Method of preparing microbial protein concentrate

Abstract

The present disclosure provides a method of purifying or preparing microbial protein concentrate from microbial cell mass. More particularly, the present disclosure provides a method of preparing a microbial protein concentrate, comprising: subjecting a microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated microbial cell mass; and separating protein from the treated microbial cell mass to obtain microbial protein concentrate with reduced nucleic acid content and color & odor removed. Additionally, the microbial protein concentrate obtained by the present method has high crude protein content and improved functional properties.

Description

METHOD OF PREPARING MICROBIAL PROTEIN CONCENTRATETECHNICAL FIELD

[0001] The present disclosure is in the field of alternate proteins including microbial proteins. The disclosure relates to a method of purifying or preparing microbial protein concentrate from microbial cell mass.BACKGROUND

[0002] Increase in incomes, changes in lifestyle and urbanization are continually resulting in significant changes in food consumption patterns by humans. Protein is one of the key ingredients to the heart of food security. Meeting the demand for protein within environmental limits is one of the biggest challenges for the global food system in the 21stcentury. Demand for protein in all its forms is expected to grow significantly with the increase in global population. At the same time, the supply chain is being constrained due to the increased pressure on land and water resources, and the impact of climate change.

[0003] While animal-derived and plant-derived proteins are widely employed protein sources, they come with limitations. For instance, animal-derived protein production alone contributes to 12% of greenhouse gas emissions and 30% of human- induced terrestrial biodiversity loss. Additionally, land use is another major concern - for example, two third of total agricultural area globally is used for livestock production. With regard to plant-based protein sources, they often lack one or more amino acids in sufficient quantity to meet human nutritional needs. Other environmental issues such as water use, soil degradation and heavy use of agricultural inputs like fertilizers and protection products are also key drawbacks associated with plant protein production. Overall, all our food systems - agriculture, animal husbandry and aquaculture - are grappling with degradation of land, declining water use efficiency and increasing impact on climate change. These trends clearly illustrate the need for increased production of sustainable protein to meet the growing demand.

[0004] Microbial proteins have the potential to enhance, improve or even replace the currently available alternatives. They are highly resilient due to their decentralized nature and are independent of environmental limitations, such as temperature, weather, and other climactic fluctuations. Given the ecological and nutritional benefits, there is a renewed demand for microbial proteins. However, to fully leverage microbial proteins in food, there are some technical difficulties that must be overcome.

[0005] One of the main nutritional drawbacks in microbial proteins is the high content of nucleic acids (RNA and DNA). Ingestion of excessive quantities of nucleic acids such as RNA increases the quantity of uric acid in the body which is a risk factor for gout and renal calculi as well as a strong risk factor for metabolic syndromes and cardiovascular diseases. Second, microbial proteins have a non-typical odor and texture. In addition, functional properties such as viscosity, clarity and solubility required for optimal food formulations based on microbial proteins are lacking. The present disclosure tries to address these limitations associated with microbial proteins.SUMMARY

[0006] The present disclosure provides a method of preparing microbial protein concentrate from microbial cell mass.

[0007] In some embodiments, the disclosure provides a method of preparing a microbial protein concentrate, comprising subjecting a microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated microbial cell mass; and separating protein from the treated microbial cell mass. In some embodiments, the present disclosure provides a method with a unique combination of steps and conditions / parameters that result in preparation of microbial protein concentrate with reduced nucleic acid content and color & odor removed. Additionally, the microbial protein concentrate obtained by the present method has high crude protein content and improved functional properties.

[0008] In some embodiments, the present disclosure provides an efficient downstream processing method for microbial cell biomass to obtain microbial protein concentrate. More particularly, the present disclosure provides a method of preparing a microbial protein concentrate, comprising: subjecting a microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain a treated microbial cell mass; and separating protein from the treated microbial cell mass.

[0009] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to 10 wt% or less.

[0010] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar and at a pH of about 4 to 9 reduces color and odor of the microbial cell mass.

[0011] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor.

[0012] In some embodiments of the method, separating protein (or protein separation) from the treated microbial cell mass is performed by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and antisolvent precipitation.

[0013] In some embodiments of the method, the separated protein from the treated microbial cell mass is subjected to drying by at least one of spray drying, drum drying, freeze drying tray drying, microwave drying and rotary vacuum drying.

[0014] In some embodiments of the method, the treated microbial cell mass is optionally subjected to a step of color removal, odor removal, or both before separating protein from the treated microbial cell mass.

[0015] In some embodiments of the method, the treated microbial cell mass is subjected to color and odor removal before separating protein.

[0016] In some embodiments of the method, said color removal, odor removal, or both is performed by treatment with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof.

[0017] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated microbial cell mass; separating protein from the treated microbial cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0018] In some embodiments of the method, the microbial cell mass is a bacterial cell mass, and the microbial protein concentrate is a bacterial protein concentrate.

[0019] In some embodiments of the method, the method comprises:subjecting the bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated bacteria cell mass; and separating protein from the treated bacteria cell mass.

[0020] In some embodiments of the method, the method comprises: subjecting the bacteria cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C, a pressure of about 0.1 bar to 20 bar for a time period of about 1 minute to 60 minutes and at a pH of about 4 to 9 to obtain the treated obtain the treated bacteria cell mass; subjecting the treated bacteria cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0021] In some embodiments of the method, the bacterial cell mass is a methanotrophic bacterial cell mass, and the bacterial protein concentrate obtained by the present method is a methanotrophic bacterial protein concentrate.

[0022] In some embodiments, the present method comprises: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

[0023] In some embodiments, the present method comprises: culturing methanotrophic bacterial cells by continuous fermentation in the presence of a carbon source to obtain a methanotrophic bacteria cell mass;subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

[0024] In some embodiments, the present method comprises: subjecting the Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated Methylococcus capsulatus cell mass; and separating protein from the treated Methylococcus capsulatus cell mass.

[0025] In some embodiments, the microbial protein concentrate is a methanotrophic bacterial protein concentrate.

[0026] In some embodiments, the methanotrophic bacteria is Methylococcus capsulatus. In some embodiments, the microbial protein concentrate is Methylococcus capsulatus protein concentrate.

[0027] In some embodiments, the microbial protein concentrate comprises at least 70 wt.% crude protein. In some embodiments, the microbial protein concentrate has less than 10 wt.% nucleic acid content, preferably less than 2 wt.% nucleic acid content.

[0028] In some embodiments, the microbial protein concentrate is colorless or of white / off- white color and is odorless.

[0029] In some embodiments, the microbial protein concentrate has improved properties, such as reduced water absorption capacity, reduced oil absorption capacity, high foaming capacity, and low water solubility index.BRIEF DESCRIPTION OF FIGURES

[0030] FIGURE 1 illustrates a general method of preparing microbial protein concentrate from microbial cell mass in accordance with some embodiments of the present disclosure.

[0031] FIGURE 2 illustrates a method of preparing microbial protein concentrate by steam explosion in accordance with some embodiments of the present disclosure.

[0032] FIGURE 3 illustrates a method of preparing microbial protein concentrate by single stage ultra-high temperature (UHT) process in accordance with some embodiments of the present disclosure.

[0033] FIGURE 4 illustrates a method of preparing microbial protein concentrate by two stage ultra-high temperature (UHT) process in accordance with some embodiments of the present disclosure.

[0034] FIGURE 5 illustrates a method of preparing microbial protein concentrate by steam explosion in accordance with some embodiments of the present disclosure.

[0035] FIGURE 6 are photographs of spray dried microbial protein obtained: a) after UHT treatment; b) after UHT and color and odor removal treatment.DESCRIPTION

[0036] With respect to the use of substantially any plural and / or singular terms herein (such as “a,” “an” and “the”), those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

[0037] The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0038] Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0039] As used herein, the term “comprising” or “comprises” when placed before the recitation of steps in a method means that the method could encompass one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” or “comprises” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprisingsteps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

[0040] Throughout this specification, the term “combination thereof’ or “combinations thereof’ or “and combinations thereof’ or “any combination thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.

[0041] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / - 5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0042] As used herein, the term ‘methanotrophic bacteria’ or ‘methanotroph bacteria’ or ‘methanophile bacteria’ refers to bacteria that can grow aerobically or anaerobically and metabolize single-carbon compounds (such as methane) as their source of carbon and chemical energy for growth / survival.

[0043] As used herein, the terms ‘cell mass’ or ‘biomass’ or ‘solid biomass’ are used interchangeably and refer to a mass or collection of cells and extracellular molecules. In some embodiments, the cell mass refers to the product of fermentation or cell culturing process, wherein said product comprises but is not limited to cells, cell metabolites, secreted molecules, culture media components etc. In some embodiments, the methanotrophic bacteria cell mass refers to the product of methanotrophic bacteria fermentation comprising methanotrophic bacteria cells, cell metabolites, extracellular / secreted molecules, culture media components etc.

[0044] As used herein, the term “crude protein” refers to the total amount of protein present in a sample. It is a measurement of nitrogen in a protein sample. The amount of nitrogen is indicative of the amount of protein in a sample. In some embodiments, the protein concentrate obtained from methanotrophic bacterial cell mass subjected to the method of the present disclosure has at least 70 wt.% crude protein.

[0045] As used herein, the term “microbial protein concentrate” refers to the composition obtained from the bacterial biomass after subjecting said biomass to the method of the present invention. It primarily comprises mixture of isolated, purified or extracted from the bacterial biomass and in which other solid components such as cell debris etc. are removed. In some embodiments, the bacterial biomass is of methanotrophic bacterium.

[0046] The present disclosure is in relation to production of microbial protein concentrate. As discussed above, the key challenges in the application of microbial protein for human consumption are higher level of nucleic acid content and sub-optimal odor, taste and / or color. The disclosure aims at achieving at least one or more objectives related to microbial protein concentrate production including but not limiting to:1. reducing nucleic acid (DNA and / or RNA) content of microbial protein to obtain an optimal protein concentrate;2. obtaining microbial protein concentrate having improved textural and sensory properties (such as texture, color, taste, and odor). Microbial proteins with neutral odor and / or color enable optimal blending of such proteins during preparation of food formulations;3. producing microbial protein concentrate having diverse functional properties such as viscosity (low to high), solubility (low to high), clarity (clear to turbid), water absorption capacity, oil absorption capacity and / or foamability. These properties could help in optimizing food formulations based on microbial proteins during their preparation;4. preparing microbial protein concentrate from cell biomass obtained by fermentation of Cl substrates like methane. For example, as bacteria such as methanotrophic bacteria leverages methane (a potent greenhouse gas) as the sole carbon source for its growth, the preparation of methanotrophic bacteria protein concentrate from cell biomass derived from Cl based fermentation process would combine two vital features, namely, nutrient supplementation / optimization in the protein concentrate along with environmental sustainability;5. producing microbial protein concentrate which retains the nutrition / protein content along with achieving above mentioned objectives.

[0047] Accordingly, the present disclosure provides an efficient downstream processing method for microbial cell biomass to obtain microbial protein concentrate. More particularly, the present disclosure provides a method of preparing a microbial protein concentrate, comprising:- subjecting a microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain a treated microbial cell mass; and- separating protein from the treated microbial cell mass.

[0048] In some embodiments, the present disclosure provides a method with a unique combination of steps and parameters that results in preparation of microbial protein concentrate with reduced nucleic acid content and color and odor removed. Additionally, the microbial protein concentrate obtained by the present method has high crude protein content and improvedfunctional properties such as water absorption capacity, oil absorption capacity, foamability and water absorption index.

[0049] In some embodiments of the method, the microbial protein concentrate is a bacterial protein concentrate.

[0050] In some embodiments of the method, the microbial protein concentrate is a methanotrophic bacteria protein concentrate.

[0051] In some embodiments of the method, the microbial protein concentrate is a gammaproteobacterial methanotroph protein concentrate.

[0052] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C, 105°C, 110°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C, 200°C, 205°C, or 210°C, including any value therebetween.

[0053] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a pressure of about 0.1 bar, 0.5 bar, 1 bar, 1.5 bar, 2 bar, 2.5 bar, 3 bar, 3.5 bar, 4 bar, 4.5 bar, 5 bar, 5.5 bar, 6 bar, 6.5 bar, 7 bar, 7.5 bar, 8 bar, 8.5 bar, 9 bar, 9.5 bar, 10 bar, 10.5 bar, 11 bar, 11.5 bar, 12 bar, 12.5 bar, 13 bar, 13.5 bar, 14 bar, 14.5 bar, 15 bar, 15.5 bar, 16 bar, 16.5 bar, 17 bar, 17.5 bar, 18 bar, 18.5 bar, 19 bar, 19.5 bar, or 20 bar, including any value therebetween.

[0054] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar for a suitable period of time ranging from about 1 minute to 60 minutes.

[0055] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar for a suitable period of time ranging from about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes, including any value therebetween. A person skilled in the art would know how long the microbial cell mass has to be treated at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated microbial cell mass.

[0056] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar at a pH of about 4 to 9.

[0057] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure ofabout 1 bar to 20 bar at a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9, including any value therebetween.

[0058] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to 10 wt% or less.

[0059] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 9 wt%.

[0060] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 8 wt%.

[0061] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 7 wt%.

[0062] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 6 wt%.

[0063] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 5 wt%.

[0064] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 4 wt%.

[0065] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 3 wt%.

[0066] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0.1 bar to 20 bar, and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 2 wt%.

[0067] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C, a pressure of about 0. 1 bar to 20 bar and at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to less than 1 wt%.

[0068] In some embodiments of the method, the microbial protein concentrate comprises the nucleic acid component in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% with respect to weight of the microbial protein concentrate.

[0069] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar and at a pH of about 4 to 9 reduces color and odor of the microbial cell mass.

[0070] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor.

[0071] In some embodiments of the method, said process is selected from at least one of ultra- high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor is performed at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar and at a pH of about 4 to 9.

[0072] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to steam explosion.

[0073] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to ultra-high temperature (UHT).

[0074] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to steam explosion at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar.

[0075] In some embodiments of the method, the treated microbial cell mass is obtained by subjecting the microbial cell mass to ultra-high temperature (UHT) at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar.

[0076] In some embodiments of the method, separating protein (or protein separation) from the treated microbial cell mass is performed by a solid-liquid separation process.

[0077] In some embodiments of the method, separating protein (or protein separation) from the treated microbial cell mass is performed by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and antisolvent precipitation.

[0078] In some embodiments of the method, the protein separation from the treated microbial cell mass is performed by centrifugation. In some embodiments of the method, the protein separation from the treated microbial cell mass is performed by sedimentation. In someembodiments of the method, the protein separation from the treated microbial cell mass is performed by filtration. In some embodiments of the method, the protein separation from the treated microbial cell mass is performed by precipitation. In some embodiments of the method, the protein separation from the treated microbial cell mass is performed by decantation. In some embodiments of the method, the protein separation from the treated microbial cell mass is performed by chromatography.

[0079] In some embodiments of the method, the filtration is a membrane filtration process selected from microfiltration, ultrafiltration and nanofiltration.

[0080] In some embodiments of the method, the chromatography is selected from ion exchange chromatography, size exclusion chromatography and adsorptive chromatography.

[0081] In some embodiments of the method, the centrifugation is performed at 1000 rpm to 20000 rpm.

[0082] In some embodiments of the method, the protein separation from the treated microbial cell mass provides: i) a pellet or a solid mass or a concentrated mass or a retentate or a precipitate or a residue; and i) a supernatant or a liquid or a filtrate or a permeate.

[0083] As used herein, the terms ‘pellet’ or ‘solid mass’ or ‘concentrated mass’ or ‘retentate’ or ‘precipitate’ or ‘residue’ with respect to protein separation are used interchangeably depending on the protein separation process employed and refers to the solid fraction obtained by employing the protein separation process. In some embodiments, said pellet or solid mass or the concentrated mass or the retentate or the precipitate or the residue comprises insoluble protein.

[0084] As used herein, the terms ‘supernatant’ or ‘liquid’ or ‘filtrate’ or ‘permeate’ with respect to protein separation are used interchangeably depending on the protein separation process employed and refers to the liquid fraction obtained by employing the protein separation process. In some embodiments, the supernatant or the liquid or the filtrate or the permeate comprises soluble protein.

[0085] In some embodiments of the method, the separated protein from the treated microbial cell mass is the microbial protein concentrate. In some embodiments of the method, the pellet obtained after the protein separation process comprises insoluble protein which is the microbial protein concentrate.

[0086] In some embodiments of the method, the supernatant obtained after the protein separation process is optionally processed by steps selected from protein purification, protein concentration, drying, or combinations thereof to obtain the microbial protein concentrate comprising soluble protein. In some embodiments of the method, the supernatant obtained afterthe protein separation process is subjected to protein purification, or protein concentration, or both, by techniques selected from filtration, reverse osmosis, precipitation, chromatography, coagulation, and anti-solvent precipitation, or combinations thereof, protein. In some embodiments of the method, the supernatant after protein purification, or protein concentration, or both, is subjected to drying by at least one of spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0087] In some embodiments of the method, the separated protein from the treated microbial cell mass is subjected to drying by at least one of spray drying, drum drying, freeze drying tray drying, microwave drying and rotary vacuum drying. In some embodiments of the method, the pellet comprising insoluble protein obtained after protein separation is subjected to drying by at least one of spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0088] In some embodiments of the method, the drying is performed at about 80 °C to 200 °C for a time-period of about 1 to 300 minutes.

[0089] In some embodiments of the method, subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar at a pH of about 4 to 9 reduces nucleic acid content of the microbial cell mass to 10 wt% or less, preferably less than 2 wt% along with color and odor removal of the microbial cell mass.

[0090] In some embodiments of the method, the treated microbial cell mass is optionally subjected to a step of color removal, odor removal, or both before separating protein from the treated microbial cell mass.

[0091] In some embodiments of the method, the treated microbial cell mass is subjected to color and odor removal before separating protein.

[0092] In some embodiments of the method, the microbial cell mass is subjected to color removal, odor removal, or both after treatment at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar and before separating protein.

[0093] In some embodiments of the method, said color removal, odor removal, or both is performed by treatment with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof. In some embodiments of the method, metal sulfite is sodium sulfite, potassium sulfite, or both. In someembodiments of the method, metal bisulfite is sodium bisulfite, potassium bisulfite, or both. In some embodiments of the method, metal metabisulfite is sodium metabisulfite, potassium metabisulfite, or both.

[0094] In some embodiments of the method, said color removal, odor removal, or both are performed by treatment with hydrogen peroxide (H2O2).

[0095] In some embodiments of the method, said color removal, odor removal, or both is performed by treatment with one or more of activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, and solvent.

[0096] In some embodiments of the method, the color of the obtained microbial concentrate varies from brown color, light brown color, off white color, or white color.

[0097] In some embodiments of the method, the odor of the microbial concentrate is significantly reduced by 10% to 90% compared to the odor of the microbial concentrate before subjecting to odor removal step.

[0098] In some embodiments of the method, the odor of the microbial concentrate is reduced by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to the odor of the microbial concentrate before subjecting to odor removal step.

[0099] In some embodiments, the method comprises: culturing microbial cells by continuous fermentation in the presence of a carbon source to obtain a microbial cell mass; subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain a treated microbial cell mass; and separating protein from the treated microbial cell mass.

[0100] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated microbial cell mass; separating protein from the treated microbial cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0101] In some embodiments of the method, the method comprises:subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated microbial cell mass; separating protein from the treated microbial cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0102] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C, a pressure of about 0.1 bar to 20 bar and at a pH of about 4 to 9 to obtain the treated microbial cell mass; subjecting the treated microbial cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0103] In some embodiments, the method comprises: subjecting microbial cell mass to steam explosion at a temperature of about 135°C to 170°C and pressure of about 4 bar to 8 bar for time period of about 1 minute to 60 minutes and at pH of about 4 to 9; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0104] In some embodiments, the method comprises:subjecting microbial cell mass to a single stage ultra-high temperature (UHT) process at a temperature of about 110°C to 170°C, pressure of about 1 bar to 8 bar and at pH of about 7 to 8; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0105] In some embodiments, the method comprises: subjecting microbial cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated microbial cell mass to color and odor removal by treating with hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0106] In some embodiments, the method comprises: subjecting microbial cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated microbial cell mass to color and odor removal by treating with one or more of activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, and solvent; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0107] In some embodiments, the method comprises: subjecting microbial cell mass to steam explosion at a temperature of about 140°C to 160°C and pressure of about 4 bar to 8 bar and at pH of about 4 to 9; separating protein by at least one process selected from centrifugation, filtration, sedimentation, decantation, coagulation, and anti-solvent precipitation; and subjecting the separated protein to spray drying.

[0108] In some embodiments, the method comprises: subjecting microbial cell mass to steam explosion at a temperature of about 130°C to 160°C and pressure of about 3 bar to 6 bar and at pH of about 4 to 9;separating protein by centrifugation to obtain a pellet and a supernatant; and subjecting the pellet to spray drying to obtain the microbial protein concentrate, and further subjecting the supernatant to filtration followed by sequential drying, enzyme hydrolysis and drying to obtain the microbial protein concentrate.

[0109] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bar obtain the treated microbial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 2 wt.%.

[0110] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bar to obtain the treated microbial cell mass; subjecting the treated microbial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed.

[0111] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to ultrahigh temperature at a temperature of about 145°C and a pressure of about 4 bar to obtain the treated microbial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 3 wt.%.

[0112] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to ultrahigh temperature at a temperature of about 121°C and a pressure of about 2 bars to obtain the treated microbial cell mass; subjecting the treated microbial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; andsubjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 1 wt.% and color and odor removed.

[0113] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to steam explosion at a temperature of about 170°C and a pressure of about 8 bars to obtain the treated microbial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 2 wt.%.

[0114] In some embodiments of the method, the method comprises: subjecting the microbial cell mass to single stage ultra-high temperature treatment at a temperature of about 135°C and a pressure of about 3 bar obtain the treated microbial cell mass; subjecting the treated microbial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain microbial protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed; wherein the microbial protein concentrate has water absorption capacity of 235%, oil absorption capacity of 130%, foaming capacity of 78%, water solubility index of 9.6%.

[0115] In some embodiments of the method, the microbial cell mass is a bacterial cell mass, and the microbial protein concentrate is a bacterial protein concentrate.

[0116] In some embodiments, the method comprises: culturing bacterial cells by continuous fermentation in the presence of a carbon source to obtain a bacterial cell mass; subjecting the bacterial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain a treated bacterial cell mass; and separating protein from the treated bacterial cell mass.

[0117] In some embodiments of the method, the method comprises: subjecting the bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated bacteria cell mass; and separating protein from the treated bacteria cell mass.

[0118] In some embodiments of the method, the method comprises:subjecting the bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated bacteria cell mass; and separating protein from the treated bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0119] In some embodiments of the method, the method comprises: subjecting the bacteria cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high-pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C, a pressure of about 0.1 bar to 20 bar for a time period of about 1 minute to 60 minutes and at a pH of about 4 to 9 to obtain the treated obtain the treated bacteria cell mass; subjecting the treated bacteria cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0120] In some embodiments, the method comprises: subjecting bacterial cell mass to steam explosion at a temperature of about 135°C to 170°C and pressure of about 4 bar to 8 bar for time period of about 1 minute to 60 minutes and at pH of about 4 to 9; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0121] In some embodiments, the method comprises: subjecting bacterial cell mass to a single stage ultra-high temperature (UHT) process at a temperature of about 110°C to 170°C, pressure of about 1 bar to 8 bar and at pH of about 7 to 8;separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0122] In some embodiments, the method comprises: subjecting bacterial cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated bacterial cell mass to color and odor removal by treating with hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0123] In some embodiments, the method comprises: subjecting bacterial cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated bacterial cell mass to color and odor removal by treating with one or more of activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, and solvent; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0124] In some embodiments, the method comprises: subjecting bacterial cell mass to steam explosion at a temperature of about 140°C to 160°C and pressure of about 4 bar to 8 bar and at pH of about 4 to 9; separating protein by at least one process selected from centrifugation, filtration, sedimentation, decantation, coagulation, and anti-solvent precipitation; and subjecting the separated protein to spray drying.

[0125] In some embodiments, the method comprises: subjecting bacterial cell mass to steam explosion at a temperature of about 130°C to 160°C and pressure of about 3 bar to 6 bar and at pH of about 4 to 9; separating protein by centrifugation to obtain a pellet and a supernatant; and subjecting the pellet to spray drying to obtain the bacterial protein concentrate, and further subjecting the supernatant to filtration followed by sequential drying, enzyme hydrolysis and drying to obtain the bacterial protein concentrate.

[0126] In some embodiments of the method, the method comprises: subjecting the bacterial cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bars to obtain the treated bacterial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 2 wt.%.

[0127] In some embodiments of the method, the method comprises: subjecting the bacterial cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bars to obtain the treated bacterial cell mass; subjecting the treated bacterial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed.

[0128] In some embodiments of the method, the method comprises: subjecting the bacterial cell mass to ultrahigh temperature at a temperature of about 145°C and a pressure of about 4 bars to obtain the treated bacterial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 3 wt.%.

[0129] In some embodiments of the method, the method comprises: subjecting the bacterial cell mass to ultrahigh temperature at a temperature of about 121 °C and a pressure of about 2 bar to obtain the treated bacterial cell mass; subjecting the treated bacterial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 1 wt.% and color and odor removed.

[0130] In some embodiments of the method, the method comprises:subjecting the bacterial cell mass to steam explosion at a temperature of about 170°C and a pressure of about 8 bars to obtain the treated bacterial cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 2 wt.%.

[0131] In some embodiments of the method, the method comprises: subjecting the bacterial cell mass to single stage ultra-high temperature treatment at a temperature of about 135°C and a pressure of about 3 bar obtain the treated bacterial cell mass; subjecting the treated bacterial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain bacterial protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed; wherein the bacterial protein concentrate has water absorption capacity of 235%, oil absorption capacity of 130%, foaming capacity of 78%, water solubility index of 9.6%.

[0132] In some embodiments of the method, the bacterial cell mass is a methanotrophic bacterial cell mass, and the bacterial protein concentrate obtained by the present method is a methanotrophic bacterial protein concentrate.

[0133] In some embodiments, the methanotrophic bacteria subjected to the present method is a gammaproteobacterial methanotroph belonging to a genus selected from a group comprising Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methylovulum, Methylomicrobium, Methylosarcina, Methylosphaera, Methyloprofundus, Methylosoma, Methylocucumis, Methylocaldum, Methyloparacoccus, Methylogaea, Methylomagnum, Methyloterricola, Methylothermus, Methylohalobius, Methylomarinovum,Methylomarinum, Crenothrix and combinations thereof.

[0134] In some embodiments, the methanotrophic bacteria subjected to the present method is a gammaproteobacterial methanotroph selected from a group comprising Methylococcus capsulatus, Methylococcus mobilis, Methylomicrobium kenyense, Methylomicrobium alcaliphilum, Methylomicrobium alcaliphilum 20Z, Methylomicrobium buryatense 5G, Methylomicrobium buryatense 4G, Halomonas pantelleriensis, Methylomicrobium album, Methylomonas methanica, MB 126, Methylobacter tundripaludum, Methylovulum miyakonense, Methylomonas rubra, Methylomonas koyamae, Methylomonas methancia, Methylomonasdenitrificans, Methylomonas paludis, Methylomonas lenta, Methylomarinum vadi, Methylococcus thermophilus, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacter vinelandii, Methylomicrobium japanense, Methylococcaceae bacterium, Methylocystis methanolicus, Methylocucumis oryzae, Methylogaea oryzae, Methylosarcina lacus, Methylosoma difficile and combinations thereof.

[0135] In some embodiments, the methanotrophic bacteria subjected to the present method is Methylococcus capsulatus. Accordingly, in some embodiments of the method, the microbial protein concentrate is a Methylococcus capsulatus protein concentrate.

[0136] In some embodiments, the present method comprises: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

[0137] In some embodiments, the present method comprises: culturing methanotrophic bacterial cells by continuous fermentation in the presence of a carbon source to obtain a methanotrophic bacteria cell mass; subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

[0138] In some embodiments, the present method comprises: subjecting the Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated Methylococcus capsulatus cell mass; and separating protein from the treated Methylococcus capsulatus cell mass.

[0139] In some embodiments, the present method comprises: culturing Methylococcus capsulatus cells by continuous fermentation in the presence of a methane (CH4) carbon source to obtain Methylococcus capsulatus cell mass; subjecting Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated Methylococcus capsulatus cell mass; and separating protein from the treated Methylococcus capsulatus cell mass.

[0140] In some embodiments, the present method comprises: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0141] In some embodiments, the present method comprises: subjecting the Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated Methylococcus capsulatus cell mass; separating protein from the treated Methylococcus capsulatus cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying tray drying, microwave drying and rotary vacuum drying.

[0142] In some embodiments, the present method comprises: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated methanotrophic bacteria cell mass; subjecting the treated methanotrophic bacteria cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein from the treated methanotrophic bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0143] In some embodiments, the present method comprises:subjecting the Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain treated Methylococcus capsulatus cell mass; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein from the treated Methylococcus capsulatus cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying tray drying, microwave drying and rotary vacuum drying.

[0144] In some embodiments, the present method comprises: subjecting the methanotrophic bacteria cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor comprising temperature of about 100°C to 170°C and pressure of about 1 bar to 8 bar for time period of about 1 minute to 60 minutes and at pH of about 4 to 9, to obtain treated methanotrophic bacteria cell mass; separating protein from the treated methanotrophic bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0145] In some embodiments, the present method comprises: subjecting the Methylococcus capsulatus cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor comprising temperature of about 100°C to 170°C and pressure of about 1 bar to 8 bar for time period of about 1 minute to 60 minutes and at pH of about 4 to 9, to obtain treated Methylococcus capsulatus cell mass; separating protein from the treated Methylococcus capsulatus cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; andsubjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

[0146] In some embodiments, the present method comprises: subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 135°C to 170°C and pressure of about 4 bar to 8 bar for time period of about 1 minute to 60 minutes and at pH of about 4 to 9; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0147] In some embodiments, the present method comprises: subjecting Methylococcus capsulatus cell mass to a single stage ultra-high temperature (UHT) process at a temperature of about 110°C to 170°C, and pressure of about 1 bar to 8 bar for time period of about 1.0 minutes to 60 minutes and at pH of about 7 to 8; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0148] In some embodiments, the present method comprises: subjecting Methylococcus capsulatus cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0149] In some embodiments, the present method comprises: subjecting Methylococcus capsulatus cell mass to a two stage ultra-high temperature (UHT) process at a temperature of about 110°C to 135°C and pressure of about 1 bar to 4 bar and at pH of about 4 to 9; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with one or more of activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, and solvent; separating protein by centrifugation; and subjecting the separated protein to spray drying.

[0150] In some embodiments, the present method comprises:subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 140°C to 160°C and pressure of about 4 bar to 8 bar and at pH of about 4 to 9; separating protein by at least one process selected from centrifugation, filtration, sedimentation, decantation, coagulation, and anti-solvent precipitation; and subjecting the separated protein to spray drying.

[0151] In some embodiments, the present method comprises:- subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 130°C to 160°C and pressure of about 3 bar to 6 bar and at pH of about 4 to 9;- separating protein by centrifugation to obtain a pellet and a supernatant; and- subjecting the pellet to spray drying to obtain the Methylococcus capsulatus protein concentrate, and further subjecting the supernatant to filtration followed by sequential drying, enzyme hydrolysis and drying to obtain the Methylococcus capsulatus protein concentrate.

[0152] In some embodiments of the method, the method comprises: subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bars to obtain the treated Methylococcus capsulatus cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 2 wt.%.

[0153] In some embodiments of the method, the method comprises: subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 135°C and a pressure of about 3 bars to obtain the treated Methylococcus capsulatus cell mass; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed.

[0154] In some embodiments of the method, the method comprises:subjecting Methylococcus capsulatus cell mass to ultrahigh temperature at a temperature of about 145°C and a pressure of about 4 bars to obtain the treated Methylococcus capsulatus cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 3 wt.%.

[0155] In some embodiments of the method, the method comprises: subjecting Methylococcus capsulatus cell mass to ultrahigh temperature at a temperature of about 121°C and a pressure of about 2 bar to obtain the treated Methylococcus capsulatus cell mass; subjecting the treated bacterial cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 1 wt.% and color and odor removed.

[0156] In some embodiments of the method, the method comprises: subjecting Methylococcus capsulatus cell mass to steam explosion at a temperature of about 170°C and a pressure of about 8 bar to obtain the treated Methylococcus capsulatus cell mass; separating protein by centrifugation; and subjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 2 wt.%.

[0157] In some embodiments of the method, the method comprises: subjecting Methylococcus capsulatus cell mass to single stage ultra-high temperature treatment at a temperature of about 135°C and a pressure of about 3 bar obtain the treated Methylococcus capsulatus cell mass; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with 100 ppm to 1000 ppm hydrogen peroxide (H2O2); separating protein by centrifugation; andsubjecting the separated protein to spray drying to obtain Methylococcus capsulatus protein concentrate with nucleic acid content less than 2 wt.% and color and odor removed; wherein the Methylococcus capsulatus protein concentrate has water absorption capacity of 235%, oil absorption capacity of 130%, foaming capacity of 78%, water solubility index of 9.6%.

[0158] In some embodiments, the microbial protein concentrate obtained by the present method comprises insoluble protein, or soluble protein, or both. In some embodiments of the present method, the microbial protein concentrate comprises insoluble protein which is the pellet obtained after the protein separation process. In some embodiments of the present method, the microbial protein concentrate comprises soluble protein which is derived by further processing the supernatant obtained after the protein separation process.

[0159] In some embodiments, the microbial protein concentrate is a methanotrophic bacterial protein concentrate.

[0160] In some embodiments, the methanotrophic bacteria is Methylococcus capsulatus. In some embodiments, the microbial protein concentrate is Methylococcus capsulatus protein concentrate.

[0161] In some embodiments, the microbial protein concentrate comprises at least 70 wt.% crude protein. In some embodiments, the Methylococcus capsulatus protein concentrate comprises at least 70 wt.% crude protein.

[0162] In some embodiments, the microbial protein concentrate has less than 10 wt.% nucleic acid content. In some embodiments, the Methylococcus capsulatus protein concentrate has less than 10 wt.% nucleic acid content, preferably less than 2 wt.% nucleic acid content.

[0163] In some embodiments, the microbial protein concentrate is colorless or of white / off- white color and is odorless.

[0164] In some embodiments, the microbial protein concentrate has improved properties, such as reduced water absorption capacity, reduced oil absorption capacity, high foaming capacity, and low water solubility index. In some embodiments, the Methylococcus capsulatus protein concentrate has improved properties, such as reduced water absorption capacity, reduced oil absorption capacity, high foaming capacity, and low water solubility index.

[0165] In some embodiments, the microbial protein concentrate comprises water absorption capacity of 235%, oil absorption capacity of 130%, foaming capacity of 78%, water solubilityindex of 9.6%. In some embodiments, the Methylococcus capsulatus protein concentrate comprises water absorption capacity of 235%, oil absorption capacity of 130%, foaming capacity of 78%, water solubility index of 9.6%.

[0166] In some embodiments, the microbial cell mass subjected to the present method is obtained by any means. In some embodiments, the microbial cell mass subjected to the present method is obtained by microbial fermentation or cell culturing. Such fermentation or cell culturing processes are well known in the art and any such known fermentation or cell culturing processes to obtain cell biomass can be employed in the present invention.

[0167] In some embodiments, the methanotrophic bacteria cell mass subjected to the present method is obtained by fermentation or cell culturing. In some embodiments, the Methylococcus capsulatus cell mass subjected to the present method of preparing a microbial protein concentrate is obtained by microbial fermentation or cell culturing. Such fermentation or cell culturing processes are well known in the art and any known fermentation or cell culturing processes can be employed to obtain the cell biomass in the present invention. In general, the fermentation or cell culturing process comprises culturing microbial cells in a growth media under suitable culturing conditions to produce cell biomass. In some embodiments, the methanotrophic bacteria cell mass is obtained by culturing methanotrophic bacteria cells in a growth media under suitable culturing conditions and in presence of methane. In some embodiments, the Methylococcus capsulatus cell mass is obtained by culturing Methylococcus capsulatus cells in a growth media under suitable culturing conditions and in presence of methane.

[0168] In some embodiments, the methanotrophic bacterial cell mass may be grown by batch culture fermentation or continuous fermentation. A batch culturing method is a closed system in which the media composition is set when the culture is started and is not altered during the culture process. That is, media is inoculated at the beginning of the culturing process with one or more microorganisms of choice and then are allowed to grow without adding anything to the system. The continuous fermentation is an open system that involves the addition of the substrate in the fermenter and the removal of fermentation products continuously. Continuous fermentation starts as a batch process. At a certain point, for example, when the culture reaches the exponential growth phase, or when the culture becomes substrate limited, a feed with fresh growth medium is started, and an equal volume of culture broth is removed. Continuous fermentation generally maintains the cells at a constant high, liquid phase density where cells are primarily in logarithmic growth phase.

[0169] In some embodiments, the Methylococcus capsulatus cell mass is obtained by culturing Methylococcus capsulatus cells in a growth media by continuous fermentation and in presence of methane.

[0170] Thus, the present method of obtaining microbial protein concentrate, preferably methanotrophic bacteria protein concentrate, addresses some of the major drawbacks which currently limit the use of microbial proteins for human nutrition / food. For instance, one of the main challenges in the application of microbial protein for human consumption is the higher levels of nucleic acid content which lead to health disorders. The present method reduces the nucleic acid (DNA / RNA) content significantly to obtain microbial protein concentrate which can be used for human consumption. Also, the major difficulty associated with alternative proteins such as microbial proteins is the off flavor. The present method is able to prepare microbial protein concentrate with improved textural and sensory properties such as neutral flavor, odor and / or color which can easily blend with other food ingredients. Additionally, the presently prepared microbial protein concentrate has diverse functional properties (viscosity, solubility, clarity, water absorption capacity, oil absorption capacity and / or foamability) which enables easy blending with other food ingredients / compositions during the preparation of various food products.

[0171] It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

[0172] Descriptions of well-known / conventional methods / steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

[0173] EXAMPLES

[0174] Materials ... Employed: The Methylococcus capsulatus strain used in the present experiments / examples has a source and geographical origin of United Kingdom (UK) and has also been deposited in accordance with the Budapest Treaty with the Microbial Type Culture Collection and Gene Bank (MTCC 25398). Upon procurement from UK, the strain was maintained at String Bio Private Limited and used for the experiments / examples described below.

[0175] Example 1: General method of preparing microbial protein concentrate from microbial cell mass

[0176] Microbial biomass was produced by growing methanotrophic bacteria in a fermenter on a feedstock containing Cl substrate such as methane. The source of methane for the fermentation can be pure methane, or methane from natural gas or biogas. Fermentation broth containing solid biomass was drawn out.

[0177] Fermentation broth was collected for preparing the microbial protein concentrate, and the pH of the collected broth was adjusted anywhere between 4 to 9 and when its required. The pH adjusted broth was subjected to high temperature of anywhere between about 100°C to 210°C and high pressure of anywhere between about 0.1 bar to 20 bar by employing process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor.

[0178] The treated methanotrophic bacteria biomass was then cooled and subjected to protein separation technique selected from one or more of centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation to separate impurities from the methanotrophic bacteria biomass and obtain a pellet and a supernatant. The obtained pellet comprising insoluble protein was the methanotrophic bacteria protein concentrate which was further dried using a drying technique selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying, or combinations thereof.

[0179] The supernatant obtained from the protein separation technique can be optionally processed by protein purification and / or protein concentration selected from filtration (e.g., ultrafiltration, nanofiltration), reverse osmosis, precipitation, chromatography, coagulation, and anti-solvent precipitation, or combinations thereof, to obtain methanotrophic bacteria protein concentrate comprising soluble protein. Said methanotrophic bacteria protein concentrate comprising soluble protein can be further dried using a drying technique selected from spraydrying, drum drying, freeze drying tray drying, microwave drying and rotary vacuum drying, or combinations thereof.

[0180] The above-described general method of preparing microbial protein concentrate is depicted in Figure 1. High temperature and high-pressure treatment described above enables significant removal / reduction of nucleic acids below 2 wt% and also removes / neutralizes color and odor. However, an optional color and odor removal step can be further performed before protein separation step to specifically remove or reduce any residual color and odor remaining (if any) after the high temperature and high-pressure treatment.

[0181] Example 2: Method of preparing microbial protein concentrate by steam explosion

[0182] Microbial cell mass was produced by growing Methylococcus capsulatus in a continuous fermenter on a feedstock containing methane. The continuous fermentation process carried out was as follows:

[0183] Fermenter was filled with growth media composition prepared in accordance with the Applicant’s previous patent no. IN421950 (Palabhanvi et. al). The said growth media was inoculated with a starter culture of M. capsulatus. The fermenter run was initiated with sparging about 0.1 1pm of about 99.9% pure methane and 0.05 1pm of about 99.9% pure oxygen at the bottom of the impeller. Fermentation broth containing solid biomass was continuously drawn out.

[0184] Said fermentation broth containing solid biomass was collected from continuous fermentation and subjected to concentration by continuous centrifugation process. The centrifuged broth was subjected to a steam explosion process by passing them through a steam explosion chamber at a temperature of 135 °C and pressure of about 3 bars.

[0185] Steam explosion-treated broth was cooled down to 70°C and centrifuged to separate impurities from the microbial biomass. Continuous centrifugation was performed at 9000 g force and concentrated cell mass from centrifuge was subjected to spray drying.

[0186] A flow chart showing the production of protein concentrate using steam explosion, centrifugation and spray drying is depicted in Figure 2.

[0187] The dried protein concentrate was characterized for DNA and RNA levels in the samples by Diphenyl amine (DPA) and Orcinol method, respectively. Nucleic acid content (DNA and RNA levels) remained low at 1 to 2% of the total weight of the protein concentrate.

[0188] The dried protein concentrate obtained by method in accordance with the present invention (with steam explosion) was further characterized for crude protein content, amino acid content and nucleic acid content and compared with control, i.e. dried protein concentrate obtained without stem explosion as given in Table 1 below.Table 1 : Comparison of control microbial protein concentrate (obtained without stem explosion) and microbial protein concentrate (with steam explosion - in accordance with the method of the present invention)As can be seen from the above table, the treatment of microbial cell mass to high temperature (steam explosion treatment) does not affect the microbial crude protein content. However, the treatment significantly reduces the nucleic acid content of the resulting microbial protein concentrate upon subjecting the microbial cells to steam explosion in accordance with the method of the present invention. Thus, the combination of method steps and parameters such as temperature and pressure of the present method results in significant reduction of nucleic acid content in the microbial protein concentrate.

[0189] Example 3: Method of preparing microbial protein concentrate by single stage ultra-high temperature (UHT) process

[0190] Microbial cell mass was produced by growing Methylococcus capsulatus in a continuous fermentation process as described in Example 2. Fermentation broth containing solid biomass from continuous fermentation was collected and the cell mass was subjected to single stage ultra- high temperature (UHT) process at a temperature of about 145 °C and a pressure of about 4 bar pressure for a suitable amount of time ranging from about 1 to 60 minutes. The treated cell mass was then subjected to centrifugation for the removal of non-protein nitrogen compounds andother impurities. The concentrated cell mass (pellet) obtained post centrifugation was subjected to drying by spray drying.

[0191] A flow chart showing the production of protein concentrate using single stage ultra-high temperature (UHT) process, centrifugation and spray drying is depicted in Figure 3.

[0192] Further, the dried protein concentrate obtained by single stage UHT method in accordance with the present method, has the following composition as given in Table 2:Table 2: Composition of microbial concentrateAs evident from Table 2, the microbial protein concentrate obtained by subjecting microbial cell mass to single stage ultra-high temperature (UHT) in accordance with the present method has high amount of crude protein (over 70 wt.%) and energy, and reduced amounts of ash and nucleic acid content (less than 3 wt.%). Thus, the combination of method steps and parameters such as temperature and pressure results in significant reduction of the nucleic acid content to below 3 wt% and also imparts other desirable properties such as high energy, low ash content and high amount of crude protein to the resulting microbial protein concentrate.

[0193] Example 4: Method of preparing microbial protein concentrate by two stage ultra- high temperature (UHT) process

[0194] Biomass obtained from Methylococcus capsulatus fermentation process as described in Example 2 was subjected to two stage ultra-high temperature (UHT) process at a high temperature of about 121 °C and a high pressure of about 2 bar for a suitable amount of time ranging from about 1 to 60 minutes. Particularly, stage-one UHT comprised treating the initial cell mass at said temperature, pressure, pH, and time-period, followed by subjecting the pellet to centrifugation. The obtained pellet was subjected to stage-two UHT. Said combination oftemperature, pressure and time removed RNA completely and DNA to less than 1%. The stage- two UHT treated cell mass was cooled and additionally subjected to odor and color removal at 70°C by adding hydrogen peroxide (about 100 to 1000 ppm). The cell mass after hydrogen peroxide treatment was subjected to centrifugation for the removal of non-protein nitrogen compounds and other impurities. The concentrated cell mass (pellet) was subjected to drying by spray drying. The dried protein powder showed less than 1 wt% nucleic acid and was lighter in color.

[0195] A flow chart showing the production of protein concentrate using two stage ultra-high temperature (UHT) process, additional hydrogen peroxide treatment for color and odor removal, centrifugation and spray drying is depicted in Figure 4.

[0196] A comparison of the total nucleic acid content was carried out between the microbial protein concentrate obtained by single stage UHT method of Example 3 and two stage UHT method of Example 4, provided in Table 3 below.Table 3: Nucleic acid content in microbial protein concentrate obtained by single stage UHT method and two stage UHT method in accordance with the present inventionAs demonstrated in the above table, the microbial protein concentrate obtained by the method of the present invention has significantly less total nucleic acid content, less than 4.5 wt.%. In other words, the combination of method steps and parameters, i.e., temperature and pressure results in significant reduction of the nucleic acid content to below 4.5 wt%.

[0197] Example 5: Method of preparing microbial protein concentrate b

[0198] Biomass was produced by fermentation of Methylococcus capsulatus as described in Example 2. Fermentation broth containing solid biomass was subjected to steam explosion at high temperature (about 170°C) and high pressure (about 8 bar). At said high temperature andpressure, nucleic acids, carbohydrates and some of the proteins are solubilized. Also, the unpalatable odors and volatile organic compounds are removed from the cell mass during steam explosion process due to exposure at high temperature. The cell mass after steam explosion process was subjected to protein separation by centrifugation process. The concentrated cell mass (pellet) was then subjected to spray drying. The dried protein powder exhibited less than 2 wt% nucleic acid and was pale white color.

[0199] Example 6: ..Method of .preparing microbial protein conc^

[0200] This experiment involves steam explosion operation along with various steps including centrifugation, hydrolysis, microfiltration, ultrafiltration, and spray drying among others as shown in Figure 5. Biomass was produced by fermentation of Methylococcus capsulatus as described in Example 2. The obtained fermentation broth containing solid biomass was first subjected to a concentration step by centrifugation. The concentrated cell mass (initial cell mass) was subjected to a steam explosion at a temperature of about 170°C and a pressure of about 8 bar. After the steam explosion, the cell mass was subjected to centrifugation which separated the high viscous protein concentrate (pellet) from the soluble protein stream (supernatant). The high viscous protein concentrate (pellet) was subjected to spray drying.

[0201] The soluble protein stream (supernatant) was next subjected to microfiltration for the removal of non-protein nitrogen impurities. A portion of the concentrated cell mass (retentate) was thereafter subjected to spray drying to obtain a dried protein concentrate comprising soluble / medium viscous protein. The remaining portion of the concentrated cell mass (retentate) from filtration was subjected to hydrolysis using endo and exo-protease enzymes. The hydrolyzed protein was further subjected to clarification, color, and odor removal steps. The purified hydrolyzed protein concentrate (low viscous protein concentrate) was finally subjected to spray drying. Thus, three protein concentrates with varying viscosities (low, medium, and high viscosities) were obtained in the present experiment.

[0202] Example 7: Method of preparing microbial protein concentrate with improved colorMicrobial cell mass was produced by growing Methylococcus capsulatus in a continuous fermentation process as described in Example 2. Fermentation broth containing solid biomass from continuous fermentation was collected and the cell mass was subjected to centrifugationfollowed by single stage ultra-high temperature (UHT) process at a temperature of about 145°C and a pressure of about 4.0 bar pressure. In one case, the treated cell mass was then subjected to centrifugation for the removal of non-protein nitrogen compounds and other impurities. The concentrated cell mass (pellet) obtained post centrifugation was subjected to drying by spray drying. In the second case, the UHT treated cell mass was cooled and additionally subjected to odor and color removal at 70°C by adding hydrogen peroxide (H2O2) at about 100 ppm to 1000 ppm). The cell mass after hydrogen peroxide treatment was subjected to centrifugation for the removal of non-protein nitrogen compounds and other impurities. The microbial protein treated with hydrogen peroxide shows improved color and taste compared with UHT treated spray dried protein powder. Further, Figure 6 shows the microbial protein concentrate obtained without (Fig. 6A) and with color and odor removal step (Fig. 6B).

[0203] Example 8: Method of preparing microbial protein concentrate by single stage ultra-high temperature (UHT) process

[0204] Microbial cell mass was produced by growing Methylococcus capsulatus in a continuous fermentation process as described in Example 2. Fermentation broth containing solid biomass from continuous fermentation was collected and the cell mass was subjected to single stage ultra-high temperature (UHT) process at a temperature of about 135°C and a pressure of about 3.0 bar. The UHT treated cell mass was cooled and additionally subjected to odor and color removal by adding hydrogen peroxide (H2O2) at about 100 ppm to 1000 ppm. The cell mass after hydrogen peroxide treatment was subjected to centrifugation. The concentrated cell mass (pellet) was subjected to drying by spray drying. Said combination of method steps and parameters such as temperature, pressure treatment produces microbial concentrate with improved functional properties, provided in the following Table 4.Table 4: Composition and functional properties of microbial protein concentrate

[0205] As evident from Table 4, the microbial protein concentrate obtained by subjecting microbial cell mass to single stage ultra-high temperature (UHT) in accordance with the present invention has high amount of crude protein (over 70 wt.%) and energy, and reduced amounts of ash and nucleic acid content (less than 2 wt.%). Additionally, the microbial protein concentrate has improved functional properties such as reduced water absorption capacity and oil absorption capacity, less gelation concentration, high foaming capacity, and low water solubility index.

[0206] Example 9: Effect of subjecting microbial cell mass to different temperatures and pressure

[0207] Microbial cell mass was produced by growing Methylococcus capsulatus in a continuous fermentation process as described in Example 2. Fermentation broth containing solid biomass from continuous fermentation was collected and the cell mass was subjected to centrifugation. The centrifuge cell mass was further subjected to heat treatment at a temperature between 70 to 135°C at pressure ranging from 0 to 2 bars. The treated cell mass was then subjected to centrifugation. The concentrated cell mass (pellet) obtained post-centrifugation was subjected to drying by tray drying. This was followed by measuring the nucleic acid content of the microbial protein concentrate of each sample obtained at different temperatures, provided in Table 5 below.Table 5: Effect of varying temperature on microbial protein concentrateThe above data / result demonstrates the criticality of temperature range and pressure range in the present method. If the present method is carried out at a temperature less than the claimed value of 100°C and at pressure of less than 0.1 bar, the resulting microbial protein concentrate has high nucleic acid content (over 10 wt.%), which is undesirable. On the other hand, if the method is carried out within the claimed range of 100 to 201 °C and at pressure between 0.1 bar to 20 bar, the resulting microbial protein concentrate obtained has significantly less nucleic acid content, i.e. less than 5 wt.% and, preferably less than 2 wt.%.

[0208] Example 10: Effect of subjecting microbial cell mass to different pH

[0209] Microbial cell mass was produced by growing Methylococcus capsulatus in a continuous fermentation process as described in Example 2. Fermentation broth containing solid biomass from continuous fermentation was collected and the cell mass was subjected to a heat treatment at a temperature of 121°C at a pH between 4 to 7. The treated cell mass was then subjected to centrifugation. The concentrated cell mass (pellet) obtained post centrifugation was subjected to drying by tray drying. This was followed by measuring the nucleic acid content of the microbial protein concentrate of each sample obtained at different pH, provided in Table 6 below.Table 6: Effect of varying pH on microbial protein concentrateThe above data / result demonstrates the criticality of the pH range in the present method. If the method of the present invention is carried out at a pH of less than claimed value of pH 4, then the resulting microbial protein concentrate has high nucleic acid content (over 10 wt.%), which is undesirable. On the other hand, if the method is carried out within the claimed range of pH 4 to pH 9, the resulting microbial protein concentrate obtained has significantly less nucleic acid content, i.e. less than 9 wt.%.

[0210] Example 11: Method of preparing microbial protein concentrate by twin screw extruderThis experiment involves twin screw extruder with modified screw configuration along with various steps including centrifugation, homogenization, and spray drying, among others. Biomass was produced by fermentation of Methylococcus capsulatus as described in Example 2. The obtained fermentation broth containing solid biomass was first subjected to a concentration step by centrifugation. The concentrated cell mass (12% of total solids) was subjected to cell lysis by known techniques such as dynomill, bead mill, high pressure homogenizer, ultrasonication, followed by homogenization in twin screw extruder. The microbial protein concentrate obtained had significantly less nucleic acid content, i.e., less than 2 wt.%.

[0211] Example 12: Comparison of microbial protein concentrate with other plant protein concentrates

[0212] The functional properties of the microbial protein (MP) concentrate produced in Example 8 were compared with the functional properties of soya protein isolate (SPI) and pea protein isolate (PPI). The comparative results are provided in Table 7 below:Table 7: Comparative analysis of properties of different sources of proteinThe results in the above Table 7 clearly indicate that the microbial protein concentrate obtained by the method of present invention exhibits improved properties, i.e., reduced water and oil absorption capacity, reduced water solubility index, and high foaming capacity as compared to other plant proteins.

[0213] Reference throughout this specification to “some embodiments”, “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0214] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0215] Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0216] As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0217] Any discussion or reference of documents, acts, materials, devices, articles, and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application. Particularly, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

WE CLAIM:

1. A method of preparing a microbial protein concentrate, comprising: subjecting a microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain a treated microbial cell mass; and separating protein from the treated microbial cell mass.

2. The method as claimed in claim 1, wherein the microbial cell mass is a bacterial cell mass, and the microbial protein concentrate is a bacterial protein concentrate.

3. The method as claimed in claim 2, wherein the bacterial cell mass is a methanotrophic bacterial cell mass, and the bacterial protein concentrate is a methanotrophic bacteria protein concentrate.

4. The method as claimed in any one of claims 1 to 3, wherein the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar for a time period of about 1 minute to 60 minutes.

5. The method as claimed in any one of claims 1 to 4, wherein the treated microbial cell mass is obtained by subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar, at a pH of about 4 to 9.

6. The method as claimed in any one of claims 1 to 5, wherein the treated microbial cell mass is obtained by subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor.

7. The method as claimed in any one of claims 1 to 6, wherein separating protein from the treated microbial cell mass is performed by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and antisolvent precipitation.

8. The method as claimed in any one of claims 1 to 7, wherein the treated microbial cell mass is subjected to color and odor removal before separating protein; and wherein said color and odor removal is performed by treatment with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof.

9. The method as claimed in any one of claims 1 to 8, wherein the separated protein is subjected to drying by at least one of spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

10. The method as claimed in any one of claims 1 to 9, comprising: subjecting the microbial cell mass to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated microbial cell mass; and separating protein from the treated microbial cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

11. The method as claimed in any one of claims 1 to 10, comprising: subjecting the microbial cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor at a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated microbial cell mass; subjecting the treated microbial cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof;separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

12. The method as claimed in any one of claims 1 to 11, comprising: subjecting the bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

13. The method as claimed in any one of claims 1 to 12, comprising: subjecting the bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated bacteria cell mass; and separating protein from the treated bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

14. The method as claimed in any one of claims 1 to 13, comprising: subjecting the bacteria cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated bacteria cell mass; subjecting the treated bacteria cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; andsubjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

15. The method as claimed in any one of claims 1 to 14, comprising: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated methanotrophic bacteria cell mass; and separating protein from the treated methanotrophic bacteria cell mass.

16. The method as claimed in any one of claims 1 to 15, comprising: subjecting the methanotrophic bacteria cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated methanotrophic bacteria cell mass; separating protein from the treated methanotrophic bacteria cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

17. The method as claimed in any one of claims 1 to 16, comprising: subjecting the methanotrophic bacteria cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C and a pressure of about 0. 1 bar to 20 bar to obtain the treated methanotrophic bacteria cell mass; subjecting the treated methanotrophic bacteria cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; andsubjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

18. The method as claimed in any one of claims 1 to 17, wherein the methanotrophic bacteria is a gammaproteobacterial methanotroph belonging to a genus selected from a group comprising Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methylovulum, Methylomicrobium, Methylosarcina, Methylosphaera, Methyloprofundus, Methylosoma, Methylocucumis, Methylocaldum, Methyloparacoccus, Methylogaea, Methylomagnum, Methyloterricola, Methylothermus, Methylohalobius, Methylomarinovum,Methylomarinum, Crenothrix and combinations thereof.

19. The method as claimed in any one of claims 1 to 18, wherein the methanotrophic bacteria is a gammaproteobacterial methanotroph selected from a group comprising Methylococcus capsulatus, Methylococcus mobilis, Methylomicrobium kenyense, Methylomicrobium alcaliphilum, Methylomicrobium alcaliphilum 20Z, Methylomicrobium buryatense 5G, Methylomicrobium buryatense 4G, Halomonas pantelleriensis, Methylomicrobium album, Methylomonas methanica, MB 126, Methylobacter tundripaludum, Methylovulum miyakonense, Methylomonas rubra, Methylomonas koyamae, Methylomonas methancia, Methylomonas denitrificans, Methylomonas paludis, Methylomonas lenta, Methylomarinum vadi, Methylococcus thermophilus, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacter vinelandii, Methylomicrobium japanense, Methylococcaceae bacterium, Methylocystis methanolicus, Methylocucumis oryzae, Methylogaea oryzae, Methylosarcina lacus, Methylosoma difficile and combinations thereof.

20. The method as claimed in any one of claims 1 to 19, comprising: subjecting Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated methanotrophic bacteria cell mass; and separating protein from the treated Methylococcus capsulatus cell mass.

21. The method as claimed in any one of claims 1 to 20, comprising:subjecting the Methylococcus capsulatus cell mass to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated Methylococcus capsulatus cell mass; separating protein from the treated Methylococcus capsulatus cell mass by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

22. The method as claimed in any one of claims 1 to 21, comprising: subjecting the Methylococcus capsulatus cell mass to at least one process selected from ultra-high temperature (UHT), steam explosion, high pressure cooker, single screw extruder, twin screw extruder and high temperature plug flow reactor to a temperature of about 100°C to 210°C and a pressure of about 0.1 bar to 20 bar to obtain the treated Methylococcus capsulatus cell mass; subjecting the treated Methylococcus capsulatus cell mass to color and odor removal by treating with a bleaching agent or deodorizing agent selected from hydrogen peroxide (H2O2), activated carbon, metal sulfite, metal bisulfite, metal metabisulfite, ammonium bisulfite, sulfur dioxide, natural lemon flavor, acidic clay, bentonite, silica, diatomaceous earth, pozzolan, sepiolite, saponite, kaolinite, or solvent, or any combination thereof; separating protein by at least one process selected from centrifugation, sedimentation, precipitation, filtration, decantation, chromatography, coagulation, and anti-solvent precipitation; and subjecting the separated protein to drying by at least one process selected from spray drying, drum drying, freeze drying, tray drying, microwave drying and rotary vacuum drying.

23. The method as claimed in any of claims 1 to 22, wherein the microbial cell mass is obtained by culturing microbial cells in a growth media by continuous fermentation in the presence of a carbon source.

24. The method as claimed in any of claims 1 to 23, wherein the methanotrophic bacterial cell mass is obtained by culturing Methylococcus capsulatus cells in a growth media by continuous fermentation in the presence of methane (CH4) carbon source.

25. The method as claimed in any one of claims 1 to 24, wherein the microbial protein concentrate comprises insoluble protein, or soluble protein, or both.

26. The method as claimed in any one of claims 1 to 25, wherein the microbial protein concentrate comprises 10 wt% or less nucleic acid content, preferably less than 2 wt.% nucleic acid content along with color and odor removed.

27. The method as claimed in any one of claims 1 to 27, wherein the microbial protein concentrate comprises at least 70 wt.% crude protein.