Method for producing casein and its use

Abstract

The present invention relates to a novel method for producing a casein composition by increasing the proportion of casein in the composition by heating the composition for a short period of time through a pipe, and to the use thereof, particularly for producing cheese substitutes.

Description

Technical Field

[0001] The present invention relates to the food industry, and relates to a novel method for producing a casein composition and its use, particularly for the production of cheese substitutes (especially vegan cheese).

Background Art

[0002] The use of milk as a nutrient rich in protein, sugar and lipids has become almost universal in traditional societies. Furthermore, the conversion of milk into various derivatives is one of the oldest examples of human agriculture. Today, the cheese market accounts for 20 million tons of products annually, with a value of approximately $140 billion.

[0003] However, dairy products are associated with several issues or concerns from a health perspective, as well as from an environmental and ethical standpoint. These concerns highlight the need for dairy alternatives that mitigate these various problems. Health issues include lactose intolerance, allergies (Mousan and Kamat (2016) Cow's Milk Protein Allergy. Clin Pediatr (Phila), Volume 55(11) pages 1054-63.; Manuyakorn and Tanpowpong (2018) Cow milk protein allergy and other common food allergies and intolerances. Paediatr Int Child Health, Volume 39(1), pages 32-40), and others such as high levels of saturated fatty acids, which are known to have potential adverse health effects. (Micha and Mozaffarian (2010) Saturated fat and cardiometabolic risk factors, coronary heart disease, stroke, and diabetes: A fresh look at the evidence. Lipids, Volume 45, pages 893-905; Jakobsen et al (2009) Major types of dietary fat and risk of coronary heart disease: A pooled analysis of 11 cohort studies. Am. J. Clin. Nutr; Volume 89, pages 1425-1432; Nettleton et al (2017) Saturated fat consumption and risk of coronary heart disease and ischemic stroke: A science update. Ann Nutr Metab Volume 70, pages 26-33).Lactose intolerance is caused by a deficiency of lactase, the enzyme that breaks down lactose in the stomach and small intestine, leading to the accumulation of lactose in the colon and its digestion by bacteria (Ugidos-Rodriguez et al (2018) Lactose malabsorption and intolerance: a review. Food Funct, Volume 9(8), pages 4056-4068). This is a very common characteristic in humans, is genetically determined, and leads to the need for a dairy-free diet. Dairy substitutes have been able to be formulated to avoid lactose, saturated fatty acids, and even allergenic proteins.

[0004] Furthermore, environmental and ethical concerns related to animal-derived food products have increased over the past few decades. The burden of raising livestock for a population of 7 billion (11 billion by 2050) is becoming increasingly heavy. The environmental impact is serious. These are currently being examined primarily in terms of anthropogenic greenhouse gas (GHG) emissions, water consumption, wastewater pollution, and land use.

[0005] Cattle farming is considered one of the primary sources of GHG emissions today, accounting for an estimated 7.1 gigatons of CO2 equivalent per year and representing 14.5% of all anthropogenic greenhouse gas (GHG) emissions (Rotz (2017) Modeling greenhouse gas emissions from dairy farms. J Dairy Science, Volume 101, pages 6675-6690). However, a UN study estimated the contribution of the dairy sector alone to global anthropogenic GHG emissions in 2010 at 4% (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS, Greenhouse Gas Emissions from the Dairy Sector. A Life Cycle Assessment).

[0006] Extremely high water consumption is also linked to livestock farming (Sultana et al (2014). Comparison of water use in global milk production for different typical farms. Agricultural Systems, Volume 129, pages 9-21; Ercin and Aldaya (2012) The water footprint of soy milk and soy burger and equivalent animal products. Ecological Indicators, Volume 18, pages 392-402). Furthermore, agricultural drainage also has a strong environmental impact. While the impact of crop fertilizers on groundwater should not be ignored, the impact of livestock drainage is often massive and has proven to be catastrophic in many parts of the world (https: / / www.nrdc.org / issues / livestock-production). Moreover, livestock occupy nearly 80% of the world's agricultural land but produce less than 20% of the world's calorie supply, indicating a very high level of stress that livestock farming places on land resources.

[0007] Finally, animal welfare is becoming an increasingly important concern. The scaling up of meat and dairy production and processing has transformed it into an intensive industrial process, which is increasingly recognized as ethically unacceptable.

[0008] Therefore, there is a strong need for dairy alternatives that alleviate the problems mentioned above.

[0009] Plant-based alternatives are a potential alternative to traditional dairy products. However, these often come from starch, soy, almonds, or coconut milk, and can be far from mimicking the taste of dairy. Furthermore, they are fundamentally different in terms of composition in any case.

[0010] Therefore, today, (i) Free from undesirable compounds (ii) The appearance, texture, and taste are similar to the original product. (iii) It is equivalent to or better than the original product in terms of nutrition. There is a need for dairy alternatives, and especially for cheese alternatives.

[0011] Table 1 shows the typical composition of cow's milk. More detailed compositions of cow's milk and milk from other animals, including lists of different lipids, proteins, salts, vitamins, and other nutrients, can be found in numerous sources (https: / / en.wikipedia.org / wiki / Milk#Cow's_milk; Haug et al (2007) Bovine milk in human nutrition - a review, Lipids Health Dis.; Volume 6, pages 25; Dominguez-Salasa et al (2019) Contributions of Milk Production to Food and Nutrition Security; Encyclopedia of Food Security and Sustainability, Volume 3, pages 278-291).

[0012] [Table 1]

[0013] Lipids and carbohydrates (excluding lactose) can be obtained from plants, and calcium can be recovered from inorganic, animal, or plant sources (such as seaweed). For proteins, animal proteins have a different amino acid content than plant proteins, so other sources must be considered. Milk proteins can also be produced by fermentation, which is another source of non-animal components.

[0014] Furthermore, proteins isolated from milk are also used individually as nutritional supplements and for other purposes. Therefore, milk proteins produced by fermentation independently of other milk components can also be used for purposes other than the production of dairy substitutes.

[0015] Fermentation production is based on the growth of bacteria or fungi that produce the desired compound in a fermenter, followed typically by the recovery and purification of the compound. Fermentation-based protein production has been a widely used method in the food industry. Several studies have described the production of milk constituent proteins or homologs by fermentation in various microorganisms (see below), and the assembly of components containing fermented proteins to produce dairy substitutes has been described several times (U.S. Patent No. 6,270,827; U.S. Patent No. 5,942,274; International Publication No. 2018 / 039632; International Publication No. 2020 / 223700; International Publication No. 2020 / 081789; International Publication No. 2020 / 219596; International Publication No. 2022 / 098835).

[0016] Fermentation production is typically carried out using sugar raw materials to support the growth of microorganisms and the production of the desired compound. Depending on the ability of the microorganisms to metabolize different sugars, glucose, sucrose, or other sugars such as lactose can be used.

[0017] For example, Escherichia coli is known to consume glucose very efficiently, but it can also consume a range of other sugars, including glucose, maltose, lactose, trehalose, fructose, xylose, and arabinose, with a set of priorities (Luo et al. (2014) The transport and mediation mechanisms of the common sugars in Escherichia coli, Biotechnology Advances volume 32, pages 905-919; Postma et al. (1993) Phosphoenolpyruvate:Carbohydrate Phosphotransferase Systems of Bacteria, Microbiolgical Reviews Sept 1993, pages 543-594). Sucrose can also be metabolized by certain Escherichia coli strains that express specific genes corresponding to the Scr or Csc system (Bruschi et al. (2012) A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains. Biotechnology Advances 30 (2012) 1001-1010).Sugar-rich raw materials can offer varying levels of purity, including glucose syrup, sucrose syrup, molasses, and vinasse (Suhaili et al. (2018) Potential of sugar beet vinasse as a feedstock. J Chem Technol Biotechnol volume 94, pages 739-751; Naspolini et al. (2017, Bioconversion of Sugarcane Vinasse into High-Added Value Products and Energy, BioMed Research International, vol. 2017, Article ID 8986165, 11 pages) and other sugar beet processing products (Tomaszewska et al. (2018) Products of sugar beet processing as raw materials for chemicals and biodegradable polymers. Royal Society of Chemistry Advances, volume 8, pages 3161-3177).

[0018] Microbial growth and metabolism require not only sugar raw materials but also nitrogen input. For this purpose, various compounds, including liquid or gaseous ammonia, urea, amino acids, peptones, and yeast extracts, can be added.

[0019] Dairy-derived alternatives such as whey have also been used in fermentation with various microorganisms, including Escherichia coli (Carranza-Saavedra (2021) Kinetic analysis and modeling of L-valine production in fermentation batch from E. coli using glucose, lactose and whey as carbon sources; Biotechnology Reports 31 (2021) e00642; Chaparro et al. (2021) Whey as an Alternative Nutrient Medium for Growth of Sporosarcina pasteurii and its Effect on CaCO3 Polymorphism and Fly Ash Bioconsolidation. Materials volume 14, 2470; Christensen et al. (2011) Production of bioethanol from organic whey using Kluyveromyces marxianus J Ind Microbiol Biotechnol volume 38, pages 283-289; Hausjell et al. Valorisation of cheese whey as substrate and inducer for recombinant protein production in E.coli HMS174(DE3) Bioresource Technology Reports 8 (2019) 100340; Loaste and Elourtassi (2020) Succinic acid production from whey and lactose by Actinobacillus succinogenes 130Z in batch fermentation; Biotechnology Reports 27 (2020) e00481; Zou and Chang (2022) Past, Present, and Future Perspectives on Whey as a Promising Feedstock for Bioethanol Production by Yeast; Journal of Fungi, volume 8, page 395). .

[0020] Whey is rich in lactose and other nutrients, and intensive research has been conducted to add value to whey, especially acid whey (Rocha-Mendoza et al (2021) Invited review: Acid whey trends and health benefits; J. Dairy Sci. volume 104, pages 1262-1275). Using this type of raw material is not suitable for the production of animal-free products, given the origin of whey. Nevertheless, using waste as a raw material can be very useful in achieving good economics and a favorable life cycle analysis.

[0021] During feed-batch fermentation, the pH can be adjusted to maintain it within a specific range, and for this purpose, acidic or basic compounds including hydrochloric acid, sulfuric acid, lactic acid, and phosphoric acid (acidic compounds) or liquid or gaseous ammonia, sodium hydroxide, or potassium hydroxide (basic compounds) can be added.

[0022] All of these solutions are not mutually exclusive and can be combined.

[0023] However, for the industrial production of milk proteins, not only is it necessary to produce the required proteins at a high level of purity in a grade suitable for use in the food industry, but also a compatible purification procedure that is easily scalable at the industrial level and has a cost compatible with development, particularly the energy cost, is required. Ideally, such a procedure should be as simple as possible and should avoid the use of non-edible chemicals and the use of processes that are expensive when carried out on a very large scale.

[0024] In the case of recombinant proteins produced by fermentation, WO 2022 / 253816 describes a procedure for isolating recombinant casein proteins from biomass and culture broth by heating the composition. In this application, the microbial composition is heated in a container at a temperature of 75 °C to 105 °C. The examples show that the duration of heating is at least 10 minutes, rather at least 30 minutes or even 120 minutes. It is recommended to heat the composition for at least 1 hour.

[0025] The conditions proposed in WO 2022 / 253816 require energy to overcome the inertia of the stationary composition and the heating duration is long. The applicant has shown that it is possible to shorten the heating duration and maintain a good casein recovery rate by heating a composition containing casein and other proteins for several minutes at a high temperature while circulating the composition from one container to another. The heating is carried out during the transfer of casein from the first container to the second container. It is also envisaged that there may be further purification steps (such as centrifugation and / or filtration etc.) after the heating step before the composition is fed into the second container. This method makes it possible to shorten the duration of the casein production process so as to be more suitable for incorporation into an industrial process. This also enables energy savings as heating the pipes through which the composition moves consumes less energy than heating the container in which the fermentation process was carried out.

[0026] Ren et al (Journal of Biotechnology 129 (2007) 668-673) found that heat treatment of Escherichia coli (E. coli) is effective in disrupting the integrity of the bacterial cell wall and releasing intracellular proteins, as exemplified by recombinant hyperthermophilic esterase.

[0027] WO 90 / 00200 discloses a method for isolating a substantially pure polypeptide from a recombinant bacterial host by culturing the host under recombinant protein production conditions and then heating the aqueous nutrient medium to 50°C to 100°C for a time not exceeding 1 hour and recovering the substantially pure polypeptide thus produced.

[0028] Wang et al () evaluated the potential of lignocellulosic biomass as a carbon source for the growth of various Escherichia coli strains. As a proof of concept, they successfully produced recombinant bovine and human αS1-casein.

[0029] In the present invention, "casein" is any casein protein or a mixture of casein proteins. Thus, "casein" is alpha-S1 casein, alpha-S2 casein, beta casein, or kappa casein. To some extent, this can also refer to any mixture of such proteins. The terms "casein" or "caseins" can be used to discuss casein proteins in general.

[0030] In the present invention, the term "between" includes the limiting values.

[0031] In the present invention, the term "cheese substitute" means a food having the essential characteristics of cheese in terms of nutritional value, appearance, texture and taste.

[0032] The term "fresh cheese" refers to cheese with a fat-free base that has a moisture content of more than 80% (the percentage of water relative to the total mass of the fat-free product) and a protein content of 2% to 15% of the total weight (the percentage of protein relative to the total mass of the product).

[0033] The terms "soft cheese" or "semi-soft cheese" refer to cheeses with a fat-free base moisture content of 62% to 80% (the percentage of water relative to the total mass of the product) and a protein content of 15% to 30% of the total weight (the percentage of protein relative to the total mass of the product). Soft cheeses have a fat-free base moisture content of 67% to 80%, while semi-soft cheeses have a fat-free base moisture content of 62% to 67%.

[0034] In the present invention, the term “liquid pre-curd composition” or “LpCC” refers to a composition containing at least casein and at least one other component, including at least one of water, calcium, lipids, and carbohydrates, prior to the addition of rennet and fermentation and coagulation (particularly casein coagulation). The casein concentration in LpCC is higher than that in milk. The concentrations of the other components in LpCC are also higher than those in milk. This allows for some water savings when using LpCC containing recombinant casein as disclosed herein, since LpCC does not represent artificial (cow's) milk. The concentrations of proteins, lipids, calcium salts, and / or carbohydrates are adjusted to suit the final desired composition of the cheese substitute.

[0035] In this invention, the term "coagulant" refers to a chemical or biochemical composition capable of causing coagulation. Coagulation can be achieved by adding an acid solution, adding a starter culture (a fermented product whose growth in the presence of carbohydrates causes a decrease in pH), heat treatment, adding a calcium chelating agent, adding natural or recombinant rennet, adding a rennet substitute (such as an animal protease or a plant-derived coagulating enzyme), or a combination of these steps. The coagulant may be any additive, compound, composition, or treatment, used alone or in combination with another or other additive, compound, composition, or treatment, whose addition results in coagulation. When preparing animal-free food compositions, it is preferable to use an acidifying agent (such as an acid or starter culture) so that the coagulant does not contain any animal-derived elements, in order to avoid the use of rennet. Acidic chemicals can also be used, although it is advantageous if the acidifying agent contains lactic acid bacteria.

[0036] In the present invention, the term "curd" means a composition in which casein coagulates or precipitates under the action of a coagulant, which, if present, can be separated from the liquid phase, for example, by draining on a cheesecloth. The curd also includes other components, including water and lipids (if present).

[0037] In this invention, the term "fermented product" refers to a composition containing at least one microbial strain that is added during the production of a cheese substitute. The lactic acid fermented product is responsible for lactic acid fermentation, particularly resulting in the acidification of the culture medium. As a result, these can be used as coagulants.

[0038] Other fermented products are used in cheesemaking for curd processing, resulting in changes to texture, taste, aroma, and chemical composition (particularly involving the cleavage of proteins into smaller peptides). These fermented products can be called “aging fermented products,” “matured fermented products,” or “mature fermented products” and are not typically used in the production of fresh cheese. Such aging fermented products can be added along with coagulants.

[0039] In this invention, the term "non-animal" means a compound or composition that is not obtained directly from an animal, is not produced from animal cells in culture, and is not isolated from animal products such as milk. Therefore, a compound or composition produced by microbial fermentation is "non-animal," even if some animal-derived products, such as bactopeptone, may be involved in the fermentation. Accordingly, in this invention, a protein naturally produced in an animal is called non-animal if it is produced within a microbial (such as a bacterium or yeast) cell or plant cell, even if its sequence or structure may be identical to that of a protein that would be isolated from an animal.

[0040] In the present invention, "animal-free" means a compound or composition that is not obtained from animals, from animal cells in culture, or from animal products such as milk, and whose manufacturing method does not involve any animal-derived raw materials or additives.

[0041] In the present invention, the term "texturing agent" means any gelling agent, including emulsifiers such as lecithin, and hydrocolloids such as cassia gum, sesbania gum, tamarind gum, guar gum, fenugreek gum, gum arabic, agar (or agar-agar), carrageenan, tragacanth gum, xanthan gum, carob (locust bean) gum, and cellulose gum.

[0042] In the first embodiment, a method for solubilizing casein from a composition comprising a soluble fraction and an insoluble fraction containing casein and other proteins, wherein the casein is mainly present in the insoluble fraction of the composition. i) A composition comprising a soluble fraction and an insoluble fraction containing casein and other proteins, wherein casein is mainly present in the insoluble fraction of the composition, and the pH of the composition is 6.5 or higher, preferably less than 9, prepared in a first container, ii) Fluidizing the composition from a first container in an apparatus equipped with pipes, and heating the composition while it is flowing through the pipes, wherein the heating is performed at a temperature of 95°C or higher for a maximum of 5 minutes, iii) Recovering the soluble fraction of the flowed composition, Includes, Casein is enriched in the recovered fraction. A method is disclosed herein. A portion of the casein that was mainly present in the insoluble fraction is solubilized thereby. In a preferred embodiment, the casein is mainly present in the soluble fraction after the method is carried out (and therefore the soluble fraction is enriched with casein). The soluble fraction may be recovered after the heated composition has cooled.

[0043] The composition is a liquid solution containing casein and other proteins. It also presents soluble and insoluble fractions. In certain embodiments, the composition also contains transgenic microorganisms, such as bacteria that produce casein as a result of the presence of a transgene encoding casein in their genome. Such bacteria are present in the insoluble fraction (whether lysed or not).

[0044] Compositions containing transgenic microorganisms can be obtained by fermentation using various sugars and nitrogen sources as raw materials. In preferred embodiments, fermentation is carried out in a medium containing glucose, sucrose, or lactose. In preferred embodiments, fermentation is carried out in a medium containing glucose, sucrose, or lactose as a carbon source. In preferred embodiments, fermentation is carried out in a medium containing molasses or vinas as a carbon source. In preferred embodiments, fermentation is carried out in a medium containing molasses as a carbon source. In preferred embodiments, liquid or gaseous ammonia, urea, amino acids, peptone, or yeast extract is present in or added to the medium as a nitrogen source. In preferred embodiments, fermentation is carried out in a medium containing ammonia, urea, amino acids, peptone, or yeast extract as a nitrogen source. In preferred embodiments, fermentation is carried out on a medium containing whey as a raw material. In preferred embodiments, fermentation is carried out on a medium containing acidic whey as a raw material. In preferred embodiments, fermentation is carried out on a medium containing whey and another sugar as an additional sugar source. In a preferred embodiment, fermentation is carried out on a culture medium containing acidic whey and glucose, sucrose, or lactose as an additional sugar source.

[0045] Before heating, the pH can be adjusted by adding liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or other basic compounds. The pH should be greater than 6.5, preferably less than 9.

[0046] Therefore, a composition containing casein and other proteins (and potentially microorganisms) is flowed from a first container through an apparatus including pipes or tubes, such a tubular exchanger having both the function of moving the composition and heating it. Thus, there is heat transfer (or heat exchange) as the composition passes through the pipes so as to heat the composition, and such transfer occurs in at least a portion of the pipes.

[0047] The soluble fraction may be recovered by centrifuging the liquid composition flowing from the pipe.

[0048] The concentration of casein, and / or the proportion of casein to other proteins, is higher in the recovered soluble fraction than in the initial composition, thereby enriching the soluble recovered fraction with casein.

[0049] As shown, composition i) contains a soluble fraction and an insoluble fraction. The soluble fraction represents the fraction that is not pelletized by centrifugation. The insoluble fraction is the pellet obtained after centrifugation. Centrifugation may be performed at approximately 3000 g for 20-30 minutes. On an industrial scale, the soluble fraction may be recovered using continuous flow centrifugation or other methods such as filtration. In the case of filtration, different types of membranes may be used.

[0050] The desired technical effect is obtained by heating while passing through the tube, and preferably in the absence of or without the addition of an organic solvent.

[0051] The apparatus may include a zone (such as a chamber or the length of the pipe) prior to the heating pipe zone for preheating the composition before it flows through the pipe to a desired temperature. For example, if the composition is at room temperature, preheating may raise the composition temperature to about 80°C. The apparatus may also provide a zone after the heating pipe zone for cooling the composition to a temperature below 100°C (preferably about 60 to a maximum of 80°C) so that the soluble fraction can be recovered.

[0052] When the relative amount of casein increases compared to the total amount of protein, the proportion of casein in the soluble fraction increases.

[0053] The inventors have shown that when the composition passes through a pipe, and therefore heating for a very short period (less than 5 minutes, preferably less than 2 minutes, more preferably less than 1 minute), it is possible to increase or maintain the amount of casein in the soluble fraction of the composition while decreasing the amount of other proteins in the soluble fraction. Therefore, the recovered fraction is enriched with casein.

[0054] Other proteins are non-casein proteins. Therefore, this method makes it possible to obtain a casein-enriched composition. The enrichment of casein in the composition represents an increase in the soluble fraction of casein compared to the amount present in the soluble fraction of the initial composition before flow through the pipe.

[0055] Such a method can be used to isolate or purify casein from compositions containing casein and other proteins. The method can also be used to enrich a protein composition with casein, i.e., to increase the proportion of casein in the protein fraction of the protein composition.

[0056] In the methods described herein, “casein composition” refers to a composition containing casein. The casein composition must contain a soluble fraction in order to use a purification process based on the separation of soluble and insoluble fractions. However, this casein composition can then be dried in a further step. In some embodiments, casein accounts for more than 25% of the protein in the composition. In other embodiments, casein accounts for more than 50% of the protein in the composition. In some embodiments, casein accounts for the majority (more than 50%) of the dry weight. Such a casein composition may also contain other compounds, particularly calcium, other proteins, lipids, and others. A casein composition produced by microbial culture is a non-animal derived casein composition.

[0057] In view of this, the recovered soluble fraction may contain only casein at the end of the method, or it may also contain other proteins (if the precipitation and / or degradation of other proteins during heating is not complete). However, as described above, it is preferable that casein accounts for at least 50% (by weight) of the protein in the recovered soluble fraction obtained after performing the method. In preferred embodiments, casein accounts for at least 60%, at least 62%, at least 65%, at least 67%, or at least 70%, at least 71%, at least 72%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the protein in the recovered soluble fraction obtained after performing the method. The amount of protein can be measured by any method known in the art (using gels, protein quantification).

[0058] As described above, the casein and other protein-containing composition used in (i) may also contain other components such as carbohydrates or lipids. This is especially true when the casein and other protein-containing composition results from the culture of recombinant microorganisms (including eukaryotic cells) that express casein. Such other components (all or part of them) will be present in the casein composition.

[0059] (i) The composition containing casein and other proteins used in (i) may contain microorganisms suspended in a suitable buffer or solution, more preferably water, or, if casein is secreted by the microorganisms, the supernatant resulting from the culture of the microorganisms.

[0060] In certain embodiments, the initiation composition (a composition containing casein and other proteins) contains alpha-S1 casein. In one embodiment, alpha-S1 casein is the only casein in the initiation composition.

[0061] In certain embodiments, the initiation composition (a composition containing casein and other proteins) contains alpha-S2 casein. In one embodiment, alpha-S2 casein is the only casein in the initiation composition.

[0062] In certain embodiments, the initiation composition (a composition containing casein and other proteins) contains beta-casein. In one embodiment, beta-casein is the only casein in the initiation composition.

[0063] In certain but undesirable embodiments, the initiating composition (a composition containing casein and other proteins) contains kappa-casein. Therefore, in preferred embodiments, the initiating composition (a composition containing casein and other proteins) does not contain kappa-casein.

[0064] In certain embodiments, the initiation composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 casein and beta-casein. In one embodiment, alpha-S1 casein and beta-casein are the only caseins in the initiation composition.

[0065] In certain embodiments, the initiating composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 casein and alpha-S2 casein. In one embodiment, alpha-S1 casein and alpha-S2 casein are the only caseins in the initiating composition.

[0066] In certain embodiments, the initiation composition (a composition containing casein and other proteins) contains a mixture of alpha-S2 casein and beta-casein. In one embodiment, alpha-S2 and beta-casein are the only caseins in the initiation composition.

[0067] In certain embodiments, the initiating composition (a composition containing casein and other proteins) contains a mixture of alpha-S1, alpha-S2 casein, and beta-casein. In one embodiment, alpha-S1 casein, alpha-S2 casein, and beta-casein are the only caseins in the initiating composition.

[0068] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 casein, alpha-S2 casein, beta-casein, and kappa-casein.

[0069] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of combinations of two to three caseins selected from alpha-S1 casein, alpha-S2 casein, beta-casein, and kappa-casein.

[0070] If the initial composition is obtained after the production of casein by culturing microorganisms, such microorganisms are transgenic to the genes that enable the production of the casein(s) desired in the composition.

[0071] The soluble fraction may be subjected to further purification steps, such as filtration (especially ultrafiltration, nanofiltration, or reverse osmosis), centrifugation, chromatography, or other precipitation, such as acid precipitation of casein, to further purify and / or enrich the casein. In particular, acid precipitation may be carried out at a pH of 4-5 (e.g., pH=4.6) at about 90°C, but is preferably carried out at room temperature (about 20°C). Multiple other purification steps can be performed.

[0072] In certain embodiments, the microorganism is a bacterial cell. In preferred embodiments, the microorganism is Escherichia coli. In other embodiments, the microorganism is a fungal cell (including yeast cells). In yet another embodiment, the microorganism is a eukaryotic cell, particularly a plant cell. It is certainly preferable that the casein is produced in a non-animal organism.

[0073] Microorganisms can be cultured by fermentation using various sugars and nitrogen sources as raw materials. In a preferred embodiment, fermentation is carried out on a medium containing glucose, sucrose, or lactose. In a preferred embodiment, fermentation is carried out on a medium containing glucose, sucrose, or lactose as a carbon source. In a preferred embodiment, fermentation is carried out on a medium containing liquid or gaseous ammonia, urea, amino acids, peptone, or yeast extract as a nitrogen source. In a preferred embodiment, fermentation is carried out on a medium containing whey. In a preferred embodiment, fermentation is carried out on a medium containing acidic whey. In a preferred embodiment, fermentation is carried out on a medium containing acidic whey and another sugar as an additional sugar source. In a preferred embodiment, fermentation is carried out on a medium containing acidic whey and glucose, sucrose, or lactose as an additional sugar source.

[0074] When casein is produced by culturing or fermenting microorganisms (especially bacteria) transformed to express casein, the properties of casein (remaining soluble at high temperatures, while other proteins precipitate and / or degrade) are utilized. i) preparing a microbial composition (preferably a bacterial composition), wherein the microbial composition comprises a microorganism (preferably a bacterium) transformed with at least one nucleic acid encoding casein, the microorganism is cultured under culture conditions to express and produce casein, and the pH of the composition is 6.5 or higher, preferably less than 9. ii) If necessary, wash the microbial composition to remove the culture medium, iii) If necessary, dissolve the microbial composition to obtain a liquid composition, wherein the liquid composition is contained in the first container. iv) Flowing the liquid composition from the first container through a device equipped with a pipe, wherein the liquid composition is heated to a temperature of 75°C or higher during the flow, v) To recover the soluble fraction from the pipe and thereby obtain a casein composition, A method for producing a casein composition containing the above can be carried out.

[0075] When this embodiment is performed (casein produced by transgenic microorganisms), the casein often remains in an insoluble fraction (such as inclusion bodies within bacteria). Therefore, performing the method disclosed herein makes it possible to solubilize the casein and move it from the insoluble fraction to the soluble fraction, thereby providing a liquid soluble casein composition.

[0076] The soluble fraction may be recovered by centrifuging the liquid composition flowing from the pipe using a conventional centrifuge or a disk-stacked centrifuge. Alternatively, it can be obtained by microfiltration or diafiltration.

[0077] The soluble fraction recovered in v) may be subjected to one further step as needed, selected from the following: addition of activated carbon, chemical resin, membrane filtration (ultrafiltration, nanofiltration, or reverse osmosis), centrifugation, chromatography, or casein precipitation.

[0078] In iv), heating the microbial composition (preferably a bacterial composition) also induces cell lysis to produce a microbial extract if the cells have not yet lysed, and the soluble fraction is enriched with casein and / or the amount of other proteins in the soluble fraction decreases.

[0079] Therefore, if the microbial composition contains microorganisms that have already been dissolved, heating in iv) enriches casein in the soluble fraction and / or reduces the amount of other proteins in the soluble fraction, and if the microorganisms present in the microbial composition have not yet been dissolved, a further effect of microbial dissolution will be observed. In fact, it is foreseeable that microbial dissolution can be carried out by any method known in the art, preferably by chemical substances (such as detergents), or by enzymatic dissolution using, for example, lysozyme or proteinase K, or mechanically by using a French press.

[0080] The enrichment of casein or a decrease in other proteins in the soluble fraction is observed compared to the soluble fraction of the microbial composition in which microorganisms were dissolved before heating.

[0081] Therefore, such a method makes it possible to obtain a casein composition and to isolate a casein-rich (or casein-enriched) composition from a microbial culture. A casein-rich composition is a composition in which casein constitutes the major protein. In a preferred embodiment, casein accounts for more than 50% of the protein in the casein-rich composition. In a more preferred embodiment, casein accounts for more than 80%, or 90% or 95%, of the protein in the casein-rich composition.

[0082] Indeed, as shown in the examples, when heated, casein moves from the insoluble fraction (pellet) to the soluble fraction (supernatant). The examples also demonstrate that, even if the duration of heating is shorter than that described in International Publication No. 2022 / 253816, this is essentially casein that is transferred to the soluble fraction by the action of heat.

[0083] The microbial composition contains microbial cells suspended in a suitable liquid (such as water or other buffer, as described below). Therefore, centrifugation can yield an insoluble (pellet) fraction and a soluble fraction (supernatant). However, it is preferable to perform the method before centrifugation. In this embodiment, if heating is performed, the insoluble fraction may be suspended within the soluble fraction. Therefore, centrifugation or another method (such as filtration) may be used to recover the soluble fraction.

[0084] In one embodiment, the microbial composition (preferably a bacterial composition) was obtained by centrifugation, washing, and resuspension of the cultured microbial cells (preferably bacteria) in a suitable liquid or fluid (a suitable buffer, more preferably water, more preferably in the absence of an organic solvent). The buffer is non-acidic (6.5 or higher) pH, preferably near neutral (6.5 to 7.5) or basic (preferably less than 9). In fact, casein may precipitate at acidic pH, and the process is further accelerated by heating, as shown in Example 7. It is also preferable that the buffer is non-ionic or has low ionic strength. The soluble fraction of iii) can be obtained by centrifugation of the composition heated in ii), or by other methods known in the art.

[0085] Therefore, when the composition of i) in the method disclosed above passes through the pipe, it is heated to a temperature higher than 75°C (heating temperature). More preferably, the temperature is higher than about 80°C, more preferably higher than about 85°C, more preferably higher than about 90°C, more preferably higher than about 95°C, and most preferably higher than about 100°C or higher. It may be recommended to use temperatures of 105°C or higher, or 110°C or higher. Higher temperatures of 140°C or even 150°C are possible. It is preferable to heat the composition for a maximum of 3 minutes, or even more preferably a maximum of 1 minute. In this case, a suitable temperature is preferably 95°C to 115°C, preferably 100°C to 115°C (including the limit). A temperature of about 110°C would be suitable. Such temperatures allow for technical and functional effects of removing other proteins, thereby reducing their amounts and / or moving casein from the insoluble fraction to the soluble fraction, while heating for a short time (up to 5 minutes, preferably up to 2 minutes, but rather 90 seconds or less, or 75 seconds or less, more preferably 60 seconds or less, or even 45 seconds or less). Examples have shown that durations of 30 seconds or even 2 seconds can be applied. However, it is preferable to heat the composition flowing through the pipe for at least 5 seconds or at least 10 seconds.

[0086] The pressure inside the pipe is typically higher than 3 bar, more preferably 4 bar or more, and more preferably 4.5 bar or more. This is typically 7 bar or less, preferably 6.5 bar or less, and more preferably 6 bar or less. Pressures between 4.5 bar and 5.5 bar (including the limit) are well-suited.

[0087] When referring to measurable values, the term "about" means that a variation of ±3% from the specified value is included, where such variation is appropriate for performing the disclosed method.

[0088] The examples illustrate various conditions that enable the purification of casein from recombinant Escherichia coli (E. coli). While these examples also describe specific conditions, it is worth noting the specific temperature / holding time lengths at these temperatures (110°C / 75 sec; 110°C / 30 sec; 120°C / 30 sec; 140°C / 30 sec; 140°C / 2 sec) disclosed to address the technical problem of purifying casein. It may be recommended to avoid overheating the composition and to use longer times rather than very short times.

[0089] As described above, it is recommended to preheat the composition coming from the first vessel before carrying out the heating process in the pipe. This is done in a chamber outside the first vessel, or in the pipe that guides the composition from the first vessel to the pipe to be heated for a desired time and at a desired temperature. The preheating temperature may be 20°C or 30°C lower than the target heating temperature, or about 20°C to about 60°C lower than the heating temperature. Preheating to 75°C to 90°C (e.g., 80°C) is well suitable.

[0090] To obtain the desired temperature within the pipe, especially when heating by heat exchange, heating is performed at a temperature higher than the desired temperature (usually about 5°C higher) to account for the temperature gradient from the outside of the pipe to the center of the pipe. Heating can be carried out by one of the following methods:

[0091] Direct steam injection: Steam is injected directly into the pipe to heat it. The steam is generated in a boiler and sent to the pipe, where it transfers heat to the pipe walls and heats the product flowing through it.

[0092] Indirect heating by heat exchange: Double-walled pipes are used. The composition flows through the inner tube, and the steam circulates in the space between the two walls. Heat from the steam is transferred through the pipe walls, heating the product.

[0093] Electric heating: A heating element is incorporated into the pipe to heat it, using electricity to generate heat directly on the pipe wall.

[0094] Inductive beam heating: This method uses electromagnetic induction to heat a pipe by the Joule effect, by using an alternating current induced in the pipe wall with the help of a magnetic field.

[0095] In some embodiments, the composition is circulated through the pipe at a flow rate of 15 L / hour to 30 L / hour.

[0096] The composition is usually cooled and, after flowing, supplied to a second container. In some embodiments, the soluble fraction recovered by centrifugation and / or filtration after flowing and heating is supplied to the second container. Further purification or concentration of casein (e.g., by evaporation) and / or quantification of casein in the soluble fraction can be performed.

[0097] As described above, certain embodiments include situations in which the initial composition (a composition containing casein and other proteins) is obtained from a bacterial culture. In such embodiments, the bacteria are transformed with one or more nucleic acids encoding two or more caseins.

[0098] In particular, in one embodiment, bacteria are transformed with one or more nucleic acids encoding beta-casein.

[0099] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding alpha-S1 casein.

[0100] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding alpha-S2 casein.

[0101] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding both beta-casein and alpha-S1 casein.

[0102] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding beta-casein, alpha-S1 casein, and alpha-S2 casein, or a combination of two of these.

[0103] It should be noted that when multiple proteins are produced, bacteria can be transformed with different nucleic acids (each encoding a different protein) or with a single nucleic acid (containing elements that enable the production of various proteins). Methods for such transformation of microorganisms, and especially bacteria, to produce one or more proteins are known in the art.

[0104] Nucleic acids are those that contain all the elements (promoters, enhancers, terminators, and so on) that enable the production of the casein they encode. A person skilled in the art knows all these elements and can select the most appropriate ones. A person skilled in the art can also select the microorganism to use to produce casein.

[0105] In particular, suitable prokaryotic hosts for casein expression include Escherichia coli (E. coli), Bacillus subtilis, Salmonella typhimurium, Lactococcus lactis, and various species within the genera Lactococcus, Pseudomonas, Streptomyces, and Staphylococcus.

[0106] Suitable eukaryotic hosts for casein expression include fungi such as Saccharomyces cerevisae, Kluyveromyces lactis, Pichia pastoris, or Trichoderma reesei.

[0107] Plant cells may be used to produce proteins in cell culture or in the plant as a whole.

[0108] Further steps such as the addition of chemical resins, activated carbon, membrane filtration (especially ultrafiltration, nanofiltration, or reverse osmosis), centrifugation, chromatography, or casein precipitation can be added, resulting in: i) preparing a microbial composition (preferably a bacterial composition), wherein the microbial composition comprises a microorganism (preferably a bacterium) transformed with at least one nucleic acid encoding casein, the microorganism is cultured under culture conditions to express and produce casein, and the pH of the composition is 6.5 or higher, preferably less than 9. ii) If necessary, wash the microbial composition to remove the culture medium, iii) If necessary, dissolve the microbial composition to obtain a liquid composition, wherein the liquid composition is contained in the first container. iv) Flowing the liquid composition from the first container through a device equipped with a pipe, wherein the liquid composition is heated at a temperature of 75°C or higher for a maximum of 5 minutes while it is flowing through the pipe, v) The soluble fraction is recovered from the pipe, vi) Further processing of the soluble fraction by adding at least one step selected from activated carbon addition, membrane filtration, chromatography, or casein precipitation, thereby obtaining a casein composition. A method including this can be obtained.

[0109] In this embodiment, heating during the flow in iv) further induces the dissolution of microorganisms, resulting in a microbial extract in which casein is enriched and / or other proteins are reduced.

[0110] In addition, the heating process is performed independently of cell lysis. i) Preparing a microbial extract in a first container, wherein the microbial extract is obtained from the lysis of a microbial culture containing microorganisms transformed with nucleic acids encoding casein and cultured to express and produce casein, ii) Fluidizing the microbial extract from the first container in an apparatus equipped with a pipe, wherein the liquid composition is heated at a temperature of 75°C or higher for a maximum of 5 minutes for at least a portion of the time the fluid is flowing in the pipe. iii) The soluble fraction is recovered from the pipe, iv) Further processing the soluble fraction by adding at least one step selected from the addition of a chemical substance, activated carbon, membrane filtration, centrifugation, chromatography, or casein precipitation, thereby obtaining a casein composition. It can be used in methods that include [specific methods].

[0111] The soluble fraction recovered in iii) can be further processed to increase the purity of casein.

[0112] Activated carbon can be added to the soluble fraction and stirred. The activated carbon can be used to adsorb impurities (particularly organic impurities or chlorine) present in the soluble fraction that were not removed when the soluble fraction was recovered in iii). Stirring with the activated carbon is preferable, and the activated carbon is removed before another method (such as those disclosed below) is performed on the soluble fraction.

[0113] Chromatographic methods are widely used in purification, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and others.

[0114] Membrane filtration, particularly ultrafiltration and nanofiltration, is commonly used in protein purification (Saxena et al. (2009) Membrane-based techniques for the separation and purification of proteins: An overview. Advances in colloids and Interface Science Volume 145, pages 1-22), and membrane filtration techniques are widely used in the dairy industry. Interestingly, these can be used to separate different caseins from each other (see above).

[0115] Furthermore, the specific properties of casein (Post et al. (2012) Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of caseins. J. Dairy Sci. Volume 95: pages 1603-1616), and in particular, their tendency to precipitate under acidic conditions, can be used for further purification.

[0116] In a preferred embodiment, the soluble fraction is further treated by casein precipitation under acidic conditions. After precipitation, the casein can be resuspended and resolubilized using a suitable basic buffer (Post et al. (2012) Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of caseins. J. Dairy Sci. Volume 95: pages 1603-1616). In a preferred embodiment, the soluble fraction is further treated by casein precipitation at a pH in the range of pH=4 to pH=5. In a more preferred embodiment, casein precipitation is carried out at a pH of about 4.6. Various acids, including lactic acid, hydrochloric acid, and sulfuric acid, can be used.

[0117] Next, the casein can be recovered by methods such as centrifugation or tangential flow filtration. A washing step may be added using water adjusted to the pH used for precipitation. The washed casein can be separated using the methods cited above.

[0118] Precipitation under acidic conditions is particularly interesting when isolating casein from microbial cultures, as it allows for the removal or degradation of any nucleic acids that may be present in the casein composition. When casein is used to obtain edible compositions intended for human consumption (such as cheese substitutes), the presence of microbial DNA or RNA may prove harmful (at least from a regulatory standpoint). The pH is approximately 4.6 and its range, the temperature is in the range of 80°C to 140°C, and the time can vary from a few minutes to two hours.

[0119] Alternatively, recombinant DNA can be removed using nucleases such as DNAse, which can be removed by heating.

[0120] When using microorganisms such as Gram-negative bacteria, additional steps may be taken to remove lipopolysaccharide (LPS), and high-temperature treatment under acidic or basic conditions, use of activated carbon at high temperatures, or filtration methods (10-20 kDa separation, or various extraction methods may be used).

[0121] Next, the pH can be neutralized to obtain the casein salt. For this purpose, various basic compounds such as liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, or calcium hydroxide can be added. The target pH may be in the range of 6.5 to 9. The kinetics of pH adjustment may be continuous or follow a series of plateaus to achieve a stable target pH. Before drying, the casein can be concentrated by filtration or evaporation. The casein can then be dried by spray drying, flash drying, or other methods known in the art.

[0122] In a preferred embodiment, the pH is adjusted using sodium hydroxide. In another embodiment, the pH is adjusted using potassium hydroxide. In yet another embodiment, the pH is adjusted using calcium hydroxide.

[0123] In certain embodiments, the casein composition is dried. When obtained from a bacterial culture, such a composition should contain about 15% (by weight) to 30% (by weight) of casein and carbohydrates in the dried composition.

[0124] It is preferable that a small amount of water remains to promote future rehydration. Therefore, drying should be understood as reducing the amount of water. In some embodiments, the amount of water is about 50% (by weight) or less.

[0125] The insoluble fraction, which contains the majority of the biomass produced during fermentation, can be processed separately and given added value. This biomass can be used for feed or food applications after being treated in the same way as casein to remove recombinant DNA: the pH is in the range of 4.6, the temperature is in the range of 80°C to 140°C, and the time can vary between a few minutes and 2 hours; or the recombinant DNA can be removed using a nuclease such as DNAse, which can be removed by heating.

[0126] In certain embodiments, the present invention is i) preparing a bacterial composition in a first container, wherein the bacterial composition comprises bacteria transformed with at least one nucleic acid encoding casein, and the bacteria are cultured to express and produce casein, ii) Fluidizing the bacterial composition from a first container in a device equipped with a pipe, and heating the composition while it is flowing through the pipe, wherein the heating is performed at a temperature of 75°C or higher for a maximum of 5 minutes, iii) Recovering the soluble fraction from the heated cell composition of ii), iv) If necessary and preferably, the soluble fraction is further processed by adding at least one step selected from the addition of activated carbon, membrane filtration, chromatography, or casein precipitation, thereby obtaining a casein composition in the soluble fraction. This invention relates to a method for producing a casein composition, including the above.

[0127] If the bacteria in the bacterial composition are not lysed, heating in ii) also induces cell lysis, thereby producing a bacterial extract.

[0128] In other embodiments, the present invention is i) preparing a bacterial composition in a first container, wherein the bacterial composition comprises bacteria transformed with at least one nucleic acid encoding casein, and the bacteria are cultured to express and produce casein, ii) Fluidizing the bacterial composition from a first container in a device equipped with a pipe, and heating the composition while it is flowing through the pipe, wherein the heating is performed at a temperature of 75°C or higher for a maximum of 5 minutes, iii) Recovering the soluble fraction from the heated cell composition of ii), iv) Further treatment of the soluble fraction by precipitation of casein under acidic conditions, thereby obtaining a casein composition (in the precipitate), This invention relates to a method for producing a casein composition, including the above.

[0129] In this embodiment, if the bacteria in the bacterial composition are not dissolved, the heating in ii) also induces the dissolution of the bacteria, thereby producing a bacterial extract.

[0130] In other embodiments, the present invention is i) preparing a bacterial composition in a first container, wherein the bacterial composition comprises bacteria transformed with at least one nucleic acid encoding casein, and the bacteria are cultured to express and produce casein, ii) Fluidizing the bacterial composition from a first container in a device equipped with a pipe, and heating the composition while it is flowing through the pipe, wherein the heating is performed at a temperature of 75°C or higher for a maximum of 5 minutes, iii) Recovering the soluble fraction from the heated cell composition of ii), iv) Further treatment of the soluble fraction by precipitation of casein by heating at 90°C at pH=4, thereby obtaining a casein composition (in the precipitate), This invention relates to a method for producing a casein composition, including the above.

[0131] In a preferred embodiment, heating the bacterial composition induces cell lysis.

[0132] Therefore, the methods disclosed herein make it possible to obtain a casein composition. Such a casein composition that can be obtained (or obtained) by the methods disclosed herein is a further subject of the present invention. Such a composition can be characterized as defined above.

[0133] In milk, casein is present in the micelle form found in milk, and these micelles include alpha-S1 casein, alpha-S2 casein, beta-casein, and kappa-casein assembled within the same particle. In this invention, casein may not be assembled in the micelle form. However, this does not prevent the ability to obtain curd from the casein composition by adding a suitable coagulant.

[0134] In fact, as demonstrated in the examples, such casein compositions can be used to produce cheese substitutes, and in particular animal-free cheese substitutes, i.e., cheese substitutes that do not contain any animal-derived products (especially when the casein is isolated from a bacterial or yeast culture).

[0135] In summary, simply heating a composition containing casein and other proteins through a pipe for a short time (less than 5 minutes, or preferably less than 2 minutes) allows for enrichment of the casein in the composition by reducing the amount of the other proteins in the soluble fraction. Heating also allows for the solubilization of casein found in the insoluble fraction of bacterial cultures. The resulting casein, while not under optimal conditions for micelle formation, can be properly coagulated by the action of a coagulant. This finding was unexpected.

[0136] In particular, as shown in International Publication No. 2022 / 253816, despite the fact that recombinant casein does not have post-translational modifications (such as phosphorylation) and despite the fact that kappa-casein is known to play an important role in the formation of milk casein micelles, coagulation can be achieved using bacterial recombinant casein in the absence of kappa-casein.

[0137] Therefore, compositions disclosed herein and obtained or obtainable by the above methods are, i) Preparing the casein composition disclosed herein, ii) Mixing the casein composition with at least one other component, which is selected from the group consisting of water, calcium, lipids, and carbohydrates, to obtain a liquid precard composition (LpCC), iii) Adding at least one coagulant to the liquid composition in order to obtain a card, It can be used in methods for obtaining cards, including [mention specific methods / methods].

[0138] Such embodiments are disclosed in particular in International Publication No. 2022 / 058573 and International Publication No. 2022 / 253816.

[0139] As indicated, the coagulant is preferably an acidifying agent, particularly lactic acid bacteria, or lactic acid, citric acid, or acetic acid, rather than rennet. The casein composition may contain only alpha-S1 casein (as casein protein), or only alpha-S2 casein (as casein protein), or only beta-casein (as casein protein), or only alpha-S1 casein and beta-casein (as casein protein), or only alpha-S2 casein and beta-casein (as casein protein), or only alpha-S1 casein and alpha-S2 casein (as casein protein), or alpha-S1 casein, alpha-S2 casein, and beta-casein.

[0140] The curd may be processed to obtain edible compositions, in particular cheese substitutes, as disclosed in International Publication No. 2022 / 058573 and International Publication No. 2022 / 253816. These two documents are incorporated herein by reference in part, in particular, in the parts describing obtaining LpCC or edible compositions starting from compositions containing casein, in particular bacterial casein, and / or casein without kappa-casein, in particular, in the parts relating to obtaining cheese substitutes having the essential characteristics of fresh cheese and cheese substitutes having the essential characteristics of soft or semi-soft cheese. The parts of these documents, in particular International Publication No. 2022 / 253816, relating to calcium and other salts, lipids, emulsifiers and gelling agents, carbohydrates, vitamins, coagulants, fermented products, moisture, and processing of the curd are also incorporated herein by reference.

[0141] It is conceivable that a gelling agent (especially agar) can be added along with other components before or after solidification.

[0142] b) is preferable if all other components added are of non-animal origin.

[0143] The components added in ii) are: i. Proteins other than casein as needed ii. Lipids iii. Water, and iv. Carbohydrates It includes; in a preferred embodiment, the carbohydrates are lactose-free so that the edible composition is more acceptable to customers with lactose intolerance.

[0144] The selection and quantity of ingredients are adjusted to ensure that the composition of LpCC conforms to the desired final composition for the edible composition, taking into account water loss due to coagulation and maturation (which can be controlled by those skilled in the art by changing the duration and conditions of maturation).

[0145] The present invention i) preparing a bacterial composition in a first container, wherein the bacterial composition comprises bacteria transformed with at least one nucleic acid encoding casein (preferably not kappa-casein), and the bacteria are cultured to express and produce casein, ii) Fluidizing the bacterial composition from a first container in a device equipped with a pipe, and heating the composition while it is flowing through the pipe, wherein the heating is performed at a temperature of 75°C or higher for a maximum of 5 minutes, iii) Recovering the soluble fraction from the heated cell composition of ii) iv) If necessary and preferably, the soluble fraction is further processed by adding at least one step selected from the addition of activated carbon, membrane filtration, chromatography, or casein precipitation, thereby obtaining a casein composition. v) Mixing the casein composition with at least one other component, which is selected from the group consisting of water, calcium, lipids, and carbohydrates, to obtain a liquid precard composition (LpCC), vi) Adding at least one coagulant to the liquid composition in order to obtain a card, This also includes methods. [Brief explanation of the drawing]

[0146] [Figure 1] Structure of a plasmid expressing beta-casein and alpha-S1 casein. [Figure 2] Product monitoring of supply pressure and temperature during the heating process and at the outlet of the sterilization chamber (tubular heat exchanger). Solid line: Product heating temperature. Thin dotted line: Product temperature at the outlet of the sterilization chamber. Thick dotted line: Supply pressure. A. Test 1: Holding temperature: 110℃ - Heating time: 75 seconds B. Test 2: Holding temperature: 110℃ - Heating time: 30 seconds C. Test 3: Holding temperature: 120℃ - Heating time: 30 seconds D. Test 4: Holding temperature: 140℃ - Heating time: 30 seconds E. Test 5: Holding temperature: 140℃ - Heating time: 2 seconds [Figure 3]SDS-PAGE analysis of αs1-casein and β-casein in soluble and insoluble fractions of the dissolved solution, before and after heat treatment. Lane 1: Size marker, Precision Plus Protein (trademark) Unstained Standards (Bio-Rad, 1610363EDU) - Lane 2: Cell suspension before heat treatment, soluble fraction - Lane 3: Lysate after heat treatment (Test 1), soluble fraction - Lane 4: Lysate after heat treatment (Test 2), soluble fraction - Lane 5: Lysate after heat treatment (Test 3), soluble fraction - Lane 6: Lysate after heat treatment (Test 4), soluble fraction - Lane 7: Lysate after heat treatment (Test 5), soluble fraction - Lane 8: Cell suspension before heat treatment, insoluble fraction - Lane 9: Lysate after heat treatment (Test 1), insoluble fraction - Lane 10: Lysate after heat treatment (Test 2), insoluble fraction - Lane 11: Lysate after heat treatment (Test 3), insoluble fraction - Lane 12: Lysate after heat treatment (Test 4), insoluble fraction - Lane 13: Lysate after heat treatment (Test 5), insoluble fraction. [Figure 4] SDS-PAGE analysis of αs1-casein and β-casein in soluble and insoluble fractions of lysates before and after heat treatment. Analysis of 3-fold diluted samples. For each panel, Lane 1: Size marker, Precision Plus Protein (trademark) Unstained Standards (Bio-Rad, 1610363EDU) - Lane 2: Cell suspension before heat treatment, soluble fraction - Lane 3: Cell suspension before heat treatment, insoluble fraction - Lane 4: Lysate after heat treatment, soluble fraction - Lane 5: Lysate after heat treatment, insoluble fraction. Panel A: 110°C, 75 sec; B: 110°C, 30 sec; C: 120°C, 30 sec; D: 140°C, 30 sec; E: 140°C, 2 sec. [Examples]

[0147] Example 1: Production of alpha-S casein and beta-casein Synthetic genes encoding alpha-S1 casein and beta-casein (corresponding to the native genes P02662 and P02666, respectively) were modified to remove the signal peptide, and the sequences of the novel synthetic open reading frames (SEQ ID NOs. 2 and 4) are shown in the last column of Table 1. These were cloned into pET25b+ (Figure 1), and the resulting plasmids were transformed into the BL21(DE3) strain (Novagene). Individual transformed clones were isolated, and for each synthetic gene, one clone was used to inoculate into LB medium.

[0148] [Table 2]

[0149] To produce batches of alpha-S1 casein and beta-casein, the obtained strains were cultured as follows: Five concentrated stocks of each strain (1 mL each, stored in 10% DMSO at -80°C) were thawed and used to inoculate five pre-cultures of 1 L each in reactivated medium Y15 consisting of yeast extract (15 g / L) and NaCl (5 g / L), supplemented with 100 μg / mL ampicillin (inoculation rate of 0.1% (volume / volume)). The five pre-cultures were grown in 4 L or 5 L Erlenmeyer flasks at 30°C for 9 hours at 170 rpm (swirl diameter: 25 mm). After 9 hours, an optical density in the range of 3 (60 nm) was achieved.

[0150] Using 2.4 L of the initial pre-culture, it was inoculated to a volume of 120 L in optimized culture medium supplemented with 100 μg / mL ampicillin and 15 g / L glucose (inoculation rate of 2% (volume / volume)). Growth parameters (oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density (600 nm) in the range of 15.

[0151] Using 90 L of this culture, it was inoculated into a 1500 L initial volume fermenter in optimized culture medium supplemented with 100 μg / mL ampicillin (inoculation rate of 6% (volume / volume)). Growth parameters (glucose feed, oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density in the range of 90 (600 nm). When the optical density reached 20, IPTG was added at a final concentration of 0.2 mM. This yielded approximately 2 tons of culture medium with 3.4% dry biomass.

[0152] Example 2: Thermal dissolution of bacteria by ultra-high temperature (UHT) treatment At extremely high temperatures, α s1 We established protocols for the extraction and solubilization of casein and β-casein.

[0153] Biomass concentration and washing The culture medium obtained as described in Example 1 was concentrated using a self-cleaning disk stacking centrifuge (SSE20, GEA, centrifugal force: 20000G, bowl rotation speed: 12000rpm, bowl filling: 80% of capacity, flow rate: 500L / hour, back pressure: 5 bar, temperature <20°C, sedimentation time: 54 seconds for the first solid-liquid separation (biomass concentration), and 40 seconds for the three biomass washing steps). 298 kg of concentrated cells were recovered, while 1670 kg of fermentation medium was discarded.

[0154] Next, the concentrated biomass was washed three times with leachate using the same centrifuge. The concentrated biomass was mixed with water until a homogeneous suspension was obtained (11.17% of the dry mass). The suspension was then separated by centrifugation, and the first fraction of the wash water (800.4 kg) was removed. This washing procedure was repeated twice, and finally a pellet of concentrated cells weighing 239.1 kg was obtained. The color of the supernatant gradually faded from yellow until a slightly colored yellow wash water was obtained after the third wash.

[0155] Thermal dissolution of bacteria Next, the washed concentrated biomass was resuspended in 4.2 times its volume of leachate until a homogeneous suspension was obtained. 60 L of this suspension was stored at +4°C for 4 days without stirring. After 4 days, the pH of the suspension was 6.654 ± 0.057. The suspension was then divided into four 10 kg fractions and one 8.15 kg fraction. The pH of each suspension was adjusted to 8.5 using a 2 M sodium hydroxide (NaOH) solution. The pH and temperature of the different fractions recovered before and after pH adjustment are shown in Table 2.

[0156] [Table 3]

[0157] Next, five fractions were heated using a UHT sterilization device (sterilization device, OMVE, HTST / UHT system, HT220-DSI, initial product temperature: 20°C, configuration: tubular heat exchanger, flow rate: 16.66~27.2 L / hour, holding time: 2~75 seconds, preheating temperature: 80°C, heating temperature: 113~145°C, temperature inside sterilization chamber: 110~140°C, precooling temperature: 80°C, cooling temperature: 40°C, pressure: 5 bar), by monitoring the holding time at a predetermined temperature inside the sterilization chamber.

[0158] To determine the optimal and rapid heat treatment that enables efficient bacterial lysis, E. coli protein precipitation, and casein solubilization, five different conditions were tested on five bacterial suspensions. Table 3 lists the applied flow rate, holding time, preheating and heating temperatures, as well as the precooling and cooling temperatures for each case. The different set heating temperatures were slightly higher than the temperatures applied in the sterilization chamber to ensure these final temperatures were achieved. Test 1: Temperature = 110°C, Holding time = 75 seconds Test 2: Temperature = 110°C, Holding time = 30 seconds Test 3: Temperature = 120°C, Holding time = 30 seconds Test 4: Temperature = 140°C, Holding time = 30 seconds Test 5: Temperature = 140°C, holding time = 2 seconds, on a micropilot scale.

[0159] General conditions are described in detail in Table 3. All of these different conditions allow for the complete inactivation of the E. coli (E. coli) strain used in fermentation.

[0160] [Table 4-1] [Table 4-2]

[0161] Product: The quantity of the sample to be processed. Initial product temperature: The initial temperature of the product to be processed. Configuration: A tubular chamber (sterilization chamber) preceded by two heat exchangers (one heat exchanger that heats the product to be processed at the processing outlet - one heat exchanger to complete heating before the tubular chamber), followed by one heat exchanger that pre-cools and cools the processed product. Flow rate: The flow rate of the product through the sterilization chamber. Holding time: The time the product spends in the sterilization chamber. Preheating temperature: The temperature the product reaches at the outlet of the first heat exchanger. Heating temperature: The highest temperature the product reaches at the inlet of the chamber. Temperature in sterilization chamber: The temperature of the product in the tubular chamber. Pre-cooling temperature: The temperature the processed product reaches at the coldest outlet of the first heat exchanger. Cooling temperature: The temperature of the processed product reached at the outlet of the final cooling heat exchanger. The total duration of the process (preheating + heating + chamber holding + precooling + cooling) was approximately 150 to 350 seconds, preferably 150 to 320 seconds, or 200 to 320 seconds.

[0162] In each case, the product temperature was controlled during the heating process and at the outlet of the sterilization chamber, while the supply pressure was measured throughout the entire process. Monitoring of these parameters is shown in Figures 2 to 6.

[0163] For Tests 1 through 5, the weight, pH, and temperature of each fraction were measured before and after the heating process. Furthermore, the amount of 2M NaOH solution added to adjust the pH to 8.5 was also determined (Table 4).

[0164] [Table 5-1] [Table 5-2]

[0165] After the applied different heat treatments, the pH of each fraction decreased from 7.790 to 7.593, i.e., a decrease of 0.71 to 0.90 pH units. From 10 kg of product, 8.9 kg to 9.165 kg were recovered in tests 1 to 4. In the final test, a fraction of 7.115 kg was obtained from an initial weight of 8.15 kg.

[0166] To investigate the efficiency of these five different heat treatments, samples were collected before and after each test and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a polyacrylamide gel (4-20% Criterion® TGX Stain-Free Protein Gel, Bio-Rad, 5678093) using Precision Plus Protein® Unstained Standards (Bio-Rad, 1610363EDU) as the ladder.

[0167] For each test, 1 mL of bacterial suspension was taken before and after the applied heat treatment and centrifuged (13000 rpm, 5 minutes, room temperature) to separate the soluble and insoluble fractions. The supernatant was separated from the pellet and mixed in a 1:1 ratio with a 2x sample buffer consisting of 0.1 M Tris-HCl, 4% vol:vol SDS, 0.2% wt:vol bromophenol blue, and 20% vol:vol glycerol. The cell pellet was mixed with 1 mL of 2% vol:vol SDS solution until a homogeneous suspension was obtained, in the same ratio as the 2x sample buffer described above. Each resulting solution was heated at 95°C for 5 minutes and loaded into the wells of a polyacrylamide gel using a running buffer consisting of Tris-HCl (25 mM), glycine (250 mM), and SDS (0.1% vol:vol). The gel was then visualized under UV light in a Gel-Doc® EZ Imager (Bio-Rad). To achieve better quantification, diluted samples (3-fold) were also used. The ratio of casein content to total protein content was determined by analyzing each SDS-PAGE gel using Imagelab software, comparing the band intensity of casein with that of other proteins from Escherichia coli (E. coli).

[0168] The results are summarized in Figures 3 and 4, showing the undiluted (Figure 3) and diluted (Figure 4) samples. Under all test conditions, E. coli cells were efficiently dissolved for sterilization, and α s1 Most of the casein and β-casein were solubilized: the total amount of casein was very similar before and after sterilization, but these caseins were essentially in the insoluble fraction before sterilization (Figure 3, lane 8) and in the soluble fraction after sterilization (Figure 3, lanes 3-7), indicating a very high yield of the method (at least >80%).

[0169] The proportion of casein to total protein in the soluble fraction of the lysate (Figure 4, lane 4 in each panel) was significantly the same across all five studies, ranging from 75% to 77%. This proportion was significantly higher than that in the insoluble fraction of the cells before lysis (Figure 4, lane 3 in each panel), which showed many additional protein bands, as seen in Figure 4 (lane 3 in each panel). This proportion is naturally higher than that in the cell composition before lysis (insoluble + soluble), indicating that the soluble fraction of the lysate was enriched with casein compared to the initial composition.

[0170] In addition, the average αs1 casein / β casein ratio was very similar across the five studies, ranging from 47% to 53%, and was similar to the initial intracellular composition before treatment, under the conditions used by the inventors. s1 This indicates that there was no bias in the purification of casein versus β-casein.

[0171] Other conditions (using heating temperatures from 95°C to 140°C and holding times in the heated chamber from 2 seconds to 120 seconds) were also tested. All of these allowed for obtaining moderately concentrated casein with good purity in the soluble fraction after heat treatment.

Claims

1. A method for obtaining a casein composition, i) Preparing a microbial composition, wherein the microbial composition comprises a microorganism transformed with at least one nucleic acid encoding casein, the microorganism is cultured under culture conditions to express and produce casein, and the pH of the composition is 6.5 or higher, preferably less than 9. ii) If necessary, wash the microbial composition to remove the culture medium, iii) If necessary, dissolve the microbial composition to obtain a liquid composition, wherein the liquid composition is contained in the first container. iv) Flowing the liquid composition from the first container through a device equipped with a pipe, wherein the liquid composition is heated at a temperature of 95°C or higher for a maximum of 5 minutes for at least a portion of the flow, v) The soluble fraction is recovered from the pipe, thereby obtaining a casein composition in the soluble fraction. Methods that include...

2. The method according to claim 1, wherein the casein accounts for at least 60%, preferably at least 62%, preferably at least 65%, preferably at least 67%, and preferably at least 70% of the total protein in the soluble fraction.

3. The method according to claim 1 or 2, wherein the proportion of casein compared to the total protein is increased in the soluble fraction compared to the proportion in the microbial composition.

4. The method according to any one of claims 1 to 3, wherein the heating is performed at a temperature of 140°C or lower.

5. The method according to any one of claims 1 to 4, wherein heating is performed for a maximum of 2 minutes.

6. The method according to any one of claims 1 to 5, wherein heating is performed for at least 2 seconds.

7. The method according to any one of claims 1 to 6, wherein heating is performed for a maximum of 60 seconds.

8. The method according to any one of claims 1 to 8, wherein the composition is preheated at a temperature 20°C to 60°C lower than the heating temperature before being flowed at the aforementioned heating temperature.

9. The method according to any one of claims 1 to 8, wherein the composition is circulated in the pipe at a flow rate in the range of 15 L / hour to 30 L / hour.

10. The method according to any one of claims 1 to 9, wherein the composition is supplied to a second container after it has flowed.

11. The method according to any one of claims 1 to 9, wherein the soluble fraction is recovered by fluidization and heating, then by centrifugation and / or filtration, and then supplied to the second container.

12. The method according to any one of claims 1 to 11, wherein the microorganisms in the microbial composition are not dissolved, and heating in iv) dissolves the microorganisms.

13. The method according to any one of claims 1 to 12, wherein the microorganism is transformed with one or more nucleic acids encoding two or more caseins.

14. The method according to any one of claims 1 to 13, wherein the casein is selected from beta-casein, alpha-S1 casein, alpha-S2 casein, and mixtures thereof.

15. The method according to any one of claims 1 to 13, wherein the casein is selected from beta-casein, alpha-S1 casein, and mixtures thereof.

16. The method according to any one of claims 1 to 15, further comprising the step of further purifying the casein of the soluble fraction obtained in iii).

17. The method according to claim 16, wherein the further step applied to the soluble fraction includes at least one step selected from the group consisting of the addition of activated carbon, membrane filtration, chromatography, and casein precipitation.

18. The method according to claim 17, wherein the further step is acid precipitation of casein.

19. The method according to claim 18, wherein the further step of acid precipitation of casein is carried out by heating at 90°C at pH = 4.

6.

20. The method according to any one of claims 1 to 19, wherein the microbial composition is a bacterial composition.

21. The method according to claim 20, wherein the bacterium is Escherichia coli (E. coli).

22. A casein composition that can be obtained by the method described in any one of claims 1 to 21.

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