Food or edible material: processes – compositions – and products – Fermentation processes – Of milk or milk product
Patent
1995-12-29
1998-09-01
Wong, Leslie
Food or edible material: processes, compositions, and products
Fermentation processes
Of milk or milk product
426 34, 426 38, 426580, 426582, A23C 912
Patent
active
058008497
DESCRIPTION:
BRIEF SUMMARY
This is a National stage filing of PCT/DK94/00163 filed Apr. 22, 1994.
FIELD OF INVENTION
This invention relates to a process for producing cheese in improved yields.
BACKGROUND OF THE INVENTION
In the production of cheese it is necessary to coagulate the milk in order to be able to separate the casein from the whey. Products containing rennin, which is a milk coagulating enzyme isolated from calf stomachs, have for many years been used for this purpose. Shortage of calf stomachs has in the last decades resulted in intense searches for other milk coagulating enzymes. Today, bovine pepsin, porcine pepsin, as well as microbial enzymes are being used commercially. The most useful among the microbial rennets are Rhizomucor miehei rennet and Rhizomucor pusillus rennet.
SUMMARY OF THE INVENTION
In this invention it is surprisingly found that the glycosylation of the aspartic protease can give an increase in cheese yield of 0.2% compared with the native enzyme. An increase in cheese yield of 0.2% would mean an extra 2 kg of cheese per one ton of cheese having a value of about 8-10 US$ (prices of the dairy).
Accordingly, the present invention relates to a process of producing cheese in improved yields, wherein a recombinant aspartic protease is added to milk in sufficient amounts to effect clotting of the milk, after which the resulting curd is processed in a manner known per se for making cheese.
The term "recombinant aspartic protease" is applied to (pro) chymosin or microbial aspartic protease produced in a host organism transformed with DNA coding for the protease. The host organism may conveniently be a fungus, e.g. a yeast or a filamentous fungus.
The term "filamentous fungus" is intended to include fungi belonging to the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes or fungi imperfecti, including Hyphomycetes such as the genera Asperaillus, Trichoderma, Penicillium, Fusarium or Humicola.
DETAILED DISCLOSURE OF THE INVENTION
According to this invention it is preferred to use recombinant aspartic protease from Mucorales (e.g. Rhizomucor, in particular Rhizomucor miehei or Rhizomucor pusillus) expressed in Aspergillus or Trichoderma in a process of producing cheese in improved yields.
A DNA sequence encoding bovine prochymosin or preprochymosin may for instance be obtained as described in EP 215 594. A DNA sequence encoding Rhizomucor miehei aspartic protease may be isolated as described in EP 238 023. The nucleotide sequence encoding Rhizomucor miehei aspartic protease is set forth herein as SEQ ID NO: 2. The nucleotide sequence encoding Rhizomucor miehei aspartic protease is set forth herein as SEQ ID NO: 1. The deduced amino acid sequence is set forth as SEQ ID NO:2.
DNA sequences encoding functions facilitating gene expression typically comprise a promoter, transcription initiation sites, and transcription termination and polyadenylation functions.
The promoter which may be preceded by upstream activating sequences and enhancer sequences as known in the art may be any DNA sequence exhibiting a strong transcriptional activity in filamentous fungi and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase or a cellulase.
Examples of suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral .alpha.-amylase, A. niger acid stable .alpha.-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease or A. oryzae triose phosphate isomerase.
The filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Asperaillus sp., such as A. niger, A. nidulans or A. oryzae. The use of A. oryzae in the production of recombinant proteins is extensively described in e.g. EP 238 023.
Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
The techniques used to transform a fungal
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Budtz Peter
Heldt-Hansen Hans Peter
Gist-Brocades B.V.
Wong Leslie
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