WO2017080511A1

WO2017080511A1 - Agitation, aeration and /or fermentation processes with reduced foam

Info

Publication number: WO2017080511A1
Application number: PCT/CN2016/105477
Authority: WO
Inventors: Zhen Long; Yu Zhang; Hua Ye
Original assignee: Novozymes A/S
Priority date: 2015-11-12
Filing date: 2016-11-11
Publication date: 2017-05-18

Abstract

Disclosed herein is a process of producing a fermentation product from protein-containing culture medium, and/or a process of preventing or reducing foam generated by an agitation, aeration and/or fermentation process from protein-containing material, comprising using a S1 protease or S8 protease.

Description

AGITATION, AERATION AND /OR FERMENTATION PROCESSES WITH REDUCED FOAM

FIELD OF THE INVENTION

The present invention relates to fermentation enhancing and/or reducing foaming in agitation, aeration and/or fermentation processes.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Agitation, aeration /or fermentation processes are widely used in various industries. In many chemical or biological operations, which are conducted in a liquid containing material it is common practice to agitate, aerate and/or ferment the liquid containing material. Agitation refers to making liquid containing material move around by agitating, stirring or shaking. Aeration refers to adding a gas, for example air, oxygen, or carbon dioxide, to liquid containing material under pressure. Fermentation refers to subjecting the liquid containing material to chemical change by the action of Fermenting organisms. In agitation, aeration and/or fermentation process, foaming is a serious problem. Foaming results in broth overflow, and therefore product loss. Currently certain chemical antifoam agents are used to avoid foaming or reduce foaming. However, the added foreign chemical antifoam agents decrease the purity of the end products and may interfere with the recovery of the desired end products.

US 3,959,175 discloses an aqueous defoamer composition containing liquid polybutene. The defoamer composition can further comprise in part hydrophobic silica and silicone oils. US 5,288,789 discloses the use of a condensate of alkylphenol and aldehyde that has been polyoxyalkylated to reduce foam in a fermentation broth. US 6,083,998 concerns defoamer compositions for alcoholic fermentations which as aqueous based and comprise polydimethylsiloxane oils, ethylene oxide/propylene oxide block copolymers and a silicone/silica blend.

Even though chemical defoamers can be used there is still a desire and need for improved processes for agitation, aeration and/or fermentation.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an improved agitation, aeration and/or fermentation processes. The inventors surprisingly found that a serine protease can be used to effectively solve the foaming problem and/or improve the yield of fermentation product.

In one aspect, the present invention relates to a process of producing a fermentation product from protein-containing culture medium, comprising

i) feeding the protein-containing culture medium into a fermentation vat；

ii) fermenting the protein-containing culture medium by fermenting organisms into a fermentation product；

wherein a S1 protease or a S8 protease is added

a) before, during or after feeding of step i) , and/or

b) before or during fermentation in step ii) .

In other aspect, the present invention relates to a method to prevent or reduce foam generated by agitation, aeration and/or fermentation process from protein-containing material, comprising contacting the protein-containing material with a serine protease.

In other aspect, the present invention relates to a method to treat protein-containing material, comprising contacting the protein-containing material with a serine protease.

In other aspects, the present invention relates to use of a serine protease for preventing or reducing foam generated by agitation, aeration and/or fermentation process from protein-containing material, and relates to an antifoam agent, comprising a chemical antifoam agent and a S1 protease or a S8 protease.

In a preferred embodiment the protease is a S1E protease or a S8A protease.

In a preferred embodiment the protease is derived from Nocardiopsis sp., preferably from Nocardiopsis sp. NRRL 18262, or the protease is from Bacillus licheniformis.

In a preferred embodiment the protease is selected from the group consisting of:

(a) a protease comprises or consists of amino acids of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(d) a fragment of the polypeptide of (a) , (b) , or (c) that has protease activity.

(a) a protease comprises or consists of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

DETAILED DESCRIPTION OF THE INVENTION

i) feeding the protein-containing culture medium into a fermentation vat；

wherein a S1 protease or a S8 protease is added

a) before, during or after feeding of the step i) , and/or

b) before or during fermentation in step ii) .

According to the invention the term “fermentation vat” means and includes any type of fermentation vat, fermentation vessel, fermentation tank, or fermentation container, or the like, in which fermentation is carried out.

In a preferred embodiment, a S1 protease or a S8 protease is incubated with the protein-containing culture medium before, during or after feeding in step i) . In a further embodiment, the incubation is carried out for about 0.1-48 hours, preferably about 0.5-30 hours, more preferably about 1-20 hours, much more preferably about 10-12 hours.

According to the present invention, the protease can be inactivated, for example, by autoclaving, before fermentation in step ii) . In one embodiment, the protease does not maintain any protease activity after it is inactivated. Alternatively, the protease can still maintain some degree of protease activity, after it is inactivated.

“Protein-containing culture medium” refers to any culture medium comprising protein-containing material, which can be used for food, pharmaceuticals and the biotechnology industry.

“Protein-containing material” refers to any material in the art, which comprises a protein or polypeptide, oligopeptide or peptide. For example, it can be selected from the group consisting of yeast infusion/extract, fish infusion/extract, casein hydrolysate, peptone, soyabean cake, lupin cake, apeseed cake, soyabean meal, lupin meal, rapeseed meal, peanut cake, peanut meal, cottonseed cake, cottonseed meal, corn (maize) steep liquor, rice steep liquor, barley steep liquor, sorghum steep liquor, fruit-hull steep liquor, wheat steep liquor, corn protein, potato protein, corn protein, distillers's olubles, and a cell lysate with or without cellular debris. A variety of corns are included in the term “corn” , including, e.g., dent corn, flint corn, pod corn, striped maize, sweet corn, waxy corn and the like. In an embodiment, the corn is yellow dent corn kernel.

In a preferred embodiment, the protein-containing material is not pretreated by for example, acidic hydrolysis or salting out before it is added to the culture medium as a nitrogen source.

In a preferred embodiment, the protein-containing material is pretreated by a protease before it is added to the culture medium as a nitrogen source.

In a preferred embodiment, the protease is mixed with the protein-containing material before step i) .

In the present invention, the protein-containing material, for example, corn steep liquor, can be pretreated by for example, acidic hydrolysis or salting out before it is added to the culture medium as a nitrogen source. Protein-containing material, for example, corn steep liquor is conventionally pretreated with diluted sulfuric acid or diluted hydrochloric acid (for example, pressure 0.2 Bar, 100℃ for around 16 hours) . Considerable amount of acid will be consumed and the acid steam will be produced during the process. The purpose of the pretreatment of the corn steep liquor is to stabilize fermentation by countering corn steep liquor batch quality variation and increasing accessible free amino nitrogen. However, the pretreatment is very harsh and environmentally unfriendly. It also has very strict requirement for the equipments. In the present invention, it is not necessary to pretreat the protein-containing material, for example, corn steep liquor, due to the presence of the protease. In the presence of the protease of the present invention, the protein-containing material, for example, corn steep liquor is not pretreated by for example acidic hydrolysis or salting out before it is added to the culture medium as a nitrogen source.

Today huge amount of biochemical products is produced through fermentation process and today’s fermentation industry is highly consolidated, driven by cost effectiveness and environmental requirement. Complex protein substrates, for example, soyabean cake, lupin cake, apeseed cake, soyabean meal, lupin meal, rapeseed meal, peanut cake, peanut meal, cottonseed cake, cottonseed meal, corn steep liquor, rice steep liquor, barley steep liquor, sorghum steep liquor, fruit-hull steep liquor, wheat steep liquor, corn protein, potato protein, corn protein, distillers's olubles, and a cell lysate with or without cellular debris, are widely used as nitrogen source in various fermentation products due to low cost and easy accessibility. However, there are two problems with those complex protein substrates: 1) . Low/slow accessibility by fermenting organisms, which leads a long fermentation time； 2) . Foam formation during fermentation. These probleims are solved in the present invention by adding a protease in the agitation, aeration and/or fermentation process. In one embodiment, complex protein substrates are used as protein-containing materials of the present invention.

In addition to the protein-containing materials, the protein-containing culture medium of the present invention can further comprise other protein source matter, for example, protein, ammonia, nitrate, or phosphorus source. Furthermore, the protein-containing culture medium of the present invention can further comprise carbon source matters, which can be glucose, sucrose, glycerol, starch, maltodextrine, lactose, fats, hydrocarbons, corn sugar, starch, cellulose, sugarcane, sugar beet molasses, milk whey, vegetable oils, or petroleum fractions. The protein-containing culture medium can further comprises trace elements, for example, Fe, Zn, Cu, Mn, Mo, Co； Buffers, for example, calcium carbonate, phosphates； growth factors, for example, thiamine, biotin, calcium pentothenate.

“Fermenting organism” refers to any organism including microorganism, for example bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable Fermenting organisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product.

The fermenting organisms can be bacteria, for example, Gram positive bacteria such as Acidothermus, Arthrobacter, Bacillus, Brevibacterium, Caldicellulosiruptor, Clostridium, Corynebacterium sp. but not limited to, Corynebacterium glutamicum, Diplococcus including but not limited to, Diplococcus glycinophilus, Lactobacillus including but not limited to Lactobacillus rhamnosus, Lactococcus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Geobacillus, Microbacterium, Thermobifidia, or Oceanobacillus, or Gram negative bacteria such as an Anaerobiospirillum including but not limited to Anaerobiospirillum succiniciproducens, Campylobacter, Escherichia including but not limited to Escherichia coli, Flavobacterium, Fusobacterium, Ilyobacter, Methylobacterim Pseudomonas, Salmonella, Helicobacter, Neisseria, Streptomyces, Ureaplasma, or Zygomonas but not limited to Zygomonas mobilis.

In one embodiment, the fermenting organisms are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis. In a further embodiment, the fermenting organisms are Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus. In a still further embodiment, fermenting organisms are Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

The fermenting organisms may also be fungi, and more preferably a yeast such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia； or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

In one embodiment, fermenting organisms are Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis. In one aspect, fermenting organisms are Pichia sp. including, but not limited to, Pichia pastoris, Pichia stipites. In one aspect, fermenting organisms are Schizosaccharomyces sp. including but not limited to Schizosaccharomyces pombe.

In a further embodiment, fermenting organisms are Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata.

Commercially available yeast suitable for ethanol production include, e.g., BIOFERM^TM AFT and XR (NABC -North American Bioproducts Corporation, GA, USA) , ETHANOL RED^TM yeast (Fermentis/Lesaffre, USA) , FALI^TM (Fleischmann’s Yeast, USA) , FERMIOL^TM (DSM Specialties) , GERT STRAND^TM (Gert Strand AB, Sweden) , and SUPERSTART^TM and THERMOSACC^TM fresh yeast (Ethanol Technology, WI, USA) .

In a preferred embodiment, the Fermenting organism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.

The cloning of heterologous genes into various Fermenting organisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147； Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859； Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783； Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61: 4184-4190； Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664； Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303； Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214； Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243； Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470； WO 2003/062430, xylose isomerase) .

In a preferred embodiment, the genetically modified Fermenting organism is Candida sonorensis. In another preferred embodiment, the genetically modified Fermenting organism is Escherichia coli. In another preferred embodiment, the genetically modified Fermenting organism is Klebsiella oxytoca. In another preferred embodiment, the genetically modified Fermenting organism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified Fermenting organism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified Fermenting organism is Zymomonas mobilis.

The fermenting organisms can be algae, for example, green algae, and spiral algae.

In one aspect, the yeast and/or another microorganism are applied to the protein-containing culture medium and the fermentation is performed for about 2 to about 120 hours, for example about 4 to about 96 hours, such as 5-50 hours, typically 6-35 hours. In another aspect, the temperature is preferably between about 20℃ to about 60℃, e.g., about 25℃ to about 50℃, about 32℃ to about 50℃, or about 32℃ to about 50℃, and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is preferably applied in amounts of approximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰, especially approximately 2 x 10⁸ viable cell count per ml of fermentation broth.

A fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the Fermenting organism, such as, rate enhancement and product yield. A “fermentation stimulator” refers to a stimulator for growth of the Fermenting organisms. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1, 3-propanediol [propylene glycol] , butanediol, glycerin, sorbitol, and xylitol) ； an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane) , a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane) , an alkene (e.g., pentene, hexene, heptene, and octene) ； an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, threonine, phenylalanine, and tryptophan) ； a gas (e.g., methane, hydrogen (H2) , carbon dioxide (CO2) , and carbon monoxide (CO) ) ； isoprene； a ketone (e.g., acetone) ； an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2, 5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid) ； polyketide and a polypeptide (e.g., enzyme) .

In one aspect, the fermentation product is an alcohol. The term “alcohol” encompasses a substance that contains one or more hydroxyl moieties. The alcohol can be, but is not limited to, n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol, glycerin, glycerol, 1, 3-propanediol, sorbitol, xylitol. See, for example, Gong et al., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241； Silveira and Jonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408； Nigam and Singh, 1995, Process Biochemistry 30 (2) : 117-124； Ezeji et al., 2003, World Journal of Microbiology and Biotechnology 19 (6) : 595-603.

In another aspect, the fermentation product is an alkane. The alkane may be an unbranched or a branched alkane. The alkane can be, but is not limited to, pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.

In another aspect, the fermentation product is a cycloalkane. The cycloalkane can be, but is not limited to, cyclopentane, cyclohexane, cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene may be an unbranched or a branched alkene. The alkene can be, but is not limited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. The organic acid can be, but is not limited to, aspartic acid, glutamic acid, glycine, lysine, serine, threonine, phenylalanine, or tryptophan. See, for example, Richard and Margaritis, 2004, Biotechnology and Bioengineering 87 (4) : 501-515.

In another aspect, the fermentation product is a gas. The gas can be, but is not limited to, methane, H₂, CO₂, or CO. See, for example, Kataoka et al., 1997, Water Science and Technology 36 (6-7) : 41-47； and Gunaseelan, 1997, Biomass and Bioenergy 13 (1-2) : 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term “ketone” encompasses a substance that contains one or more ketone moieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. The organic acid can be, but is not limited to, acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2, 5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

In another aspect, the fermentation product is a polypeptide. A polyptide can be, but not limited to an enzyme. In another aspect, the fermentation product is the fermenting organisms. In another aspect, the fermentation product is an end product such as monosodium L-glutamate (MSG) , threonine, Vitamin, antibiotics, polylactic acid (PLA) , etc.

The fermentation product (s) can be optionally recovered from the fermentation culture medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol or amino acid is separated from the protein containing material and purified by conventional methods of distillation. Alternatively, the desired fermentation product may be extracted from the fermentation culture medium by micro or membrane filtration techniques. The fermentation product may also be recovered by stripping or other method well-known in the art.

Nowadays a chemical antifoam agent or defoaming agent is widely used to prevent or reduce foam. These antifoam agents are generally classified as fats, oils, aliphatic acids or esters, alcohols, sulfates, sulfonates, fatty acid soaps, waxes, phosphates, sulfides, thio compounds, organosilicones, halogenated, and inorganic compounds. Alcohols (octanol, etc. ) are good defoamers. Antifoam and defoaming are used interchangeable in the present invention.

In the present invention, the protease can be used alone or together with a chemical antifoam agent. In a preferred embodiment, the addition amount of the chemical antifoam agent is reduced compared to a corresponding process where no protease is added.

In one embodiment of the present invention, a chemical antifoam agent is added

a) before, during or after feeding of the step i) , and/or

b) before or during fermentation in step ii) ；

and wherein the addition amount of a chemical antifoam agent is reduced compared to a corresponding process where no protease is added. In one embodiment, a chemical antifoam agent is added before, during or after the addition of the S1 protease or the S8 protease.

In one embodiment, a chemical antifoam agent is reduced by about 10-80％, preferably 20-70％, more preferably 30-60％. For example, 2-8 kg chemical antifoam agent in average is needed to produce 1 ton of amino acid in a conventional process. With the addition of the protease of the present invention, the addition amount of the antifoam agent can be reduced by about 10-80％, preferably 20-70％, more preferably 30-60％.

After fermentation in step ii) the (used) fermenting organisms are collected/isolated, e.g., by centrifugation. According to the invention from 50-100％, such as 70-95％, such as about 90％of the fermenting organisms are collected and returned to the fermentation vat, and (re-) used for fermentation in one or more subsequent fermentation cycles. The fermenting organisms, such as yeast, are collected after fermentation in step ii) , acid washed, and recycled to the fermentation vat. The fermenting organisms are acid washed with sulfuric acid, e.g., at pH 1.5–3.0, such as 2.0-2.5, e.g., for 1–2 hours. The process of the invention may be carried out as a batch or fed-batch fermentation. However, the process of the invention may also be done as a semi-contineous or continuous process.

In a preferred embodiment, foaming in the fermentation vat is reduced compared to a corresponding process where no protease is added. In a further embodiment, adding the protease results in boosted growth of microorganisms, compared to a corresponding process where no protease is present or added. In a still further embodiment, adding the protease results in increased yields, e.g., ethanol or amino acid yield, compared to a corresponding process where no protease is present or added. In a further embodiment, adding the protease results in improved separation and/or recovery of the fermentation product in a fermentation process, compared to a corresponding process where no protease is present or added. In a further embodiment, the fermentation period is reduced compared to a corresponding process where no protease is added.

In one embodiment, the fermentation product is produced from protein-containing culture medium by fermentation in a fermentation vat, the process comprises adding a protease to a protein-containing material, inculating the protease with the protein-containing material, adding the incubated protein-containing material as nitrogen sorce to a culture medium； feeding the protein-containing culture medium into the fermentation vat； fermenting the protein-containing culture medium by a slurry of fermenting organisms into the fermentation product.

In a further embodiment, the fermentation product is produced from protein-containing culture medium by fermentation in a fermentation vat, the process comprises adding a protease to the protein-containing culture medium before feeding； feeding the protein-containing culture medium containing the protease into the fermentation vat； fermenting the protein-containing culture medium by a slurry of fermenting organisms into the fermentation product.

In a still further embodiment, ethanol or amino acid is produced in a batch, fed batch, semi continuous or continuous fermentation process in a fermentation vat, comprising adding the protease to protein-containing culture medium before feeding； feeding the protein-containing culture medium containing the protease into the fermentation vat comprising a slurry of Saccharomyces cerevisae or Pichia pastoris yeast or Corynebacterium glutamicum； and fermenting protein-containing culture medium into ethanol or amino acid.

In a still further embodiment, the fermentation product is produced from protein-containing culture medium by fermentation in a fermentation vat, wherein the process comprises: feeding protein-containing culture medium into the fermentation vat comprising a slurry of fermenting organisms； feeding the protease into the fermentation vat comprising protein-containing culture medium； fermenting the protein-containing culture medium into the fermentation product.

In a still further embodiment, the fermentation product is produced from protein-containing culture medium by fermentation in a fermentation vat, wherein the process comprises: feeding protein-containing culture medium into the fermentation vat comprising a slurry of fermenting organisms； adding a protease into the fermentation vat during fermention of protein-containing culture medium into a desired fermentation product.

Treatment of protein-containing material

In another aspect, the present invnetion relates to a process of treating protein-containing material, comprising subjecting the protein-containing material to a S1 protease or a S8 protease.

In one embodiment, the protein-containing material is a component of a protein-containing culture medium.

In a further embodiment, the protein-containing material is used for an agitation, aeration and/or fermentation process.

In a still further embodiment, the S1 protease or the S8 protease is incubated with the protein-containing material before, or during agitation, aeration and/or fermentation process； preferably, the protease is inactivated before agitation, aeration and/or fermentation process. In a preferred embodiment, the incubation is carried out for 0.1-48 hours, preferably 0.5-30 hours, more preferably 1-20 hours.

In a still further embodiment, the protein-containing material is selected from the group consisting of yeast infusion/extract, fish infusion/extract, casein hydrolysate, peptone, trypton, soyabean cake, lupin cake, apeseed cake, soyabean meal, lupin meal, rapeseed meal, peanut cake, peanut meal, cottonseed cake, cottonseed meal, corn (maize) steep liquor, rice steep liquor, barley steep liquor, sorghum steep liquor, fruit-hull steep liquor, wheat steep liquor, corn protein, potato protein, corn protein, distillers’s olubles and a cell lysate with or without cellular debris. In a preferred embodiment, the protein-containing material is not pretreated by for example, acidic hydrolysis or salting out before it is subjected to a S1 protease or a S8 protease.

In a still further embodiment, a chemical antifoam agent is added before, during or after the addition of the protease. In a still further embodiment, the addition amount of the chemical antifoam agent is reduced compared to a corresponding process where no protease is added. In a preferred embodiment, the chemical antifoam agent is reduced by 10-80％, preferably 20-70％, more preferably 30-60％.

In a still further embodiment, the S1 protease or the S8 protease is the only enzyme added. In a still further embodiment, the S1 protease or the S8 protease is added together with one or more enzymes selected from the group consisting of: cellulase, hemicellulase (for example, xylanase) , glucoamylase, alpha-amylase, oxidase, peroxidase, catalase, laccase, beta-glucosidase, other carbohydrases, and oxidases.

Use of a protease for preventing or reducing foam

In one aspect, the present invention relates to use of a S1 protease or a S8 protease for preventing or reducing foam generated by agitation, aeration and/or fermentation process from a protein-containing material. According to the present invention, foaming in the agitation, aeration and/or fermentation process is reduced compared to a corresponding process where no protease is added.

Use of a protease for boosting the growth of microorganisms

In another aspect, the present invention relates to use of a S1 protease or a S8 protease for boosting the growth of microorganisms. According to the present invention, adding the protease results in boosted growth of microorganisms, compared to a corresponding process where no protease is present or added.

Use of a protease for improving yield or capacity of an agitation, aeration and/or fermentation process

In a further aspect, the present invention relates to use of a S1 protease or a S8 protease for improving yield or capacity of an agitation, aeration and/or fermentation process. According to the present invention, adding the protease results in increased yields, e.g., ethanol or amino acid yield, compared to a corresponding process where no protease is present or added. According to the present invention, adding the protease results in increased capacity of the agitation, aeration and/or fermentation process compared to a corresponding process where no protease is present or added.

Use of a protease for improving the separation and/or recovery of a fermentation product in a fermentation process

In a further aspect, the present invention relates to use of a S1 protease or a S8 protease for improving the separation and/or recovery of a fermentation product in a fermentation process. According to the present invention, adding the protease results in improved separation and/or recovery of the fermentation product in an agitation, aeration and/or fermentation process, compared to a corresponding process where no protease is present or added.

In a further aspect, the present invention relates to use of a S1 protease or a S8 protease for reducing fermentation period. According to the present invention, the fermentation period is reduced compared to a corresponding process where no protease is added.

In one embodiment the uses of the present invention are related to any of a process of the invention.

A protein-containing material composition

In a still further aspect, the present invention relates to a protein-containing material composition comprising a protein-containing material and a S1 protease or a S8 protease.

A protein-containing culture medium

In a still further aspect, the present invention relates to a protein-containing culture medium, comprising a protein-containing material as as a nitrogen source and a S1 protease or a S8 protease. In one embodiment, the protein-containing culture medium, further comprises a carbohydrate source.

An antifoam agent

In a still further aspect, the present invention relates to an antifoam agent, comprising a chemical antifoam agent and a S1 protease or a S8 protease.

Protease used during agitation, aeration and/or fermentation

A process of the invention, as defined above, includes addition of a S1 protease or a S8 protease. In a still further aspect, the protease is a S1E protease or a S8A protease. In a still further aspect, the protease is derived from Nocardiopsis sp., preferably from Nocardiopsis sp. NRRL 18262, or the protease is from Bacillus licheniformis.

In one aspect, the protease is added in a dosage of 0.001 to 50 mg enzyme protein/g protein of protein-containing culture medium, preferably 0.01-40 mg enzyme protein/g protein of protein-containing culture medium, 0.05-20 mg enzyme protein/g protein of protein-containing culture medium.

In another aspect, the protease is added in a dosage of 0.001 to 50 mg enzyme protein/g protein of the protein-containing material, preferably 0.01-40 mg enzyme protein/g protein of the protein-containing material, 0.05-20 mg enzyme protein/g protein of the protein-containing material.

In one aspect, the protease is incubated with the protein-containing culture medium before, during or after feeding.

In one aspect, the chemical antifoam agent is reduced by 10-80％, preferably 20-70％, more preferably 30-60％.

Enzymes

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity” .

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) , preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100) / (Length of Alignment –Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra) , preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100) / (Length of Alignment –Total Number of Gaps in Alignment)

The term “variant” means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid； a deletion means removal of the amino acid occupying a position； and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In describing variants, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed. Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A” . Multiple mutations are separated by addition marks ( “+” ) , e.g., “Gly205Arg + Ser411Phe” or “G205R + S411F” , representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) , respectively.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*” . Multiple deletions are separated by addition marks ( “+” ) , e.g., “Gly195*+ Ser411*” or “G195*+ S411*” .

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK” . An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2； etc. ] . For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA” .

In such cases the inserted amino acid residue (s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue (s) . In the above example, the sequence would thus be:

Parent:	Variant:
195	195 195a 195b
G	G - K - A

Multiple alterations. Variants comprising multiple alterations are separated by addition marks ( “+” ) , e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Different alterations. Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “Arg170Tyr, Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly, Ala + Arg170Gly, Ala” designates the following variants: “Tyr167Gly+Arg170Gly” , “Tyr167Gly+Arg170Ala” , “Tyr167Ala+Arg170Gly” , and “Tyr167Ala+Arg170Ala” .

Protease

The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof, these enzymes being in the following referred to as “belonging to the EC 3.4. -. -group” ) . The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5； Eur. J. Biochem. 1995, 232, 1-6； Eur. J. Biochem. 1996, 237, 1-5； Eur. J. Biochem. 1997, 250, 1-6； and Eur. J. Biochem. 1999, 264, 610-650； respectively. The nomenclature is regularly supplemented and updated； see e.g. the World Wide Web at http: //www. chem. qmw. ac. uk/iubmb/enzyme/index. html.

In one embodiment, the protease is a protease with broad specificity. “A protease with broad specificity” as used herein refers to a protease which can be able to break several specific peptide bonds of a protein or peptide. “A protease with narrow specificity” as used herein refers to a protease which can able to break a few specific peptide bonds of a protein or peptide.

The proteases according to the invention are serine proteases. The term serine protease refers to serine peptidases and their clans as defined in the above Handbook. In the 1998 version of this handbook, serine peptidases and their clans are dealt with in chapters 1-175. Serine proteases may be defined as peptidases in which the catalytic mechanism depends upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. In one aspect, the protease is a protease with broad specificity.

Serine proteases of peptidase family S1E is described in Biochem. J. 290: 205-218 (1993) and in MEROPS protease database. The database is described in Rawlings, N.D., O'Brien, E.A. &Barrett, A.J. (2002) MEROPS: the protease database. Nucleic Acids Res. 30, 343-346.

Members of family S8 have a catalytic triad in the order Asp, His and Ser in the sequence. The term “S8A protease” means an S8 protease belonging to subfamily A. Subtilisins, EC 3.4.21.62, are a subgroup in subfamily S8A.

In subfamily S8A, the active site residues frequently occur in the motifs Asp-Thr/Ser-Gly (which is similar to the sequence motif in families of aspartic endopeptidases in clan AA (AA) ) , His-Gly-Thr-His and Gly-Thr-Ser-Met-Ala-Xaa-Pro.

The Nocardiopsis proteases and Bacillus licheniformis protease disclosed herein are serine proteases.

In a still further embodiment, the protease of the invention is a subtilisin and/or derived from the subtilisin family. The terms subtilisin or subtilisin family include all Clan SB serine proteases, in particular Family S8 thereof (Clan SB is dealt with in Chapter 93 of the handbook) . For determining whether a given protease is a subtilisin or not, reference is made to the handbook and the principles indicated therein. Such determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases； or genetically engineered or synthetic proteases. In a particular embodiment, the order of the catalytic triad in the protease of the invention is Asp-His-Ser. In another particular embodiment, the tertiary structure of the protease of the invention includes both alpha-helices and beta sheets. Clan SB includes endoproteases and exoproteases.

In a still further particular embodiment the protease of the invention is an endoprotease. Endoproteases show activity on N-and C-terminally blocked peptide substrates that are relevant for the specificity of the protease in question.

Endoprotease or endoproteinase are proteolytic proteases that break peptide bonds of nonterminal amino acids (i.e. within the molecule) , in contrast to exoproteases, which break peptide bonds from their end-pieces. An exoprotease is any protease that catalyzes the cleavage of the terminal (or the penultimate) peptide bond； the process releases a single amino acid or dipeptide from the peptide chain. Depending on whether the amino acid is released from the amino or the carboxy terminal, an exopeptiase is further classified as an aminoprotease or a carboxyprotease, respectively.

In another embodiment, the protease of the present invention is an endoprotease； preferably the protease is a combination of an endoprotease and an exoprotease. In a more preferably embodiment, the endoprotease is about 40％-99％of the combination of endoprotease and exoprotease, preferably, about 50％-95％of the combination of endoprotease and exoprotease, more preferably, about 60％-90％of the combination of endoprotease and exoprotease.

Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 5, 6, 7, 8, 9, 10, or 11. Examples of assay-temperatures are 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, or 70℃.

Examples of protease substrates are casein, and pNA-substrates, such as Suc-AAPF-pNA (available e.g. from Sigma S-7388) . The capital letters in this pNA-substrate refers to the one-letter amino acid code. Another example is Protazyme AK (azurine-dyed crosslinked casein prepared as tablets by Megazyme T-PRAK) . For pH-activity and pH-stability studies, the pNA-substrate is preferred, whereas for temperature-activity studies, the Protazyme AK substrate is preferred.

Examples of protease assays are described in the experimental part.

The protease may be of any origin. In a preferred embodiment, the protease is a microbial protease. In an embodiment the protease is of bacterial origin. In a preferred embodiment, the protease is of Gram-positive bacteria origin, e.g. bacteria of the phylum Actinobacteria phy. nov., e.g. of class I: Actinobacteria, e.g. of the Subclass V: Actinobacteridae, e.g. of the Order I: Actinomycetales, e.g. of the Suborder XII: Streptosporangineae, e.g. of the Family II: Nocardiopsaceae, e.g. of the Genus I: . Examples of the protease are the protease from bacteria, e.g. bacteria of the phylum Actinobacteria phy. nov., e.g. of class I: Actinobacteria, e.g. of the Subclass V: Actinobacteridae, e.g. of the Order I: Actinomycetales, e.g. of the Suborder XII: Streptosporangineae, e.g. of the Family II: Nocardiopsaceae, e.g. of the Genus I.

In a more preferred embodiment the protease is derived from Nocardiopsis, e.g. Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba； or mutants or variants thereof exhibiting protease activity. In a most preferrd embodiment, the protease comprises or consists of amino acids of SEQ ID NO: 1 herein, which is also disclosed as SEQ ID NO: 26 in WO2004/072279, or a variant thereof.

In another embodiment the protease has at least 60％, such as at least 70％, such as at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to SEQ ID NO: 26 in WO2004/072279 or SEQ ID NO: 1 herein.

In another embodiment the protease is a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279.

In a more preferred embodiment, the protease is a serine protease from Bacillus licheniformis, which is also designated subtilisin Carlsberg. In a most preferrd embodiment, the protease comprises or consists of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160, or a variant thereof.

In another embodiment the protease has at least 60％, such as at least 70％, such as at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to SEQ ID NO: 2 in WO2006/136160 or SEQ ID NO: 3 herein.

In another embodiment the protease is a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

The proteases include not only natural or wild-type proteases, but also any mutants, variants, fragments etc. thereof exhibiting protease activity, as well as synthetic proteases, such as shuffled proteases, and consensus proteases. Genetically engineered metallo proteases can be prepared as is generally known in the art, e.g., by Site-directed Mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers in the PCR reactions) , or by Random Mutagenesis. The preparation of consensus proteins is described in, e.g., EP 897,985. The term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source is present. In a preferred embodiment, the polypeptide is secreted extracellularly.

In another embodiment the variant of the protease of the present invention is a fragment of the protease thereof. The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain； wherein the fragment has protease activity. In one embodiment a fragment contains at least 75 amino acid residues, or at least 100 amino acid residues, or at least 125 amino acid residues, or at least 150 amino acid residues, or at least 160 amino acid residues, or at least 165 amino acid residues, or at least 170 amino acid residues, or at least 175 amino acid residues.

An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Other enzymes

One or more of the following enzyme activities may be added together with the protease or present and/or added during fermentation.

Alpha-Amylases

According to the invention an alpha-amylase may be added together with the protease or present and/or added during fermentation. The alpha-amylase may be of, e.g., bacterial or fungal origin.

Bacterial Alpha-Amylases

Examples of suitable bacterial alpha-amylases include the below mentioned. Preferred bacterial alpha-amylases may be derived from a strain the genus Bacillus (sometimes referred to as Geobacillus) , including a strain of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, or Bacillus subtilis. Other bacterial alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988) , pp. 25-31 (hereby incorporated by reference) .

The Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference) . Specifically contemplated alpha-amylase variants are disclosed in US patent nos. 6,093,562, 6,297,038 or US patent no. 6,187,576 (hereby incorporated by reference) and include Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, preferably a double deletion disclosed in WO 1996/023873 –see e.g., page 20, lines 1-10 (hereby incorporated by reference) , preferably corresponding to delta (181-182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference) . Even more preferred are Bacillus alpha-amylases, especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta (181-182) and further comprise a N193F substitution (also denoted I181*+ G182*+ N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467.

In an embodiment the Bacillus stearothermophilus alpha-amylase is one disclosed in WO 2011/082425, such as one selected from the group of:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V； and

-I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S.

The truncated Bacillus stearothermophilus alpha-amylase is typically naturally truncated to be about from 485-495 amino acids long, such as 491 amino acids. In a preferred embodiment the truncation is at the C-terminal. A hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467) , with the following substitution: G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the numbering in SEQ ID NO: 4 in WO 99/19467) . Especially preferred are variants having one or more of the mutations H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467) .

Commercially available bacterial alpha-amylase products and products containing alpha-amylases include TERMAMYL^TM SC, LIQUOZYME^TM SC, BAN (Novozymes A/S, Denmark) DEX-LO^TM, SPEZYME^TM XTRA, SPEZYME^TM AA, SPEZYME FRED-L, SPEZYME^TM ALPHA, GC358, SPEZYME RSL, SPEZYME HPA and SPEZYME^TM DELTA AA (from DuPont, USA) , FUELZYME^TM (Verenium, USA) .

Bacterial alpha-amylase may be added in concentrations well-known in the art. When measured in KNU units (described below in the Materials &Methods” -section) the alpha-amylase activity is preferably present in the range from 0.5-50 KNU/L fermentation medium, such as 1-25 KNU/L fermentation medium, or more preferably in an amount of 2-10 KNU/L fermentation medium.

Fungal Alpha-Amylases

Fungal alpha-amylases (EC 3.2.1.1) are preferably of filamentous fungus origin. The fungal alpha-amylase may be a fungal acid alpha-amylase.

Fungal acid alpha-amylases include acid alpha-amylases derived from a strain of the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger alpha-amylases.

A preferred fungal alpha-amylase is a Fungamyl-like alpha-amylase which is preferably derived from a strain of Aspergillus oryzae. In the present disclosure, the term "Fungamyl-like alpha-amylase" indicates an alpha-amylase which exhibits a high identity, i.e. more than 70％, more than 75％, more than 80％, more than 85％more than 90％, more than 95％, more than 96％, more than 97％, more than 98％, more than 99％or even 100％identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strain Aspergillus niger. In a preferred embodiment the acid fungal alpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 (Example 3) . The acid Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by reference. Also variants of said acid fungal amylase having at least 70％identity, such as at least 80％or even at least 90％identity, such as at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％identity to SEQ ID NO: 1 in WO 2004/080923 are contemplated. A suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark) .

The fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., a none-hybrid) , or a variant thereof. In an embodiment the wild-type acid fungal alpha-amylase is derived from a strain of Aspergillus kawachii.

Commercial available compositions comprising fungal alpha-amylase include FUNGAMYL^TM and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark) .

In an embodiment the fungal acid alpha-amylase is a hybrid alpha-amylase. Preferred examples of fungal hybrid alpha-amylases include the ones disclosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or US patent application no. 60/638,614 (Novozymes) which is hereby incorporated by reference. A hybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM) , such as a starch binding domain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in co-pending US patent application no. 60/638,614, including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 2 herein and SEQ ID NO: 100 in US 60/638,614) , Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 3 herein and SEQ ID NO: 101 in US 60/638,614) , Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO: 20 SEQ ID NO: 72 and SEQ ID NO: 96 in US application no. 11/316,535 and further as SEQ ID NO: 13 herein) , and Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in US 60/638,614) . Other specifically contemplated hybrid alpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 in Example 4 in US application no. 11/316,535 or (WO 2006/069290) (hereby incorporated by reference) . Other specific examples of contemplated hybrid alpha-amylases include those disclosed in U.S. Patent Publication no. 2005/0054071, including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain.

An acid alpha-amylases may be added in an amount of 0.1 to 250 FAU (F) /L fermentation medium, preferably 1 to 100 FAU (F) /L fermentation medium.

Glucoamylase

Contemplated glucoamylases include those from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984) , EMBO J. 3 (5) , p. 1097-1102) , or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark) ； the A. awamori glucoamylase disclosed in WO 84/02921, A. oryzae glucoamylase (Agric. Biol. Chem. (1991) , 55 (4) , p. 941-949) , or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996) , Prot. Eng. 9, 499-505) ； D257E and D293E/Q (Chen et al. (1995) , Prot. Eng. 8, 575-582) ； N182 (Chen et al. (1994) , Biochem. J. 301, 275-281) ； disulphide bonds, A246C (Fierobe et al. (1996) , Biochemistry, 35, 8698-8704； and introduction of Pro residues in position A435 and S436 (Li et al. (1997) , Protein Eng. 10, 1199-1204.

Other glucoamylases contemplated include glucoamylase derived from a strain of Athelia, preferably a strain of Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see US patent no. 4,727,026 and (Nagasaka, Y. et al. (1998) “Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50: 323-330) , Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448) , Talaromyces leycettanus (US patent no. Re. 32,153) , Talaromyces duponti, Talaromyces thermophilus (US patent no. 4,587,215) . Also contemplated are the Trichoderma reesei glucoamylases disclosed as SEQ ID NO: 4 in WO 2006/060062 and glucoamylases being at least 80％or at least 90％identical thereto and further the glucoamylase derived from Humicola grisea disclosed as SEQ ID NO: 3 in US 7,262,041-B2 (US10/992,187) (hereby incorporated by reference) or sequences having at least 80％or at least 90％identity thereto.

In a preferred embodiment the glucoamylase is derived from a strain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae； or a strain of Trichoderma, preferably T. reesei； or a strain of Talaromyces, preferably T. emersonii.

Other contemplated glucoamylases include glucoamylase derived from a strain of Trametes, preferably a strain of Trametes cingulata disclosed in WO 2006/069289 (which is hereby incorporated by reference) . Also hybrid glucoamylase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference. ) .

Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135, 138) , and C. thermohydrosulfuricum (WO 86/01831) .

Commercially available compositions comprising glucoamylase include AMG 200L； AMG 300 L； SAN^TM SUPER, SAN^TM EXTRA L, SPIRIZYME^TM PLUS, SPIRIZYME^TM FUEL, SPIRIZYME ULTRA, SPIRIZYME EXCEL, SPIRIZYME^TM B4U and AMG^TM E (from Novozymes A/S) ； OPTIDEX^TM 300 (from Genencor Int. ) ； AMIGASE^TM and AMIGASE^TM PLUS (from DSM) ； G-ZYME^TM G900, G-ZYME^TM and G990 ZR (from Genencor Int. ) .

Glucoamylases may in an embodiment be added in an amount of 1-5, 000 AGU/L fermentation medium, preferably 10-1,000 AGU/L fermentation medium.

The present invention is described in further detail in the following examples which are offered to illustrate the present invention, but not in any way intended to limit the scope of the invention as claimed. All references cited herein are specifically incorporated by reference for that which is described therein.

Materials &Methods

Enzymes

Nocardiopsis protease: Serine 1E protease derived from Nocardiopsis sp. NRRL 18262 disclosed as amino acids of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279.

Metalloprotease A (MPA) : Metallo protease derived from Thermoascus aurantiacus CGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein and amino acids 1-177 in SEQ ID NO: 2 in WO 2003/048353.

Bacillus licheniformis protease A: Serine 8A protease derived from Bacillus licheniformis disclosed as amino acids 1-274 of SEQ ID NO: 3 herein and SEQ ID NO: 2 in WO2006/136160.

Bacillus licheniformis protease B: Serine 8A protease derived from Bacillus licheniformis which is Genecor Commercial Protex 6L enzyme.

Bacillus amyloliquefaciens protease: Protease derived from Bacillus amyloliquefaciens which is Genecor Commercial Protex 7L enzyme.

PfuS protease: Protease derived from Pyrococcus furiosus shown in SEQ ID NO: 4 herein.

Corn steep liquor

“Corn steep liquor” is a by-product of the corn wet milling process in starch industry. It is a concentrate obtained by the evaporation of the water used to soak shelled corn prior to the first stage of milling. (Steeping: the corn is soaked in water, called steepwater, at 50℃ for between 20 and 30 hours, during which time it doubles in size. Sulphur dioxide is added to the water to prevent excessive bacterial growth. As the corn swells and softens, the mildly acidic steepwater starts to loosen the gluten bonds with the corn, and to release the starch. The corn goes on to be milled. ) Corn steep liquor is a viscous concentrate of corn solubles, rich in vitamins, amino acids, minerals and other growth stimulants, contains approx. 35-45％ (w/w) solid. It’s an important constituent of some growth media. It is an excellent source of organic nitrogen.

Methods

Protease Assay method -AU (RH)

The proteolytic activity may be determined with denatured hemoglobin as substrate. In the Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA) . The amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU-RH) is defined as the amount of enzyme which under standard conditions (i.e. 25℃, pH 5.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.

The AU (RH) method is described in EAL-SM-0350 and is available from Novozymes A/SDenmark on request.

Protease assays

AZCL-casein assay

A solution of 0.2％of the blue substrate AZCL-casein is suspended in Borax/NaH2PO4 buffer pH9 while stirring. The solution is distributed while stirring to microtiter plate (100 microL to each well) , 30 microL enzyme sample is added and the plates are incubated in an Eppendorf Thermomixer for 30 minutes at 45℃ and 600 rpm. Denatured enzyme sample (100℃ boiling for 20min) is used as a blank. After incubation the reaction is stopped by transferring the microtiter plate onto ice and the coloured solution is separated from the solid by centrifugation at 3000rpm for 5 minutes at 4℃. 60 microL of supernatant is transferred to a microtiter plate and the absorbance at 595nm is measured using a BioRad Microplate Reader.

pNA-assay

50 microL protease-containing sample is added to a microtiter plate and the assay is started by adding 100 microL 1mM pNA substrate (5 mg dissolved in 100 microL DMSO and further diluted to 10 mL with Borax/NaH2PO4 buffer pH 9.0) . The increase in OD405 at room temperature is monitored as a measure of the protease activity.

Example 1: Antifoaming effect in tubes with corn steep liquor as substrate

All Corn steep liquor and H₂O were mixed in the ratio of 1: 1. pH of the mixture was adjusted to 7.0. Three ml or 5 ml of the mixture were transferred into a 14 ml tube with cap. 0.75 mg (to 3 ml) or 1.25 mg (to 5 ml) enzyme proteins were added to the mixture and incubated under shaking at 50 ℃ overnight. Then the tubes were taken out from the shaker, shaked at full tilt. The total volume (foam and liquid) was measured at once. The results were shown in the following Table 1. From Table 1, it was observed Nocardiopsis protease and Bacillus licheniformis protease A results in a better foam reduction.

Table 1 Foam Reduction in tubes with corn steep liquor as substrate

Example 2: Antifoaming effect in a bioreactor in presense of Nocardiopsis protease

The fermentation medium was composed of 8％glucose, 0.5％corn steep liquor, 0.5％soybean meal hydrolysate, 0.05％MgSO₄ and 0.1％K₂HPO₄, pH7.0～7.2.7.14 mg Nocardiopsis protease was added to 2 L fermentation medium and incubated at 50 ℃ overnight. By control, no enzyme was added to the medium. Then the medium was filled in a 7L bioreactor, autoclaved and inoculated with seed culture of Corynebacterium glutamicum. The fermentation lasted for 24 hours and foaming was observed. The foam volume was 2.5 L by the enzyme treated culture, while more than 4.5 L foam was created by control.

Example 3: Antifoaming effect with Nocardiopsis protease in large-scale fermenters

2.5L corn steep liquor were diluted with 2.5L water in 10L seed tank, pH was adjusted to 7.0. Nocardiopsis protease was added to the diluted corn steep liquor and incubated at 65 ℃for 16 hours. Two dosages of Nocardiopsis protease were used for treatment: 0.018％＝ 18 mg pure enzyme protein/100 ml corn steep liquor； 0.054％＝ 54 mg pure enzyme protein/100 ml corn steep liquor. After enzyme treatment, the hydrolyzed corn steep liquor was sterilized. 1.04L above treated corn steep liquor was used for 25L medium in 50L fermenter for threonine fermentation. The fermentation lasted 34 hours at 37 ℃, pH 7.0. At the same time a conventional process (without enzyme treatment) was conducted as a control.

Table 2 Fermentation results in a 50L fermenter

Item	Blank	0.054％dosage	0.018％dosage
Time (h)	34	34	34
Threonine production rate (％)	10.65％	14％	11.62％
Defoaming agent consumption (g) *	121.81	58.55	76.58
Saving Defoaming agent (％)	0	51.93％	37.13％

*Defoaming agent (Bubble-enemy, a common polyether antifoam agent in China, 1％water solution) is diluted 20 times before adding to a fermenter.

From above 50L threonine fermentation result (table 2) , it can be seen that with 0.018％and 0.054％enzyme dosage, the consumption of chemical deforming agent was saved 37.13％and 51.93％, the threonine yield was increased 0.6％and 18.3％respectively.

In addition, the fermentation process was speeded up by using enzyme hydrolysis, as can be seen in table 3 the threonine yield at 24 hour reached to the same level as 34 hour of conventional process/

Table 3. Fermentation speed in a 50L fermenter

Example 4: Analysis of hydrolyzed corn steep liquor

1.0L corn steep liquor were diluted with 1.0L water in 5L seed tank, pH was adjusted to 7.50. Bacillus licheniformis protease A was added to the diluted corn steep liquor and incubated at 60 ℃ for 16 hours. The dosage of alcalase was 0.375g enzyme protein/g CSL protein. At different time interval 10 ml corn steep liquor were taken for peptide distribution analysis. All the samples were diluted by 1: 20, filtrated and measured through high performance liquid chromatography (HPLC-SEC) . Proportion of different molecular sizes were calculated from diagram and presented in table 4.

Table 4 Different molecular weight of hydrolyzed corn steep liquor

From the above moleculare weight analysis, it can be seen that the big MW proteins (>3000) in the corn steep liquor were degraded eventually from 6.15％to around 0.10％along with hydrolysis time, and in the meantime the small MW proteins/peptide (<1000) which is normally considered having less impact on foam formation and easily to be digested by microorganisms were increased from 78.26％to 91.70％.

Example 5: The antifoaming effects of different proteases

Corn steep liquor hydrolysis was carried out as follows. 7.0 grams of corn steep liquor (CSL) was mixed with H₂O and the pH was adjusted to 7.0 by 2mol/L KOH before enzyme hydrolysis. Then the diluted CSL was hydrolyzed by different proteases at 50℃ for 16 hours. The control sample was diluted and adjusted pH before the culture medium preparation but was not incubated overnight, as incubation of overnight resulted in more foam. The final weight was adjusted to 14.0 grams. The enzyme dosing and culture medium preparation were carried out as shown below in Table 5 and Table 6.

Table 5 Enzyme dosing for samples

Culture medium preparation was carried out as follows (table 6) . Then the diluted CSL was mixed with other materials to prepare the culture medium.

Table 6 Culture medium composition

Then the medium was autoclaved at 121℃ for 20 min and 150ml glucose solution (contain glucose 70.0 grams) was added to the medium.

Foam character evaluation was carried out as follows (see table 7) . The foam characters of medium sample (A to D) were analyzed. 200ml medium was added into 1000ml container and air was bubbled into the medium with stirring. Then the time required for the foam generated from 200ml to 1000ml was recorded as t₁. The stirring and bubbling was stopped immediately and the time required for the generated foam to collapse from 1000ml to 200ml was recorded as t₂. Each sample was carried out in duplicate batches and 6 tests were conducted on each batch. The average data of these 12 tests were shown in Table 7, it was observed that Bacillus licheniformis protease A and Bacillus licheniformis protease B achieved better antifoaming effects.

Table 7 Foam generation time and foam collapse time for the samples

Sample	Foam generation time (t₁, s)	Foam collapse time (t₂, s)
Culture medium A	31.8	222.3
Culture medium B	37.7	79.0
Culture medium C	38.7	75.3
Culture medium D	33.5	141.5

Example 6: Comparison between Bacillus licheniformis protease A and PfuS protease on antifoaming effects

Corn steep liquor hydrolysis was carried out as follows. 7.0 grams of corn steep liquor (CSL) was mixed with H₂O and the pH was adjusted to 7.0 by 2mol/L KOH before enzyme hydrolysis. Then the diluted CSL was hydrolyzed by different proteases at 50℃ for 16 hours. The control sample was diluted and adjusted pH before the culture medium preparation but was not incubated overnight, as incubation of overnight resulted in more foam. The final weight was adjusted to 14.0 grams. The enzyme dosing and culture medium preparation were carried out as shown below in Table 8 and Table 9.

Table 8 on enzyme dosing for samples

Culture medium preparation was carried out as follows. Then the diluted CSL was mixed with other materials to prepare the culture medium.

Table 9 Culture medium composition

Foam character evaluation was carried out as follows. The foam characters of medium samples were analyzed. 200ml medium was added into 1000ml container and air was bubbled into the medium with stirring. Then the time required for the foam generated from 200ml to 1000ml was recorded as t₁. The stirring and bubbling was stopped immediately and the time required for the generated foam to collapse from 1000ml to 200ml was recorded as t₂. Each sample was carried out in duplicate batches and 6 tests were conducted on each batch. The average data of these 9 tests were shown in Table 10, Bacillus licheniformis protease A achieved a better antifoaming effect.

Table 10 Foam generation time and foam collapse time for the samples

Sample	Foam generation time (t₁, s)	Foam collapse time (t₂, s)
Culture medium B	44.5	78.0
Culture medium E	43.5	114.8

Example 7: Fermentation of glutamic acid in presence of protease

The dry weight content and pH of untreated CSL was 37.2％and 3.90.15m³ untreated CSL was added into enzymatic hydrolyzation tank (total volume 30m³) , and turn on the mechanic agitation which the velocity was 60rpm. Then 30％NaOH solution was added into the enzymatic hydrolyzation tank to adjust pH to 7.30 at 60℃, and maintained the temperature 58-62 ℃ by jacket heating. Bacillus licheniformis protease A was then added into the tank, which the dose was 0.50g enzyme protein/g CSL protein. The temperature of the solution was maintained 58-62℃ with agitation for 16hr.

After that, soy bean hydrolytes and/or H₃PO₄ were added into the tank according to the dosage in the fermentation medium. The solution in the tank was then used for fermentation medium preparation according to the requirements of medium preparation.

The medium made by enzymatic hydrolyzed CSL was continuous sterilized at 123℃ for 18min, and cooled to 33.5℃ in 500m³ fermenter. 37m³ seed of Corynebacterium Glutamicum (OD600nm was 0.93 when it was 20X diluted. ) was then inoculated into the fermenter and the total volume was 240m³. Agitation and ventilation was adjusted and maintained to ensure DO in the broth was greater than 5％during the total fermentation process. 30hr later the fermentation was ended； the conversion rate from glucose to glutamic acid was 69.02％.

At the same time a conventional process (without enzyme treatment) was conducted as a control. 15m³ untreated CSL was used to prepare the medium and apply in the fermentation A, which the dry weight content and pH of the CSL was 37.2％and 3.90. After the fermentation, the conversion rate from glucose to glutamic acid was only 68.24％.

Claims

A process of producing a fermentation product from protein-containing culture medium, comprising

i) feeding the protein-containing culture medium into a fermentation vat；

ii) fermenting the protein-containing culture medium by fermenting organisms into a fermentation product；

wherein a S1 protease or a S8 protease is added

a) before, during or after feeding of the step i) , and/or

b) before or during fermentation in step ii) .
The process of claim 1, wherein the protease is a S1E protease or a S8A protease.
The process of claim 1, wherein the protease is derived from Nocardiopsis sp., preferably from Nocardiopsis sp. NRRL 18262, or the protease is from Bacillus licheniformis.
The process of claim 1, wherein the protease is selected from the group consisting of:

(a) a protease comprises or consists of amino acids of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(d) a fragment of the polypeptide of (a) , (b) , or (c) that has protease activity.
The process of claim 1, wherein the protease is selected from the group consisting of:

(a) a protease comprises or consists of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(d) a fragment of the polypeptide of (a) , (b) , or (c) that has protease activity.
The process of any of claims 1-5, wherein protein-containing culture medium comprise a protein-containing material which is preferably selected from the group consisting of yeast infusion/extract, fish infusion/extract, casein hydrolysate, peptone, soyabean cake, lupin cake, apeseed cake, soyabean meal, lupin meal, rapeseed meal, peanut cake, peanut meal, cottonseed cake, cottonseed meal, corn (maize) steep liquor, rice steep liquor, barley steep liquor, sorghum steep liquor, fruit-hull steep liquor, wheat steep liquor, corn protein, potato protein, corn protein, distillers's olubles, and a cell lysate with or without cellular debris； preferably wherein the protein-containing material is not pretreated by for example, acidic hydrolysis or salting out, or preferably the protein-containing material is pretreated by the said protease before it is added to the culture medium as a nitrogen source, or preferably the said protease is mixed with the protein-containing material before step i) .
The process of any of claims 1-6, wherein the fermentation product is an alcohol (e. g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1, 3-propanediol [propylene glycol] , butanediol, glycerin, sorbitol, and xylitol) ； an alkane (e. g. , pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane) , a cycloalkane (e. g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane) , an alkene (e. g. , pentene, hexene, heptene, and octene) ； an amino acid (e. g. , aspartic acid, glutamic acid, glycine, lysine, serine, threonine, phenylalanine, and tryptophan) ； a gas (e. g. , methane, hydrogen (H2) , carbon dioxide (CO₂) , and carbon monoxide (CO) ) ； isoprene； a ketone (e. g. , acetone) ； an organic acid (e. g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2, 5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid) ； polyketide, and a polypeptide (e.g. , enzyme) .
A method to prevent or reduce foam generated by agitation, aeration and/or fermentation process from protein-containing material, or a method to treat protein-containing material, comprising contacting the protein-containing material with a S1 protease or a S8 protease.
The method of claim 8, wherein the protease is a S1E protease or a S8A protease.
The process of claim 8, wherein the protease is derived from Nocardiopsis sp. , preferably from Nocardiopsis sp. NRRL 18262, or the protease is from Bacillus licheniformis.
The process of claim 8, wherein the protease is selected from the group consisting of:

(a) a protease comprises or consists of amino acids of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of SEQ ID NO: 1 herein or SEQ ID NO: 26 in WO2004/072279；

(d) a fragment of the polypeptide of (a) , (b) , or (c) that has protease activity.
The process of claim 8, wherein the protease is selected from the group consisting of:

(a) a protease comprises or consists of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(b) a protease has at least 80％, such as at least 85％, such as at least 90％, such as at least 95％, such as at least 96％, such as at least 97％, such as at least 98％, such as at least 99％sequence identity to amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(c) a protease variant comprising one or more (several) substitution, deletion, and/or insertion in the amino acid sequence of amino acids 1-274 of SEQ ID NO: 3 herein, which is also disclosed as SEQ ID NO: 2 of WO2006/136160；

(d) a fragment of the polypeptide of (a) , (b) , or (c) that has protease activity.
The process of any of claims 8-12, wherein protein-containing material is selected from the group consisting of yeast infusion/extract, fish infusion/extract, casein hydrolysate, peptone, soyabean cake, lupin cake, apeseed cake, soyabean meal, lupin meal, rapeseed meal, peanut cake, peanut meal, cottonseed cake, cottonseed meal, corn (maize) steep liquor, rice steep liquor, barley steep liquor, sorghum steep liquor, fruit-hull steep liquor, wheat steep liquor, corn protein, potato protein, corn protein, distillers's olubles, and a cell lysate with or without cellular debris； preferably wherein the protein-containing material is not pretreated by for example, acidic hydrolysis or salting out.
Use of a serine protease for preventing or reducing foam generated by agitation, aeration and/or fermentation process from protein-containing material, preferably, the process is related to a process as defined in any of the preceding claims, and/or the protease is defined in any of the preceding claims.
An antifoam agent, comprising a chemical antifoam agent and a S1 protease or a S8 protease, preferably, the protease is defined in any of the preceding claims.