle sammensatte kulturmedier er det ved hjelp av Streptomyceter oppnåelig ved en f ermenteringstid på 1-7 dager, et høyt utbytte av bakterielyserende. enzymprodukt. Fortrinnsvis anvendes imidlertid idet i henhold til tysk Offenlegungs-schrift 34 40 735 fra Streptomyces coelicolor DSM 3030 samt dets varianter og.mutanter,.fremstilte.bakterielyserende enzymprodukt. In principle, according to the invention, any lysozyme-active product from Streptomyceter can be used. In simple compound culture media, with the help of Streptomyceter, a high yield of bacteria lyser can be achieved with a fermentation time of 1-7 days. enzyme product. Preferably, however, it is used according to German Offenlegungs-schrift 34 40 735 from Streptomyces coelicolor DSM 3030 as well as its variants and.mutants,.produced.bacteria-lysing enzyme product.
Spesielt har det vist seg egnet for dyrkning av Streptomyces coaelicolor DSM 3030 en tilsetning av sukkerroemelasse i en mengde fra 5-50 g, fortrinnsvis 10 - 20 g, pr. liter kulturmedium.. En ytterligere utbytteøkning oppnår man til kulturmediet setter kalsiumioner i form av litt oppløselig ugiftig kalsiumsalter, fortrinnsvis i form av det prisguns-tige kalsiumklorid. Fordelaktig er en 0,051 molar kalsium-ionkonsentrasjon, spesielt foretrukket er konsentrasjoner på 100-500 mol, eksempelvis 1 form av en tilsetning av 0,2 -0,5 vekt-# av dikalsiumklorid-dihydrat. In particular, an addition of sugar beet molasses in an amount of 5-50 g, preferably 10-20 g, per liter of culture medium.. A further yield increase is achieved by adding calcium ions to the culture medium in the form of slightly soluble non-toxic calcium salts, preferably in the form of the inexpensive calcium chloride. A 0.051 molar calcium ion concentration is advantageous, particularly preferred are concentrations of 100-500 mol, for example 1 form of an addition of 0.2-0.5 wt-# of dicalcium chloride dihydrate.
Da det fra Streptomyceter utvunnede bakterielyserende enzymprodukt er fulgt av proteaser, må det før anvendelsen som konserveringsmiddel i ost først befries for disse. Denne oppdeling kan foregå ved ioneutvekslerkromatografi fortrinnsvis på svakt sure mikroporøse kationeutvekslere som f.eks. kryssbundet metakrylater ("Amberlite" ). Det ønskede enzymprodukt forblir omtrent kvantitativt bundet til utvekslingshar-piksen mens hoveddelen av proteasen forblir i kulturoppløs-ningen. Eluering med 0,05 - 0,5 molar saltoppløsning, fortrinnsvis NaCl oppløsning, eluereres den gjenblivne rest av proteasene fra søylen. Deretter resorberes det bakterielyserende enzymprodukt med 0,5 til 2 molar saltoppløsning, eksempelvis en NaCl-acetat eller -laktatoppløsning fra søylen. As the bacteria-lysing enzyme product extracted from Streptomyceters is followed by proteases, it must first be freed from these before being used as a preservative in cheese. This division can take place by ion exchange chromatography, preferably on weakly acidic microporous cation exchangers such as e.g. cross-linked methacrylates ("Amberlite"). The desired enzyme product remains roughly quantitatively bound to the exchange resin while the bulk of the protease remains in the culture solution. Elution with 0.05 - 0.5 molar salt solution, preferably NaCl solution, the remaining residue of the proteases is eluted from the column. The bacteria-lysing enzyme product is then resorbed from the column with a 0.5 to 2 molar salt solution, for example a NaCl acetate or lactate solution.
Ved etterfølgende membranfi 1trering, fortrinnsvis ultrafiltrering, eksempelvis på celluloseacetatmembraner med en gjennomtrengelighet på 5000 - 10 000 dalton, avsaltes produktet og konsentreres. Enzymkonsentratet er i denne form lagringsstabilt og kan allerede således anvendes som konserveringsmiddel. Det kan da dessuten tørkes før det anvendes som konserveringsmiddel i ost. Man får ifølge oppfinnelsen et enzymprodukt med en spesifik aktivitet på ca. 20 000 til 60 000 U/mg protein, fortrinnsvis på 30 000 til 40 000 U/mg protein. By subsequent membrane filtration, preferably ultrafiltration, for example on cellulose acetate membranes with a permeability of 5,000 - 10,000 daltons, the product is desalted and concentrated. In this form, the enzyme concentrate is storage-stable and can thus already be used as a preservative. It can also be dried before it is used as a preservative in cheese. According to the invention, an enzyme product with a specific activity of approx. 20,000 to 60,000 U/mg protein, preferably of 30,000 to 40,000 U/mg protein.
Enzymproduktet ifølge oppfinnelsen kan fortrinnsvis settes til kjelemelken allerede før løpetilsetningen. Det er riktignok også mulig å innarbeide enzymet i ferdige oste-brudd, der en jevn fordeling i bruddet 1 imidlertid vanskelig. The enzyme product according to the invention can preferably be added to the boiled milk already before the addition of the run. Admittedly, it is also possible to incorporate the enzyme into finished cheese curds, where an even distribution in the curd 1 is however difficult.
Bakterielyserende enzymprodukt kan anvendes såvel som vanlig konsentrert oppløsning som også i forskjellige formuleringer. Således kan enzymet eksempelvis påføres på en bærer, hvilket har den fordel at ved tykklegning av melken, forblir enzymet omtrent kvantitativt i bruddet. "Coaten" av enzymproduktet har dessuten den ekstra fordel at enzymet langsomt frigjøres i det..- medium som skal konserveres, derved er det sikret lengre virksom aktivitet. Som bærematerialer kan det anvendes melkeproteiner som foreligger i en nativ tilstand. Bærere av enzym oppløses i forhold 20:1 til 200:1 i vann og sprøytes med hverandre deretter i handelsvanlig forstøvnings-tørkere. Også til "Powder Coaten" eksempelvis etter det kjente "Wurster-Prinzip" kan det anvendes vandige oppløsnin-ger av native melkeproteiner som f.eks. kaseinater eller myseproteiner eller andre vanlige næringsmiddelhjelpestoffer som hydrokolloide proteiner, fett (mono-, di- og triglyceri-der), voks etc. Coating-Material og enzym anvendes i et forhold på 10:1 til 1:20. Coating-fremgangsmåten etter Wurster-prinsippet er omtalt i tallrike patenter, således f.eks. i US-PS 32 41 520, 34 45 120, 36 98 133 eller 3-950 891. Bacteria-lysing enzyme product can be used as well as a normal concentrated solution as well as in different formulations. Thus, for example, the enzyme can be applied to a carrier, which has the advantage that when the milk is thickened, the enzyme remains approximately quantitatively in the break. The "coat" of the enzyme product also has the added advantage that the enzyme is slowly released in the medium to be preserved, thereby ensuring longer effective activity. As carrier materials, milk proteins that exist in a native state can be used. Enzyme carriers are dissolved in a ratio of 20:1 to 200:1 in water and then sprayed with each other in commercial spray dryers. Aqueous solutions of native milk proteins such as e.g. caseinates or whey proteins or other common food additives such as hydrocolloid proteins, fats (mono-, di- and triglycerides), waxes etc. Coating material and enzyme are used in a ratio of 10:1 to 1:20. The coating method according to the Wurster principle is discussed in numerous patents, such as e.g. in US-PS 32 41 520, 34 45 120, 36 98 133 or 3-950 891.
Doseringen av enzymproduktet kan svinge innen vide grenser.The dosage of the enzyme product can fluctuate within wide limits.
Et overskudd er ikke skadelig, imidlertid bør hensiktsmessig doseringen dimensjoneres etter melkens forurensning. Derfor er det fordelaktig på forhånd etter i og for seg kjente metoder å fastslå melkens mikrobielle forurensning, og deretter å tilsette den tilsvarende mengde av det bakterielyserende enzymprodukt som er tilstrekkelig for å hindre de skadelige mikroorganismer i en ytterligere formering. Konsentrasjonene kan velges således at det befinner seg 1000 til 15 000 U enzymprodukt, fortrinnsvis 1000 til 10 000 U, spesielt 2000 til 5000 U, respektivt 1 g osteprodukt. Etter tilsetning og fordeling av det bakterielyserende enzymprodukt i melken eller også i ostesbruddet, får man frem videre som vanlig ved ostefremstilling...Starter- og modningskulturer hvorved melken i løpet av ostefremstillingen podes, beskadi-ges ikke. An excess is not harmful, however, the dosage should be sized according to the contamination of the milk. Therefore, it is advantageous to determine the milk's microbial contamination in advance according to methods known per se, and then to add the corresponding quantity of the bacteria-lysing enzyme product which is sufficient to prevent the harmful microorganisms from further multiplying. The concentrations can be chosen so that there are 1000 to 15,000 U of enzyme product, preferably 1,000 to 10,000 U, especially 2,000 to 5,000 U, respectively 1 g of cheese product. After the addition and distribution of the bacteria-lysing enzyme product in the milk or also in the cheese curd, one proceeds as usual in cheesemaking... Starter and ripening cultures by which the milk is inoculated during cheesemaking are not damaged.
Det kan prinsippielt behandles alle ostetyper som f.eks. hårdost, skjæreost, halvfast skjæreost, mykost og friskost, med det bakterielyserende enzymprodukt. Fortrinnsvis anvendes det imidlertid ved fremstilling av hårdost som eksempelvis Emmentaler, bergost, eller Chester, skjæreost som f.eks. Edamer, Gauda eller Tilsiter, og halvfast skjæreost som eksempelvis Wilstermarsch, smørost eller Steinbucher. In principle, all types of cheese can be processed, such as e.g. hard cheese, cut cheese, semi-hard cut cheese, soft cheese and fresh cheese, with the bacteria-lysing enzyme product. Preferably, however, it is used in the production of hard cheese such as Emmentaler, rock cheese, or Chester, cutting cheese such as e.g. Edamer, Gauda or Tilsiter, and semi-hard cheeses such as Wilstermarsch, butter cheese or Steinbucher.
Osteprodukter som inneholder det bakterielyserende enzymprodukt fra Streptomyceter, viser alt etter ostetyper etter 6-12 ukers lagring, også ved høyere temperaturer inntil 20"C, ingen fordervelsesforeteelser. Smaken av den således fremstilte ost er ikke påvirket. Dessuten er det å fremheve at disse produkter kan fremstilles uten tilsetning respektive bare med små mengder av kaliumnitrater. Bakterielyserende enzymprodukter av Streptomyceter lar seg prinsippielt fremstille mere prisgunstig enn de nevnte produkter som fremstilles av sekretene av dyr eller planter. En kostguns-tig fremstilling er forutsetning for en bre anvendelse av enzymet i osteindustrien. Cheese products containing the bacteria-lysing enzyme product from Streptomyceter show, depending on the type of cheese, after 6-12 weeks of storage, even at higher temperatures of up to 20"C, no spoilage phenomena. The taste of the cheese produced in this way is not affected. Furthermore, it should be emphasized that these products can be produced without addition or only with small amounts of potassium nitrates. Bacteria-lysing enzyme products from Streptomyceters can in principle be produced more cost-effectively than the aforementioned products that are produced from the secretions of animals or plants. A cost-effective production is a prerequisite for a wide application of the enzyme in the cheese industry .
Streptomycet-enzymproduktet virker i motsetning til de sammenlignbare kjente produtker også mot gram-negative fordervelsesfrembringere i melk. Mot gram-positive sporedan-nere virkere det mere effektivt enn de andre nevnte produkter. Det må ved samme resultat anvendes forholdsvis mindre Streptomycet-enzymprodukt ved ostefremstillingen enn av de kjente andre lysosymaktige produkter. The streptomycete enzyme product, unlike the comparable known products, also works against gram-negative spoilage agents in milk. Against gram-positive spore formers, it worked more effectively than the other mentioned products. With the same result, comparatively less Streptomycet enzyme product must be used in cheese production than other known lysozyme-like products.
Oppfinnelsen skal forklares nærmere ved hjelp av noen eksempler,hvor prosentangivelsene refererer seg til vekt når intet annet er angitt. The invention shall be explained in more detail with the help of some examples, where the percentages refer to weight when nothing else is stated.
Eksempel 1.Example 1.
Isolering og rensning av det bakterielyserende enzymprodukt. Isolation and purification of the bacteria-lysing enzyme product.
Etter fermenteringen Ifølge eksempel 3 ifølge tysk Offenleg-ungsschrift 35 40 735, blandes fermenteringsvæsken med en til Ca<2+>ekvimolar -mengde av-. Na-oksalat for å utfelle det- i næringsmediet tilstedeværende kalsium. Deretter frasentri-fugeres kalsiumoksalatet med soppmycelet. Kulturoverstående innstilles med. eddiksyre på pH 7,0. 300 ml kryssbundet metakrylat ("Amberlite" IRC-50) I H+<->form ekvilibreres med 0,05 molar Na-acetatbuffer ved pH 6,0. Etter ifylling av utveksleren i en kromatografisøyle, has 10 1 kulturoverstående med 2-3 BV/time (BV=bettvolum) på søylen. Ved vasking av utveksleren med 3-5 BV 0,05 molar Na-acetatbuffer (pH 6) og 5-10 BV 0,15 mol NaCl i 0,05 molar Na-acetatbuffer (pH 6), fjernes proteasen. Deretter elueres det bakterielyserende enzymprodukt med 34 BV, 1 mol NaCl i 0,05 molar N-acetatbuffer (pH 6). Før frystørkning avsaltes den enzymholdige oppløsning ved hjelp av ultrafiltrering (10 000 dalton) og oppkonsentreres. Den- således dannede enzymkonsentrat viser ingen målbar proteaseaktivitet, og har en spesifik lytisk aktivitet ved gjennomsnittlig 40 000 U/mg protein. After the fermentation According to example 3 according to German Offenleg-ungsschrift 35 40 735, the fermentation liquid is mixed with a Ca<2+>equimolar amount of-. Na-oxalate to precipitate the calcium present in the nutrient medium. The calcium oxalate is then centrifuged with the mushroom mycelium. Culture superiors are set with. acetic acid at pH 7.0. 300 ml of cross-linked methacrylate ("Amberlite" IRC-50) in H+<-> form is equilibrated with 0.05 molar Na-acetate buffer at pH 6.0. After filling the exchanger into a chromatography column, have 10 1 culture supernatant with 2-3 BV/hour (BV=bed volume) on the column. By washing the exchanger with 3-5 BV 0.05 molar Na-acetate buffer (pH 6) and 5-10 BV 0.15 mol NaCl in 0.05 molar Na-acetate buffer (pH 6), the protease is removed. The bacteria-lysing enzyme product is then eluted with 34 BV, 1 mol NaCl in 0.05 molar N-acetate buffer (pH 6). Before freeze-drying, the enzyme-containing solution is desalted using ultrafiltration (10,000 daltons) and concentrated. The enzyme concentrate thus formed shows no measurable protease activity, and has a specific lytic activity at an average of 40,000 U/mg protein.
Aktivitetsbestemmelse av bakterielyserende enzymprodukt:Activity determination of bacteria-lysing enzyme product:
Til 2,8 ml av en suspensjon av 0,2 mg Micrococcus luteus ATCC 4698 (Boehringer Mannheim) pr. ml 0,1 molar natriumacetatbuf-fer (pH 5,0), pipetterer 0,2 ml bakterielyserende enzympro-duktholdige prøver, og ved 25'C bestemmes nedgangen av uklarheten ved måling av ekstinksjonen ved 450 nm. Som 1 U defineres ekstinksjonsnedgangen av 0,001 fotometrisk-skalaen-heter pr. minutt. To 2.8 ml of a suspension of 0.2 mg Micrococcus luteus ATCC 4698 (Boehringer Mannheim) per ml of 0.1 molar sodium acetate buffer (pH 5.0), pipette 0.2 ml of bacteria-lysing enzyme product-containing samples, and at 25°C the decrease in turbidity is determined by measuring the extinction at 450 nm. 1 U is defined as the extinction decrease of 0.001 photometric scale units per minute.
Eksempel 2.Example 2.
Anvendelse av bakterielyserende enzymprodukt som konserveringsmiddel i ost. Application of bacteria-lysing enzyme product as a preservative in cheese.
Til 100 1 kjelemelk settes 100 g syrevekker (Streptococcus diacetylactis og Streptococcus cremori, mesofile blandings-kulturer fra firma Wiesby, Nibull) og 50 ml av en 20#-ig vandig CaClg-oppløsning samt e-karotin. Blandingen bringes til en temperatur på 32°C. 5 g av det bakterielyserende produkt ifølge eksempel 1 oppløses i 2 liter kjelemelk og under omrøring i 100 1 melk. Etter ytterligere tilsetning av 120 g av ovennevnte syrevekker , utkobles røreverket for å la det modne i ca. 20 minutter. Modningen er avsluttet når det nådd en pH-verdi på 5,6-6,3, og en Soxlett-Henkel tall på 7-8. To 100 liters of boiled milk, add 100 g of acidifiers (Streptococcus diacetylactis and Streptococcus cremori, mesophilic mixed cultures from the company Wiesby, Nibull) and 50 ml of a 20# aqueous CaClg solution and e-carotene. The mixture is brought to a temperature of 32°C. Dissolve 5 g of the bacteria-lysing product according to example 1 in 2 liters of boiled milk and, while stirring, in 100 1 milk. After a further addition of 120 g of the above-mentioned acid raiser, the mixer is switched off to allow it to mature for approx. 20 minutes. Ripening is finished when a pH value of 5.6-6.3 has been reached, and a Soxlett-Henkel number of 7-8.
Deretter tilsettes melken 30 ml løpe under omrøring. Koaguleringstiden utgjør 30 minutter. I denne tid stoppes røreren. Then add 30 ml of rennet to the milk while stirring. The coagulation time is 30 minutes. During this time, the stirrer is stopped.
Deretter kuttes brudd med en bruddkniv loddrett og vannrett. For å få en jevn bruddstørrelse omrøres igjen ca. 30 minutter. Etter uttømning av mysen, (ca. 40 liter), vaskes bruddet 30 minutter med 30 liter varmt vann (60°C). Etter adskill-else av vaskemysen, presses bruddet ved 3 bar over et tidsrom på 3-4 timer, og legges 24 timer i et saltbad (20$ NaCl). Fractures are then cut vertically and horizontally with a fracture knife. To get a uniform fracture size, stir again approx. 30 minutes. After draining the whey (approx. 40 litres), the fracture is washed for 30 minutes with 30 liters of warm water (60°C). After separation of the whey, the fraction is pressed at 3 bar over a period of 3-4 hours, and placed in a salt bath (20$ NaCl) for 24 hours.
Den etterfølgende modning foregår 6 uker ved 16-20°C. Selv ved lengre modning, inntil 12 uker kan det ikke iakttas fordervelsesforeteeIser. The subsequent ripening takes place for 6 weeks at 16-20°C. Even with longer ripening, up to 12 weeks, no spoilage events can be observed.
Eksempel 3.Example 3.
Fiksering av det bakterielyserende enzymprodukt på en bærer. Fixation of the bacteria-lysing enzyme product on a carrier.
1000 g av et handelsvanlig ikke-denaturert Na-caseinat dispergeres i 49 liter vann. Denne suspensjons pH-verdi innstilles ved hjelp av melkesyre på pH 5,5. Deretter tilsettes 1000 ml av et konsentrat, da det ifølge eksempel 1 fremstilte enzymprodukt med en aktivitet på 1000000 U/ml. Oppløsningen tørkes med en handelsvanlig forstøvningstørker som er utrystet med en forstøvningsdyse ved en lufttemperatur på 80°C. 1000 g of a commercial non-denatured Na-caseinate is dispersed in 49 liters of water. The pH value of this suspension is set to pH 5.5 using lactic acid. 1000 ml of a concentrate is then added, as the enzyme product produced according to example 1 has an activity of 1000000 U/ml. The solution is dried with a commercially available spray dryer equipped with a spray nozzle at an air temperature of 80°C.
Eksempel 4Example 4
1000 g av et ifølge eksempel 1 fremstillet enzymprodukt (forstøvningstørket), haes i en handelsvanlig Powder-Coater (hvirvelsjikttørker med Wurster-innretning og Bottom-Spray-innretning). Ved en omgivelsesluft på 20-30"C påsprøytes den samme mengde av et spesialfett med smp.( >40°C), f.eks. en kakaosmørerstatning ut på smeiten. Sluttproduktet er ennu risledyktig. 1000 g of an enzyme product produced according to example 1 (spray-dried) is placed in a commercially available Powder-Coater (fluidized bed dryer with Wurster device and Bottom-Spray device). At an ambient air of 20-30"C, the same amount of a special fat with a melting point (>40°C), e.g. a cocoa butter substitute, is sprayed onto the melt. The end product is still runnable.
Claims (11)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873701835 DE3701835A1 (en) | 1987-01-23 | 1987-01-23 | BACTERIA LYSING ENZYME PRODUCT FROM STREPTOMYCETE, ITS PRODUCTION AND USE FOR THE PRESERVATION OF CHEESE |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NO880279D0 NO880279D0 (en) | 1988-01-22 |
| NO880279L true NO880279L (en) | 1988-07-25 |
Family
ID=6319326
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO880279A NO880279L (en) | 1987-01-23 | 1988-01-22 | BACTERY LYZING ENZYM PRODUCT FROM STREPTOMYCETS, ITS MANUFACTURING AND USE FOR CONSERVATION OF CHEESE. |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0280032A3 (en) |
| JP (1) | JPS63192386A (en) |
| AU (1) | AU602949B2 (en) |
| DE (1) | DE3701835A1 (en) |
| DK (1) | DK31188A (en) |
| FI (1) | FI880262A (en) |
| IE (1) | IE880163L (en) |
| IL (1) | IL85160A0 (en) |
| NO (1) | NO880279L (en) |
| NZ (1) | NZ223254A (en) |
| PT (1) | PT86606B (en) |
| ZA (1) | ZA88445B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3701565A1 (en) * | 1987-01-21 | 1988-08-04 | Hoechst Ag | USE OF STREPTOMYCETE BACTERIA-LYING ENZYMPRODUCT FOR THE PRESERVATION OF FRESH MEAT |
| AU631803B2 (en) * | 1988-06-22 | 1992-12-10 | Applied Microbiology, Inc | Nisin compositions for use as enhanced, broad range bactericides |
| EP0368224A3 (en) * | 1988-11-10 | 1991-12-27 | Hoechst Aktiengesellschaft | Streptomycete lysozyme gene, method for its isolation and its use |
| NL1020716C2 (en) * | 2002-05-30 | 2003-12-02 | Tno | Antimicrobial packaging. |
| CN113897344B (en) * | 2021-11-03 | 2023-09-08 | 浙江莱康生物工程有限公司 | Lysozyme composition with anti-inflammatory effect |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2475857A1 (en) * | 1980-02-14 | 1981-08-21 | France Ingelheim Ste Financier | Mfr. of cheese stabilised against butyric swelling - by adding lysozyme during mfr. |
| FR2569722B1 (en) * | 1984-08-28 | 1986-09-19 | Agronomique Inst Nat Rech | PROCESS FOR OBTAINING LYSOZYME BY MICROFILTRATION FROM AN EGG WHITE MATERIAL |
| DE3440735A1 (en) * | 1984-11-08 | 1986-05-15 | Hoechst Ag, 6230 Frankfurt | BACTERIA-LYING ENZYMPRODUCT FROM STREPTOMYCETE, METHOD FOR THE PRODUCTION THEREOF, AND STRUCTURE SUITABLE FOR THIS |
-
1987
- 1987-01-23 DE DE19873701835 patent/DE3701835A1/en not_active Withdrawn
-
1988
- 1988-01-20 EP EP88100716A patent/EP0280032A3/en not_active Withdrawn
- 1988-01-21 IL IL85160A patent/IL85160A0/en unknown
- 1988-01-21 FI FI880262A patent/FI880262A/en not_active Application Discontinuation
- 1988-01-21 NZ NZ223254A patent/NZ223254A/en unknown
- 1988-01-22 AU AU10710/88A patent/AU602949B2/en not_active Expired - Fee Related
- 1988-01-22 IE IE880163A patent/IE880163L/en unknown
- 1988-01-22 JP JP63011028A patent/JPS63192386A/en active Pending
- 1988-01-22 NO NO880279A patent/NO880279L/en unknown
- 1988-01-22 DK DK031188A patent/DK31188A/en not_active Application Discontinuation
- 1988-01-22 PT PT86606A patent/PT86606B/en not_active IP Right Cessation
- 1988-01-22 ZA ZA880445A patent/ZA88445B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0280032A3 (en) | 1989-11-15 |
| DK31188D0 (en) | 1988-01-22 |
| PT86606B (en) | 1991-12-31 |
| IE880163L (en) | 1988-07-23 |
| JPS63192386A (en) | 1988-08-09 |
| DE3701835A1 (en) | 1988-08-04 |
| FI880262A (en) | 1988-07-24 |
| EP0280032A2 (en) | 1988-08-31 |
| NO880279D0 (en) | 1988-01-22 |
| FI880262A0 (en) | 1988-01-21 |
| DK31188A (en) | 1988-07-24 |
| AU602949B2 (en) | 1990-11-01 |
| PT86606A (en) | 1988-02-01 |
| AU1071088A (en) | 1988-07-28 |
| ZA88445B (en) | 1988-07-22 |
| IL85160A0 (en) | 1988-06-30 |
| NZ223254A (en) | 1990-06-26 |
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