DK143714B - METHOD FOR HYDROLYSE OF LACTOSE UNDER GLUCOSE AND GALACTOSE CREATION - Google Patents

METHOD FOR HYDROLYSE OF LACTOSE UNDER GLUCOSE AND GALACTOSE CREATION Download PDF

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DK143714B
DK143714B DK545175A DK545175A DK143714B DK 143714 B DK143714 B DK 143714B DK 545175 A DK545175 A DK 545175A DK 545175 A DK545175 A DK 545175A DK 143714 B DK143714 B DK 143714B
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lactose
lactase
growth
galactose
hydrolysis
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M E Long
C K Lee
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Reynolds Tobacco Co R
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23CDAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00Whey; Whey preparations
    • A23C21/02Whey; Whey preparations containing, or treated with, microorganisms or enzymes
    • A23C21/023Lactose hydrolysing enzymes, e.g. lactase, B-galactosidase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/78Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y305/00Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02006Beta-lactamase (3.5.2.6)

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Description

09) DANMARK (^|)09) DENMARK (^ |)

|p (12) FREMUEGGELSESSKRIFT (ni 143714B| p (12) PREFACE SCRIPTURE (ni 143714B

DIREKTORATET FOR PATENT- OG VAREMÆRKEVÆSENETDIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Ansøgning nr. 5^51/75 (51) lnt.CI.3 C 12 P 19/14 (22) Indleveringsdag 3· dec. 1975 (24) Løbedag 5· dec. 1975 (41) Aim. tilgængelig 5· jun. 1976 (44) Fremlagt 28. sep. 1981 (86) International ansøgning nr.(21) Application No. 5 ^ 51/75 (51) lnt.CI.3 C 12 P 19/14 (22) Filing Day 3 · Dec. 1975 (24) Race day 5 · dec. 1975 (41) Aim. available 5 Jun. 1976 (44) Presented 28 Sep. 1981 (86) International application no.

(86) International indleveringsdag (85) Videreførelsesdag - (62) Stamansøgning nr. -(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Prioritet 4. dec. 1974, 529329, US(30) Priority 4 Dec. 1974, 529329, US

(71) Ansøger R. J. REYNOLDS TOBACCO COMPANY, Winston-Salem, US.(71) Applicant R. J. REYNOLDS TOBACCO COMPANY, Winston-Salem, US.

(72) Opfinder Margaret E. Long, US: Chin K. Lee, US.(72) Inventor Margaret E. Long, US: Chin K. Lee, US.

(74) Fuldmægtig Ingeniørfirmaet Hofman-Bang & Bout ard.(74) Associate Engineer Hofman-Bang & Bout ard.

(54) Fremgangsmåde til hydrolyse af lactose under dannelse af glu»= cose og galactose.(54) Process for hydrolysis of lactose to form glu »= cose and galactose.

Den foreliggende opfindelse angår en fremgangsmåde til hydrolyse af lactose under dannelse af glucose og galactose, og fremgangsmåden ifølge opfindelsen er ejendommelig ved, at man bringer lac-tosen i kontakt med en lactase produceret ved dyrkning, af organismen Bacillus coagulans NRRL B-8100 i et lactoseholdigt næringsmedium.The present invention relates to a process for hydrolysis of lactose to form glucose and galactose, and the process of the invention is characterized by contacting the lactose with a lactase produced by cultivation of the organism Bacillus coagulans NRRL B-8100 in a lactose-containing nutrient medium.

q Der har i de senere år været betydelig interesse for at ud- j. vikle metoder til reduktion af lactoseindholdet i mælk og “ produkter afledt af mælk. Denne interesse er blevet forøget r) ved nylige påvisninger af, at en stor procentdel af den men- £ neskelige befolkning lider af lactasemangel, hvilket enten er en nedarvet egenskab eller et resultat af "en ældningsproces.q In recent years, there has been considerable interest in pursuit. develop methods for reducing the lactose content of milk and “products derived from milk. This interest has been heightened by recent evidence that a large percentage of the human population suffers from lactase deficiency, which is either an inherited property or a result of "an aging process.

t 2 143714 Sådanne lactasemangler fører til forstyrrelser i tarmkanalen, hvor fødens indhold af lactose er højt. Endvidere har man observeret en lignende lactoseintolerance hos visse husdyr.t 2 143714 Such lactase deficiency leads to intestinal tract disorders where the lactose content of the food is high. Furthermore, similar lactose intolerance has been observed in some domestic animals.

Hydrolysen af lactose i mælk og produkter afledt af mælk under dannelse af glucose og galactose er et attraktivt mål, ikke bare fordi det ville løse lactoseintoleranceproblemet, men også fordi det ville forøge sødheden af produkterne og reducere de såkaldte sandagtige texturer i visse mælkeafledte produkter, forårsaget af lactosekrystallisation. Fagfolk har længe erkendt ønskeligheden af at fremkalde denne hydrolyse ved anvendelse af lactase. Til trods for det forhold, at lactase forekommer relativt udbredt i naturen og produceres af mange mikroorganismer, har anvendelsen af lactase ved kommerciel produktion af mælk og produkter afledt af mælk været meget begrænset. En af årsagerne til den begrænsede kommercielle anvendelse af lactase er, at mange af de almindelige lactaser, såsom lactaser afledt af gær, udviser en optimal enzymaktivitet ved temperaturer, der også fører til bakterievækst. Som følge heraf har der været en stigende interesse i at finde lactase med en høj grad af termostabilitet. En sådan termo-stabil lactase ville tillade udførelse af lactosehydrolysen under betingelser, som er ikke-favorable for væksten af visse bakterier, der hyppigt er til stede i mælk eller produkter afledt af mælk. En sådan lactase afledt af Streptomyces coelicolor er beskrevet i USA-patentskrift nr. 3.816.259.The hydrolysis of lactose in milk and milk-derived products to form glucose and galactose is an attractive target not only because it would solve the lactose intolerance problem, but also because it would increase the sweetness of the products and reduce the so-called sandy textures in certain milk-derived products, caused of lactose crystallization. Those skilled in the art have long recognized the desirability of inducing this hydrolysis using lactase. Despite the fact that lactase is relatively widespread in nature and produced by many microorganisms, the use of lactase in the commercial production of milk and milk-derived products has been very limited. One of the reasons for the limited commercial use of lactase is that many of the common lactases, such as yeast derived lactases, exhibit optimal enzyme activity at temperatures also leading to bacterial growth. As a result, there has been a growing interest in finding lactase with a high degree of thermostability. Such thermostable lactase would allow lactose hydrolysis to be carried out under conditions which are unfavorable to the growth of certain bacteria frequently present in milk or milk derived products. Such a lactase derived from Streptomyces coelicolor is described in U.S. Patent No. 3,816,259.

Det er på dette tidspunkt ikke klart, hvorvidt lactase fra S. coelicolor kan anvendes uden risiko, fordi visse medlemmer af denne art har vist sig at producere antibiotika.It is not clear at this time whether S. coelicolor lactase can be used without risk because some members of this species have been shown to produce antibiotics.

Produktionen af lactase af medlemmer af Bacillus-slægten er tidligere beskrevet. Således har P.J. Anema, Biochem. Biophys.The production of lactase by members of the Bacillus genus has been described previously. Thus, P.J. Anema, Biochem. Biophys.

Acta, §2 (3), 495-502 (1964) beskrevet isolation af lactase fra B. subtilis. Lactase fra B. megaterium er beskrevet af S.R. Rohlfing og I.P. Crawford, J. Bacteriology 92 (4), 1258-9 (1966). Ingen af disse organismer kan imidlertid betragtes som varmetolerante, og den dannede lactase må i reglen anvendes ved temperaturer under 50° C for at bevare en nyttig 3 143714 enzymaktivitet i længere tid.Acta, §2 (3), 495-502 (1964) described the isolation of lactase from B. subtilis. B. megaterium lactase is described by S.R. Rohlfing and I.P. Crawford, J. Bacteriology 92 (4), 1258-9 (1966). However, none of these organisms can be considered heat-tolerant, and the lactase formed must generally be used at temperatures below 50 ° C to maintain useful enzyme activity for an extended period of time.

Det er formålet med den foreliggende opfindelse at angive en fremgangsmåde til hydrolyse af lactose under dannelse af glucose og galactose ved relativt høje temperaturer på op til 70^0, især mellem 45 og 65°C, og dette opnås ved, at man bringer lac-tosen i kontakt med en lactase, der er produceret ved dyrkning af en hidtil ukendt organisme af slægten Bacillus med særligt fordelagtige egenskaber. Således udviser den en optimal vækst ved en temperatur på mindst ca. 45^0. Organismen, der betegnes Bacillus coagulans NRRL B-8100 dyrkes i et næringsmedium indeholdende lactose, hvorefter den dannede lactase isoleres.It is the object of the present invention to provide a process for the hydrolysis of lactose to form glucose and galactose at relatively high temperatures of up to 70 ° C, especially between 45 and 65 ° C, and this is achieved by toxin in contact with a lactase produced by the cultivation of a novel organism of the genus Bacillus with particularly advantageous properties. Thus, it exhibits optimum growth at a temperature of at least approx. 45 ^ 0th The organism, designated Bacillus coagulans NRRL B-8100, is grown in a nutrient medium containing lactose and then the lactase formed is isolated.

Den anvendte organisme til dette formål er isoleret fra en jordprøve. Dyrkning af organismen i et passende næringsmedium indeholdende lactose fører til dannelsen af den ønskede lactase intracellulært. Der er foretaget en karakteristik af kulturen, og den er identificeret som faldende inden for arten Bacillus coagulans i overensstemmelse med klassifikationen efter Bergey's Manual of Determinative Bacteriology, 7th Edition. Denne organisme er deponeret i kultursamlingen i U.S.D.A. Northern Regional Research Laboratory under betegnelsen NRRL B-8100. De taxonomiske egenskaber af denne stamme er beskrevet i nedenstående tabel 1.The organism used for this purpose is isolated from a soil sample. Cultivation of the organism in a suitable nutrient medium containing lactose leads to the formation of the desired lactase intracellularly. A characteristic of the culture has been made and it has been identified as declining within the species Bacillus coagulans according to the classification according to Bergey's Manual of Determinative Bacteriology, 7th Edition. This organism is deposited in the cultural collection of the U.S.D.A. Northern Regional Research Laboratory under the designation NRRL B-8100. The taxonomic properties of this strain are described in Table 1 below.

TABEL 1 A. Morphologiske karakteristika: 1. Vegetative stave: Mindre end 0,9 um i diameter, læng den varierende i reglen op til 5,0 - 6,0 μιη. Enkelte filamenter. Ingen kæder. Gram positiv, ensartet farvning. Bevægelig.TABLE 1 A. Morphological characteristics: 1. Vegetative rods: Less than 0.9 µm in diameter, length varying in general up to 5.0 - 6.0 µιη. Some filaments. No chains. Gram positive, uniform staining. Movable.

2. Sporangier: I reglen ikke opsvulmede, men enkelte opsvulmede bronchier kan findes.2. Sporangia: As a rule, not swollen, but some swollen bronchi can be found.

3. Sporer: 0,9 x 1,2 - 1,5 pm, ellipsoidiske, subterminale Indtil terminale.3. Spores: 0.9 x 1.2 - 1.5 µm, ellipsoidal, subterminal Until terminal.

4 U37U4 U37U

B. Dyrkningsmæssige karakteristika: 1. Gelatine-agar Ingen hydrolyse, stregkultur: 2. Agarkolonier: Uigennemsigtige, små, runde.B. Cultural characteristics: 1. Gelatin agar No hydrolysis, line culture: 2. Agar colonies: Opaque, small, round.

Ikke skelnelige.Not distinguishable.

3. Agarskråkulturer: Spredt til moderat vækst. Flad, glat, gennemsigtig.3. Agar slant cultures: Scattered to moderate growth. Flat, smooth, transparent.

4. Glucose-agar Væksten kraftigere end på nærings- skråkultur: agar. Glat, hvid.4. Glucose agar Growth more powerful than nutritional culture: agar. Smooth, white.

5. Glucose-asparagin Ingen vækst i 24 timer, agarskråkultur: Moderat vækst efter 48 timer.5. Glucose asparagine No growth for 24 hours, agar culture: Moderate growth after 48 hours.

6. Sure proteose- God vækst, bedre end på nærings- pepton-agarskrå- agar.6. Acid Proteosis- Good growth, better than on nutrient peptone agar goblet.

kulturer: 7. Sojabønneagar- Moderat vækst, lidt stærkere end skråkulturer: på næringsagar.cultures: 7. Soybean agar- Moderate growth, slightly stronger than oblique cultures: on nutrient agar.

8. Skråkultur på Spredt vækst efter 24 timer, agar til opbe- lige så god som næringsagar ved varing: 48 timer.8. Slant culture on Scattered growth after 24 hours, agar for living as good as nutritional agar for lasting: 48 hours.

9. Kødsuppe: Ringe vækst efter 24 timer.9. Meat Soup: Poor growth after 24 hours.

10. Natriumchlorid- Ingen vækst i 7 % natriumchlorid. kødsuppe: 11. Mælkeagar streg- Ingen hydrolyse, kultur: 12. Kartoffel: Spredt, tør, rynket.10. Sodium Chloride- No growth in 7% sodium chloride. meat soup: 11. Milk agar line- No hydrolysis, culture: 12. Potato: Scattered, dry, wrinkled.

C. Fysiologiske karakteristika: 1. Under anvendelse af pepton som nitrogehkilde producerede organismen syre, men ingen gasser ud fra glucose, lactose, arabinose, xylose, mannitol og maltose.C. Physiological characteristics: 1. Using peptone as a nitrogen source, the organism produced acid but no gases from glucose, lactose, arabinose, xylose, mannitol and maltose.

Neutral reaktion på saccharose og glycerol.Neutral reaction to sucrose and glycerol.

2. pH i glucosenæringsvæske er 5,0 eller mindre på 7 dage.2. The pH of glucose nutrient fluid is 5.0 or less in 7 days.

3. Ingen citratudnyttelse.3. No citrate utilization.

4. Tomatgærmælk løb sammen efter 24 timer ved 45° C, 5. Ingen produktion af nitriter fra nitrater.4. Tomato yeast milk ran out after 24 hours at 45 ° C, 5. No production of nitrates from nitrates.

6. Negativ Voges-Proskauer-test. pH i Voges-Proskauer-væsken var 4,2.6. Negative Voges-Proskauer test. The pH of the Voges-Proskauer liquid was 4.2.

7· Positiv stivelseshydrolyse.7 · Positive starch hydrolysis.

5 143714 8, Positiv katalasereaktion.Positive catalase reaction.

9. Ingen vaskst på nitratholdigt medium under anaerobe betingelser. Vækst på glucosenæringsvæske under anaerobe betingelser fører til pH under 5,2 i løbet af 7 dage.9. No washing on nitrate-containing medium under anaerobic conditions. Growth on glucose nutrient fluid under anaerobic conditions leads to pH below 5.2 over 7 days.

10. Aerob, fakultativt anaerobt, 11. Minimumstemperatur for væksten er 25° C, Maksimumstemperaturen for vækst er 60° G. Optimalvækst ved 45 - 50° C.10. Aerobic, optional anaerobic, 11. Minimum temperature for growth is 25 ° C, Maximum temperature for growth is 60 ° G. Optimal growth at 45 - 50 ° C.

Som angivet under de fysiologiske karakteristika i tabel 1 udviser den beskrevne Baeillus-organisme optimal vækst ved temperaturer på cirka 45 - 50° C og kan derfor opfattes som en varmetolerant organisme i sammenligning med andre Bacillus*· o arter, der udviser optimal vækst ved cirka 37 C. En varmetolerant Baeillus-organisme defineres her som en organisme, der udviser optimal vækst ved temperaturer på cirka 45° C og derover.As indicated under the physiological characteristics of Table 1, the described Baeillus organism exhibits optimum growth at temperatures of about 45 - 50 ° C and can therefore be perceived as a heat tolerant organism in comparison to other Bacillus * o species exhibiting optimum growth at ca. 37 C. A heat-tolerant Baeillus organism is defined herein as an organism that exhibits optimum growth at temperatures of about 45 ° C and above.

Opfindelsen illustreres nærmere ved nedenstående eksempel.The invention is further illustrated by the following example.

EKSEMPELEXAMPLE

pH-optimum for den af organismen producerede lactase bestemtes ved at analysere hele celler i nærvær af o-nitrophenyl-β-galactosid (ONPG) som substrat. Man gik frem i det væsentlige som beskrevet af J. Lederberg, J. Bacteriology 60, 381 (1950). Cellerne blev først behandlet med toluen, der anvendtes en phosphatpuffer, og analysetemperaturen var 37° C, Lactaseaktiviteten observeredes i intervallet pH cirka 4,5 - 8,0, med optimal aktivitet ved cirka pH 6,0.The pH optimum for the lactase produced by the organism was determined by analyzing whole cells in the presence of o-nitrophenyl-β-galactoside (ONPG) as a substrate. It proceeded essentially as described by J. Lederberg, J. Bacteriology 60, 381 (1950). The cells were first treated with toluene using a phosphate buffer and the assay temperature was 37 ° C.

Stabiliteten af lactasen produceret af B, coagulans vurderedes ved at anvende ONPG som substrat i en modificeret Leder-berg-fremgangsmåde. Til denne undersøgelse suspenderedes udvaskede celler i 0,05 M phospha tpuf fer ved pH 7,0 i nærvær 6 143714 af ONPG-substratet. Suspensionen holdtes ved 60° C i fire dage, idet prøver periodisk udtoges til bestemmelse af den tilbageværende lactaseaktivitet. Temperaturen ved denne stabilitetsprøve valgtes som tilnærmelsesvis de anvendte temperaturniveauer ved lavtemperatur-pasteurisering. Resultaterne af prøverne er vist i tabel 2.The stability of the lactase produced by B, coagulans was assessed by using ONPG as a substrate in a modified Leder-Berg method. For this study, washed out cells were suspended in 0.05 M phosphate buffer at pH 7.0 in the presence of the ONPG substrate. The suspension was kept at 60 ° C for four days, taking samples periodically to determine residual lactase activity. The temperature of this stability test was chosen as approximately the temperature levels used in low temperature pasteurization. The results of the tests are shown in Table 2.

TABEL 2TABLE 2

Tid (timer) Lactaseaktivitet som pot.Time (hours) Lactase activity as pot.

af oprindelig aktivitet 0 100 21 92 45 51 71 36 99 20of initial activity 0 100 21 92 45 51 71 36 99 20

Lactasen produceret af Bacillus coagulans udviser nyttig enzymaktivitet op til 70° C. Optimal aktivitet synes at forekomme ved temperaturer på 60 - 65° C. Den specielle temperatur, der vælges til gennemførelse af lactosehydrolyse ved anvendelse af dette enzym, vil afhænge noget af det anvendte substratmedium. Generelt foretrækkes imidlertid temperaturer på 45 - 65° C.The lactase produced by Bacillus coagulans exhibits useful enzyme activity up to 70 ° C. Optimal activity appears to occur at temperatures of 60-65 ° C. The particular temperature selected for carrying out lactose hydrolysis using this enzyme will depend on some of the enzyme used. substratmedium. Generally, however, temperatures of 45 - 65 ° C are preferred.

Et typisk medium til dyrkning af Bacillus coagulans under produktion af lactase har følgende sammensætning:A typical medium for growing Bacillus coagulans during lactase production has the following composition:

Proteose-pepton.......................... 1,0 % Gærekstrakt................i............. 1,0 %Proteose-peptone .......................... 1.0% Yeast Extract ................ i ............. 1.0%

Kaliumdihydrogenphosphat ................. 0,8 %Potassium dihydrogen phosphate ................. 0.8%

Lactose (steriliseret separat) ........... 2,0 % pH....................................... 6,0Lactose (sterilized separately) ........... 2.0% pH ............................. .......... 6.0

Kulturer inkuberes på en roterende ryster i 48 timer ved 45° C. Efter afslutning af inkubationsperioden tilsættes toluenCultures are incubated on a rotary shaker for 48 hours at 45 ° C. After completion of the incubation period, toluene is added.

7 14371A7 14371A

til væsken (0,5 % på volumeribasis), og blandingen omrøres i 30 minutter. Cellerne udvindes derpå ved flokkulering, som beskrevet i USA-patentskrift nr. 3*821.086, og det opnåede flokkulerede celleaggregat tørres ved 55° C. Lactase-aktiviteten af de tørrede aggregerede partikler er 38,5 enheder pr. gram, hvor en enhed defineres som den mængde enzym, der er nødvendig til dannelse af et mikromol dextrose pr. minut under prøvebetingelserne. Den anvendte prøvemetode er beskrevet af Weetall et al., Biotechnology and Bioengineering 16, 295 (1974).to the liquid (0.5% by volume) and the mixture is stirred for 30 minutes. The cells are then recovered by flocculation, as described in U.S. Patent No. 3 * 821,086, and the resulting flocculated cell aggregate is dried at 55 ° C. The lactase activity of the dried aggregate particles is 38.5 units per minute. grams, where one unit is defined as the amount of enzyme needed to generate one micromole of dextrose per gram. minute under the test conditions. The test method used is described by Weetall et al., Biotechnology and Bioengineering 16, 295 (1974).

Effektiviteten af den af Bacillus coagulans producerede lactase demonstreredes ved hydrolyse af lactose i en sød lagret valle. Organismen dyrkedes, og cellerne udvandtes som beskrevet ovenfor. De tørrede aggregerede partikler frasigtedes, og 5 g af 16-20 mesh portionen (0,017-0,024/um) hydratiseredes i en 50% lactoseopløsning pufret til pH 7,0 med en 0,05 phosphatpuf-fer. De hydratiserede partikler anbragtes derpå i en lille glaskolonne, der holdtes ved en temperatur på 60° C. Gennem denne pakkede kolonne ledtes kontinuerligt en vandig opløsning indeholdende 70 gram af et kommercielt tørret sødt vallepulver pr. liter, idet opløsningen pufredes med 0,05 M phosphat til pH 7,0. Opløsningen indeholdt cirka 5 vægtprocent lactose, beregnet på lactoseindholdet i den søde valle, og der tilsattes 100 mg/liter methyl-p-hydroxybenzoat som konserveringsmiddel. Strømningshastigheden gennem kolonnen holdtes på 375 ml/dag, og graden af lactosehydrolyse bestemtes dagligt ved rutineanalyse. Begyndelsesgraden for lactosehydrolysen viste sig at være 90 pct. Efter 3 ugers kontinuert drift var hydrolysegraden faldet til 80 pct.The efficacy of the lactase produced by Bacillus coagulans was demonstrated by the hydrolysis of lactose in a sweet stored whey. The organism was cultured and the cells were watered as described above. The dried aggregate particles were sieved and 5 g of the 16-20 mesh portion (0.017-0.024 µm) hydrated in a 50% lactose solution buffered to pH 7.0 with a 0.05 phosphate buffer. The hydrated particles were then placed in a small glass column maintained at a temperature of 60 ° C. Through this packed column, an aqueous solution containing 70 grams of a commercially dried sweet whey powder was continuously passed. The solution is buffered with 0.05 M phosphate to pH 7.0. The solution contained about 5% by weight of lactose, calculated on the lactose content of the sweet whey, and 100 mg / liter of methyl p-hydroxybenzoate was added as a preservative. The flow rate through the column was maintained at 375 ml / day and the degree of lactose hydrolysis was determined daily by routine analysis. The initial rate of lactose hydrolysis was found to be 90 per cent. After 3 weeks of continuous operation, the degree of hydrolysis had dropped to 80 per cent.

Det er klart, at den fremstillede lactase kan anvendes enten til batchvis eller kontinuert behandling ved lactoseholdige substrater. Endvidere kan lactasen anvendes ved direkte anvendelse af cellerne, eller den kan anvendes i form af det cellefri enzym ved i sig selv kendte metoder.It will be appreciated that the lactase produced can be used either for batch or continuous treatment with lactose-containing substrates. Furthermore, the lactase can be used by direct use of the cells or it can be used in the form of the cell-free enzyme by methods known per se.

DK545175A 1974-12-04 1975-12-03 METHOD FOR HYDROLYSE OF LACTOSE UNDER GLUCOSE AND GALACTOSE CREATION DK143714C (en)

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JPS53148591A (en) 1977-05-31 1978-12-25 Daiwa Kasei Kk Production of novel lactase
NZ190603A (en) * 1978-06-07 1982-03-23 Nat Res Dev Heat-stable -galactosidase derived from bacillus stearothermophilus hydrolysis of lactose
DK146325C (en) * 1979-01-10 1984-02-20 Novo Industri As BETA-GALACTOSIDASE, METHOD OF PREPARING IT AND USING THEREOF
US6428786B1 (en) 1993-09-28 2002-08-06 Mcneil-Ppc, Inc. Composition and method for lactose hydrolysis
US6057139A (en) 1995-06-29 2000-05-02 Mcneil-Ppc, Inc. Preblend of microcrystalline cellulose and lactase for making tablets

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DK545175A (en) 1976-06-05
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