SI8510914A8

SI8510914A8 - Process for producing microbiological rennet with submersive aerobic fermentation

Info

Publication number: SI8510914A8
Application number: SI8510914A
Authority: SI
Inventors: Jozica Friedrich; Nina Gunde-Cimerman; Aleksa Cimerman; Branko Filipovic
Original assignee: Kemijski Inst; Krka Tovarna Zdravil
Priority date: 1985-05-30
Filing date: 1985-05-30
Publication date: 1996-08-31
Also published as: YU91485A; YU43410B

Description

Postopek za proizvodnjo mikrobiološkega sirišča s submerzno aerobno fermentacijoProcess for the production of microbial rennet by submerged aerobic fermentation

Področje tehnike, v katero spada izumFIELD OF THE INVENTION

Predloženi izum se nanaša na nov postopek za proizvodnjo mikrobiološkega sirišča s submerzno aerobno fermentacijo. Siriščni encim ali sirišče se uporablja pri proizvodnji sira v fazi koagulacije kazeina v mleku.The present invention relates to a new process for the production of microbial rennet by submerged aerobic fermentation. The rennet enzyme or rennet is used in the production of cheese during the coagulation phase of casein in milk.

Tehnični problemA technical problem

Obstajala je potreba, da bi našli ugoden mikrobiološki postopek za pripravo sirišča, s katerim bi nadomestili zelo drag postopek za proizvodnjo tega encima iz telečjih želodcev.There was a need to find a favorable microbiological process for rennet preparation to replace a very expensive process for the production of this enzyme from calf stomachs.

Stanje tehnikeThe state of the art

Do šestdesetih let so siriščni encim pridobivali izključno z ekstrakcijo želodcev neodstavljenih telet, starih 10 do 5θ dni. Zaradi omejitve zakola tako mladih telet je postal preparat težje dostopen in se je zelo podražil. Po drugi strani je zaradi vedno večje proizvodnje sira potreba po tem encimu močno narasla. Zato so začeli pred približno 20 leti intenzivno iskati nadomestne vire sirišČnega encima.Until the 1960s, the rennet enzyme was obtained solely by the extraction of the stomachs of unattached calves aged 10 to 5θ days. Due to the restriction of slaughter of such young calves, the preparation became more difficult to access and became very expensive. On the other hand, due to the increasing production of cheese, the need for this enzyme has increased dramatically. Therefore, about 20 years ago, they began intensively searching for alternative sources of rennet enzyme.

Ena od bistvenih zahtev uspešnega preparata je, da ima visoko specifično koagulacijsko aktivnost in čim nižjo nespecifično proteolitično aktivnost. Tej zahtevi so do sedaj najbolj zadostili encimi, pridobljeni z glivami Rhizomucor pusillus, Endothia parasitica in Rhizomucor miehei.One of the essential requirements of a successful preparation is that it has high specific coagulation activity and the lowest non-specific proteolytic activity. To date, enzymes derived from Rhizomucor pusillus, Endothia parasitica and Rhizomucor miehei have been the most satisfying of these requirements.

Opis rešitve tehničnega problema z izvedbenimi primeriDescription of solution to a technical problem with implementation examples

Ugotovili smo, da navedeni tehnični problem ugodno rešimo s postopkom za proizvodnjo sirišča s submerzno aerobno fermentacijo z glivo Rhizomucor miehei (NRRL 5285) ali Rhizomucor Pusillus (NRRL 2545).We have found that the above technical problem is advantageously solved by a process for the production of rennet by submerged aerobic fermentation with Rhizomucor miehei (NRRL 5285) or Rhizomucor Pusillus (NRRL 2545).

Postopek v smislu izuma je označen s tem, da steriliziran substrat, ki ga sestavlja vodovodna voda z 1,5 do 5,5 % pšeničnih otrobov in 2 do 4 % sirotke iz neposnetega mleka, z vrednostjo pH od 4,5 do 6,0 cepimo s suspenzijo spor glive RhizomucoiThe process of the invention is characterized in that the sterilized substrate consisting of tap water with 1.5 to 5.5% wheat bran and 2 to 4% whey from whole milk, with a pH of 4.5 to 6.0 vaccine with Rhizomucoi spore suspension

Miehei (NRRL 5285)ali glive Rhizomucor pusillus (NRRL 2545) s 6 7 koncentracijo spor v vcepku od 10 do 10^r spor/ml, fermentiramo pri temperaturi od 50 do 58°C in fermentacijsko brozgo filtriramo.Miehei (NRRL 5285) or Rhizomucor pusillus fungus (NRRL 2545) with a 6 7 concentration of spores in an injection of 10 to 10 ^r spores / ml were fermented at 50 to 58 ° C and the fermentation broth was filtered.

- 5 Kot smo že omenili, je fermentacijski substrat sestavljen iz 1,5 do 5,5 %, prednostno 5 %, pšeničnih otrobov, do 4 %, prednostno 2 %, sirotke iz neposnetega mleka, ostalo pa je vodovodna voda. pH substrata naravnamo s kislino, prednostno s HCl, na pH 4,5 do 6,0, prednostno 5,0· Tako pripravljen substrat steriliziramo pri 121°C 2 uri oz. pri 155°C 1,5 ure, ohladimo na 50 do 40°C in cepimo s suspenzijo spor glive Rhizomucor miehei ali Rhizomucor pusillus, vzgojene na krompirglukoznem agarju oz. katerikoli podlagi, ki omogoča primerno sporulacijo. Kot produkcijski sev je najugodnejši sev Rhizomucor miehei, deponiran pod oznako NRRL 5285· Fermentacijo lahko izvedemo na stresalniku ali v fermentorju pri temperaturi od 5θ do 5θ°θ, prednostno 55°θ· Postopek traja 3 do 4 dni.- 5 As mentioned earlier, the fermentation substrate consists of 1.5 to 5.5%, preferably 5%, wheat bran, up to 4%, preferably 2%, whey made from whole milk and the rest is tap water. The pH of the substrate is adjusted with acid, preferably HCl, to pH 4.5 to 6.0, preferably 5.0 · The substrate thus prepared is sterilized at 121 ° C for 2 hours or more. at 155 ° C for 1.5 hours, cooled to 50 to 40 ° C and vaccinated with a spore suspension of Rhizomucor miehei or Rhizomucor pusillus grown on potato aglucose agar or. any basis that allows for proper sporulation. As the production strain, the most favorable Rhizomucor miehei strain deposited under NRRL code 5285 · Fermentation can be carried out on a shaker or in a fermenter at a temperature of 5θ to 5θ ° θ, preferably 55 ° θ · The process takes 3 to 4 days.

Za streseno kulturo fermentiramo po 100 ml substrata, cepljenega z 2 ml suspenzije spor, v 5θθ ml erlenmajericah. Stresamo pri 80 do 110 obr/min, prednostno 100 obr/min.For shaken culture, ferment 100 ml of substrate grafted with 2 ml of spore suspension in 5θθ ml conical flasks. Shake at 80 to 110 rpm, preferably 100 rpm.

Pri fermentaciji v fermentorju z mešalom cepimo substrat po sterilizaciji in ohladitvi s suspenzijo spor v količini 1 % glede na substrat. Mešamo s 400 do 500 obr/min, prednostno 500 obr/min, zračimo z 0,5 do 0,75 1/1/min, prednostno 0,5 1/1/min.For fermentation in a stirred fermenter, the substrate is cleaved after sterilization and cooling with a spore suspension of 1% relative to the substrate. It is mixed with 400 to 500 rpm, preferably 500 rpm, aerated from 0.5 to 0.75 1/1 / min, preferably 0.5 1/1 / min.

Po končani fermentaciji fermentacijsko brozgo prefiltriramo in tako dobljeni filtrat uporabimo za pripravo sira, lahko pa ga zgostimo, npr. z ultrafiltracijo, oz. izoliramo.After fermentation is complete, the fermentation broth is filtered and the filtrate thus obtained is used to make the cheese, but it can be concentrated, e.g. by ultrafiltration, respectively. we isolate.

Prednosti postopka v smislu predloženega izuma so enostavnejši in cenejši substrat ter krajši čas fermentacije (za primerjavo glej US-PS 4,156,201), visoka koagulacijska in specifična aktivnost surovega preparata (primerjaj Hung.teljes 12,417, in Jap. 22,179) in uporabnost preparata brez dodatnega koncentriranja in čiščenja.The advantages of the process of the present invention are a simpler and cheaper substrate and a shorter fermentation time (for comparison, see US-PS 4,156,201), high coagulation and specific activity of the crude preparation (cf. Hung.Teljes 12,417, and Jap. 22,179), and the usefulness of the preparation without additional concentration. and cleaning.

Torej dobimo po predloženem postopku že brez izolacije takoj uporaben, visoko aktiven siriščni encim, ki ima visoko razmerje koagulacijske aktivnosti proti nespecifični proteolitični aktivnosti (specifična aktivnost SA od 7000 do 9500).Thus, by the present method, an immediately useful, highly active rennet enzyme having a high ratio of coagulation activity to non-specific proteolytic activity (specific activity of SA from 7000 to 9500) is obtained without isolation.

Predloženi izum pojasnjujemo z naslednjima izvedbenima primeroma.The present invention is illustrated by the following embodiments.

- 5 PRIMER 1- 5 EXAMPLE 1

Fermentacija v stresani kulturiFermentation in shaken culture

Substrat vsebuje 2 % sirotke in 3 % pšeničnih otrobov v vodovodni vodi. pH naravnamo s HC1 na 5,0. V erlenmajerice (5θθ ml) nalijemo po 100 ml substrata, steriliziramo 2 uri pri 121°C in ohladimo na 35°θ· Za vcepek pripravimo suspenzijo spor glive Rhizomucor miehei (NRRL 5283), ki smo jo gojili 1 teden na krompir-glukoznem agarju pri 3θ°θ· Koncentracija spor v vcepku je 10' spor/ml suspenzije. Po 100 ml substrata cepimo z 2 ml inokuluma. Fermentiramo 3 dni pri temperaturi 35°θ na stresalniku s 100 obr/min. Že po 24 urah fermentacije dobimo specifično morfološko obliko submerznega micelija v obliki številnih drobnih peletov. Po 3 dneh fermentacije substrat prefiltriramo. Surovi encimski preparat ima koagulacijsko aktivnost, ki znaša od 1200 do 2000 Soxhletovih enot (SU), proteolitično aktivnost (Εθθθ) od 0,150 do 0,230 in specifično aktivnost (SA) od 6600 do 8?00. Filtrat je v takem stanju direktno uporaben za koagulacijo mleka pri proizvodnji sira in ni nujno potrebno nadaljnje koncentriranje in čiščenje. Preparat lahko shranjujemo do 3 mesece pri 4°C brez padca aktivnosti.The substrate contains 2% whey and 3% wheat bran in tap water. The pH was adjusted from HCl to 5.0. Pour 100 ml of substrate into flasks (5θθ ml), sterilize for 2 hours at 121 ° C and cool to 35 ° θ. at 3θ ° θ · The concentration of spores in the clamp is 10 'spore / ml of suspension. Vaccine 100 ml of substrate with 2 ml of inoculum. Ferment for 3 days at 35 ° θ on a 100 rpm shaker. After 24 hours of fermentation, a specific morphological form of submerged mycelium is obtained in the form of numerous small pellets. After 3 days of fermentation, the substrate is filtered. The crude enzyme preparation has a coagulation activity of 1200 to 2000 Soxhlet units (SU), proteolytic activity (Εθθθ) of 0.150 to 0.230 and specific activity (SA) of 6600 to 8? 00. The filtrate is in this state directly useful for milk coagulation in cheese production and does not necessarily require further concentration and purification. The preparation can be stored for up to 3 months at 4 ° C without any decrease in activity.

- 6 PRIMER 2- 6 EXAMPLE 2

Fermentacija v laboratorijskem fermentorju z mešalom.Fermentation in a laboratory fermenter with agitator.

Sestava substrata in sterilizacija sta enaki kot pri fermentaciji v stresani kulturi. Vcepek pripravimo na isti način kot pri stresani kulturi, le da je količina za polovico zmanjšana (100 ml suspenzije spor na 10 1 substrata). Pogoji fermentacije: temperatura 35°G, zračenje 0,5 1/1/min, mešanje 500 obr/min. Fermentiramo 5 do 4 dni, fermentacijsko brozgo prefiltriramo in filtrat uporabimo kot surov encimski preparat. Koagulacijska aktivnost filtrata je od 1200 do 2500 Soxhletovih enot (SU), proteolitična aktivnost (Εθθθ) od 0,200 do 0,500, specifična aktivnost (SA) od 6600 do 8800. Tak surov encimski preparat lahko uspešno nadomešča telečje sirišče.Substrate composition and sterilization are the same as in fermentation in shaken culture. The inoculum is prepared in the same manner as in shaken culture except that the amount is reduced by half (100 ml of spore suspension per 10 l of substrate). Fermentation conditions: temperature 35 ° G, irradiation 0.5 1/1 / min, stirring 500 rpm. Ferment for 5 to 4 days, filter the fermentation broth and use the filtrate as a crude enzyme preparation. The coagulation activity of the filtrate is from 1200 to 2500 Soxhlet units (SU), proteolytic activity (Εθθθ) from 0.200 to 0.500, specific activity (SA) from 6600 to 8800. Such a crude enzyme preparation can successfully replace calf rennet.

Do podobnih rezultatov pridemo, Če v gornjih primerih uporabimo glivo Rbizomucor pusillus (NRRL 2543).Similar results are obtained if Rbizomucor pusillus (NRRL 2543) is used in the above cases.

Najboljši način za gospodarsko izkoriščanje izumaThe best way to make economic use of the invention

Fermentacija v fermentorjuFermentation in a fermenter

Substrat vsebuje 2 % sirotke in 3 % pšeničnih otrobov v vodovodni vodi. pH naravnamo s HC1 na 5,0. Steriliziramo 2 uri pri 121°C in ohladimo na 35°O· Za vcepek pripravimo suspenzijo spor glive Rhizomucor miehei (NRRL 5283), ki smo jo gojili Ί teden na krompir-glukoznem agarju pri 3θ°^. Koncentracija spor v vcepku je 10' spor/ml suspenzije. 100 ml suspenzije uporabimo za cepljenje 10 1 substrata. Pogoji fermentacije: temperatura 35°C, zračenje 0,5 1/1/min, mešanje 500 obr/min. Fermentiramo 3 do 4 dni, fermentacijsko brozgo prefiltriramo in filtrat uporabimo kot surov encimski preparat. Koagulacijska aktivnost filtrata je od 1200 do 25OO Soxhletovih enot (SU), proteolitična aktivnost (Ε^-θθ) od 0,200 do 0,300, specifična aktivnost (SA) od 6600 do 8800. Tak surov encimski preparat lahko uspešno nadomešča telečje sirišče.The substrate contains 2% whey and 3% wheat bran in tap water. The pH was adjusted from HCl to 5.0. Sterilize for 2 hours at 121 ° C and cool to 35 ° O · Prepare a Rhizomucor miehei (NRRL 5283) spore suspension for cultivation for Ί week on potato glucose agar at 3θ °. The concentration of spores in the well is 10 'spores / ml of suspension. Use 100 ml of suspension for the administration of 10 l of substrate. Fermentation conditions: temperature 35 ° C, radiation 0.5 1/1 / min, stirring 500 rpm. Ferment for 3 to 4 days, filter the fermentation broth and use the filtrate as a crude enzyme preparation. The coagulation activity of the filtrate is from 1200 to 25OO Soxhlet units (SU), proteolytic activity (Ε ^ -θθ) from 0.200 to 0.300, specific activity (SA) from 6600 to 8800. Such crude enzyme preparation can successfully replace calf rennet.

Preparat lahko shranjujemo do 3 mesece pri 4°C brez padca aktivnosti.The preparation can be stored for up to 3 months at 4 ° C without any decrease in activity.

Claims

PATENT APPLICATION

Process for the production of microbial rennet by submerged aerobic fermentation, characterized in that a sterilized substrate consisting of tap water with 1.5 to 5.5% wheat bran and 2 to 4% whey from whole milk, with a pH of 4 , 5 to 6.0 are vaccinated with a suspension of spores of Rhizomucor miehei (NRRL 5285) or Rhizomucor pusillus fungus (NRRL 2545) with a spore concentration of 10 to 10 'spores / ml, fermented at 5θ to 58 ° C and fermented we filter the broth.