Microorganism protein isolate

Abstract

The present application relates to protein derived from microorganisms, and more specifically derived from yeasts, that possess low RNA content and high gelling capacity. This application also relates to an industrial process and industrial uses of such single cell proteins for human and animal nutrition, health and well-being.

Description

MICROORGANISM PROTEIN ISOLATE

[0001] The present disclosure relates to the field of proteins derived from microorganisms, and more specifically derived from yeasts, that can be widely used for human and animal nutrition, health and well-being.Background art

[0002] Along with carbohydrates and lipids, proteins make up a significant part of our diet. The required amount of protein is generally said to be between 12% and 20% of our daily food intake.

[0003] The proteins consumed are generally either of animal origin, such as meats, fish, eggs and milk products, or of plant origin, including cereals, oleaginous plants and leguminous plants.

[0004] In industrialized countries, protein intake comes predominantly from proteins of animal origin. It is important to note that many studies show that excessive consumption of proteins that are of animal origin, and significantly less plant proteins, is one cause of the increase in the rates of cancers and cardiovascular diseases.

[0005] Moreover, animal proteins have many disadvantages, both in terms of their allergenicity, in particular with regards to proteins from milk and eggs, along with the degradation of our environment due to the intensive farming that is necessary for animal protein production.

[0006] Given this, manufacturers have gradually turned to alternative proteins to replace animal proteins. Indeed, it is known practice to use plant, insect or single cell proteins to replace all or some of the animal proteins in food products.

[0007] Single cell proteins are obtained by culturing microbial cells which will mainly convert sugars into proteins. After growing, proteins are directly recovered from culture media or after additional cell lysis step.

[0008] A first drawback of single cell proteins consists in their nucleic acid content. This content must be low because one of the end products of the metabolism ofnucleic acids is uric acid which the human body, lacking the enzyme uricase required for catabolism thereof, is unable to degrade. Such technical problems have been already worked and some solutions already exist, mainly via chemical and / or physical hydrolysis or via enzymatic treatments with RNase.

[0009] Another drawback is the low functionality of single cell protein obtained. Indeed replacement of animal protein is not always easy, because the functional properties of single cell proteins are different from those of animal proteins. In this case functional properties refer to the physical or physicochemical properties, like emulsifying capacity, foaming capacity or especially gelling capacity, that will help to deliver the right sensory qualities to the food that will be made with.

[0010] Patents US 3,867,555 or US 3,848,812 disclose a method for producing low nucleic acids protein extracts via chemical and / or physical hydrolysis. After cell lysis, nucleic acids are hydrolyzed thanks to heat treatment at alkaline pH (e.g. about 9.5 to about 12.5 and a temperature of about 50°C to about 120°C for less than 4 hours in US 3,867,555). As it will be disclosed in the following chapters of application, such treatment at high pH and temperature will damage protein, e.g. by promoting Maillard reaction or hydrolysis, and will alter their functionalities including gelling capacity. Isoelectric precipitation which traditionally follows in order to recover single cell protein is also known to reduce protein functionalities including gelling capacity. Last point to mention, the protein content obtained via this method is also below 85%.

[0011] Patent application US 2022 / 0071231 discloses a method for producing low nucleic acids protein extracts via enzymatic hydrolysis with RNase. Such enzymatic treatment allows to reduce RNA content without heating too much at high pH. RNA content of single cell protein obtained is disclosed to be below 11 %. As it will be exemplified in the following chapters of this application, if RNA is effectively reduced via this method, it will not be lower than 5%.

[0012] With such technical background, the purpose of the present invention is to overcome or reduce at least one of the disadvantages of the prior art, and / or to provide a single cell protein with low content of RNA, high functionalities including gelling capacity and high protein content.Brief Description of DrawingsFigures

[0013] [Fig. 1] shows egg white protein after cooking in a frying pan.

[0014] [Fig. 2] shows SCP from Example 1 after cooking in a frying pan.General description of Embodiments

[0015] A first embodiment is a single cell protein characterized in that its content of protein is comprised between 80% and 99% expressed on dry matter of single cell protein, its gelling capacity measured with Test A is comprised between 10000 and 50000, preferably between 15000 and 50000, and its content of RNA is comprised between 2% and 6%, preferably between 2% and 5%, expressed on dry matter of single cell protein.

[0016] The first embodiment is preferably characterized in that it is a yeast protein.

[0017] The first embodiment is preferably characterized in that it is protein from Saccharomyces cerevisiae.

[0018] The first embodiment is preferably characterized in that its protein content is comprised between 80% and 99%, preferably between 83% and 99%, preferably between 85% and 97% expressed on dry matter of single cell protein.

[0019] The first embodiment is preferably characterized in that its gelling capacity measured with Test A is comprised between 10000 and 40000, preferably between 20000 and 40000.

[0020] The first embodiment is preferably characterized in that its RNA content is comprised between 3% and 5% expressed on dry matter of single cell protein.

[0021] The first embodiment is preferably characterized in that its solubility measured with Test B from pH 4 to 8 is comprised between 60% and 99%.

[0022] The first embodiment is preferably characterized in that its solubility measured with Test B from pH 6 to 8 is comprised between 85% and 99%.

[0023] The first embodiment is preferably characterized in that its gelling capacity is comprised between 10000 and 25000, preferably between15000 and 25000, and its RNA content is comprised between 2% and 4%.

[0024] The first embodiment is preferably characterized in that its gelling capacity is comprised between 25001 and 50000, preferably between 40000 and 50000, and its RNA content is comprised between 4% and 6%, preferably between 4% and 5%.

[0025] The first embodiment is preferably characterized in that said single cell protein is obtainable by a process according to the second embodiment.

[0026] A second embodiment is a process for producing a single cell protein comprising the steps of:1 . Producing a microorganism suspension containing proteins,2. Lysing of the microorganism suspension produced in step 1 , resulting in a lysate,3. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,4. Ultrafiltration of the microfiltration permeate obtained in previous step 3, resulting in an ultrafiltration permeate and an ultrafiltration retentate,5. Optional sterilizing of said ultrafiltration retentate obtained in previous step 4,6. Optional drying of sterilized retentate obtained in previous step 5.

[0027] The second embodiment is preferably characterized in that the microorganism of step 1 is a yeast.

[0028] The second embodiment is preferably characterized in that the yeast is selected from the list of Saccharomyces, Yarrowia, Hansenula, Candida, and Pichia.

[0029] The second embodiment is preferably characterized in that crude protein content of microorganism is comprised between 40% and 60% expressed on dry matter of microorganism.

[0030] The second embodiment is preferably characterized in that step 1 includes a concentration step before step 2 selected from the list of centrifugation and press-filter in order to obtain a concentrated microorganism suspension with dry matter comprised between 10% and 30%. Before the concentration step, said dry matter content is usually below 10%.

[0031] The second embodiment is preferably characterized in that lysis of step 2 is performed using process selected from list of ball mill and high-pressure homogenization. At the end of step 2, a lysate is obtained.

[0032] The second embodiment is preferably characterized in that pH during lysis of step 2 is adjusted and regulated between 7,0 and 10,0, such as between 7,5 and 9,5.

[0033] The second embodiment is preferably characterized in that step 3 is done by succession of a centrifugation of the lysate obtained in previous step 2 resulting in an underflow and an overflow, followed by a microfiltration with a permeability gradient membrane of said overflow resulting in a microfiltration permeate and a microfiltration retentate.

[0034] The second embodiment is preferably characterized in that the centrifugation of step 3 produce an underflow which is recycled back in the microorganism suspension of step 2 to be lysed.

[0035] The second embodiment is preferably characterized in that pH of the lysate processed during permeability gradient microfiltration in step 3 is adjusted and regulated between 7,0 and 10,0, such as between 7,5 and 9,5.

[0036] The second embodiment is preferably characterized in that pH of the lysate processed during permeability gradient microfiltration in step 3 is adjusted and regulated between 8,0 and 9,5.

[0037] The second embodiment is preferably characterized in that microfiltration with a permeability gradient membrane of step 3 is done with a membrane having a porosity comprised between 0,1 microns and 0,2 microns, and at transmembrane pressure comprised between 0,50 bars and 1 ,50 bars.

[0038] The second embodiment is preferably characterized in that transmembrane pressure of microfiltration with a permeability gradient membrane of step 3 is comprised between 0,75 bars and 1 ,25 bars.

[0039] The second embodiment is preferably characterized in that ultrafiltration of step 4 is done with membrane porosity comprised between 3 to 10 KDa.

[0040] The second embodiment is preferably characterized in that sterilizing step of step 5 is done via a process selected in the list of pasteurization, HTST (High Temperature Short Time) pasteurization, ultraviolet treatment and filtration.

[0041] The second embodiment is preferably characterized in that drying step of step 6 is done via spray-drier.

[0042] The second embodiment is preferably characterized in that it consists only of all steps recited.

[0043] A third embodiment is the use of said single cell protein from first embodiment or produced via process from second embodiment in several applications comprised in the list of food applications, feed applications, cosmetic applications and pharmaceutical applications.

[0044] The third embodiment is preferably selecting from the list of egg or egg white replacers in bakery, especially for producing angel cake, cream cake, and meringues.

[0045] The third embodiment is preferably for food applications as binders for meat or fish analogue.

[0046] The third embodiment is preferably for food applications as milk protein replacers in dairy (milk, yogurt, cheese production).

[0047] A fourth embodiment relates to a single cell, preferably yeast, protein concentrate that can be obtained by the following process:1. Producing a microorganism suspension containing protein,2. Lysing of the microorganism suspension produced in step 1 , resulting in a lysate,3. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,4. Optionally centrifugating the microfiltration retentate obtained in step 3, resulting in an underflow and an overflow,5. Optionally drying the microfiltration retentate obtained in step 3 or the overflow obtained in step 4.

[0048] A fifth embodiment is a single cell, preferably a yeast, more preferably a Saccharomyces protein isolate that can be obtained by any process of the second embodiment.Detailed description of Embodiments

[0049] A first embodiment is a single cell protein characterized in that its content of protein is comprised between 80% and 99% expressed on dry matter of single cell protein, its gelling capacity measured with Test A is comprised between 10000 and 50000, preferably between 15000 and 50000 and its content of RNA is comprised between 2% and 6%, preferably between 2% and 5%, expressed on dry matter of single cell protein.

[0050] By “single cell protein”, it is understood in the present application a protein that is produced by culture of microorganisms. Microorganisms can be microalgae, yeast, bacteria or fungi. Such single cell protein can be excreted by microorganisms or extracted after lysis. Single cell protein can also be referred in literature as “SCP”.

[0051] As used herein, “between 2% and 6%” is to be understood as “between 2%- 5% and between 5%-6%”.

[0052] In a specific embodiment, the microorganism is bacteria, and in a preferred embodiments strains are chosen among Lactococcus lactis, Lactobacillus delbrueckii (bulgaricus), Lactobacillus helveticus, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus curvatus, Lactobacillus casei, Lactobacillus salivarius, Pediococcus pentosaceus, Oenococcus oeni, Corynebacterium glutamicum, Cupriavidus necator, Brevibacillus agri, Xanthomonas campestris, Staphylococcus carnosus, Staphylococcus xylosus, Ideonella sakaiensis, Bacillus subtilis and Bacillus (Priestia) megaterium.

[0053] By “protein”, it is understood in the present application the molecules corresponding to a sequence of amino-acid residues. In this application, proteins are understood in their native form or in a modified form, including hydrolyzed proteins. In a specific embodiment of the invention, native form of the protein ispreferred. These proteins can be used from compositions of different concentrations, including isolates having a protein content of above 80% on dry matter or concentrates having a protein content of between 50% and 80% on dry matter. In this application, isolates in which the protein content is above 80% on dry matter are particularly preferred.

[0054] The first embodiment is preferably characterized in that it is a yeast protein.

[0055] By “yeast”, it is understood in the present application a single-cell microorganism belonging to the fungus kingdom. Yeasts are widely utilized in various industrial processes and biological research due to its ability to ferment sugars into alcohol and carbon dioxide through the process of anaerobic respiration. Such industrial processes include bakery, brewery, wine, ethanol, dairy and livestock feed industries.

[0056] Yeasts are widely studied since many years and following books offer deeper knowledge and definition:- The Yeast Handbook Volume 2. Yeasts in Food and Beverages. Editors: Amparo Querol, Graham Fleet. 2006- The Yeasts, Vol 5. Yeast technology. Eds: A.H. Rose, J.S. Harrison. 1993- The yeast in the Brewery. G. Annemuller, H.J. Manger, P. Lietz. 2018

[0057] In a specific embodiment yeast types are chosen among S. cerevisiae, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces bayanus, Saccharomyces elipsoides, Saccharomyces uvarum, Saccharomyces ludwigii, Pichia pastoris (Komagataella), Candida utilis (Torula, Cyberlindnera jadinii) and Yarrowia (Candida) Hpolytica.

[0058] The first embodiment is preferably characterized in that it is a protein from Saccharomyces cerevisiae.

[0059] Any Saccharomyces cerevisiae can be used in the present application. More preferably, Saccharomyces cerevisiae are selected from public culture collections like DSM, ATCC for being produced by fermentation or commercially bought like LaKFerm® No1 from Lallemand.

[0060] The first embodiment is preferably characterized in that its protein content is comprised between 80% and 99%, preferably 83% and 99%, more preferably between 85% and 97% expressed on dry matter of single cell protein.

[0061] Protein content is measured by all conventional methods well known by a person skilled in the art. More preferably, protein content is measured by quantifying nitrogen content using Kjeldahl or Dumas method. Preferably the nitrogen content is measured using Kjeldahl method and said obtained nitrogen content is multiplied by 6,25. Such method is described in Codex Guidelines on Nutrition Labelling CAC / GL 2-19851 or in Ell Regulation 1169 / 2011. It corresponds to Test D. Obtained protein content is commonly known as “crude protein” content.

[0062] Dry matter is measured using any conventional methods known by a person skilled in the art. More preferably, analysis is done using moisture analyzer. Preferably, moisture analyzer is MX-50 from A&D. Principle of moisture analyzer it to weigh a sample (called W1 ), heating it in order to evaporate water until weight is stable and weighing it after heating (called W2). Dry matter is then calculated by dividing W2 by W1 and multiplying by 100. In this application, heating is done at 130°c until weight is stable.

[0063] The first embodiment is preferably characterized in that its gelling capacity measured with Test A is comprised between 10000 and 40000, preferably between 20000 and 40000.

[0064] By “Gelling capacity”, it is understood in the present application the functional property which corresponds to the capacity of a protein composition for forming a gel or a network, which increases the viscosity and generates a state of matter between the liquid and solid states. The term “gel strength” or “gel power” may also be used. To quantify this gelling capacity, it is thus necessary to generate this network and to evaluate its strength. To perform this quantification, in the present invention, Test A is used, the description of which is as follows.

[0065] The measurement of gelling capacity requires the measurement of viscoelastic properties, thus elastic modulus (G’) is measured before and after gel setting. The elastic modulus is the modulus which characterizes solids. Thus, the gelling power is determined by the difference of G’ before and after heat treatment which set gel.

[0066] In a preferred embodiment, gelling capacity is measured on a sample which is solubilized at 15%+ / - 0.5% of dry matter in water, wherein following steps are:- a first elastic modulus G’1 is measured at 20°C with the help of a rheometer,- sample is heated from 20°C to 80°C in 2 hours,- sample is cooled from 80°C to 20°C in 30 minutes,- a second elastic modulus G’2 is measured at 20°C with the help of a rheometerThe gelling capacity expressed in pascals (Pa) being equal to G’2 minus G’1.

[0067] Devices used for Test A are a balance having a precision of 0.1 mg such as Balance Mettler MS240S, a magnetic stirring plate and a rheometer such as DHR- 2 from TA Instruments and MCR (Modular Compact Rheometer) 301 or 302 from Anton Paar.

[0068] Samples are prepared by solubilization of 15% of dry matter in water. 50g of solution are used to carry out the analysis (7.5g of protein for 42.5g of water).1 . Weigh the water (42.5g) in a glass vial,2. Weigh the protein (7.5g),3. Stir at around 350 rpm + / - 30 minutes on a stirring plate at room temperature. The samples are prepared 3 times, therefore, 3 measurements are made in order to calculate mean and standard deviation.

[0069] Gel measurement consists in 4 steps:G’1 is measured.G’2 is measured.

[0070] Gelling capacity (expressed in Pa) = G’2 - G’1 .

[0071] Gelling capacity is preferably 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600,11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700,12800, 12900, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13700, 13800,13900, 14000, 14100, 14200, 14300, 14400, 14500, 14600, 14700, 14800, 14900,15000, 15100, 15200, 15300, 15400, 15500, 15600, 15700, 15800, 15900, 16000,16100, 16200, 16300, 16400, 16500, 16600, 16700, 16800, 16900, 17000, 17100,17200, 17300, 17400, 17500, 17600, 17700, 17800, 17900, 18000, 18100, 18200,18300, 18400, 18500, 18600, 18700, 18800, 18900, 19000, 19100, 19200, 19300,19400, 19500, 19600, 19700, 19800, 19900, 20000, 20100, 20200, 20300, 20400,20500, 20600, 20700, 20800, 20900, 21000, 21100, 21200, 21300, 21400, 21500,21600, 21700, 21800, 21900, 22000, 22100, 22200, 22300, 22400, 22500, 22600,22700, 22800, 22900, 23000, 23100, 23200, 23300, 23400, 23500, 23600, 23700,23800, 23900, 24000, 24100, 24200, 24300, 24400, 24500, 24600, 24700, 24800,24900, 25000, 25100, 25200, 25300, 25400, 25500, 25600, 25700, 25800, 25900,26000, 26100, 26200, 26300, 26400, 26500, 26600, 26700, 26800, 26900, 27000,27100, 27200, 27300, 27400, 27500, 27600, 27700, 27800, 27900, 28000, 28100,28200, 28300, 28400, 28500, 28600, 28700, 28800, 28900, 29000, 29100, 29200,29300, 29400, 29500, 29600, 29700, 29800, 29900, 30000, 30100, 30200, 30300,30400, 30500, 30600, 30700, 30800, 30900, 31000, 31100, 31200, 31300, 31400,31500, 31600, 31700, 31800, 31900, 32000, 32100, 32200, 32300, 32400, 32500,32600, 32700, 32800, 32900, 33000, 33100, 33200, 33300, 33400, 33500, 33600,33700, 33800, 33900, 34000, 34100, 34200, 34300, 34400, 34500, 34600, 34700,34800, 34900, 35000, 35100, 35200, 35300, 35400, 35500, 35600, 35700, 35800,35900, 36000, 36100, 36200, 36300, 36400, 36500, 36600, 36700, 36800, 36900,37000, 37100, 37200, 37300, 37400, 37500, 37600, 37700, 37800, 37900, 38000,38100, 38200, 38300, 38400, 38500, 38600, 38700, 38800, 38900, 39000, 39100,39200, 39300, 39400, 39500, 39600, 39700, 39800, 39900, 40000, 40100, 40200,40300, 40400, 40500, 40600, 40700, 40800, 40900, 41000, 41100, 41200, 41300,41400, 41500, 41600, 41700, 41800, 41900, 42000, 42100, 42200, 42300, 42400,42500, 42600, 42700, 42800, 42900, 43000, 43100, 43200, 43300, 43400, 43500,43600, 43700, 43800, 43900, 44000, 44100, 44200, 44300, 44400, 44500, 44600,44700, 44800, 44900, 45000, 45100, 45200, 45300, 45400, 45500, 45600, 45700,45800, 45900, 46000, 46100, 46200, 46300, 46400, 46500, 46600, 46700, 46800,46900, 47000, 47100, 47200, 47300, 47400, 47500, 47600, 47700, 47800, 47900,48000, 48100, 48200, 48300, 48400, 48500, 48600, 48700, 48800, 48900, 49000,49100, 49200, 49300, 49400, 49500, 49600, 49700, 49800, 49900, 50000 and also ranges using previously cited values as borders.

[0072] As it will be disclosed in the examples chapter below, gelling capacity of the single cell protein of the first embodiment is equivalent to egg white protein. “Equivalent” must be understood as a gelling capacity value that is comprised between 25% and 200%, preferably between 50% and 150%, more preferably between 75% and 125% of egg white protein, preferably powder, gelling capacity.

[0073] By “RNA”, it is understood in the present application ribonucleic acids of any molecular weight (MW): from high MW macromolecules packed in ribosomes to transfer RNA, matrix RNA, non-coding RNA, to monomeric nucleotides.

[0074] RNA content is preferably 2,0%, 2,1 %, 2,2%, 2,3%, 2,4%, 2,5%, 2,6%, 2,7%, 2,8%, 2,9%, 3,0%, 3,1%, 3,2%, 3,3%, 3,4%, 3,5%, 3,6%, 3,7%, 3,8%, 3,9%, 4,0%, 4,1 %, 4,2%, 4,3%, 4,4%, 4,5%, 4,6%, 4,7%, 4,8%, 4,9%, 5,0%, 5,1 %, 5,2%, 5,3%, 5,4%, 5,5%, 5,6%, 5,7%, 5,8%, 5,9%, 6,0% and also ranges using previously cited values as borders.

[0075] All methods for RNA determination well-known to a person skilled in the art can be used. A preferred method called Test C in this application was adapted according to Rut M. (1973) “Determination of nucleic acids on yeast and yeast related products." (Kvasny Prumysl, 19, 131-133). The samples were treated with 0.5M HCIO4 for 30 min at 90°C by mixing. Then, the samples were cooled and subsequently centrifuged at 17000 xg for 5 min. The supernatants were neutralized with 1 M NaOH and the amount of total RNA was determined by measuring the absorbance at 260 nm using a spectrophotometer, such as the NanoDrop 2000c spectrophotometer.

[0076] The first embodiment is preferably characterized in that its RNA content is comprised between 3% and 4% expressed on dry matter of single cell protein.

[0077] The first embodiment is preferably characterized in that its solubility measured with Test B from pH 4 to 8 is comprised between 60% and 99%.

[0078] The first embodiment is preferably characterized in that its solubility measured with Test B from pH 6 to 8 is comprised between 85% and 99%.

[0079] “Solubility” is understood to mean the quantification of the percentage of water-soluble material in a powder by diluting the powder in distilled water in controlled conditions especially temperature and pH, centrifuging the resulting suspension and analyzing the amount of solubilized material in the supernatant. In this application, the following Test B is preferred.

[0080] Test B: 150 g of distilled water are introduced into a 400ml beaker at 20°C + / -2°C by stirring with a magnetic stirrer bar, and precisely 5.00g of single cell protein sample to be tested are added. If required, the pH is adjusted to the desired value with 0.1 N NaOH or O.I N HCI. In this application preferred pH values for Test B are especially 4,0; 6,0; 7,0 and 8,0. The content is supplemented with water to reach 200g of water. Mixing is carried out for 30 minutes at 1000 rpm and centrifugation is carried out for 15 minutes at 3000 g. 25g of the supernatant are collected and introduced into a crystallizing dish dried and fared beforehand. The crystallizing dish is placed in an oven at 103°C + / -2°C for 1 hour until all water evaporate. It is then placed in a desiccator (with desiccant) to cool to ambient temperature. The sample is collected, then is weighed.

[0081] The solubility corresponds to the content of soluble dry matter, expressed as % by weight relative to the weight of the sample. The solubility is calculated with the following formula:(ml — m2) * (200 + P) * 100 P1 * PIn which:• P = weight, in g, of the sample = 5 g• ml = weight, in g, of the crystallizing dish after drying• m2 = weight, in g, of the empty crystallizing dish• P1 = weight, in g, of the sample collected = 25 g

[0082] It is the merit of Applicant to have reach to produce a single cell protein, especially from yeast, that have protein content comprised between 80% and 99%, RNA content between 2% and 6%, such as between 2% and 5%, while having also gelling capacity between 15000 and 50000. Processes from prior art are incompatible to both produce such high gelling and protein content, while having such low RNA content. Treatment at high pH and temperature are well known to decrease RNA but, as it will be exemplified in the application below, it will also damage protein and alter their functionalities including gelling capacity. RNase or ribonucleases allow milder conditions for decreasing RNA but, as it will be also demonstrated below in the present application, RNA level lower than 5% are not reachable.

[0083] In one embodiment, the first embodiment is characterized in that its gelling capacity is comprised between 10000 and 25000, preferably between 15000 and 25000, preferably between 15000 and 20000, and its RNA content is comprised between 2% and 5%.

[0084] The first embodiment is preferably characterized in that its gelling capacity is comprised between 10000 and 15000, preferably between 15000 and 25000, and its RNA content is comprised between 2% and 4%.

[0085] The first embodiment is preferably characterized in that its gelling capacity is comprised between 25001 and 50000, preferably between 40000 and 50000, and its RNA content is comprised between 4% and 6%, preferably between 4% and 5%.

[0086] In one embodiment, the first embodiment is also preferably characterized in that its gelling capacity is comprised between 30000 and 40000, and its RNA content is comprised between 4% and 6%, preferably between 4% and 5%.

[0087] As used herein, “between 25001 and 50000” is to be understood “between 25001-40000 and between 40000-50000”.

[0088] As used herein, “between 4% and 6%” is to be understood as “between 4%- 5% and between 5%-6%”.

[0089] A second embodiment is a process for producing single cell protein comprising steps of:1 . Producing a microorganism suspension containing protein,2. Lysis of the microorganism suspension produced in step 1 , resulting in a lysate,3. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,4. Ultrafiltration of the microfiltration permeate obtained in previous step 3, resulting in an ultrafiltration permeate and an ultrafiltration retentate,5. Optional sterilizing of said ultrafiltration retentate obtained in previous step 4,6. Optional drying of sterilized retentate obtained in previous step 5.

[0090] Step one of the second embodiment corresponds to the production of a microorganism suspension that contains proteins.

[0091] Relevant type of microorganisms has been already defined and discussed in the description part of first embodiment.

[0092] The second embodiment is preferably characterized in that the microorganism of step 1 is a yeast.

[0093] The second embodiment is preferably characterized in that yeast belongs to the genus Saccharomyces, such as S. cerevisiae, S. pastorianus, S. elipsoides, S. ludwigii, S. chevalieri, S. boulardii, S. bayanus, S. italicus, S. delbrueckii, S. rosei, S. microellipsodes, S. carlsbergensis, S. bisporus, S. fermentati, S. rouxii, or S.uvarunr, a yeast belonging to the genus Schizosaccharomyces, such as S. japonicus, S. kambucha, S. octosporus, orS. pombe', a yeast belonging to the genus Hansenula, such as H. wingei, H. ami, H. henricii, H. americana, H. canadiensis, H. capsulata, or H. polymorpha', a yeast belonging to the genus Candida, such as C. albicans, C. utilis, C. boidinii, C. stellatoidea, C. famata, C. tropicalis, C. glabrata, or C. parapsilosis', a yeast belonging to the genus Pichia, such as P. pastoris, P. kluyveri, P. polymorpha, P. barkeri, P. cactophila, P. rhodanensis, P. cecembensis, P. cephalocereana, P. eremophilia, P. fermentans, or P. kudriavzevir', a yeast belonging to the genus Kluyveromyces, such as K. marxianus; a yeast belonging to the genus Yarowia, such as Y. lipolytica and a yeast belonging to the genus Torn lopsis, such as T. bovina, or T. glabrata.

[0094] The second embodiment is even more preferably characterized in that yeast is Saccharomyces cerevisiae.

[0095] Production of microorganism suspension containing protein can be firstly done by cultivating a selected strain in a reactor, preferably a fermenter. Selected strains can be bought in a collection (e.g. ATCC, DSM) or screened in nature. Strains can be used as native or after being modified using GMO techniques like molecular biology tools or mutagenesis. Unique strain, mix of strains or even cocultivation of strains can be used.

[0096] Culture media and methods for growing microorganisms, preferably yeasts, are well known in the art. Suitable media comprise, for example, the YPD medium of Sigma Aldrich (Taufkirchen, Germany).

[0097] Microorganism suspension containing protein can secondly be obtained on the market, produced by commercial companies. In a preferred embodiment, microorganism suspension containing protein are byproducts left in the reactor after production of another main products like beer, ethanol, fuel, enzymes. Such products are mainly obtained in dried and / or compressed storage form. Water is added in order to obtain microorganism suspension containing proteins.

[0098] The second embodiment is preferably characterized in that crude protein content of microorganism is comprised between 40% and 60% expressed on dry matter of microorganism. Ways to measure the protein content was described in previous description of first embodiment above.

[0099] The second embodiment is preferably characterized in that step 1 includes a concentration step before step 2 selected from the list of centrifugation and pressfilter in order to obtain a concentrated microorganism suspension with dry matter comprised between 10% and 30%, preferably between 10% and 20%. The dry matter percentages cited herein refer to wt. % based on the total weight of the suspension. The dry matter content of a suspension can be determined in accordance with standard procedures using commercially available devices, for example, the Moisture Analyzer MX-50 from A&D. Once the dry matter content of a starting suspension has been determined, this suspension can be adjusted to a predetermined value either by diluting or concentrating the suspension.

[0100] Any relevant centrifuge apparatus based on separation of water and solid contents using sedimentation under gravity field can be used.

[0101] Any relevant filter based on separation of water and solid contents can be used. Press-filter is preferably used.

[0102] In a preferred embodiment, the cells (microorganisms) are washed with a buffer, such as water, before lysing step according to step two of second embodiment. Such washing step is useful to remove unwanted compounds from the microorganism suspension and / or reduce the taste of products obtained.

[0103] Step two of second embodiment corresponds to the lysis of the microorganism suspension containing proteins obtained in step one. Microorganisms commonly comprise inner soluble compounds (e.g. proteins, sugars) and insoluble compounds contained in a space called cytoplasm which is separated from external media via cell wall. Lysis consists of mechanical and / or chemical ways to break cell wall and release inner soluble compounds including proteins in order to make them available for next steps.

[0104] Yeast species like Saccharomyces cerevisiae have cell walls that mainly consists of [3-glucans, mannoproteins, and chitin in a covalently linked matrix. Beneath the cell walls, yeast cells have a cell membrane of a lipid bilayer. To release proteins from the cell interior, both of these protective barriers need to be disrupted.

[0105] Lysis efficiency (LE) expressed in percentage is calculated based on the following protocol called Test C:- lysate is centrifuged at 16639 G for 10m in,- dry matter & weight of underflow & overflow is measured

[0106] LE = (1 - (Qu x DMu) / (QI x DMI)) *100%Where:- QI - amount of lysate before centrifugation, expressed in g;- DMI - dry matter of lysate, expressed in %.- Qu - amount of underflow (intact yeast) after centrifugation, expressed in g;- DMu - dry matter of underflow, expressed in %.

[0107] The second embodiment is preferably characterized in that lysis of step 2 is obtained using process preferably selected from list of ball mill, and high-pressure homogenization.

[0108] According to the process of the invention, cell lysis will be performed preferably mechanically, but use of other enzymes like e.g. glucanases or mannanases, which are commonly employed for yeast cell lysis, can be used if temperature does not exceed 40°C in order to keep protein functionalities.

[0109] In addition, microorganism cells contain a wide variety of proteases which are able to hydrolyze proteins into amino acids. When microorganism cells are disrupted, the proteases are released and could decompose the other protein molecules which are released by the microorganism cells. Accordingly, in a preferred embodiment of the invention, the cell lysis is performed at ambient temperatures. In this embodiment the activity of proteases is thus reduced.

[0110] According to a preferred embodiment, second step of second embodiment ( / .e. the lysis) is performed at temperatures of below 40°C, more preferably below 30°C, and even more preferably below 20°C. A temperature of below 20°C, such as 15°C or 10°C, is particularly preferred to avoid an undesired decomposition of the proteins by proteases released from the microorganism cells. For precision, temperatures of 10°C, 11 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C and ranges obtained with these values as borders.

[0111] According to a preferred embodiment, microorganism cell lysis is achieved by use of a bead mill. On one hand, this ensures that the proteins remain native upon cell rupture. On the other hand, the use of a bead mill does not require any additives that could impair the food grade quality of the resulting protein compositions obtained. A bead mill normally comprises a chamber that is filled with beads which are moved around by a set of impeller fins. When a cell-containing suspension is passed through the chamber, the cells are disrupted upon collision with the beads. The efficiency of cell rupture can be adjusted by routine measures, for example, by changing the flow rate which determines how fast the microorganism suspension (such as yeast suspension) is passed through the chamber of the bead mill. Normally, a low flow rate leads to high lysis efficiency, since the cells have more time to collide with the grinding beads. Dependent on the volume of the chamber of the bead mill, flow rates of at least 10 kg / h can be selected, such at least 20 kg / h, at least 50 kg / h, or at least 100 kg / h. It is particularly preferred that during operation the bead mill is cooled to temperatures below 20°C, such 18°C, 16°C, 14°C, 12°C or 10°C. Bead mills are offered by different manufacturers, for example, the Dyno®-Mill Multi Lab Wab.

[0112] Another factor that has a direct impact on the efficiency of lysis is the bead material and size. Beads can be made of different materials, e.g. glass, ceramic or plastic. Particularly good results have been achieved with zirconium oxide beads. In addition, these beads are significantly more durable compared to glass beads. The beads used for disrupting the yeast cells, e.g. the zirconium oxide beads, may have different sizes which normally range from 0.2-2.0 mm. It has been found that for disrupting the yeast cells, a bead size of 0.25-0.35 mm leads to particularly good results. Accordingly, a bead size of 0.25-0.35 mm is particular preferred. Bead filling volumes of between 30-80% may be used, for example, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%. A bead filling volume of between 50-60% is particularly preferred according to the invention.

[0113] Another way of influencing the efficiency of cell rupture is to adapt the impeller architecture. The impeller is normally mounted with plastic fins to move the beads. For improved cell rupture, it is for example possible to provide accelerators onto the rotor. Accelerators are designed to force the beads to collide more often.The impeller rotation speed is another parameter that may be adjusted to improve cell rupture. A rotor speed of between 1 -20 m / s can be used, for example, about 1 m / s, about 2 m / s, about 3 m / s, about 4 m / s, about 5 m / s, about 6 m / s, about 7 m / s, about 8 m / s, about 9 m / s, about 10 m / s, about 11 m / s, about 12 m / s, about 13 m / s, about 14 m / s, about 15 m / s, about 16 m / s, about 17 m / s, about 18 m / s, or about 19 m / s. A skilled person would be readily able by routine experimentation to find optimum parameters for disrupting a yeast suspension in a bead mill.

[0114] Yet another way of mechanically lysing the microorganism cells uses high- pressure homogenization (HPH). High pressure homogenization is a method commonly used in the pharmaceutical, chemical and food industry to stabilize emulsions, and the like. It can however also be used to disrupt bacteria and yeast in order to extract intracellular products from the cells. During HPH, the cells to be disrupted are passed through a narrow slit under high pressure. As the yeast cells pass this narrow slit, the flow rate sharply increases and the pressure abruptly decreases. The resulting shear forces cause cell disruption.

[0115] HPH devices for use in the method of the present invention can be obtained from different manufacturers. For example, the EmulsiFlex-C3 of Avestin Europe GmbH (Mannheim, Germany) or the 1000 / 2000 Homogenizer of SPX Flow Technology Germany GmbH (Moers, Germany) can be used. The microorganism suspension can be passed through the HPH device one time or several times. If the suspension is passed through the device several times, the efficiency of cell rupture increases. The pressure of the device can be set to a pressure of at least 700 bar. Preferably, the pressure will be at least 1000 bar, at least 1100 bar, at least 1200 bar, at least 1300 bar, at least 1400 bar, at least 1500 bar, at least 1600 bar, at least 1700 bar, at least 1800 bar, at least 1900 bar, or at least 2000 bar or more.

[0116] Microfluidics Microfluidizer 7250-10 equipment is preferred for cell lysis.

[0117] After or during lysis of the cells as described above, the pH of the lysate may approach a value of 6.0 or below. It is preferred that the pH of the lysate is readjusted to a value between 7.0 and 10.0. For example, the pH of the lysate is readjusted to about 7,0; 7,1 ; 7,2; 7,3; 7,4; 7,5; 7,6; 7,7; 7,8; 7,9; 8,0; 8,1 ; 8,2; 8,3; 8,4; 8,5; 8,6; 8,7; 8,8; 8,9; 9,0; 9,1 ; 9,2; 9,3; 9,4; 9,5; 9,6; 9,7; 9,8; 9,9; 10,0 and all rangesthat can be obtained with these values as borders. More preferably, pH will be adjusted between 8,5 and 9,5.

[0118] Step three of second embodiment corresponds to a microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate.

[0119] As it will be described below, the microfiltration with a permeability gradient membrane is key in this step in order to separate soluble proteins (containing the functional proteins) in its permeate on one side and insoluble proteins containing main part of RNA in its retentate in the other side.

[0120] Microfiltration is a type of physical filtration process where a fluid is passed through a pore-sized membrane filter to separate suspended particles from process liquid. Microfiltration usually serves as a pre-treatment for other separation processes such as ultrafiltration, and a post-treatment for granular media filtration. In terms of approximate molecular weight these membranes can separate macromolecules of molecular weights generally less than 100,000 g / mol.

[0121] The fluid is passed through at a relatively high velocity, parallel or tangential to the membrane in a sheet or tubular form. A pump is commonly fitted onto the processing equipment to allow the fluid to pass through the membrane filter. There are also two pump configurations, either pressure driven or vacuum. A differential or regular pressure gauge is commonly attached to measure the pressure drop between the outlet and inlet streams.

[0122] Microfiltration in the present application use a particular membrane which is commonly called “permeability gradient” or “permeability gradient membrane”. That is why the microfiltration according to the present disclosure is called a “microfiltration with a membrane having a permeability gradient” or a “permeability gradient microfiltration”.

[0123] Permeability gradient membrane is designed for optimizing compounds transfer across the microfiltration membrane. In conventional microfiltration membranes, the natural pressure drop creates asymmetric transmembrane pressure (TMP) from the inlet to the outlet of the flow channel. Permeability gradient membranes are concepted with permeability gradient built into the support structure which allows a stable microfiltration regime all along the membrane. Suchmembranes are well known in industry. Pall® Membralox® GP is an example of commercial membrane suitable for application. Applicant has surprisingly demonstrated that microfiltration membranes with a permeability gradient allows to retain efficiently RNA in retentate while allowing purified soluble protein to pass in filtrate.

[0124] Permeability gradient membrane are composed of at least two layers or structures: the support layer and the filtration layer. The filtration layer which is designed to perform the separation is deposited at the surface of the support layer which aims to support it.

[0125] In a preferred embodiment, the permeability gradient is built into the support layer of the permeability gradient membrane used in step three of second embodiment. If the permeability gradient is built into the filtration layer of the permeability gradient membrane, the overall yield of soluble proteins will be not efficient enough. The second embodiment is preferably characterized in that step 3 is done by the succession of a centrifugation of the lysate obtained in previous step 2, resulting in a centrifugation underflow and a centrifugation overflow, followed by a microfiltration with a permeability gradient membrane of said overflow resulting in a microfiltration permeate and a microfiltration retentate.

[0126] Said centrifugation overflow is free of intact cell or cell wall debris, preferably less than 1 %, more preferably less than 0,5%, more preferably less than 0,1 %.

[0127] The second embodiment is preferably characterized in that the centrifugation of step 3 produces an underflow which is recycled back in lysis of step 2. Said underflow contains intact cells and / or cell wall debris and can be recycled back in the microorganism suspension of step 2, to be lysed, therefore improving the yield.

[0128] The second embodiment is preferably characterized in that pH of the lysate obtained in step 2 is adjusted and regulated between 7,0 and 10,0 (such as between 7,5 and 9,5) while feeding microfiltration of step 3.

[0129] The second embodiment is preferably characterized in that pH of the lysate obtained in step 2 is adjusted and regulated between 8,0 and 9,5 while feeding microfiltration of step 3.

[0130] As it will be exemplified, precise control of pH during the microfiltration with a permeability gradient membrane will allow to optimize RNA separation between permeate and retentate.

[0131] pH can be adjusted with any of well-known reagents selecting in basic and acid reagents. In a preferred embodiment, NaOH, KOH and their blend will be used as basic reagents. In a preferred embodiment, HCI, H2SO4 and their blend will be used as acid reagents.

[0132] The second embodiment is preferably characterized in that permeability gradient microfiltration of step 3 is done with a membrane having a porosity comprised between 0,1 microns and 0,2 microns, at transmembrane pressure comprised between 0,50 bars and 1 ,50 bars.

[0133] The second embodiment is preferably characterized in that transmembrane pressure is comprised between 0,75 bars and 1 ,25 bars.

[0134] Microfiltration membrane porosity can be 0,10; 0,11 ; 0,12; 0,13; 0,14; 0,15; 0,16; 0,17; 0,18; 0,19; 0,20 microns and all ranges that can be obtained with these values as borders.

[0135] Transmembrane pressure (TMP) is to be understood as the pressure difference between two sides of membranes. This TMP is the power allowing membrane filtration.

[0136] Transmembrane pressure can be 0,50; 0,55; 0,60; 0,65; 0,70; 0,75; 0,80; 0,85; 0,90; 0,95; 1 ,00; 1 ,05; 1 ,10; 1 ,15; 1 ,20; 1 ,25; 1 ,30, 1 ,35; 1 ,40; 1 ,45; 1 ,50 bars and all ranges that can be obtained with these values as borders.

[0137] In a preferred embodiment, the permeability gradient microfiltration is carried out in a tangential filtration module.

[0138] In an even more preferred embodiment, said microfiltration is followed by a diafiltration step. In this embodiment, diafiltration corresponds to successive steps of concentration of microfiltration retentate and addition of water to increase the purity of retentate. Preferably, diafiltration will be carried out one, two or three times with one, two or three volumes of water. More preferably, diafiltration will be carried out one time with three volumes of water.

[0139] Microfiltration retentate obtained in step 3 can also undertake a separation process that will allow production of a protein concentrate. Such additional step allows to maximize overall yield by proposing also a protein concentrate and a composition containing RNA products (including inosinate and guanylate as well as others ribotides).

[0140] According to a preferred embodiment, the third step of second embodiment ( / .e. microfiltration) is performed at temperatures of below 40°C, more preferably below 30°C, more preferably below 22°C and even more preferably below 20°C. A temperature of below 22°C, preferably below 20°C, such as 15°C or 10°C, is particularly preferred to avoid an undesired decomposition of the proteins by proteases released from the microorganism cells. For precision, this encompasses temperatures such as 10°C, 11 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C and ranges obtained with these values as borders.

[0141] Step 4 corresponds to an ultrafiltration of the permeate obtained with a permeability gradient microfiltration of step 3. The obtained ultrafiltration retentate contains single cell proteins.

[0142] Ultrafiltration refers to the membrane separation method, which is distinguished from microfiltration or nanofiltration by the size of suspended or solution particles that may pass through. For ultrafiltration this size is between 1 and 100 nanometers (nm).

[0143] The second embodiment is preferably characterized in that the ultrafiltration of step 4 is done with a membrane porosity comprised between 3 to 10 KDa.

[0144] The cutoff thresholds can therefore be 3 KDa, 4 KDa, 5 KDa, 6 KDa, 7 KDa, 8 KDa, 9 KDa and 10 KDa, as well as all the ranges formed by these values. The preferred filtration temperature is 5°C to 15°C. The filtration temperature can therefore be 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11 °C, 12°C, 13°C, 14°C, 15°C, and all ranges formed by these values.

[0145] Preferably, the transmembrane pressure will be between 1 and 10 bars, preferably between 2 and 4 bars. The transmembrane pressure values may be 1 bar, 2 bars, 3 bars or 4 bars, as well as all the ranges formed by these values. Thetransmembrane pressure is a parameter well known to the skilled person which consists of the pressure difference on either side of the ultrafiltration membrane.

[0146] In a preferred embodiment, said ultrafiltration is carried out in a tangential filtration module.

[0147] In an even more preferred embodiment, said ultrafiltration is followed by a diafiltration step. In this embodiment, diafiltration corresponds to successive steps of concentration of ultrafiltration retentate and addition of water to increase the purity of retentate. Preferably, diafiltration will be carried out in order to achieve at least a protein richness of 70%. Preferably, diafiltration will be carried out in order to achieve at least a protein richness of 80%. Protein richness can be 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and all ranges formed by these values.

[0148] According to a preferred embodiment, the fourth step of second embodiment ( / .e. ultrafiltration) is performed at temperatures of below 40°C, more preferably below 30°C, and even more preferably below 20°C, such as below 10°C. A temperature of below 20°C, such as 15°C or 10°C, is particularly preferred to avoid an undesired decomposition of the proteins by proteases released from the microorganism cells. For precision, this encompasses temperatures such as 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C and ranges obtained with these values as borders.

[0149] Ultrafiltration permeate can also be highly valorized as it contains, especially in case of yeast as starting material, yeast peptides that confers kokumi taste. Kokumi is predominantly found in the realm of Japanese cuisine, where its taste sensation occurs naturally in fermented foods like alcohol, soy sauce, fish sauces and shrimp paste. Ultrafiltration permeate can allow easy use of protein product to confer kokumi taste in food applications, without use of fermentation.

[0150] The second embodiment is preferably characterized in that sterilizing step of step 5 is done via a process selected in the list of pasteurization, HTST, ultraviolet treatment and filtration.

[0151] Preferably, the process may then include an additional heat treatment of ultrafiltration retentate. Temperature and time conditions can vary widely in this step, for example from 70 to 140°C and from 0.1 seconds to several minutes. Main driver will be to allow reduction of microorganism without altering protein functionalities. According to a first variant of this additional heat treatment step, the temperature ranges from 70 to 90°C and its duration ranges from 0.1 seconds to 30 minutes. According to a second variant of this additional heat treatment step, the temperature ranges from 90 to 110°C and its duration ranges from 0.1 seconds to 5 minutes. In another variant, this additional heat treatment step is performed at a temperature ranging from 110 to 140°C for a time ranging from 0.1 to 30 seconds, preferably from 0.2 to 15 seconds, for example from 0.3 to 10 seconds. This step may aim to sanitize the single cell protein, preferably from yeast. To achieve this additional heat treatment step, single cell protein, preferably from yeast can be in the form of an aqueous dispersion, preferentially having a dry matter ranging from 10 to 25%, for example from 15 to 20%. Advantageously, following heat treatment step, a cooling step may occur. According to a preferred variant, this cooling step is obtained by rapid cooling («flash-cooling»). At the end of this step, the temperature can range from 60 to 100°C, for example between 70 and 90°C. Similarly, this rapid cooling step ("flash-cooling") is performed by applying a vacuum to the aqueous dispersion of single cell protein, preferably yeast, the vacuum applied being determined according to the chosen cooling temperature.

[0152] The second embodiment is preferably characterized in that drying step of step 6 is done via a spray-drier.

[0153] The principle of spray drying is based on the dispersion of a solution into fine droplets which are introduced into a flow of hot air. The solvent evaporates from the substrate droplets so that dry product clusters remain. Spray drying is usually performed at temperatures that are normally higher than the melting temperature of proteins. Standard spray drying devices can be used, such as the Mini Spray Dryer B-290 from Buchi Labortechnik GmbH (Essen, Germany) or the Mobile Minor™ Spray Dryer from GEA (Berlin, Germany).

[0154] Freeze drying or lyophilization is a process which removes water from a product to extend shelf life. Freeze drying encompasses freezing the product, reducing the pressure and adding heat to allow the frozen water in the material tosublimate. Various methods can be applied for freezing the product. For example, freezing can be achieved by using a standard freezer or a chilled bath. Cooling the product below its triple point ensures that sublimation will occur upon heating. To prevent the formation of large crystals that may damage the structure of the product to be dried, freezing is done rapidly. About 95% of the water in the product is removed when the frozen water sublimates. Most materials can be dried to 1 -5% residual moisture. Standard freeze-drying devices can be used, such as the Lyovac™ devices from GEA (Berlin, Germany), the Gamma 2-20 Freeze dryer LCM-1 from Christ (Osterode am Harz, Germany), or the Christ Martin™ Alpha 1 -2 Lyophilisator from Fisher Scientific GmbH (Schwerte, Germany).

[0155] The second embodiment is preferably characterized in that it consists only of all steps described and / or claimed.

[0156] In a preferred embodiment, the second embodiment is a process for producing a single cell protein consisting in the following steps of:1 . Producing a microorganism suspension containing protein,2. Lysing of the microorganism suspension produced in step 1 , resulting in a lysate.3. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,4. Ultrafiltration of the microfiltration permeate obtained in previous step 3, resulting in an ultrafiltration permeate and an ultrafiltration retentate,5. Optional sterilizing of said ultrafiltration retentate obtained in previous step 4,6. Optional drying of sterilized retentate obtained in previous step 5.

[0157] In a preferred embodiment, steps 1 to 4, preferably steps 1 to 5, more preferably steps 1 to 6 of the process of the second embodiment are carried out under temperature controlled between 4°C and 22°C, preferably between 5°C and 20°C, more preferably between 10°C and 15°C. The controlled temperature of steps 1 to 4, preferably steps 1 to 5, more preferably steps 1 to 6 of the process of the second embodiment can therefore be 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11 °C,12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C and all ranges formed by two of these values as lower and upper values. It is well known in the field that such temperature ranges will help to protect protein from thermal and / or enzymatic denaturation. Using such controlled temperature will help also to obtain good gelling capacity and low RNA content.

[0158] It is a considerable advantage of the process to allow a production of a functional isolate (e.g. single cell protein isolate), but potentially also a protein concentrate (e.g. single cell protein concentrate), RNA products and a kokumi peptide starting from a unique microorganism. Such multiple products allow a better valorization.

[0159] A third embodiment is uses of single cell protein as described above or produced via a process as described above in industrial applications comprised in the list of food applications, feed applications, cosmetic applications and pharmaceutical applications.

[0160] The food and feed industry products are understood to mean industrial confectionery (for example, chocolate, caramel, jelly sweets), bakery products (for example, bread, brioches, muffins), the meat and fish substitutes industry (for example, sausages, hamburgers, fish nuggets, chicken nuggets), sauces substitutes (for example, bolognaise, mayonnaise), substitutes of products derived from milk (for example, cheese, plant milk), beverages (for example, high protein beverages, powdered beverages to be reconstituted).

[0161] The invention will be of particular interest in the field of analogs of meat, fish, sauces, soups. A particular application relates to the use of the composition according to the invention for manufacturing meat substitutes, in particular minced meat, and also bolognaise sauce, steak for hamburgers, meat for tacos and pitta, “Chili sin carne”.

[0162] The third embodiment is preferably selecting from the list of egg or egg white replacers in bakery especially for producing angel food cake, cream cake, meringues.

[0163] The third embodiment is preferably useful for producing binders for meat or fish analog.

[0164] The third embodiment is preferably for food as milk protein replacers in dairy (milk, yogurt, cheese production).

[0165] A fourth embodiment relates to a single cell, preferably yeast, protein concentrate obtained from a microfiltration retentate as described above.

[0166] Said single cell, preferably yeast, protein concentrate can be obtained by the following process:6. Producing a microorganism suspension containing protein,7. Lysing of the microorganism suspension produced in step 1 , resulting in a lysate,8. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,9. Optionally centrifugating the microfiltration retentate obtained in step 3, resulting in an underflow and an overflow,10. Optionally drying the microfiltration retentate obtained in step 3 or the overflow obtained in step 4.

[0167] All definitions, preferred embodiments and so on defined in relation with the second embodiment also applies herein for the fourth embodiment.

[0168] In a preferred embodiment, microfiltration retentate obtained in step 3 is centrifuged, preferably at force 20,000xg. Centrifugation may be beneficial in order to remove insoluble particles including cell walls debris, and / or for a better protein concentration and less carbohydrates contained in the single cell, preferably yeast, protein concentrate. Standard centrifugation devices can be used such as Beckman Coulter Avanti J-26S Series, Thermo Scientific Sorvall LYNX 6000, Eppendorf 581 OR, Hettich ROTINA 420R, Sigma 3-30KS, or Alfa Laval AS26 centrifuge, preferably Alfa Laval AS26 centrifuge.

[0169] In a preferred embodiment, microfiltration retentate obtained in step 3 or overflow obtained in step 4, may be dried using common technologies including spray-drying or heat treatment precipitation combined with oven drying.

[0170] Heat treatment precipitation combined with oven drying may comprise or consist of the following steps:i) Acid-thermal precipitation of the microfiltration retentate obtained in step 3 or the overflow obtained in step 4, preferably wherein the precipitation is done at an isoelectric pH by heating the microfiltration retentate, preferably at temperatures between 80°C and 90°C, more preferably between 80 and 85°C, resulting in a protein precipitate and a supernatant; ii) Optionally a step of cooling down of the protein precipitate of step i), preferably to a temperature under 25°C, more preferably until 20°C; iii) Centrifugation of the protein precipitate obtained in step i) or the protein precipitate obtained in step ii), preferably at a force under 10,000xg, resulting in an underflow and an overflow; or iii’) Filtration of the protein precipitate obtained in step i) or the protein precipitate obtained in step ii); iv) Oven drying of the overflow obtained in step iii) or the filtrated protein precipitate obtained in step iii’), resulting in a single cell, preferably yeast, protein concentrate.

[0171] In a preferred embodiment of the fourth embodiment, steps 1 to 3 are carried out, followed by optionally centrifugating the microfiltration retentate obtained in step 3, and drying the microfiltration retentate obtained in step 3 or the overflow obtained in step 4, preferably wherein the drying may be either spray-drying or heat treatment precipitation combined with oven drying, to obtain a single cell, preferably yeast, protein concentrate.

[0172] In one embodiment, spray-drying is preferred as it may lead to improve gel strength (gelling capacity) and / or solubility of the single cell, preferably yeast, protein concentrate.

[0173] Standard spray drying devices can be used, such as the Mini Spray Dryer B-290 from Buchi Labortechnik GmbH (Essen, Germany), the Mobile Minor™ Spray Dryer from GEA (Berlin, Germany), or Fujisaki MDL 050MG spray-drier. In a preferred embodiment, Fujisaki MDL 050MG spray-drier is used.

[0174] In one embodiment heat treatment precipitation combined with oven drying is preferred as it may lead to an increase in the dry matter (DM) obtained.

[0175] In one embodiment, and in order to obtain over 97% of dry matter (DM), the microfiltration retentate obtained in step 3 or overflow obtained after centrifugation of step 4 may be dried using heat treatment precipitation combined with oven drying.

[0176] In a particular embodiment, the invention relates to a single cell, preferably yeast, protein concentrate obtained by the process described above.

[0177] In a particular embodiment, the invention relates to a single cell, preferably yeast, protein concentrate characterized in that its content of protein is over 60% of the total weight of dry matter, and preferably its content of carbohydrates is inferior to 10% of the total weight of dry matter. Such single cell, preferably yeast, protein concentrate is preferably obtained by the process comprising or consisting of steps 1 to 3 of the fourth embodiment, as recited above, followed by a centrifugation step and a drying such as spray-drying or heat treatment precipitation combined with oven drying, preferably heat treatment precipitation combined with oven drying.

[0178] In another embodiment, the invention relates to a single cell, preferably yeast, protein concentrate characterized in that its content of protein is over 50% of the total weight of dry matter, preferably its content of carbohydrates is at least 30% of the total weight of dry matter. Such single cell, preferably yeast, protein concentrate is preferably obtained by the process comprising or consisting of steps 1 to 3 of the fourth embodiment, as recited above, followed by an optional centrifugation step, and a drying such as spray-drying or heat treatment precipitation combined with oven drying, preferably heat treatment precipitation combined with oven drying.

[0179] In another embodiment, the invention relates to a single cell, preferably yeast, protein concentrate characterized in that its content of carbohydrates is at least 30% of the total weight of dry matter, preferably above 33%. Such single cell, preferably yeast, protein concentrate is preferably obtained by the process comprising or consisting of steps 1 to 3 of the fourth embodiment, as recited above, directly followed by a drying such as spray-drying or heat treatment precipitation combined with oven drying, preferably spray-drying.

[0180] In another embodiment, the invention relates to a single cell, preferably yeast, protein concentrate characterized in that its solubility measured with Test B is of at least 10%, preferably of at least 25% and a content of carbohydrates of atleast 40% of the total weight of dry matter. Such single cell, preferably yeast, protein concentrate is preferably obtained by the process comprising or consisting of steps 1 to 3 of the fourth embodiment, as recited above, followed by an optional centrifugation step, and a spray-drying. In this preferred embodiment, said centrifugation is herein not carried out.

[0181] In another embodiment, the invention relates to a single cell, preferably yeast, protein concentrate characterized in that its solubility measured with Test B is at least 10%, preferably its content of proteins is of at least 60% of protein of the total weight of dry matter. Such single cell, preferably yeast, protein concentrate is preferably obtained by the process comprising or consisting of steps 1 to 3 of the fourth embodiment, as recited above, followed by an optional centrifugation step, and a spray-drying. In this preferred embodiment, said centrifugation is herein carried out.

[0182] Another embodiment of the invention is also the uses of said single cell, preferably yeast, protein concentrate as described above or produced via a process as described above in industrial applications.

[0183] In a non-exhaustive listing, single cell, preferably yeast, protein concentrate obtained from microfiltration retentate is interesting for all industrial applications including food industries, feed industries, pharmaceutical industries, cosmetic industries, plant care industries. Such applications can be beverage, food, feed, nutraceutical, dietary supplement or functional food, beverage ingredients, food ingredients, animal feed ingredients, pet food ingredients, nutraceutical ingredients, dietary supplement ingredients, and functional food ingredients. In particular, it concerns a product comprising the single cell, preferably yeast, protein concentrate according to the present invention and at least one component selected from the group consisting of beverage ingredients, food ingredients, animal feed ingredients, pet food ingredients, nutraceutical ingredients, dietary supplement ingredients, and functional food ingredients. It is in particular of interest in feed and pet-food industries.

[0184] Single cell, preferably yeast, protein concentrate is particularly dedicated to produce feed or pet-food compositions destinated to animals.

[0185] The term "animal" means a human or other animal, including avian, bovine, canine, equine, feline, hircine, murine, ovine, and porcine animals. The animal can be a ruminant, pig, avian species (e.g., chicken, duck, or turkey), horse, aquaculture animal, companion animal, or wild game. The animal can be a companion animal, most preferably a canine or feline such as a dog or a cat. A "companion animal" herein is a vertebrate animal of any species that is kept by a human owner as a domestic pet, or for work related to sensory abilities or useful behavioral attributes of the animals (for example, hunting dogs, guard dogs, sheepdogs, guide dogs, etc.). In most cases the species is mammalian.

[0186] Feed or pet-food compositions can be formulation for use in any suitable life stage of the animal, such as during at least one of breeding, gestation, or a lactation phase of a female animal or pregnant female animal.

[0187] Feed or pet-food compositions may be formulated as a food, drink, food additive, drink additive, animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral feeding product, soluble, slurry, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet or bolus. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

[0188] Feed or pet-food compositions may be prepared in a canned or wet form using conventional food preparation processes known to skilled artisans. Typically, proteinaceous tissues are mixed with the other ingredients such as oils, vitamins, or inorganic salts and water in amounts sufficient for processing. These ingredients are mixed in a vessel suitable for heating while blending the components. When heated, the material will typically be in the form of a thick liquid that is filled into cans. A lid is applied, and the container is hermetically sealed. The sealed can is then placed into conventional equipment designed to sterilize the contents. The compositions of the present invention can be added to the food compositions before, during, or after preparation.

[0189] Feed or pet-food compositions may be prepared in a dry form using conventional processes known to skilled artisans. Typically, dry ingredients including protein are ground and mixed together. Moist or liquid ingredients, including fats, oils, animal protein, water, and the like are then added to and mixedwith the dry mix. The mixture is then processed into kibbles or similar dry pieces. Kibble is often formed using an extrusion process in which the mixture of dry and wet ingredients is subjected to mechanical work at a high pressure and temperature and forced through small openings and cut off into kibble by a rotating knife. The wet kibble is then dried and optionally coated with one or more topical coatings such as flavors, fats, oils, powders, and the like. Kibble also can be made from the dough using a baking process, rather than extrusion, wherein the dough is placed into a mold before dry-heat processing. The single cell, preferably yeast, protein concentrate of the present invention can be added to the food compositions before, during, or after preparation.

[0190] Single cell, preferably yeast, protein concentrate can be used in a non- exhaustive list in feed or pet-food composition for nutritive, functional or organoleptic goals. As non-exhaustive examples, single cell, preferably yeast, protein concentrate can bring various amino-acids from its protein or organoleptic compounds improving its palatability for animals, bringing umami flavors.

[0191] A fifth embodiment is a single cell, preferably a yeast, more preferably a Saccharomyces protein isolate that can be obtained by the of the second embodiment.As it will be disclosed in the examples chapter below, gelling capacity of the single cell protein of the fifth embodiment is equivalent to egg white protein. “Equivalent” must be understood as a gelling capacity value that is comprised between 25% and 200%, preferably between 50% and 150%, more preferably between 75% and 125% of egg white protein gelling capacity.

[0192] Invention will be better understood by reading the following non-exhaustive examples.Examples

[0193] Example 1 : Process of the invention

[0194] Commercial bakery yeast (S. cerevisiae) La Ferm® No1 from Lallemand is used to produce a suspension in water in a range of 17% DM (dry matter).

[0195] The suspension is transferred to a high-pressure homogenizer Microfluidics Microfluidizer® 7250-10 working at least at 600 bar and cooled in order not toexceed 17°C in the suspension. Recirculation through the homogenizer is carried out to reach at least 70% of disrupted cells, as measured by Test C described in description above.

[0196] Suspension with disrupted cells (a lysate) is transferred to a microfiltration (MF) system comprising ceramic membranes having a permeability gradient (such as Pall Membralox®) of 0.1 pm pore size installed for separation of the suspension into permeate (called MF-P) and retentate (called MF-R). After microfiltration is completed, diafiltration with 3 volume water is carried out and a diapermeate (MF_DP) and a diaretentate (MF-DR) are obtained. MF-P is mixed with MF-DP and is transferred to the next step of ultrafiltration.

[0197] During all microfiltration the transmembrane pressure is maintained at 1 bar; pH is maintained in the MF system at 8.5 with controlled addition of NaOH 2% solution; the temperature is maintained in the range of 18-22°C.

[0198] Mixed permeates from MF (MF-P plus MF-DP) undergo protein concentration and purification in an ultrafiltration step. 5 kDa hollow-fiber membranes Fluid Systems Romicon 6" Hollow Fiber PM 5 from Koch are used. Ultrafiltration (UF) process is carried out at 4-8°C temperature to restrain proteolysis by yeast endogenous proteases. The retentate obtained at the end of the UF process (after concentration from 900L until 20L) is diafiltered in the same UF system by water (3 volume). The obtained UF diaretentate as a purified yeast protein concentrate may be transferred for a sterilization step or directly to drying by a spray dryer.

[0199] Spray-dryer is operated at 200°C air temperature at an inlet and 70°C at an outlet, with 0.25 bar pressure.

[0200] Product obtained is called “Product of the invention” or “SCP of Example 1 ”.

[0201] Example 2: Prior art process with heat and alkali treatment to hydrolyze RNA

[0202] Example 2 aims to reproduce Example 1 of US 3,867,555. Yeast suspension was prepared, homogenized, and centrifuged as recommended in Example 1 of US 3,867,555 in order to produce an alkali extract. Then the alkaliextract was adjusted to pH 12 by the addition of 10 N NaOH. The extract was incubated for 1 hour at 60°C with gentle agitation. Then pH was adjusted to 4.5 with 85% phosphoric acid and filtered. The filtrate was washed and spray-dried.

[0203] Product obtained is called “Prior art product #1”

[0204] Example 3: Prior art process with RNase

[0205] Example 3 aims to reproduce Example 1 from US2022 / 0071231 A1 , combined with preferred use of RNase teached in paragraph 23 of this application US2022 / 0071231.

[0206] Saccharomyces cerevisiae yeast cells were suspended in water at 14% dry matter content and pH 7.5 using NaOH. Yeast cells were then lysed using a Dyno®- Mill Multi Lab Wab using yttria-stabilized zirconium oxide beads with a size of 0.25- 0.35 mm. The flow rate was set to 7 kg / h and the rotor speed to 8 m / s. The bead filling volume was set to 65%. The pH of the lysate obtained from the bead mill was adjusted to 7.6 using NaOH. Subsequently, the lysate was subjected to centrifugation for 120 minutes at 25000g. The supernatant was homogenized, treated by RNase A obtained from Thermo Fisher Scientific used at the recommended dosage of 0.01 % for 30 min at 8°C with gentle agitation. Supernatant treated by RNase was then subjected to activated carbon filtration using an active carbon filtration cartridge filled with steam activated Norit® SX Plus. The solution obtained from activated carbon filtration was sterilized at a temperature of 130°C for 3 seconds and subsequently spray-dried.

[0207] RNA level originally assessed to 12% is reduced to 8,5% which indicates that RNase treatment was active.

[0208] Product obtained is called “Prior art product #2”

[0209] Example 4: Influence of pH on gradient MF

[0210] Example 1 is reproduced but pH of feed of permeability gradient microfiltration is adjusted and controlled at pH 5,5; pH 7,5; pH 9,5; pH 9,8 and pH 11.

[0211] Products obtained are called “Comparative product #1 (pH 5,5) and #2 (pH 11 ), product of the invention #2 (pH 7,5), product of the invention #3 (pH 9,5) and product of the invention #4 (pH 9,8).

[0212] Example 5: Summary of all previous examples to compare performances

[0213] Example 6: Comparison of functional properties between egg white protein and SCP from invention

[0214] First, the minimal concentration leading to coagulation has been evaluated, using following procedure:- 10%, 7,5%, 5% and 2,5% protein solution were prepared (at iso protein content between SCP protein from the invention and egg white protein)- 10g of the solutions are put in test tube and place in water bath at 80°C- Observations made were: o Coagulation: Yes or no o Time to coagulate

[0215] Results are summarized in Table 1 below :Results show that minimal concentration leading to coagulation is the same (5%).

[0216] As second comparison, behavior in a frying pan was observed. 80g of the 10% solution used for coagulation test is heated in a pan to observe coagulation capacity. The idea of this simplified test is to imitate the making of an omelet.

[0217] Figure 1 shows the results with egg white protein and Figure 2 shows the results using SCP from Example 1 . External aspect is very close. After consumption by 10 people, 8 on 10 found SCP (from Example 1 ) very close to egg white protein.

[0218] Example 7: Use of SCP from invention in angel food cake

[0219] Angel food cake is an interesting application to highlight how single-cell protein of the invention properties are close to egg white ones. The quantity of egg white used in the recipe is important compared to other ingredients.

[0220] Recipes are the following ones, in Table 2:

[0221] Process is:Mix, in Hobart planetary mixer egg white protein or SCP from Example 1 with water, at speed 9 during 3 minutes,Add slowly sucrose during 30 seconds and continue to whip during 1 ’30 minutes,Add other ingredients slowly so as not to break the foam,Put in a fairly high mold,Heat in the oven, at 170°C for 30 minutes.

[0222] After consumption by 10 people, everyone found that both cakes are very close.

[0223] Example 8: Use of SCP from invention in cream cake

[0224] Cream cake is also an interesting application to highlight how single-cell protein of the invention properties are close to egg white ones.

[0225] Recipes are the following ones, in Table 3:

[0226] Process is:- Whip protein, sucrose and water for 5 minutes at speed 3 in a planetary mixer (Hobart type),- Add other ingredients, mix for 1 minute at speed 2,- Add oil, and mix for 1 min speed 2,- Put in a greased aluminum mold (300g),Bake in an oven at 170°C for 40 minutes.

[0227] Example 9: Production of a protein concentrate

[0228] Microfiltration diaretentate (MF-DR) produced in example 1 is separated into two equal parts.

[0229] A first half is centrifuged in an Alfa Laval AS26 centrifuge at 20,0000g in order to remove cell wall debris: overflow is harvested and is called MF-DR-CF. The second half is left as is and is called MF-DR-NCF.

[0230] MF-DR-CF and MF-DR-NCF are respectively separated into two equal parts. First half of MF-DR-CF and MF-DR-NCF are concentrated to 15% dry matter in a rotary evaporator and dried using a Fujisaki MDL 050MG spray-drier (Inlet temperature 200°c, Outlet temperature 65°C). Second halfs of MF-DR-CF and MF- DR-NCF are dried using heat treatment precipitation combined with oven drying (acid-thermal precipitation with a pH adjusted to pH 4,5 with 10% HCI, then heated up to 82-85°C, cooled down until 20°C, centrifugated at 7,000g for 10min and oven dried at 85°C during 16h).

[0231] In final, 4 samples are produced and named:- YPC 1 : Microfiltration retentate, centrifuged and spray dried- YPC 2: Microfiltration retentate, not centrifuged and spray dried- YPC 3: Microfiltration retentate, centrifuged, heat treatment precipitated then oven dried- YPC 4: Microfiltration retentate, not centrifuged, heat treatment precipitated then oven dried.

[0232] Table 4 below summarizes samples produced in example 9 with chemical and functional properties:

[0233] A person skilled in the art will undoubtfully learn that all these concentrates are of interest, especially for feed and pet food industries.

[0234] Depending on application needs, the person skilled in the art will choose the best one based on chemical composition and functionalities.

[0235] Centrifuged concentrates such as YPC 1 and YPC 3 are richer in proteins than non-centrifuged concentrates YPC2 and YPC4. Spray-dried concentrates YPC 1 and YPC 2 seem more soluble and seem to have a better gel strength than heat treatment precipitated + oven dried concentrates YPC3 and YPC4. Centrifuged and thermal precipitated concentrates like YPC 3 are less functional but less rich in carbohydrates.

[0236] Example 10 : Production of a protein of the invention starting from brewery spent yeast

[0237] Spent brewery yeast (S. cerevisiae) is obtained from a brewery. It is adjusted at 17% DM (dry matter).

[0238] The process is then the same as the process described in Example 1 .

[0239] Analysis are summarized in Table 5 below :

Claims

Claims

1. Single cell protein characterized in that its content of protein is comprised between 80% and 99% expressed on dry matter of single cell protein, its gelling capacity measured with Test A is comprised between 10000 and 50000, preferably between 15000 and 50000, and its content of RNA is comprised between 2% and 6%, preferably between 2% and 5% expressed on dry matter of single cell protein.

2. Single cell protein from claim 1 characterized in that it is a yeast protein.

3. Single cell protein from claim 2 characterized in that it is a protein from Saccharomyces cerevisiae.

4. Single cell protein from anyone of claims 1 to 3 characterized in that its protein content is comprised between 85% and 97% expressed on dry matter of single cell protein.

5. Single cell protein from anyone of claims 1 to 4 characterized in that its solubility measured with Test B from pH 4 to 8 is comprised between 60% and 99%.

6. Single cell protein from anyone of claims 1 to 5 characterized in that its gelling capacity is comprised between 15000 and 25000, and its RNA content is comprised between 2% and 4%.

7. Process for producing a single cell protein comprising the steps of:

1. Producing a microorganism suspension containing protein,2. Lysing of the microorganism suspension produced in step 1 , resulting in a lysate,3. Microfiltration of the lysate obtained in step 2 with a permeability gradient membrane, resulting in a microfiltration permeate and a microfiltration retentate,4. Ultrafiltration of the microfiltration permeate obtained in previous step 3, resulting in an ultrafiltration permeate and an ultrafiltration retentate,5. Optional sterilizing of retentate obtained in previous step 4,6. Optional drying of sterilized retentate obtained in previous step 5.

8. Process of claim 7 characterized in that the microorganism of step 1 is a yeast.

9. Process of anyone of claims 7 to 8 characterized in that crude protein content of microorganism is comprised between 40% and 60% expressed on dry matter of microorganism.

10. Process of anyone of claims 7 to 9 characterized in that step 1 includes a concentration step before step 2 selected from the list of centrifugation and press-filter in order to obtain a concentrated microorganism suspension with dry matter comprised between 10% and 30%.

11. Process of anyone of claims 7 to 10 characterized in that step 3 is done by succession of a centrifugation of the lysate obtained in previous step 2, resulting in an underflow and an overflow, followed by a permeability gradient microfiltration of said overflow resulting in a microfiltration permeate and a microfiltration retentate.

12. Process of anyone of claims 7 to 11 characterized in that said centrifugation of step 3 produces an underflow which is recycled back in the microorganism suspension of step 2.

13. Process of anyone of claims 7 to 12 characterized in that pH of the lysate obtained in step 2 is adjusted and regulated between 7,0 and 10,0.

14. Process of anyone of claims 7 to 13 characterized in that the microfiltration of step 3 is done with a membrane having a porosity comprised between 0,1 microns and 0,2 microns, at transmembrane pressure comprised between 0,50 bars and 1 ,50 bars.

15. Process of anyone of claims 7 to 14 characterized in that the ultrafiltration of step 4 is done with a membrane having a porosity comprised between 3 to 10 KDa.

16. Process of anyone of claims 7 to 15 characterized in that it consists only of all steps claimed.

17. Uses of a single cell protein from claim 1 to 6 or produced via a process from claims 7 to 16 for food applications, feed applications, cosmetic applications or pharmaceutical applications.

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